JP2003509011A - Angiogenic proteins and their use - Google Patents

Abstract

  (57) [Summary] Disclosed are human angiogenic polypeptides, their biologically active, diagnostically or therapeutically useful fragments, analogs, or derivatives, and DNA (RNA) encoding such angiogenic polypeptides. . Also provided are procedures for producing such polypeptides by recombinant techniques, as well as antibodies, agonists and antagonists to such polypeptides. Such polypeptides and polynucleotides can be used therapeutically to stimulate wound healing and for vascular tissue repair. Also provided are methods of using the antibodies and antagonists to inhibit tumor angiogenesis, and thus tumor growth, inflammation, diabetic retinopathy, rheumatoid arthritis, and psoriasis. Further provided are methods of using the antibodies and agonists to promote angiogenesis and lymphangiogenesis.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to angiogenic proteins and agonists or antagonists of angiogenic proteins. The invention further relates to screening methods for identifying agonists and antagonists of angiogenic protein activity.

RELATED ART The formation of new blood vessels, or angiogenesis, is essential for embryonic development, subsequent growth and tissue repair. Angiogenesis is also an integral part of certain pathological conditions such as neoplasms (ie tumors and gliomas). Abnormal angiogenesis is inflammation,
Associated with other diseases such as rheumatoid arthritis, psoriasis and diabetic retinopathy (F
olkman, J .; And Klagsbrun, M .; , Science 235
: 442-447 (1987)).

Both acidic and basic fibroblast growth factor molecules are
It is a mitogen for endothelial cells and other cell types. Angiotropin
otropin) and angiogenin can induce angiogenesis, but their mechanism has not been elucidated (Folkman, J.,.
Cancer Medicine, Lea and Febiger Pres
s, 153-170 (1993)). Highly selective mitogens for vascular endothelial cells include vascular endothelial growth factor or VEGF (Ferrara, N. et al., E.
ndocr. Rev. 13: 19-32 (1992)) (also known as vascular infiltration factor (VPF)).

Vascular endothelial growth factor is a secretory angiogenic mitogen whose target cells appear to be specifically restricted to vascular endothelial cells. The mouse VEGF gene was characterized and its expression pattern in embryogenesis was analyzed. Persistent expression of VEGF was observed in epithelial cells adjacent to the fenestrated endothelium (eg choroid plexus and renal glomerulus). This data is consistent with the role of VEGF as a multifunctional regulator of endothelial cell proliferation and differentiation (Breier, G. et al., Deve).
Lopment 114: 521-532 (1992)).

VEGF shares sequence homology with the human platelet-derived growth factors PDGFa and PDGFb (Leung, DW, et al., Science 246: 1306).
-1309 (1989)). The degree of homology is about 21% and 23%, respectively. The eight cysteine residues that contribute to disulfide bond formation are tightly conserved in these proteins. Although they are similar, there are specific differences between VEGF and PDGF. PDGF is the major connective tissue growth factor, whereas VEGF is highly specific to endothelial cells. Alternatively, splice mRN
A has been identified for both VEGF, PLGF and PDGF, and these different splicing products differ in biological activity and receptor binding specificity. VEGF and PDGF bind to receptors that function as homodimers or heterodimers and induce endogenous tyrosine kinase activity after receptor dimerization.

VEGF has four different forms of 121, 165, 189 and 206 amino acids due to alternative splicing. VEGF121 and VEGF
165 is soluble and can promote angiogenesis, while VEGF189 and VEGF206 bind heparin-containing proteoglycans on the cell surface.
Temporal and spatial expression of VEGF is associated with physiological growth of blood vessels (Gaj
duty, C.I. M. And Carbon, S .; J. , Cell Physio
l. 139: 570-579 (1989); McNeil, P .; L. Et al., J. C
ell. Biol. 109: 811-822 (1989)). Its high affinity binding site is localized only on endothelial cells in tissue sections (Jakeman, L.
． B. Et al., Clin. Invest. 89: 244-253 (1989)). This factor can be isolated from pituitary cells and several tumor cell lines and is associated with several human gliomas (Plate, KH, Nature 359).
: 845-848 (1992)). Interestingly, VEGF121 or VE
Expression of GF165 confers the ability of Chinese hamster ovary cells to form tumors in nude mice (Ferrara, N. et al., J. Clin. Inve.
st. 91: 160-170 (1993)). Inhibition of VEGF function by VEGF monoclonal antibody has been shown to inhibit tumor growth in immunodeficient mice (Kim, KJ, Nature 362: 841-844 (1993).
)). Furthermore, dominant negative mutations in the VEGF receptor have been shown to inhibit glioblastoma growth in mice.

Vascular invasion factor (VPF) is also responsible for persistent microvascular permeability to plasma proteins, even after cessation of injury, which is a characteristic feature of normal wound healing. It was found. This suggests that VPF is an important factor in wound healing. Brown, L .; F. Et al., J. Exp. Me
d. 176: 1375-1379 (1992).

VEGF expression is high in vascularized tissues (eg lung, heart, placenta and solid tumors) and is associated with angiogenesis both temporally and spatially. VEG
F has also been shown to induce angiogenesis in vivo. Angiogenesis is
Since it is essential for repair of normal tissue (particularly vascular tissue), VEGF has been proposed for use in promoting vascular tissue repair (eg, atherosclerosis).

US Pat. No. 5,073,492 (issued to Chen et al., Dec. 17, 1991) discloses a method for synergistically enhancing endothelial cell proliferation in a suitable environment,
The method involves adding VEGF, effector and serum derived factors to the environment. In addition, the DNA of the subunit of vascular endothelial growth factor C
Prepared by the polymerase chain reaction technique. This DNA encodes a protein that can exist as either a heterodimer or a homodimer. This protein is a mitogen of mammalian vascular endothelial cells, and in such cases, it is involved in vascular development and repair as disclosed in European Patent Application No. 92302750.2 (published September 30, 1992). Useful for promotion.

A recent addition to the vascular endothelial growth factor family is VEGF-.
3, VEGF-D, and VEGF-E. VEGF-3 growth factor is described in WO 96/39421 (Hu et al., Dec. 12, 1996).
As disclosed in (Publication Date), it is a strong mitogen that promotes proliferation of vascular endothelial cells and / or mesodermal cells. VEGF-D is another member of the vascular endothelial growth factor family, and WO 98/07832 (19).
(Published February 26, 1998) and have the ability to stimulate and / or enhance endothelial cell proliferation or differentiation. More recently, Meyer et al. Published a new member of the vascular endothelial growth factor family, designated VEGF-E. VEGF-E is a potent angiogenic factor that selectively binds to VEGF receptor 2 (KDR) (Meyer et al., EMBO J, 18: 363-374 (1).
999)).

Accordingly, there is a need for treatments that promote or inhibit angiogenic proteins.

SUMMARY OF THE INVENTION The present invention provides members of the VEGF / PDGF family of proteins and related proteins (collectively referred to herein as “angiogenic proteins” or “angiogenic proteins”).
Angiogenic polypeptides "), as well as agonists and / or antagonists of angiogenic proteins.

According to a further aspect of the invention, for therapeutic purposes (eg for stimulating angiogenesis, wound healing, proliferation of damaged bone and tissue, and promoting vascular tissue repair), and so on. Processes for utilizing different polypeptides or polynucleotides encoding such polypeptides are provided. In particular, processes are provided that utilize such polypeptides, or polynucleotides encoding such polypeptides, for the treatment of peripheral arterial disease, such as severe limb ischemia and coronary disease. According to yet another aspect of the invention there is provided an agonist for such a polypeptide, which agonist promotes the action of such a polypeptide (eg, angiogenesis, lymphangiogenesis (ly).
mphangiogenesis) or to promote vascular tissue repair). The invention further relates to variants of the above-mentioned polypeptide, which encode an angiogenic or immunogenic epitope of this polypeptide.

According to yet another aspect of the invention there is provided an agonist for such a polypeptide, wherein the agonist enhances or enhances the action of such a polypeptide (eg inhibiting tumor angiogenesis). To inhibit tumor growth,
Or to treat diabetic retinopathy, inflammation, rheumatoid arthritis and psoriasis). According to yet another aspect of the invention there is provided an agonist of such a polypeptide, which agonist enhances the action of such polypeptide (eg, promotes angiogenesis, lymphangiogenesis). Or to promote vascular tissue repair).

According to yet another aspect of the invention, there are provided antibodies against such polypeptides and processes for producing such antibodies. In accordance with still further aspects of the invention, for therapeutic purposes (eg, to stimulate angiogenesis, wound healing, proliferation of damaged bone and tissue, to promote vascular tissue repair, and to stimulate lymphangiogenesis. In order to do so, a process for utilizing such an antibody is provided. In particular, processes are provided that utilize such antibodies for the treatment of peripheral arterial disease, such as severe limb ischemia and coronary disease.

According to yet another aspect of the invention, to inhibit the action of such polypeptides (eg to inhibit tumor angiogenesis to inhibit tumor growth, or to diabetic retinopathy, inflammation). , For treating rheumatoid arthritis and psoriasis) are provided.

According to one aspect of the invention, novel mature polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs thereof,
And derivatives are provided. The polypeptides of the present invention are of human origin.

According to another aspect of the invention, there is provided an isolated nucleic acid molecule comprising a polynucleotide encoding a full length or truncated angiogenic polypeptide having the amino acid sequence shown in Table 1. The invention also relates to biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives of angiogenic proteins.

According to yet another aspect of the invention there is provided a process for producing such a polypeptide by recombinant technology, which process promotes expression of the protein and subsequent recovery of the protein. Culturing a recombinant prokaryotic and / or eukaryotic host cell comprising a nucleic acid sequence encoding a polypeptide of the invention under facilitating conditions.

According to another aspect of the invention there is provided a nucleic acid probe comprising a nucleic acid molecule of sufficient length to specifically hybridize to the nucleic acid sequence of the invention. According to another aspect of the invention there is provided a method of diagnosing a disease or susceptibility to such a disease associated with mutations in the nucleic acid sequences of the invention and the proteins encoded by such nucleic acid sequences.

According to still further aspects of the invention, scientific research, DNA synthesis and DNA
Processes are provided for utilizing such polypeptides or polynucleotides encoding such polypeptides for in vitro purposes associated with the production of vectors.

According to yet another aspect of the invention, there is provided a method of screening for angiogenic protein agonists and / or antagonists that may enhance or inhibit a desired activity.

These and other aspects of the invention will be apparent to those skilled in the art from the teachings herein.

Detailed Description of the Preferred Embodiments Definitions According to one aspect of the invention there is provided a polypeptide molecule comprising members of the PDGF and VEGF families. More specifically, PDGF and VEGF
Members of the family include: European Patent EP-6821.
Platelet-derived growth factor A (PDGF-A) as disclosed in No. 10 (SEQ ID NO: 1)
~ 2); Platelet-derived growth factor B (PDGF-B) (SEQ ID NOs: 3-4) as disclosed in European Patent EP-283217; Placental proliferation as disclosed in WO92 / 06194. Factor (PlGF) (SEQ ID NOs: 5-6); Hause
r et al., Placental Growth Factor 2 (PIGF-2) (SEQ ID NOs: 7-8) as disclosed in Gorwt Factors 4: 259-268 (1993); nerves as disclosed in EP-399816. Glioma-derived growth factor (GDGF) (
SEQ ID NOs: 9-10); Vascular Endothelial Growth Factor (VEGF) as disclosed in WO 90/13649 (SEQ ID NOs: 11-12); European Patent EP-5064.
Vascular Endothelial Growth Factor A (VEGF-A) as disclosed in No. 77 (SEQ ID NO: 13)
~ 14); Vascular Endothelial Growth Factor 2 (VEGF-2) as disclosed in WO 96/39515 (SEQ ID NOs: 15-16); WO 96/394.
21. Vascular Endothelial Growth Factor 3 (VEGF-3) (SEQ ID NO: 1).
7-18); vascular endothelial growth factor D (VEGF-D) (SEQ ID NOs: 19-20) as disclosed in WO98 / 07832; WO98 / 02.
Vascular endothelial growth factor-D1 (VEGF-D1) (SEQ ID NOS: 21-22) as disclosed in DE 543; and vascular endothelial growth factor E (VEGF-E) as disclosed in German Patent DE19639601. (SEQ ID NOS: 23-24) (these are collectively referred to herein as "angiogenic proteins" or "angiogenic polypeptides"). The above references are incorporated herein by reference in their entireties.

“Agonist” as used herein refers to any polynucleotide, polypeptide, antibody or molecule (including small molecules) that promotes or enhances the desired activity of an angiogenic protein. ..

As used herein, an “antagonist” is any polynucleotide, polypeptide, antibody or molecule that reduces or inhibits the desired activity of an angiogenic protein, including small molecules. Say.

As used herein, “desired activity” means that an agonist and / or an antagonist refers to endothelial cell proliferation (angiogenesis or lymphangiogenesis) relative to the natural activity of angiogenic proteins. ) Or inhibit endothelial cell proliferation (angiogenesis or lymphangiogenesis).

Angiogenic Protein Agonists and Antagonists The present invention relates to angiogenic protein agonists and / or antagonists.

One embodiment of an angiogenic protein agonist includes an antibody that binds to a polypeptide and promotes or enhances its desired activity. Examples of these agonists include those that bind to angiogenic polypeptides and promote the angiogenic activity of those polypeptides (eg, by stabilizing dimeric forms of angiogenic proteins. , Or by condensing the polypeptide that binds to the antibody and concentrating or localizing the activity of the angiogenic polypeptide) to a specific angiogenic polypeptide, a monoclonal or polyclonal antibody.

Another potential angiogenic protein agonist includes small molecules that promote or enhance the activity of angiogenic proteins. For example, such small molecule agonists bind to angiogenic protein receptors or related receptors,
It may thus be involved in enhancing the activity of angiogenic proteins.

In a further embodiment, angiogenic protein agonists include polypeptides that promote the activity of angiogenic proteins. For example, such polypeptide agonists may be involved in binding angiogenic proteins or related receptors, thereby enhancing the activity of angiogenic proteins.

Another example includes antisense constructs that enhance or enhance the desired activity of angiogenic proteins.

Potential angiogenic protein antagonists include antibodies or (in some cases) oligonucleotides that bind to the polypeptide and effectively eliminate angiogenic protein activity. Alternatively, the potential antagonist may be intimately associated with a protein that binds to the angiogenic protein receptor,
These are inactive forms of the polypeptide and thereby interfere with the action of angiogenic proteins. Examples of these antagonists include negative dominant variants of angiogenic protein polypeptides, eg, one chain of angiogenic protein in heterodimeric form is predominant and biologically active. It can be mutated so that it is not retained. Examples of negative dominant mutants include truncated versions of dimeric angiogenic proteins, which may interact with another dimer to form wild-type angiogenic proteins. The resulting homodimer is inactive and is unable to exhibit the characteristic desired activity.

Another potential angiogenic protein antagonist is an antisense construct prepared using antisense technology. Antisense technology can be used to control gene expression via triple helix formation or antisense DNA or RNA, each of which methods is based on the binding of a polynucleotide to DNA or RNA. For example, the 5'coding region of the polynucleotide sequence encoding the mature polypeptide of the invention is used to design an antisense RNA oligonucleotide of about 10-40 base pairs in length. DNA oligonucleotides are designed to be complementary to the gene regions involved in transcription (triple helix-Lee et al., Nu.
cl. Acids Res. 6: 3073 (1979); Cooney et al., Sc.
ience 241: 456 (1988); and Dervan et al., Science.
251: 1360 (1991)), thereby interfering with the transcription and production of angiogenic proteins. Antisense RNA oligonucleotides hybridize to mRNA in vivo and block the translation of mRNA molecules into angiogenic protein polypeptides (Antisense-Okano, J.
． Neurochem. 56: 560 (1991); Oligodeoxynu.
cleotides as Antisense Inhibitors of
Gene Expression, CRC Press, Boca Rato
n, FL (1988)). The above oligonucleotides are also antisense RN
A or DNA can be delivered to cells such that it can be expressed in vivo to inhibit the production of angiogenic proteins.

In a further embodiment, angiogenic protein antagonists also include small molecules, which bind to and occupy the active site of the polypeptide, thereby rendering its catalytic site a substrate. Inaccessible, thus impeding normal biological activity. Examples of small molecules include, but are not limited to, small peptide molecules or small peptide-like molecules.

Antagonists can be utilized to limit the angiogenesis required for solid tumor metastasis. Since angiogenesis and neovascularization are essential steps in solid tumor growth, inhibition of the angiogenic activity of angiogenic proteins interferes with further growth and thus delays solid tumors. , Or even very useful for eliminating solid tumors.

The present invention also relates to methods of screening compounds to identify those that are agonists or antagonists of angiogenic proteins. An example of such a method utilizes the ability of angiogenic proteins to significantly stimulate the proliferation of human endothelial cells in the presence of comitogen Con A. Endothelial cells were obtained and in 96-well flat bottom culture plates (Costar, C
Ambridge, MA), Con-A (Calbiochem, La.
(Jolla, CA) is incubated in the reaction mixture supplemented. Con-A, the polypeptide of the invention and the compound to be screened are added. After incubation at 37 ° C., the cultures contained 1 FCi of ³ [H] thymidine (5 Ci / m 2
mol; 1Ci = 37BGq; NEN) and pulsed for a period of time sufficient to incorporate ³ [H], and a glass fiber filter (Cambridge).
e Technology, Watertown, MA). Mean ³ [H] thymidine incorporation (cpm) of triplicate cultures was measured by liquid scintillation counter (Beckman Instruments, Irvine, CA).
Was measured using. Significant ³ [H] thymidine incorporation compared to control assay (when compound was excluded) is indicative of stimulation of endothelial cell proliferation.

To assay for agonists, the above assay was performed and the compound was in the presence of angiogenic proteins. ^The ability to promote ³ [H] thymidine uptake indicates that this compound is an agonist for angiogenic proteins. Conversely, the above assay is performed to assay for antagonists, and the compound is in the presence of angiogenic proteins. ^The ability to inhibit ³ [H] thymidine uptake indicates that this compound is an antagonist for angiogenic proteins. Alternatively, the antagonist to the angiogenic protein combines the angiogenic protein and potential antagonist with a membrane-bound, angiogenic protein receptor or recombinant receptor under conditions suitable for competitive inhibition assays. Can be detected. Angiogenic proteins can be labeled, such as by radioactivity, so that the number of angiogenic protein molecules bound to this receptor can determine the efficacy of potential antagonists.

Alternatively, the response of a known second messenger system after interaction of angiogenic proteins and receptors is measured and compared in the presence or absence of the compound. As such a second messenger system, cAM
P-guanylate cyclase, ion channels or phosphoinositide hydrolysis, but is not limited thereto. In another method, a mammalian cell or membrane preparation expressing an angiogenic protein receptor is incubated with a labeled angiogenic protein in the presence of the compound. The ability of the compound to enhance or block this interaction can then be measured.

Epitopes and Antibodies The present invention is encoded by the epitope of a polypeptide having the amino acid sequence of SEQ ID NO: 2 or 4, or the polynucleotide sequence contained in ATCC Deposit No. 97149 or 75698, or SEQ ID NO: 1. Or 3 or a polynucleotide complementary to the complementary strand of the sequence of ATCC Deposit No. 97149 or 75698 under stringent or lower stringency hybridization conditions as defined above. Contains an epitope of the polypeptide sequence, or
Alternatively, it includes a polypeptide consisting of them. A representative clone containing the sequence of SEQ ID NO: 2 or 4 was the American TypeCul on March 4, 1994.
Deposited to the Pure Collection ("ATCC"), was given ATCC deposit no. 75698, and was deposited on May 12, 1995, was given ATCC deposit no. 97149. ATCC is Virginia 20, USA
Located at 11001 Boulevard University, 110-2209, Manassas. ATC
The C deposit was made in accordance with the Budapest Treaty Convention on International Recognition. The present invention further provides a polynucleotide sequence encoding an epitope of the polypeptide sequence of the present invention (eg, the sequence disclosed in SEQ ID NO: 1 or 3), a polynucleotide of a complementary strand of the polynucleotide sequence encoding the epitope of the present invention. Nucleotide sequences, and polynucleotide sequences that hybridize to complementary strands under stringent or lower stringency hybridization conditions as defined above.

As used herein, the term "epitope" refers to that portion of a polypeptide that has antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably a human. In a preferred embodiment, the present invention includes a polypeptide comprising an epitope as well as a polynucleotide encoding this polypeptide. As used herein, an "immunogenic epitope" is defined as the portion of a protein that elicits an antibody response in an animal and can be any method known in the art (eg, antibody production as described below). Method). (For example, Geysen et al., Proc. Natl. Acad. Sci. USA.
81: 3998-4002 (1983)). As used herein, the term "antigenic epitope" refers to an antibody as defined by any method known in the art, such as the immunoassays described herein. It is defined as the part of the protein that is capable of immunospecifically binding to an antigen. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes do not necessarily require immunogenicity.

Fragments that function as epitopes can be generated by any conventional means. (For example, Houghten, Proc. Natl. Acad. Sci.
USA 82: 5131-5135 (1985) (U.S. Pat. No. 4,631,21).
1)).

In the present invention, the antigenic epitope is preferably at least 4, at least 5, at least 6, at least 7, more preferably at least 8,
At least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and most It preferably comprises a sequence of between about 15 and about 30 amino acids. Preferred polypeptides are at least 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100
It includes immunogenic or antigenic epitopes of amino acid residue length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies (including monoclonal antibodies) that specifically bind to this epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as target molecules in immunoassays. (For example, Wilson et al., Cell 37: 767-778 (19
84); Sutcliffe et al., Science 219: 660-666 (1).
983)).

Similarly, immunogenic epitopes can be used to induce antibodies, eg, according to methods well known in the art. (For example, Sutcliffe et al. (Supra); Wils
on et al. (supra); Chow et al., Proc. Natl. Acad. Sci. USA
82: 910-914; and Bittle et al., J. Am. Gen. Virol. 6
6: 2347-2354 (1985)). Preferred immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes. A polypeptide comprising one or more immunogenic epitopes may be presented to elicit an antibody response against an animal system (eg rabbit or mouse) with a carrier protein (eg albumin), or the polypeptide may be If sufficiently long (at least about 25 amino acids), the polypeptide can be presented without a carrier. However, immunogenic epitopes containing as few as 8-10 amino acids
It has been shown to be sufficient (e.g. in Western blotting) to raise antibodies capable of (at least) binding to the linear epitope of the denatured polypeptide.

The epitope-bearing polypeptides of the present invention can be used to induce antibodies according to methods well known in the art. This method includes, but is not limited to, in vivo immunization, in vitro immunization, and phage display methods.
For example, Sutcliffe et al., Supra; Wilson et al., Supra; and Bitt.
le et al. Gen. Virol. , 66: 2347-2354 (1985). When in vivo immunization is used, animals can be immunized with free peptides; however, anti-peptide antibody titers are dependent on the macromolecule carrier (eg, keyhole limpet hemacyanin (KLH) or tetanus toxoid) peptide. Can be boosted by combining. For example, peptides containing cysteine residues can be linked to carriers using linkers such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS). On the other hand, other peptides can be bound to the carrier using more common binding agents such as glutaraldehyde. Animals such as rabbits, rats, and mice use, for example, emulsions (about 100 μg of peptide or carrier protein and Freund's adjuvant or any known to stimulate an immune response) with either free peptide or carrier-bound peptide. Immunization by intraperitoneal injection (including other adjuvants) and / or intradermal injection. Several booster injections may be needed to provide a useful titer of anti-peptide antibody, for example, at intervals of about 2 weeks. This titer can be detected, for example, by an ELISA assay using free peptide adsorbed on the solid surface. Titer of anti-peptide antibody in serum from immunized animals is increased by selection of anti-peptide antibody (eg, by adsorption of peptide on solid support and elution of selected antibody according to methods well known in the art). You can

As will be appreciated by those in the art, and as discussed above, the polypeptides of the present invention that include immunogenic or antigenic epitopes can be fused to other polypeptide sequences. For example, the polypeptides of the present invention are immunoglobulin (Ig
A, IgE, IgG, IgM) constant domains or parts thereof (CH1, C)
H2, CH3, or any combination thereof and portions thereof), resulting in a chimeric polypeptide. Such fusion proteins may facilitate purification and increase half-life in vivo. This has been shown for a chimeric protein consisting of the first two domains of the human CD4-polypeptide and various domains of the constant region of the heavy or light chain of mammalian immunoglobulins. For example, EP 394,827; Traunecker et al., Nature,
331: 84-86 (1988). Enhanced delivery of antigens across epithelial disorders to the immune system has been demonstrated for antigens bound to FcRn binding partners such as IgG or Fc fragments (eg insulin) (
See, for example, PCT publications WO 96/2024 and WO 99/04813). IgG fusion proteins having a disulfide-bonded dimer structure due to disulfide bonds of the IgG moiety are also more effective in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone Was found. For example, Fountoulakis et al. Biochem.
, 270: 3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (eg, a hemagglutinin (“HA”) tag or a flag tag) for detection and purification of the expressed polypeptide. Can assist. For example, Janknecht
The system described by et al. Allows easy purification of native fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Na.
tl. Acad. Sci. USA 88: 8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid so that the open reading frame of this gene is translationally fused to an amino-terminal tag consisting of 6 histidine residues. This tag serves as the substrate binding domain for the fusion protein. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2 + nitriloacetic acid-agarose columns, and histidine-tagged proteins can be selectively eluted with imidazole-containing buffer.

Further fusion proteins of the invention may be produced through the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). D
NA shuffling can be utilized to modulate the activity of the polypeptides of the invention and this method can be used to produce polypeptides with modified activity, as well as agonists and antagonists of these polypeptides. Generally, US Pat. Nos. 5,605,793; 5,811,238; 5,83.
No. 0,721; No. 5,834,252; and No. 5,837,458,
And Patten et al., Curr. Opinion Biotechnol.
8: 724-33 (1997); Harayama, Trends Biote.
chnol. 16 (2): 76-82 (1998); Hansson et al., J. Am. M
ol. Biol. 287: 265-76 (1999); and Lorenzo
And Blasco, Biotechniques 24 (2): 308-13.
(1998), each of which is incorporated herein by reference in its entirety. In one embodiment, modification of the polynucleotides corresponding to SEQ ID NO: 1 or 3 and the polypeptides encoded by these polynucleotides can be accomplished by DNA shuffling. DNA shuffling involves assembling two or more DNA segments to produce changes in polynucleotide sequence by homologous or site-specific recombination. In another embodiment, the polynucleotide of the invention or its encoded polypeptide is error prone prior to recombination.
-Prone) by subjecting it to random mutagenesis by PCR, random nucleotide insertion or other methods. In another embodiment, one or more components, motifs, sections, portions, domains, fragments, etc. of the polynucleotides encoding the polypeptides of the invention are of one or more heterologous molecules,
It can be recombined with one or more components, motifs, sections, parts, domains, fragments and the like.

Antibodies In addition, the polypeptides of the invention may be polypeptides, polypeptide fragments of the invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding). , Or variants of SEQ ID NO: 2 or 4, and / or antibodies and T cell antigen receptors (TCRs) that immunospecifically bind to an epitope. The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ') fragments, fragments produced by Fab expression libraries. , But not limited to, anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies against the antibodies of the invention), and epitope binding fragments of any of the above. As used herein, the term "
"Antibody" refers to an immunologically active portion of an immunoglobulin molecule and an immunoglobulin molecule, ie, a molecule containing an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecule of the present invention may be any type of immunoglobulin molecule (eg IgG
, IgE, IgM, IgD, IgA, and IgY), any class (eg,
IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass.

Most preferably, the antibody is a human antigen-binding antibody fragment of the invention, which comprises Fab, Fab 'and F (ab') 2, Fd, single chain Fvs (sc).
Fv), single chain antibodies, disulfide-bonded Fvs (sdFv) and VL or V
Examples include, but are not limited to, fragments that include any of the H domains. Antigen-binding antibody fragments, including single chain antibodies, may include variable regions alone or in combination with all or part of the following: hinge region, CH1 domain, C
H2 domain and CH3 domain. Also included in the invention are antigen binding fragments that also include any combination of variable and hinge regions, CH1 domains, CH2 domains and CH3 domains. Antibodies of the invention may be derived from any animal origin including birds and mammals. Preferably, the antibody is human, murine (eg, mouse and rabbit), donkey, ship rabbit (
ship rabbit), goat, guinea pig, camel, horse, or chicken. As used herein, "human" antibody includes antibodies having the amino acid sequence of human immunoglobulin and is transgenic for a human immunoglobulin library or one or more human immunoglobulins and is endogenous. Antibodies isolated from animals that do not express sex immunoglobulin (as described below and, for example, in US Pat. No. 5,939,598 to Kucherlapati et al.).

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. A multispecific antibody may be specific for different epitopes of a polypeptide of the invention, or both a polypeptide of the invention and a heterologous epitope (eg, a heterologous polypeptide or a solid support material). Can be specific to. For example, PCT Publication WO 93/17715; WO 9
2/08802; WO 91/00360; WO 92/05793; Tu
tt et al. Immunol. 147: 60-69 (1991); U.S. Pat. No. 4
, 474,893, 4,714,681, 4,925,648,
No. 5,573,920, No. 5,601,819; Kostelny et al.
J. Immunol. 148: 1547-1553 (1992).

The antibodies of the invention may be described or specified with respect to the epitopes or parts of the polypeptides of the invention to which they recognize or specifically bind. Portions of this epitope or polypeptide may be identified, for example, by N-terminal and C-terminal positions, by size in contiguous amino acid residues, as described herein, or as listed in the tables and figures. Can be transformed.

In one embodiment, antigenic polypeptides or peptides that can be used to generate PDGF-A-specific antibodies include: about Lys-72 to about Leu- of SEQ ID NO: 2. A polypeptide comprising 80 amino acid residues, a polypeptide comprising amino acid residues from approximately Ile-82 to approximately Ile-88 of SEQ ID NO: 2, approximately Pro-106 to approximately Ser-114 in SEQ ID NO: 2.
A polypeptide comprising amino acid residues of SEQ ID NO: 2, a polypeptide comprising amino acid residues of approximately Lys-139 to Val-145 of SEQ ID NO: 2, approximately Ar of SEQ ID NO: 2.
A polypeptide comprising amino acid residues from g-159 to about Lys-165, and a polypeptide containing amino acid residues from about Ser-183 to about Thr-211 of SEQ ID NO: 2. These polypeptide fragments were determined to carry an antigenic epitope of the PDGF-A protein by analysis of the Jameson-Wolf antigenic index using default parameters.

In a preferred embodiment, the antigenic epitope is approximately I of SEQ ID NO: 2.
It contains amino acid residues from le-82 to approximately Ile-88.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate PDGF-B specific antibodies include: about Arg-39 to about Gln- of SEQ ID NO: 4. A polypeptide containing 45 amino acids, SEQ ID NO: 4
Of about Asp-51 to about Asp-56 of amino acids, SEQ ID NO: 4 of about Thr-65 to about Gly-71 amino acids of polypeptide, SEQ ID NO: 4 of about Leu-110 to about Ala-116. Of the amino acid sequence of SEQ ID NO: 4, approximately Ser-131 to approximately Thr-144
A polypeptide containing amino acids of SEQ ID NO: 4, from about Pro-188 to about T
A polypeptide comprising the amino acid of hr-206, approximately Arg-21 of SEQ ID NO: 4.
A polypeptide comprising 6 to about Lys-227 amino acids, and a polypeptide comprising about Thr-229 to about Leu-235 amino acids of SEQ ID NO: 4.
These polypeptide fragments are Jam using the default parameters.
Analysis of the eson-Wolf antigenic index determined that it carried an antigenic epitope of the PDGF-B protein.

In a preferred embodiment, the antigenic epitope is approximately A of SEQ ID NO: 4.
rg-39 to about Gln-45 amino acid residues, about Asp-of SEQ ID NO: 4
51 to approximately Asp-56 amino acid residues, and SEQ ID NO: 4 approximately Thr-
65 to approximately containing Gly-71 amino acid residues.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate PIGF-specific antibodies include: approximately Va of SEQ ID NO: 6.
a polypeptide comprising amino acid residues 1-46 to approximately Cys-52, SEQ ID NO: 6
Of about Arg-110 to about Ser-116, and a polypeptide containing about Val-126 to about Asp-144 of SEQ ID NO: 6. These polypeptide fragments were labeled PI by analysis of the Jameson-Wolf antigenicity index using default parameters.
It was determined to carry an antigenic epitope of the GF protein.

In a preferred embodiment, the antigenic epitope is approximately V of SEQ ID NO: 6.
It contains amino acid residues from al-46 to approximately Cys-52.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate PIGF-2 specific antibodies include: approximately Val-46 to approximately Cys- of SEQ ID NO: 8. A polypeptide comprising the amino acid residue of 52, a polypeptide comprising the amino acid residue of about Arg-110 to about Ser-116 of SEQ ID NO: 8, and the amino acid residue of about Val-126 to about Asp-159 of SEQ ID NO: 8. A polypeptide comprising. These polypeptide fragments were determined to carry an antigenic epitope of the PIGF-2 protein by analysis of the Jameson-Wolf antigenic index using default parameters.

In a preferred embodiment, the antigenic epitope is approximately V of SEQ ID NO: 8.
It contains amino acid residues from al-46 to approximately Cys-52.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate GDGF-specific antibodies include: approximately T of SEQ ID NO: 10.
a polypeptide containing amino acid residues from hr-30 to about Ala-36, a polypeptide containing amino acid residues from about Tyr-46 to about Cys-51 of SEQ ID NO: 10, about Glu-63 to about Ile of SEQ ID NO: 10. A polypeptide comprising -68 amino acid residues, approximately Ser-125 to approximately Cys-1 of SEQ ID NO: 10
A polypeptide comprising 42 amino acid residues, approximately Pro-144 of SEQ ID NO: 10
~ A polypeptide comprising amino acid residues of about His-151, a polypeptide comprising amino acid residues of about Gln-155 to about Lys-172 of SEQ ID NO: 10, and about Asn-179 to about Arg-190 of SEQ ID NO: 10. A polypeptide comprising the amino acid residue of. These polypeptide fragments were analyzed for GDG by the Jameson-Wolf antigenic index analysis using default parameters.
It was determined to carry an antigenic epitope of the F protein.

In a preferred embodiment, the antigenic epitope is about Thr-30 to about Ala-36 amino acid residues of SEQ ID NO: 10 and about Tyr-46 to Cys-51 amino acid residues of SEQ ID NO: 10. including.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate VEGF-specific antibodies include: approximately G of SEQ ID NO: 12.
A polypeptide containing amino acid residues from lu-64 to about Ile-69, a polypeptide containing amino acid residues from about Gly-85 to about Gly-91 of SEQ ID NO: 12, about His-125 to about Cys with SEQ ID NO: 12. -143, a polypeptide comprising amino acid residues, approximately Pro-145 to approximately His of SEQ ID NO: 12.
A polypeptide comprising the amino acid residue of -152, approximately Gln-1 of SEQ ID NO: 12
A polypeptide comprising amino acid residues from 56 to about Lys-173, and a polypeptide comprising amino acid residues from about Asn-180 to about Arg-191 of SEQ ID NO: 12. These polypeptide fragments were determined to carry an antigenic epitope of the VEGF protein by analysis of the Jameson-Wolf antigenic index using default parameters.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate VEGF-A specific antibodies include: about Thr-30 to about Ala- of SEQ ID NO: 14. A polypeptide containing 36 amino acid residues, a polypeptide containing amino acid residues of about Tyr-46 to about Cys-51 of SEQ ID NO: 14, an amino acid residue of about Glu-63 to about Ile-68 of SEQ ID NO: 14. A polypeptide comprising, about Ser-125 to about Val of SEQ ID NO: 14
A polypeptide comprising an amino acid residue of -143, approximately Gly-1 of SEQ ID NO: 14
A polypeptide comprising amino acid residues from 45 to approximately Cys-166, SEQ ID NO: 14
Of about Pro-168 to about His-175, a polypeptide containing about Gln-179 to about Lys-196 of SEQ ID NO: 14, and about Asn-203 of SEQ ID NO: 14. A polypeptide comprising about Arg-214 amino acid residues. These polypeptide fragments were determined to carry an antigenic epitope of the VEGF-A protein by analysis of the Jameson-Wolf antigenic index using default parameters.

In a preferred embodiment, the antigenic epitope comprises the following amino acid residues: approximately Thr-30 to approximately Ala-36 of SEQ ID NO: 14, and approximately Tyr-46 to approximately Cys- of SEQ ID NO: 14. 51.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate VEGF-2 specific antibodies include polypeptides that include the following amino acid residues: approximately to SEQ ID NO: 16. Asp-40 to about Ala-45, about Lys-54 to about Leu-60 of SEQ ID NO: 16, about G of SEQ ID NO: 16
In-84 to about Gly-89, about Arg-94 to about Thr-106 of SEQ ID NO: 16, about Ile-122 to about Cys-131 of SEQ ID NO: 16.
SEQ ID NO: 16, about Asn-175 to about Leu-181, SEQ ID NO: 16
About Gln-237 to about Pro-244, about Asp of SEQ ID NO: 16
-267 to about Phe-276, about Pro-282 to about Asp-287 of SEQ ID NO: 16, about Ser-303 to about Ser-31 of SEQ ID NO: 16.
4, about Ala-330 to about Thr-338 of SEQ ID NO: 16, SEQ ID NO: 1
6 about Arg-345 to about Cys-358, SEQ ID NO: 16 about Gl
y-373 to approximately Cys-395, and approximately Pro-41 of SEQ ID NO: 16.
0 to approximately Ser-419. These polypeptide fragments were identified as VE by analysis of the Jameson-Wolf antigenic index using default parameters.
It was determined to carry an antigenic epitope of the GF-2 protein.

In a preferred embodiment, the antigenic epitope comprises the following amino acid residues: approximately Asp-40 to approximately Ala-45 of SEQ ID NO: 16, SEQ ID NO: 1.
6 about Lys-54 to about Leu-60, SEQ ID NO: 16 about Gln-.
84 to about Gly-89, about Arg-94 to about Thr of SEQ ID NO: 16
-106, and about Ile-122 to about Cys-131 of SEQ ID NO: 16.
.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate VEGF-3-specific antibodies include polypeptides that include the following amino acid residues: SEQ ID NO: 18 approximately. Ser-24 to approximately Lys-34, SEQ ID NO: 18 approximately Ala-45 to approximately Arg-50, SEQ ID NO: 18 approximately A
rg-125 to approximately Ala-138, SEQ ID NO: 18 to approximately Pro-141 to
Approximately Ser-149, and approximately His167 to approximately Pr of SEQ ID NO: 18.
o-172. These polypeptide fragments were determined to carry an antigenic epitope of the VEGF-3 protein by analysis of the Jameson-Wolf antigenic index using default parameters.

In a preferred embodiment, the antigenic epitope comprises the following amino acid residues: approximately Ser-24 to approximately Lys-34 of SEQ ID NO: 18, and approximately Ala-45 to approximately Arg- of SEQ ID NO: 18. Fifty.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate VEGF-D specific antibodies include polypeptides that include the following amino acid residues: SEQ ID NO: 20 approximately. Met-1 to approximately Gln-14, SEQ ID NO: 20 approximately His-29 to approximately Arg-41, SEQ ID NO: 20 approximately Me
t-48 to about Thr-58, about Glu-75 to about T of SEQ ID NO: 20
hr-87, about Leu-95 to about Phe-103 of SEQ ID NO: 20, about Pro-181 to about Lys-191 of SEQ ID NO: 20, about Trp-199 to about Cys-204 of SEQ ID NO: 20, About Asp-28
1 to about Thr-293, about Pro-310 to about Al of SEQ ID NO: 20
a-316, and about Gly-318 to about Pro-32 of SEQ ID NO: 20.
5. These polypeptide fragments are labeled with Ja using the default parameters.
Analysis of the meson-Wolf antigenic index determined that it carries an antigenic epitope of the VEGF-D polypeptide.

In a preferred embodiment, the antigenic epitope comprises the following amino acid residues: approximately Met-1 to approximately Gln-14 of SEQ ID NO: 20, SEQ ID NO: 20.
About His-29 to about Arg-41 of SEQ ID NO: 20.
8 to about Thr-58, and about Glu-75 to about T of SEQ ID NO: 20.
hr-87.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate VEGF-D1-specific antibodies include polypeptides that include the following amino acid residues: SEQ ID NO: 22 approximately. Ser-22 to about Gln-43, about His-58 to about Arg-70 of SEQ ID NO: 22, about Met-77 to about Thr-87 of SEQ ID NO: 22, about Glu-104 to about Thr-116 of SEQ ID NO: 22. , About Leu-124 to about Phe-1 of SEQ ID NO: 22
32, about Pro-210 to about Lys-220 of SEQ ID NO: 22, about Trp-228 to about Cys-233 of SEQ ID NO: 22, about A of SEQ ID NO: 22
sp-310 to about Thr-322, about Pro-339 of SEQ ID NO: 22 to
Approximately Ala-345, and approximately Gly-347 to approximately P of SEQ ID NO: 22.
ro-354. These polypeptide fragments were determined to carry an antigenic epitope of the VEGF-D protein by analysis of the Jameson-Wolf antigenic index using default parameters.

In a preferred embodiment, the antigenic epitope comprises the following amino acid residues: about Ser-22 to about Gln-43 of SEQ ID NO: 22, SEQ ID NO: 2.
2 from about His-58 to about Arg-70, and about M of SEQ ID NO: 22.
et-77 to approximately Thr-87.

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate VEGF-E specific antibodies include polypeptides comprising the following amino acid residues: SEQ ID NO: 24 approximately Asp-21 to approximately Trp-26, SEQ ID NO: 24 approximately Asp-46 to approximately Phe-56, SEQ ID NO: 24 approximately G
ly-68 to approximately Ser-74, and approximately Pro-121 of SEQ ID NO: 24.
~ Approximately Arg-132. These polypeptide fragments were labeled with VEG by analysis of the Jameson-Wolf antigenic index using default parameters.
It was determined to carry an antigenic epitope of the FE protein.

In a preferred embodiment, the antigenic epitope comprises amino acid residues from about Asp-21 to about Trp-26 of SEQ ID NO: 24.

Antibodies that specifically bind any epitope or polypeptide of the invention may also be excluded. Therefore, the present invention specifically binds the polypeptide of the present invention,
It also includes antibodies that allow the elimination of the polypeptides of the invention.

[0076]   The antibodies of the invention are also described or specified by their cross-reactivity.
Can be Any other analog of the polypeptides of the invention, ortholog
Antibodies that do not bind log) or homologs are included. Polypeptide of the invention
At least 95%, at least 90%, at least 85%, at least
80%, at least 75%, at least 70%, at least 65%, at least
60%, at least 55% and at least 50% identity (known in the art
Method and when calculated using the methods described herein)
Antibodies that bind the polypeptide are also included in the invention. In particular embodiments
Thus, the antibody of the present invention is a human homologue of a human protein, a rat homologue, and a rat homologue.
And / or rabbit homologues and corresponding epitopes thereof
. Less than 95%, less than 90%, less than 85%, 80% to the polypeptide of the present invention
Less than, less than 75%, less than 70%, less than 65%, less than 60%, less than 55% and 5
Less than 0% identity (methods known in the art and those described herein)
Antibodies that do not bind to a polypeptide having (as calculated using the method) are also
Included in the invention. In certain embodiments, the above cross-reactivity is referred to herein
Any of the disclosed monospecific antigenic or immunogenic polypeptides, or
A set of 2, 3, 4, 5 or more specific antigenic and / or immunogenic polypeptides
Regarding the alignment. Further, the present invention includes striations (as described herein).
Polynucleotides of the invention are hybridized under
Antibodies that bind the polypeptide encoded by the bridging polynucleotide
The body is included. The antibody of the present invention also has its binding parent to the polypeptide of the present invention.
It may be described or specified by its compatibility. The preferred binding affinity is 5 x 10 ^-2 M, 10^-2M, 5 x 10^-3M, 10^-3M, 5 x 10^-FourM, 10^-FourM, 5 x 10 ^-Five M, 10^-FiveM, 5 x 10^-6M, 10^-6M, 5 x 10^-7M, 10^-7M, 5 x 10 ^-8 M, 10^-8M, 5 x 10^-9M, 10^-9M, 5 x 10^-TenM, 10^-TenM, 5 x 1
0^-11M, 10^-11M, 5 x 10^-12M, 10^-12M, 5 x 10^-13M, 10^-13M,
5 x 10^-14M, 10^-14M, 5 x 10^-15M or 10^-15Dissociation constant less than M
Nawachi K_dAnd binding affinity with.

The present invention also includes any method known in the art for determining competitive binding (
Antibodies that competitively inhibit the binding of antibodies to the epitopes of the invention as determined by, for example, the immunoassays described herein) are provided. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%. .

The antibody of the present invention may act as an agonist or antagonist of the polypeptide of the present invention. For example, the invention includes antibodies that disrupt the interaction of the receptor / ligand with either or all of the polypeptide moieties of the invention. Preferably, the antibodies of the present invention bind to the antigenic epitopes disclosed herein or a portion thereof. The invention features both receptor-specific and ligand-specific antibodies. The present invention also features receptor-specific antibodies that do not interfere with ligand binding but prevent receptor activation. Activation of the receptor (ie,
Signal transduction) can be determined by the techniques described herein or otherwise known in the art. For example, activation of the receptor may be due to receptor phosphorylation (eg tyrosine or serine / threonine) or (eg
It can be determined by immunoprecipitation (as described above), followed by detection of its substrate by Western blot analysis. In certain embodiments, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%, ligand activity of activity in the absence of antibody. Alternatively, an antibody that inhibits receptor activity is provided. The present invention also recognizes receptor-specific antibodies that interfere with both ligand binding and receptor activation, as well as receptor-ligand complexes, and, preferably, specifically binds unbound receptor or unbound ligand. Characterize antibodies that do not recognize. Similarly, the invention includes neutralizing antibodies that bind to the ligand and prevent it from binding to the receptor, as well as binding to the ligand, thereby preventing receptor activation, but preventing the ligand from binding to the receptor. No antibodies included. Further included in the invention are antibodies that activate the receptor. These antibodies may act like receptor agonists, ie, all or a subset of the biological activity of ligand-mediated receptor activation (
any of the subsets) is enhanced or activated, for example by inducing receptor dimerization. The antibody may be specified as an agonist, antagonist or inverse agonist for a biological activity, including the specific biological activity of the peptides of the invention disclosed herein.
The antibody agonists described above can be made using methods known in the art. For example, PCT Publication WO 96/40281; US Pat. No. 5,811,097; Den.
g et al., Blood 92 (6): 1981-1988 (1998); Chen et al., Cancer Res. 58 (16): 3668-3678 (1998); H.
arrop et al. Immunol. 161 (4): 1786-1794 (19
98); Zhu et al., Cancer Res: 58 (15): 3209-3214.
(1998); Yoon et al. Immunol. 160 (7): 3170-3
179 (1998); Prat et al. Cell. Sci. 111 (Pt2):
237-247 (1998); Pitard et al. Immunol. Meth
ods 205 (2): 177-190 (1997); Liautard et al., C.
ytokine 9 (4): 233-241 (1997); Carlson et al.,
J. Biol. Chem. 272 (17): 11295-11301 (1997)
); Taryman et al., Neuron 14 (4): 755-762 (1995).
); Muller et al., Structure 6 (9): 1153-1167 (1).
998); Bartunek et al., Cytokine 8 (1): 14-20 (1.
996) (all of the above references are incorporated herein by reference in their entirety).

The antibodies of the invention can be used to purify, detect and target the polypeptides of the invention, including, but not limited to, diagnostic and therapeutic methods both in vitro and in vivo. . For example, the antibody has use in immunoassays for qualitatively and quantitatively measuring the levels of polypeptides of the invention in biological samples. For example, Harlow et al., An
tibodies: A Laboratory Manual, (Cold S
pring Harbor Laboratory Press, 2nd edition, 19
88), which is incorporated herein by reference in its entirety.

As discussed in more detail below, the antibodies of the invention can be used either alone or in combination with other compositions. The antibody may further be recombinantly fused at its N-terminus or C-terminus to a heterologous polypeptide, or may be chemically linked (including covalent and non-covalent) to the polypeptide or other composition. . For example, the antibodies of the invention can be recombinantly fused or conjugated to molecules and effector molecules useful as labels in detection assays, such as heterologous polypeptides, drugs, radionuclides or toxins. For example, PCT publication WO92 / 0
8495; WO91 / 14438; WO89 / 12624; US Pat. No. 5,31.
4,995; and EP 396,387.

The antibodies of the invention may be modified (ie, covalent attachment of any type of molecule to the antibody such that the antibody does not prevent the antibody from producing an anti-idiotypic response). Derivatives (by covalent attachment). For example, without limitation, antibody derivatives include, for example, glycosylations, acetylations, pegylations, phosphorylations.
), Amidation, derivatization with known blocking / blocking groups, proteolytic cleavage, ligation to cellular ligands or other proteins, and the like. Numerous optional chemical modifications
It can be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-classical amino acids.

The antibodies of the invention can be produced by any suitable method known in the art.
Polyclonal antibodies to the antigen of interest can be produced by various procedures well known in the art. For example, the polypeptides of the invention can be administered to a variety of host animals, including but not limited to rabbits, mice, rats, etc., to induce the production of serum containing polyclonal antibodies specific for the antigen. Various adjuvants can be used to increase the immunological response, depending on the host species, and Freund's (complete and incomplete), mineral gels such as aluminum hydroxide (mi
general gel), surface-active substances such as lysolecithin, pluronic (p
luuronic) polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and BCG (calumet-
Bacillus gellan) and potentially useful human adjuvants such as corynebacterium parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display techniques, or a combination thereof. For example, monoclonal antibodies can be produced using the hybridoma technology known in the art, including: Har
Low et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press
, 2nd edition, 1988); Hammerling et al., Monoclonal An.
tibodies and T-Cell Hybridomas 563-6
81 (Elsevier, NY 1981), which is incorporated by reference in its entirety. The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which the monoclonal antibody is produced.

Methods for producing and screening for particular antibodies using hybridoma technology are routine and well known in the art and are discussed in detail in the Examples. In a non-limiting example, mice can be immunized with a polypeptide of the invention or cells expressing such a peptide. Once an immune response is detected (eg, antibodies specific for the antigen are detected in mouse serum), the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable melanoma cells (eg cells from cell line SP20 available from the ATCC). Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed for cells secreting antibodies capable of binding the polypeptides of the invention by methods known in the art. Ascites fluid, which generally contains high levels of antibodies, can be produced by immunizing mice with positive hybridoma clones.

Accordingly, the invention provides a method of producing an antibody produced by a method comprising culturing a monoclonal antibody and a hybridoma cell secreting the antibody of the invention, wherein, preferably, this antibody The hybridoma is a hybridoma clone which secretes an antibody capable of binding to the polypeptide of the present invention by fusing splenocytes isolated from the immunized mouse with the antigen of the present invention using myeloma cells. Produced by screening the hybridomas resulting from the fusion.

Antibody fragments that recognize particular epitopes can be produced by known techniques. For example, Fab and F (ab ′) 2 fragments of the invention are (Fa
It can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produce the b fragment) or pepsin (to produce the F (ab ') 2 fragment). F (ab ′) 2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (eg human or mouse). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with the antigen (eg, using labeled antigen or antigen bound or supplemented to a solid surface or bead). Phage used in these methods are typically phage having the antibody domain of Fab, Fv or disulfide-stabilized Fv recombinantly fused to either the phage gene III protein or the phage gene VIII protein. Is a filamentous phage containing the fd and M13 binding domains expressed from E. coli. Examples of phage display methods that can be used to generate the antibodies of the invention include those disclosed below: Brinkman et al. Immunol. Method
s 182: 41-50 (1995); Ames et al., J. Am. Immunol. Me
thods 184: 177-186 (1995); Kettleboroug.
h et al., Eur. J. Immunol. 24: 952-958 (1994); Pe.
rsic et al., Gene 187 9-18 (1997); Burton et al., Ad.
vances in Immunology 57: 191-280 (1994)
); PCT application number PCT / GB91 / 01134; PCT publication WO 90/0
2809; WO 91/10737; WO 92/01047; WO 92/1.
8619; WO 93/11236; WO 95/15982; WO 95/2.
0401; and U.S. Patent Nos. 5,698,426; 5,223,409.
No. 5,403,484; No. 5,580,717; No. 5,427.
No. 908; No. 5,750,753; No. 5,821,047; No. 5,5.
No. 71,698; No. 5,427,908; No. 5,516,637; No. 5,780,225; No. 5,658,727; No. 5,733,743 and No. 5,969,108 (the above references are incorporated herein by reference in their entirety).

As described in the above references, after phage selection, the region encoding the antibody derived from the phage can be used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment. It can be separated and used and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria (eg, as described in detail below). For example, techniques for recombinantly producing Fab, Fab 'and F (ab') 2 fragments can also be used using methods known in the art. Such methods are disclosed, for example, in: PCT Publication WO 92/22324; Mullinax et al., BioT.
techniques 12 (6): 864-869 (1992); and Saw.
ai et al., AJRI 34: 26-34 (1995); and Better et al., S.
science 240: 1041-1043 (1998) (these references are incorporated by reference in their entirety).

Examples of techniques that can be used to produce single chain Fvs and antibodies include US Pat. Nos. 4,946,778 and 5,258,498; Huston.
Et al., Methods in Enzymology 203: 46-88 (19.
91); Shu et al., PNAS 90: 7995-7999 (1993); and Skerra et al., Science 240: 1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, the use of chimeric, humanized or human antibodies may be preferred. A chimeric antibody is a molecule in which different parts of the antibody are derived from different animal species (for example, an antibody having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region). Methods for making chimeric antibodies are known in the art. For example, Morris
on, Science 229: 1202 (1985); Oi et al., BioTec.
hnies 4: 214 (1986); Gillies et al., (1989) J.
． Immunol. Methods 125: 191-202; US Pat. No. 5,
807,715; 4,816,567; and 4,816,397.
No., which are hereby incorporated by reference in their entireties. A humanized antibody is an antibody molecule from a non-human species antibody that binds to a desired antibody having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. . Often, framework residues in the human framework regions will be replaced with the corresponding residue from the CDR donor antibody to alter (preferably improve) antigen binding. Substitutions of these frameworks are identified by methods well known in the art, for example modeling CDR and framework residue interactions to identify framework residues important for antigen binding, and at specific positions. By sequence comparison to identify unusual framework residues. (For example, Queen et al., US Pat. No. 5,585,089; Riechmann. Et al.
See Nature 332: 323 (1988). These are incorporated herein by reference in their entirety). Antibodies can be humanized using various techniques known in the art including the following: eg, CDR-grafting (European Patent No. 23).
No. 9,400; PCT Publication WO 91/09967; US Pat. No. 5,225,5.
39; 5,530,101 and 5,585,089), veneering or resurfacing.
ng) (European Patent Nos. 592,106; 519,596; Padlan,
Molecular Immunology 28 (4/5): 489-498
(1991); Studnicka et al., Protein Engineeri.
ng 7 (6): 805-814 (1994); Roguska et al., PNAS.
91: 969-973 (1994)), and chain shuffling (cha).
in shuffling (US Pat. No. 5,565,332).

Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT
Published WO 98/46645, WO 98/50433, WO 98/2489
3, WO 98/16654, WO 96/34096, WO 96/3373
5, and WO 91/10741 ;, each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins but are capable of expressing human immunoglobulin genes. For example, a complex of human heavy and light chain immunoglobulin genes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, human variable regions, constant regions and diversity regions (diversity)
region) can be introduced into mouse embryonic stem cells in addition to the human heavy chain gene and human light chain gene. The mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletions in the JH region interfere with endogenous antibody production. The modified embryonic stem cells are developed and microinjected into undifferentiated embryo cells to produce chimeric mice. Next, the chimeric mouse
Breed to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the normal fashion with a selected antigen (eg, all or part of a polypeptide of the invention). Monoclonal antibodies directed against the antigen are obtained from immunized transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes are masked during B cell differentiation by rearrangement of transgenic mice and are followed by class switching and somatic mutations. Thus, the use of such techniques allows for the production of therapeutically useful IgG, IgA, IgM and IgE antibodies.
For an overview of this technology for producing human antibodies, see Lonberg and H.
uszar, Int. Rev. Immunol. 13: 65-93 (1995)
checking ... For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, eg, PCT Publication WO98 / 24893; WO92 / 01047; WO
96/34096; WO96 / 33735; European Patent 0 598 877; U.S. Patents 5,413,923; 5,625,126; 5,633.
No. 425; No. 5,569,825; No. 5,661,016; No. 5,5.
45,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598 (these are incorporated by reference in their entirety). Are incorporated herein by reference). In addition, Abgenix,
Inc. (Freemont, CA) and Genpharm (San Jos.
e., CA) may engage in providing human antibodies directed against the selected antigen using techniques similar to those described above.

Human antibodies that fully recognize the selected epitope were "guided (gui).
ed) selection ”. In this approach,
The selected non-human monoclonal antibody (eg, mouse antibody) is used to guide the selection of human antibodies that fully recognize the same epitope. (Jespers
Et al., Bio / technology 12: 899-903 (1988)) Furthermore, antibodies to the polypeptides of the invention produce anti-idiotypic antibodies that "mimic" the polypeptides of the invention using techniques well known to those of skill in the art. Can be used in sequence to do. (Eg Greenspan and Bona, FASEB
J. 7 (5): 437-444; (1989) and Nissinoff, J.
． Immunol. 147 (8): 2429-2438 (1991). ) For example, binding and multimerization of polypeptides
ization) and / or antibodies that competitively inhibit the binding of the polypeptides of the invention to ligands can be used to produce anti-idiotypes that “mimic” the multimerization and / or binding domains of the polypeptides, And consequently binds and neutralizes the polypeptide and / or its ligand.
Neutralization of such anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligands. For example, such anti-idiotypic antibodies can be used to bind to a polypeptide of the invention and / or to bind its ligand / receptor, thereby blocking its biological activity.

Polynucleotides Encoding Antibodies The present invention further provides polynucleotides comprising nucleotide sequences encoding the antibodies of the present invention and fragments thereof. The invention also provides for stringent or low stringency hybridization conditions (eg,
A polypeptide encoding an antibody (as defined above), preferably an antibody which specifically binds to a polypeptide of the invention, preferably which binds to a polypeptide having the amino acid sequence of SEQ ID NO: 2 or 4. It includes a polynucleotide that hybridizes to the nucleotide.

The polynucleotide may be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide determined. For example, if the nucleotide sequence of the antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (eg, Kutmeier et al., B.
ioTechniques 17: 242 (1994)),
Briefly, this involves the following steps: synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of these oligonucleotides, followed by PCR of the ligated oligonucleotides. amplification.

Alternatively, the antibody-encoding polynucleotide can be made from nucleic acids from suitable sources. No clone containing a nucleic acid encoding a particular antibody is available, but if the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin is
It can be chemically synthesized or from a suitable source (eg, an antibody cDNA library, or any tissue or cell expressing the antibody (eg, a hybridoma cell selected for expression of an antibody of the invention)). The generated cDNA library, or a nucleic acid isolated from it (preferably polyA + RNA), is used, for example, to identify a cDNA clone from a cDNA library encoding an antibody at the 3'end or 5'end of the sequence. It can be obtained by PCR amplification using synthetic primers hybridizable to the ends or by cloning using oligonucleotide probes specific for particular gene sequences. The amplified nucleic acid produced by PCR can then be cloned into a replicable cloning vector using any method known in the art.

Once the nucleotide sequence of the antibody and the corresponding amino acid sequence have been determined,
Nucleotide sequences of antibodies can be prepared by methods well known in the art for manipulation of nucleotide sequences (eg, recombinant DNA technology, site-directed mutagenesis, PCR, etc. (eg,
Sambrook et al., 1990, Molecular Cloning, AL.
Laboratory Manual, 2nd Edition, Cold Spring Har
bor Laboratory, Cold Spring Harbor, NY
And Ausubel et al., 1998, Current Protocols.
in Molecular Biology, John Wiley & Sons
, NY. Both are incorporated herein by reference in their entirety. )) Can be engineered with to produce antibodies with different amino acid sequences to generate, for example, amino acid substitutions, deletions, and / or insertions.

In certain embodiments, the amino acid sequences of the variable domains of the heavy and / or light chains are identified by methods well known in the art for identification of sequences of complementarity determining regions (CDRs) (eg, sequence Other heavy chains to determine hypervariable regions,
And by comparing the variable region of the light chain with a known amino acid sequence). Using conventional recombinant DNA techniques, one or more CDRs may be inserted within the framework regions as described above (eg, into the human framework regions for humanizing non-human antibodies). . This framework region can be naturally occurring or can be a consensus framework region, and preferably a human framework region (eg, for the listed human framework regions, Ch
othia et al. Mol. Biol. 278: 457-479 (1998)). Preferably, the polynucleotide produced by the combination of the framework regions and CDRs encodes an antibody that specifically binds to a polypeptide of the invention. Preferably, as discussed above, one or more amino acid substitutions may be made within the framework regions, and preferably the amino acid substitutions improve the binding of the antibody to its antigen. Further, such methods provide for amino acid substitutions or deletions of cysteine residues in one or more variable regions involved in intrachain disulfide bonds, so as to produce antibody molecules lacking one or more intrachain disulfide bonds. It can be used to create losses. Other changes to the polynucleotide are encompassed by the present invention and in the art.

In addition, techniques developed to produce "chimeric antibodies" by splicing genes from mouse antibody molecules of suitable antigen specificity with genes from human antibody molecules of suitable biological activity. (Morrison et al., Proc. N.
atl. Acad. Sci. 81: 851-855 (1984); Neuber.
ger et al., Nature 312: 604-608 (1984); Takeda.
Et al., Nature 314: 452-454 (1985)). As mentioned above, a chimeric antibody is a molecule in which different portions are derived from different animal species, such molecules having variable regions derived from the constant regions of mouse mAb and human immunoglobulin (eg, human Antibody).

Alternatively, techniques described for the production of single chain antibodies (US Pat. No. 4,946,46)
778; Bird, Science 242: 423-42 (1988); H.
uston et al., Proc. Natl. Acad. Sci. USA 85: 587.
9-5883 (1988); and Ward et al., Nature 334: 544.
-54 (1989)) can be adapted to the production of single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region through amino acid bridges, yielding single chain polypeptides. E. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Sk
erra et al., Science 242: 1038-1041 (1988)).

Methods of Producing Antibodies The antibodies of the invention are produced by any method known in the art for synthesizing antibodies, particularly by chemical synthesis or, preferably, by recombinant expression techniques. Can be done.

An antibody of the invention, or a fragment, derivative or analog thereof (eg,
Recombinant expression of the heavy or light chain of the antibody of the invention or the single chain antibody of the invention)
Construction of an expression vector containing a polynucleotide encoding the antibody is required. Once the polynucleotide encoding the antibody molecule of the invention or the heavy or light chain of the antibody, or a portion thereof (preferably containing the variable domain of the heavy or light chain) is obtained, production of the antibody molecule Vectors for can be produced by recombinant DNA technology using techniques well known in the art. Accordingly, methods for preparing a protein by expression of a polynucleotide containing an antibody encoding a nucleotide sequence are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example:
In vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination are included. Accordingly, the invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. To do. Such vectors include constant regions of antibody molecules (eg, PCT Publication WO 86/0580).
7; PCT Publication WO 89/01036; and US Pat. No. 5,122,464) and antibodies that can be cloned into such vectors for full expression of heavy or light chains. Variable domains of

The expression vector is transferred into a host cell by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibody of the invention. Accordingly, the invention includes a host cell containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter. In a preferred embodiment for expression of double-chain antibodies, the vectors encoding both heavy and light chains are co-expressed in a host cell for total expression of immunoglobulin molecules, as detailed below. Can be done.

A variety of host expression vector systems may be utilized to express the antibody molecule of the present invention. Such host expression systems represent vehicles in which the coding sequence of interest may be produced and subsequently purified, but also in situ when transformed or transfected with sequences encoding the appropriate nucleotides. 2 represents a cell capable of expressing an antibody molecule of the invention. These include bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody-encoding sequences (eg E. coli, B. subtilis).
To a yeast transformed with a recombinant yeast expression vector containing a sequence encoding an antibody (eg, Saccharomyces, Pichia); a recombinant viral expression vector containing a sequence encoding an antibody (eg, baculovirus) Insect insect cell line; plant cell line infected with a recombinant viral expression vector (eg cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or a recombinant plasmid expression vector containing a sequence encoding an antibody (eg Ti plasmid ) Plant cell line transformed with); or a promoter derived from the genome of a mammalian cell (eg, metallothionein promoter)
Alternatively, a mammalian cell line (eg, COS, CHO, BHK, 29) carrying a recombinant expression construct containing a promoter derived from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K promoter).
3, 3T3 cells), but are not limited thereto. Preferably Esc
bacterial cells such as herichia coli, and more preferably, especially
Eukaryotic cells for the expression of whole recombinant antibody molecules are used for the expression of recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) in combination with vectors such as the major immediate early gene promoter element from human cytomegalovirus have been shown to be effective expression systems for antibodies. Yes (Föcking et al., Gene 45: 101 (1986); C
Ockett et al., Bio / Technology 8: 2 (1990)).

In bacterial systems, a number of expression vectors may be advantageously selected depending on the use intended for the expression of the antibody molecule. For example, if large quantities of such proteins are to be produced, vectors may be desired that direct high levels of expression of the fusion protein product, which are easily purified for the production of pharmaceutical compositions of antibody molecules. In such a vector, the antibody-encoding sequence may be separately ligated in frame to the vector along with the lacZ-encoding region, such that a fusion protein is produced.
coli expression vector pUR278 (Ruther et al., EMBO J. 2:17).
91 (1983)); pIN vector (Inouye & Inouye, Nucl.
eic Acids Res. 13: 3101-3109 (1985); Van.
Heeke & Schuster, J. Biol. Chem. 24: 5503-
5509 (1989)) and the like, but are not limited thereto. pGEX
Vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general,
Such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vector is designed to contain a thrombin or factor Xa protease cleavage site so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (Aut
ographa California nuclear polyhedr
osis virus) (AcNPV) is used as a vector to express foreign genes. The virus is Spodoptera frugi
Proliferates in perda cells. The antibody coding sequence may be individually cloned into a nonessential region of the virus (eg, the polyhedrin gene) and placed under control of an AcNPV promoter (eg, the polyhedrin promoter).

Many viral-based expression systems can be utilized in mammalian host cells. When an adenovirus is used as an expression vector, the sequence of interest encoding the antibody can be linked to an adenovirus transcription / translation control complex (eg, a late promoter and a tripartite leader sequence). This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion into a nonessential region of the viral genome (eg region E1 or E3) results in a recombinant virus that survives in the infected host and is capable of expressing antibody molecules. (For example, Logan & Shenk, Proc. Natl. Ac
ad. Sci. USA 81: 355-359 (1984)). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, this start codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be increased by including appropriate transcription enhancer elements, transcription terminators, etc. (Bittner et al., Meth).
ods in Enzymol. 153: 51-544 (1987)).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can be important for protein function. Different host cells have characteristic and unique mechanisms for post-translational processing and modification of protein and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells may be used that have the cellular machinery for proper processing of the primary transcript, glycosylation of the gene product, and phosphorylation. Such mammalian host cells include CHO, VERY, BHK, Hela, COS, M
DCK, 293, 3T3, WI38 and, in particular, breast cancer cell lines (eg BT
483, Hs578T, HTB2, BT20 and T47D) as well as normal mammary gland cell lines such as CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred.
For example, cell lines that stably express the antibody molecule can be engineered. Rather than the use of expression vectors containing viral origins of replication, the host cell is controlled by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.) and selectable markers. It can be transformed with DNA. Following the introduction of exogenous DNA, engineered cells can be grown for 1-2 days in concentrated medium and then switched to selective medium. A selectable marker in the recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into their chromosomes and proliferate, forming a lesion that can then be cloned and propagated into the cell line. . This method can be advantageously used to engineer cell lines expressing antibody molecules. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

Many selection systems may be used, including herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223 (1977)), which may be used in tk-, hgprt-, or aprt- cells, respectively. Hypoxanthine-guanine phosphoribosyl transferase (Szybalska and S
zybalski, Proc. Natl. Acad. Sci. USA 48: 2
02 (1992)), and adenine phosphoribosyl transferase (Lo
wy et al., Cell 22: 817 (1980)) gene, but is not limited thereto. Antimetabolite resistance can also be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al.,
Natl. Acad. Sci. USA 77: 357 (1980)); O'Ha.
Re et al., Proc. Natl. Acad. Sci. USA 78: 1527 (1
981); gpt which confers resistance to mycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA 78: 2072 (
1981)); conferring resistance to aminoglycoside G-418 neo (Clin
ic Pharmacy 12: 488-505; Wu and Wu, Bio.
therapy 3: 87-95 (1991); Tolstosev, Ann.
． Rev. Pharmacol. Toxicol. 32: 573-596 (19
93); Mulligan, Science 260: 926-932 (199).
3); and Morgan and Anderson, Ann. Rev. Bio
chem. 62: 191-217 (1993); May 1993, TIB TE.
CH 11 (5): 155-215); as well as hygro conferring resistance to hygromycin (Santerre et al., Gene 30: 147 (1984)).
. Recombinant DNA technology methods generally known in the art can be routinely applied to the selection of desired recombinant clones, and such methods are described, for example, in Ausub.
el et al. (ed.), Current Protocols in Molecular
r Biology, John Wiley & Sons, NY (1993); K.
riegler, Gene Transfer and Expression
, A Laboratory Manual, Stockton Press,
NY (1990); and Dracopoli et al. (Eds.), Current P.
rotocols in Human Genetics, John Wile
y & Sons, NY (1994) chapters 12 and 13; Colberre-Ga.
rapin et al. Mol. Biol. 150: 1 (1981), which are incorporated herein by reference in their entirety.

Expression levels of antibody molecules can be increased by vector amplification (for a review, Bebbington and Hentschel, The use of).
vectors based on gene amplification
for the expression of cloned genes i
n mammalian cells in DNA cloning, Volume 3 (Academic Press, New York, 1987)). If the marker can be amplified in the vector system expressing the antibody, increasing the level of inhibitor present in the culture medium of the host cell will increase the number of copies of the marker gene. The production of this antibody is also increased due to the binding of this amplified region to the antibody gene (Crouse et al., Mol. Cell. Biol. 3: 257.
(1983)).

The host cell is co-expressed with two expression vectors of the invention, a first vector encoding a polypeptide derived from the heavy chain and a second vector encoding a polypeptide derived from the light chain. Can be transfected. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides and allows for expression thereof. In such a situation, this light chain should be placed before the heavy chain to avoid excess toxic free heavy chain (Proudfoot, Nature 322: 52 (1986);
Kohler, Proc. Natl. Acad. Sci. USA 77: 219.
7 (1980)). The coding sequences for the heavy and light chains may be cDNA or genomic DN.
A may be included.

Once the antibody molecule of the present invention is produced by an animal, chemically synthesized, or recombinantly expressed, any of the art-known methods for the purification of immunoglobulin molecules. Methods such as chromatography (eg, ion exchange,
It can be purified by affinity (particularly by affinity for protein A followed by specific antigen), and size column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification.
Furthermore, the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

The present invention relates to a polypeptide of the invention (or a portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 of this polypeptide,
Or 100 amino acids), recombinantly fused or chemically linked (including both covalent and non-covalent bonds) to produce a fusion protein. The fusion need not be direct, but can occur via a linker sequence. The antibody is a polypeptide of the invention (or a portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70,
(80, 90, or 100 amino acids). For example, by fusing or conjugating a polypeptide of the invention to an antibody specific for a particular cell surface receptor, either in vitro or in vivo, the polypeptide of the invention is directed against a particular cell type. To target
Antibodies can be used. Antibodies fused or conjugated to the polypeptides of the invention can also be used in in vitro immunoassays and purification methods using methods known in the art. See, eg, Harbor et al., Supra, and PCT Publication WO93.
/ 21232; EP 439,095; Naramura et al., Immunol. L
ett. 39: 91-99 (1994); US Patent No. 5,474,981; G.
illies et al., PNAS 89: 1428-1432 (1992); Fell.
Et al., J. Immunol. 146: 2446-2452 (1991). These are incorporated by reference in their entirety.

The invention further includes compositions comprising a polypeptide of the invention fused or linked to a domain other than the variable region of an antibody. For example, the polypeptides of the invention can be fused or conjugated to the Fc region of an antibody, or a portion thereof. The part of this antibody fused to the polypeptide of the invention comprises the constant region, hinge region, CH1 domain, C
It may include the H2 domain, and the CH3 domain, or any combination of whole domains or portions thereof. These polypeptides can also be fused or conjugated to portions of the above antibodies to form multimers. For example, an Fc portion fused to a polypeptide of the invention can form a dimer through a disulfide bond between the Fc portions. Higher multimeric forms can be made by fusing the polypeptide to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the invention to antibody moieties are known in the art. For example, US Pat. Nos. 5,336,603; 5,622,929; 5,359,04.
No. 6, No. 5,349,053; No. 5,447,851; No. 5,112.
, 946; EP 307,434; EP 367,166; PCT publication WO9.
6/04388; WO91 / 06570; Ashkenazi et al., Proc. N
atl. Acad. Sci. USA 88: 10535-10539 (1991).
); Zheng et al. Immunol. 154: 5590-5600 (199
5); and Vil et al., Proc. Natl. Acad. Sci. USA 89
: 11337-11341 (1992) (the above references are incorporated by reference in their entireties).

As discussed above, the polypeptide corresponding to the polypeptide, polypeptide fragment, or variant of SEQ ID NO: 2 or 4, is intended to increase the in vivo half-life of this polypeptide. Alternatively, it can be fused or conjugated to the antibody moieties described above for use in immunological assays using methods known in the art. In addition, the polypeptide corresponding to SEQ ID NO: 2 or 4 may be fused or conjugated to the antibody portion described above to facilitate purification. One reported example is
A chimeric protein consisting of the first two domains of the human CD4 polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins has been described (EP 394,827; Traunecker et al., Nature).
331: 84-86 (1988)). Polypeptides of the invention fused or conjugated to an antibody having a disulfide-linked dimer structure (due to IgG) also bind to other molecules than monomeric secreted proteins or protein fragments alone. And can be more efficient in neutralizing (Funtoulaki
S., et al. Biochem. 270: 3958-3964 (1995)). In many cases, the Fc portion of the fusion protein will be beneficial in therapy and diagnosis, and thus may result, for example, in improved pharmacokinetic properties (EP A 232,
262). Alternatively, it may be desirable to delete the Fc portion after the fusion protein has been expressed, detected and purified. For example, if the fusion protein is used as an antigen for immunization, the Fc portion may interfere with therapy and diagnosis. For example, in drug discovery, human proteins such as hIL-5 are
Has been fused to the Fc portion for the purpose of high throughput screening assays to identify antagonists of -5 (Bennett et al., J. Molec.
urular Recognition 8: 52-58 (1995); Johann.
Son et al. Biol. Chem. 270: 9459-9471 (1995).
checking).

In addition, the antibodies of the invention or fragments thereof may be fused to a marker sequence, such as a peptide that facilitates purification. In a preferred embodiment, the marker amino acid sequence is inter alia a hexa-histidine peptide (eg pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chats.
The tag, provided by W. Worth, CA, 91311), and many of these marker amino acid sequences are commercially available. For example, Gentz et al., Proc
． Natl. Acad. Sci. Hexa-histidine provides convenient purification of fusion proteins, as described in USA 86: 821-824 (1989). Another peptide tag useful for purification is the "HA" tag (Wilson et al., Ce, which corresponds to an epitope derived from the influenza hemagglutinin protein.
ll37: 767 (1984)), and "flag" tags.

The invention further includes an antibody or fragment thereof conjugated to a diagnostic or therapeutic agent. Antibodies can be used diagnostically, for example, to monitor tumor development or progression as part of a clinical testing procedure (eg, to determine the efficacy of a given treatment regimen). Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radioactive substances,
Positron emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. The detectable substance is either directly or indirectly directed to the antibody (or fragment thereof) by a mediator using techniques known in the art, such as linkers known in the art. Can be linked or conjugated via. See, eg, US Pat. No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent substances include luminol; bioluminescent substances Examples of luciferase include luciferase, luciferin and aequorin; and examples of suitable radioactive substances include 125I, 131I, 111In or 99Tc.

Furthermore, the antibody or fragment thereof may be used in therapeutic moieties (eg cytotoxins (eg cytostatic or cytotoxic agents)), therapeutic agents or radioactive metal ions (eg α-emitters (eg 213Bi)). Etc.). Cytotoxic or cytotoxic agents include any agent that is harmful to cells. Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide (t
Enoposide), vincristine, vinblastine, colchicine (col)
chicin), doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,
Glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologues thereof. The therapeutic agent may be an antimetabolite (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine (5
-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa (thioepa) chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide (cyclothosphamide), busulfan, dibromomannitol, streptozotocin, mitomycin C, And cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (eg daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg dactinomycin (formerly actinomycin), bleomycin, mithramycin, And anthramycin (AMC),
And anti-mitotic agents such as vincristine and vinblastine, but are not limited thereto.

The conjugates of the invention can be used to modify a given biological response and the therapeutic agent or drug moiety is not construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide that has the desired biological activity. Such proteins include, for example, toxins (eg, abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin); proteins (eg,
Tumor necrosis factor, a-interferon, β-interferon, nerve growth factor,
Platelet-derived growth factor, tissue plasminogen activator, apoptotic drug (eg TNF-α, TNF-β, AIM I (International Publication No. WO97 / 338).
99), AIM II (see WO 97/34911), Fas ligand (Takahashi et al., Int. Immunol).
． 6: 1567-1574 (1994)), VEGI (International Publication No. WO99 / 2).
3105)), thrombotic or anti-angiogenic agents (eg, angiostatin or endostatin); or biological response modifiers (eg, lymphokines, interleukin-1 (“IL-1”)). , Interleukin-2 ("
IL-2 "), interleukin-6 (" IL-6 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), granulocyte colony stimulating factor (" G-CS ").
F "), or other growth factors).

The antibody can also be attached to a solid support, which is particularly useful for immunoassay or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moieties to antibodies are well known, eg Arn.
on et al., “Monoclonal Antibodies For Immun.
targeting Of Drugs In Cancer Therap
y ”Monoclonal Antibodies And Cancer T
cure, Reisfeld et al. (eds.), pp. 243-56 (Alan RL
iss, Inc. 1985); Hellstrom et al., "Antibodies.
For Drug Delivery "Controlled Drug D
release (2nd edition), Robinson et al. (eds.), pp. 623-53 (Ma.
rcel Dekker, Inc. 1987); Thorpe, "Antibo
dy Carriers Of Cytotoxic Agents In C
cancer Therapy: A Review "Monoclonal An
tibodies'84: Biological And Clinical
Applications, Pinchera et al. (Eds.), Pp. 475-506 (1
985); "Analysis, Results, And Future Pr.
Descendant Of The Therapeutic Use Of
Radiolabeled Antibody In Cancer Ther
apy ”Monoclonal Antibodies For Cancer
Detection And Therapy, Baldwin et al. (Ed.), 3
03-16 (Academic Press 1985), and Thorp
e, et al., "The Preparation And Cytotoxic Pr.
operations of Antibody-Toxin Conjugate
s ", Immunol. Rev. 62: 119-58 (1982).

Alternatively, the antibody is conjugated to a second antibody, and is described in US Pat. No. 4,676,980 (
This may form an antibody heteroconjugate as described by Segal in (incorporated herein in its entirety).

Antibodies, with or without a therapeutic moiety that binds to the antibody, administered alone or in combination with cytotoxic factors and / or cytokines, can be used as therapeutic agents.

Immunophenotyping The antibodies of the invention can be utilized for immunophenotyping cell lines and biological samples. The translation products of the genes of the present invention are useful as cell-specific markers, or more particularly as cell markers that are differentially expressed at various stages of differentiation and / or maturation of particular cell types. obtain. Monoclonal antibodies against specific epitopes, or combinations of epitopes, allow screening of cell populations that express the marker. Various techniques can be utilized with monoclonal antibodies to screen for cell populations that express markers, and include magnetic separation using antibody-coated magnetic beads, solid matrices (ie, "Panning" with antibodies attached to plates), as well as flow cytometry (see, eg, US Pat. No. 5,985,660; and Morrison et al., Cell, 96: 737-49 (1999)). To be

These techniques apply to hematological malignancies (ie, minimal residual disease (MRD) in patients with acute leukemia).
And allows screening of specific populations of cells as may be found with "non-self" cells in transplantation to prevent graft versus host disease (GVHD). Alternatively, these techniques allow for the screening of hematopoietic stem and progenitor cells that may undergo proliferation and / or differentiation as may be found in human cord blood.

Assays for Antibody Binding The antibodies of the invention can be assayed for immunospecific binding by any method known in the art. Immunoassays that can be used include Western blots, radioimmunoassays, to name just a few.
ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, precipitation reaction, gel diffusion precipitation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric assay, fluorescent immunoassay, protein A Included, but not limited to, competitive and non-competitive assay systems that use techniques such as immunoassays. Such assays are routine and well known in the art (eg, Ausubel et al. Eds., 1994, Current Protocol), which is incorporated by reference herein in its entirety.
ls in Molecular Biology, Volume 1, John Wil
ey & Sons, Inc. , New York). Exemplary immunoassays are briefly described below (although these are not intended to be limiting).

Immunoprecipitation protocols generally involve RIPA buffer (1% NP-40 or Triton X-100, 1, 1% NP-40 or Triton X-100, supplemented with protein phosphatase inhibitors and / or protease inhibitors (eg, EDTA, PMSF, aprotinin, sodium vanadate). % Sodium deoxycholate, 0.1%
SDS, 0.15M NaCl, 0.01M sodium phosphate (pH 7.2)
) Lysing a population of cells in a lysis buffer such as 1% Transylol), adding the antibody of interest to the cell lysate, and incubating at 4 ° C. for a period of time (eg 1-4 hours) Adding protein A sepharose beads and / or protein G sepharose beads to the cell lysate, incubating at 4 ° C. for about 1 hour or more, washing the beads in lysis buffer, and SDS / sample buffer Resuspending the beads in. The ability of the antibody of interest to immunoprecipitate a particular antigen can be determined, for example, by Western blot analysis.
Can be assayed. One of skill in the art will be aware of parameters that can be modified to increase binding of antibody to antigen and to reduce background (eg, preclearing cell lysates with Sepharose beads). obtain. For further discussion on immunoprecipitation protocols, see, eg, Ausub.
el et al., 1994, Current Protocols in Molec.
ural Biology, Volume 1, John Wiley & Sons, Inc.
． , New York, 10.16.1.

Western blot analysis generally involves the steps of preparing a protein sample, polyacrylamide gel (eg 8% -20% SDS-depending on the molecular weight of the antigen).
Electrophoresis of the protein sample in PAGE, transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking solution (eg 3% BSA or PBS with skim milk powder) ), Blocking the membrane in a wash buffer (eg,
Washing the membrane in PBS-Tween 20), blocking the membrane with a primary antibody (antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer , A secondary antibody conjugated to an enzyme substrate (eg, horseradish peroxidase or alkaline phosphatase) or a radioactive molecule (eg, 32P or 125I) diluted in blocking buffer (which recognizes the primary antibody, eg, anti-body). Human antibody) to block the membrane, wash the membrane in wash buffer, and detect the presence of the antigen. One of skill in the art will be aware of parameters that can be modified to increase the signal detected and to reduce background noise. For further discussion on Western blot protocols, see, eg, Ausubel et al., Eds., 1994, Current Protocols i.
n Molecular Biology, Volume 1, John Wiley & S
ons, Inc. , New York, 10.8.1.

ELISA involves preparing an antigen, coating the wells of a 96-well microtiter plate with the antigen, conjugating to a detectable compound such as an enzyme substrate (eg horseradish peroxidase or alkaline phosphatase). The antibody of interest is added to the wells and incubated for a period of time, and the presence of the antigen is detected. In the ELISA, the antibody of interest need not be bound to a detectable compound; instead, a second antibody conjugated to the detectable compound (which recognizes the antibody of interest) will bind to the wells. Can be added. Further, instead of coating the well with the antigen, the antibody can be coated to the well. In this case, a second antibody conjugated to a detectable compound can be added following the addition of the antigen of interest to the coated wells. One of ordinary skill in the art will be aware of parameters that can be modified to increase the signal detected and other variations of the ELISA known in the art. For more discussion on ELISA, see, for example, Aus.
ubel et al., 1994, Current Protocols in Mol.
electrical Biology, Volume 1, John Wiley & Sons, I
nc. , New York, 11.2.1.

[0130] The binding affinity of the antibody for the antigen and the off-rate of the antibody-antigen interaction (of
f-rate) can be determined by competitive binding assay. One example of a competitive binding assay is a radioimmunoassay, which comprises incubating a labeled antigen (eg, 3H or 125I) with an antibody of interest in the presence of increasing amounts of unlabeled antigen, and Includes detection of antibody conjugated to labeled antigen. The affinity of the antibody of interest for a particular antigen, and the binding off-rate can be determined from the data by Scatchard plot analysis. Competition with the second antibody can also be determined using a radioimmunoassay. In this case, the antigen is labeled compound (eg, 3H or 125) in the presence of increasing amounts of unlabeled second antibody.
Incubated with the antibody of interest bound to I).

Therapeutic Uses The present invention further relates to antibody-based therapies for treating an animal, preferably a mammal, and most preferably for treating one or more of the disclosed diseases, disorders, or conditions. Preferably, the step of administering the antibody of the present invention to a human patient is included. Therapeutic compounds of the invention include antibodies of the invention (including fragments, analogs and derivatives thereof, as described herein) and nucleic acids encoding the antibodies of the invention (described herein). (Including, but not limited to, fragments, analogs and derivatives thereof and anti-idiotypic antibodies). Antibodies of the invention include diseases, disorders or conditions associated with aberrant expression and / or activity of the polypeptides of the invention (including any one or more of the diseases, disorders, or conditions described herein). Can be used to treat, inhibit or prevent). Aberrant expression of the polypeptide of the invention and /
Alternatively, treating and / or preventing a disease, disorder or condition associated with activity includes, but is not limited to, ameliorating the symptoms associated with the disease, disorder or condition. The antibodies of the present invention are known in the art or can be provided in a pharmaceutically acceptable composition as described herein.

A summary of how the antibodies of the invention may be used therapeutically is mediated locally or systemically in the body (eg, mediated by complement (CDC), or by effector cells (ADCC)). By direct binding of the polynucleotide or polypeptide of the invention by the direct cytotoxicity of the antibody. Some of these approaches are described in more detail below. Given the teachings provided herein, one of ordinary skill in the art would be able to determine, without undue experimentation, how to use the antibodies of the invention for diagnostic, monitoring or therapeutic purposes. Recognize.

The antibodies of the invention may be other monoclonal or chimeric antibodies, or lymphokines or hematopoietic growth factors (eg IL-2, IL-2, IL-2, IL-2, IL-2, IL-2, IL-2, IL-2, IL-2 IL-3 and IL
(Such as −7).

The antibodies of the invention can be administered alone or in combination with other types of treatments such as radiation therapy, chemotherapy, hormone therapy, immunotherapy and anti-tumor agents.
Generally, it is preferred to administer a species-sourced or species-reactive product that is the same species as the patient's species (in the case of antibodies). Thus, in a preferred embodiment, human antibodies,
The fragment derivative, analog, or nucleic acid is administered to a human patient for treatment or prevention.

A high affinity and / or Alternatively, it is preferred to use potent in vivo inhibitory and / or neutralizing antibodies, fragments thereof, or regions thereof. Such antibodies, fragments or regions preferably have an affinity for the polynucleotides or polypeptides of the present invention, including fragments thereof. The preferred binding affinity is 5 × 10 ⁻² M, 10 ⁻² M, 5 × 10 ⁻³ M, 10 ⁻³ M, 5 × 10 ⁻⁴ M,
10 ⁻⁴ M, 5 × 10 ⁻⁵ M, 10 ⁻⁵ M, 5 × 10 ⁻⁶ M, 10 ⁻⁶ M, 5 × 10 ⁻⁷ M,
10 ^-7 M, 5 x 10 ^-8 M, 10 ^-8 M, 5 x 10 ^-9 M, 10 ^-9 M, 5 x 10 ^-10 M
10 ^-10 M, ^{5x10 -11} M, 10 ^-11 M, ^{5x10 -12} M, 10 ^-12 M, ^{5x1
0 -13 M, 10 -13 M,} 5 × 10 -14 M, 10 -14 M, 5 × 10 -15 M, and 10 ^{- 15} binding affinity are exemplified with small dissociation constant or Kd than M.

Gene Therapy In certain embodiments, a nucleic acid comprising a sequence encoding an antibody or functional derivative thereof treats a disease or disorder associated with aberrant expression and / or activity of a polypeptide of the invention, Administered for the purpose of gene therapy to inhibit or prevent. Gene therapy refers to a therapy that is performed by administering an expressed or expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acids produce their encoded protein, which mediates a therapeutic effect.

Any method available in the art for gene therapy can be used in accordance with the present invention. An exemplary method is described below.

For a general review of methods of gene therapy, see Goldspiel et al., Cli.
Nal Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstos.
hev, Ann. Rev. Pharmacol. Toxicol. 32: 573
-596 (1993); Mulligan, Science 260: 926-.
932 (1993); and Morgan and Anderson, Ann.
Rev. Biochem. 62: 191-217 (1993); May, TIB.
See TECH 11 (5): 155-215 (1993). Methods generally known in the recombinant DNA art that can be used are described in Ausubel.
Et al., Current Protocols in Molecular
Biology, John Wiley & Sons, NY (1993); and Kriegler, Gene Transfer and Express.
Ion, A Laboratory Manual, Stockton Pre
ss, NY (1990).

In a preferred aspect, the compound comprises a nucleic acid sequence encoding an antibody, said nucleic acid sequence being an expression vector expressing the antibody, or a fragment or chimeric protein thereof, or a heavy or light chain thereof in a suitable host. Is part of. In particular, such nucleic acid sequences have a promoter operably linked to the antibody coding region, said promoter being inducible or constitutive, and optionally tissue-specific. In another particular embodiment, nucleic acid molecules are used that flank the region encoding the antibody and any other desired sequence that facilitates homologous recombination at the desired site in the genome, whereby the antibody is Provides intrachromosomal expression of a nucleic acid encoding (Koller and Smithies, Proc
． Natl. Acad. Sci. USA 86: 8932-8935 (1989).
); Zijlstra et al., Nature 342: 435-438 (1989).
). In a particular embodiment, the expressed antibody molecule is a single chain antibody; or the nucleic acid sequence comprises sequences encoding both the heavy and light chains of the antibody or fragments thereof.

Delivery of the nucleic acid to the patient can be direct (where the patient is directly exposed to the nucleic acid or nucleic acid-bearing vector) or indirect (where the cells are first transfected with the nucleic acid in vitro). Converted and then transplanted to the patient). These two approaches are known as in vivo gene therapy or as ex vivo gene therapy, respectively.

In certain embodiments, the nucleic acid sequences are directly administered in vivo, where they are expressed to produce the encoded product. This can be accomplished by a number of methods known in the art, such as constructing them as part of a suitable nucleic acid expression vector,
And by administering it intracellularly (eg, defective or attenuated retroviruses or other viral vectors (US Pat. No. 4,980,
286)), or by direct injection of naked DNA, or microparticle bombardment (eg gene gun; Bioli).
Stic, Dupont), or by coating with lipids or cell surface receptors, or by transfecting drugs, encapsulation in liposomes, microparticles, or microcapsules, or by entering them into the nucleus. By administering in combination with a known peptide, by administering in combination with a ligand that undergoes receptor-mediated endocytosis (eg Wu and Wu, J. Biol. Chem. 262: 4429-
4432 (1987)) (which can be used to target cell types that specifically express the receptor) and the like. In another embodiment, a nucleic acid-ligand complex can be formed where the ligand comprises a fusogenic viral peptide that disrupts endosomes, allowing the nucleic acid to avoid lysosomal degradation. In yet another embodiment, the nucleic acids can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (eg, PCT Publication No. WO92 / 06180; WO92 /). No. 22635; No. WO 92/20
316; WO93 / 14188, WO93 / 20221). Alternatively, the nucleic acid can be introduced inside the cell and by homologous recombination,
Can be incorporated into host cell DNA for expression (Koller and Smit
hies, Proc. Natl. Acad. Sci. USA 86: 8932-
8935 (1989); Zijlstra et al., Nature 342: 435-.
438 (1989)).

In a specific embodiment, viral vectors that contain nucleic acid sequences that encode the antibodies of the invention are used. For example, a retroviral vector can be used (Mi
ller et al., Meth. Enzymol. 217: 581-599 (1993).
checking). These retroviral vectors contain the necessary components for the correct packaging of the viral genome and integration into host cell DNA. The nucleic acid sequence encoding the antibody used in gene therapy is cloned into one or more vectors, which facilitates delivery of the gene into patients. For more details on retroviral vectors, see Boesen et al., Biothe.
rapid 6: 291-302 (1994), which describes the use of retroviral vectors to deliver the mdr1 gene to hematopoietic stem cells to make the stem cells more resistant to chemotherapy. Can be issued. Other references describing the use of retroviral vectors in gene therapy are: Clo
wes et al. Clin. Invest. 93: 644-651 (1994);
Kiem et al., Blood 83: 1467-1473 (1994); Salmo.
ns and Gunzberg, Human Gene Therapy 4: 1.
29-141 (1993); and Grossman and Wilson, C.
urr. Opin. in Genetics and Devel. 3: 110
-114 (1993).

Adenovirus is another viral vector that can be used in gene therapy. Adenoviruses are particularly attractive vehicles for delivering genes to respiratory epithelia. Adenovirus naturally infects respiratory epithelia where it causes mild disease. Other targets of adenovirus-based delivery systems are the liver, central nervous system, endothelial cells, and muscle. Adenovirus has the advantage that it can infect non-dividing cells. Kozarsky and Wilson, Curr
ent Opinion in Genetics and Developopm
ent 3: 499-503 (1993) provides an overview of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5: 3.
-10 (1994) demonstrated the use of adenovirus vectors to transfer genes into the respiratory epithelia of rhesus monkeys. Other examples of the use of adenoviruses in gene therapy are described by Rosenfeld et al., Science 252: 431-434.
(1991); Rosenfeld et al., Cell 68: 143-155 (19.
92); Mastrangeli et al., J. Am. Clin. Invest. 91:22
5-234 (1993); PCT Publication No. WO94 / 12649; and Wan.
G et al., Gene Therapy 2: 775-783 (1995). In a preferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 2).
04: 289-300 (1993); U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection. Generally, the method of transfer will involve transfer of the selectable marker to the cells. The cells are then subjected to selection to isolate cells that have taken up and are expressing the transferred gene. The cells are then delivered to the patient.

In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be performed by any method known in the art including, but not limited to, transfection, electroporation, microinjection, viral vectors containing nucleic acid sequences. Alternatively, infection with a bacteriophage vector, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion and the like. Many techniques are known in the art for introducing foreign genes into cells (eg, Loeffler and Behr, Meth. Enzymol. 217: 599-618 (1993).
); Cohen et al., Meth. Enzymol. 217: 618-644 (19
93); Cline, Pharmac. Ther. 29: 69-92m (198
5)), and can be used according to the invention if the necessary developmental and physiological functions of the recipient cells are not disrupted. This technique should provide for stable transfer of the nucleic acid to the cell so that the nucleic acid is expressible by the cell and preferably heritable and expressible by the progeny of the cell.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. Recombinant blood cells (eg, hematopoietic stem cells or hematopoietic progenitor cells) are preferably administered intravenously. The amount of cells contemplated for use depends on the desired effect, patient condition, etc. and can be determined by one of ordinary skill in the art.

Cells into which nucleic acids can be introduced for purposes of gene therapy include any desired available cell type and include, but are not limited to: epithelial cells, endothelial cells, keratinocytes, fibers. Blood cells such as blast cells, muscle cells, hepatocytes; T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, especially hematopoietic Stem cells or hematopoietic progenitor cells (eg, cells such as those obtained from bone marrow, cord blood, peripheral blood, fetal liver, etc.).

In a preferred embodiment, the cells used for gene therapy are autologous to the patient.

In embodiments in which recombinant cells are used in gene therapy, the nucleic acid sequence encoding the antibody is introduced into the cells such that the nucleic acid sequences are expressible by the cells or their progeny, and then the recombinant cells are , Administered in vivo for therapeutic effects. In certain embodiments, stem cells or progenitor cells are used. Any stem and / or progenitor cells that can be isolated in vitro and maintained in vitro can potentially be used according to this embodiment of the invention (eg, PCT).
Publication No. WO94 / 08598: Temple and Anderson, Ce
ll 71: 973-985 (1992); Rheinwald, Meth. C
ell Bio. 21A: 229 (1980); and Pitelkow and Scott, Mayo Clinic Proc. 61: 771 (1986
)checking).

In a particular embodiment, the nucleic acid to be introduced for the purpose of gene therapy contains an inducible promoter operably linked to the coding region so that expression of the nucleic acid is controlled by the appropriate transcription inducer. Can be controlled by controlling the presence or absence of.

Demonstration of Therapeutic or Prophylactic Activity The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo, for the desired therapeutic or prophylactic activity prior to use in humans. It For example, in vitro assays to demonstrate therapeutic or prophylactic utility of a compound or pharmaceutical composition include the effect of the compound on cell lines or patient tissue samples. The effect of a compound or composition on cell lines and / or tissue samples can be determined utilizing techniques known to those of skill in the art, including but not limited to rosette formation assays and cytolytic assays. In vitro assays that can be used in accordance with the present invention to determine if administration of a particular compound is indicated include in vitro cell culture assays, in which a patient tissue sample is grown in culture and the compound Or otherwise administered the compound, and the effect of such compound on the tissue sample is observed.

Therapeutic / Prophylactic Administration and Compositions The present invention provides for treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention. To provide a method. In a preferred aspect, the compound is substantially purified (eg, substantially free of substances that limit its effect or produce undesired side effects). The subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably a human.

Formulations and methods of administration that can be used when the compound comprises nucleic acids or immunoglobulins are described above; additional suitable formulations and routes of administration are selected from among those described herein below. Can be done.

Various delivery systems are known and can be used to administer a compound of the invention (eg, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound). , Receptor-mediated endocytosis (
For example, Wu and Wu, J .; Biol. Chem. 262: 4429-443
2 (1987)), the construction of nucleic acids as part of retroviruses or other vectors). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition may be administered by any convenient route, such as by infusion or bolus injection, by absorption through the epithelial or mucosal lining, such as the oral, rectal and intestinal mucosa, and other It can be administered with a biologically active agent. Administration can be systemic or local. Further, the pharmaceutical compounds or compositions of the invention may be administered by any suitable route, including intraventricular and intrathecal injections; intraventricular injection may be for example, intraventricular injection attached to a reservoir, such as the Ommaya reservoir. Introduction to the central nervous system by means of a catheter). Pulmonary administration may also be employed, eg, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In certain embodiments, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this is for example, but not limited to, surgery. By local injection, topical application (eg in combination with post-surgical wound dressing), injection, by catheter, by suppository, or by an implant (the implant being a membrane such as a sialastic membrane or Porous, non-porous, or glue-like materials, including fibers). Preferably, when administering proteins of the invention, including antibodies, care must be taken to use materials that do not absorb proteins.

In another embodiment, the compound or composition may be delivered into vesicles, particularly liposomes (Langer, Science 249: 1527-1533 (
1990); Treat et al., Liposomes in the Therap.
y of Infectious Disease and Cancer, L
opez-Berestein and Fidler (ed.), Liss, New
York, pp. 353-365 (1989); Lopez-Berestein,
Ibid, pages 317-327; see ibid.).

In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (Lang.
er (supra); Sefton, CRC Crit. Ref. Biomed. En
g. 14: 201 (1987); Buchwald et al., Surgery 88:
507 (1980); Saudek et al., N. et al. Engl. J. Med. 321: 5
74 (1989)). In another embodiment, polymeric materials may be used (Medical Applications of Control).
led Release, Langer and Wise (ed.), CRC Pre
s. , Boca Raton, Florida (1974); Control
ed Drug Bioavailability, Drug Product
Design and Performance, Smolen and Bal
1 (eds.), Wiley, New York (1984); Ranger and P.
eppas, J .; , Macromol. Sci. Rev. Macromol. C
hem. 23:61 (1983); Levy et al., Science.
228: 190 (1985); During et al., Ann. Neurol. 25:
351 (1989); Howard et al., J. Am. Neurosurg. 71: 105
(1989) also). In yet another embodiment, the controlled release system can be placed near the therapeutic target, ie, the brain, thus requiring only a portion of the systemic dose (eg, Goodson, Medical Applica).
Tions of Controlled Release (supra), Volume 2,
115-138 (1984)).

Other controlled release systems are discussed in the review by Langer (Science 249: 1527-1533 (1990)).

In certain embodiments, where the compound of the invention is a protein-encoding nucleic acid, the nucleic acid constitutes it as part of a suitable nucleic acid expression vector and is administered so that it is intracellular. (Eg, by using a retroviral vector (see US Pat. No. 4,980,286), or by direct injection, or microparticle bombardment (eg, gene gun; Biolist).
ic, Dupont), or by coating with lipids or cell surface receptors or transfectants, or in combination with homeobox-like peptides known to enter the nucleus (eg, Joliot et al. , Proc. Natl. Acad. Sci. USA 88: 1.
864-1868 (1991)) and the like, so as to promote the expression of the encoded protein in vivo. Alternatively, the nucleic acid is
It can be introduced intracellularly and integrated into the host cell DNA for homologous recombination for expression.

The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier. In certain embodiments,
The term "pharmaceutically acceptable" is licensed by the federal regulatory agency or state government for use in animals, and more particularly in humans, or is listed in the United States Pharmacopeia or other generally recognized pharmacopoeia. Means that The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers include sterile liquids such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin (eg, peanut oil, soybean oil, mineral oil, sesame oil, etc.). Can be. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions, and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene. , Glycol, water, ethanol and the like. The composition can also, if desired,
Small amounts of wetting or emulsifying agents, or pH buffering agents may be included. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutical carriers are E.I. W. "Remington" by Martin
's Pharmaceutical Sciences ". Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated according to conventional procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. If desired, the composition can also include a solubilizer and a local anesthesia such as lignocaine to relieve pain at the site of injection. In general, the ingredients are either dried separately in a unit dosage form or mixed together, for example in a dry container in an airtight container, such as an ampoule or sachet, which shows a quantity of the active agent. Supplied as frozen powder or water-free concentrate. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

The compounds of the present invention may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with anions, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and sodium, potassium, ammonium, calcium, ferric hydroxide. , Isopropylamine, triethylamine, 2
-Salts formed with cations such as salts derived from ethylaminoethanol, histidine, procaine and the like.

The amount of a compound of the invention that is effective in treating, inhibiting and preventing a disease or disorder associated with aberrant expression and / or activity of a polypeptide of the invention can be determined by standard clinical techniques. . In addition, in vitro assays can optionally be used to help identify optimal dosage ranges. Precise dosages to be used in the formulation will also depend on the route of administration, and the severity of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1 mg to 100 mg per kg patient body weight. Preferably, the dosage administered to a patient is between 0.1 mg and 20 mg / kg of the patient's body weight, more preferably 1 mg to 10 mg / kg of the patient's body weight. In general, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, low dosages and infrequent administrations of human antibodies are often possible. Further, the dosage and frequency of administration of the antibodies of the invention may be altered (eg,
It can be reduced by enhancing antibody uptake by lipidation and the like and tissue penetration (eg into the brain).

The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more ingredients of the pharmaceutical compositions of the invention. Notifications in the form prescribed by governmental agencies regulating the manufacture, use or sale of pharmaceutical or biological products may optionally be accompanied by such containers. This notice
It reflects the agency's approval of manufacture, use or sale for human administration.

Diagnostic and Imaging Labeled antibodies that specifically bind to a polypeptide of interest, as well as its derivatives and analogs thereof, have been used for diagnostic purposes to detect abnormal expression and expression of a polypeptide of the invention. Diseases and / or disorders associated with activity can be detected, diagnosed or monitored. The present invention provides for the detection of aberrant expression of a polypeptide of interest, which comprises (a) using one or more antibodies specific for the polypeptide of interest to target in a cell or body fluid of an individual. Assaying the expression of the polypeptide of
And (b) comparing the level of gene expression to the level of standard gene expression, whereby increasing or decreasing the level of assayed polypeptide gene expression compared to the standard level of expression. Shows abnormal expression.

The present invention provides a diagnostic assay for diagnosing a disorder, which assay comprises (
a) assaying the expression of the polypeptide of interest in cells or fluids of an individual using one or more antibodies specific for the polypeptide of interest, and (b) this gene expression level and standard Comparing the level of gene expression, whereby an increase or decrease in the assayed polypeptide gene expression level compared to its standard expression level is indicative of a particular disorder. With respect to cancer, the presence of relatively high amounts of transcripts in biopsied tissue from an individual may indicate a predisposition for the development of the disease or may provide a means for detecting the disease prior to the onset of actual clinical symptoms. Can be provided. A more definitive diagnosis of this type may allow health professionals to use preventative measures or early aggressive treatment, thereby preventing the development or further progression of cancer.

Antibodies of the invention can be used to assay protein levels in biological samples using classical immunohistological methods known to those of skill in the art (eg, Jalk).
anen et al. Cell. Biol. 101: 976-985 (1985);
Jalkanen et al. Cell. Biol. 105: 3087-3096 (
1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known in the art and are enzymatic labels (eg glucose oxidase); radioisotopes (eg iodine (125I, 1, 1
21I), carbon (14C), sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc)); luminescent labels (eg luminol); and fluorescent labels (eg fluorescein and rhodamine), and Examples include biotin.

One aspect of the present invention is the detection and diagnosis of diseases or disorders associated with aberrant expression of the polypeptide of interest in animals, preferably mammals, and most preferably humans. In one embodiment, diagnosis is a) administering to a subject an effective amount of a labeled molecule that specifically binds to a polypeptide of interest (eg, parenterally, subcutaneously, or intraperitoneally). B) to allow the labeled molecule to preferentially concentrate at the site in the subject where the polypeptide is expressed (and to remove unbound labeled molecule to background levels) A) waiting for a time interval after administration; c) determining a background level; and d) detecting a labeled molecule in the subject, so that the background level is exceeded. Detection of the labeled molecule indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest. Background level can be determined by a variety of methods, including comparing the amount of labeled molecule detected to standard values previously determined for a particular system.

It will be appreciated in the art that the size of the subject and the imaging system used will determine the amount of imaging moiety needed to produce a diagnostic image. In the case of a radioisotope moiety, the amount of radioactivity injected for a human subject will usually be about 5
It is in the range of 20 millicuries of 99mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the particular protein.
In vivo tumor imaging is described by S. W. Burchiel et al., "Immunophar
macokinetics of Radiolabeled Antibod
ies and Their Fragments "(Tumor Image
ng: The Radiochemical Detection of Ca
ncer, Chapter 13, S. W. Burchiel and B.I. A. Rhodes, Masson Publishing Inc. (1982).

Depending on a number of variables, including the type of label used and the mode of administration, the labeled molecule will preferentially concentrate at the site of the subject and will not be bound. The time interval after administration is 6-48 hours or 6-24 hours or 6-12 hours, which allows the C. to be cleared to background levels.
In another embodiment, the post-administration time interval is 5-20 days or 5-10 days.

In one embodiment, monitoring a disease or disorder comprises repeating the method for diagnosing the disease or disorder (eg, 1 month after the first diagnosis, 6 months after the first diagnosis, the first diagnosis). 1 year later).

The presence of labeled molecule can be detected from the patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One of ordinary skill in the art can determine the appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods of the invention include, but are not limited to, computer tomography (CT), whole body scanning such as position emission tomography (PET), magnetic resonance imaging (MRI). , And ultrasonic diagnostic methods.

In a specific embodiment, the molecule is labeled with a radioisotope and is detected in a patient using a radiation responsive surgical instrument (Thurston et al., US Pat. No. 5,441,050). . In another embodiment, the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument. In another embodiment, the molecule is labeled with a positron emitting metal and is detected in the patient using proton emission tomography. In yet another embodiment, the molecule is labeled with a paramagnetic label and is detected in the patient using magnetic resonance imaging (MRI).

(Kit) The present invention provides a kit that can be used in the above method. In one embodiment, the kit comprises an antibody of the invention (preferably a purified antibody) in one or more containers. In certain embodiments, the kits of the invention comprise a substantially isolated polypeptide comprising an epitope that is specifically immunoreactive with the antibodies included in the kit. Preferably, the kit of the present invention further comprises a control antibody that does not react with the polypeptide of interest. In another specific embodiment, a kit of the invention provides for the binding of an antibody to a polypeptide of interest (e.g., the antibody has a detectable substrate (e.g., the antibody is a fluorescent compound, an enzyme substrate, a radioactive compound or A luminescent compound) or a second antibody which recognizes the first antibody can be conjugated with a detectable substrate).

In another specific embodiment of the invention, the kit is for diagnostic use in screening serum containing antibodies specific for proliferative and / or cancerous polynucleotides and polypeptides. It's a kit. Such a kit may include a control antibody that does not react with the polypeptide of interest. Such a kit may comprise a substantially isolated polypeptide antigen that comprises an epitope that is specifically immunoreactive with at least one anti-polypeptide antigen antibody. Further, such kits are for detecting the binding of the antibody to an antigen (eg, the antibody can be conjugated with a fluorescent compound such as fluorescein or rhodamine, which can be detected by flow cytometry). Means are provided. In certain embodiments, the kit may comprise a recombinantly produced polypeptide antigen or a chemically synthesized polypeptide antigen. The polypeptide antigens of this kit can also be attached to a solid support.

In a more particular embodiment, the detection means of the above kit comprises a solid support to which the polypeptide antigen is attached. Such a kit also comprises a non-attached reporter-labeled anti-human antibody. In this embodiment, binding of the antibody to the polypeptide antigen can be detected by binding of the reporter-labeled antibody.

In a further embodiment, the invention comprises a diagnostic kit for use in screening serum containing an antigen of a polypeptide of the invention. The diagnostic kit comprises a substantially isolated antibody that is specifically immunoreactive with a polypeptide or polynucleotide antigen, and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. . In one embodiment, the antibody is
Attached to a solid support. In a particular embodiment, the antibody may be a monoclonal antibody. The detection means of this kit may include a second labeled monoclonal antibody. Alternatively, or additionally, the detection means may include a labeled, competing antigen.

In one diagnostic configuration, the test serum is reacted with a solid phase reagent having surface bound antigens obtained by the method of the invention. After binding the specific antigen antibody with this reagent and removing the unbound serum components by washing, in order to bind the reporter with this reagent, depending on the amount of anti-antigen antibody bound on the solid support, This reagent is reacted with a reporter-labeled anti-human antibody. The reagent is washed again to remove unbound labeled antibody and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme detected by incubating this solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).

The solid surface reagents in the above assay are used to convert protein material to solid support material (
For example, prepared by known techniques for attachment to polymeric beads, dipsticks, 96-well plates or filtration material). These methods of attachment generally include non-specific adsorption of proteins to the support or chemically active groups (eg, activated carboxyl groups, hydroxyl groups, or aldehyde groups) on the solid support. Covalent attachment of proteins (typically via free amine groups). Alternatively, streptavidin-coated plates can be used with biotinylated antigen.

Accordingly, the present invention provides an assay system or kit for performing this diagnostic method. The kit generally comprises a support having surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.

Polynucleotides Isolated nucleic acid molecules comprising a polynucleotide of the invention include: Platelet-derived growth factor A (PDGF-A) as disclosed in European Patent No. EP-682110. (SEQ ID NO: 1); Platelet-derived growth factor B (PDGF-B) as disclosed in European Patent No. EP-283217 (SEQ ID NO: 3); Placental growth factor as disclosed in International Publication No. WO 92/06194. (PIGF) (SEQ ID NO: 5); Hauser et al., Gorth Factors, 4: 259-26.
8 (1993) placental growth factor 2 (PIGF-2) (SEQ ID NO: 7); glioma-derived growth factor (GDGF) (SEQ ID NO: 9) as disclosed in European Patent No. EP-399816. ); Vascular Endothelial Growth Factor (VEGF) (SEQ ID NO: 11) as disclosed in WO 90/13649; European Patent No. EP.
Vascular Endothelial Growth Factor A (VEGF-A) (SEQ ID NO: 13) as disclosed in WO-506477; Vascular Endothelial Growth Factor 2 (VEGF-2) (SEQ ID NO: SEQ ID NO: as disclosed in WO 96/39515). 15); International Publication Number WO96 / 394
Vascular Endothelial Growth Factor-3 (VEGF-3) as disclosed in No. 21 (SEQ ID NO: 1)
7); Vascular Endothelial Growth Factor D (VEGF-D) as disclosed in International Publication No. WO98 / 07832 (SEQ ID NO: 19); Vascular Endothelial Growth Factor D1 as disclosed in International Publication No. WO98 / 02543. (VEGF-D1) (SEQ ID NO: 21)
And vascular endothelial growth factor E (VEGF-E) (SEQ ID NO: 23) as disclosed in German Patent No. DE19639601. The above references are incorporated herein by reference in their entireties.

The angiogenic proteins of the present invention are all structurally related. It is especially important that all eight cysteines are conserved within all members of the VEGF / PDGF family (see boxed regions in Figures 3A-3C). Furthermore, PDG
F / VEGF family signature, PXCVXXXRCXGCC (SEQ ID NO: 29
) Is conserved in all angiogenic proteins disclosed herein.

The angiogenic polypeptides of the present invention include full length polypeptides as well as polynucleotide sequences encoding any leader sequences and active fragments of the full length polypeptides.

The polynucleotides of the invention may be in the form of RNA or in the form of DNA (eg c
(Including DNA, genomic DNA and synthetic DNA). The DNA can be double-stranded or single-stranded, and the single-stranded can be the coding strand or non-coding (antisense) strand. The coding sequence encoding the mature polypeptide may be identical to the coding sequence of the nucleotide sequence of Table 1 or, as a result of duplication or degeneracy of the genetic code, SEQ ID NOs: 1, 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21
Alternatively, it may be a different coding sequence that codes for the same mature polypeptide as the 23 DNA.

The polynucleotide encoding each mature polypeptide of FIGS. 3A-3C may include: coding sequence of each mature polypeptide only; coding sequence of each mature polypeptide and additional coding sequences (eg, A leader or secretory sequence, or a proprotein sequence); the coding sequence (and optionally further coding sequence) and non-coding sequence of each mature polypeptide (
(For example, introns, or non-coding sequences 5'and / or 3'of the mature polypeptide coding sequence).

Thus, the term “polynucleotide encoding a polypeptide” includes polynucleotides that contain only the coding sequence for that polypeptide, as well as polynucleotides that contain additional coding and / or non-coding sequences.

The present invention further relates to variants of the polynucleotides described herein above which encode fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequences of Figures 3A-3C. The variant of the polynucleotide can be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Accordingly, the present invention thus encodes the same polynucleotides encoding each of the mature polypeptides shown in FIGS. 3A-3C, as well as fragments, derivatives or analogs of the polypeptides of FIGS. Various polynucleotide variants. Such nucleotide variants include deletion variants, substitution variants, and addition or insertion variants.

As indicated herein above, the polynucleotides are SEQ ID NOs: 1, 3, 5
, 7, 9, 11, 13, 15, 17, 19, 21, or 23 coding sequences that are naturally occurring allelic variants of the coding sequences. As is known in the art, allelic variants are alternative forms of polynucleotide sequences that have one or more nucleotide substitutions, deletions or additions, which substitutions, deletions or additions, are encoded polypeptides. Does not substantially change the function of.

The invention also provides that the coding sequence for the mature polypeptide functions as a polynucleotide (eg, a secretory sequence to control the transport of the polypeptide from the cell) that aids expression or secretion of the polypeptide from the host cell. A leader sequence), which can be fused in the same reading frame. A polypeptide having a leader sequence is a preprotein, and its leader sequence may be cleaved by the host cell to form the mature form of the polypeptide. The polynucleotide also
It may encode a mature protein plus a proprotein which is an additional 5'amino acid residue. A mature protein with a prosequence is a proprotein and is an inactive form of that protein. Once the prosequence is cleaved, the active mature protein remains.

Thus, for example, a polynucleotide of the invention may encode a mature protein, or a protein having a prosequence, or a protein having both a prosequence and a presequence (leader sequence).

The polynucleotides of the invention may also have a coding sequence fused in frame to a marker sequence, which marker sequence allows for purification of the polypeptide of the invention. The marker amino acid sequence can be a hexa-histidine tag provided by the pQE vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence can be a mammalian sequence. When using an animal host (eg, COS-7 cells), hemagglutinin "
HA "tag. This HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell 37:76.
7 (1984)).

A further embodiment of the present invention is a single embodiment comprising a polynucleotide having a nucleotide sequence which is at least 95% identical to the following nucleotide sequences, and more preferably at least 96%, 97%, 98% or 99% identical. Including isolated nucleic acid molecules: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2; (b) a polypeptide having the amino acid sequence of SEQ ID NO: 2 but lacking the N-terminal methionine. (C) a nucleotide sequence encoding a polypeptide having the amino acid sequence from about position 1 to about position 211 of SEQ ID NO: 2; or (d) (a), (b) or (c) the nucleotide sequence A nucleotide sequence that is complementary to any of the sequences.

A further embodiment of the present invention is a single polynucleotide comprising a polynucleotide having a nucleotide sequence that is at least 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical to the following nucleotide sequences: Including isolated nucleic acid molecules: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 4; (b) a polypeptide having the amino acid sequence of SEQ ID NO: 4 but lacking the N-terminal methionine. (C) a nucleotide sequence encoding a polypeptide having the amino acid sequence of about 1 to about 241 of SEQ ID NO: 4; or (d) (a), (b) or (c) the nucleotide sequence A nucleotide sequence that is complementary to any of the sequences.

A further embodiment of the present invention is a single embodiment comprising a polynucleotide having a nucleotide sequence which is at least 95% identical to the following nucleotide sequences, and more preferably at least 96%, 97%, 98% or 99% identical. Including isolated nucleic acid molecules: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 6; (b) a polypeptide having the amino acid sequence of SEQ ID NO: 6 but lacking the N-terminal methionine. (C) a nucleotide sequence encoding a polypeptide having an amino acid sequence from about position 1 to about 149 of SEQ ID NO: 6; or (d) (a), (b) or (c) the nucleotide sequence A nucleotide sequence that is complementary to any of the sequences.

A further embodiment of the invention is a single polynucleotide comprising a polynucleotide having a nucleotide sequence which is at least 95% identical to the following nucleotide sequences, and more preferably at least 96%, 97%, 98% or 99% identical. Including isolated nucleic acid molecules: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 8; (b) a polypeptide having the amino acid sequence of SEQ ID NO: 8 but lacking the N-terminal methionine. (C) a nucleotide sequence encoding a polypeptide having an amino acid sequence from about position 1 to about position 170 of SEQ ID NO: 8; or (d) (a), (b) or (c) the nucleotide sequence A nucleotide sequence that is complementary to any of the sequences.

A further embodiment of the present invention is a polynucleotide having a nucleotide sequence that is at least 95% identical, and more preferably at least 96%, 97%, 98%, or 99% identical to the following nucleotide sequences: An isolated nucleic acid molecule comprising nucleotides is included: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 10; A nucleotide sequence encoding a peptide; (c) a nucleotide sequence encoding a polypeptide having the sequence of amino acids from about 1 to about 190 in SEQ ID NO: 10; or (d) (a), (b), or (
A nucleotide sequence complementary to any of the nucleotide sequences in c).

Further embodiments of the invention are polys that have a nucleotide sequence that is at least 95% identical, and more preferably at least 96%, 97%, 98%, or 99% identical to the following nucleotide sequences: An isolated nucleic acid molecule comprising nucleotides is included: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 12; (b) a polynucleic acid having the amino acid sequence of SEQ ID NO: 12 but lacking the N-terminal methionine. A nucleotide sequence encoding a peptide; (c) a nucleotide sequence encoding a polypeptide having the sequence of amino acids from about 1 to about 191 in SEQ ID NO: 12; or (d) (a), (b), or (
A nucleotide sequence complementary to any of the nucleotide sequences in c).

A further embodiment of the present invention is a poly nucleotide sequence having at least 95% identity, and more preferably at least 96%, 97%, 98% or 99% identity to the following nucleotide sequences: An isolated nucleic acid molecule comprising nucleotides is included: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 14; (b) a polynucleotide having the amino acid sequence of SEQ ID NO: 14 but lacking the N-terminal methionine. A nucleotide sequence encoding a peptide; (c) a nucleotide sequence encoding a polypeptide having the sequence of amino acids from about 1 to about 214 in SEQ ID NO: 14; or (d) (a), (b), or (
A nucleotide sequence complementary to any of the nucleotide sequences in c).

A further embodiment of the present invention is a polynucleotide having a nucleotide sequence that is at least 95% identical, and more preferably at least 96%, 97%, 98%, or 99% identical to the following nucleotide sequences: An isolated nucleic acid molecule comprising nucleotides is included: (a) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 16; (b) a polynucleotide having the amino acid sequence of SEQ ID NO: 16 but lacking the N-terminal methionine. A nucleotide sequence encoding a peptide; (c) a nucleotide sequence encoding a polypeptide having the sequence of amino acids from about 1 to about 419 in SEQ ID NO: 16; or (d) ATCC Deposit No. 971.
A nucleotide sequence encoding a polypeptide having the amino acid sequence encoded by the cDNA clone contained in No. 49; (e) ATCC Deposit No. 971.
A nucleotide sequence encoding a mature VEGF2 polypeptide having an amino acid sequence encoded by the cDNA clone contained in No. 49; (f) encoded by the cDNA clone contained in ATCC Accession No. 97149;
A nucleotide sequence encoding a polypeptide lacking a terminal methionine; or (g
) A nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), or (f).

A further embodiment of the present invention is a polynucleotide having a nucleotide sequence that is at least 95% identical, and more preferably at least 96%, 97%, 98%, or 99% identical to the following nucleotide sequences: An isolated nucleic acid molecule comprising nucleotides is included: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 18; A nucleotide sequence encoding a peptide; (c) a nucleotide sequence encoding a polypeptide having the sequence of amino acids from about 1 to about 207 in SEQ ID NO: 18; or (d) (a), (b), or (
A nucleotide sequence complementary to any of the nucleotide sequences in c).

Further embodiments of the invention are polys that have a nucleotide sequence that is at least 95% identical, and more preferably at least 96%, 97%, 98%, or 99% identical to the following nucleotide sequences: An isolated nucleic acid molecule comprising nucleotides is included: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 20; (b) a polynucleotide having the amino acid sequence of SEQ ID NO: 20 but lacking the N-terminal methionine. A nucleotide sequence encoding a peptide; (c) a nucleotide sequence encoding a polypeptide having the sequence of amino acids from about 1 to about 325 in SEQ ID NO: 20; or (d) (a), (b), or (
A nucleotide sequence complementary to any of the nucleotide sequences in c).

Further embodiments of the invention are polys that have a nucleotide sequence that is at least 95% identical, and more preferably at least 96%, 97%, 98%, or 99% identical to the following nucleotide sequences: An isolated nucleic acid molecule comprising nucleotides is included: (a) a nucleotide sequence that encodes a polypeptide having the amino acid sequence of SEQ ID NO: 22; (b) a polynucleotide having the amino acid sequence of SEQ ID NO: 22 but lacking the N-terminal methionine. A nucleotide sequence encoding a peptide; (c) a nucleotide sequence encoding a polypeptide having the sequence of amino acids from about 1 to about 354 in SEQ ID NO: 22; or (d) (a), (b), or (
A nucleotide sequence complementary to any of the nucleotide sequences in c).

A further embodiment of the present invention is a polynucleotide having a nucleotide sequence which is at least 95% identical, and more preferably at least 96%, 97%, 98% or 99% identical to the following nucleotide sequences: An isolated nucleic acid molecule comprising nucleotides is included: (a) a nucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 24; (b) a polynucleotide having the amino acid sequence of SEQ ID NO: 24 but lacking the N-terminal methionine. A nucleotide sequence encoding a peptide; (c) a nucleotide sequence encoding a polypeptide having the sequence of amino acids from about 1 to about 132 in SEQ ID NO: 24; or (d) (a), (b), or (
A nucleotide sequence complementary to any of the nucleotide sequences in c).

A polynucleotide having a nucleotide sequence that is, for example, at least 95% “identical” to a reference nucleotide sequence that encodes an angiogenic polypeptide is a reference nucleotide whose polynucleotide sequence encodes an angiogenic polypeptide. Except that it may contain up to 5 point mutations for each 100 nucleotides of the sequence,
The nucleotide sequence of the polynucleotide is intended to be identical to the reference sequence. In other words, up to 5% of the nucleotides of the reference sequence can be deleted or replaced by another nucleotide in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence. Alternatively, up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These variations in the reference sequence may occur at the 5'or 3'ends of the reference nucleotide sequence, or at any position between their terminal positions, individually between the nucleotides in the reference sequence. , Or as one or more contiguous groups in the reference sequence.

As a practical matter, any particular nucleic acid molecule may be labeled, for example, in SEQ ID NO: 1, 3, 5, 7,
, 9, 11, 13, 15, 17, 19, 21, or 23 are known to be at least 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown. Computer programs (eg, Bestfit)
program (Wisconsin Sequence Analysis)
Package, Version 8 for Unix (registered trademark), Gen
etics Computer Group, University Rese
arch Park, 575 Science Drive, Madison,
WI53711) and can be conventionally determined. Bestfit is Sm
Find the best segment of homology between two sequences using the local homology algorithm of ith and Warterman (Advances in Ap).
plied Mathematics 2: 482-489 (1981)). B
It will be appreciated that if estfit or any other sequence alignment program is used to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the invention, the identity will be the same. Is calculated over the entire length of the reference nucleotide sequence and the parameters are set such that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

The angiogenic “polynucleotide” also has the sequence of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 17, 19 under stringent hybridization conditions.
, 21 or 23, or, for example, ATCC Deposit No. 97149.
Or a cDNA clone contained in 75698, or a polynucleotide capable of hybridizing to the complement thereof. “Stringent hybridization conditions” means 50% formamide, 5 × SSC (750 mM NaCl
, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6)
5x Denhardt's solution, 10% dextran sulfate, and 20 µg / ml denatured sheared salmon sperm DNA at 42 ° C overnight incubation, then wash filters in 0.1xSSC at about 65 ° C. It means to do.

Nucleic acid molecules that hybridize to the angiogenic polynucleotides at lower stringency hybridization conditions are also contemplated. Changes in hybridization stringency and signal detection are primarily achieved through manipulation of formamide concentrations (lower percentages of formamide produce reduced stringency); salt conditions, or temperature. For example, lower stringency conditions are 6 × SSPE (20 × SSPE = 3M NaCl;
2M NaH ₂ PO ₄ ; 0.02M EDTA, pH 7.4), 0.5% SDS
, 30% formamide, 100 μg / ml salmon sperm blocking DNA, incubated overnight at 37 ° C .; then 1 × SSPE, 0.1% S
Includes washing with DS at 50 ° C. Furthermore, in order to achieve even lower stringency, the washes performed after stringent hybridization can be performed at higher salt concentrations (eg 5 × SSC).

Note that changes in the above conditions may be achieved by the inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Representative blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercial product formulations. The inclusion of specific blocking reagents may require modification of the above hybridization conditions due to compatibility issues.

Of course, the poly A + sequence (eg, any 3'of the cDNA shown in the sequence listing)
A polynucleotide that hybridizes only to the terminal poly A + region) or to a complementary stretch of T (or U) residues is any such polynucleotide comprising a poly (A) stretch or its complement. Nucleic acid molecule (eg, virtually any double-stranded cDNA generated using oligo dT as a primer)
Clone) and is not included in the definition of "polynucleotide".

A polynucleotide that hybridizes to a “portion” of a polynucleotide is at least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, even more preferably at least about 30 nt.
, And even more preferably, a polynucleotide (either DNA or RNA) that hybridizes to about 30-70 (eg, 50) nt. These are useful as diagnostic probes and diagnostic primers, as discussed above and in more detail below.

[0214] For example, a portion of a "at least 20 nt long" polynucleotide refers to a 2'derived from the nucleotide sequence of a reference polynucleotide (eg, the nucleotide sequence of Table 1).
0 or more consecutive nucleotides are intended. Of course, a polynucleotide that hybridizes only to a poly A sequence (eg, the 3'terminal poly (A) tract of SEQ ID NO: X, or a complementary stretch of T (or U) residues) is such a polynucleotide. Hybridizes to any nucleic acid molecule comprising a poly (A) stretch, or its complement (eg, virtually any double-stranded cDNA clone), and thus is used for hybridizing to a portion of a nucleic acid of the invention. The present invention is not included in the polynucleotide of the present invention.

However, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21
, Or 23, or indeed at least 9 in the nucleic acid sequence of the deposited cDNA encoding a polypeptide having angiogenic protein activity.
Nucleic acid molecules having 5%, 96%, 97%, 98% or 99% sequence identity are preferred. By "polypeptide having angiogenic activity" is intended a polypeptide that exhibits angiogenic activity in a particular biological assay. For example, angiogenic protein activity can be measured using, for example, mitogenic and endothelial cytophoretic assays. For example, Olofsson et al., Proc. Natl.
Acad. Sci. USA 93: 2576-2581 (1996) and Jo.
ukov et al., EMBO J .; 5: 290-298 (1996). "
A polypeptide having lymphangiogenic activity "
By is intended a polypeptide that exhibits lymphangiogenic activity in a particular biological assay. For example, angiogenic protein activity can be measured using, for example, mitogen and endothelium assay. For example, Olofss
on et al., Proc. Natl. Acad. Sci. USA 93: 2576-2.
581 (1996) and Joukov et al., EMBO J. 5: 290-298
See (1996).

Of course, due to the degeneracy of the genetic code, for example, the deposited cD
NA nucleic acid sequence, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19
, 21, or 23, or a fragment thereof, has a sequence of at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a number of nucleic acid molecules. One of skill in the art will immediately understand that it encodes a “polypeptide having tube forming functional activity”. In fact, since all degenerate variants of any of these nucleotide sequences encode the same polypeptide, this will often be apparent to one of skill in the art without performing the comparative assay described above. Is. It is further understood in the art that, for nucleic acid molecules that are not degenerate variants, a reasonable number also encodes a polypeptide having functional activity. Amino acid substitutions that cause the protein to function significantly or are unlikely to function (eg, replacing one aliphatic amino acid with a second aliphatic amino acid), as described further below. Because I know completely.

For example, guidance on how to make phenotypically silent amino acid substitutions includes:
Bowie, J .; U. Et al., "Deciphering the Message"
in Protein Sequences: Tolerance to A
"Mino Acid Substitutions", Science 247
1306-1310 (1990), where the authors show that the protein is surprisingly tolerant of amino acid substitutions.

Therefore, the present invention provides SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18.
, 20, 22 or 24 polypeptides and fragments thereof (these fragments have at least 30 bases, and preferably at least 50 bases relative to the polypeptide encoded by such a polynucleotide). At least 70% identity, preferably at least 90% identity, and more preferably at least 95% identity to the polynucleotide,
It relates to polypeptides having 96%, 97% or 98% identity.

“Identity”, by itself, has its art-recognized meaning and can be calculated using published techniques. (For example, COMPUTATIONAL MO
LECULAR BIOLOGY, Lesk, A .; M. Hen, Oxford Un
diversity Press, New York, (1988); BIOCO
MPUTING: INFORMATION AND GENOME PROJE
CTS, Smith, D.M. W. Hen, Academic Press, New Y
ork, (1993); COMPUTER ANALYSIS OF OF SEQU
ENCE DATA, PART I, Griffin, A .; M. , And Gri
ffin, H.F. G. Hen, Humana Press, New Jersey, (
1994); SEQUENCE ANALYSIS IN MOLECULAR
BIOLOGY, von Heinje, G .; , Academic Pres
s, (1987); and SEQUENCE ANALYSIS PRIME
R, Gribskov, M .; And Devereux, J .; Hen, M Stock
Ton Press, New York, (1991)). Although there are numerous ways to determine the identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to those of skill in the art. (Carillo, H .;
, And Lipton, D .; , SIAM J .; Applied Math. 48
: 1073 (1988). ) A commonly used method for determining identity or similarity between two sequences is the “Guide to Huge Comp”.
Uters, "Martin J. Bishop, ed., Academic Pr
ess, San Diego, (1994), and Carillo, H .; , And Lipton, D .; , SIAM J .; Applied Math. 48: 1
No. 073 (1988), but not limited thereto.
Methods for aligning polynucleotides or polypeptides are described by computer programs (GCG program package (Devereux, J., et al., Nucle.
ic Acids Research 12 (1): 387 (1984)), B
LASTP, BLASTN, FASTA (Atschul, SF, et al., JM.
olec. Biol. 215: 403 (1990), Bestfit prog.
ram (Wisconsin Sequence Analysis Pack)
age, Version 8 for Unix (registered trademark), Genetic
s Computer Group, University Research
Park, 575 Science Drive, Madison, WI 5
3711 (Advances in Applied Mathematics 2 using the local homology algorithm of Smith and Waterman.
: 482-489 (1981))). By a polynucleotide having a nucleotide sequence that is at least 95% “identical” to a reference nucleotide sequence of the invention, the following is intended. The nucleotide sequence of the polynucleotide is identical to the reference sequence except for the following. The polynucleotide sequence comprises 100 each of the reference nucleotide sequences encoding the angiogenic polypeptide.
Up to 5 point mutations can be included per nucleotide. That is, up to 5% of the nucleotides in the reference sequence may be deleted or replaced by another nucleotide or a reference nucleotide may be obtained in order to obtain a polynucleotide having a nucleotide sequence which is at least 95% identical to the reference nucleotide sequence. Up to 5% of the total nucleotides within can be inserted into the reference sequence. Inquiries are SEQ ID Nos. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23
, ORF (open reading frame), or any of the specific fragments described herein.

As a practical matter, will any particular nucleic acid molecule or polypeptide be at least 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the invention? Whether or not it can be determined conventionally using known computer programs. A preferred method for determining the best overall compatibility (also referred to as global sequence alignment) between a query sequence (sequence of the invention) and a subject sequence is described by Brutlag et al. (Comp. App. Biosci
． 6: 237-245 (1990)) based on the FASTDB computer program. In a sequence alignment, both the query and subject sequences are DNA sequences. RNA sequences are U to T
Can be compared by converting to. The results of this global sequence alignment are expressed as percent identity (%). The preferred parameters used in the FASTDB alignment of DNA sequences to calculate percent identity are: Matrix =
Unity, k-tuple = 4, Mismatch Penalty = 1
, Joining Penalty = 30, Randomization Gr
up Length = 0, Cutoff Score = 1, Gap Pena
lty = 5, Gap Size Penalty 0.05, Windows S
size = 500 or the length of the nucleotide sequence of interest (whichever is shorter).

If the subject sequence is shorter than the query sequence due to the 5'or 3'deletions (as opposed to due to internal deletions), manual correction must be made to the results. This is because the FASTDB program does not consider 5'and 3'truncations of the subject sequence when calculating percent identity. For a subject sequence that is cleaved at the 5'or 3'end, the percent identity to the query sequence is the 5'and 3'of the subject sequence that is not matched / aligned as a percentage of the total bases of the query sequence. Corrected by calculating the number of bases in. Whether the nucleotides are matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity and calculated by the FASTDB program above using the specified parameters to arrive at the final percent identity score. This corrected score is what is used for the purposes of the present invention. Only the bases outside the 5'and 3'bases of the subject sequence that are not matched / aligned with the query sequence, as indicated by the FASTDB alignment, are calculated for the purpose of manually adjusting the percent identity score.

For example, a 90 base subject sequence is aligned with a 100 base query sequence to determine percent identity. The deletion occurs at the 5'end of the subject sequence, so the FASTDB alignment shows no match / alignment at the first 10 bases at the 5'end. 1
0 unpaired bases represent 10% of the sequence (number of bases at the non-matching 5'and 3'ends / total number of bases in the query sequence), so 10% is FASTD
Subtracted from the percent identity score calculated by the B program.
If the remaining 90 bases were an exact match, the final percent identity would be 90%. In another example, a 90 base subject sequence is compared to a 100 base query sequence. In this case, the deletion is an internal deletion, so that there are no bases at the 5'or 3'of the subject sequence that are not matched / aligned with the query. In this case, FASTD
The percent identity calculated by B is not manually corrected. Again, only the 5'or 3'bases of the subject sequence that do not match / align with the query sequence are manually corrected. No other manual correction is made for the purposes of the present invention.

An amino acid sequence of a subject polypeptide may be one in which the subject polypeptide sequence has It is intended to be identical to the query sequence, except that changes of up to 5 amino acids per 100 amino acids may be included. In other words, up to 5% of the amino acid residues in the sequence of interest are inserted, deleted, (indels) in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the query amino acid sequence.
Or it may be replaced by another amino acid. These modifications of the reference sequence can occur at the amino-terminal or carboxy-terminal positions of the reference amino acid sequence, or anywhere between those terminal positions, either individually between the residues in the reference sequence or the reference sequence. Interspersed in any of one or more consecutive groups within.

As a practical matter, any particular polypeptide will be at least 90%, 95%, 96%, 97% relative to, for example, the amino acid sequence shown in Table 1 or the amino acid sequence encoded by the deposited DNA clone. Whether it is%, 98%, or 99% identical can be conventionally determined using known computer programs. A preferred method for determining the best overall compatibility (also called global sequence alignment) between a query sequence (the sequence of the invention) and a subject sequence is Br.
utlag et al. (Comp. App. Biosci. (1990) 6: 237-2.
45) can be determined using the FASTDB computer program based on the algorithm. In a sequence alignment, the query and subject sequences are either both nucleotide sequences or both amino acid sequences. The results of this global sequence alignment are expressed as percent identity. The preferred parameters used in the FASTDB amino acid alignment are: Matrix = PAM0, kt
apple = 2, Mismatch Penalty = 1, Joining Pe
nalty = 20, Randomization Group Length =
0, Cutoff Score = 1, Window Size = sequence length, Gap
Penalty = 5, Gap Size Penalty = 0.05, Win
dow Size = 500 or the length of the target amino acid sequence (whichever is shorter).

If the subject sequence is shorter than the query sequence (rather than due to internal deletions) due to N-terminal or C-terminal deletions, a manual correction must be made to the results. This is because the FASTDB program does not consider N- and C-terminal truncations of the subject sequence when calculating the overall percent identity. For subject sequences that are truncated at the N-terminus and the C-terminus, the percent identity to the query sequence is the N-terminus of the subject sequence that is not matched / aligned with the corresponding subject residue as a percent of the total bases of the query sequence and Corrected by calculating the number of residues in the query sequence that are C-terminal. Whether the residues are matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated by the FASTDB program above using the specified parameters to arrive at the final percent identity score. This final percent identity score is what is used for the purposes of the present invention. Only residues at the N- and C-termini of the subject sequence that are not matched / aligned with the query sequence are considered for the purpose of manually adjusting the percent identity score.
It is only the query residues at positions outside the furthest N- and C-terminal residues of the subject sequence.

For example, a 90 amino acid subject sequence is used to determine percent identity to determine 10
Aligned to a 0 residue query sequence. Deletions occur at the N-terminus of the subject sequence, so the FASTDB alignment shows no match / alignment at the first 10 residues at the N-terminus. 1
0 unpaired residues represent 10% of the sequence (number of non-matching N- and C-terminal residues / total number of residues in the query sequence), so 10% are FASTDB
Subtracted from the percent identity score calculated by the program. If the remaining 90 bases were an exact match, the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared to a 100 residue query sequence. In this case, the deletion is an internal deletion so that there are no residues at the N-terminus or C-terminus of the subject sequence that are not matched / aligned with the query. In this case, FAST
The percent identity calculated by DB is not corrected manually. Also, F
Only residues outside the N- and C-termini of the subject sequence that do not match / align with the query sequence are manually corrected, as shown in the ASTDB alignment. No other manual correction is made for the purposes of the present invention.

Angiogenic Polypeptides The present invention further relates to polypeptides having the deduced amino acid sequences of Figures 3A-3C, as well as fragments, analogs and derivatives of such polypeptides.

In the present invention, “polypeptide fragment” refers to SEQ ID NO: 2, 4, 6, 8
It refers to the short amino acid sequence encoded by the cDNA contained in 10, 12, 14, 16, 18, 18, 22, 22 or 24 or contained in the deposited clone. Protein fragments are "free-standing."
Or it may be contained within a larger polypeptide forming part or region of this fragment, most preferably as a single contiguous region. Representative examples of polypeptide fragments of the invention are, for example, approximately amino acid number 1 to
20, 21-40, 41-60, 61-80, 81-100, 102-120,
121-140, 141-160, 161-180, 181-200, 201-
220, 221-240, 241-260, 261-280 or 281 from the end of the coding region. Furthermore, the peptide fragment is
Approximately 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 1
It can be 20, 130, 140 or 150 amino acids long. In this context, "approximately" means some (5, 4, 3,
2 or 1) amino acids larger or smaller, including the specifically listed ranges (see Table 1).

[0229]

[Table 1] Preferred polypeptide fragments include secreted angiogenic proteins as well as their mature forms. Further preferred polypeptide fragments include secreted angiogenic proteins or mature forms thereof with a series of deleted residues from the amino or carboxy terminus, or both. For example, any number of amino acids in the range of 1-60 can be deleted from the amino terminus of either the secreted angiogenic polypeptide or the mature form. Similarly, any number of amino acids in the range 1-30 may be deleted from the carboxy terminus of the secreted angiogenic protein or mature form. Moreover, any combination of the above amino-terminal deletions and carboxy-terminal deletions is also preferred. Similarly, polypeptide nucleotide fragments encoding these angiogenic polypeptide fragments are also preferred.

Fragments of angiogenic polypeptides and polynucleotides characterized by structural or functional domains are also preferred. Such fragments include α-helix and α-helix forming regions, β-sheet and β-sheet forming regions, turns and turn-forming regions, coils and coil-forming regions, hydrophilic regions, hydrophobic regions, α-amphipathic regions. , Β-amphipathic region, flexible region, surface forming region, substrate binding region and high antigenicity indicator region. Polypeptide fragments of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 contained in the conserved domain are specifically contemplated by the present invention (FIG. 3).
See A-3C). Moreover, polynucleotide fragments encoding these domains are also contemplated.

Other preferred fragments are biologically active angiogenic protein fragments. Biologically active fragments are fragments that exert an activity that is similar to one of the disclosed angiogenic polypeptides, but need not be the same. The biological activity of this fragment may include an improved desired activity, or a decreased undesired activity.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

It is recognized in the art that some amino acid sequences of angiogenic polypeptides may be altered without having a significant effect on protein structure or function. If such differences in sequence are intended, it should be remembered that there are important regions on the protein that determine activity.

Accordingly, the invention further includes modifications of angiogenic polypeptides that exhibit substantial angiogenic polypeptide activity or include regions of angiogenic proteins such as the protein moieties described below. . Such variants include deletions, insertions, and rearrangements (in
version), iteration and type substitution. As noted above, guidelines for amino acid changes being phenotypically silent are provided by Bowie, J. et al. U. "De
ciphering the Message in Protein Seq
uences: Tolerance to Amino Acid Subst
"Motions" Science 247: 1306-1310 (1990)
Can be found in.

Accordingly, fragments, derivatives or analogs of the polypeptides of Table 1 can be: (I) one or more amino acid residues being conservative or non-conservative (preferably conservative). Amino acid residue) (such a substituted amino acid residue may or may not be an amino acid residue encoded by the genetic code), or (ii) 1 Having one or more amino acid residues having a substituent, or (iii) fusing the mature polypeptide with another compound (eg, a compound for increasing the half-life of the polypeptide (eg,
Polyethylene glycol)), or (iv) those in which additional amino acids are fused to the mature polypeptide (eg, leader or secretory sequences, or sequences used to purify the mature polypeptide or proprotein sequences), or (v
) Containing a small number of amino acid residues shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24, and retaining a conserved motif and poly in the VEGF / PDGF family. Those that still retain the active properties of the peptide. Such fragments, derivatives and analogs are considered to be within the skill of those in the art given the teachings herein.

Of particular desirability is the replacement of a charged amino acid with another charged amino acid and a neutral or negatively charged amino acid. The latter results in proteins with a reduced positive charge, improving the properties of angiogenic proteins. Prevention of agglomeration is highly desirable. When a pharmaceutical formulation is prepared, protein aggregation not only results in diminished activity, but is also problematic. This is because aggregates are immunogenic (Pinckard et al., Clin. Exp. Immunol. 2: 3.
31-340 (1967); Robbins et al., Diabetes 36:83.
8-845 (1987); Cleland et al., Crit. Rev. Therap
eutic Drug Carrier Systems 10: 307-37
7 (1993)).

Amino acid substitutions may also alter the selectivity of binding to cell surface receptors. O
Stade et al., Nature 361: 266-268 (1993) describe that certain mutations result in the selective binding of TNF-α to only one of the two known forms of the TNF receptor. Thus, the angiogenic proteins of the present invention may contain one or more amino acid substitutions, deletions or additions, either by natural mutations or artificial manipulations.

As shown, charge is preferably a minor property, such as a conservative amino acid substitution, that does not have a significant effect on protein folding or activity (see Table 2).

[0239]

[Table 2] Of course, the number of amino acid substitutions that one of ordinary skill in the art will make will depend on many factors, including those described above. Generally, the number of substitutions for any given angiogenic polypeptide is 50, 40, 30, 25, 20, 15, 10, 5 or 3 or less.

Amino acids in the angiogenic proteins of the invention that are essential for function can be generated by methods known in the art (eg, site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:
1081-1085 (1989)). The latter procedure induces a single alanine mutation at any residue in the molecule. The resulting mutant molecule is then tested for biological activity (eg, receptor binding or proliferative activity in vitro or in vivo). Sites essential for ligand-receptor binding can also be determined by structural analysis (eg, crystallization, nuclear magnetic resonance, or photoaffinity labeling) (Smith et al., J. Mol. Biol. 224: 899-9).
04 (1992) and de Vos et al., Science 255: 306-3.
12 (1992)).

The polypeptides and polynucleotides of the invention are preferably provided in isolated form and are preferably purified to homogeneity.

The term “isolated” means that the material is removed from its original environment (eg, its natural environment if naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but is the same polynucleotide or DNA or polypeptide that is separated from some or all of the coexisting materials in the natural system. The peptide is isolated. Such polynucleotides may be part of the vector, and / or such polynucleotides or polypeptides may be part of the composition. And when such a vector or composition is not part of its natural environment, it is further isolated.

In a particular embodiment, the polynucleotide of the invention is 300 kb, 200
The length is kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb or less. In a further embodiment, the polynucleotide of the invention comprises at least 15 contiguous nucleotides of the disclosed angiogenic protein coding sequence, but includes all or part of any intron of the particular angiogenic protein. Absent. In another embodiment, the nucleic acid comprising a sequence encoding a particular angiogenic protein does not include the coding sequence of a genomic flanking gene (ie, 5 ′ or 3 ′ of the particular angiogenic protein gene in the genome). .

The polypeptides of the present invention have the sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16.
, 18, 20, 22 or 24 polypeptides (and particularly the mature form of the polypeptides), and SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18
, 20, 22 or 24 polypeptides having at least 70% similarity (preferably at least 70% identity), and SEQ ID NOs: 2, 4,
Polypeptides having at least 90% similarity (preferably at least 95% identity) to 6,8,10,12,14,16,18,20,22 or 24 polypeptides, and even more preferably SEQ ID NO: 2, 4, 6, 8, 10, 1
At least 9 in 2, 14, 16, 18, 20, 22, or 24 polypeptides
Polypeptides having 5% similarity (even more preferably at least 90% identity), and at least 30 amino acids and more preferably at least 5 amino acids.
Also included are portions of such polypeptides that have such portions of the polypeptide that generally contain 0 amino acids.

As is known in the art, “similarity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide and its conservative amino acid substitutions with the sequence of a second polypeptide. To be done.

A fragment or portion of a polypeptide of the invention can be used to produce the corresponding full-length polypeptide by peptide synthesis; thus, the fragment can be used as an intermediate to produce the full-length polypeptide. . Fragments or portions of the polynucleotides of the invention can be used to synthesize the full length polynucleotides of the invention.

Amino-Terminal Deletions and Carboxy-Terminal Deletions In addition, protein engineering can be used to improve or modify one or more properties of native angiogenic proteins. Deletion of the carboxy terminal amino acid may enhance the activity of the protein. One example is interferon gamma, by deleting 10 amino acid residues from the carboxy terminus of this protein,
It showed up to 10-fold higher activity (Dobeli et al., J. of Biotec.
Hnology 7: 199-216 (1988)). Accordingly, one aspect of the invention is that such a pulse exhibits enhanced stability (eg, when exposed to typical pH, temperature conditions, or other storage conditions) relative to the native angiogenic protein. To provide polypeptide analogs of tube forming proteins and nucleotide sequences encoding such analogs.

Deletion variants with amino acids deleted from both the N- and C-termini are also included by the invention. Such variants include all combinations of N-terminal deletion variants and C-terminal deletion variants described by the definitions below. These combinations can be made using recombinant techniques known to those of skill in the art.

In particular, the N-terminal deletion of angiogenic polypeptides can be described by the following general formula: ) M ₂ ~211, wherein, m ₂ is an integer of from 1 to 210, wherein, m ₂
Corresponds to the position of the amino acid residue identified in SEQ ID NO: 2. Furthermore, C
Terminal deletion also can be described by the general formula 1 to n _2, where, n ₂ is 2 to 21
Is an integer of 1, where n ₂ corresponds to the position of the amino acid residue identified in SEQ ID NO: 2. Furthermore, the invention also provides a polypeptide in which one or more amino acids have been deleted from both the amino and carboxyl termini, which generally comprises residues m _{2 to} n ₂ of SEQ ID NO: 2. Can be described, where n ₂ and m ₂ are integers as described above.

2. ) M ₄ ~241, wherein, m ₄ is an integer of 1 to 240, wherein, m ₄
Corresponds to the position of the amino acid residue identified in SEQ ID NO: 4. Furthermore, C
Terminal deletions can also be described by the general formulas 1 to n ₄ , where n ₄ is 2 to 24
Is an integer of 1, where n ₄ corresponds to the position of the amino acid residue identified in SEQ ID NO: 4. Furthermore, the invention also provides a polypeptide in which one or more amino acids have been deleted from both the amino and carboxyl termini, which generally has residues m _{4 to} n ₄ of SEQ ID NO: 4. Can be described, where n ₄ and m ₄ are integers as described above.

3. ) M _{4 to} 149, where m ₆ is an integer from 1 to 148, where m ₆
Corresponds to the position of the amino acid residue identified in SEQ ID NO: 6. Furthermore, C
Terminal deletion also can be described by the general formula 1 to n _6, wherein, n ₆ is 2 to 14
Is an integer of 9, where n ₆ corresponds to the position of the amino acid residue identified in SEQ ID NO: 6. Furthermore, the present invention may also include one or more amino acids providing the deleted polypeptide from both the amino and carboxyl termini, which generally as having residues m ₆ ~n ₆ of SEQ ID NO: 6 Can be described, where n ₆ and m ₆ are integers as described above.

4. ) M ₈ to 170, where, m ₈ is an integer of from 1 to 169, where, m ₈
Corresponds to the position of the amino acid residue identified in SEQ ID NO: 8. Furthermore, C
Terminal deletion also can be described by the general formula 1 to n _8, wherein, n ₈ is 2 to 17
Is an integer of 0, where n ₈ corresponds to the position of the amino acid residue identified in SEQ ID NO: 8. Furthermore, the present invention may also include one or more amino acids providing the deleted polypeptide from both the amino and carboxyl termini, which generally as having residues m ₈ ~n ₈ of SEQ ID NO: 8 Can be described, where n ₈ and m ₈ are integers as described above.

[0253]   5. ) M_Ten~ 190, where m_TenIs an integer from 1 to 189, where m _Ten Corresponds to the position of the amino acid residue identified in SEQ ID NO: 10. further
, C-terminal deletion also has the general formula 1-n_TenCan be described by where n_TenIs 2
Is an integer from ~ 190, where n_TenIs the amino acid identified in SEQ ID NO: 10.
Corresponds to the position of the no acid residue. Furthermore, the present invention also provides that one or more amino acids are amino
Providing a polypeptide deleted from both the terminal and carboxyl termini, which
Is generally residue m of SEQ ID NO: 10_Ten~ N_TenCan be described as having n _Ten And m_TenIs an integer as described above.

[0254]   6. ) M₁₂~ 191, where m₁₂Is an integer from 1 to 190, where m ₁₂ Corresponds to the position of the amino acid residue identified in SEQ ID NO: 12. further
, C-terminal deletion also has the general formula 1-n₁₂Can be described by where n₁₂Is 2
˜191, where n₁₂Is the amino acid identified in SEQ ID NO: 12.
Corresponds to the position of the no acid residue. Furthermore, the present invention also provides that one or more amino acids are amino
Providing a polypeptide deleted from both the terminal and carboxyl termini, which
Is generally residue m of SEQ ID NO: 12₁₂~ N₁₂Can be described as having n ₁₂ And m₁₂Is an integer as described above.

[0255]   7. ) M₁₄~ 214, where m₁₄Is an integer from 1 to 213, where m ₁₄ Corresponds to the position of the amino acid residue identified in SEQ ID NO: 14. further
, C-terminal deletion also has the general formula 1-n₁₄Can be described by where n₁₄Is 2
Is an integer from ˜214, where n₁₄Is the amino acid identified in SEQ ID NO: 14.
Corresponds to the position of the no acid residue. Furthermore, the present invention also provides that one or more amino acids are amino
Providing a polypeptide deleted from both the terminal and carboxyl termini, which
Is generally residue m of SEQ ID NO: 14₁₄~ N₁₄Can be described as having n ₁₄ And m₁₄Is an integer as described above.

[0256]   8. ) M₁₆~ 419, where m₁₆Is an integer from 1 to 418, where m ₁₆ Corresponds to the position of the amino acid residue identified in SEQ ID NO: 16. further
, C-terminal deletion also has the general formula 1-n₁₆Can be described by where n₁₆Is 2
Is an integer from ˜419, where n₁₆Is the amino acid identified in SEQ ID NO: 16.
Corresponds to the position of the no acid residue. Furthermore, the present invention also provides that one or more amino acids are amino
Providing a polypeptide deleted from both the terminal and carboxyl termini, which
Is generally residue m of SEQ ID NO: 16₁₆~ N₁₆Can be described as having n ₁₆ And m₁₆Is an integer as described above.

Particularly preferred is a polypeptide fragment comprising amino acid residues F-32 to R-226 of SEQ ID NO: 16, as well as a polynucleotide encoding this polypeptide. Yet another preferred embodiment is amino acid S-2 of SEQ ID NO: 16.
28 to S-419 are included. Also preferred are polynucleotides encoding these polypeptides. Furthermore, this F-3 of the polypeptide of SEQ ID NO: 16
2 to R-226 are preferably S-228 to S- of the polypeptide of SEQ ID NO: 16.
Meet with 419. Association may be through disulfide, covalent or non-covalent interactions, by linkage through a linker (eg, serine, glycine, proline linkage) or by an antibody.

[0258]   9. ) M₁₈~ 207, where m₁₈Is an integer from 1 to 206, where m ₁₈ Corresponds to the position of the amino acid residue identified in SEQ ID NO: 18. further
, C-terminal deletion also has the general formula 1-n₁₈Can be described by, where n₁₈Is
Is an integer from 2 to 207, where n₁₈Is the amino acid identified in SEQ ID NO: 18.
Corresponds to the position of the no acid residue. Furthermore, the present invention also provides that one or more amino acids are amino
Providing a polypeptide deleted from both the terminal and carboxyl termini, which
Is generally residue m of SEQ ID NO: 18₁₈~ N₁₈Can be described as having n ₁₈ And m₁₈Is an integer as described above.

10. ) M _{20 to} 325, where m ₂₀ is an integer from 1 to 324, and
m ₂₀ corresponds to the position of the amino acid residue identified in SEQ ID NO: 20. Furthermore, C-terminal deletions can also be described by the general formulas 1 to n ₂₀ , where n ₂₀ is
Is an integer from 2 to 325, where n ₂₀ corresponds to the position of the amino acid residue identified in SEQ ID NO: 20. Furthermore, the invention also provides a polypeptide in which one or more amino acids have been deleted from both the amino and carboxyl termini, which generally comprises residues m _{20 to} n ₂₀ of SEQ ID NO: 20. Can be described, where
n ₂₀ and m ₂₀ are integers as described above.

11. ) M ₂₂ ~354, wherein m ₂₂ is an integer of from 1 to 353, where
m ₂₂ corresponds to the position of the amino acid residue identified in SEQ ID NO: 22. Further, C-terminal, deletions also can be described by the general formula 1 to n _22, wherein, n ₂₂ is
Is an integer from 2 to 354, where n ₂₂ corresponds to the position of the amino acid residue identified in SEQ ID NO: 22. Furthermore, the present invention may also include one or more amino acids providing the deleted polypeptide from both the amino and carboxyl termini, which generally as having residues m ₂₂ ~n ₂₂ of SEQ ID NO: 22 Can be described, where
n ₂₂ and m ₂₂ are integers as described above.

12. ) M ₂₄ to 132, wherein m ₂₄ is an integer of from 1 to 131, where
m ₂₄ corresponds to the position of the amino acid residue identified in SEQ ID NO: 24. In addition, C-terminal deletions may also be described by the general formulas 1 to n ₂₄ , where n ₂₄ is
Is an integer from 2 to 132, where n ₂₄ corresponds to the position of the amino acid residue identified in SEQ ID NO: 24. Furthermore, the invention also provides a polypeptide in which one or more amino acids have been deleted from both the amino and carboxyl termini, which generally comprises residues m _{24 to} n ₂₄ of SEQ ID NO: 24. Can be described, where
n ₂₄ and m ₂₄ are integers as described above.

In yet another aspect, the invention also includes deletion mutants in which amino acids have been deleted from both the N-terminal residue and the C-terminal residue. Such variants include all combinations of N-terminal deletion variants and C-terminal deletion variants described above.

In another aspect of the invention, the disclosed variants of the angiogenic polypeptide sequences are useful as screening agents for detecting agonists and / or antagonists of angiogenic activity, or Can be modified to be used as an agonist and / or antagonist agent for the angiogenic activity of the disclosed angiogenic polypeptide, or can be modified by another agent (eg, to the disclosed angiogenic polypeptide). Can be used in a co-treatment application with a monoclonal antibody (eg, the result of which can be either an agonist and / or an antagonist of the angiogenic activity of the disclosed angiogenic polypeptides) specific for the same. .

In another aspect of the invention, fragments of other reference and disclosed angiogenic polypeptides of the invention (SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18).
, 20, 22 or 24) can be generated to give variants that may be useful as agonist and / or antagonist agents that modulate the activity of the disclosed angiogenic polypeptides. In addition, these polypeptide variants may be used in co-treatment applications with another agent (eg, a monoclonal antibody specific for the disclosed angiogenic polypeptides) to, for example, enhance or reduce certain desired activities. You can

[0265] The term "gene" means the segment of DNA involved in the production of polypeptide chains; that is, the region before the coding region and the region after the coding region (leader and trailer), and It contains intervening sequences (introns) between individual coding segments (exons).

The present invention further relates to fragments of the isolated nucleic acid molecules described herein. SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21
Or at least about 15 nt, and more preferably at least about 20 nt, useful as a diagnostic probe and primer as discussed herein, with a fragment of the isolated nucleic acid molecule having the nucleotide sequence set forth in 23.
, Even more preferably at least about 30 nt, and even more preferably at least about 40 nt long fragments are contemplated. Of course 50, 75
, 100, 125, 150, 175, 200, 225, 250, 275, 300
325, 350, 375, 400, 425, 450, 475, 500, 525
550, 575, 600, 625, 650, 675, 700, 725, 750
, 775, 800, 825, 850, 875, 900, 925, 950, 975
, 1000, 1025, 1050, 1075, 1100, 1125, 1150,
1175, 1200, 1225, 1250, 1275, 1300, 1325, 1
350, 1375, 1400, 1425, 1450, 1475, 1500, 15
Larger fragments of 25, 1550, 1575, 1600, 1625 or 1650 nt in length are also SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15,
, 17, 19, 21 or 23 are useful according to the invention, as are the fragments corresponding to most, if not all, of the nucleotide sequences shown. For example, with fragments of at least 20 nt in length, SEQ ID NOs: 1, 3, 5, 7
, 9, 11, 13, 15, 17, 19, 21, or 23 are intended to be fragments containing 20 or more contiguous bases from the nucleotide sequence shown. In this context, "about" means some (5, 4, 3,
2 or 1) inclusive of the specifically quoted ranges, larger or smaller nucleotides. Preferably, these fragments encode a polypeptide having biological activity.

Fragments of the full-length gene of the present invention can be used as hybridization probes for cDNA libraries to isolate full-length cDNAs and have high sequence similarity to this gene or similar organisms. Other cDNAs with biological activity can be isolated. This type of probe preferably has at least 30 bases and can include, for example, 50 or more bases. This probe can also be used to identify cDNA clones corresponding to full length transcripts as well as genomic clones containing the complete gene including regulatory or promoter regions, exons and introns. An example of a screen involves isolating the coding region of a gene by synthesizing an oligonucleotide probe with a known DNA sequence. Using a labeled oligonucleotide having a sequence complementary to the sequence of the gene of the present invention, a library of human cDNA, genomic DNA or mRNA is screened to determine which member of the library the probe hybridizes to. It is determined.

Fusion Proteins Any of the disclosed angiogenic polypeptides can be used to make fusion proteins. For example, the angiogenic polypeptide can be used as an antigenic tag when fused to a second protein. Antibodies raised against the angiogenic polypeptide can be used to indirectly detect the second protein by binding to the angiogenic polypeptide. Moreover, since secreted proteins target cell locations based on transport signals, angiogenic polypeptides, once fused to other proteins, can be used as targeting molecules.

Examples of domains that can be fused to the disclosed angiogenic polypeptides include not only heterologous signal sequences, but also other heterologous functional regions. The fusion does not necessarily need to be direct, but can occur through linker sequences.

In addition, fusion proteins can also be engineered to improve the characteristics of one or more of the disclosed angiogenic polypeptides. For example, a region of additional amino acids (especially charged amino acids) may be added to the N-terminus of an angiogenic polypeptide to provide stability and persistence during purification from host cells or subsequent handling and storage. Can be improved. Also, peptide moieties can be added to any of the disclosed angiogenic polypeptides to facilitate purification. Such regions may be removed prior to final preparation of the particular angiogenic polypeptide. Addition of peptide moieties to facilitate handling of polypeptides is well known and routine in the art.

Further, the disclosed angiogenic polypeptides, including fragments and particularly epitopes, are combined with portions of the constant domain of immunoglobulins (IgG),
Chimeric polypeptides can be produced. These fusion proteins facilitate purification and exhibit increased half-life in vivo. One reported example describes a chimeric protein consisting of the first two domains of the human CD4 polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EP
A 394,827; Traunecker et al., Nature 331: 84-.
86 (1988)). Disulfide-bonded dimer structure (due to IgG)
A fusion protein with a protein may also be more efficient at binding to and neutralizing other molecules than monomeric secretory proteins or protein fragments alone (Funtoulakis et al., J. Biochem. 270: 395.
8-3964 (1995)).

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)
Disclosed are fusion proteins that include various portions of the constant regions of immunoglobulin molecules, along with another human protein or portion thereof. In many cases, the Fc part in the fusion protein may be beneficial in therapy and diagnosis, thus leading to, for example, improved pharmacokinetic properties (EP-A 0232 262). Alternatively, it may be desirable to delete the Fc portion after the fusion protein has been expressed, detected and purified. For example, the Fc portion can interfere with therapy and diagnosis when the fusion protein is used as an antigen for immunization. In drug searches, for example, human proteins (eg, hIL-5) were fused to the Fc portion for the purpose of high throughput screening assays to identify antagonists of hIL-5 (D. Bennett et al., J. Molecular). Recogni
8: 52-58 (1995); Johnson et al. Bio
l. Chem. 270: 9459-9471 (1995)).

Further, the disclosed angiogenic polypeptides can be fused to a marker sequence (eg, a peptide that facilitates purification of a particular angiogenic polypeptide). In a preferred embodiment, the marker amino acid sequence is, inter alia, a hexa-histidine peptide (eg pQE vector (QIAGEN, Inc., 9259 Eto.
n Avenue, Chatsworth, CA, 91311), and many of these marker amino acid sequences are commercially available. For example, Gentz et al., Proc. Natl. Acad. Sci. USA 86:
Hexa-histidine provides convenient purification of the fusion protein, as described in 821-824 (1989). Another peptide tag useful for purification, the "HA" tag, corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37: 767 (1984)).

Accordingly, any of these above fusions may be engineered using the disclosed angiogenic polynucleotides or angiogenic polypeptides.

Vectors and Host Cells The present invention also relates to vectors containing the disclosed angiogenic protein-encoding polynucleotides, host cells, and recombinantly produced polypeptides. The vector can be, for example, a phage vector, a plasmid vector, a viral vector, or a retroviral vector. The retroviral vector may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.

Polynucleotides encoding the disclosed angiogenic proteins can be ligated into a vector containing a selectable marker for growth in a host. Generally, the plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in complex with a charged lipid. If the vector is a virus, it can be packaged in vitro using a suitable packaging cell line and then transduced into host cells.

The disclosed polynucleotide inserts encoding angiogenic proteins of interest are phage λPL promoter, E. coli, to name a few. coli l
ac promoter, E. E. coli trp promoter, E. coli. coli pho
A promoter, and E. It should be operably linked to a suitable promoter, such as the E. coli tac promoter, the SV40 early promoter and the SV40 late promoter, and the promoter of the retroviral LTR.
Other suitable promoters are known to those of skill in the art. The expression construct further comprises a site for transcription initiation, a site for transcription termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the transcript expressed by this construct is preferably the translation initiation codon at the beginning of the translated polypeptide and a stop codon appropriately located at the end of the translated polypeptide (UAA, UG).
A or UAG).

As indicated, the expression vector preferably contains at least one selectable marker. Such markers include dihydrofolate reductase, G418, or neomycin resistance for eukaryotic cell culture, and E. coli. coli
For culturing in and other bacteria, a tetracycline resistance gene, a kanamycin resistance gene, or an ampicillin resistance gene can be mentioned. Representative examples of suitable hosts include bacterial cells (eg, E. coli cells, Streptomyces.
s cells, and Salmonella typhimurium cells); fungal cells (eg yeast cells); insect cells (eg Drosophila S2 cells and Spodoptera Sf9 cells); animal cells (eg CHO cells,
COS cells, 293 cells, and Bowes melanoma cells); as well as plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among the preferred vectors for use in bacteria are pQE70, pQE60, and pQE-9 (available from QIAGEN, Inc.); pBluescri.
pt vector, Phasescript vector, pNH8A, pNH16a,
pNH18A, pNH46A (Stratagene Cloning Systems)
tems, Inc. ); And ptrc99a, pKK223-.
3, pKK233-3, pDR540, pRIT5 (Pharmacia Bi
otech, Inc. Available from). Among the preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (
(Available from Stratagene); and pSVK3, pBPV, pMS
G, and pSVL (available from Pharmacia). Other suitable vectors will be readily apparent to those of skill in the art.

Introduction of the construct into the host cell is carried out by calcium phosphate transfection, DE
It can be provided by AE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals (eg, Davis et al., Basic Methods In Molecu.
lar Biology (1986)). The disclosed angiogenic polypeptides include
It is specifically contemplated that it may actually be expressed by a host cell that lacks the recombinant vector.

The disclosed angiogenic polypeptides can be ammonium sulfate or ethanol precipitated, acid extracted, anion exchange or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite. It can be recovered and purified from recombinant cell culture by well known methods including chromatography, and lectin chromatography. Most preferably high performance liquid chromatography ("HPLC") is used for purification.

The disclosed angiogenic polypeptides, preferably in secreted form, can also be recovered from: Natural sources, including body fluids, tissues and cells, whether directly isolated or cultured. Products purified from sources; products of chemical synthetic procedures; and recombinant techniques from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells. The product produced by. Depending on the host used in a recombinant production procedure, the disclosed angiogenic polypeptides may be glycosylated or non-glycosylated.
In addition, the disclosed angiogenic polypeptides may also, in some cases, include an initial modified methionine residue as a result of host-mediated processes. Therefore, it is well known in the art that, in general, the N-terminal methionine encoded by the translation initiation codon is highly efficiently removed from any post-translational protein in all eukaryotic cells. The N-terminal methionine in most proteins is also efficiently removed in most prokaryotes, but for some proteins this prokaryotic removal process depends on the nature of the amino acids to which the N-terminal methionine is covalently attached. And is inefficient.

In addition to including host cells that include the vector constructs discussed herein, the present invention also includes primar (primarily mammalian) vertebrate sources.
y) Includes host cells, secondary host cells, and immortalized host cells. These host cells have been engineered to delete or replace endogenous genetic material (eg, angiogenic protein coding sequences) and / or encode the disclosed angiogenic proteins of the invention. To include genetic material (eg, a heterologous polynucleotide sequence) that is operably linked to the polynucleotide and that activates, alters, and / or amplifies the endogenous polynucleotide encoding the disclosed angiogenic proteins. Is being operated by. For example, techniques known in the art operatively link heterologous regulatory regions (eg, promoters and / or enhancers) and endogenous polynucleotides encoding the disclosed angiogenic protein sequences via homologous recombination. Can be used to (eg, 199
US Patent No. 5,641,670 issued June 24, 1995; International Publication No. WO 96/29411 published September 26, 1996; International published August 4, 1994 Publication WO 94/12650; Koller et al., Proc.
． Natl. Acad. Sci. USA 86: 8932-8935 (1989).
); And Zijlstra et al., Nature 342: 435-438 (19).
89). Each of these disclosures is incorporated by reference in its entirety).

Further, the polypeptides of the present invention can be chemically synthesized using techniques known in the art (eg, Creighton, 1983, Proteins: Stru).
cultures and Molecular Principles, W.C. H.
Freeman & Co. , N .; Y. And Hunkapiller, M .; Et al., 1984, Nature 310: 105-111). For example,
Peptides corresponding to fragments of the angiogenic polypeptides of the present invention can be synthesized by using a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the polynucleotide sequence encoding the angiogenic protein. Non-classical amino acids include, but are not limited to, the normal amino acid D isomer, 2,4-
Diaminobutyric acid, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine , Norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-
Butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoroamino acids, designer amino acids (eg, b-methyl amino acids), Ca-methyl amino acids, Na-methyl amino acids, and common amino acid analogs. In addition, the amino acids can be D (dextrorotatory) or L (levorotatory).

The invention can be used, for example, by glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to antibody molecules or other cellular ligands, and the like. It includes angiogenic polypeptides that are differentially modified during or after translation. Any number of chemical modifications are known in the art (cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaB
Specific chemical cleavage by H ₄ ; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin, and the like).

Further post-translational modifications encompassed by the present invention include, for example, N-linked or O-linked carbohydrate chains, N- or C-terminal processing, attachment of chemical moieties to amino acid backbones, N-linked or O-linked. Chemical modification of linked carbohydrate chains, and addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression,
For example: The polypeptide may also be modified with a detectable label such as an enzymatic label, a fluorescent label, a radioisotope label, or an affinity label to allow detection and isolation of the protein.

The present invention also provides chemically modified derivatives of angiogenic polypeptides that may provide additional benefits, such as increased solubility, stability and circulation time of the polypeptide, or decreased immunogenicity. (See US Pat. No. 4,179,337). The chemical moieties for derivatization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, and the like. The polypeptide may be modified at random positions within the molecule or at predetermined positions within the molecule and may contain one, two, three or more attached chemical moieties.

The polymer can be a polymer of any molecular weight and can be branched or unbranched. With respect to polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about”) for polyethylene glycol preparations, due to ease of handling and manufacturing. Are shown to be heavier than the indicated molecular weight and some are less than the indicated molecular weight). The desired therapeutic profile (eg, desired sustained release duration, effect on biological activity, if any, ease of handling, degree of antigenicity or lack of antigenicity, and therapeutic protein or analog). Other sizes may be used, depending on other known effects of polyethylene glycol).

A polyethylene glycol molecule (or other chemical moiety) should be attached to the protein in view of its effect on the functional or antigenic domains of the protein. There are numerous coupling methods available to those of skill in the art (eg, EP 0 401 384 (G-CSF, incorporated herein by reference).
To PEG), Malik et al., Exp. Hematol. 20: 102
8-1035 (1992) (tresyl chloride)
Report also the PEGylation of GM-CSF with). For example, polyethylene glycol can be covalently attached to an amino acid residue via a reactive group, such as a free amino or carboxyl group. Reactive groups are groups to which activated polyethylene glycol molecules can be attached. The amino acid residue having a free amino group may include a lysine residue and an N-terminal amino acid residue; the amino acid residue having a free carboxyl group may include an aspartic acid residue, a glutamic acid residue and a C-terminal amino acid residue. Residues may be mentioned. Sulfhydryl residues are also
It can be used as a reactive group for attaching polyethylene glycol molecules. Preferred for therapeutic purposes is a bond at the amino group, eg at the N-terminus or at the lysine group.

Proteins that are chemically modified at the N-terminus may be particularly desirable. Using polyethylene glycol as an example of the composition of the present invention, the ratio of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mixture should be performed from various polyethylene glycol molecules (by molecular weight, branching, etc.). The type of PEGylation reaction and method of obtaining the selected N-terminal PEGylated protein can be selected. The method of obtaining the N-terminal PEGylated preparation (ie, separating this moiety from other mono-PEGylated moieties, if necessary) is performed by purification of the N-terminal PEGylated material from a population of PEGylated protein molecules. obtain. The selective protein chemically modified by N-terminal modification is
It can be achieved by reductive alkylation, which takes advantage of the differential reactivity of different types of primary amino groups (lysine vs. N-terminus) available for derivatization in a particular protein. Under appropriate reaction conditions, substantially selective derivatization of proteins at the N-terminus with a carbonyl group-containing polymer is achieved.

Angiogenic polypeptides of the present invention may be monomeric or multimeric (ie, dimers,
Trimers, tetramers and higher multimers). Accordingly, the invention relates to monomeric and multimeric disclosed angiogenic polypeptides of the invention, their preparations and compositions (preferably pharmaceutical compositions) containing them. In particular embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers.
In a further embodiment, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the present invention can be homomers or heteromers. As used herein, the term homomer refers to a multimer comprising only angiogenic polypeptides of the invention (angiogenic protein fragments, variants, splicing variants, and fusions described herein). (Including protein). These homomers can include angiogenic polypeptides having the same or different amino acid sequences. In certain embodiments, homomers of the invention are multimers that include only polypeptides having the same amino acid sequence of one of the disclosed angiogenic proteins. In another particular embodiment, the homomers of the invention are multimers that include angiogenic polypeptides having different amino acid sequences. In certain embodiments, the multimers of the invention are homodimers (eg, including angiogenic polypeptides having the same or different amino acid sequences) or homotrimers (eg, the same and / or different amino acid sequences). Including angiogenic polypeptides). In a further embodiment, the homomeric multimers of the invention are at least homodimers, at least homotrimers or at least homotetramers.

The term heteromer, as used herein, refers to a multimer comprising one or more heterologous polypeptides (ie, polypeptides of different proteins) in addition to the angiogenic polypeptide of the invention. Say. In a particular embodiment, the multimers of the invention are
It is a heterodimer, a heterotrimer or a heterotetramer. In a further embodiment, the homomeric multimers of the invention are at least homodimers, at least homotrimers or at least homotetramers.

The multimers of the invention can be the result of hydrophobic, hydrophilic, ionic and / or covalent linkages, and / or can be indirectly linked, for example by liposome formation. Thus, in one embodiment, multimers of the invention (eg, homodimers or homotrimers, etc.) are formed when the polypeptides of the invention contact each other in solution. In another embodiment, a heteromultimer of the invention (eg, a heterotrimer or heterotetramer, etc.) is a polypeptide of the invention in solution in which the antibody to the polypeptide of the invention (of the invention Formed when contacted with antibodies (including antibodies against heterologous polypeptide sequences in a fusion protein). In other embodiments, the multimers of the invention are formed by covalent attachment to and / or between angiogenic polypeptides of the invention. Such covalent linkages may be obtained by polypeptide sequence (eg, SEQ ID NOs: 2, 4, 6, 8, 10, 12).
, 14, 16, 18, 20, 22, or 24).
It may include one or more amino acid residues contained therein. In one example, the covalent bond is a bridge between cysteine residues located within the interacting polypeptide sequence in the native (ie, naturally occurring) polypeptide. In another example, this covalent bond is the result of chemical or recombinant manipulation. Alternatively, such a covalent bond may comprise one or more amino acid residues contained in the heterologous polypeptide sequence in the angiogenic fusion protein. In one example, the covalent bond is between the heterologous sequences contained in the fusion protein of the invention (eg, US Pat. No. 5,478,925).
See No.). In a particular example, the covalent bond is between a heterologous sequence contained in an angiogenic polynucleotide-Fc fusion protein of the invention (as described herein). In another particular example, the covalent attachment of a fusion protein of the invention may be accomplished by the formation of another TNF family ligand / receptor member (eg, osteoprotegerin) (eg, International Publication No. WO. 98/49305, the contents of which are incorporated herein by reference in its entirety).

The multimers of the invention can be produced using chemical techniques known in the art. For example, the polypeptides desired to be included in the multimers of the present invention can be chemically crosslinked using linker molecules and linker molecule length optimization techniques known in the art (eg, US Pat. , 478,925, which is incorporated herein by reference in its entirety). Furthermore, the multimers of the invention are known in the art to form one or more intermolecular bridges between cysteine residues located in the sequence of the polypeptide desired to be included in the multimer. (See, eg, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). Furthermore, the polypeptides of the present invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide, and techniques known in the art modified one or more of these. It can be applied to generate multimers containing polypeptides (see, eg, US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety). In addition, techniques known in the art can be applied to produce liposomes containing the polypeptide components desired to be included in the multimers of the invention (see, eg, US Pat. No. 5,478,925). See
Which is hereby incorporated by reference in its entirety).

Alternatively, the multimers of the present invention can be produced using genetic engineering techniques known in the art. In one embodiment, the polypeptides comprised in the multimers of the invention are recombinantly produced using the fusion protein techniques described herein or otherwise known in the art. (For example, US Pat. No. 5,478,925.
No., which is hereby incorporated by reference in its entirety). In a particular embodiment, the polynucleotide encoding the homodimer of the invention is
A polynucleotide sequence encoding a polypeptide of the invention is ligated to a sequence encoding a linker polypeptide and then the translation product of the polypeptide is transferred in the reverse direction from the original C-terminus to the N-terminus (lacking the leader sequence). Produced by further ligation to a synthetic polynucleotide that encodes (eg, US Pat.
478,925, which is hereby incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art have been applied to include a set of the invention comprising a transmembrane domain (or hydrophobic peptide or signal peptide). A modified polypeptide, which can be incorporated into liposomes by membrane reconstitution techniques (eg,
See US Pat. No. 5,478,925, which is incorporated herein by reference in its entirety).

Biological Activity of Angiogenic Polypeptides Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used in an assay for one or more biological processes. Can be tested for biological activity. Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, are active in a particular assay, the angiogenic polynucleotides or polypeptides are associated with a disease associated with their biological activity. Seems to be involved in. Therefore, using angiogenic polypeptides,
Related diseases can be treated. The biological activities described below are included in the "desired activity" of an angiogenic protein.

(Immune Activity) Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, activate or inhibit the proliferation, differentiation, or recruitment (chemotactic) of immune cells. By doing so, it may be useful in the treatment of immune system deficiencies or disorders. Immune cells develop through a process called hematopoiesis, from pluripotent stem cells to myeloid cells (platelets, red blood cells,
It produces neutrophils and macrophages) and lymphoid cells (B and T lymphocytes). The etiology of these immune deficiencies or disorders may be genetic, physical (s
may be acquired (eg, cancer or some autoimmune disorder), acquired (eg, due to chemotherapy or toxin) or infectious. Further, angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used as markers or detectors of certain immune system diseases or disorders.

Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be useful in treating or detecting hematopoietic cell defects or disorders. Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, are pluripotent in attempts to treat disorders associated with the reduction of certain (or many) types of hematopoietic cells. It can be used to increase the differentiation and proliferation of hematopoietic cells, including stem cells. Examples of immunological deficiency syndromes include blood protein disorders (eg, agammaglobulinemia, hypogammaglobulinemia), ataxia-telangiectasia, unclassified immunodeficiency, Di George syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocytic bactericidal dysfunction, severe combined immunodeficiency (SCID), viscot-oldrich disorder, anemia, thrombocytopenia, or hemoglobinuria. , But not limited to these.

In addition, angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, may also be used to induce hemostatic activity (stop bleeding) or thrombolytic activity (clot formation). )
Can be adjusted. For example, by increasing hemostatic or thrombolytic activity,
Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, are used to impair blood coagulation (eg, fibrinogenemia, factor deficiency), blood platelet disorders (eg, , Thrombocytopenia), or wounds resulting from trauma, surgery or other causes. Alternatively, angiogenic polynucleotides or polypeptides that can reduce hemostatic or thrombolytic activity, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used to inhibit or lyse blood clots. These molecules may be important in the treatment of heart attack (infarction), stroke or scarring.

Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, may also be useful in treating or detecting autoimmune disorders. Many autoimmune disorders
It results from inappropriate recognition of self as a foreign substance by immune cells.
This improper recognition triggers an immune response that results in the destruction of host tissue. Therefore, administration of angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, capable of inhibiting the immune response, particularly T cell proliferation, differentiation or chemotaxis, is associated with autoimmune disorders. It can be an effective treatment in prevention.

Examples of autoimmune disorders that may be treated or detected include Addison's disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's syndrome, Graves. Disease, multiple sclerosis, myasthenia gravis, neuritis, ophthalmitis, bullous pemphigoid, pemphigus, multiple endocrine disorders, purpura, Reiter's disease, Stiffman syndrome, autoimmune thyroiditis, systemic Lupus erythematosus, autoimmune pulmonary inflammation, Guyan-Barre syndrome, insulin-dependent diabetes mellitus, and autoimmune inflammatory eye disease.

Similarly, asthma (particularly allergic asthma) or other respiratory problems,
Allergic reactions and conditions can also be treated with angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides. In addition, these molecules may be used to treat anaphylaxis, hypersensitivity to antigenic molecules or blood group incompatibility.

Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, are also used to treat and / or prevent organ rejection or graft versus host disease (GVHD). obtain. Organ rejection occurs by host immune cell destruction of transplanted tissue via an immune response. Similarly, the immune response is also involved in GVHD, where foreign transplanted immune cells destroy host tissue. Administration of angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, that inhibit the immune response, particularly T cell proliferation, differentiation or chemotaxis, prevents organ rejection or GVHD Can be an effective treatment in.

Similarly, angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, may also be used to modulate inflammation. For example, angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can inhibit proliferation and differentiation of cells involved in the inflammatory response. Using these molecules, infection-related inflammation (eg, septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury-related inflammation, endotoxin lethal-related inflammation, arthritis Inflammation associated with, inflammation associated with complement-mediated hyperacute rejection, inflammation associated with nephritis, inflammation associated with cytokine- or chemokine-induced lung injury, inflammation associated with inflammatory bowel disease, Crohn's disease or cytokine ( For example, inflammatory conditions, both chronic and acute, can be treated, including inflammation resulting from overproduction of TNF or IL-1).

Hyperproliferative Disorders Using angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides,
Hyperproliferative disorders, including neoplasms, can be treated or detected. Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can inhibit the growth of this disorder through direct or indirect interactions. Alternatively, angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can proliferate other cells that can inhibit hyperproliferative disorders.

For example, by increasing the immune response, in particular by increasing the antigenic quality of the hyperproliferative disorder, or by proliferating, differentiating or recruiting T cells.
Hyperproliferative disorders can be treated. This immune response can be augmented by either enhancing an existing immune response or initiating a new immune response. Alternatively, reducing the immune response may also be a method of treating hyperproliferative disorders, such as chemotherapeutic agents.

Examples of hyperproliferative disorders that may be treated or detected by angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides include abdomen, bone, breast, digestive system, liver, Pancreas, peritoneum, endocrine glands (adrenal gland, parathyroid gland, pituitary gland, testis, ovary, thymus, thyroid gland),
Eyes, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue,
Includes, but is not limited to, neoplasms located in the spleen, thorax, and genitourinary organs.

Similarly, other hyperproliferative disorders can also be treated or detected with angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides. Examples of such hyperproliferative disorders include hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sézary syndrome, Waldenstrom macroglobulinemia, Goscher's disease, histiocytes Proliferative diseases, and any other hyperproliferative disease, as well as neoplasms located in the organ systems listed above, are not limited.

Cardiovascular disorders Using angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides,
Cardiovascular disorders may be treated, including peripheral arterial disease such as limb ischemia.

Cardiovascular disorders include arterio-arterial fistulas.
stula), arteriovenous fistula, cerebral arteriovenous birth defects, congenital heart defects (congeni)
cardiovascular abnormalities such as tal heart defects, pulmonary valve atresia, and Scimitar syndrome. Congenital heart defects include aortic coarctation, tricentric heart, coronary vessel anomalies
, Cross heart, right thoracic heart, patent ductus arteri
oss), Ebstein's anomaly, Eisenmenger complex, left ventricular underdevelopment syndrome, left thoracic heart, tetralogy of Fallot, transposition of the great arteries, bilateral right ventricle origin, tricuspid valve closure, residual arterial canal , And heart septal deficiency
ts) (e.g., aortopulmonary separation)
eptal defect, endocardial bed deficiency, Luthambache syndrome, trilogy of Fallot, ventricular heart septal defect (ventricular heart sep
tal defects)).

Cardiovascular disorders also include arrhythmias, carcinoid heart disease, high cardiac output (hi
gh cardiac output, low cardiac output (low cardiac)
output), cardiac tamponade, endocarditis (including bacterial), cardiac aneurysm,
Cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, edema, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction (post-inf)
arcion heart rupture), ventricular septal rupture, heart valve disease,
Myocardial disease, myocardial ischemia, endocardial effusion, epicarditis (including infarct and tuberculosis),
Pneumopericardial disease, postpericardiotomy syndrome, right heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications
and heart diseases such as scimitar syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

The arrhythmia includes sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, premature contraction, Adams-Stokes syndrome, leg block, sinoatrial block, long QT syndrome (long).
QT syndrome), collateral contraction, Lone-Ganning-Levine syndrome, Maheme-type pre-excita
tion syndrome), Wolff-Parkinson-White syndrome, sinus dysfunction syndrome, tachycardia, and ventricular fibrillation. As tachycardia, paroxysmal tachycardia,
Supraventricular tachycardia, ventricular intrinsic rhythm promotion, atrioventricular nodal reentry tachycardia (atrioventri
circular nodal reentry tachycardia), ectopic atrial tachycardia, ectopic junction tachycardia, sinoatrial nodal reentry tachycardia
dal reentry tachycardia), sinus tachycardia, torsade
De pointe, and ventricular tachycardia.

Heart valve diseases include aortic insufficiency, aortic stenosis, and murmur (hea).
r murmurs), aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve dysfunction, mitral valve stenosis, pulmonary valve atresia, pulmonary valve dysfunction, pulmonary valve stenosis, tricuspid Includes atresia, tricuspid dysfunction, and tricuspid stenosis.

Cardiomyopathy includes alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, subvalvular aortic stenosis, subvalvular pulmonary stenosis, restricted cardiomyopathy, Chagas cardiomyopathy, intracardiac Membrane fibroelasticity, endocardial myocardial fibrosis, Keens syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemia includes angina, coronary aneurysm, coronary atherosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction, and myocardial stunnin.
g) such as coronary artery disease.

Cardiovascular diseases also include aneurysms, angiogenesis, hemangiomatosis, bacterial hemangiopathies, Hippel-Lindau Disease.
), Klipel-Tornone-Weber syndrome, Sturgi-Weber syndrome,
Vasomotor edema, aortic disease, Takayasu arteritis, aortitis, Leuriche syndrome,
Arterial occlusive disease, arteritis, arteritis, polyarteritis nodosa, cerebrovascular disease, diabetic vasculopathy, diabetic retinopathy, embolism, thrombosis, erythromelalgia, hemorrhoids, liver Venous obstruction disorder, hypertension, hypotension, ischemia, peripheral vascular disorder, phlebitis, pulmonary vein occlusion disease, hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary vein occlusion disease, Raynaud's disease, CREST syndrome, Retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, ataxia tela
vascular diseases such as hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose ulcers, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, pseudoaneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms. Is mentioned.

Arterial occlusion disease includes arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasia, mesenteric vascular occlusion, Moyamoya disease, renal artery occlusion, retinal artery occlusion, and occlusive thrombosis. Sexual vasculitis.

Cerebrovascular disorders include carotid artery disease, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous congenital abnormality, cerebral artery disease, cerebral embolism and thrombosis, Carotid artery thrombosis, sinus thrombosis, Wallenberg syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subarachnoid hemorrhage
Morrage), cerebral infarction, cerebral ischemia (including transient), subclavian stealing syndrome, periventricular leukomalac
ia), vascular headache, cluster headache, migraine, and vertebro.
basallar) dysfunction.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, toe cyanosis syndrome, fat embolisms, pulmonary embolisms, and thromboembolisms. As thrombosis, coronary artery thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis,
Includes sinus thrombosis, Wallenberg syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, and partition syndrome (compartme).
nt syndrome, anterior compar
ment syndrome), myocardial ischemia, reperfusion injury, and peripheral limb ischemia. Vasculitis includes aortitis, arteritis, Behcet.
t) Syndrome, Churg-Strauss Syndrome, Mucocutaneous Lymph Node Syndrome, Obstructive Thrombotic Vasculitis, Hypersensitivity Vasculitis, Schoenline-Henoho Purpura (Schoenl)
ein-Henoch purpura), allergic cutaneous vasculitis and Wegener's granulomatosis.

Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, are particularly effective for the treatment of dangerous limb ischemia and coronary artery disease. As shown in the Examples, experimentally induced administration of angiogenic polynucleotides and polypeptides to the hindlimb of ischemic rabbits can restore blood pressure ratio, blood flow, angiographic score, and capillary density. .

Angiogenic polypeptides can be administered using any method known in the art, including direct needle injection at the site of delivery, intravenous injection, topical administration, catheter infusion. , Biolistic injection, particle accelerator,
Gel foam sponge depots, other commercial depot substances, osmotic pumps, oral or suppository solid pharmaceutical formulations, intra-operative decanting or topical application, aerosol delivery include, but are not limited to. Such methods are known in the art. Angiogenic polypeptides can be administered as part of a pharmaceutical composition, described in more detail below. Methods of delivering polynucleotides are described in more detail herein.

Anti-Angiogenic Activity The naturally occurring equilibrium between endogenous stimulators and inhibitors of angiogenesis is the equilibrium in which the inhibitory effects predominate. Rastinejad et al., Cell 5
6: 345-355 (1989). In rare cases where neovascularization occurs under normal physiological conditions (eg, wound healing, organ regeneration, embryogenesis, and female reproductive processes), angiogenesis is tightly regulated and spatial and temporal. It is specified. Conditions for pathological angiogenesis (eg, characterizing solid tumor growth)
Below, there is no control over these adjustments. Unregulated angiogenesis becomes pathological and maintains the progression of many neoplastic and non-neoplastic diseases. Many serious diseases are caused by abnormal neovascularization, including solid tumor growth and metastasis.
It is dominated by arthritis, some types of eye disorders, and psoriasis. For example, Mos
es et al., Biotech. 9: 630-634 (1991); Folkman et al., N. et al. Engl. J. Med. 333: 1757-1763 (1995); A
uerbach et al. Microvasc. Res. 29: 401-411 (
1985); Folkman, Advances in Cancer Res.
Edit, Klein and Weinhouse, Academic Pr
ess, New York, pp. 175-203 (1985); Patz, Am.
J. Opthalmol. 94: 715-743 (1982); and Folk.
See review by man et al., Science 221: 719-725 (1983). In many pathological conditions, the process of angiogenesis contributes to the disease state. For example, significant data has been accumulated that suggests that solid tumor growth depends on angiogenesis. Folkman and Klagsbrun, Sc
ience 235: 442-447 (1987).

The present invention provides for the treatment of diseases or disorders associated with neovascularization by the administration of angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides. Malignant and metastatic conditions that can be treated with the polynucleotides and polypeptides of the invention, or agonists or antagonists, include malignant diseases, solid tumors, and cancers described herein, as well as others known in the art. (For a review of such disorders, see Fishman et al., Medicine, 2nd Edition, JBL.
ippincott Co. , Philadelphia (1985)), but is not limited thereto.

Ocular disorders associated with neovascularization that can be treated with the angiogenic polynucleotides and polypeptides of the present invention, including angiogenic agonists and / or antagonists, include: Without limitation: neovascular glaucoma, diabetic retinopathy, retinoblastoma, post lens fibroplasia, uveitis, retinopathy of prematurity, corneal transplant neovascularization, as well as other inflammatory diseases of the eye, ocular tumor,
And diseases associated with choroidal or iris neovascularization. For example, Waltma
n et al., Am. J. Ophthal. 85: 704-710 (1978) and G.
artner et al., Surv. Ophthal. 22: 291 to 312 (1978)
) See the review article.

Further, disorders that can be treated with the angiogenic polynucleotides and polypeptides of the present invention (including angiogenic agonists and / or antagonists) include:
These include but are not limited to: hemangiomas, arthritis, psoriasis, angiofibromas, atherosclerotic plaques, delayed wound healing, granulation, hemophilic joints, hyperplastic scars, pseudoarthral fractures, and Aussler. -Osler-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesion.

Further, disorders and / or conditions that may be treated with the angiogenic polynucleotides and polypeptides of the present invention, including angiogenic agonists and / or antagonists, include, but are not limited to: : Solid tumors, blood born tumors (eg leukemia), tumor metastases, Kaposi's sarcoma, benign tumors (eg hemangiomas, acoustic neuromas, neurofibromas, trachoma and purulent granulomas), chronic joints Rheumatism, psoriasis, ocular angiogenic disorders (eg, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal transplant rejection, neovascular glaucoma, post lens fibroplasia, rubeosis, retinoblastoma, and uveal membrane) Flame (uvieti
s)), delayed wound healing, endometriosis, angiogenesis, granulation, hyperplastic scar (keloid), pseudoarticular fracture, scleroderma, trachoma, vascular adhesion, myocardial angiogenesis, coronary Collaterals, cerebral collaterals, arteriovenous malformations, ischemic extremity angiogenesis, Austler-Weber syndrome, plaque neovascularization, telangiectasia, hemophilic joints, angiofibromas, Fibromuscular dysplasia, wound granulation, Crohn's disease, atherosclerosis, birth control drugs (to control menstruation,
By preventing angiogenesis necessary for embryo implantation, a consequence of pathogenicity (eg, disease with angiogenesis as cat scratch disease (Rochele minalia quintosa), ulcer (Helicobacter pylor).
i), Bartonellosis and bacterial hemangiomas). Diseases at the cellular level Diseases associated with increased cell survival or inhibition of apoptosis that can be treated or detected by angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides include: Cancer (eg, follicular lymphoma, carcinoma with p53 mutation, and hormone-dependent tumor, which are: colon cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestine Cancer, testicular cancer, gastric cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelial tumor, osteoblastoma, giant cell tumor of the bone, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovary Cancer, including but not limited to; autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto thyroiditis, gall) Juvenile cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) and viral infections (eg herpesvirus, poxvirus and adenovirus), Inflammation, graft versus host disease, acute graft rejection, as well as chronic graft rejection. In a preferred embodiment, the angiogenic polynucleotides, polypeptides, and / or antagonists of the invention are used to inhibit the growth, progression, and / or metastasis of cancer, particularly those listed above. It

Additional diseases or conditions associated with increased cell survival that can be treated or detected by angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides include:
Malignant disease progression and / or metastasis and related disorders such as, but not limited to, leukemia (acute leukemia (eg, acute lymphocytic leukemia, acute myelogenous leukemia (myeloblastic, promyelocytic, promyelocytic). Including leukemia, myelomonocytic, monocytic and erythroleukemia) and chronic leukemia (eg chronic myelogenous (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma (Eg, Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, and solid tumors (sarcomas and carcinomas (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma). , Osteogenic sarcoma, chordoma, hemangiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelioma, periosteoma, mesothelioma, Tumors, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary gland Cancer, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, Bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineocytoma, hemangioblastoma, acoustic neuroma,
(Including but not limited to oligodendrogliomas, menangiomas, melanomas, neuroblastomas, and retinoblastomas)).

Diseases associated with increased apoptosis that can be treated or detected by angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, include: AIDS; neurodegeneration Disorders (eg, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, cerebellar degeneration and brain tumors or previously associated disorders); autoimmune disorders (eg, multiple sclerosis, Sjogren's syndrome, Hashimoto) Thyroiditis, biliary cirrhosis, Behcet's disease,
Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis), myelodysplastic syndrome (eg, aplastic anemia),
Graft-versus-host disease, ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury (eg hepatitis-related liver injury, ischemia / reperfusion injury, cholestosis ( Bile duct injury) and liver cancer); toxic-induced liver disease (as caused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation According to yet a further aspect of the invention, an angiogenic polynucleotide or polypeptide, or an agonist or antagonist of an angiogenic polynucleotide or polypeptide, for therapeutic purposes, eg, Processes are provided for utilization to stimulate epithelial cell proliferation and basal keratinocytes for the purpose of wound healing, as well as to stimulate hair follicle production and skin wound healing. Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be clinically useful in stimulating wound healing, including: surgical wounds, excisional wounds, Deep wounds (including dermal and epidermal damage), ocular tissue wounds, tooth tissue wounds, oral wounds, diabetic ulcers, skin ulcers, elbow ulcers, arterial ulcers, venous stasis ulcers, fever Exposure to or chemical burns, and other abnormal wound healing conditions (eg, uremia, malnutrition, vitamin deficiency, and systemic treatment with steroids, radiotherapy and antineoplastic drugs and antimetabolites). Related complications)
. Angiogenic polynucleotides or polypeptides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used to promote skin recovery after skin loss.

Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used in wound bed (
It can be used to increase the adhesion of skin grafts to the wound bed and to stimulate re-epithelialization from the wound bed. The following are types of implants in which angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides can be used to increase adhesion to the wound bed: autograft, Artificial skin, allograft
), Autograft, autoepdermographic graft,
Avascular graft, Blair-Brown graft, bone graft, embryonic tissue graft, dermal graft, delayed graft, skin graft, epidermal graft, fascia graft, full-thickness skin graft. , Xenografts, xenografts, homografts
ft), proliferative implants, lamellar implants, reticular implants, mucosal implants, Olie-Tielsch implants, omental grafts, patch implants, stalk implants, whole. Layered grafts, split-thickness skin grafts, split-thickness skin grafts. Angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides can be used to promote skin strength and improve the appearance of aged skin.

Angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides, can also induce hepatocyte proliferation,
And is thought to result in changes in epithelial cell proliferation in the lung, breast, pancreas, stomach, small intesting, and colon. Angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides, can bind to epithelial cells (eg, sebocytes (se
follicles, hair follicles, liver parenchymal cells, alveolar epithelial cells type II p
neuocytes, mucin-producing goblet cells, and other epithelial cells, and their progenitors contained in the skin, lungs, liver, and gastrointestinal tract). Angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides can promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.

Angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides can also be used to reduce the toxic side effects of intestine resulting from irradiation, chemotherapy treatments or viral infections. . Angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides may have a cytoprotective effect on the small intestinal mucosa. Angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides may also be associated with mucositis resulting from chemotherapy and viral infections.
This can stimulate healing of the tis) (oral mucosa).

Angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides, provide sufficient regeneration of the skin in full and partial thickness skin defects, including burns (ie, Hair follicles,
Sweat glands, and regrowth of the sebaceous glands), and other skin defects such as psoriasis. Angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides, cause epidermolysis bullosa, a frequent open and painful blistering by promoting re-epithelialization of these lesions It can be used to treat defects in the adhesion of the epidermis to the underlying dermis). Angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides, also treat gastric and duodenal ulcers, and scar formation of the mucosal lining and more rapid glandular and duodenal mucosa. Can be used to help healing by regeneration of the inner lining of. Inflammatory bowel disease (eg, Coulomb's disease and ulcerative colitis) are diseases that result in the disruption of the mucosal surface of the small or large intestine, respectively. Accordingly, angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides, promote resurfacing of mucosal surfaces to aid in faster healing and in the inflammatory bowel. It can be used to prevent the progression of the disease. Treatment with angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides is expected to have a significant effect on mucosal production throughout the gastrointestinal tract, and , Can be used to protect against harmful substances that have been ingested or after surgery. Angiogenic polynucleotides or polypeptides, agonists or antagonists of angiogenic polynucleotides or polypeptides can be used to treat diseases associated with the expression of angiogenic polypeptides.

Further, the angiogenic polynucleotide or polypeptide, or an agonist or antagonist of the angiogenic polynucleotide or polypeptide,
It can be used to prevent and cure damage to the lungs due to various pathological conditions. Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can stimulate proliferation and differentiation to prevent or treat acute or chronic lung injury, and alveoli and It may promote repair of bronchiola epithelium. For example, emphysema (which results in the progressive loss of alveoli) and inhalation injuries (ie, resulting from smoke inhalation and burns), which results in necrosis of bronchial epithelium and aveoli, are angiogenic. Polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be effectively treated. Also, angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used to stimulate the proliferation and differentiation of alveolar epithelial cell type II, which is immature. It can help treat or prevent diseases such as hyaline membrane disease in infants (eg, infant respiratory distress syndrome and bronchopulmonary dysplasia).

Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can stimulate hepatocyte proliferation and differentiation, and, thus, liver diseases and conditions (eg,
Fulminant liver failure caused by cirrhosis, hepatitis virus and toxic substances (ie acetaminophen, carbon tetrachloride,
And other known in the art hepatic toxins) resulting in liver damage).

Further, the angiogenic polynucleotide or polypeptide, or an agonist or antagonist of the angiogenic polynucleotide or polypeptide,
It can be used to treat or prevent the onset of diabetes mellitus. Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, in patients newly diagnosed with type I diabetes and type II diabetes if some islet cell function remains Is
It can be used to maintain the islet function so as to alleviate, delay or prevent the persistent onset of the disease. Also, angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used as an adjunct in islet cell transplantation to improve or promote islet cell function.

Infectious Diseases Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used to treat or detect infectious agents. For example, infectious diseases can be treated by increasing the immune response, particularly by increasing the proliferation and differentiation of B and / or T cells. The immune response can be elevated by either raising an existing immune response or initiating a new immune response. Alternatively, the angiogenic polynucleotide or polypeptide, or an agonist or antagonist of the angiogenic polynucleotide or polypeptide, may also directly inhibit the infectious agent without necessarily eliciting an immune response.

A virus is an example of an infectious agent that can cause a disease or condition that can be treated or detected by an angiogenic polynucleotide or polypeptide, or an agonist or antagonist of an angiogenic polynucleotide or polypeptide. Examples of viruses include, but are not limited to, the following DNA and RNA viruses and viridae: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Calcivirus. Family, Sarcoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (hepatitis), Herpesviridae (eg cytomegalovirus, herpes simplex, herpes zoster)
, Mononegavirus (for example, Paramyxoviridae, measles virus, Rhabdoviridae), Orthomyxoviridae (for example, influenza), Papovaviridae, Parvoviridae, Picornavirus, Poxviridae (for example, , Smallpox or vaccinia), Reoviridae (
For example, rotavirus), retroviridae (HTLV-I, HTLV-II,
Lentivirus), and Togaviridae (eg, Rubivirus). Viruses that enter these families can cause a variety of diseases or conditions, including but not limited to: arthritis, bronchiolitis.
, Encephalitis, ocular infections (eg conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, chronic active, delta), meningitis, opportunistic infections (eg ,
AIDS), pneumonia, Burkitt lymphoma, chickenpox, hemorrhagic fever, measles, mumps, parainfluenza, rabies, common cold, polio, leukemia, rubella, sexually transmitted diseases, skin diseases (eg, capoge, warts), And viremia. Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used to treat or detect any of these conditions or diseases.

Similarly, a bacterial or fungal agent that can cause a disease or condition and that can be treated or detected by an angiogenic polynucleotide or polypeptide, or an agonist or antagonist of an angiogenic polynucleotide or polypeptide is , But not limited to the following Gram-negative and Gram-positive bacteria and bacteria families and fungi: Actinomycetales (eg, Corynebacterium, Mycobacterium, Nor.
cardia), Aspergillosis, Bacilraceae (eg, Anthrax, Clostridium), Bacteroidacea
e, Blastomycosis, Bordetella, Borrelia,
Brucellosis, Candidiasis, Campylobacter
r, Coccidioidomycosis, Cryptococcosis,
Dermatocycleses, Enterobacteriaceae (Kl
ebsiella, Salmonella, Serratia, Yersini
a), Erysipelothrix, Helicobacter, Legio
nellosis, Leptospirosis, Listeria, Myco
plasmales, Neisseriaceae (eg, Acinet
obter, Gonorrhea, Menigococcal), Past
eurellacea infections (eg Actinobacillus, He
amophilus, Pasteurella), Pseudomonas, R
ickettsiaceae, Chlamydiaceae, and Staph
ylococcal ,. These bacterial or fungal families can cause diseases or conditions including, but not limited to: bacteremia, endocarditis, eye infections (
Conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (eg, AIDS-related infections), periungual inflammation, prosthesis-related infections, Reiter's disease, respiratory tract infections (eg, pertussis or empyema), sepsis , Lyme disease, cat scratch disease, dysentery, paratyphoid fever, food poisoning, typhoid fever, pneumonia, gonorrhea, meningitis, chlamydia, syphilis, diphtheria, lei disease, paratuberculosis, tuberculosis, lupus, botulinum poisoning, gangrene, tetanus, impetigo Rash, rheumatic fever, scarlet fever, sexually transmitted diseases, skin diseases (eg cellulitis, dermatomycosis (de)
rmatocycles)), toxemia, urinary tract infection, wound infection. Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used to treat or detect any of these conditions or diseases.

In addition, the parasite factor-caused diseases or conditions that may be treated or detected by angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides include the following families or classes: Include, but are not limited to: amebiasis, babesiosis, coccidiosis, cryptosporidiosis, dinuclear amebiasis (Dientamoebiasis), copulation, ectoparasite, giardia flagellosis, helminthosis, leishmaniasis, Theileriosis, toxoplasmosis, trypanosomiasis, and Trichomonas disease. These parasites can cause a variety of diseases or conditions including, but not limited to: scabies, scrub typhus, ocular infections, intestinal diseases (eg, dysentery, giardia giardiasis), liver diseases, lungs. Diseases, opportunistic infections (eg, AIDS-related), malaria, pregnancy complications, and toxoplasmosis. Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used to treat or detect any of these conditions or diseases.

Preferably, the treatment with the angiogenic polynucleotide or polypeptide, or an agonist or antagonist of the angiogenic polynucleotide or polypeptide, administers to the patient an effective amount of the angiogenic polypeptide, or Either by removing the cells from the patient, supplying the cells with angiogenic polynucleotides, and returning the engineered cells to the patient (ex vivo treatment). In addition, angiogenic polypeptides or polynucleotides can be used as antigens in vaccines to elicit an immune response against infectious diseases.

Regeneration Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, are used to differentiate, proliferate, and attract cells to induce tissue regeneration. Get (Scien
Ce 276: 59-87 (1997)). With tissue regeneration
Birth defects, trauma (wounds, burns, incisions, or ulcers), aging, diseases (eg, osteoporosis, osteoarthritis, periodontal disease, liver failure)
, Surgery including cosmetic surgery, fibrosis, reperfusion injury, or repair, replacement, or protection of tissue damaged by systemic cytokine damage.

Tissues that can be regenerated using the present invention include the following: Organs (eg,
Pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle, or myocardium),
Tissues of the vascular system (including blood vessels and lymph vessels), nerves, hematopoiesis, and skeleton (bones, cartilage, tendons, and ligaments). Preferably, regeneration occurs without or with reduced scarring. Regeneration can also include angiogenesis.

Further, the angiogenic polynucleotide or polypeptide, or an agonist or antagonist of the angiogenic polynucleotide or polypeptide,
It can increase the regeneration of tissues that are difficult to heal. For example, increasing tendon / ligament regeneration speeds recovery time after injury. The angiogenic polynucleotides or polypeptides of the invention, or agonists or antagonists of angiogenic polynucleotides or polypeptides, may also be used prophylactically in an attempt to avoid injury. Specific diseases that can be treated include tendinitis, carpal tunnel syndrome,
And other tendon or ligament defects. Further examples of tissue regeneration of non-healing wounds include ulcers associated with decubitus ulcers, vascular insufficiency, surgical wounds, and traumatic wounds.

Similarly, nerve and brain tissue is also regenerated by using angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, to proliferate and differentiate neural cells. Can be done. Diseases that can be treated using the present methods include central and peripheral nervous system diseases, neuropathy, or mechanical and traumatic disorders (eg,
Spinal cord disorders, head trauma, cerebrovascular disease, and stroke).
In particular, diseases associated with peripheral nerve injury, peripheral neuropathy (eg resulting from chemotherapy or other medical therapies), localized neuropathy, and central nervous system diseases (eg Alzheimer's disease, Parkinson's disease, Huntington). Disease, amyotrophic lateral sclerosis,
And Shy-Drager syndrome) can all be treated with angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides.

Chemotaxis Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can have chemotactic activity. Chemotactic molecules allow cells (eg, monocytes, fibroblasts, neutrophils, T cells, mast cells, eosinophils, epithelial cells and / or endothelial cells) to move to specific sites in the body (eg, Site of inflammation, infection, or hyperproliferation). The recruited cells may then repel and / or heal the particular trauma or abnormality.

Angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can increase the chemotactic activity of particular cells. These chemotactic molecules are then used to treat inflammation, infection, hyperproliferative disorders, or any immune system disorder by increasing the number of cells targeted to specific locations in the body. obtain. For example, chemotactic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Angiogenic polypeptides as chemotactic molecules can also attract fibroblasts, which can be used to treat wounds.

It is also contemplated that angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, may inhibit chemotactic activity. These molecules can also be used to treat disorders. Thus, angiogenic polynucleotides or polypeptides, or agonists or antagonists of angiogenic polynucleotides or polypeptides, can be used as inhibitors of chemotaxis.

Binding Activity Angiogenic polypeptides can be used to screen for molecules that bind, or are bound by, angiogenic polypeptides. The binding of the polypeptide to the molecule can activate (agonist), increase, inhibit (antagonist), or decrease the activity of the bound angiogenic polypeptide or molecule. Examples of such molecules include antibodies, oligonucleotides, proteins (eg receptors), or small molecules.

Preferably, screening for these molecules involves producing appropriate cells that express the angiogenic polypeptide, either as a secreted protein or on the cell membrane. Preferred cells include mammals, yeast, Dr
osophila, or E. cells derived from E. coli. The cells that express the angiogenic polypeptide (or the cell membrane containing the expressed polypeptide) are then preferably conjugated, stimulated, or activated with either the angiogenic polypeptide or the molecule. A test compound potentially containing a molecule for observing inhibition is contacted.

The assay may simply test binding of a candidate compound to an angiogenic polypeptide,
Here binding is detected by the label or in an assay for competition with the labeled competitor. In addition, the assay can test whether a candidate compound produces a signal generated by binding to this polypeptide.

Alternatively, the assay can be performed with cell-free preparations, polypeptides / molecules attached to a solid support, chemical libraries, or a mixture of natural products. The assay also comprises mixing the candidate compound with a solution containing the angiogenic polypeptide, measuring the activity or binding of the angiogenic polypeptide / molecule, and the activity or binding of the angiogenic polypeptide / molecule. , Can simply include the step of comparing to a standard.

Preferably, the ELISA assay may use monoclonal or polyclonal antibodies to measure the level or activity of angiogenic polypeptides in a sample (eg, a biological sample). An antibody may measure the level or activity of an angiogenic polypeptide by either directly or indirectly binding to the angiogenic polypeptide or by competing with the angiogenic polypeptide for a substrate.

In addition, the receptors to which angiogenic polypeptides bind are determined by a number of methods known to those of skill in the art (eg, ligand panning and FACS sorting (Coliga).
n et al., Current Protocols in Immun. , 1 (2),
Chapter 5 (1991)). For example, expression cloning is used when polyadenylated RNA is prepared from cells responsive to this polypeptide, eg, NIH3T3 cells, which are known to contain multiple receptors for FGF family proteins, And SC-3 cells, and a cDNA library made from this RNA are pooled and used to transfect COS cells or other cells that are not responsive to the polypeptide. Transfected cells growing on glass slides are exposed to the polypeptides of the invention after labeling them. The polypeptide may be labeled by various means, including iodination or encapsulation of recognition sites for site-specific protein kinases.

After immobilization and incubation, slides are subjected to autoradiographic analysis. Positive pools are identified and subpools are prepared and retransfected using an iterative subpooling and rescreening process to ultimately yield a single clone encoding the putative receptor.

As an alternative approach for receptor identification, labeled polypeptides can be photoaffinity-linked or extracted with a preparation expressing the receptor molecule, photoaffinity. The crosslinked material is separated by PAGE analysis and exposed to X-ray film. A labeled complex containing the receptor for the polypeptide can be excised,
It can be separated into peptide fragments and subjected to protein microsequencing. The amino acid sequence obtained from microsequencing is used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.

The invention further provides methods of screening compounds to identify those that modulate the action of the polypeptides of the invention. An example of such an assay involves combining mammalian fibroblasts, a polypeptide of the invention, the compound to be screened and ³ [H] thymidine under cell culture conditions in which the fibroblasts normally grow. . Control assays are performed in the absence of the compound to be screened to obtain, and compared to the amount of fibroblast proliferation in the presence of the compound, ³ in each case [H] thymidine incorporation of The determination can determine whether the compound stimulates proliferation. The amount of fibroblast proliferation is ³ [H
] Measured by liquid scintillation chromatography, which measures thymidine incorporation. Both agonist and antagonist compounds can be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the invention is incubated with a labeled polypeptide of the invention in the presence of the compound. The ability of the compound to enhance or block this interaction can then be measured. Alternatively, the response and receptor of a known second messenger system according to the interaction of the compound to be screened is measured and the ability of the compound to bind to the receptor and elicit a second messenger response is measured to Determine if it is a potential agonist or antagonist. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognostic markers. Molecules discovered using these assays can be used to treat or treat diseases by activating or inhibiting angiogenic polypeptides / molecules or to produce a particular result in a patient (eg, blood vessel growth). Can be used. In addition, the assay can discover factors that can inhibit or enhance the production of angiogenic polypeptides from properly engineered cells or tissues. Accordingly, the invention includes a method of identifying a compound that binds to an angiogenic polypeptide, comprising the steps of:
a) incubating the candidate binding compound with an angiogenic polypeptide;
And (b) determining whether binding has occurred. Further, the invention includes a method of identifying an agonist / antagonist comprising the steps of: (a) incubating a candidate compound with an angiogenic polypeptide, (b) assaying biological activity, And (b) determining whether the biological activity of the angiogenic polypeptide is modified.

Antisense and Ribozymes (Antagonists) In certain embodiments, the antagonists according to the invention are SEQ ID NOs: 1, 3,
A nucleic acid corresponding to the sequence contained in 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 or its complementary strand. In one embodiment, the antisense sequences are produced internally by the organism, and in another embodiment the antisense sequences are administered separately (eg, O'Connor, J. Neurochem.
56: 560 (1991)). Oligodeoxynucleo
tides as Antisense Inhibitors of Gen
e Expression, CRC Press, Boca Raton, FL
(1988). Antisense DNA or RN using antisense technology
Gene expression may be regulated through A or through the formation of triple helices. Antisense technology is described in, for example, Okano, J. et al. Neurochem. 56: 560
(1991); Oligodeoxynucleotides as Anti
sense Inhibitors of Gene Expression,
CRC Press, Boca Raton, FL (1988).
Triple helix formation is described, for example, in Lee et al., Nucleic Acids Rese.
arch 6: 3073 (1979); Cooney et al., Science, 24.
1: 456 (1988); and Dervan et al., Science, 251: 1.
300 (1991). These methods are based on the binding of polynucleotides to complementary DNA or RNA.

For example, the 5'coding portion of the polynucleotide encoding the mature polypeptide of the invention can be used to design an antisense RNA oligonucleotide of about 10-40 base pairs in length. DNA oligonucleotides are designed to be complementary to regions of the gene involved in transcription, thereby inhibiting transcription and production of receptors. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into a receptor polypeptide.

In one embodiment, the angiogenic antisense nucleic acids of the invention are produced intracellularly by transcription from a foreign sequence. For example, the vector or a portion thereof is transcribed to produce the antisense nucleic acid (RNA) of the invention. Such vectors include sequences encoding angiogenic antisense nucleic acids. Such a vector
It may remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. The vector can be a plasmid, virus, etc. known in the art used for replication and expression in vertebrate cells. Expression of sequences encoding angiogenic proteins or fragments thereof may be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include the SV40 early promoter region (Bernoist and Chamber, Nature, 29:30.
4-310 (1981)), a promoter contained in the 3'long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell, 22: 787-797 (1980).
)), The herpes thymidine promoter (Wagner et al., Proc. Natl.
Acad. Sci. U. S. A. 78: 1441-1445 (1981)), regulatory sequences of the metallothionein gene (Brinster et al., Nature, 296).
: 39-42 (1982)) and the like, but are not limited thereto.

The antisense nucleic acids of the invention include sequences complementary to at least a portion of the RNA transcript of the angiogenic gene. However, perfect complementarity, although preferred, is not necessary. A sequence “complementary to at least a portion of RNA” referred to herein means a sequence that has sufficient complementarity to hybridize with RNA and forms a stable duplex; Thus, in the case of double-stranded antisense nucleic acids that target RNA transcripts that encode angiogenic proteins, single strands of double-stranded DNA can be tested or triplex formation can be assayed. The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. Typically,
The longer the hybridizing nucleic acid, the more bases will mismatch with the angiogenic RNA, which may include stable duplexes (or possibly triplexes) and still form. One of skill in the art can ascertain the degree of mismatch tolerance by using standard procedures to determine the melting temperature of the hybridizing complex.

Oligonucleotides that are complementary to the 5'end of the message (eg, 5'untranslated sequences up to and including the AUG start codon) should work most efficiently in inhibiting translation. is there. However, sequences complementary to the 3'untranslated sequences of mRNAs have likewise been shown to be effective in inhibiting translation of mRNAs. Generally, Wagner, R .; , 1994, Nature 372: 333-33.
See 5. Thus, oligonucleotides complementary to either the 5'or 3'untranslated noncoding regions of the angiogenic proteins shown in Figures 3A-3C inhibit translation of the endogenous mRNA encoding the angiogenic protein. It can be used for antisense approaches. Oligonucleotides complementary to the 5'untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region, although less efficient inhibitors of translation, can be used in accordance with the present invention. Whether designed to hybridize to the 5'region, 3'region or coding region of angiogenic mRNA, the antisense nucleic acid should be at least 6 nucleotides in length, and preferably 6 Is an oligonucleotide ranging from about 50 nucleotides in length. In certain aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
It is a nucleotide.

The polynucleotides of the present invention may be DNA, or RNA, or chimeric mixtures, or derivative or modified versions thereof, single-stranded, or double-stranded. The oligonucleotide may be modified, for example, with a base moiety, sugar moiety, or phosphate backbone to improve molecular stability, hybridization, and the like.
This oligonucleotide can be attached to other additional groups such as peptides (eg, to target host cell receptors in vivo), or to factors that facilitate transport across cell membranes (eg, Letsinger et al., 1989, Proc. Natl.
． Acad. Sci. U. S. A. 86: 6553-6556; Lemaitr.
e et al., 1987, Proc. Natl. Acad. Sci. 84: 648-65
2; PCT Publication No. WO88 / 09810 (published December 15, 1988), or the blood-brain barrier (eg, PCT Publication No. WO89 / 10134).
(Published April 25, 1988)), hybridization-triggered cleaving agent.
agent) (eg, Krol et al., 1988, BioTechniques,
6: 958-976), or an intercalating agent (eg, Zo.
n, 1988, Pharm. Res. 5: 539-549). To this end, the oligonucleotide can be conjugated to another molecule, such as a peptide, hybridization-triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, and the like.

Antisense oligonucleotides may include at least one modified base moiety, which is selected from the group including, but not limited to: 5-fluorouracil, 5-bromouracil, 5 -Chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2
-Thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylcueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D- Mannosyl cuocosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2
-Methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v
), Wybutoxosine, pseudouracil, queocin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxy. Acetic acid (v), 5-methyl-2-thiouracil,
3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3)
w, and 2,6-diaminopurine.

Antisense oligonucleotides may also include at least one modified sugar moiety selected from the group including, but not limited to: arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to: phosphorothioate, phosphorodithioate, phosphoro. Amidothioates, phosphoramidates, phosphorodiamidates, methylphosphonates, alkyl phosphotriesters, and formacetals or analogs thereof.

In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. The a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA, whose strands run parallel to each other as opposed to the normal b-unit (Gautier et al., 1987, Nucl. Ac.
ids Res. , 15: 6625-6641). This oligonucleotide is
Is it a 2'-0-methyl ribonucleotide (Inoue et al., 1987, Nuc.
l. Acids Res. , 15: 6131-6148), or chimeric RNA.
-A DNA analog (Inoue et al., 1987, FEBS Lett. 21).
5: 327-330).

Polynucleotides of the invention may be prepared using standard methods known in the art (eg, automated D
NA synthesizer (such equipment is Biosearch, Applied Bios
commercially available from systems, etc.). For example, phosphorothioate oligonucleotides are described by Stein et al.
8, Nucl. Acids Res. 16: 3209) and methyl phosphonate oligonucleotides can be synthesized as controlled pore glass (contr).
Old porous glass polymer support (Sarin et al., 1988)
, Proc. Natl. Acad. Sci. U. S. A. 85: 7448-74
51) and the like.

On the other hand, antisense nucleotides complementary to the coding region sequences encoding angiogenic proteins may be used, with antisense nucleotides complementary to the transcribed, untranslated region being most preferred.

Potential antagonists according to the present invention also include catalytic RNA, ie ribozymes (eg International Application Publication No. WO published Oct. 4, 1990).
90/11364; Sarver et al., Science 247: 1222-12.
25 (1990)). Ribozymes that cleave mRNAs at site-specific recognition sequences can be used to destroy mRNAs encoding angiogenic proteins, although the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at positions dictated by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA has the following two base sequences: 5'-UG-3 '. The construction and production of hammerhead ribozymes is well known in the art, and Haselo
ff and Gerlach, Nature 334: 585-591 (1988).
) Is more fully described in. Angiogenic proteins (SEQ ID NOs: 1, 3, 5,
There are a number of potential hammerhead ribozyme cleavage sites in the nucleotide sequences encoding 7, 9, 11, 13, 15, 17, 17, 19, 21 and 23). Preferably, the ribozyme is such that the cleavage recognition site is located near the 5'end of the mRNA encoding the angiogenic protein; ie, increased efficiency and intracellular accumulation of non-functional mRNA transcripts. Are manipulated to minimize.

As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (eg, for improved stability, targeting, etc.) and in vivo angiogenic proteins. Should be delivered to cells expressing. The ribozyme-encoding DNA construct can be introduced into cells in a manner similar to that described above for the introduction of antisense-encoding DNA. A preferred method of delivery uses a DNA construct that "encodes" the ribozyme under the control of a strong constitutive promoter (eg, the pol III or pol II promoter), so that the transfected cells are angiogenic. It involves destroying the endogenous messages encoding proteins and producing sufficient amounts of ribozymes to interfere with their translation. Ribozymes, unlike antisense molecules, are catalytic, so lower intracellular concentrations are required for efficiency.

Antagonist / agonist compounds are utilized to grow and proliferate the polypeptides of the invention on neoplastic cells and tissues.
eration) effect may be hindered. That is, it stimulates tumor angiogenesis, thereby slowing or preventing aberrant cell growth and proliferation (eg, in tumor formation or proliferation).

Antagonists / agonists may also be utilized to prevent hypervascular disorders,
It also prevents the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the invention may also be required in cases such as restenosis after balloon angioplasty.

Antagonists / agonists may also be utilized to prevent the growth of scar tissue during wound healing.

Antagonists / agonists may also be utilized to treat the diseases described herein.

Other Activities The polypeptides of the invention are responsible for various disease states (eg, thrombosis, arteriosclerosis, and other cardiovascular conditions) as a result of their ability to stimulate vascular endothelial cell proliferation. Can be used in a procedure for stimulating the revascularization of ischemic tissue. These polypeptides may also be utilized to stimulate angiogenesis and limb remodeling as discussed above.

The polypeptides of the present invention may also be utilized in the treatment of wounds due to injury, burns, post-operative tissue repair, and scarring. Because they are mitogenic for a variety of cells of different origin (eg fibroblasts and skeletal muscle cells) and therefore have the ability to promote repair or replacement of damaged or diseased tissue. Is.

The polypeptides of the present invention may also be used to stimulate neuronal growth and to inhibit neuronal development in specific neuronal disorders or neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and AIDS-related complexes. Damage can be treated and prevented. Angiogenic proteins, as well as agonists and / or antagonists to angiogenic proteins, may have the ability to stimulate chondrocyte proliferation, thus enhancing bone and periodontal remodeling, and tissue graft or bone. Can be used for assistance in implants.

The polypeptides of the present invention may also be utilized to prevent aging of the skin due to sunburn by stimulating keratinocyte proliferation.

Angiogenic proteins, as well as agonists and / or antagonists to angiogenic proteins, can also be utilized to maintain pre-transplant organs or can be used to support cell culture of primordial tissue.

The polypeptides of the present invention may also be utilized to induce tissues of mesodermal origin for differentiation in early embryos.

Angiogenic proteins, as well as agonists and / or antagonists to angiogenic proteins, can also increase or decrease differentiation or proliferation of embryonic stem cells in addition to hematopoietic lineages, as discussed above.

Angiogenic proteins, as well as agonists and / or antagonists to angiogenic proteins, can also be used to characterize mammals (eg, height, weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation). , Size, and shape (eg, cosmetic surgery). Similarly, angiogenic proteins, as well as agonists and / or antagonists to angiogenic proteins, are used to regulate mammalian metabolism that affects catabolism, anabolic activity, processing, utilization, and energy storage. obtain.

Angiogenic proteins, as well as agonists and / or antagonists to angiogenic proteins, are found in biorhythms, caricadi.
c) Rhythm, depression (including depressive disorder), tendency to violence, resistance to pain, fertility (preferably by activin or inhibin-like activity), hormone or endocrine levels, appetite, libido, memory, stress , Or other effects on cognitive quality, and may be used to alter a mammal's mental or physical condition.

Angiogenic proteins, as well as agonists and / or antagonists to angiogenic proteins, can also, for example, store capacity, fat content, lipids, proteins, carbohydrates, vitamins, minerals, cofactors, or other nutritional components. It can be used as a food additive or preservative to increase or decrease.

The applications listed above are used in a wide variety of hosts. Such hosts include, but are not limited to, humans, mice, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, micro pigs, chickens, goats, cows. , Sheep, dogs, cats,
Non-human primates, and humans. In a particular embodiment, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In a preferred embodiment, the host is a mammal. In the most preferred embodiment, the host is human.

Therapeutic Uses The angiogenic proteins of the present invention are potent mitogens for vascular and lymphatic endothelial cells. Thus, angiogenic polypeptides or biologically active portions thereof may be used with agonists and / or antagonists to treat vascular trauma by promoting angiogenesis. For example, to stimulate the growth of transplanted tissue when coronary artery bypass surgery is performed. Angiogenic polypeptides for promoting wound healing, especially for revascularizing damaged tissue or for stimulating collateral blood flow during ischemia and when new capillary formation is desired. Alternatively, biologically active portions thereof may also be used with agonists and / or antagonists. Quite thick (full-thickn
ess) for treating wounds (eg, skin ulcers including pressure ulcers, venous ulcers, and diabetic ulcers), an angiogenic polypeptide or a biologically active portion thereof together with an agonist and / or antagonist, Can be used. further,
Angiogenic polypeptides or biologically active portions thereof may be used as agonists and / or
Alternatively, it can be used with an antagonist to repair fairly thick burns and injuries, where a skin graft or tissue flap is used to repair such burns and injuries. Angiogenic polypeptides or biologically active portions thereof may also be used with agonists and / or antagonists for use in plastic surgery (eg, for repair of lacerations, burns, or other trauma). obtain. Additionally, angiogenic polypeptides or biologically active portions thereof may be used with agonists and / or antagonists to promote healing of wounds and injuries to the eye and to treat ocular diseases.

Along these same systems, angiogenic polypeptides or biologically active portions thereof are also used with agonists and / or antagonists to repair damaged bone, periodontal or ligament tissue. It can induce proliferation. Angiogenic polypeptides or biologically active portions thereof, along with agonists and / or antagonists, also regenerate tooth supporting tissue (including cementum and periodontal ligament) damaged, for example, by periodontal disease or trauma. Can be used to

Since angiogenesis is important in maintaining the wound clean and free of infection, the angiogenic polypeptide or biologically active portion thereof, along with agonists and / or antagonists, may be used in conjunction with surgery. It can be used in association and after repair of incisions and cuts. Angiogenic polypeptides or biologically active portions thereof may also be used with agonists and / or antagonists to treat abdominal wounds at high risk of infection.

Angiogenic polypeptides or biologically active portions thereof, along with agonists and / or antagonists, may be endothelized in vascular transplant surgery.
It can be used to promote eliaization. In the case of vascular grafts using either transplant or synthetic materials, the angiogenic polypeptide or biologically active portion thereof may be combined with an agonist and / or antagonist,
It can be applied to the surface of the graft or at the junction to promote proliferation of vascular endothelial cells. The angiogenic polypeptides or biologically active portions thereof may also be used with agonists and / or antagonists to repair myocardial tissue damage resulting from myocardial infarction. Angiogenic polypeptides or biologically active portions thereof, also used with agonists and / or antagonists,
It can repair the cardiovascular system after ischemia. Angiogenic polypeptides or biologically active portions thereof, also used with agonists and / or antagonists, to treat vascular tissue damaged as a result of coronary artery disease and peripheral vascular disease and CNS vascular disease. obtain.

Angiogenic polypeptides or biologically active portions thereof, also used with agonists and / or antagonists, to coat artificial prostheses or natural organs to be implanted in the body to reject transplanted materials. Can be minimized and stimulate angiogenesis of the implanted material.

Angiogenic polypeptides or biologically active portions thereof are also used with agonists and / or antagonists, eg, for vascular tissue repair of injuries resulting from trauma occurring during atherosclerosis. And may be needed after balloon angioplasty where vascular tissue is damaged.

Angiogenic polypeptides or biologically active portions thereof may also be used with agonists and / or antagonists to treat peripheral arterial disease.
Thus, in a further aspect, there is provided a process for utilizing an angiogenic polypeptide or biologically active portion thereof together with an agonist to treat peripheral arterial disease. Preferably, the angiogenic polypeptide co-treated with the agonist is administered to an individual for the purpose of alleviating or treating peripheral arterial disease. Suitable doses, formulations and routes of administration are described below.

Angiogenic polypeptides or biologically active portions thereof may also be used with agonists and / or antagonists to induce endothelial function in lymphoid tissues and vessels (eg, loss of lymphatic vessels, blockage of lymphatic vessels). And to treat lymphangioma). Angiogenic polypeptides can also be used with agonists to stimulate lymphopoiesis.

Angiogenic polypeptides or biologically active portions thereof may also be used with agonists and / or antagonists to treat hemangiomas in newborns. Thus, in a further aspect there is provided a process for utilizing angiogenic polypeptides co-treated with an agonist and / or antagonist to treat hemangiomas in a neonate. Preferably, the angiogenic polypeptide co-treated with the agonist is administered to an individual to reduce or treat hemangiomas in the newborn. Suitable doses, formulations and routes of administration are described below.

Angiogenic polypeptides or biologically active portions thereof may also be used with agonists and / or antagonists to prevent or treat abnormal retinal development in immature neonates. Thus, in a further aspect, an angiogenic polypeptide is administered to treat abnormal retinal development in an immature neonate.
Provide a process for utilization with either agonists or antagonists. Preferably, the angiogenic polypeptide co-treated with an agonist or antagonist is administered to an individual to reduce or treat abnormal retinal development in immature neonates. Suitable doses, formulations and routes of administration are described below.

Angiogenic polypeptides or biologically active portions thereof are used with agonists and / or antagonists to treat primary (idiopathic) lymphedema, including Milroy's disease and early-onset lymphedema. obtain. Thus, in a further aspect, there is provided a process for using angiogenic polypeptides with agonists to treat primary (idiopathic) lymphedema, including Milroy's disease and early-onset lymphedema. Preferably, the angiogenic polypeptide co-treated with the agonist is administered to an individual to reduce or treat primary (idiopathic) lymphedema, including Milroy's disease and early-onset lymphedema. Angiogenic polypeptides can also be used with agonists to treat edema and affect blood pressure in animals. Suitable doses, formulations and routes of administration are described below.

Angiogenic polypeptides or biologically active portions thereof, also used in conjunction with agonists and / or antagonists, (I) removal of lymph nodes and vessels, (ii) radiation therapy in the treatment of cancer. And surgery, and (iii)
Secondary (closed) lifespans can be treated, including the lifetime resulting from trauma and infection. Thus, in a further aspect, (I) removal of lymph nodes and vessels, (ii) radiation therapy and surgery in the treatment of cancer, and (i
ii) Provide a process for utilizing angiogenic polypeptides with agonists to treat secondary (closed) life span, including life time resulting from trauma and infection. Preferably, the angiogenic polypeptide has (I
) Removal of lymph nodes and vessels, (ii) radiation therapy and surgery in the treatment of cancer, and (iii) lifetime resulting from trauma and infection
Due to secondary (closed) life span including Suitable doses, formulations and routes of administration are described below.

Angiogenic polypeptides or biologically active portions thereof may also be used with agonists and / or antagonists to treat Kaposi's sarcoma. Accordingly, in a further aspect, there is provided a process for utilizing an angiogenic polypeptide with an agonist to treat Kaposi's sarcoma. Preferably, the angiogenic polypeptide is administered to an individual to reduce or treat Kaposi's sarcoma. Suitable doses, formulations and routes of administration are described below.

Antagonists of angiogenic polypeptides can be used to treat cancer by inhibiting the angiogenesis necessary to support cancer and tumor growth.

Gene Therapy Methods Another aspect of the present invention relates to gene therapy methods for treating disorders, diseases, and conditions. This method of gene therapy uses nucleic acid (DNA, RNA, and antisense DNA or RNA) to achieve expression of the angiogenic polypeptides of the invention.
Introducing the sequence into an animal. This method requires a polynucleotide encoding an angiogenic polypeptide operably linked to a promoter and any other genetic element necessary for the expression of this polypeptide by the target tissue. Techniques for such gene therapy and delivery are known in the art, see, eg, WO 90/11092, incorporated herein by reference.

Thus, for example, a cell derived from a patient may have a polynucleotide (DNA or R that contains a promoter operably linked to an angiogenic polynucleotide ex vivo.
NA) and the engineered cells are then provided to the patient to be treated with the polypeptide. Such methods are well known in the art.
For example, Belldegrun, A .; Et al., J. Natl. Cancer Ins
t. , 85: 207-216 (1993); Ferrantini, M .; Et al., C
ancer Research, 53: 1107-1112 (1993); Fe
rrantini, M .; Et al., J. Immunology 153: 4604-4.
615 (1994); Kaido, T .; Et al., Int. J. Cancer 60:
221-229 (1995); Ogura, H .; Et al, Cancer Rease
rch 50: 5102-106 (1990); Santodonato, L.
． Et al., Human Gene Therapy 7: 1-10 (1996); S.
antodonato, L .; Et al., Gene Therapy 4: 1246-1.
255 (1997); and Zhang, J. et al. -F. Et al, Cancer Gen
e Therapy 3: 31-38 (1996). These documents are incorporated herein by reference. In one embodiment, the engineered cells are arterial cells. The arterial cells can be reintroduced into the patient by direct injection into the arteries, tissues surrounding the arteries, or by catheter injection.

As discussed in more detail below, angiogenic polynucleotide constructs can be delivered by any method that delivers an injectable substance to cells of an animal, such as tissue (heart, muscle, skin, lung, liver, etc. ) Injection into the interstitial space)). The angiogenic polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

[0409] In one embodiment, the angiogenic polynucleotide is delivered as a naked polynucleotide. The term "naked" polynucleotide, DNA or RNA refers to any delivery vehicle (that acts to assist, facilitate or facilitate entry into cells.
(Including viral sequences, viral particles, liposomal formulations, lipofectin, precipitants, etc.). However, angiogenic polynucleotides can also be delivered in liposomal formulations, and lipofectin formulations and the like can be prepared by methods well known to those of skill in the art. Such methods are described, for example, in US Pat. Nos. 5,593,972 and 5,589, incorporated herein by reference.
, 466 and 5,580,859.

The angiogenic polynucleotide vector construct used in the gene therapy method is preferably not integrated into the host genome and does not contain sequences that allow replication.
It is a construct. Suitable vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; Phar
pSVK3, pBPV, pMSG and pSVL available from Macia; and pEF1 / V5, pcDNA3.1 available from Invitrogen.
, And pRc / CMV2. Other suitable vectors will be readily apparent to those of skill in the art.

Any strong promoter known to those of skill in the art can be used to drive the expression of angiogenic DNA. Suitable promoters include adenovirus promoters (eg, adenovirus major late promoter); or heterologous promoters (eg, cytomegalovirus (CMV) promoter); RS virus (
RSV) promoter; inducible promoter (eg, MMT promoter, metallothionein promoter); heat shock promoter; albumin promoter; ApoAI promoter; human globin promoter; viral thymidine kinase promoter (eg, herpes simplex thymidine kinase promoter).
Retrovirus LTR; b-actin promoter; and human growth hormone promoter; The promoter can also be the native promoter for each gene encoding a given angiogenic protein.

Unlike other gene therapy techniques, one major advantage of introducing a naked nucleic acid sequence into a target cell is the transient nature of polynucleotide synthesis in that cell. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide for production of the desired polypeptide for a period of up to 6 months.

Angiogenic polynucleotide constructs are used in tissues in animals (muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, Includes intestines, testes, ovaries, uterus, rectum, nervous system, eyes, glands, and connective tissue)
Can be delivered to the interstitial space of the. This interstitial space is the space between the reticulated fibers of organ tissue,
Liquid mucopolysaccharide matrix between cells, elastic fibers in the walls of blood vessels or chambers, collagen fibers of fibrotic tissue, or connective tissue sheath muscle cells (connective cells).
Includes the same matrix within the issue enheasing muscle cell) or in the lacunae of the bone. This is likewise the space occupied by circulating plasma and lymphatic fluid of the lymphatic channels. Delivery of muscle tissue to the interstitial space
Preferred for reasons discussed below. The polynucleotide construct of the present invention comprises
It can be conveniently delivered by injection into the tissue containing these cells. The polynucleotide constructs of the present invention are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, which delivery and expression may be in undifferentiated cells or cells that are not fully differentiated ( (Eg, blood stem cells or dermal fibroblasts). Muscle cells in vivo take up a polynucleotide,
And it is particularly qualified in its ability to develop.

For naked nucleic acid sequence injections, an effective dosage of DNA or RNA is about 0.05.
It ranges from mg / kg body weight to about 50 mg / kg body weight. Preferably, this dosage is about 0.005 mg / kg to about 20 mg / kg, and more preferably about 0.05 mg / kg to about 5 mg / kg. Of course, as the skilled artisan will appreciate, this dosage will vary depending on the tissue site of injection. Suitable and effective dosages of nucleic acid sequences can be readily determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. However, other parenteral routes may also be used, including, for example, inhalation of aerosol formulations, especially for delivery to lung or bronchial tissues, throat or mucous membranes of the nose, among others. In addition, the naked angiogenic DNA construct can be delivered to the arteries during angioplasty by the catheter used in this procedure.

Naked polynucleotides can be any method known in the art including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter injection, and so-called “gene gun”. Delivered by. These methods of delivery are known in the art.

The naked VEGF-2 nucleic acid sequence can be administered in vivo and has been shown to produce successful expression of VEGF-2 polypeptide in the femoral artery of rabbits.

The constructs can also be delivered using delivery vehicles such as viral sequences, viral particles, liposomal formulations, lipofectin, precipitants and the like. Such delivery methods are known in the art.

[0419] In certain embodiments, the angiogenic polynucleotide construct is complexed in a liposome preparation. Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic liposomes are particularly preferred because they can form a robust charge complex between the cationic liposomes and the polyanionic nucleic acid. Cationic liposomes, in functional form, are plasmid DNA (Felgner et al., Proc. Natl. Acad. S, incorporated herein by reference).
ci. USA (1987) 84: 7413-7416); mRNA (Malone et al., Proc. Natl. Acad. S, incorporated herein by reference).
ci. USA (1989) 86: 6077-6081); and purified transcription factors (Debs et al., J. Biol. Che, incorporated herein by reference).
m. (1990) 265: 10189-10192) have been shown to mediate intracellular delivery.

Cationic liposomes are readily available. For example, N [1-2,3-dioleyloxy) propyl] -N, N, N-triethylammonium (DOTM
A) Liposomes are particularly useful, and are available under the trademark Lipofectin® from GIBCO BRL, Grand Island, N.F. Y. (Felgner et al., Proc. Natl. Acad. Sc, incorporated herein by reference.
i. USA (1987) 84: 7413-7416). Other commercially available liposomes include transfectase (tra
nsfectace) (DDAB / DOPE) and DOTAP / DOPE (B
oehringer).

Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. DOTAP (1,2-bis (oleoyloxy)-
For a description of the synthesis of 3- (trimethylammonio) propane) liposomes, see, eg, PCT Publication No. WO 90/11092, which is incorporated herein by reference. The preparation of DOTMA liposomes is described in the literature and is described, for example, in P. et al. Felgner et al., Proc.
Natl. Acad. Sci. USA, 84: 7413-7417. Similar methods can be used to prepare liposomes from other cationic lipid substances.

Similarly, anionic and neutral liposomes can be prepared, for example, by Avant.
It is readily available from i Polar Lipids (Birmingham, Ala.) or can be easily prepared using readily available materials.
Such substances include, among others, phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC).
), Dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE). These substances also
It can be mixed with DOTMA starting material and DOTAP starting material in suitable proportions. Methods of using these materials to form liposomes are well known in the art.

For example, commercially, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) were used in various combinations with the addition of cholesterol. Even without, conventional liposomes can be produced. Therefore,
For example, DOPG / DOPC vesicles can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas into a sonicated vial. The sample is placed under vacuum pump overnight and hydrated the next day with deionized water. This sample was then placed in a capped vial using a Heat Systems Model 350 sonicator equipped with an inverted cup (bath type) probe at maximum setting while the bath was circulating at 15 EC. Sonicate for 2 hours. Alternatively, negatively charged vesicles can be prepared without sonication to generate multilamellar vesicles or extruded through nucleopore membranes to separate unilamellar vesicles of different sizes. Can be generated. Other methods are known and available to those of skill in the art.

As the liposome, there are multilamellar vesicles (MLV), small unilamellar vesicles (SUV).
Alternatively, large unilamellar vesicles (LUVs) may be mentioned, SUVs being preferred. Various liposome-nucleic acid complexes are prepared using methods well known in the art. For example,
Straubinger et al., Method, incorporated herein by reference.
Sof Immunology (1983), 101: 512-527. For example, MLVs containing nucleic acids can be prepared by depositing a thin film of phospholipids on the walls of glass tubes and then hydrating with a solution of the substance to be encapsulated. SUVs are prepared by long-term sonication of MLVs, producing a homogeneous population of unilamellar vesicles. Pre-formed M to be encapsulated
Add to suspension of LV, then sonicate. When using liposomes containing cationic lipids, dry lipid membranes should be treated with sterile water or 10 mM Tris / N.
Resuspend in a suitable solution, such as an isotonic buffered solution such as aCl, sonicate, then mix the preformed liposomes directly with the DNA. Due to the binding of the positively charged liposomes to the cationic DNA, the liposomes and DNA form a very stable complex. SUVs find use with small nucleic acid fragments. LUVs are prepared by many methods well known in the art. A commonly used method is Ca ²⁺ -EDTA chelation (Papahadjopou).
los et al., Biochim. Biophys. Acta (1975) 394: 4.
83; Wilson et al., Cell (1979)) 17:77; ether injection (D.
eamer, D.E. And Bangham, A .; , Biochim. Biophy
s. Acta (1976) 443: 629; Ostro et al., Biochem. B
iophys. Res. Commum. (1977) 76: 836; Fulle.
y et al., Proc. Natl. Acad. Sci. USA (1979) 76:33.
48); detergent dialysis (Enoch, H. and Strittmatter,
P. , Proc. Natl. Acad. Sci. USA (1979) 76:14.
5); and reverse phase evaporation (REV) (Fraley et al., J. Biol.
． Chem. (1980) 255: 10431; Szoka, F .; And Pap
ahadjopuulos, D.A. , Proc. Natl. Acad. Sci. U
SA (1978) 75: 145; Schaefer-Ridder et al., Scie.
No. (1982) 215: 166), which are incorporated herein by reference.

Generally, the ratio of DNA to liposomes is from about 10: 1 to about 1:10. Preferably, the ratio is about 5: 1 to about 1: 5. More preferably, the ratio is from about 3: 1 to about 1: 3. Even more preferably, the ratio is about 1: 1.

US Pat. No. 5,676,954 (incorporated herein by reference) reports the injection of genetic material complexed with cationic liposome carriers into mice. U.S. Pat. Nos. 4,897,355, 4,946,787, 5,049,386, 5,459,127, 5,589,466.
No. 5,693,622, No. 5,580,859, No. 5,703.
055 and WO 94/9469, which are incorporated herein by reference, provide cationic lipids for use in transfecting DNA into cells and mammals. U.S. Pat. No. 5,589,466,
No. 5,693,622, No. 5,580,859, No. 5,703,05
No. 5 and WO 94/9469, which are incorporated herein by reference, provide methods of delivering DNA-cationic lipid complexes to mammals.

In a particular embodiment, an RNA comprising a sequence encoding an angiogenic protein
Are used to engineer cells ex vivo or in vivo. The retrovirus capable of inducing a retrovirus plasmid vector includes Moloney murine leukemia virus, splenic necrosis virus, Rous sarcoma virus, Harvey sarcoma virus, chicken leukemia virus, gibbon ape leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and Breast cancer viruses are included, but are not limited to.

This retroviral plasmid vector is used to transduce packaging cell lines to form producer cell lines. Examples of packaging cells that can be transfected are incorporated herein by reference in their entirety,
Miller, Human Gene Therapy, 1: 5-14 (1990)
) PE501, PA317, R-2, R-AM, PA12 as described in
, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP + E
-86, GP + envAm12 and DAN cell lines, but are not limited thereto. The vector can transduce the packaging cells by any means known in the art. Such means, electroporation, the use of liposomes, and CaPO ₄ precipitation include, but are not limited to. In one alternative, the retroviral plasmid vector can be encapsulated in liposomes or can be lipid bound and then administered to a host.

This producer cell line produces infectious retroviral vector particles containing a polynucleotide encoding an angiogenic protein. Such retroviral vector particles can then be used to transduce eukaryotic cells either in vitro or in vivo. The transduced eukaryotic cells express angiogenic proteins.

In certain other embodiments, angiogenic polynucleotides contained in adenovirus vectors are used to engineer cells ex vivo or in vivo. Adenovirus can be engineered such that it encodes and expresses angiogenic proteins and at the same time is inactivated with respect to its ability to replicate in the normal lytic viral life cycle. Adenovirus expression is achieved without integration of the viral DNA into the host cell chromosome, resulting in less worry about insertional mutagenesis. In addition, adenovirus has been used for many years as a live intestinal vaccine with excellent safety features (Schwartzet, AR et al., (
1974) Am. Rev. Respir. Dis. , 109: 233-238).
. Finally, adenovirus-mediated gene transfer leads to α-1 in the lungs of cotton rats.
-Demonstrated in a number of examples including the transfer of antitrypsin and CFTR (Rosenfeld, MA et al., (1991) Science, 252:
431-434; Rosenfeld et al., (1992) Cell 68: 143.
~ 155). Moreover, extensive research seeking to establish adenovirus as a causative agent in human cancers was uniformly negative (Green, M. et al. (1979).
) Proc. Natl. Acad. Sci. USA, 76: 6606).

Suitable adenovirus vectors useful in the present invention are, for example, Koz.
arsky and Wilson, Curr. Opin. Genet. Devel
． 3: 499-503 (1993); Rosenfeld et al., Cell, 68.
: 143-155 (1992); Engelhardt et al., Human Gen.
et. Ther. 4: 759-769 (1993); Yang et al., Nature.
e Genet, 7: 362-369 (1994); Wilson et al., Natu.
Re, 365: 691-692 (1993); and US Pat. No. 5,652,2.
24, which are hereby incorporated by reference. For example, the adenovirus vector Ad2 is useful and can be propagated in human 293 cells. These cells contain the adenovirus E1 region and constitutively express Ela and Elb, which complement the defective adenovirus by providing the product of the gene deleted from this vector. . In addition to Ad2, a variety of other adenoviruses (eg, Ad3, Ad5, and Ad7) are also useful in the present invention.

[0432] Preferably, the adenovirus used in the present invention is replication defective. Replication-defective adenoviruses require the help of helper viruses and / or packaging cell lines to form infectious particles. The resulting virus is capable of infecting cells and contains a promoter (eg, HARP of the invention).
It is capable of expressing the polynucleotide of interest operably linked to a promoter) but is incapable of replicating in most cells. Replication-defective adenoviruses have the following genes:
It may be deleted at one or more of E1a, E1b, E3, E4, E2a or all or part of L1 to L5.

In certain other embodiments, adeno-associated virus (AAV) is used to engineer the cells ex vivo or in vivo. AAV is a naturally occurring defective virus that requires a helper virus to produce infectious particles (
Muzyczka, N .; Curr. Topics in Microbiol
． Immunol. , 158: 97 (1992)). AAV is also one of the few viruses that can integrate its DNA into cells that are not dividing.
Vectors containing AAV as small as 300 base pairs can be packaged and can integrate, but space for exogenous DNA is limited to about 4.5 kb. Methods of generating and using such AAV are known in the art. For example, US Pat. Nos. 5,139,941, 5,173,414 and 5,354,67.
No. 8, No. 5,436, 146, No. 5,474, 935, No. 5, 478.
, 745 and 5,589,377.

For example, an AAV vector suitable for use in the present invention contains all the sequences necessary for DNA replication, encapsidation, and host cell integration. Sam
Brook et al., Molecular Cloning: A Laborator.
y Manual, Cold Spring Harbor Press (19
The angiogenic polynucleotide construct is inserted into this AAV vector using standard cloning methods, such as those found in 89). The recombinant AAV vector is then transfected into packaging cells infected with helper virus using any standard technique, including lipofection, electroporation, calcium phosphate precipitation and the like. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus, or herpes virus. Once the packaging cells are transfected and infected, they produce infectious AAV viral particles that contain the angiogenic polynucleotide construct. These viral particles are then used to transduce eukaryotic cells, either ex vivo or in vivo.
The transduced cell contains an angiogenic polynucleotide construct integrated into its genome and expresses an angiogenic protein.

Another method of gene therapy is homologous recombination (see, eg, US Pat. No. 5,641,6).
70, published June 24, 1997; International Publication No. WO 96/29411, 19
Published September 26, 1996; International Publication No. WO94 / 12650, August 4, 1994
Published: Koller et al., Proc. Natl. Acad. Sci. USA, 8
6: 8932-8935 (1989); and Zijlstra et al., Nature.
342: 435-438 (1989)), operably linking the heterologous regulatory region and the endogenous polynucleotide sequence (eg, a sequence encoding an angiogenic protein). Including. The method involves activation of a gene that is present in the target cell but is normally not expressed in that cell or is expressed at a lower level than desired.

Polynucleotide constructs are made using standard techniques known in the art. This construct contains a promoter with targeting sequences flanking the promoter. Suitable promoters are described herein. The targeting sequence is sufficiently complementary to the endogenous sequence to allow homologous recombination between the promoter-targeting sequence and the endogenous sequence. The targeting sequence is located sufficiently close to the 5'end of the desired endogenous polynucleotide sequence of the angiogenic protein of the invention so that upon homologous recombination, the promoter will render the endogenous sequence operable. Be connected.

The promoter and targeting sequence can be amplified using PCR. Preferably, the amplified promoter contains additional restriction enzyme sites at the 5'and 3'ends. Preferably, the 3'end of the first targeting sequence contains the same restriction enzyme sites as the 5'end of the amplified promoter, and the 5'end of the second targeting sequence contains the 3'end of the amplified promoter. Includes the same restriction sites as the termini. The amplified promoter and targeting sequences are digested and ligated together.

Either as a naked polynucleotide or with a transfection facilitating agent such as liposomes, viral sequences, viral particles, whole viruses, lipofections, precipitants, etc. as described in more detail above. And deliver the promoter-targeting sequence construct to cells. The P promoter-targeting sequence may be delivered by any method, including direct needle injection, intravenous injection, topical administration, catheter infusion, particle accelerators and the like. This method is described in more detail below.

The promoter-targeting sequence construct is taken up by cells. Homologous recombination occurs between this construct and the endogenous sequence, such that the endogenous sequence encoding the angiogenic protein is placed under the control of this promoter. This promoter then drives the expression of the endogenous angiogenic sequence.

Polynucleotides encoding angiogenic proteins of the invention can be administered with other polynucleotides that encode other angiogenic proteins.

Preferably, the polynucleotide encoding the angiogenic polypeptide of the present invention comprises a secretory signal sequence that facilitates secretion of the protein. Typically, the signal sequence is located in the coding region of the polynucleotide, expressed towards or at the 5'end of the coding region of the polynucleotide. The signal sequence may be homologous or heterologous to the polynucleotide of interest and homologous or heterologous to the transfected cells. In addition, the signal sequence can be chemically synthesized using methods known in the art.

Any mode of administration of any of the polynucleotide constructs described above may be used, so long as the mode of administration expresses one or more molecules in an amount sufficient to provide a therapeutic effect. This includes direct needle injection, systemic injection, catheter injection, biolistic injector, particle accelerator (ie "gene gun"), gel foam sponge depot, other commercially available depot materials, osmotic pressure. Pumps (eg Alza minipumps), solids for oral or suppository (
Tablets or pills) pharmaceutical formulations, and intra-operative decanting or topical applications. For example, direct injection of calcium phosphate-precipitated naked plasmid into rat liver and rat spleen or direct injection of protein-coated plasmid into the portal vein resulted in gene expression of foreign genes in rat liver (Kaneda).
Et al., Science, 243: 375 (1989)).

The preferred method of local administration is by direct injection. Preferably, the recombinant molecule of the present invention complexed with the delivery vehicle is administered by direct injection into the arterial region or locally within the arterial region. Topical administration of the composition within the arterial region refers to injection of the composition into the artery several centimeters, and preferably several millimeters.

Another method of topical administration is to contact the polynucleotide constructs of the invention within or around a surgical wound. For example, a patient can undergo surgery and the polynucleotide construct can be coated on the tissue surface inside the wound or the construct can be injected into the tissue area inside the wound.

Therapeutic compositions useful for systemic administration include a recombinant molecule of the present invention complexed with a targeted delivery vehicle of the present invention. Suitable delivery vehicles for use in systemic administration include liposomes containing a ligand for targeting the vehicle to a particular site.

Preferred methods of systemic administration include intravenous injection, aerosol, oral and transdermal (topical) delivery. Intravenous injections may be performed using methods standard in the art. Aerosol delivery may also be performed using methods standard in the art (eg, Stribling, incorporated herein by reference).
Et al., Proc. Natl. Acad. Sci. USA, 189: 11277-1.
1281 (1992)). Oral delivery can be accomplished by complexing the polynucleotide constructs of the present invention to a carrier capable of withstanding degradation by digestive enzymes in the intestine of the animal. Examples of such carriers include:
Mention may be made of plastic capsules or tablets, such as those known in the art. Topical delivery may be accomplished by mixing a lipophilic reagent (eg DMSO) that is capable of passing into the skin with a polynucleotide construct of the present invention.

Determining the effective amount of a substance to be delivered can be determined, for example, by the chemical structure and biological activity of the substance, the age and weight of the animal, the exact condition requiring treatment and its severity, and administration. It may depend on a number of factors, including the pathway. The frequency of treatment is
It depends on many factors such as the amount of polynucleotide construct administered per dose and the health and medical history of the subject. The exact amount, frequency of dosing and timing of dosing will be determined by the attending physician or veterinarian.

The therapeutic compositions of the present invention may be administered to any animal, preferably mammals and birds. Preferred mammals include humans, dogs, cats, mice, rats,
Rabbits, sheep, cows, horses and pigs are mentioned, with humans being particularly preferred.

Pharmaceutical Compositions Angiogenic polypeptides, polynucleotides, and agonists and antagonists can be used in combination with a suitable pharmaceutical carrier. Such compositions comprise a therapeutically effective amount of the polypeptide or agonist or antagonist and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The present invention also provides a pharmaceutical pack or kit comprising one or more containers containing one or more ingredients of the pharmaceutical compositions of the invention. Such containers may be accompanied by a notice in the form prescribed by a governmental agency that oversees the manufacture, use or sale of pharmaceuticals or biological products. This notice states that manufacturing, use,
And reflects the approval of the marketing authority. In addition, the pharmaceutical composition can be used with other therapeutic compounds.

The pharmaceutical composition may be administered in a convenient fashion (eg, topical, intravenous, intraperitoneal,
Can be administered intramuscularly, intratumorally, subcutaneously, intranasally, or intradermally). The pharmaceutical composition is administered in an amount that is effective for the treatment and / or prevention of specific symptoms. Generally, the pharmaceutical composition is administered in an amount of at least about 10 mg / kg body weight, and in most cases, no more than about 8 gm / Kg body weight per day. In most cases, this dosage will be from about 10 mg / kg body weight to about 1 mg / kg body weight daily, taking into account the route of administration, symptoms, etc.

Angiogenic polypeptides, as well as polypeptides, agonists or antagonists can also be used in accordance with the present invention by in vivo expression of such polypeptides. This is often referred to as "gene therapy", as described above.

Thus, for example, a cell (eg, bone marrow cell) can be engineered ex vivo with a polynucleotide (DNA or RNA) encoding the polypeptide,
The engineered cells are then provided to the patient treated with the polypeptide. Such methods are well known in the art. For example, cells can be engineered by procedures known in the art by the use of retroviral particles containing RNA encoding the polypeptides of the present invention.

Similarly, cells may be engineered in vivo for expression of the polypeptide in vivo, eg, by procedures known in the art. As is known in the art, producer cells for producing retroviral particles containing RNA encoding a polypeptide of the invention may be used to engineer cells in vivo and for expression of the polypeptide in vivo. Can be administered to. These and other methods for administering the polypeptides of the present invention by such methods should be apparent to those of ordinary skill in the art in view of the teachings of the present invention. For example, the expression vehicle for the engineered cell can be other than a retroviral particle, such as an adenovirus, which can be used to engineer the cell in vivo after combination with a suitable delivery vehicle.

The retroviruses from which the retroviral plasmid vectors referred to herein above may include, but are not limited to: Moloney murine leukemia virus, splenic necrosis virus, retroviruses (eg, Rous sarcoma virus). , Hervey Sarcoma Virus, Avian Leukemia Virus, Gibbon Leukemia Virus, Human Immunodeficiency Virus), Adenovirus, Myeloproliferative Sarcoma Virus, and Mammalian Tumor Virus. In one embodiment, the retroviral plasmid vector is derived from Moloney murine leukemia virus.

Vectors of the invention include one or more promoters. Suitable promoters that may be used include, but are not limited to: Retroviral LTR:
SV40 promoter: and human cytomegalovirus (CMV) promoter (Miller et al., Biotechniques 7: 980-990 (19).
89)), or any other promoter (eg cellular promoter such as eukaryotic cell promoter (histone promoter, p.
ol III promoter, and b-actin promoter). Other viral promoters that can be used include, but are not limited to: adenovirus promoter, thymidine kinase (T
K) promoter, and B19 parvovirus promoter. Selection of the appropriate promoter will be apparent to those of skill in the art from the teachings contained herein.

The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters that can be used include, but are not limited to, adenovirus promoters (eg, adenovirus major late promoter); or heterologous promoters (eg, cytomegalovirus (CMV)).
) Promoter); RS virus (RSV) promoter; inducible promoter (for example, MMT promoter); metallothionein promoter; heat shock promoter; albumin promoter; ApoAI promoter; human globin promoter; viral thymidine kinase promoter (for example, herpes simplex thymidine kinase promoter) ); Retrovirus LTRs (including modified retrovirus LTRs herein above); b-actin promoter; and human growth hormone promoter. The promoter can also be the native promoter that controls the gene encoding the polypeptide of the invention.

Retroviral plasmid vectors are used to transduce packaging cell lines to form producer cell lines. Examples of packaging cells that can be transfected include, but are not limited to: Mille
R., et al., Human Gene Therapy 1: 5-14 (1990), which is incorporated herein by reference in its entirety.
E501, PA317, y-2, y-AM, PA12, T19-14X, VT-
19-17-H2, yCRE, yCRIP, GP + E-86, GP + envAm
12, and the DAN cell line. The vector may transduce the packaging cell line through any means known in the art. Such means, electroporation, the use of liposomes, and are included CaPO ₄ precipitated without limitation. As one alternative, the retroviral plasmid vector can be encapsulated in liposomes or associated with lipids and then administered to the host.

The producer cell line produces an infectious retroviral vector containing a nucleic acid sequence encoding a polypeptide. Such retroviral vector particles can then be used to transduce eukaryotic cells either in vitro or in vivo. Transduced eukaryotic cells express a nucleic acid sequence encoding a polypeptide. Eukaryotic cells that can be transduced include, but are not limited to, embryonic stem cells, embryonal cancer cells, as well as hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts, keratinocytes, endothelial cells, And bronchial epithelial cells.

Diagnostic Assays The invention also encodes angiogenic proteins as part of a diagnostic assay for detecting a disease or susceptibility to a disease associated with the presence of a mutation in a nucleic acid sequence encoding an angiogenic protein. The use of the gene to do.

Individuals with mutations in the gene encoding the angiogenic protein can be detected at the DNA level by a variety of techniques. Nucleic acid for diagnosis is
For example, blood), urine, saliva, tissue biopsy, and autopsy material. Genomic DNA can be used directly for detection, or PCR (Sai) prior to analysis.
ki et al., Nature 324: 163-166 (1986)). RNA or cDNA may also be used for the same purpose. As an example, PC is complementary to a nucleic acid encoding an angiogenic protein
The R primer can be used to identify and analyze the mutation. For example,
Deletions and insertions can be detected by a change in the size of the amplification product compared to the normal genotype. Point mutations allow the amplified DNA to hybridize with radiolabeled RNA encoding angiogenic proteins, or, alternatively, radiolabeled antisense DNA sequences specific for RNA encoding angiogenic proteins. Can be identified by Perfectly matched sequences can be distinguished from unmatched duplexes by differences in RNase A digestion or melting temperatures.

Genetic testing based on differences in DNA sequence was performed in the presence or absence of denaturing agents,
This can be accomplished by detection of electrophoretic mobility changes of the DNA fragment in the gel. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on a denaturing formamide gradient gel. Here, the mobilities of different DNA fragments are delayed at different positions in the gel according to their specific and partial melting temperatures (eg Myers et al., Science 230: 1242 (1985)).
checking).

Sequence changes at specific positions can also be associated with nuclease protection assays (eg, RN
ase and S1 protection) or chemical cleavage methods (eg Co
tton et al., PNAS, USA 85: 4397-4401 (1985)).

Thus, the detection of specific DNA sequences may include hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes (eg, restriction fragment length polymorphism (RFLP)), and genomic DNA. It can be achieved by methods such as Southern blotting.

In addition to the more conventional gel electrophoresis and DNA sequencing, mutagenesis can also be detected by in situ analysis.

The present invention also relates to diagnostic assays for detecting altered levels of angiogenic proteins in various tissues. This is because overexpression of the protein compared to normal control tissue samples can detect the presence or susceptibility to a disease (eg, abnormal cell proliferation). Assays used to detect levels of angiogenic proteins in a sample from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, western blot analysis, ELISA assays, and A "sandwich" assay is included. ELISA assay (Coligan et al., Current Pro
tocols in Immunology 1 (2), Chapter 6, (1991)
) Initially comprises the step of preparing an antibody (preferably a monoclonal antibody) specific for the angiogenic protein antigen. In addition, a reporter antibody is prepared against the monoclonal antibody. A detectable reagent (eg, radioactivity, fluorescence, or horseradish peroxidase enzyme in this case) is attached to the reporter antibody. The sample is removed from the host and incubated on a solid support (eg, polystyrene dish) that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubation with a non-specific protein (eg bovine serum albumin). The monoclonal antibody is then incubated in the dish for the time that the monoclonal antibody binds to any angiogenic protein bound to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. A reporter antibody conjugated to horseradish peroxidase is placed in the dish, which results in the binding of the reporter antibody to the monoclonal antibody bound to the angiogenic protein. Unbound reporter antibody is then washed away. The peroxidase substrate is then added to the dish and the amount of color developed over a period of time is the amount of angiogenic protein present in a given volume of patient sample when compared against a standard curve. Is a measure of.

Competitive assays may be utilized in which an antibody specific for angiogenic protein is attached to a solid support. A polypeptide of the invention is then labeled, eg, by radioactivity, and a sample from the host is passed through a solid support, and the amount of label detected (eg, detected by liquid scintillation chromatography) is , Can be correlated with the amount of angiogenic protein in the sample.

The “sandwich” assay is similar to the ELISA assay. In the "sandwich" assay, angiogenic proteins pass through the solid support and bind to the antibody bound to the solid support. The second antibody then binds to the angiogenic protein. A third antibody, labeled and specific for the second antibody, then passes through the solid support and binds to the second antibody, and the amount can then be quantified.

Polynucleotide Identification The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
Moreover, there is a current need to identify specific sites on the chromosome. Chromosome marking reagents based on actual sequence data (data for repeat polymorphisms) are currently scarcely available for marking chromosomal locations. The mapping of DNA to chromosomes according to the present invention is an important first step in correlating disease-associated genes with their sequences.

Briefly, the sequences are PCR primers derived from cDNA (preferably 15-2
5 bp) can be mapped to the chromosome. Computer analysis of the cDNA is used to rapidly select primers that do not span more than one exon in genomic DNA, thereby complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only hybrids containing the human gene corresponding to the primer will produce an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning specific DNA to specific chromosomes. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved in a similar fashion with panels of fragments from specific chromosomes or pools of large genomic clones. Other mapping strategies that may also be used to map to that chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes, and construction of chromosome-specific cDNA libraries. Preselection by hybridization to

Fluorescence in situ hybridization (“FISH”) of cDNA clones to metaphase chromosome spreads can be used to provide the correct chromosomal location in one step. This technique can be used with probes derived from cDNA as short as 50 or 60 bp. For a review of this technology, see Verma et al., Human.
Chromosomes: A Manual Of Basic Techni
ques, Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, the physical position of that sequence on the chromosome can be correlated to genetic map data. Such data can be found, for example, in V.I. McKusick, Mendelian Inheritance
In Man (Johns Hopkins University, Welc
h Medical Library) (available online from Medical Library). The relationship between genes and diseases that map to the same chromosomal region is then identified via linkage analysis (co-inheritance of physically adjacent genes).

[0474] It is then necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If the mutation is observed in some or all of the affected individuals but not in normal individuals, the mutation may be the causative agent of the disease.

Using the current resolution of physical and genetic mapping techniques,
A cDNA correctly located in a chromosomal region associated with disease can be one of between 50 and 500 potential causative genes. (This assumes a mapping resolution of 1 megabase and one gene per 20 kb.) Comparisons of affected and unaffected individuals generally show structural alterations in chromosomes (eg, deletions). Or translocation), the alteration being visible from the chromosomal spread or detectable using PCR based on its cDNA sequence. Finally, complete sequencing of genes from some individuals is needed to confirm the presence of mutations and distinguish mutations from polymorphisms.

Having generally described the invention, the invention will be more readily understood by reference to the following examples. This example is provided for purposes of illustration and not intended to be limiting.

Examples Example 1: In Vitro Expression of Angiogenic Proteins Example 1A: Bacterial Expression and Purification of Angiogenic Proteins DNA sequences encoding angiogenic proteins were processed. Amplification was carried out using PCR oligonucleotide primers corresponding to the 5'sequence of the protein (lacking the signal peptide sequence) and the vector 3'sequence for this gene.
Additional nucleotides corresponding to this gene are added to the 5'and 3'sequences, respectively. This PCR product can be cloned into pQE60 (Qiag
en, Inc. Chatsworth, CA 91311).

The restriction enzyme site corresponds to the restriction enzyme site in the bacterial expression vector pQE60. pQE-60 is antibiotic resistant (Amp ^r ), bacterial origin of replication (ori)
, An IPTG regulatable promoter (P / O), a ribosome binding site (RBS),
It encodes a 6-His tag and a restriction enzyme site. Next, change qQE60 to Nco
Digested with I and BglII. The amplified sequence was ligated into pQE60,
It is then inserted in frame with the sequences encoding the histidine tag and the ribosome binding site (RBS). This ligation mixture was then used to
mbrook, J.M. Et al., Molecular Cloning: A Labor.
atory Manual, Cold Spring Laboratory
Press, (1989), by the procedure described in E. coli available from Qiagen. The E. coli chain M15 / rep4 was transformed. M15 / rep4 contains the replication of multiple plasmids pREP4, which expresses the lacI repressor and also confers kanamycin resistance (Kan ^r ). Transformants were identified by their ability to grow on LB plates and ampicillin / kanamycin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired construct were cloned into Amp (100 μg /
ml) and Kan (25 μg / ml) in LB medium liquid medium supplemented overnight (O / N). Using this O / N culture, 1: 100 to 1: 2
Inoculate a large culture at a ratio of 50. The cells are grown to an absorbance 600 (OD ⁶⁰⁰ ) between 0.4 and 0.6. IPTG (“Isopropyl-BD-thiogalactopyranoside”) is then added to a final concentration of 1 mM. IPT
G induces by clearing P / O by inactivating the lacI repressor, leading to increased gene expression. Allow cells to grow for another 3-4 hours.
The cells were then harvested by centrifugation. The cell pellet is dissolved in the chaotropic agent 6M guanidine HCl. After clarification, the solubilized angiogenic protein is purified from this solution by chromatography on a nickel chelate column under conditions that allow it to be tightly bound by a protein containing a 6-His tag (H
ochuli, E .; Et al., J. Chromatography 411: 177-
184 (1984)). This protein was added to 6M guanidine HCl pH 5.0.
Eluted from the column in, and for regeneration purposes, 3 molar concentrations of guanidine HC
1, 100 mM sodium phosphate, 10 mM glutathione (reduced) and 2 mM glutathione (oxidized). After 12 hours of incubation in this solution, the protein is dialyzed against 10 millimolar sodium phosphate.

In addition to the above vector, the present invention further provides an expression vector (referred to as pHE4a) (referred to as pHE4a) that includes a phage promoter and promoter elements operably linked to a polynucleotide encoding an angiogenic polypeptide. No. 209645, deposited on February 25, 1998). This vector
Includes: 1) Neomycin phosphotransferase gene as a selectable marker, 2) E. coli for replication. E. coli origin, 3) T5 phage promoter sequence, 4
) 2 rack operator sequences, 5) Shine-Delgarno sequence, and 6) lactose operon receptor gene (lacIq). Origin of replication (o
riC) is derived from pUC19 (LTI, Gaithersburg, MD). The promoter and operator sequences are made synthetically.

DNA was transformed with NdeI and XbaI, BamHI, XhoI or Asp71.
It can be inserted into pHEa by restricting the vector with 8 and running the restricted product on a gel and isolating the larger fragment (the stuffer fragment should be about 310 base pairs). The DNA insert was digested with NdeI (5 ′ primer) and XbaI, Ba according to the PCR protocol above.
It is generated using PCR primers with restriction sites for mHI, XhoI or Asp718 (3 'primer). The PCR insert is gel purified and restricted with compatible enzymes. The insert and vector are ligated according to standard protocols.

Engineered vectors can be readily replaced in the above protocol to express proteins in bacterial systems.

Example 1B: Cloning and Expression of Angiogenic Proteins in Baculovirus Expression System A DNA oligonucleotide encoding a full-length angiogenic protein was generated by PCR oligonucleotides corresponding to the 5 ′ and 3 ′ sequences of this gene. Amplify using primers. The amplified sequence was transferred to a commercial kit (“Geneclean” BIO 101
Inc. , La Jolla, Ca. ) Is used to isolate from a 1% agarose gel. The fragments were then each digested with endonuclease and repurified on a 1% agarose gel. This fragment was named F2.

The vector pA2 (discussed below, a modified version of the pVL941 vector) is used for expression of this protein using the baculovirus expression system (for review, Summers, MD and Smith, G. E., 1987, A.
manual of methods for baculovirus v
vectors and insect cell culture process
dues, Texas Agricultural Experimenta
l Station Bulletin No. 1555). This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus (AcMNPV), followed by recognition sites for the restriction endonucleases Bam HI and Xba I. The polyadenylation site of simian virus SV40 is used for efficient polyadenylation. For easy selection of recombinant viruses, E. The β-galactosidase gene from E. coli was inserted in the same direction as the polyhedrin promoter, followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequence was flanked on both sides by viral sequences for cell-mediated homologous recombination of co-transfected wild type viral DNA. Many other baculovirus vectors, such as pRG1, pAc373, pVL941 and pAcIM1 may be used in place of pA2 (Luckow, VA and Summers, MD, V.
irology, 170: 31-39).

This plasmid is digested with restriction enzymes and then dephosphorylated with calf intestinal phosphatase using procedures known in the art. Then, a commercially available kit ("
Geneclean "BIO 101 Inc. , La Jolla, Ca) is used to isolate DNA from a 1% agarose gel. This vector DNA is designated herein as V2.

Fragment F2 and dephosphorylated plasmid V2 are ligated with T4 DNA ligase. Then E. E. coli DH5α cells are transformed and the plasmid-containing bacteria are identified using the respective restriction enzymes. The sequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of plasmid was treated with the lipofection method (Felgner et al., Proc.
Natl. Acad. Sci. USA, 84: 7413-7417 (1987).
) Of 1.0 μg of commercially available linearized baculovirus (“BaculoGo
ld baculovirus DNA ", Pharmingen, San D
iego, CA. ) And co-transfection with.

1 μg BaculoGold viral DNA and 5 μg plasmid
50 μl of serum-free Grace's medium (Life Technologies Inc.
． , Gaithersburg, MD) in sterile wells of a microtiter plate. Afterwards 10 μl Lipofectin and 90 μl Grace's medium were added, mixed and incubated for 15 minutes at room temperature. The transfection mixture is then added to 3 ml with 1 ml Grace's medium without serum.
Sf9 insect cells (ATCC CRL 1 seeded in 5 mm tissue culture plates
711). The plate is rocked back and forth to mix the newly added solution. The plates are then incubated at 27 ° C for 5 hours. After 5 hours, the transfection solution is removed from the plate and 1 ml Grace's insect medium supplemented with 10% fetal bovine serum is added. The plate is returned to the incubator and the culture is continued at 27 ° C. for 4 days.

After 4 days, the supernatants are collected and plaque assayed as described by Summers and Smith (supra). As a variant, "Blue Gal" (Life Techno), which allows easy isolation of blue-stained plaques.
logs Inc. , Gaithersburg). (A detailed description of the "plaque assay" can also be found in Life Techn.
logistics Inc. , Gaithersburg, pages 9-10, and can also be found in user guides for insect cell culture and baculovirology).

Four days after serial dilution, the virus is added to the cells and blue-stained plaques are picked with an Eppendorf pipette tip. The agar containing the recombinant virus is then resuspended in Eppendorf tubes containing 200 μl Grace's medium. The agar is removed by brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect insect Sf9 cells seeded in 35 mm dishes. After 4 days, the supernatants of these culture dishes are harvested and then they are stored at 4 ° C.

Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS. Cells are infected with multiplicity of infection (MOI) 2 of recombinant baculovirus. 6
After hours, the medium was removed and SF9 depleted of methionine and cysteine
00 II medium (Life Technologies Inc., Gaith
ersburg). After 42 hours, 5 μCi [ ³⁵ S] -methionine and 5 μCi [ ³⁵ S] -cysteine (Amersham) are added. Cells are incubated for a further 16 hours, then cells are harvested by centrifugation and labeled proteins visualized by SDS-PAGE and autoradiography.

The amino-terminal sequence of the angiogenic protein produced can be determined using microsequences of the amino-terminal amino acid sequence of the purified protein.

Example 1C: Expression of Recombinant Angiogenic Proteins in Mammalian Cells (Cloning and Expression in COS Cells) Expression of plasmid X-HA (X represents the angiogenic protein of interest) Derived from the vector pcDNA3 / Amp (Invitrogen) containing:
(1) SV40 origin of replication; (2) ampicillin resistance gene; (3) E. coli
Origin of replication; (4) CMV promoter followed by polylinker region, SV40 intron, and polyadenylation site. The DNA fragment encoding the entire precursor of the angiogenic protein and the HA tag fused in frame to its 3'end are cloned into the polylinker region of this vector. Therefore, this recombinant protein expression is under the control of the CMV promoter. This HA tag is derived from an epitope (I. Wi) from the previously described influenza hemagglutinin protein.
lson, H .; Niman, R .; Heighten, A .; Cherenson,
M. Conony, and R.M. Lerner, 1984, Cell 37: 7.
67). Fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes this HA epitope.

The plasmid construction strategy is described as follows: The DNA sequence encoding the angiogenic polypeptide was constructed by PCR using the following two primers: 5 ′ primer, Bam HI site followed by Contains a coding sequence of 18 nucleotides starting from the start codon; , Including complementary sequences. Therefore, the PCR product contains a Bam HI site, the coding sequence, followed by the HA tag fused in frame, the translation termination stop codon next to the HA tag,
And contains the Xho I site.

This PCR amplified DNA fragment and the vector pcDNA3 / Amp
Are digested with the respective restriction enzymes and ligated. This ligation mixture was transformed into E. c
oli strain SURE (Stratagene Cloning Systems,
11099, available from La Jolla, CA 92037). The transformed culture is plated on ampicillin medium plates and resistant colonies are selected. Plasmid DNA is isolated from transformants and tested for the presence of the correct fragment by restriction analysis. For expression of recombinant angiogenic polypeptides, COS cells are transfected with this expression vector by the DEAE-DEXTRAN method. (J. Sambrook, E
． Fritsch, T .; Maniatis, Molecular Clonin
g: A Laboratory Manual, Cold Spring La
laboratory Press, (1989)). Expression of X-HA protein is detected by radiolabeling and immunoprecipitation. (E. Harlow, D. La
ne, Antibodies: A Laboratory Manual, Co
ld Spring Harbor Laboratory Press (19
89)). Cells are labeled with ³⁵ S-cysteine for 8 hours 2 days after transfection. The culture medium is then collected and the cells are washed with detergent (RIP
A buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1%
NP-40, 0.5% DOC, 50 mM Tris, pH 7.5; Wil
son, I.S. Et al., Ibid., 37: 767 (1984)). Both cell lysates and culture media are precipitated with HA-specific monoclonal antibodies. The precipitated proteins are then analyzed on a 15% SDS-PAGE gel.

Cloning and Expression in Other Mammalian Cells Angiogenic polypeptides can be expressed in mammalian cells. Representative mammalian expression vectors include promoter elements that mediate the initiation of mRNA transcription, protein coding sequences, and signals necessary for termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences, and intervening sequences flanking the donor and acceptor sites of RNA splicing. Very efficient transcription is achieved by the SV40-derived early and late promoters, retroviruses (eg RSV, HTLVI, HI).
VI) derived long terminal repeats (LTRs), and the cytomegalovirus (CMV) early promoter. However, cellular elements such as the human actin promoter can also be used.

Expression vectors suitable for use in practicing the present invention include, for example, pSVL
And pMSG (Pharmacia, Uppsala, Sweden), pR
SVcat (ATCC 37152), pSV2DHFR (ATCC 3714)
6), pBC12MI (ATCC 67109), pCMVSport2.0,
And vectors such as pCMVSport3.0. Mammalian host cells that can be used include human Hela cells, 293 cells, H9 cells and Jurkat cells, mouse NIH3T3 cells and C127 cells, Cos1 cells, Cos7 cells and CV1 cells, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells are mentioned.

Alternatively, the angiogenic polypeptide can be expressed in stable cell lines containing a polynucleotide encoding the angiogenic polypeptide integrated into a chromosome. D
Co-transfection with selectable markers such as HFR, gpt, neomycin, hygromycin allows the identification and isolation of transfected cells.

The transfected gene encoding an angiogenic polypeptide can also be amplified to express large amounts of the encoded protein. The DHFR (dihydrofolate reductase) marker is useful for developing cell lines that carry hundreds or even thousands of copies of the gene of interest. (For example, Alt et al., J. Biol. Che.
m. 253: 1357-1370 (1978); Hamlin et al., Bioche.
m. et Biophys. Acta, 1097: 107-143 (1990).
: Page et al., Biotechnology 9: 64-68 (1991)). Another useful selectable marker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227: 277-279 (199).
1); Bebbington et al., Bio / Technology 10: 169.
-175 (1992)). These markers are used to grow mammalian cells in selective medium and select the cells with the highest resistance. These cell lines contain the amplified gene integrated into the chromosome. Chinese hamster ovary (CHO) cells and NSO cells are often used for protein production.

Expression vectors pC4 (ATCC Accession No. 209646) and pC6 (AT), which are derivatives of plasmid pSV2-DHFR (ATCC Accession No. 37146).
CC Accession No. 209647) is for Rous sarcoma virus (Cullen et al., Mol.
electrical and Cellular Biology, 438-447 (
March 1985)), a strong promoter (LTR), and a fragment of the CMV enhancer (Boshart et al., Cell 41: 521-530 (1985)). For example, multiple cloning sites with BamHI, XbaI, and Asp718 restriction enzyme cleavage sites facilitate cloning of the gene encoding the angiogenic polypeptide. The vector also includes the 3'intron of the rat preproinsulin gene, polyadenylation and termination signals, and
Contains the mouse DHFR gene under the control of the SV40 early promoter.

Specifically, for example, plasmid pC6 or pC4 is digested with appropriate restriction enzymes and then calf intestinal phosphatase (
dephosphorylation using a phosphate). Then add 1% of this vector
Isolate from agarose gel.

The cDNA sequence encoding the full length angiogenic protein is amplified using PCR oligonucleotide primers corresponding to the 5'and 3'sequences of this gene. If a naturally occurring signal sequence is used to produce the secreted protein, the vector does not require a second signal peptide. Alternatively, if a naturally occurring signal sequence is not used, the vector may be modified to include a heterologous signal sequence in an attempt to secrete the protein from the cell (eg, W
O96 / 34891).

The amplified fragment was then digested with the appropriate restriction enzymes and a commercially available kit (“Geneclean”, BIO 101 Inc., La Jolla, C.
a. ) Is used to purify on a 1% agarose gel. The isolated fragment and dephosphorylated vector are then digested with T4 DNA ligase. E. c
oli HB101 or XL-1 blue cells are transformed and bacteria containing the fragment inserted into plasmid pC6 or pC4 are identified using, for example, restriction enzyme analysis.

Chinese hamster ovary cells lacking the active DHFR gene are used for transfection. 5 μg of expression plasmid pC6 or pC4 was added to 0.5 μg of plasmid pC6 using lipofectin (Felgner et al., Supra).
Co-transfect with SVneo. Plasmid pSV2-neo contains the dominant selectable marker, the Tn5-derived neo gene encoding an enzyme that confers resistance to a group of antibiotics, including G418. Cells are seeded in α-minus MEM supplemented with 1 mg / ml G418. After 2 days, cells were trypsinized and treated with 10, 25, or 50 ng / ml methotrexate and 1 m.
Seed in hybridoma cloning plates (Greiner, Germany) in α-MEM supplemented with g / ml G418. After about 10-14 days, single clones were trypsinized and then using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).
Seed in 6-well Petri dishes or 10 ml flasks. Clones growing at the highest concentration of methotrexate were then selected for higher concentrations (1 μM, 2 μM, 5 μM).
μM, 10 mM, 20 mM) in a new 6-well plate containing methotrexate. The same procedure is repeated until clones are obtained that grow at a concentration of 100-200 μM. Expression of angiogenic polypeptides is analyzed, for example, by SDS-PAGE and Western blot, or by reverse phase HPLC analysis.

Example 2 Isolation of Genomic Clones Encoding Angiogenic Polypeptides Human Genome P1 Library (Genomic Systems, Inc.)
According to the method of Sambrook et al., SEQ ID NOs: 1, 3, 5, 7, 9, 11,
Screen by PCR with primers selected for the cDNA sequence corresponding to 13, 15, 17, 19, 21 or 23, respectively.

Example 3 Tissue Distribution of Angiogenic Polypeptides Tissue distribution of mRNA expression of angiogenic proteins, particularly Sambrook.
Determine using the protocol for Northern blot analysis described by K. et al. For example, a probe specific to a gene encoding an angiogenic protein of interest is labeled with a rediprime ^™ DNA labeling system (A
Label with P ³² using the Mersham Life Science) according to the manufacturer's instructions. After labeling, the probe was loaded onto a CHROMA SPIN-100 ^™ column (Clontech Laboratories, Inc.) using
Purify according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to test various human tissues for mRNA expression.

Multiple Tissue Northern (MTN) Blots (Clontech) containing different human tissues (H) or human immune system tissues (IM) were used with ExpressHyb ^™ Hybridization Solution (Clontech), manufacturer's protocol number P.
Test with labeled probe according to T1190-1. After hybridization and washing, the blots are mounted and exposed to film overnight at -70 ° C and the film developed according to standard procedures.

Example 4 Chromosomal Mapping of Angiogenic Proteins A set of oligonucleotide primers was added to SEQ ID NOs: 1, 3, 5, respectively.
Design according to the sequence at the 5'end of 7, 9, 11, 13, 15, 17, 19, 21 or 23. This primer preferably spans about 100 nucleotides.
This primer set is then used for polymerase chain reaction under the following set of conditions: 95 ° C for 30 seconds; 56 ° C for 1 minute; 70 ° C for 1 minute. This cycle is repeated 32 times and then once at 70 ° C. for 5 minutes. Somatic cell hybrid panel containing individual chromosomes or chromosome fragments (Bios, Inc)
In addition, human, mouse, and hamster DNA are used as templates. Reactions are analyzed on either an 8% polyacrylamide gel or a 3.5% agarose gel. Chromosome mapping is performed for about 100 in specific somatic cell hybrids.
Determined by the presence of the bp PCR fragment.

Example 5: Purification of Angiogenic Polypeptides from Inclusion Bodies Another method below is that, when angiogenic polypeptides are present in the form of inclusion bodies,
E. It can be used to purify angiogenic polypeptides expressed in E. coli. Unless otherwise specified, all the following steps are performed at 4-10 ° C.

E. After completion of the production phase of the E. coli fermentation, the cell culture is cooled to 4-10 ° C. and the cells are harvested by continuous centrifugation at 15,000 rpm (Heraeus Sepatech). Based on the expected yield of protein per unit weight of cell paste and the amount of purified protein required, an appropriate amount of cell paste (by weight) was added to 100 mM Tris, 50 mM EDTA, pH 7.
4. Suspend in buffer solution containing 4. The cells are dispersed into a homogenous suspension using a high shear mixer.

Next, a microfluidizer (Mic
rofluidics, Corp. Or APV Gaulin, Inc. Lyse the cells by passing the solution twice at 4000-6000 psi. The homogenate is then treated with Na to a final concentration of 0.5 M NaCl.
Mix with Cl solution followed by centrifugation at 7000 xg for 15 minutes. The obtained pellet was treated with 0.5 M NaCl, 100 mM Tris, 50 mM EDTA,
Wash again using pH 7.4.

The resulting washed inclusion bodies were treated with 1.5M guanidine hydrochloride (GuHCl) to
Solubilize for 4 hours. After centrifugation at 7000 xg for 15 minutes, the pellet is discarded, and the polypeptide-containing supernatant is incubated at 4 ° C overnight to allow additional Gu.
Allows HCl extraction.

Following high speed centrifugation (30,000 xg) to remove insoluble particles, Gu
HCl solubilized protein was extracted with GuHCl extract, 50 mM sodium, pH
Refold by rapid mixing with 20 volumes of buffer containing 4.5, 150 mM NaCl, 2 mM EDTA with vigorous stirring. The refolded diluted protein solution is kept at 4 ° C. without mixing for 12 hours before further purification steps.

To clarify the refolded polypeptide solution, a pre-prepared tangential filtration unit (eg, with a 0.16 μm membrane filter with appropriate surface area, equilibrated with 40 mM sodium acetate, pH 6.0) (eg, Filtro
n) is used. The filtered sample is treated with a cation exchange resin (eg, Poros).
HS-50, Perseptive Biosystems). The column was washed with 40 mM sodium acetate, pH 6.0, and 250 mM, 500 mM, 1000 mM, and 1500 mM NaC in the same buffer.
With l, elute in a stepwise fashion. The absorbance of the eluate at 280 nm is continuously monitored. Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing angiogenic polypeptides are then pooled and mixed with 4 volumes of water. Then, the diluted sample was treated with a strong anion exchange resin column (Poros HQ-50, Perseptive Biosystems) prepared in advance.
) And a weak anion exchange resin column (Poros CM-20, Persept).
Ive Biosystems) in series. 40m column
Equilibrate with M sodium acetate, pH 6.0. Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl. Then CM-2
0 column to 10 column volumes linear gradient (0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, p
H6.5 range). Fractions are collected under constant A ₂₈₀ monitoring of the eluate. Fractions containing the polypeptide (eg, as determined by 16% SDS-PAGE) are then pooled.

The resulting angiogenic polypeptide is 95% soluble after the refolding and purification steps described above.
It should show a purity higher than%. When 5 μg of purified protein is loaded, there is no large contaminating band, Coomassie blue stained 16% SDS-PAGE
It should not be observed from the gel. Purified angiogenic proteins can also be tested for endotoxin / LPS contamination, and typically LPS content is LAL.
Less than 0.1 ng / ml by assay.

Example 6: Construction of N-terminal and / or C-terminal deletion mutants The following general approach can be used to clone N-terminal or C-terminal deletion mutants of angiogenic proteins. . Generally, two oligonucleotide primers of about 15-25 nucleotides are provided with SEQ ID NOS: 1, 3, 5, 7, 9, respectively.
The desired 5 of 11, 13, 15, 17, 19, 21, or 23 polynucleotides
Derived from the'position 3 '. The 5'and 3'positions of the primer are determined based on the desired polynucleotide fragment encoding the angiogenic polypeptide. A start codon and a stop codon are added to the 5'and 3'primers, respectively, if necessary to express the angiogenic polypeptide fragment encoded by the polynucleotide fragment.

Additional nucleotides, including restriction sites, were also added to the 5'and 3'primer sequences to facilitate cloning of the polynucleotide fragment encoding the angiogenic polypeptide in the desired vector. obtain. A polynucleotide fragment encoding an angiogenic polypeptide is amplified from a genomic DNA or cDNA clone using appropriate PCR oligonucleotide primers and conditions discussed herein or known in the art. . An angiogenic polypeptide fragment encoded by a suitable polynucleotide fragment of the invention may be expressed and purified in the same general manner as the full length polypeptide, but may require routine modification, which is specific fragmentation. Due to differences in chemical and physical properties between the and full-length polypeptides.

The amplified polynucleotide fragment and expression vector are digested with a restriction enzyme that recognizes the site in the primer. The digested polynucleotides are then ligated together. The polynucleotide fragment encoding the angiogenic polypeptide is inserted into the restricted expression vector, preferably in such a manner that the angiogenic polypeptide fragment coding region is located downstream of the promoter. This ligation mixture was transformed into competent E. coli using standard procedures and as described in the Examples herein. E. coli. Isolation of plasmid DNA from resistant colonies,
The identity of the cloned DNA is then confirmed by restriction analysis, PCR, and DNA sequencing.

Example 7: Protein Fusion of Angiogenic Polypeptides Angiogenic polypeptides are preferably fused to other proteins. These fusion proteins can be used in various applications. For example, fusion of an angiogenic polypeptide to a His tag, HA tag, protein A, IgG domain, and maltose binding protein facilitates purification (see Example 5; EP.
A 394,827; Traunecker et al., Nature, 331: 84-.
86 (1988) also). Similarly, fusions to IgG-1, IgG-3, and albumin increase half-life in vivo. A nuclear localization signal fused to an angiogenic polypeptide can target the protein to specific intracellular locations. On the other hand, covalent heterodimers or homodimers may increase or decrease the activity of the fusion protein. Fusion proteins can also create chimeric molecules that have more than one function. Finally, fusion proteins may increase the solubility and / or stability of fusion proteins as compared to non-fusion proteins. All forms of the above fusion proteins can be made by modifying the protocol below, which outlines the fusion of polypeptides to IgG molecules, or the protocol described in the Examples.

Briefly, the human Fc portion of an IgG molecule can be PCR amplified using primers spanning the 5'and 3'ends of the sequences described below. These primers should also have convenient restriction enzyme sites which facilitate cloning into an expression vector, preferably a mammalian expression vector.

For example, when using pC4 (accession number 209646), the human Fc portion is
It can be ligated into the BamHI cloning site. Note that the 3'BamHI site should be destroyed. Next, the vector containing the human Fc portion was added to BamHI.
Re-restrict with, linearize the vector, and ligate a polynucleotide encoding an angiogenic polypeptide isolated by PCR to this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise the fusion protein will not be produced.

When the naturally occurring signal sequence is used to produce a secreted protein, pC4 does not require a second signal peptide. Alternatively, if the naturally occurring signal sequence is not used, the vector may be modified to include a heterologous signal sequence (see, eg, WO 96/34891). Human IgG Fc region:

[0523]

[Chemical 1] (SEQ ID NO: 30).

Example 8: Production of Antibodies Example 8A. Hybridoma Technology The antibodies of the invention can be prepared by a variety of methods. (Current Pro
See tocols, Chapter 2). As one example of such a method, cells expressing an angiogenic polypeptide are administered to an animal to induce the production of serum containing polyclonal antibodies. In a preferred method, an angiogenic protein preparation is prepared and purified to be substantially free of natural contaminants.
Such preparations are then introduced into animals to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the invention are monoclonal antibodies (or protein binding fragments thereof). Such monoclonal antibodies may be prepared using hybridoma technology (Koehler et al., Nature 25.
6: 495 (1975); Koehler et al., Eur. J. Immunol. 6
: 511 (1976); Koehler et al., Eur. J. Immunol. 6:
292 (1976); Hammerling et al .: Monoclonal Ant.
ibodies and T-Cell Hybridomas, Elsevi
er, N.N. Y. , Pp. 563-681 (1981)). Generally, such a procedure immunizes an animal, preferably a mouse, with angiogenic polypeptides, or more preferably with secreted angiogenic polypeptide-expressing cells. Such polypeptide expressing cells may be cultured in any suitable tissue culture medium; supplemented with 10% fetal bovine serum (inactivated at about 56 ° C) and containing about 10 g / l non-essential amino acids, It is preferable to culture in Earle modified Eagle medium supplemented with about 1,000 U / ml penicillin and about 100 μg / ml streptomycin.

The splenocytes of such mice are extracted and fused with the appropriate myeloma cell line.
Any suitable myeloma cell line may be used in accordance with the present invention; however, it is preferred to use the parental myeloma cell line (SP2O) available from the ATCC. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium and then Wan
ds et al. (Gastroenterology 80: 225-232 (1981)
)) And clone by limiting dilution. The hybridoma cells obtained through such selection are then assayed to identify clones secreting antibodies capable of binding the angiogenic polypeptide.

Alternatively, additional antibodies capable of binding to angiogenic polypeptides may be generated using anti-idiotype antibodies in a two-step procedure. Such a method takes advantage of the fact that the antibody itself is the antigen and therefore it is possible to obtain an antibody that binds to the second antibody. According to this method, protein-specific antibodies are used to immunize animals, preferably mice. The splenocytes of such animals are then used to produce hybridoma cells. The hybridoma cells are then screened to identify clones producing antibodies that can be blocked by angiogenic polypeptides for their ability to bind to angiogenic protein-specific antibodies. Such antibodies include anti-idiotypic antibodies against angiogenic protein-specific antibodies, and can be used to immunize animals to induce the formation of additional angiogenic polyprotein-specific antibodies.

Fab and F (ab ′) 2 fragments and other fragments of the antibodies of the invention may be used according to the methods disclosed herein,
To be understood. Such fragments are typically produced by proteolytic cleavage using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F (ab ') 2 fragments). . Alternatively, secretory angiogenic protein binding fragments may be generated through the application of recombinant DNA technology or synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use "humanized" chimeric monoclonal antibodies. Such antibodies can be produced by using genetic constructs derived from hybridoma cells which produce the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. (For a review, see Morrison, Science 229: 1202.
(1985); Oi et al., BioTechniques 4: 214 (1986).
Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neube.
rger et al., WO 8601533; Robinson et al., WO 870267.
1; Boulianne et al., Nature 312: 643 (1984); Ne.
Uberger et al., Nature 314: 268 (1985)).
.

Example 8B. Isolation of Antibody Fragments Against Angiogenic Polypeptides from a Library of scFvs The naturally occurring V gene isolated from human PBL was unexposed with or without donor exposure. Construct into large libraries of antibody fragments containing reactivity to optionally angiogenic polypeptides (see, eg, US Pat. No. 5,885,793, which is incorporated herein by reference in its entirety). That).

Library Rescue From human PBL RNA to scFvs as described in WO 92/01047.
Build a library of. To rescue the phage displaying the antibody fragment, about 10 ⁹ E. coli carrying the phagemid were rescued. 50m using E.coli
1 × 2 × TY (containing 1% glucose and 100 μg / ml ampicillin) (2 × TY-AMP-GLU) and inoculated with shaking. D. = 0
． Grow to 8. Using 5 ml of this culture, 50 ml of 2xTY-AMP
-GLU was inoculated and 2 x 10 ⁸ TU of Δ gene 3 helper (M13 Δ gene II
I, see WO92 / 01047) and the cultures are incubated for 45 minutes at 37 ° C. without shaking and then for 45 minutes at 37 ° C. with shaking. This culture was incubated for 10 minutes at 4000 rpm. p. m. Centrifuge at 2 ° C., and pellet the pellet with 2 liters of 2 × TY (100 μg / ml ampicillin and 5 ml).
Reconstitute in 0 μg / ml kanamycin) and grow overnight.
Phages are prepared as described in WO92 / 01047.

M13Δ gene III is prepared as follows: M13Δ gene III helper phage does not encode gene III protein. Therefore, phage displaying antibody fragments (phagemids) have greater binding avidity for antigen. During phage morphogenesis, infectious M13Δ gene III particles are made by growing helper phage in cells carrying the pUC19 derivative that supplies the wild-type gene III protein. The culture is incubated for 1 hour at 37 ° C without shaking and then for another hour at 37 ° C with shaking. Cells were spun down (IEC-Centra 8,400 rpm for 10 minutes), 100 μg / ml ampicillin and 25 μg / ml.
2 × TY broth containing 2 kanamycin (2 × TY-AMP-KAN) 300
Resuspend in ml and grow overnight at 37 ° C with shaking. Phage particles were purified and concentrated from the culture medium by two PEG precipitations (Sambrook et al., 1990), resuspended in 2 ml PBS, and passed through a 0.45 μm filter (Minisart NML; Sartorius) to a final concentration of approximately. 1
0 ¹³ transduction units / ml (ampicillin resistant clones) are obtained.

(Paning of Library) 100 μg / ml or 10 μg / ml of Immunotubes (Nunc) was used.
4. Coat overnight in PBS with 4 ml of any of the polypeptides of the invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37 ° C and then washed 3 times in PBS. Approximately 10 ¹³ TU of phage are applied to the tubes and incubated for 30 minutes at room temperature with tumbling up and down on a turntable, then left to stand for a further 1.5 hours. Tube with PBS 0.1% Twee
Wash 10 times with n-20 and 10 times with PBS. Phages were eluted by adding 1 ml of 100 mM triethylamine and spinning up and down for 15 minutes on a rotating disc, then adding 0.5 ml of 1.0 M Tris-HCl, to the solution.
Immediately neutralize at pH 7.4. Next, the eluted phages were incubated with the bacteria at 37 ° C.
Phage was used to incubate 10 ml of mid-log phase E. E. coli TG1. Then E. E. coli is plated on TYE plates containing 1% glucose and 100 μg / ml ampicillin. The resulting bacterial library is then rescued with Δ gene 3 helper phage as described above to prepare phage for the next round of selection. This process was then repeated for all four rounds of affinity purification, for the third and fourth round the tube washes were washed with PBS, 0.1% Tween-20 for 2 times.
Increase 0 times and 20 times with PBS.

Characterization of Binders Phage eluted from the third and fourth rounds of selection were used to transform E. coli. coli
Infect HB2151 and generate soluble scFv from a single colony for assay (Marks et al., 1991). 50 mM bicarbonate, pH 9.
The ELISA is performed with microtiter plates coated with either 10 pg / ml of the polypeptide of the invention in 6. Positive clones in the ELISA are further characterized by PCR fingerprinting (see, eg, WO92 / 01047) followed by sequencing.

Example 9: Production of angiogenic proteins for screening assays The following protocol produces a supernatant containing angiogenic polypeptides to be tested. This supernatant can then be used in the screening assay described in Examples 14-21.

First, poly-D-lysine (644 587 Boehringer-Man).
nheim) stock solution (1 mg / ml in PBS) in PBS (17-516F Biowhittaker without calcium or magnesium).
Dilute to 20 to make working solution 50 μg / ml. 200 μl of this solution
Add to each well (24-well plate) and incubate for 20 minutes at room temperature. Make sure the solution is distributed over each well (Note: a 12 channel pipettor can be used with tips on every other channel). Aspirate the poly-D-lysine solution and rinse with 1 ml PBS (phosphate buffered saline). PBS should remain in the wells just prior to plating the cells, and the plates may be pre-coated with polylysine up to 2 weeks.

293T cells (passing P + 20 and carrying no cells) were treated with 2 × 10 ⁵ cells / well in 0.5 ml DMEM (Dulbecco's Modified Eagle Medium) (4.
5 G / L glucose and L-glutamine (12-604F Biowhit
10% heat-inactivated FBS (14-503F Biowhi)
ttaker) / 1 × Penstrep (17-602E Biohitter)
plate). Allow cells to grow overnight.

The next day, in a sterile solution basin, 300 μl lipofectamine (1
8324-012 Gibco / BRL) and 5 ml Optimem I (
31985070 Gibco / BRL) / 96 well plate are mixed together. Using a small volume of multi-channel pipettor, approximately 2 μg of the expression vector containing the polynucleotide insert produced by the method described in Examples 8-10 was placed in appropriately labeled 96-well round bottom plates. Aliquot. 50 μl Lipofectamine / Opti using multi-channel pipettor
Add the mem I mixture to each well. Mix gently by pipetting up and down gently. Incubate for 15-45 minutes at room temperature. After about 20 minutes, add 150 μl Optimem I to each well using a multichannel pipettor. As a control, one plate of vector DNA lacking the insert should be transfected with each set of transfections. Preferably, the transfection should be tag-teaming the following tasks. By tag teaming, the time effort is halved, and the cells do not spend too much time on PBS. First, Person A aspirates the medium from the four 24-well plates of cells, and then Person B rinses each well with 0.5-1 ml PBS.
Mr. A then aspirates the PBS rinse and Mr. B uses channel 1
Use a 12-channel pipettor with tips every other 200 μl of D
The NA / Lipofectamine / Optimem I complex is added to each row on a 24-well plate, first in the odd wells, then in the even wells. Incubate at 37 ° C for 6 hours.

While incubating the cells, 1% BSA in DMEM with 1 × penstrep or HGS CHO-5 medium (116.6).
mg / CaCl ₂ (anhydrous) of _{L; 0.00130mg / L CuSO 4 -5H} 2
O; 0.050 mg / L of _{Fe (NO 3) 3 -9H 2} O; 0.417mg / L of F
eSO ₄ -7H ₂ O; 311.80 mg / L KCl; 28.64 mg / L Mg
Cl ₂ ; 48.84 mg / L MgSO ₄ ; 6995.50 mg / L NaCl;
2400.0 mg / L NaHCO ₃ ; 62.50 mg / L NaH ₂ PO ₄ —H ₂ O; 71.02 mg / L Na ₂ HPO ₄ ; 0.4320 mg / L ZnSO ₄ −
7H ₂ O; 0.002 mg / L arachidonic acid; 1.022 mg / L cholesterol; 0.070 mg / L DL-α-tocopherol acetate; 0.0520 m
g / L linoleic acid; 0.010 mg / L linolenic acid; 0.010 mg / L myristic acid; 0.010 mg / L oleic acid; 0.010 mg / L palmitooleic acid (palmic acid); 0 0.010 mg / L palmitic acid; 100 mg / L Pluronic F-68; 0.010 mg / L stearic acid; 2.20 mg / L Tween 80; 4551 mg / L D-glucose; 130.85 mg / mL L- alanine; 147.50mg / ml of L- arginine -HCl; 7.50mg / ml of L- asparagine -H ₂ O; 6
． 65 mg / ml of L- aspartic acid; 29.56mg / ml of L- cystine _{2HCl-H 2 O; 31.29mg /} ml of L- cystine-2HCl; 7.35
mg / ml L-glutamic acid; 365.0 mg / ml L-glutamine; 18
． 75 mg / ml glycine; 52.48 mg / ml L-histidine-HCl
-H ₂ O; 106.97mg / ml of L- isoleucine; 111.45mg / m
1 L-leucine; 163.75 mg / ml L-lysine HCl; 32.34 m
g / ml L-methionine; 68.48 mg / ml L-phenylalanine; 4
0.0 mg / ml L-proline; 26.25 mg / ml L-serine; 101
． 05 mg / ml L-threonine; 19.22 mg / ml L-tryptophan; 91.79 mg / ml L-tyrosine-2Na-2.
H ₂ O; and 99.65 mg / ml L-valine; 0.0035 mg / L biotin; 3.24 mg / L D-Ca pantothenic acid; 11.78 mg / L choline chloride; 4.65 mg / L Folic acid; 15.60 mg / L i-inositol; 3
． 02 mg / L niacinamide; 3.00 mg / L pyridoxal HCl
0.031 mg / L pyridoxine HCl; 0.319 mg / L riboflavin; 3.17 mg / L thiamine HCl; 0.365 mg / L thymidine;
680 mg / L vitamin B ₁₂ ; 25 mM HEPES buffer; 2.39 mg / L
L Na hypoxanthine; 0.105 mg / L lipoic acid; 0.081 mg / L putrescine sodium-2HCl; 55.0 mg / L sodium pyruvate;
0.0067 mg / L sodium selenite; 20 μM ethanolamine;
0.122 mg / L ferric citrate; Methyl-B-cyclodextrin complexed with 41.70 mg / L linoleic acid; Methyl-B-cyclo complexed with 33.33 mg / L oleic acid Dextrin; methyl-B-cyclodextrin complexed with 10 mg / L retinal acetate). Adjust the osmolarity to 327 mOsm with 2 mM glutamine and 1 × Penstrep (1 L DM for 10% BSA stock solution).
100 g of BSA (81-068-3 Bayer) was dissolved in EM). Media is filtered and 50 μl collected in 15 ml polystyrene conical for endotoxin assay.

The transfection reaction is terminated at the end of the incubation time, preferably in tag teams. Person A aspirates off the transfection medium, while person B adds 1.5 ml of the appropriate medium to each well. Incubate at 37 ° C for 45 or 72 hours depending on the medium used:
45% for 1% BSA or 72 hours for CHO-5.

On day 4, 600 μl using a 300 μl multichannel pipettor
Aliquot into 1 ml deep well plate and 2 ml of remaining supernatant
Aliquot into deep wells of. The supernatant from each well was then added to Examples 10-1.
It can be used in the assay described in 2.

If the activity is obtained in any of the assays described below using the supernatant, then the activity may be obtained directly from the angiogenic polypeptide (eg, as a secreted protein) or other protein. It is specifically understood that it is derived from either by an angiogenic polypeptide that induces the expression of, which is then secreted into the supernatant. Thus, the invention further provides methods for identifying proteins in supernatants characterized by activity in a particular assay.

Example 10: Screening Assay to Identify Changes in Small Molecule Concentrations and Membrane Permeability Binding of ligands to receptors alters intracellular levels of small molecules such as calcium, potassium, sodium and pH. It is known to cause, as well as to change the membrane potential. These changes can be measured in assays that identify the supernatant that binds to the receptor on a particular cell. The following protocol describes an assay for calcium, but this protocol is easily modified to detect changes in potassium, sodium, pH, membrane potential, or any other small molecule detectable by a fluorescent probe. You can

The following assay measures changes in fluorescent molecules (Molecular Probes) that bind small molecules using a fluorimetric imaging plate reader (“FLIPR”). Obviously, any fluorescent molecule that detects small molecules could be used in place of the calcium fluorescent molecule, fluo-3, as used herein.

For adherent cells, seed cells at 10,000-20,000 cells / well in Co-star black 96-well plates with clear bottoms. CO _{2 the} plate
Incubate in incubator for 20 hours. The adherent cells were treated with 200 μl of HBSS (Hank's Balanced Salt) in a Biotek washer.
Wash twice with Solution, leaving 100 μl of buffer after the last wash.

A stock solution of 1 mg / ml fluo-3 was added to 10% pluronic acid (pluro acid).
nic acid) Made of DMSO. To load the cells with fluo-3,
12 μg / ml fluo-3 (50 μl) is added to each well. The plate is incubated for 60 minutes at 37 ° C. in a CO ₂ incubator. The plate is washed 4 times with HBSS in the Biotek washer, leaving 100 μl of buffer.

For non-adherent cells, the cells are spun down from the culture medium. 50 cells
Resuspend to 2-5 × 10 ⁶ cells / ml with HBSS in a ml conical tube. 4 μl of 1 mg / ml fluo-3 10% pluronic acid DMSO solution is added per 1 ml of cell suspension. Then, place the tube in a 37 ° C water bath for 30-6
Leave for 0 minutes. The cells were washed twice with HBSS and resuspended at 1 × 10 ⁶ cells / ml,
Then, dispense 100 μl / well to the microplate. Plate 100
Centrifuge at 0 rpm for 5 minutes. Next, plate the Denley Cell W
After washing once with 200 μl in ash, 100 μl final volume by aspiration step
To

For non-cell based assays, each well contains a fluorescent molecule such as fluo-3. The supernatant is added to the wells and the change in fluorescence is detected.

To measure the fluorescence of intracellular calcium, the FLIPR is set up for the following parameters: (1) System gain is 300-800 mW; (2)
Exposure time is 0.4 seconds; (3) camera F / stop is F / 2;
(4) Excitation is 488 nm; (5) Emission is 530 nm; and (6)
Sample addition is 50 μl. An increase in luminescence at 530 nm results in an extracellular signaling event resulting in an increase in intracellular Ca ⁺⁺ concentration caused by a molecule that is either an angiogenic polypeptide or a molecule induced by an angiogenic polypeptide. Indicates.

Example 11 Screening Assays to Identify Tyrosine Kinase Activity Protein tyrosine kinases (PTKs) represent a diverse group of transmembrane and cytoplasmic kinases. Within the receptor protein tyrosine kinase (RPTK) family, there is a range of receptors for mitogenic and metabolic growth factors, including PDGF, FGF, EGF, NGF, HGF and the insulin receptor subfamily. In addition, there is a large RPTK family whose corresponding ligands are unknown. RPTK ligands include primarily secreted low molecular weight proteins, but also membrane-bound and extracellular matrix proteins.

Activation of RPTK by a ligand involves ligand-mediated receptor dimerization, which results in transphosphorylation of receptor subunits and activation of cytoplasmic tyrosine kinases. As cytoplasmic tyrosine kinase, src family (
Examples include receptor-associated tyrosine kinases of src, yes, lck, lyn, fyn), and non-receptor bound and cytosolic protein tyrosine kinases such as the Jak family. These members include cytokine superfamily (eg, interleukins, interferons, GM
-CSF and leptin).

Due to the wide variety of known factors that can stimulate tyrosine kinase activity, it is not possible to identify whether angiogenic polypeptides or molecules induced by angiogenic polypeptides can activate the tyrosine kinase signaling pathway. Interesting. Therefore, the following protocol is designed to identify such molecules that can activate the tyrosine kinase signaling pathway.

Target cells (eg, primary keratinocytes) were purchased from Nalge Nunc (Naperville, IL) at a density of approximately 25,000 cells / well 96.
Seed wells Loprodyne Silent Screen Plate. The plates are sterilized by rinsing twice with 100% ethanol for 30 minutes, rinsed with water and then dried overnight. Several plates were loaded with 100 ml of cell culture grade type I collagen (50 mg / ml), gelatin (2%) or polylysine (
50 mg / ml) (all of these are Sigma Chemicals (St. L.
ouis, MO) or at Becton Dickinso
n (Bedford, MA) 10% Matrigel or calf serum for 2 hours, rinsed with PBS and stored at 4 ° C. Growth medium was seeded at 5,000 cells / well and manufactured by the manufacturer Alamar Biosie.
nces, Inc. (Sacramento, CA), as described by ala
Cell proliferation is assayed on these plates by indirect quantification of cell number after 48 hours using marBlue. Becton Dickinson
(Bedford, MA) using Falcon plate cover # 3071
Cover the Loprodyne Silent Screen Plate. Fal
The con Microtest III cell culture plate may also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded (20,000 / 200 ml / well) on nylon membranes of Loprodyne plates and cultured overnight in complete medium. The cells are incubated in serum-free basal medium for 24 hours to make them rest. 5-2 (50 ng) of EGF (60 ng / ml) or the supernatant produced in Example 12
After treatment for 0 minutes, the medium was removed and 100 ml of extraction buffer (20 mM HEP
ES pH 7.5, 0.15M NaCl, 1% Triton X-100, 0.
1% SDS, 2 mM Na ₃ VO ₄ , 2 mM Na ₄ P ₂ O ₇ and Boeheri
nger Mannheim (Indianapolis, IN) mixture of protease inhibitors (# 1836170)) is added to each well, and the plate is shaken on a rotary shaker for 5 minutes at 4 ° C. The plate is then placed in a vacuum transfer manifold (vacuum transfer manifold).
ld) and place the extract at the bottom of each well using house vacuum.
． Filter through a 45 mm membrane. The extracts are collected in a vacuum well bottom 96-well capture / assay plate and immediately placed on ice. To obtain a clarified extract by centrifugation, solubilize with detergent for 5 minutes, then remove the contents of each well and centrifuge at 16,000 xg for 15 minutes at 4 ° C.

The filtered extract is tested for levels of tyrosine kinase activity. Although many methods of detecting tyrosine kinase activity are known, one of the methods is described herein.

Generally, the tyrosine kinase activity of the supernatant is evaluated by determining its ability to phosphorylate tyrosine residues on a particular substrate (biotinylated peptide). Biotinylated peptides that can be used for this purpose include PSK1 (cell division kinase cd
and amino acids 6-20 of c2-p34) and PSK2 (corresponding to amino acids 1-17 of gastrin). Both peptides are substrates for a series of tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the following components in order. First, after adding 10 μl of 5 μM biotinylated peptide, ATP / Mg ²⁺ (
5 mM ATP / 50 mM MgCl ₂ 10 μl, 5 × assay buffer (40
mM imidazole hydrochloride, pH 7.3, 40 mM β-glycerophosphate, 1 mM
EGTA, 100 mM MgCl ₂ , 5 mM MnCl ₂ , 0.5 mg / ml
BSA) 10 μl, sodium vanadate (1 mM) 5 μl and finally water 5 μl. The components are mixed gently and the reaction mixture is preincubated for 2 minutes at 30 ° C. The reaction is started by adding 10 μl of control enzyme or filtered supernatant.

Next, the tyrosine kinase assay reaction is stopped by adding 10 μl of 120 mM EDTA and the reaction is placed on ice.

Tyrosine kinase activity is measured by transferring a 50 μl aliquot of the reaction mixture to a microtiter plate (MTP) module and incubating at 37 ° C. for 20 minutes. As a result, streptavidin (streptava)
din) Coat 96-well plate with biotinylated peptide.
Wash MTP module 4 times with 300 μl / well PBS. Next, anti-phosphotyrosine conjugated to horseradish peroxidase (phospotyro).
Sine) antibody (anti-P-Tyr-POD (0.5 u / ml)) (75 μl) is added to each well and then incubated at 37 ° C. for 1 hour. Wash wells as above.

Next, a peroxidase substrate solution (Boehringer Mannheim)
) Add 100 μl and incubate at room temperature for at least 5 minutes (up to 30 minutes). The absorbance of the sample at 405 nm is measured using an ELISA reader. The level of bound peroxidase activity was quantified using an ELISA reader, which reflects the level of tyrosine kinase activity.

Example 12: Screening Assay to Identify Phosphorylation Activity [0561] As a potential alternative and / or complement to the protein tyrosine kinase activity assay described in Example 11, major intracellular signals. Assays that detect activation (phosphorylation) of transduction intermediates can also be used.
For example, as described below, one particular assay is Erk-1 and Erk-2.
Tyrosine phosphorylation of the kinase can be detected. However, Raf, JNK, p38M
Phosphorylation of other molecules such as AP, Map Kinase Kinase (MEK), MEK Kinase, Src, Muscle Specific Kinase (MuSK), IRAK, Tec and Janus, as well as any other phosphoserine, phosphotyrosine or phosphothreonine molecule. , Erk-1 or Er in these molecules in the following assay
It can be detected by replacing k-2.

Specifically, the wells of a 96-well ELISA plate were loaded with Protein G (1 μg /
ml) 0.1 ml to coat at room temperature (RT) for 2 hours to make an assay plate. The plates are then rinsed with PBS and blocked with 3% BSA / PBS for 1 hour at RT. Next, the protein G plate was subjected to Erk-1 and E.
Two commercially available monoclonal antibodies against rk-2 (100 ng / well) (S
Ant Cruz Biotechnology) (RT for 1 hour). (This step can be easily modified by replacing the monoclonal antibody that detects any of the molecules described above to detect other molecules). After rinsing 3-5 times with PBS, plates are stored at 4 ° C until use. 20 A431 cells,
000 / well in 96-well Loprodyne filter plate,
Culture overnight in growth medium. The cells are then starved for 48 hours in basal medium (DMEM) and then treated with EGF (6 ng / well) or 50 μl of the supernatant from Example 12 for 5-20 minutes. The cells are then solubilized and the extract filtered and placed directly into the assay plate.

After incubation with the extract for 1 hour at RT, the wells are rinsed again. Commercially available MAP kinase preparation (10 ng / well) as a positive control
In place of the A431 extract. The plates are then loaded with Erk-1 and E.
Treatment with a commercially available polyclonal (rabbit) antibody (1 μg / ml) that specifically recognizes a phosphorylated epitope of rk-2 kinase (1 hour at RT). The antibody is biotinylated by standard procedures. Next, the bound polyclonal antibody was combined with Europium streptavidin and Europium fluorescence enhancer and Wallac DELF.
Quantification by continuous incubation (time-resolved fluorescence) in an IA instrument. An increase in fluorescent signal above background is indicative of phosphorylation by the angiogenic polypeptide or a molecule induced by the angiogenic polypeptide.

Example 13: Method for Determining Alterations in Genes Encoding Angiogenic Polypeptides RNA isolated from whole families or individual patients presenting a phenotype of interest (eg, disease) is isolated. To do. CDNA is then generated from these RNA samples using protocols known in the art (see Sambrook).
This cDNA is then used as a template for PCR with primers surrounding the region of interest in the nucleotide sequence encoding the respective angiogenic polypeptide. Suggested PCR conditions are described in Sitransky, D .; And S
Science 252: 706 (1991) using a buffer solution at 95 ° C.
For 30 seconds; 52-58 ° C for 60-120 seconds; and 70 ° C for 60-120 seconds for 35 cycles.

Next, the PCR product was subjected to SequiTherm Polymerase (Epi
Center Technologies) and sequenced using primers labeled with T4 polynucleotide kinase at the 5'end. The intron-exon boundaries of selected exons of the gene encoding the angiogenic polypeptide are also determined and the genomic PCR product is analyzed to confirm the results. The PCR product with the suspected mutation is then cloned and sequenced to confirm the direct sequencing results. The PCR product with the suspicious mutation in the gene encoding the angiogenic polypeptide is then cloned and sequenced to confirm the direct sequencing results.

The PCR product of the gene encoding the angiogenic polypeptide was transformed into Holton, T
． A. And Graham, M .; W. , Nucleic Acids Rese
arch, 19: 1156 (1991) and cloned into a T-tail vector and cloned into T7 polymerase (United States Biochemi).
cal). Affected individuals are identified by mutations in genes encoding angiogenic polypeptides that are not present in unaffected individuals.

Genomic rearrangements are also observed as a way to determine alterations in the gene encoding the angiogenic polypeptide. The genomic clone isolated according to Example 2 was
Digoxigenin deoxy-uridine 5'-triphosphate (Boehringer M
nick translation, and Johnson,
Cg. Et al., Methods Cell Biol. 35: 73-99 (1991).
FISH is performed as described in (). A large excess of human cot-1 due to specific hybridization to the genetic locus encoding the angiogenic polypeptide.
Hybridization with a labeled probe is performed using DNA.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C and R bands. Alignment images for accurate mapping can be obtained by triple band filter set (Chroma T
technology, Brattleboro, VT) and cooled charge coupled device cameras (Photometrics, Tucson, AZ) and tunable excitation wavelength filters (Johnson, Cv. et al., Genet. Anal. Tech. App.
l. , 8:75 (1991)). ISee Gra
physical Program System (Inovision Corp)
image acquisition, analysis, and chromosome segment length measurement using the S. Oration, Durham, NC). Chromosomal changes in the genomic region of the gene encoding the angiogenic polypeptide (hybridized by the probe) are identified as insertions, deletions and translocations. These changes are used as diagnostic markers for related diseases.

Example 14 Method for Detecting Abnormal Levels of Angiogenic Polypeptides in Biological Samples Angiogenic polypeptides can be detected in biological samples, and angiogenic polypeptide levels If an increase or decrease in is detected, the polypeptide is a marker of a particular phenotype. It is understood that there are numerous detection methods, and therefore one of skill in the art can modify the following assays to suit their particular needs.

For example, an antibody sandwich ELISA is used to detect angiogenic polypeptides in a sample, preferably a biological sample. The wells of the microtiter plate are plated with a specific antibody to the angiogenic polypeptide to a final concentration of 0.2-1.
Coat with 0 μg / ml. This antibody is either monoclonal or polyclonal and is produced by the method described in Example 8. The wells are blocked so that non-specific binding of angiogenic polypeptides to the wells is reduced.

The coated wells are then incubated with the angiogenic polypeptide-containing sample at RT for more than 2 hours. Preferably, serial dilutions of the sample should be used to confirm the results. The plate is then washed three times with deionized or distilled water to remove unbound angiogenic polypeptide.

Next, 50 μl of the specific antibody-alkaline phosphatase conjugate was added to 25-400 n.
Add at a concentration of g and incubate at room temperature for 2 hours. Plates are washed again three times with deionized or distilled water to remove unbound conjugate.

Add 75 μl of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenyl phosphate (NPP) substrate solution to each well and incubate at room temperature for 1 hour. Reactions are measured by microtiter plate reader. A standard curve is constructed using serial dilutions of control samples and the angiogenic polypeptide concentration is plotted on the X-axis (log scale) and the fluorescence or absorbance is plotted on the Y-axis (linear scale). A standard curve is used to interpolate the concentration of angiogenic polypeptides in the sample.

Example 15: Formulation The present invention also provides a disease, disorder and / or condition (eg, any one disclosed herein) by administering to a subject an effective amount of a therapeutic agent. Methods for treating and / or preventing the above diseases, disorders and / or conditions) are provided. The therapeutic agent is combined with a pharmaceutically acceptable carrier type (eg, a sterile carrier),
By polynucleotides or polypeptides of the present invention (including fragments and variants), their agonists or antagonists, and / or antibodies to them.

Therapeutic agents will take into account the clinical condition of the individual patient (particularly the side effects of treatment with the therapeutic agent alone), the site of delivery, the mode of administration, the dosing regimen and other factors known to those of skill in the art, and medical practice ( Formulated and dosed in a manner that adheres to the good medical practice. Therefore, the "effective amount" intended in the present specification is determined by taking such a consideration into consideration.

As a general proposition, the total pharmaceutically effective amount of the therapeutic agent administered parenterally per dose is in the range of about 1 μg / kg / day to 10 mg / kg / day of patient body weight,
This is at therapeutic discretion, as described above. More preferably, for the hormone, the dose is at least 0.01 mg / kg / day, most preferably between about 0.01 mg / kg / day and about 1 mg / kg / day for humans. In the case of continuous administration, the therapeutic agent is typically about 1 μg / kg / hour to about 50 μg / kg.
Administered by either 1 to 4 injections per day or by continuous subcutaneous infusion (eg using a minipump) at a dosing rate of / hour. Intravenous bag solutions may also be used. The duration of treatment required to observe changes and the post-treatment interval at which a response occurs is:
It seems to change depending on the desired effect.

The therapeutic agent may be administered orally, rectally, parenterally, intracistemally.
y), vaginal, intraperitoneal, topical (such as by powder, ointment, gel, drops, or transdermal patch), buccal or oral or nasal spray. "Pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The therapeutic agents of the present invention are also suitably administered by a sustained release system. Suitable examples of sustained release therapeutic agents include oral, rectal, parenteral, intracistema.
ly), vaginal, intraperitoneal, topical (such as by powder, ointment, gel, drops, or transdermal patch), buccal or oral or nasal spray. "Pharmaceutically acceptable carrier" means a non-toxic solid, semi-solid or liquid filler,
Diluent, encapsulant or formulation auxiliary of any type. The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

The therapeutic agents of the invention are also suitably administered by a sustained release system. Suitable examples of sustained release therapeutic agents are suitable polymeric substances (eg semipermeable polymer matrices in the form of shaped articles (eg films or microcapsules)), suitable hydrophobic substances (eg in acceptable quality oils). As an emulsion) or an ion exchange resin, and a poorly soluble derivative (eg, a poorly soluble salt).

Sustained-release matrices include polylactide (US Pat. No. 3,773,919).
No., EP 58,481), a copolymer of L-glutamic acid and γ-ethyl-L-glutamate (Sidman et al., Biopolymers 22: 547-5.
56 (1983)), poly (2-hydroxyethyl methacrylate) (Lang)
er et al. Biomed. Mater. Res. 15: 167-277 (19
81), and Langer, Chem. Tech. 12: 98-105 (19
82)), ethylene vinyl acetate (Langer et al., Ibid.) Or poly-D.
-(-)-3-hydroxybutyric acid (EP133,988) is mentioned.

Sustained-release therapeutic agents also include liposome-encapsulated compositions of the invention (generally, Langer, Science 249: 1527-1533 (1990).
); Treat et al., Liposomes in the Therapy of
Infectious Disease and Center, Lopez
-Berstein and Fidler (ed.), Liss, New Yo
rk, pages 317-327 and 353-365 (1989)). Liposomes containing therapeutic agents can be prepared by methods known per se:
DE 3,218,121; Epstein et al., Proc. Natl. Acad.
Sci. USA 82: 3688-3692 (1985); Hwang et al., Pr.
oc. Natl. Acad. Sci. USA 77: 4030-4034 (19).
80); EP52,322; EP36,676; EP88,046; EP143.
, 949; EP 142, 641; Japanese Patent Application No. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545 and EP 1
02,324. Usually, liposomes are small (about 200-800 Å) unilamellar form, where the lipid content is greater than about 30 mol% cholesterol and the selected proportion is adjusted for the optimal therapeutic agent. It

In yet a further embodiment, the therapeutic agents of the invention are delivered by pump (Langer, supra; Sefton, CRC Crit. Ref. Biome.
d. Eng. 14: 201 (1987); Buchwald et al., Surgery.
88: 507 (1980); Saudek et al., N. et al. Engl. J. Med. Three
21: 574 (1989)).

Another controlled release system is the Langer (Science 249: 1527-15).
33 (1990)).

For parenteral administration, in one embodiment, the therapeutic agent will generally be administered to the recipient in the desired degree of purity, pharmaceutically acceptable carrier, ie, the dosage and concentration employed. Formulated by mixing in a unit dosage injectable form (solution, suspension or emulsion) with a non-toxic and compatible with the other ingredients of the formulation. For example, the formulation preferably does not include oxidizing agents and other compounds known to be deleterious to therapeutic agents.

In general, the formulations are prepared by contacting the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, in a uniform and intimate contact. The product is then shaped, if desired, into the desired formulation. Preferably the carrier is a parenteral carrier, more preferably a solution that is isotonic with the blood of the recipient. Examples of such carrier vehicles include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles such as fixed oils and ethyl oleate are also useful herein, as are liposomes.

The carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability. Such substances are not toxic to the recipients at the dosages and concentrations used, and include such substances as phosphates, citrates, succinates, acetic acid and other organic acids or their salts. Buffers such as; antioxidants such as ascorbic acid; low molecular weight (less than about 10 residues) polypeptides (eg, polyarginine or tripeptides); proteins such as serum albumin, gelatin or immunoglobulins; polyvinylpyrrolidone. Hydrophilic polymers such as; glycine, glutamic acid, aspartic acid or amino acids such as arginine; cellulose or its derivatives, mono-, di-, and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA ; Sugar sugars such as mannitol or sorbitol Lumpur; counterions such as sodium; and / or polysorbate include nonionic surfactants such as poloxamers or PEG.

The therapeutic agent is typically about 0.1 mg / ml to 100 mg / ml, preferably 1
It is formulated in such a vehicle at a concentration of -10 mg / ml and a pH of about 3-8. It is understood that the use of the particular excipients, carriers or stabilizers described above will result in the formation of polypeptide salts.

Any therapeutic agent used for therapeutic administration can be sterile. Sterility is easily achieved by filtration through a sterile filtration membrane (eg 0.2 micron membrane). Generally, the therapeutic agent is placed in a container having a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.

Therapeutic agents are typically stored in unit-dose or multi-dose containers, such as sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
As an example of a lyophilized formulation, sterile filtered 1% (w / v) into 10 ml vials.
) Fill 5 ml of aqueous polypeptide solution and freeze-dry the resulting mixture.
The lyophilized polypeptide is reconstituted with bacteriostatic water for injection to prepare an infusion solution.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the therapeutic agents of the invention. A notice in the form of a government agency that regulates the manufacture, use, or sale of pharmaceuticals or biological products may accompany such containers, which notice may include government notices regarding manufacture, use, or sale for human administration. Indicates approval by the institution. In addition, therapeutic agents may be used in combination with other therapeutic compounds.

The therapeutic agents of the present invention can be administered alone or in combination with an adjuvant. Adjuvants that may be administered with the therapeutic agents of the present invention include, but are not limited to, alum, alum and deoxycholic acid (ImmunoAg), MTP-PE (Biocine Corp.), QS2.
1 (Genentech, Inc.), BCG, and MPL. In certain embodiments, therapeutic agents of the invention are administered in combination with alum. In another specific embodiment, the Therapeutic Agents of the invention are administered in combination with QS21. Additional adjuvants that may be administered with the therapeutic agents of the present invention include, but are not limited to, monophosphoryl lipid immunomodulators, Adj.
uVax 100a, QS-21, QS-18, CRL1005, aluminum salt, MF-59, and Virosomal adjuvant technology. Vaccines that may be administered with the therapeutic agents of the present invention include, but are not limited to, MMR (measles, mumps, rubella), polio, chickenpox, tetanus / diphtheria, A. Hepatitis B, hepatitis B, influenza B, whooping cough (w
Vaccines directed against protection against hoofing cough, pneumonia, influenza, Lyme disease, rotavirus, cholera, yellow fever, Japanese encephalitis, polimyelitis, rabies, typhoid fever, and pertussis. Combinations can be administered either concomitantly (eg, as a mixture, separately but simultaneously or in parallel); or sequentially. This includes a presentation in which the combined agents are administered together as a therapeutic mixture, and the combined agents are administered separately but simultaneously (eg, when administered to the same individual via separate intravenous lines). It also includes procedures. "Combination" administration further comprises the separate administration of one of the compounds or agents given first, followed by the second.

Therapeutics of the invention can be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered with the therapeutic agents of the present invention include, but are not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatory agents. , Conventional immunotherapeutic agents, cytokines and / or growth factors. The combination can be administered either, for example, simultaneously as a mixture, separately but simultaneously or in parallel; or sequentially. This includes a presentation in which the combined agents are administered together as a therapeutic mixture, and also in procedures where the combined agents are administered separately but simultaneously (eg, to the same individual via separate intravenous lines). Also includes. "Combination" administration further comprises the separate administration of one of the compounds or agents given first, followed by the second.

In one embodiment, Therapeutics of the invention are administered in combination with members of the TNF family. TNF molecules that can be administered with the therapeutic agents of the invention, T
NF-related or TNF-like molecules include, but are not limited to: soluble forms of TNF-α, lymphotoxin-α, (LT-α, TN.
Also known as F-β), LT-β (found in the complex heterotrimer LT-α2-β), OPGL, FasL, CD27L, CD30L, CD40L, 4,
-1BBL, DcR3, OX40L, TNF-γ (International Publication Number WO96 / 14
328), AIM-I (International Publication No. WO97 / 33899), endokine (
endokine) -α (International Publication Number WO98 / 07880), TR6 (International Publication Number WO98 / 30694), OPG, and Neutrokine (neut).
rokine) -α (International Publication No. WO98 / 18921), OX40, and nerve growth factor (NGF), and soluble forms of Fas, soluble forms of CD30.
, Soluble form of CD27, soluble form of CD40 and soluble form of 4-IBB
, TR2 (International Publication Number WO96 / 34095), DR3 (International Publication Number WO9)
7/33904), DR4 (International Publication Number WO98 / 32856), TR5 (International Publication Number WO98 / 30693), TR6 (International Publication Number WO98 / 3069).
4), TR7 (International Publication Number WO98 / 41629), TRANS, TR9 (International Publication Number WO98 / 56892), TR10 (International Publication Number WO98 / 542).
02), 312C2 (International Publication No. WO98 / 06842), and TR12,
And soluble form of CD154, soluble form of CD70, and soluble form of CD153.

In certain embodiments, Therapeutics of the invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors and / or protease inhibitors. Examples of nucleoside reverse transcriptase inhibitors that can be administered in combination with the therapeutic agent of the present invention include the following:
Not limited to: RETROVIR ^™ (Zidovudine / AZT), VIDE
Z ^™ (Didanocin / ddI), HIVID ^™ (Zalcitabine / ddC), ZER
IT ^™ (stavudine / d4T), EPIVIR ^™ (lamivudine / 3TC), and COMBIVIR ^™ (zidovudine / lamivudine). Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the therapeutic agents of the present invention include, but are not limited to, VIRAMUNE ^™ (nevirapine), RESCRI.
PTOR ^™ (delavirdine), and SUSTIVA ^™ (efavirenz). Protease inhibitors that can be administered in combination with the therapeutic agents of the present invention include:
Including but not limited to: CRIXIVAN ^TM (indinavir), NORVIR ^TM (ritonavir), INVIRASE ^TM (saquinavir))
, And VIRACCEPT ^™ (Nelfinavir). In certain embodiments, an antiretroviral agent, nucleoside reverse transcriptase inhibitor, non-nucleoside reverse transcriptase inhibitor, and / or protease inhibitor is for treating AIDS and / or for preventing or treating HIV infection. , And can be used in any combination with the therapeutic agents of the invention.

In other embodiments, Therapeutics of the invention may be administered in combination with anti-opportunistic infection agents. Anti-opportunistic infection agents that may be administered in combination with the therapeutic agents of the present invention include, but are not limited to, TRIMETHOP.
RIM-SULFAMETHOXAZOLE ^™ , DAPSONE ^™ , PENTA
MIDINE ^™ , ATOVAQONE ^™ , ISONIAZID ^™ , RIFAM
PIN ^™ , PYRAZINAMIDE ^™ , ETHAMBUTOL ^™ , RIFAB
UTIN ^™ , CLARITHROMYCIN ^™ , AZITHROMYCIN ^™ ,
GANCICLOVIR ^™ , FOSCARNET ^™ , CIDOFOVIR ^™ , F
LUCONAZOLE ^™ , ITRACONAZOLE ^™ , KETOCONAZO
LE ^™ , ACYCLOVIR ^™ , FAMCICOLVIR ^™ , PYRIMETH
AMINE ^™ , LEUCOVORIN ^™ , NEUPOGEN ^™ (filgrastim / G-CSF), and LEUKINE ^™ (sargramostin (sargra
mostim) / GM-CSF). In certain embodiments, the Therapeutic Agents of the present invention are used to treat or prevent opportunistic Pneumocystis carinii pneumonia infection prophylactically.
OLE ^™ , DAPSONE ^™ , PENTAMIDINE ^™ , and / or AT
Used in any combination with OVAQUONE ^™ . In another specific embodiment, the therapeutic agents of the invention are ISONIAZID ^™ , RIFA for prophylactically treating or preventing opportunistic Mycobacterium avium combined infections.
MPIN ^™ , PYRAZINAMIDE ^™ , and / or ETHAMBUTO
Used in any combination with L ^™ . In another particular embodiment, the therapeutic agents of the invention are RIFABUTIN ^™ , CLARIT for the prophylactic treatment or prevention of opportunistic Mycobacterium tuberculosis infections.
Used in combination with HRMYCIN ^™ , and / or AZITHROMYCIN ^™ . In another particular embodiment, the therapeutic agents of the invention are administered to GANCI to prophylactically treat or prevent opportunistic cytomegalovirus infections.
Used in any combination with CLOVIR ^™ , FOSCARNET ^™ , and / or CIDOFOVIR ^™ . In another particular embodiment, the therapeutic agents of the invention are FLUCONA for the prophylactic treatment or prevention of opportunistic fungal infections.
ZOLE ^™ , ITRACONAZOLE ^™ , and / or KETOCONAZ
Used in any combination with OLE ^™ . In another particular embodiment, the therapeutic agents of the invention are ACYCLOVIR ^™ and / or FA for the prophylactic treatment or prevention of opportunistic herpes simplex virus type I and / or type II infections.
Used in any combination with MCICOLVIR ^™ . In another particular embodiment, the therapeutic agents of the invention are PYRIMETHAMINE ^™ and / or for the prophylactic treatment or prevention of opportunistic Toxoplasma gondii infection.
Or used in any combination with LEUCOVORIN ^™ . In another particular embodiment, the Therapeutics of the invention are used in any combination with LEUCOVORIN ^™ and / or NEUPOGEN ^™ to prophylactically treat or prevent opportunistic bacterial infections.

In a further embodiment, the Therapeutic Agents of the invention are administered in combination with antiviral agents. Antiviral agents that may be administered with the therapeutic agents of the present invention include acyclovir, ribavirin, amantadine, and remantidine (remantidine).
Tidine), but is not limited thereto.

In a further embodiment, the therapeutic agents of the invention are administered with an antibiotic. Antibiotics that can be administered with the therapeutic agent of the present invention include amoxicillin, β
-Lactamase, aminoglycoside, β-lactam (glycopeptide), β-lactamase, clindamycin, chloramphenicol, cephalosporin, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones,
Macrolide antibiotics, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamides, tetracyclines, trimethoprim, trimethoprim-sulfamethoxazole, and vancomycin are included without limitation.

Conventional non-specific immunosuppressive agents that can be administered with the therapeutic agents of the present invention include steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxysperm. Guarin (15-deoxyspergualin), and response T
Other immunosuppressive agents that act by suppressing the function of cells include, but are not limited to.

In certain embodiments, Therapeutics of the invention are administered in combination with immunosuppressive agents. Immunosuppressant preparations that can be administered with the therapeutic agents of the invention include ORT
HOCLONE ^TM (OKT3), SANDIMMUNE ^TM / NEORAL ^TM / S
ANGDYA ^™ (cyclosporine), PROGRAF ^™ (tacrolimus), CE
Examples include, but are not limited to, LLCCEPT ^™ (mycophenolate), azathioprine, glucocorticosteroids, and RAPAMUNE ^™ (sirolimus). In certain embodiments, immunosuppressive agents may be used to prevent rejection of organ or bone marrow transplants.

In a further embodiment, the Therapeutics of the invention are administered alone or in combination with one or more intravenous immunoglobulin preparations. Intravenous immunoglobulin preparations that may be administered in combination with the therapeutic agents of the present invention include GAMMAR ^™ , IV
Examples include, but are not limited to, EEGAM ^™ , SANDOGLOBULIN ^™ , GAMMAGARD S / D ^™ and GAMIMUNE ^™ . In certain embodiments, therapeutic agents of the invention are administered in combination with an intravenous immunoglobulin preparation in transplantation therapy (eg, bone marrow transplant).

In a further embodiment, the compositions of this invention are administered in combination with an antibiotic. Antibiotics that may be administered with the compositions of the present invention include tetracycline,
Examples include, but are not limited to, metronidazole, amoxicillin, beta-lactamase, aminoglycosides, macrolides, quinolones, fluoroquinolones, cephalosporins, etilomycin, ciprofloxacin, and streptomycin.

In a further embodiment, the compositions of this invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that can be administered with the therapeutic agent of the present invention include glucocorticoids and non-steroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, Pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrin
e), bendazac, benzydamine, bucolome, diphenpyramide, ditazole, emorfazone, guaiazulene, nabumetone, nimesulide, orgothein, oxaseprol, paranyline, perisoxal,
Examples include pifoxime, procazone, proxazole, and tenidap,
It is not limited to these.

In another embodiment, the compositions of this invention are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the therapeutic agents of the invention include antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, and dactinomycin); anti-estrogens (eg, tamoxifen); antimetabolites (eg, fluorouracil, 5). -FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin
, Mercaptopurine, and 6-thioguanine); cytotoxic agents (eg, carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin). , And vincristine sulfate); hormones (eg, medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and test lactone); Nitrogen mustard derivatives (eg mephalen, chlorambucil, mechlorethamine Roger emissions mustard) and thiotepa); steroids and combinations (e.g., betamethasone sodium); and others (
Examples include, but are not limited to, dicarbazine, asparaginase, mitoten, vincristine sulfate, vinblastine sulfate, and etoposide.

In certain embodiments, Therapeutics of the invention are administered in combination with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) or in any combination of the components of CHOP. In another embodiment, the Therapeutic Agents of the invention are administered in combination with Rituximab. In a further embodiment, the therapeutic agents of the invention are Rituxmab and CHOP.
Or with any combination of Rituxmab and CHOP components.

In a further embodiment, the compositions of this invention are administered in combination with a cytokine. ILs include cytokines that can be administered with the therapeutic agents of the invention.
2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13
, IL15, anti-CD40, CD40L, IFN-γ and TNF-α, but are not limited thereto. In another embodiment, the therapeutic agent of the present invention comprises any interleukin (IL-1α, IL-1β, IL-2, IL-3, IL-4.
, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-1
1, IL-12, IL-13, IL-14, IL-15, IL-16, IL-1
7, IL-18, IL-19, IL-20 and IL-21).

In a further embodiment, the compositions of this invention are administered in combination with angiogenic proteins. Angiogenic proteins that may be administered with the compositions of the present invention include Glioma Derived Growth Factor (GDGF) as disclosed in EP-399816.
); Platelet-derived growth factor-A (as disclosed in European Patent EP-682110).
PDGF-A); Platelet Induced Growth Factor-B (PDGF-B) as disclosed in European Patent EP-283217; Placental Growth Factor (PIGF) as disclosed in WO92 / 06194; Hauser et al. , Gorth Fact
ors, 4: 259-268 (1993) placental growth factor-2.
(PIGF-2); vascular endothelial growth factor (VEGF) as disclosed in WO 90/13649; vascular endothelial growth factor-A (VEGF-A) as disclosed in European Patent EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2) as disclosed in International Publication Number WO 96/39515; International Publication Number WO
Vascular Endothelial Growth Factor B-186 (VEGF) as disclosed in 96/26736.
-B186); vascular endothelial growth factor-D (VEGF-D) as disclosed in WO98 / 02543; vascular endothelial growth factor-D (VEGF as disclosed in WO98 / 07832). -D); and German patent DE
Examples include, but are not limited to, vascular endothelial growth factor-E (VEGF-E) as disclosed in 19639601. The above references are incorporated herein by reference.

In a further embodiment, the compositions of this invention are administered in combination with fibroblast growth factor. Fibroblast growth factors that can be administered with the composition of the present invention include FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF.
-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, F
GF-12, FGF-13, FGF-14, and FGF-15 include, but are not limited to.

In a further embodiment, the compositions of this invention are administered in combination with other therapeutic or prophylactic regimes (eg, radiation treatment).

Example 16: Method for Treating Decreased Levels of Polypeptides The present invention relates to a method for treating an individual in need of increasing levels of a polypeptide of the invention in the body, the method comprising: Administering to such an individual a composition comprising a therapeutically effective amount of angiogenic polypeptides or agonists thereof.

Further, it is understood that conditions caused by reduced normal or normal expression levels of angiogenic polypeptides in an individual can be treated by administering the angiogenic polypeptide, preferably in secreted form. . Thus, the invention also provides a method of treating an individual in need of increased levels of angiogenic polypeptides. The method comprises the step of administering to such an individual a pharmaceutical composition comprising an amount of angiogenic polypeptide that increases the level of activity of the angiogenic polypeptide in such individual.

For example, a patient with reduced levels of angiogenic polypeptides may be
The daily dose of 0.1 to 100 μg / kg is taken for 6 consecutive days. Preferably the polypeptide is in secreted form. The exact details of the dosing and formulation-based regimen are:
Provided in Example 15.

Example 17: Method of Treating Elevated Levels of Angiogenic Polypeptides The present invention also provides for treating an individual in need of having reduced levels of angiogenic polypeptides of the present invention in the body. Regarding the method, the method comprises administering to such an individual a composition comprising a therapeutically effective amount of an antagonist of angiogenic polypeptides. Preferred antagonists for use in the present invention are angiogenic polypeptide-specific antibodies.

Antisense technology is used to inhibit the production of angiogenic polypeptides. This technique is one example of a method of reducing the level of angiogenic polypeptides, preferably secreted forms of angiogenic polypeptides, due to various etiologies such as cancer.

For example, in patients diagnosed with abnormally elevated levels of angiogenic polypeptides, antisense polynucleotides were administered at 0.5, 1.0, 1.5, 2.0 and 3
． It is administered intravenously at 0 mg / kg / day for 21 days. If well tolerated, the procedure is repeated after a 7 day washout period. Formulations of antisense polynucleotides are provided in Example 15.

Example 18 Treatment Method Using Gene Therapy—Ex Vivo One method of gene therapy is to implant into a patient fibroblasts capable of expressing angiogenic polypeptides. Fibroblasts are generally obtained from a subject by skin biopsy. The resulting tissue is placed in tissue culture medium and divided into small pieces. The nodule of tissue is placed on the wet surface of the tissue culture flask and approximately 10 pieces are placed in each flask. The flask is inverted and tightly closed, then left overnight at room temperature. After 24 hours at room temperature, the flask is inverted, the tissue mass remains fixed at the bottom of the flask and fresh medium (eg Ham's F12 medium containing 10% FBS, penicillin and streptomycin) is added. The flask is then incubated at 37 ° C for approximately 1 week.

At this point, fresh medium is added and then replaced every few days. After culturing for another two weeks, a monolayer of fibroblasts appears. Trypsinize the monolayer and scale up to a larger flask.

The Moloney murine sarcoma virus long flanking pMV-7 (Ki
rschmeier, P .; T. Et al., DNA, 7: 219-25 (1988)) is digested with EcoRI and HindIII and then treated with calf intestinal phosphatase. The linear vector is fractionated on an agarose gel and purified using glass beads.

The cDNAs encoding the angiogenic polypeptides correspond to the 5'and 3'terminal sequences described in Example 1, respectively, with primers and with appropriate restriction sites and start / stop codons, as required. PCR primers can be used for amplification. Preferably, the 5'primer contains an EcoRI site and the 3'primer contains a HindIII site. Equal amounts of Moloney murine sarcoma virus linear backbone and amplified EcoRI and HindIII fragments were added to T4.
Add together in the presence of DNA ligase. The resulting mixture is maintained under suitable conditions for ligating the two fragments. Then, using the ligation mixture, the bacterial HB
Transform 101. It is then plated on agar containing kanamycin to confirm that the vector has the gene of interest inserted correctly.

Amphoteric pA317 or GP + am12 packaging cells were
Dul with 10% Calf Serum (CS), Penicillin and Streptomycin
Grow to confluent density in tissue culture in becco modified Eagles medium (DMEM). Next, M containing the gene encoding the angiogenic polypeptide
The SV vector is added to the medium and the packaging cells are transduced with the vector. At this time, the packaging cells produce infectious viral particles containing a gene encoding an angiogenic polypeptide (here, the packaging cells are referred to as producer cells).

Fresh medium was added to the transduced producer cells and the medium was then added to 10c.
Harvest from m-plate confluent producer cells. The spent medium containing infectious viral particles is filtered through a Millipore filter to remove detached producer cells and then used to infect fibroblasts. Media is removed from the subconfluent plates of fibroblasts and quickly replaced with media from producer cells. This medium is removed and replaced with fresh medium. If the virus titer is high, virtually all fibroblasts will be infected and no selection is required. If the titer is very low, neo or his
It is necessary to use a retroviral vector with a selectable marker such as Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine if angiogenic proteins are being produced. The engineered fibroblasts are then transplanted into the host either alone or after being grown to confluence on Cytodex 3 microcarrier beads.

Example 19: Gene Therapy Using Endogenous Genes Corresponding to Angiogenic Polynucleotides Another method of gene therapy according to the present invention is the use of endogenous sequences encoding angiogenic polypeptides, eg, U.S. Patent No. 5,641,670 (filed June 24, 1997); International Publication No. WO 96/29411 (published September 26, 1996);
International Publication No. WO 94/12650 (published on August 4, 1994); Kolle
r et al., Proc. Natl. Acad. Sci. USA, 86: 8932-89.
35 (1989); and Zijlstra et al., Nature, 342: 435.
~ 438 (1989), operably linked to the promoter via homologous recombination. The method involves activation of a gene that is present in the target cell but is not expressed in the cell or is expressed at a lower level than desired.

A polynucleotide construct is made that contains a promoter and targeting sequence. This target sequence is homologous to the 5'non-coding sequence of the endogenous gene sequence encoding the angiogenic polypeptide, which is flanked by the promoters. The target sequence is sufficiently close to the 5'end of the gene so that the promoter is operably linked to the endogenous sequence upon homologous recombination. This promoter and targeting sequence
It can be amplified using PCR. Preferably, the amplified promoter is 5 '
It contains different restriction enzyme sites on the ends and the 3'end. Preferably, the 3'end of the first targeting sequence contains the same restriction enzyme sites as the 5'end of the amplified promoter, and the 5'end of the second targeting sequence is the 3'end of the amplified promoter. Contains the same restriction enzyme sites as. The amplified promoter and amplified targeting sequence are digested with the appropriate restriction enzymes followed by treatment with calf intestinal phosphatase. The digested promoter and digested targeting sequence are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for ligation of these two fragments. The construct is size fractionated on an agarose gel, followed by phenol extraction and ethanol precipitation.

In this example, the polynucleotide construct is administered as a naked polynucleotide via electroporation. However, the polynucleotide construct may also be administered with a transfection facilitating agent (eg, liposomes, viral sequences, viral particles, precipitating agents, etc.). Such methods for delivery are known in the art.

Once the cells have been transfected, homologous recombination will occur at the promoter and will be operably linked to the endogenous gene sequence. This results in angiogenic polypeptide expression in this cell. Expression can be detected by immunological staining or any other method known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The obtained tissue is DMEM +
Place in 10% fetal bovine serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the surface of the plastic with nutrient medium. An aliquot of cell suspension is removed for counting and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is mixed with 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137 mM NaCl, 5 mM K).
Cl, 0.7 mM Na ₂ HPO ₄ , 6 mM dextrose). The cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg / ml acetylated bovine serum albumin. This final cell suspension contains approximately 3 × 10 ⁶ cells / ml. Electroporation should be performed immediately after resuspension.

Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the locus where the gene encoding the angiogenic protein is located, the plasmid pUC18 (MBI Fermentas, Amh is used.
erst, NY) is digested with HindIII. The CMV promoter is amplified by PCR with an XbaI site at the 5'end and a BamHI site at the 3'end. Two angiogenic protein non-coding sequences are amplified via PCR: one angiogenic protein non-coding sequence (angiogenic protein fragment 1)
Amplify with a HindIII site at the'end and an XbaI site at the 3'end;
Non-angiogenic protein non-coding sequence (angiogenic protein fragment 2)
Is amplified with a BamHI site at the 5'end and a HindIII site at the 3'end. The CMV promoter and angiogenic protein fragment were loaded with the appropriate enzymes (CMV promoter-XbaI and BamHI; angiogenic protein fragment 1-XbaI; angiogenic protein fragment 2-BamHI.
) Digest and ligate together. The resulting ligation product is digested with HindIII and ligated with the HindIII digested pUC18 plasmid.

Plasmid DNA was loaded onto a sterile cuvette (B
io-Rad). Final DNA concentration is generally at least 120μ
g / ml. Then 0.5 ml of the cell suspension (containing approximately 1.5 × 10 ⁶ cells) is added to the cuvette and the cell suspension and DNA solution are gently mixed. Electroporation is performed using a Gene-Pulser instrument (Bio-Rad). The capacitance and voltage are set to 960 μF and 250-300 V, respectively. Increasing the voltage reduces cell survival, but dramatically increases the proportion of viable cells that stably integrate the introduced DNA into their genome. Given these parameters, the pulse time is about 14-2
0 mSec should be observed.

The electroporated cells are maintained at room temperature for approximately 5 minutes, then the contents of the cuvette are gently removed using a sterile transfer pipette. The cells were placed in a 10 cm dish containing pre-warmed nutrient medium (D containing 15% bovine serum).
MEM) 10 ml directly and incubate at 37 ° C. The next day, the medium was aspirated and replaced with 10 ml of fresh medium and an additional 16-24
Incubate for hours. The engineered fibroblasts were then placed in the host either alone or in cytodex 3 microcarriers.
Inject either after growing to confluency on the beads. Here, the fibroblasts produce the protein product. The fibroblasts can then be introduced into a patient as described above.

Example 20: Method of Treatment Using Gene Therapy-In Vivo Another aspect of the invention is the use of in vivo gene therapy methods to treat disorders, diseases and conditions. This gene therapy method involves the introduction of naked nucleic acid (DNA, RNA, and antisense DNA or RNA) sequences encoding angiogenic polypeptides into an animal to increase or decrease the expression of angiogenic polypeptides. Regarding The polynucleotide encoding the angiogenic polypeptide can be operably linked to a promoter, or any other genetic element required for expression of the angiogenic polypeptide by the target tissue. Techniques and methods for such gene therapy and delivery are known in the art, eg, WO 90/1109.
2, WO98 / 11779; US Pat. Nos. 5,693,622 and 5,705,151.
No. 5,580,859; Tabata et al., Cardiovasc. Res.
35 (3): 470-479 (1997); Chao et al., Pharmacol.
Res. 35 (6): 517-522 (1997); Wolff, Neurom.
uscul. Disord. 7 (5): 314-318 (1997), Schw.
artz et al., Gene Ther. 3 (5): 405-411 (1996);
Tsurumi et al., Circulation 94 (12): 3281-329.
0 (1996) (incorporated herein by reference).

A polynucleotide construct comprising a gene encoding an angiogenic polypeptide comprises:
It may be delivered by any method that delivers the injectable substance to cells of the animal, such as injecting the interstitial space of tissue (heart, muscle, skin, lung, liver, intestine, etc.). Further, the polynucleotide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA refers to any delivery vehicle (viral sequence, viral particle, liposomal formulation, lipofectin, or lipofectin that acts to assist, facilitate, or facilitate entry into cells. (Including a precipitating agent) is not included. However, the polynucleotide encoding the angiogenic polypeptide may also be a liposome formulation (which may be prepared by methods well known to those skilled in the art).
For example, Felgner P. et al. L. (1995) Ann. NY Acad. S
ci. 772: 126-139 and Abdaleh B .; Et al (1995) B
iol. Cell 85 (1): 1-7)).

The polynucleotide vector construct containing the gene encoding the angiogenic polypeptide used in this method of gene therapy is preferably one that does not integrate into the host genome and does not contain sequences that allow replication. Any strong promoter known to those of skill in the art can be used to drive the expression of DNA. Unlike other gene therapy techniques, one major advantage of introducing a naked nucleic acid sequence into a target cell is the transient nature of polynucleotide synthesis in that cell. Studies have shown that non-replicating DNA sequences can be introduced into cells to provide for the production of desired polypeptides for periods of up to 6 months.

A polynucleotide construct comprising a gene encoding an angiogenic polypeptide is
Tissues in animals (muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,
Blood, bone, cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis, ovary, uterus, rectum, nervous system, eye, gland, and connective tissue). The interstitial spaces of this tissue are intercellular fluids, mucopolysaccharide substrates (between reticulum fibers of organ tissue, elastic fibers in walls of blood vessels or chambers, collagen fibers in fibrous tissue), or connective tissue sheath muscle cells. Includes the same matrix in the interior or in the hiatus of the bone. This is likewise the space occupied by circulating plasma and lymphatic fluid of the lymphatic channels. Delivery of muscle tissue to the interstitial space is preferred for the reasons discussed below. They can be conveniently delivered by injection into the tissue containing these cells. They are preferably delivered to and expressed in differentiated, persistent, non-dividing cells, which delivery and expression can be effected on undifferentiated cells or cells that are not fully differentiated (eg, blood stem cells). Or skin fibroblasts). In vivo, muscle cells are particularly qualified for their ability to take up and express polynucleotides.

For injection of naked polynucleotides encoding angiogenic polypeptides, DN
An effective dosage of A or RNA is about 0.05 g / kg body weight to about 50 mg / kg.
In the weight range. Preferably, this dosage is from about 0.005 mg / kg to about 20 mg / kg, and more preferably from about 0.05 mg / kg to about 5.
mg / kg. Of course, as the skilled artisan will appreciate, this dosage will vary according to the tissue site of injection. Suitable and effective dosages of nucleic acid sequences can be readily determined by those of ordinary skill in the art and may depend on the condition being treated and the route of administration. The preferred route of administration is by the parenteral route of injection into the interstitial space of tissues. But,
Other parenteral routes may also be used, including inhalation of aerosol formulations, especially for delivery to the mucosa of the lungs or bronchial tissues, throat, or nose, among others. In addition, naked polynucleotide constructs containing genes encoding angiogenic polypeptides can be delivered to arteries during angioplasty by the catheter used in this procedure.

The dose response effect of infused polynucleotides encoding angiogenic polypeptides in muscle in vivo is determined as follows. Appropriate template DNA for the production of mRNA encoding an angiogenic polypeptide may be prepared using standard recombinant DNA.
Prepare according to methodology. Template DNA, which can be either circular or linear, is either used as naked DNA or complexed with liposomes. The mouse quadriceps is then injected with varying amounts of template DNA.

To 5-6 week old female and male Balb / C mice, 0.3 ml of 2.5%
Anesthetize by intraperitoneal injection of Avertin. A 1.5 cm incision is made in the anterior thigh and the quadriceps are visualized directly. Template DNA encoding angiogenic polypeptides was placed in 0.1 ml of carrier in a 1 cc syringe through a 27-gauge needle for 1 minute, about 0.5 cm from the distal insertion site of the muscle, at approximately 0 knees. Implant at a depth of 0.2 cm. Sutures are made over the injection site for future localization and the skin is closed with stainless steel clips.

After a suitable incubation time (eg 7 days), muscle extracts are prepared by cutting out whole quads. Every 5th 15 μm section of an individual quadriceps is histochemically stained for protein expression. The time course for protein expression can be done in a similar fashion, except that four animals from different mice are harvested at different times. Persistence of DNA encoding angiogenic polypeptides in muscle after injection can be determined by Southern blot analysis after preparation of total cellular DNA and HIRT supernatants from injected and control mice. The results of the above experiments in mice can be used to estimate appropriate dosages and other treatment parameters in humans and other animals using naked DNA encoding angiogenic polypeptides.

Example 21: Angiogenic Protein Transgenic Animals Angiogenic polypeptides can also be expressed in transgenic animals. Mouse, rat, rabbit, hamster, guinea pig, pig, mini pig (mic
ro-pig), goats, sheep, cows and non-human primates (eg, baboons,
Animals of any species, including but not limited to monkeys and chimpanzees), can be used to produce transgenic animals. In certain embodiments, techniques described herein or otherwise known in the art are used for expression of a polypeptide of the invention in humans as part of a gene therapy protocol. Any technique known in the art can be used to introduce a transgene (ie, a polynucleotide of the invention) into an animal to produce a founder line of transgenic animals. Such techniques are described in Pronuclear Microinjection (Patterson et al., Appl. Microbiol.
Biotechnol. 40: 691-698 (1994); Carver et al.,
Biotechnology (NY) 11: 1263-1270 (1993);
Wright et al., Biotechnology (NY) 9: 830-834 (1).
991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989).
); Germline (Van der Putten et al., Proc. Natl. Aca).
d. Sci. USA 82: 6148-6152 (1985)), retrovirus-mediated gene transfer into blastocysts or embryos; gene targeting in embryonic stem cells (Th.
Ompson et al., Cell 56: 313-321 (1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.
3: 1803-1814 (1983)); introduction of the polynucleotide of the present invention using a gene gun (for example, Ulmer et al., Science 259: 1745 (1).
993)); introduction of the nucleic acid construct into embryonic pluripotent stem cells and retransfer of these stem cells into blastocysts; and sperm-mediated gene transfer (Lavitrano et al., Cell 57: 717-723). (1989)
); And the like, but are not limited thereto. For a review of such techniques, see Gordon, "Transge," which is incorporated herein by reference in its entirety.
nic Animals ", Intl. Rev. Cytol. 115: 171-
229 (1989).

Any technique known in the art can be used to generate transgenic clones containing a polynucleotide of the present invention (eg, cultured, quiescently induced embryonic cells, fetal cells or adult cells). Nuclear Transfer of Cell-Derived Enucleated Oocytes to the Nucleus (Campell et al., Nature 380: 64-66 (1996); Wil
mut et al., Nature 385: 810-813 (1997))). The present invention provides transgenic animals that carry the transgene in all their cells, as well as animals that carry the transgene in some (but not all) of the cells (ie, mosaic animals or chimeras). The transgene can be integrated as one transgene or as multiple copies such as concatemers (eg, head-to-head tandem or head-to-tail tandem). This transgene is also described, for example, in the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sc.
i. USA 89: 6232-6236 (1992)) and can be selectively introduced into and activated by specific cell types. The regulatory sequences required for such cell type-specific activation depend on the particular cell type of interest, and they will be apparent to those of skill in the art. Targeting of the gene is preferred when it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene.

Briefly, if such a technique is utilized, a vector containing several nucleotide sequences homologous to the endogenous gene may be transferred to the endogenous gene via homologous recombination with the chromosomal sequence. Is designed to destroy the function of the nucleotide sequence. Transgenes can also be selectively introduced into specific cell types, and thus, for example, Gu et al. (Gu et al., Science.
265: 103-106 (1994)), the endogenous gene is inactivated only in the introduced cell type. The regulatory sequences required for such cell type-specific inactivation depend on the particular cell type of interest, and they will be apparent to those of skill in the art. The contents of each of the documents cited in this section are hereby incorporated by reference in their entireties.

Once a transgenic animal is produced, expression of the recombinant gene can be assayed using standard techniques. Initial screening can be accomplished by Southern blot analysis or PCR techniques to confirm that transgene integration has occurred by analysis of animal tissue. The level of transgene mRNA expression in the tissues of transgenic animals was also determined by Northern blot analysis, in situ hybridization analysis and reverse transcriptase P of tissue samples obtained from the animals.
It can be assessed using techniques including, but not limited to CR (rt-PCR). Samples of transgenic gene expressing tissues can also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once founder animals are generated, they can be crossed, inbred, outbred or crossed to generate colonies of particular animals. Examples of such mating strategies include, but are not limited to: Founder outcrosses with more than one integration site to establish another lineage; additive expression of each transgene. By effect, inbreeding of separate strains to produce composite transgenics expressing higher levels of the transgene; a given routine for both enhancing expression and eliminating the need for screening animals by DNA analysis. Hybridization of heterozygous transgenic animals that produce animals homozygous for the site of integration; crossing of separate homozygous strains to generate complex heterozygous or homozygous lines; and to the desired experimental model Mating to place the transgene on a suitable different background.

The transgenic animals of the present invention are characterized by detailed biological functions of angiogenic polypeptides, studies of conditions and / or disorders associated with aberrant angiogenic polypeptide expression, and such conditions. And / or have applications including, but not limited to, animal model systems useful in screening for compounds that are effective in ameliorating the disorder.

Example 22: Angiogenic Protein Knockout Animals Endogenous angiogenic protein gene expression also encodes angiogenic proteins and / or their promoters using targeted homologous recombination. Can be reduced by inactivating or "knocking out" a gene (eg, Sm
ities et al., Nature 317: 230-234 (1985); Tho
mas and Capecchi, Cell 51: 503-512 (1987).
See Thompson et al., Cell 5: 313-321 (1989); each of which is hereby incorporated by reference in its entirety). For example, a variant, non-functional polynucleotide (or completely unrelated DNA) of the invention flanked by DNA homologous to an endogenous polynucleotide sequence (either the coding or regulatory region of this gene). Sequence) can be used with or without a selectable marker and / or a negative selectable marker to transfect cells expressing a polypeptide of the invention in vivo. In another embodiment,
Techniques known in the art are used to generate knockouts in cells that contain the gene of interest but do not express it. This DNA via targeted homologous recombination
Insertion of the construct results in inactivation of the targeted gene. Such an approach is particularly suited to the research and agricultural fields where modifications to embryonic stem cells may be used to produce animal progeny carrying inactivated targeted genes (eg, T
home and Capecchi 1987 and Thompson 198.
9, see above). However, this approach is suitable for use in humans when recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors, which will be apparent to those skilled in the art. Can be routinely adapted.

In a further embodiment of the invention, a cell genetically engineered to express a polypeptide of the invention, or a cell genetically engineered not to express a polypeptide of the invention (eg, a knockout), Administer to patients in vivo. Such cells can be obtained from patients (ie, animals, including humans) or MHC compatible donors, and can be fibroblasts, bone marrow cells, blood cells (eg lymphocytes), adipocytes, muscle cells, endothelial cells, etc. Can be included, but is not limited to. The cells can be transformed, for example, by transduction (using a viral vector and preferably a vector that integrates the transgene into the cell genome) or transfection procedures (plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Use, including but not limited to the use of an endogenous regulatory sequence associated with the coding sequence and / or the polypeptide of the invention to introduce the coding sequence of the polypeptide of the invention into a cell. To disrupt, genetic engineering is done in vitro using recombinant DNA technology. The coding sequence of the polypeptide of the invention may be placed under the control of a strong constitutive or inducible promoter or promoter / enhancer to achieve expression and preferably secretion of the angiogenic polypeptide. Engineered cells that express and preferably secrete a polypeptide of the invention can be introduced systemically into the patient, eg, in circulation or intraperitoneally.

Alternatively, the cells can be incorporated into a matrix and transplanted into the body (
For example, genetically engineered fibroblasts can be transplanted as part of a skin graft); genetically engineered endothelial cells can be transplanted as part of a lymphoid or vascular graft (eg Anderson et al., US Patent) No. 5,399,349 and Mu
See Lligan and Wilson, US Pat. No. 5,460,959. Each of these is hereby incorporated by reference in its entirety).

If the cells to be administered are non-self or non-MHC compatible cells, they may be administered using well known techniques that interfere with the development of a host immune response against this transduced cell. For example, the cells can be introduced in a capsular form, which does not allow the introduced cells to be recognized by the host immune system, while permitting exchange of components with the immediate extracellular environment.

The angiogenic protein knockout animals of the present invention are characterized by a detailed description of the biological function of angiogenic polypeptides, studies of conditions and / or disorders associated with aberrant expression, and such conditions and / or It has applications including, but not limited to, animal model systems useful in screening compounds that are effective in ameliorating the disorder.

Example 23 Biological Effects of Angiogenic Polypeptides Fibroblast and Endothelial Cell Assays Human lung fibroblasts were cloned.
(San Diego, CA) and maintained in growth medium from Clonetics. Dermal microvascular endothelial cells were applied to Cell Applicati
ons (San Diego, CA). For proliferation assays, human lung fibroblasts and dermal microvascular endothelial cells can be cultured at 5,000 cells / well in 96 well plate growth medium for 1 day. This cell is then 0.1%
Incubate in BSA basal medium for 1 day. After replacing the medium with fresh 0.1% BSA medium, the cells are incubated with the test protein for 3 days. Alam
ar Blue (Alamar Biosciences, Sacrament
o, CA) to each well to a final concentration of 10%. 4 this cell
Incubate for hours. Cell viability is measured by reading on a CytoFluor fluorescence reader. For the PGE ₂ assay, human lung fibroblasts were added to 96
Culture in well plates for 1 day at 5,000 cells / well. 0.1% BS
After converting the medium to A basal medium, cells are incubated with FGF-2 or angiogenic polypeptide with or without IL-1α for 24 hours.
Supernatants were collected and EIA kit (Cayman, Ann Arbor, MI.
) For PGE ₂ . For the IL-6 assay, human lung fibroblasts are cultured in 96-well plates for 1 day at 5,000 cells / well. After converting the medium to 0.1% BSA basal medium, cells are incubated with FGF-2 or angiogenic polypeptides with or without IL-1α for 24 hours. Supernatant was collected and ELISA kit (Endogen,
(Cambridge, MA) for IL-6.

Human lung fibroblasts were cultured with FGF-2 or angiogenic polypeptides in basal medium for 3 days, then Alam to assess the effect on fibroblast proliferation.
Add ar Blue. FGF-2 should show a stimulation of 10 to 2500 ng / ml which could be used comparable to stimulation with angiogenic polypeptides.

Example 24 Effect of Angiogenic Polypeptides on Proliferation of Vascular Endothelial Cells On day 1, human umbilical vein endothelial cells (HUVEC) were supplemented with 4% fetal bovine serum (FBS).
), 16 units / ml heparin, and 50 units / ml endothelial cell growth supplement (ECGS, Biotechnique, Inc.).
Seed in medium at a density of 2-5 × 10 ⁴ cells per 35 mm dish. On day 2, this medium was added to M199 containing 10% FBS, 8 units / ml heparin.
Replace with. Angiogenic polypeptides and positive controls (eg VEGF
And basic FGF (bFGF)) is added at various concentrations. On days 4 and 6, the medium is replaced. On day 8, cell numbers are determined using a Coulter Counter.

An increase in HUVEC cell number indicates that angiogenic polypeptides can proliferate vascular endothelial cells.

The studies described in this example tested activity of angiogenic polypeptides. However, one of skill in the art would readily modify the exemplified studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), agonists and / or antagonists of angiogenic polypeptides. You can

Example 25: Stimulatory Effect of Angiogenic Polypeptides on Proliferation of Vascular Endothelial Cells A colorimetric assay using the electron-coupled reagent PMS (phenazine methosulfate) for evaluation of mitogenic activity of growth factors. An analytical MTS (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) 2H-tetrazolium) assay was performed (CellTiter).
96 AQ, Promega). Cells are seeded in 96-well plates (5,000 cells / well) in 0.1 mL serum-supplemented medium and allowed to attach overnight. 0
． After 12 hours serum starvation in 5% FBS, conditions (bFGF, VEGF165 or angiogenic polypeptides in 0.5% FBS) with or without Heparin (8 U / ml) were added to the wells for 48 hours. To do. 20mg MTS / PM
S mixture (1: 0.05) is added per well and allowed to incubate at 37 ° C. for 1 hour, after which the absorbance at 490 nm is measured in an ELISA plate reader. Background absorption of control wells (medium, no cells) is subtracted, and 7 wells are run in parallel for each condition. Leak et al., In Vitro Cell. Dev. Biol. 30A: 51
2-518 (1994).

The studies described in this example tested activity of angiogenic polypeptides. However, one of skill in the art would readily modify the exemplified studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), agonists and / or antagonists of angiogenic polypeptides. You can

Example 26: Inhibition of PDGF-induced vascular smooth muscle cell proliferation stimulating effect HAoSMC proliferation can be measured, for example, by BrdUrd incorporation. Briefly, subconfluent quiescent cells grown on 4-chamber slides were
Transfect with RP or FITC labeled AT2-3LP. The cells are then pulsed with 10% bovine serum and 6 mg / ml BrdUrd. 24
After hours, immunocytochemistry is performed by using the BrdUrd staining kit (Zymed Laboratories). Briefly, the cells are incubated with biotinylated mouse anti-BrdUrd antibody for 2 hours at 4 ° C. after exposure to denaturing solution, followed by streptavidin-peroxidase and diaminobenzidine.

After counterstaining with hematoxylin, the cells are mounted for microscopic examination and Brd Urd-positive cells are counted. The BrdUrd coefficient is calculated as the ratio of BrdUrd-positive cells to the total cell number. Furthermore, BrdUrd staining (for individual cells by simultaneous use of bright and dark UV fluorescence illumination (
Simultaneous detection of nuclear) and FITC uptake (cytoplasm) is performed. Hayashi
da et al. Biol. Chem. 6: 271 (36): 21985-2199.
2 (1996).

The studies described in this example tested the activity of angiogenic polypeptides.
However, one of skill in the art would readily modify the exemplified studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), agonists and / or antagonists of angiogenic polypeptides. You can

Example 27: Stimulation of Endothelial Migration This example can be used to explore the potential of angiogenic polypeptides to stimulate lymphoid endothelial cell migration.

The Endothelial Cell Migration Assay is Performed Using a 48-Well Microchemotactic Chamber (Neuroprobe Inc., Cabin John, MD; Falk).
W., J. et al. Immunological Methods 1980; 33:
239-247). Polycarbonate filter without polyvinylpyrrolidone, which has a pore size of 8 μm (Nucleopore Corp. Cam.
(Bridge, MA) coated with 0.1% gelatin for at least 6 hours at room temperature,
Then it is dried under sterile air. Test substances are diluted to the appropriate concentration in M199 supplemented with 0.25% bovine serum albumin (BSA) and 25 μl of the final dilution is placed in the bottom chamber of the modified Boyden device. Subconfluent, early passage (2-6) HUVEC or BMEC cultures are washed and trypsinized for the minimum time required to achieve cell detachment. After placing the filter between the bottom and top chamber 50 μl M19 containing 1% FBS
2.5 × 10 ⁵ cells suspended in 9 are seeded in the upper compartment. The device was then incubated for 5 hours at 37 ° C. in a chamber moistened with 5% CO ² to allow the cells to migrate. After the incubation period, the filter is removed and the top side of the filter with non-migrated cells is scraped with a rubber policeman. The filter is fixed with methanol and stained with Giemsa solution (Diff-Quick, Baxter, Mc.
(Grow Park, IL). Migration is quantified by counting 3 random high power fields (40x) of cells in each well. Then run all groups in quadruplicate.

The studies described in this example tested activity of angiogenic polypeptides.
However, one of skill in the art would readily modify the exemplified studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), agonists and / or antagonists of angiogenic polypeptides. You can

Example 28: Stimulation of nitric oxide production by endothelial cells The release of nitric oxide by vascular endothelium is believed to be a mediator of vascular endothelial relaxation. Therefore, the activity of angiogenic polypeptides can be assayed by measuring nitric oxide production by endothelial cells in response to angiogenic polypeptides.

Nitric oxide was starved for 24 hours, followed by varying levels of positive control (
For example, in a 96-well plate of confluent microvascular endothelial cells after 4 hours of exposure to VEGF-1) and angiogenic polypeptides. Nitric oxide in the medium is quantified by using the Griess reagent to measure the total amount of nitrite after reduction of nitric acid derived from nitric oxide by nitrate reductase. The effect of angiogenic polypeptides on nitric oxide release is tested in HUVEC.

Briefly, NO release from cultured HUVEC monolayers was measured using a NO meter (Iso
-NO, World Precision Instruments Inc.
) (1049) connected to a NO-specific polarographic electrode.
Calibration of the NO element is performed according to the following formula: 2KNO ₂ + 2KI + 2H ₂ SO ₄ 62NO + I ₂ + 2H ₂ O + K ₂ SO _{4 A} standard calibration curve is graded in a calibration solution containing KI and H ₂ SO _4. Concentration of KNO ₂ (0, 5, 10, 25, 5
0, 100, 250, and 500 nmol / L). The specificity of the Iso-NO electrode for NO is pre-determined by measuring NO from authentic NO gas (1050). The culture medium is removed and the HUVECs are washed twice with Dulbecco phosphate buffered saline. The cells were then treated with 5 ml of filtered Krebs in a 6-well plate.
-Dip in the Henseleit solution, and place the cell plate in a slide warmer (Lab Line Instrumen) to maintain the temperature at 37 ° C.
ts Inc. ) Hold. The NO sensor probe is inserted vertically into the well, keeping the tip of the electrode 2 mm below the surface of the solution before adding different conditions. S
-Nitrosoacetylpenicillamine (SNAP) is used as a positive control. The amount of NO released is expressed as picomolar concentration per 1 × 10 ⁶ endothelial cells. All reported values are the average of 4-6 measurements in each group (number of cell culture wells). Leak et al., Biochem. and Biophys. Res.
Comm. 217: 96-105 (1995).

The studies described in this example tested activity of angiogenic proteins.
However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), agonists, and / or antagonists of angiogenic polypeptides. obtain. Example 29: Effect of angiogenic polypeptides on cord formation in angiogenesis Another process in angiogenesis is cord (c) characterized by endothelial cell differentiation.
ord) formation. This bioassay measures the ability of microvascular endothelial cells to form capillary-like structures (hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) were used as proliferating cells (passage 2) in Cell.
Applications Inc. Buy from Cell Applicat
Cultures in Ions' CADMEC Growth Medium and used at passage 5. For in vitro angiogenesis assay, wells of a 48-well cell culture plate were placed in Cell Applications'Attachment.
Use a Factor Medium (200 ml / well) at 37 ° C for 3
Coat for 0 minutes. CADMECs are seeded in wells coated with 7,500 cells / well and cultured overnight in Growth Medium. Then this Gr
owth Medium with control buffer or angiogenic polypeptide (
300 mg Cell Applicati containing 0.1-100 ng / ml)
Replace with ons' Chord Formation Medium, and culture the cells for an additional 48 hours. The number and length of the capillary-like chords are calculated by Boeckel.
Quantification through the use of an ER VIA-170 video image analyzer. All assays were done in triplicate.

Commercially available (R & D) VEGF (50 ng / ml) is used as a positive control. b-estradiol (1 ng / ml) is used as a negative control. Appropriate buffer (no protein) is also used as a control. The studies described in this example tested activity of angiogenic proteins. However, one of ordinary skill in the art can readily modify the illustrated studies to determine the activity of a polynucleotide encoding an angiogenic polypeptide (eg, gene therapy), the activity of an agonist and / or antagonist of the angiogenic polypeptide. Can be tested.

Example 30: Angiogenic effect on chicken chorioallantoic membrane Chori chorioallantoic membrane (CAM) is a well established system for testing angiogenesis. Angiogenesis in the CAM can be easily visualized and quantified. The ability of angiogenic polypeptides to stimulate angiogenesis in the CAM can be tested.
Fertilized eggs of White Leghorn chicken (Gallus gallus) and Japanese quail (Coturnix couturnix),
Incubate at 37.8 ° C and 80% humidity. 16 day old chicken differentiated CAM and 13 day old quail embryos are studied in the following manner. On the fourth day of development, windows are made in the eggshell of chicken eggs. The embryos are examined for normal development and the eggs are sealed with Cellotape®. These are further incubated until day 13. Thermanox cover glass (Nunc, Napervi
Ile, IL) is cut into discs with a diameter of about 5 mm. Sterile and salt-free growth factor is dissolved in distilled water, and about 3.3 mg / 5 ml is pipetted onto the disc. After air drying, the inverted disc is applied to the CAM. After 3 days, the specimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsed with 0.12M sodium cacodylate buffer. These are photographed using a stereomicroscope [Wild M8] and embedded for semi-thin and ultra-thin sectioning as described above. Controls are performed with the carrier disc only.

The studies described in this example tested angiogenic protein activity. However, one of ordinary skill in the art can readily modify the illustrated studies to determine the activity of a polynucleotide encoding an angiogenic polypeptide (eg, gene therapy), the activity of an agonist and / or antagonist of an angiogenic polypeptide. You can test.

Example 31: Angiogenesis Assay Using Matrigel Implants in Mice An in vitro angiogenesis assay of angiogenic polypeptides was performed using a preexisting capillary network-like, mouse extracellular matrix material (Matrigel). ) To determine the ability to form new vessels in the implanted capsules. Liquid M at 4 ℃
It is mixed with atrigel and then the mixture is injected subcutaneously into mice, where the mixture solidifies. After 7 days, Matrigel's solid "plugs" are removed and tested for the presence of new blood vessels. Matrigel, Bec
ton Dickinson Labware / Collaborative
Purchase from Biomedical Products.

Matrigel material is a liquid when thawed at 4 ° C. This Matrix
1 is mixed with angiogenic polypeptide at 150 ng / ml at 4 ° C. and drawn into a cold 3 ml syringe. Female C57B1 / 6 mice, approximately 8 weeks old, are injected with a mixture of Matrigel and experimental protein at two sites on the midventral aspect of the abdomen (0
． 5 ml / site). After 7 days, the mice were sacrificed by cervical dislocation and Matrig
Remove the el plug and wash (ie, remove all attached membrane and fibrous tissue). Fix the same whole plug in neutral buffered 10% formamide,
After embedding in paraffin and staining with Masson's Trichrome, it is used to prepare sections for histological examination. Process cross sections from three different regions of each plug. Selected sections are stained for the presence of vWF. The positive control for this assay was bovine basic FGF (150n
g / ml). Matrigel alone is used to determine the basal level of angiogenesis.

The studies described in this example tested angiogenic protein activity. However, one of ordinary skill in the art can readily modify the illustrated studies to determine the activity of a polynucleotide encoding an angiogenic polypeptide (eg, gene therapy), the activity of an agonist and / or antagonist of an angiogenic polypeptide. You can test.

Example 32: Rescue of ischemia in a rabbit lower extremity model To study the in vivo effect of angiogenic polypeptides on ischemia, a rabbit hindlimb ischemia model was previously described (Takeshita, S et al Am J
． Pathol 147: 1649-1660 (1995)) by surgical removal of one femoral artery. Resection of the femoral artery results in retrograde propagation of thrombus and occlusion of the external iliac artery. As a result, blood flow to the ischemic limb is dependent on collateral vessels originating from the internal iliac artery (Takeshita et al. Am J. Pathol 1
47: 1649-1660 (1995)). 10 day intervals allow post-surgical recovery of rabbits and development of endogenous collateral vessels. On the 10th day after surgery (day 0),
After performing a baseline angiography, the internal iliac artery of the ischemic limb was treated with 500 mg naked expression plasmid containing the angiogenic polypeptide (Riessen, R et al.
Hum Gene Ther. 4: 749-758 (1993); Lecler.
c, R et al. Clin. Invest. 90: 936-944 (1992)).
Transfect by arterial gene transfer technique using a hydrogel coated balloon catheter as described in. DN encoding an angiogenic polypeptide
When A is used for treatment, a single bolus of 500 mg of angiogenic polypeptide or control is delivered through the infusion catheter for 1 minute into the internal iliac artery of the ischemic limb. On day 30, various parameters are measured in these rabbits: (a) BP ratio-ischemic limb systolic blood pressure to normal limb systolic blood pressure ratio; (b).
Blood Flow and Reflux-Stop FL: Blood Flow During Non-Expanding State, and Maximum FL: Blood Flow During Full Diastolic State (which is also an Indirect Measurement of Vascular Volume), and Reflux is Maximum It is reflected in the ratio of FL: stop FL; (c) angiographic value (angiograph)
ic Score) -This is measured by angiography of the collateral vessels. Scores are determined by the percentage of circles in the overlaying grid (using transverse opacified arteries divided by the total number m of rabbit thighs); (d) capillary density-number of collateral capillaries. , Determined in light microscopy sections obtained from the hind limbs.

The studies described in this example tested angiogenic protein activity. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), the activity of agonists and / or antagonists of angiogenic polypeptides. You can

(Example 33: Effect of the polypeptide of the present invention on vasodilation) Since dilation of vascular endothelium is important for lowering blood pressure, spontaneous hypertensive rats (S
The ability of angiogenic polypeptides in HR) to influence blood pressure is tested. Increasing doses (0, 10, 30, 100, 300, and 900 mg / kg) of angiogenic polypeptides are administered to 13-14 week old spontaneously hypertensive rats (SHR). Data are expressed as mean +/- SEM. Statistical analysis is performed using a two-tailed t-test and statistical significance is defined as p <0.05 vs. response to buffer alone.

The studies described in this example tested angiogenic protein activity. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), the activity of agonists and / or antagonists of angiogenic polypeptides. You can

Example 34: Rat ischemic skin flap model Evaluation parameters include skin blood flow, skin temperature, and factor VIII immunohistochemistry or endothelial alkaline phosphatase reaction. Expression of angiogenic proteins during cutaneous ischemia is studied using in situ hybridization.

[0678]   The work in this model is divided into three parts:   a) Ischemic skin   b) Ischemic skin wound   c) Normal wound.

The experimental protocol includes: a) 3 × 4 cm single stalk full thickness single pedicles.
A full-thickness random skin flap (a muscle flap over the lower back of the animal) is generated.

B) Make an excisional wound on ischemic skin (skin flap) (diameter 4-6 mm) c) Excisional wound (0, 1, 2 after wounding) with different dose ranges of angiogenic polypeptides. Local treatment on days 3, 4): 1 mg-100 mg d) for histological, immunohistochemical, and in situ studies,
Wound tissue is collected 3, 5, 7, 10, 14, and 21 days after wounding.

The studies described in this example tested angiogenic protein activity. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), the activity of agonists and / or antagonists of angiogenic polypeptides. You can

Example 35: Peripheral Arterial Disease Model Angiogenic treatment with angiogenic polypeptides is a novel therapeutic strategy to obtain restoration of blood flow around ischemia in the case of peripheral arterial disease. . The experimental protocol includes: a) One unilateral femoral artery is ligated to create the hindlimb ischemic muscle, the other unilateral hindlimb serves as a control.

B) Angiogenic protein in a dosage range of 20 mg to 500 mg intravenously and / or intramuscularly three times per week (probably more) for 2-3 weeks,
To deliver.

C) This ischemic muscle tissue is collected at 1, 2, and 3 weeks after ligation of the femoral artery for analysis of angiogenic protein expression and histology. The biopsy is also
On the normal muscle of the other contralateral hind limb.

The studies described in this example tested angiogenic protein activity. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), the activity of agonists and / or antagonists of angiogenic polypeptides. You can

Example 36: Ischemic Myocardial Disease Model Angiogenic polypeptides are evaluated as potent mitogens that can stimulate collateral vessel development and reconstitute new blood vessels after coronary artery occlusion. . Changes in the expression of angiogenic proteins will be investigated in situ. The experimental protocol includes: a) The heart is exposed via a left thoracotomy in the rat. Immediately, the left coronary artery,
Occlusate with fine suture (6-0) and close the rib cage.

B) Angiogenic proteins in a dosage range of 20 mg to 500 mg intravenously and / or intramuscularly three times per week (probably more) for 2-4 weeks,
To deliver.

C) Thirty days after surgery, the heart is removed and cross sectioned for morphometry and in situ analysis.

The studies described in this example tested angiogenic protein activity. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), the activity of agonists and / or antagonists of angiogenic polypeptides. You can

Example 37: Rat Corneal Wound Healing Model This animal model shows the effect of angiogenic polypeptides on neovascularization. The experimental protocol includes: a) making a long incision of 1-1.5 mm from the center of the cornea into the stromal layer b) inserting a spatula under the lip of the incision facing the outer edge of the eye c ) Make a pocket (the base of which is 1-1.5 mm from the edge of the eye) d) Place pellets containing 50 ng-5 μg (ug) of angiogenic polypeptide in the pocket e ) Treatment with angiogenic polypeptides may also be applied topically to corneal wounds within a dosage range of 20 mg to 500 mg (daily treatment for 5 days).

The studies described in this example tested angiogenic protein activity. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), the activity of agonists and / or antagonists of angiogenic polypeptides. You can

Example 38: Diabetic Mice and Glucocorticoid Impaired Wound Healing Model A. Diabetes db + / db + Mouse Model To demonstrate that angiogenic polypeptides promote the healing process, wound healing. The genetically diabetic mouse model of is used. The full thickness wound healing model in db + / db + mice is a well characterized, clinically relevant, and reproducible model of impaired wound healing. Healing of diabetic wounds depends on granulation tissue formation and re-epithelialization rather than contraction (Gartner, MH et al., J. Surg. Res. 52: 389 (
1992); Greenhalgh, D .; G. Et al., Am. J. Pathol. 1
36: 1235 (1990)).

This diabetic animal has many of the characteristic properties observed in type II diabetes mellitus. Homozygous (db + / db +) mice have their normal heterozygous (
db + / + m) obese compared to littermates. Mutant diabetes (db + / db +)
Mice carry a single autosomal recessive mutation on chromosome 4 (db +) (C
oleman et al., Proc. Natl. Acad. Sci. USA 77:28
3-293 (1982)). Animals exhibit polyphagia, polydipsia, and polyuria. Mutant diabetic mice (db + / db +) have elevated blood glucose, elevated or normal insulin levels, and suppressed cell-mediated immunity (Ma.
ndel et al. Immunol. 120: 1375 (1978); Debra.
y-Sachs, M .; Et al., Clin. Exp. Immunol. 51 (1): 1
-7 (1983); Leiter et al., Am. J. of Pathol. 114:
46-55 (1985)). Peripheral neuropathy, myocardial complications, and microvascular injury,
Basement membrane thickening and glomerular filtration abnormalities have been described in these animals (N
orido, F.F. Et al., Exp. Neurol. 83 (2): 221-232 (1
984); Robertson et al., Diabetes 29 (1): 60-67.
(1980); Giacomelli et al., Lab Invest. 40 (4):
460-473 (1979); Coleman, D .; L. Diabetes 3
1 (Supplement): 1-6 (1982)). These homozygous diabetic mice develop hyperglycemia and are resistant to insulin, similar to human type II diabetes (Mandel et al., J. Immunol. 120: 1375-1377).
(1978)).

The characteristics observed in these animals suggest that the healing in this model may be similar to that observed in human diabetes (Greenhalgh).
Et al., Am. J. of Pathol. 136: 1235-1246 (1990)
).

Genetically diabetic female C57BL / KsJ (db + / db +) mice and their non-diabetic (db + / + m) heterozygous littermates were used in this study (J
acksson Laboratories). These animals are purchased at 6 weeks of age. The study will start at 8 weeks of age. Animals are housed individually and given food and water ad libitum. All operations are performed using aseptic technique. This experiment, Human Ge
nome Sciences, Inc. Institutional Ani
mal Care and Use Committee and the G
uidelines for the Care and Use of La
Follow the Animal Health rules and guidelines.

The wound protocol was based on previously reported methods (Tsuboi, R. and Rif.
kin, D.M. B. J. Exp. Med. 172: 245-251 (1990)
). Briefly, on the day of wounding, animals were treated with A dissolved in deionized water.
Anesthetize with intraperitoneal injection of vertin (0.01 mg / mL), 2,2,2-tribromoethanol and 2-methyl-2-butanol. The dorsal area of the animal is shaved and the skin is washed with 70% ethanol solution and iodine. The surgical area is dried with sterile gauze before wounding. Then, an 8 mm full-thickness wound
It is prepared using a Keyes tissue punch. Immediately after wounding, the surrounding skin is gently stretched to remove the spread of the wound. The wound is left open during the experiment. The application of treatment is topically applied for 5 consecutive days from the day of wounding. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

The wounds are visually inspected and photographed at a fixed distance on the day of surgery and at 2 day intervals thereafter. Wound closure is determined by daily measurements on days 1-5 and 8. Calibrated wounds Jameson calipers (c
aliper) and measure horizontally and vertically. A wound is considered healed when granulation tissue is no longer visible and the wound is covered by continuous epithelium.

The angiogenic polypeptide is administered in the vehicle for 8 days with different dosing ranges (4 mg to 500 mg per wound per day) of the angiogenic polypeptide. The vehicle control group received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then collected for histology and immunohistochemistry. Tissue specimens are placed in 10% neutral buffered formalin in a tissue cassette between biopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls): 1) vehicle placebo control, 2) angiogenic polypeptides.

Wound closure is analyzed by measuring the area on the horizontal and vertical axes and obtaining the total area of the wound. Contraction is then assessed by establishing the difference between the area of the initial wound (day 0) and the area after treatment (day 8). This wound area on day 1 is 64 mm ² (size corresponding to the skin punch). Calculations are performed using the following formula: [Open Area on Day 8]-[Open Area on Day 1] / [Open Area on Day 1] Specimens are fixed in 10% buffered formalin and paraffin. The embedded mass is cut perpendicular to the wound surface (5 mm) and cut using a Reichert-Jung microtome. Routine hematoxylin-eosin (H & E) staining is performed on transverse sections of bisected wounds. Histological examination of wounds is used to assess whether the healing process and morphological appearance of repaired skin have been modified by treatment with angiogenic polypeptides. This assessment includes verification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization, and epidermal maturation (Greenhalgh, DG et al. Am. J. Pathol. 136: 12).
35 (1990)). Blind observer with graduated lens micrometer
used by the N. Observer.

Tissue sections are also immunohistochemically stained with a polyclonal rabbit anti-human keratin antibody using the ABC Elite detection system. Human skin is used as a positive tissue control, while non-immune IgG is used as a negative control. Keratinocyte proliferation is determined by assessing the extent of wound re-epithelialization using a calibrated lens micrometer.

Proliferating Cell Nuclear Antigen / Cyclin (PCNA) in Skin Preparations
Demonstrate by using anti-PCNA antibody (1:50) in the ite detection system. Human colon cancer can serve as a positive tissue control, and human brain tissue can be used as a negative tissue control. Each specimen contains sections with omission of primary antibody and replacement with non-immune mouse IgG. The rank of these sections is based on the extent of proliferation on a scale of 0-8 (lower scale reflecting slight proliferation to higher side reflecting vigorous proliferation).

Experimental data are analyzed using a one-tailed t-test. A p-value of <0.05 is considered significant.

B. Steroidopathic Rat Model Inhibition of wound healing by steroids has been well documented in various in vitro and in vivo systems (Wahl, SM, Glucocorticoi).
ds and Wound healing: Anti-Inflammato
ry Steroid Action: Basic and Clinical
Aspects. 280-302 (1989); Wahl, S .; M. Et al, J
． Immunol. 115: 476-481 (1975); Werb, Z .; ,
J. Exp. Med. 147: 1684-1694 (1978)). Glucocorticoids inhibit angiogenesis, vascular permeability (Ebert, RH et al.,
An. Intern. Med. 37: 701-705 (1952)), fibroblast proliferation, and collagen synthesis (Beck, L.S. et al., Grows Fact.
ors. 5: 295-304 (1991); Haynes, B .; F. Et al., J. C
lin. Invest. 61: 703-797 (1978)), as well as causing a transient reduction in circulating monocytes (Haynes, BF.
Et al., J. Clin. Invest. 61: 703-797 (1978); Wah.
l, S. M. , "Glucocorticoids and wound he
"aling": Antiflammery Steroid Acti
on: Basic and Clinical Aspects, Academ
Wound healing is delayed by ic Press, New York, 280-302 (1989)). Systemic administration of steroids for impaired wound healing
This is a well-established phenomenon in rats (Beck, L.S. et al., Grow.
th Factors. 5: 295-304 (1991); Haynes, B.
． F. Et al., J. Clin. Invest. 61: 703-797 (1978).
Wahl, S .; M. , "Glucocorticoids and woun
d healing ": Antiflammery Steroid
Action: Basic and Clinical Aspects, Ac
academic Press, New York, 280-302 (1989).
Pierce, G .; F. Et al., Proc. Natl. Acad. Sci. USA
86: 2229-2233 (1989)).

To demonstrate that angiogenic polypeptides can accelerate the healing process, multiple angiogenic polypeptides for full-thickness cutaneous wounds in rats, where healing is impaired by systemic administration of methylprednisolone. Evaluate the effect of topical application of.

Young adult male Sprague Dawley rats (weight 250-300 g)
) (Charles River Laboratories) is used in this example. The animals are purchased at 8 weeks of age. The start of the study is 9 weeks of age. The healing response in rats was determined by systemic administration of methylprednisolone (17 mg /
kg / rat, intramuscular). Keep animals individually and, if appropriate,
Give food and water. All operations are performed using aseptic technique. This research, Hum
Institutional of an Genome Sciences, Inc.
Animal Care and Use Committee and t
he Guidelines for the Care and Use o
f Follow the rules and guidelines of Laboratory Animals.

The wound protocol follows Section A above. On the day of wounding, the animals were given ketamine (5 m
g / kg) and xylazine (5 mg / kg) by intramuscular injection. The dorsal area of the animal is shaved and the skin is washed with 70% ethanol and iodine solution. The surgical area is dried with sterile gauze before wounding. 8 mm full thickness wounds are created using a Keyes tissue punch. The left side of the wound is opened during the experiment. The application of the test substance is given topically once a day for 7 consecutive days starting from the day of wounding and subsequent administration of methylmethylprednisolone. Prior to treatment, the wound is gently washed with sterile saline and gauze sponge.

Wounds are visually inspected and photographed at a fixed distance on the day of wounding and at the end of treatment. Wound closure is determined by daily measurements on days 1-5 and on day 8. Wounds are measured horizontally and vertically using a calibrated Jameson caliper. A wound is considered healed when granulation tissue is no longer visible and the wound is covered by continuous epithelium.

The angiogenic polypeptides are administered in the vehicle for 8 days with different dose ranges of angiogenic polypeptides (4 mg to 500 mg per wound per day).
The vehicle control group received 50 mL of vehicle solution. Animals are euthanized on day 8 with an intraperitoneal injection of sodium pentobarbital (300 mg / kg). The wound and surrounding skin are then collected for histology. The tissue specimens are 10% in a tissue cassette between biopsy sponges for further processing.
Place in neutral buffered formalin.

Four groups of 10 animals each (5 with methylmethylprednisolone and 5 without glucocorticoid) are evaluated: 1) untreated group, 2) vehicle placebo control, 3) vascular. Forming polypeptide treatment group.

Wound closure is analyzed by measuring the area on the vertical and horizontal axes and obtaining the total area of the wound. Closure is then assessed by establishing the difference between the area of the initial wound (day 0) and the area after treatment (day 8). This wound area on day 1 is 64 mm ² (size corresponding to the skin punch). The calculation is performed using the following formula: [Open area on day 8]-[Open area on day 1] / [Open area on day 1].

Specimens are fixed in 10% buffered formalin and paraffin-embedded sections are cut perpendicular to the wound surface (5 mm) and cut using an Olympus microtome. Routine hematoxylin-eosin (H & E) staining is performed on bisection wound cross sections. Histological examination of wounds allows evaluation of whether the healing process and morphological appearance of repaired skin was improved by angiogenic polypeptides. Blind observer with graduated lens micrometer
used by the wounded observer to determine the gap distance of the wound.

Experimental data is analyzed using a one-tailed t-test. A p-value of <0.05 is considered significant.

The studies described in this example tested activity on angiogenic proteins. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of polypeptides that encode angiogenic polypeptides (eg, gene therapy), agonists and / or antagonists of angiogenic polypeptides. obtain.

Example 39: Lymphedema Animal Model The purpose of this experimental approach was to identify lymphangiogenesis (lymph) in the rat hind limb.
to develop a suitable and consistent model of lymphedema to test the therapeutic effects of angiogenic polypeptides in reestablishing the vascular and lymphatic circulation. Efficacy is measured by quantifying the amount of swelling volume in the affected limb, lymphatic vasculature, quantifying the amount of total plasma proteins, and histopathology. Acute lymphedema is observed for 7-10 days. Perhaps more importantly, the chronic progression of edema lasts up to 3-4 weeks.

Prior to beginning surgery, blood samples are drawn for protein concentration analysis.
Male rats (weighing approximately 350 g) are dosed with pentobarbital. Then, the right limb is shaved from the knee to the hip joint. Wipe the shaved area with gauze soaked in 70% EtOH. Blood is drawn for serum total protein testing. After measuring the circumference and volume, mark two measurement levels (0.5 cm above the heel, midpoint of the back of the foot) and then inject the dye into the foot. The endothelium of both right and left dorsal foot is injected with 0.05 ml of 1% Evan's Blue. Then, after the dye is injected into the paw, circumference measurement and volume measurement are performed.

Using the knee joint as a landmark, a limb groin incision is made in an annulus so that the femoral vessels are positioned. The flaps are dissected and separated using forceps and hemostats. After positioning the femoral vessel, lymphatic vessels that run alongside and below the vessel are positioned. The major lymphatic vessels in this area are then electrocoagulated or suture ligated.

Using a microscope, the muscles of the back of the limb (semitendinosus)
And around the adductor muscle). The popliteal lymph nodes are then positioned.
The two proximal lymphatic vessels and two distal lymphatic vessels as well as the distal blood supply of the popliteal node are ligated with sutures. The popliteal lymph nodes and any associated adipose tissue are then removed by cutting connective tissue.

Take care to manage any mild bleeding that occurs with this procedure. After occlusion of lymphatic vessels, flaps are placed on the liquid skin (Vetbond) (AJ Buck).
Seal by using. Separate skin edges are sealed to the underlying muscle tissue leaving a gap of about 0.5 cm around the leg. The skin may also be anchored by suturing to the underlying muscle when needed.

To avoid infection, animals are individually housed with mesh (no bedding). Recovered animals are checked daily through the optimal edema peak. This peak typically occurs up to 5-7 days. The plateau edema peak is then observed. To assess the intensity of lymphedema, the circumference and volume of two designated locations on each limb are measured before the procedure and daily for 7 days. The effect of plasma proteins on lymphedema is determined, and it is also investigated whether protein analysis is a useful test perimeter. The weights of both control and edematous limbs are evaluated at two points. The analysis is performed in a blind manner.

Circumference measurement: Under short-term gas anesthesia to prevent movement of the limb, the circumference of the limb is measured using a tape measure. Measurements are made on the talar and dorsal paw by two different persons and then these two readings are averaged. Readings are taken from both control and edematous limbs.

Volumetric: On the day of surgery, animals are anesthetized with pentobarbital and tested prior to surgery. For daily volume measurements, animals are placed under short-term halothane anesthesia (rapid immobilization and quick recovery), both legs shaved, and the legs marked equally with water-resistant markers. The legs are first soaked in water, then soaked in the device to each significant level, and then Buxco hydrops software (Chen / Victor
or)). The data is recorded by one person, while the other is soaked in the marked area of the leg.

Blood-Plasma Protein Measurements: Blood is drawn prior to surgery, centrifuged, and serum separated, then the total protein and Ca 2+ comparisons are concluded.

Limb Weight Comparison: After blood is drawn, the animal is prepared for tissue collection.
The limb is cut with a quillitine, then both the experimental and control legs are ligated and weighed. A second weighing is performed by removing the tibio-cakaneal joint and weighing the foot.

Histological preparation: The transverse muscle located behind the knee (popliteal) area was excised and placed in a metal mold, filled with freezeGel, soaked in cold methylbutane and -80 EC until cut into labeled sample bags. Place it. Upon cutting, the muscle is observed under the fluorescent microscope for lymphatic vessels. The studies described in this example tested angiogenic polypeptide activity. However, one of ordinary skill in the art can readily modify the illustrated studies to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), agonists and / or antagonists of angiogenic polypeptides. obtain.

Example 40 Suppression of TNFα-Induced Adhesion Molecule Expression by Angiogenic Polypeptides Recruitment of lymphocytes to areas of inflammation and angiogenesis is associated with cell surface adhesion molecules (CAM) on lymphocytes. It relates to specific receptor-ligand interactions with the vascular endothelium. This adhesion process is associated with intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-) in both normal and pathological settings.
1), and following a multistep cascade involving endothelial leukocyte adhesion molecule-1 (E-selectin) expression in endothelial cells (EC). Expression of these and other molecules on the vascular endothelium determines the efficiency with which leukocytes can adhere to the local vasculature and overflow into local tissues during the development of the inflammatory response. Local concentrations of cytokines and growth factors are involved in regulating the expression of these CAMs.

Tumor necrosis factor α (TNF-α), a potent pro-inflammatory cytokine, is a stimulator of all three CMA in endothelial cells and may be involved in a wide variety of inflammatory responses, often resulting in disease. Bring physical results.

The potential of angiogenic polypeptides to mediate the suppression of TNF-α-induced CAM expression can be tested. Using a modified ELISA assay, which uses EC as a solid phase adsorbent, of CAM expression in TNF-α-processed protein EC when co-stimulated with members of the angiogenic family of proteins. Measure the quantity. To perform this experiment, human umbilical vein endothelial cell (HUVEC) cultures were
Growth medium (EGM-2; Clonetics, San) obtained from pooled cord collections and supplemented with 10% FCS and 1% penicillin / streptomycin.
Diego, CA) and maintained in a humidified incubator at 37 ° C. with 5% CO ₂ . HUVEC at a concentration of 1 × 10 ⁴ cells / well in EGM medium,
Seed in 96 well plates at 37 ° C. for 18-24 hours or until confluent. Subsequently, the monolayer was treated with 100 U / ml penicillin and 100 mg / ml.
Wash 3 times with serum free solution of RPMI-1640 supplemented with ml streptomycin and for 24 hours 37 with predetermined cytokines and / or growth factors.
Treated at ° C. After incubation, the cells are then evaluated for CAM expression.

Human umbilical vein endothelial cells (HUVEC) are grown to confluence in standard 96-well plates. Growth medium is removed from the cells and replaced with 90 μl 199 medium (10% FBS). Samples for testing and positive or negative controls are added to the plate in triplicate (10 μl volume). The plates are incubated at 37 ° C. for either 5 hours (selectin and integrin expression) or 24 hours (integrin expression only). Aspirate the plate to remove the medium and 100 μl of 0.1%
Paraformaldehyde-PBS (with Ca ++ and Mg ++) is added to each well. Hold the plate for 30 minutes at 4 ° C. The fixative is then removed from the wells and the wells are washed once with PBS (+ Ca, Mg) + 0.5% BSA and drained. Do not dry this well. 10 μl of diluted primary antibody is added to the test and control wells. Anti-ICAM
-1-Biotin, anti-VACM-1-Biotin and anti-E-selectin-
Biotin at a concentration of 10 μg / ml (1: 0.1 of 0.1 mg / ml stock antibody).
10 dilution). The cells are incubated for 30 minutes at 37 ° C in a humidified environment. Wells are washed 3 times with PBS (+ Ca, Mg) + 0.5% BSA.

Then, 20 μl of diluted ExtrAvidin-Alkaline Ph
Add osphotase (1: 5,000 dilution) to each well and at 37 ° C.
Incubate for 30 minutes. Wells with PBS (+ Ca, Mg) + 0.5
Wash 3 times with% BSA. 1 tablet of p-nitrophenol phosphate (p
NPP) is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPP substrate in glycine buffer is added to each test well. Triplicate standard wells were transferred to ExtrAvidin-Alkaline Pho in glycine buffer.
Working dilution of spotase (1: 5
000 (10 ⁰ )> 10 ^−0.5 > 10 ⁻¹ > 10 ^−1.5 ). 5 μl of each dilution was added to triplicate wells and the resulting AP content in each well was 5.
50 ng, 1.74 ng, 0.55 ng, and 0.18 ng. Then 100
μl of pNPP reagent must be added to each standard well. The plate must be incubated at 37 ° C for 4 hours. 3M NaO
Add 50 μl volume of H to all wells. The results are quantified on a plate reader at 405 nm. The background subtraction option is used on blank wells filled with glycine buffer only. The template is set up to show the concentration of AP conjugate in each standard well [5.50].
ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results are shown as the amount of bound AP conjugate in each sample.

The studies described in this example tested activity on angiogenic proteins. However, one of ordinary skill in the art can readily modify the studies illustrated to test the activity of polynucleotides encoding angiogenic polypeptides (eg, gene therapy), agonists and / or antagonists of angiogenic polypeptides. obtain.

Example 41 Mouse Immunization for Monoclonal Antibody Production Animals are housed individually and given food and water ad libitum. All operations are performed using aseptic technique. This experiment was performed on Human Genome Science.
s, Inc. Institutional Animal Care and
Use Committee and the Guidelines for
the Care and Use of Laboratory Anim
Do as per al's rules and guidelines.

Dilute the concentration of protein in 350 μl of phosphate buffer solution (PBS) (or other neutral buffer solution) to a final protein concentration of 0.43 mg / ml. 0
． 35 ml Freund's Complete Adjuvant, using two 3 cc glass syringes and a disposable 3-way cock (Bater Catalog # 2C6240), 1
Emulsify the adjuvant and protein solution for 0 minutes. To test the emulsion for properties, 50 μl of emulsion are placed on the surface of cold water in a beaker. If the emulsion does not remain as intact white droplets then further mixing is required.

All emulsions were pumped into one syringe and 4 using a 27 gauge needle.
Mice are injected subcutaneously subcutaneously in a total volume of 200 μl dispersed between ~ 8 sites (eg, the axilla and groin regions, behind the neck, and along the back).

After 2-3 weeks, the above injections are repeated, replacing with Freund's incomplete adjuvant (different from Freund's complete adjuvant).

After an additional 2-3 weeks, make sure to use Freund's incomplete adjuvant and give a third injection as outlined above.

10- 14 days after the third injection, 100-200 from mice by tail vein bleeding.
Obtain μl of blood. Incubate the blood for 60 minutes at 37 ° C, then
Allow to cool overnight at 4 ° C. Following incubation at 4 ° C, blood is centrifuged for 10 minutes. Serum is transferred to a new tube and tested for mouse serum titer. If the titer is found to be low, intraperitoneal (ip) injections may be given biweekly. For intraperitoneal injections, prepare 10-20 μg protein per mouse in a volume of 200-400 μl PBS per mouse. Mice are injected with the solution using a 1 cc syringe and a 26 gauge needle. 10-1 of injection
Four days later, a second tail bleed is taken and retested for mouse serum titer.

Example 42: Mouse Serum Titer ELISA Coat ELISA plates with 50 μl / well of purified antigen (2 μg / ml in PBS). Cover the ELISA plate with Parafilm and incubate overnight at 4 ° C. in a humid chamber. After incubation, wash the plate 4 times with 200 μl / well PBS per wash. Block with 200 μl of 3% BSA per well for 60 minutes at room temperature. Shake the blocking solution.

10 ⁻² , 10 ⁻³ , 10 ⁻⁴ diluted in PBS containing 0.1% BSA
Serum samples at dilutions of 10 ⁻⁵ , 10 ⁻⁶ and 10 ⁻⁷ are added to two wells at 50 μl / well. Includes buffer blank and positive and negative control sera at the above dilutions. Incubate at room temperature for 1-2 hours. 250 with PBST (PBS with 0.05% tween)
Wash 4 times with μl / well.

50 μl / well of biotinylated anti-mouse IgG was added to PBST (0.1% BSA).
And 2% horse serum) at a concentration of 0.5 μg / ml. Incubate for 30-60 minutes at room temperature. Wash plate 4 times with PBST.

50 μl / well ABC reagent (Vector Catalog No. PK-6100)
Is added to the plate and incubated for 30 minutes at room temperature. Wash plate 6 times with PBST.

The substrate for ELISA detection is prepared by dissolving one tetramethylbenzidine (TMB) dihydrochloride (TMB) tablet (Sigma catalog number T-3405) in 5 ml ddH ₂ O. 5 ml of 0.1 M phosphate citrate buffer (25.7 m
l 0.2 M sodium phosphate (dibasic), 24.3 ml 0.1 M citric acid (
Monohydrate), pH 5.0) is added. Add 2 μl of fresh 30% hydrogen peroxide, vortex and use immediately. Following incubation and washing of the plate, 100 μl of substrate solution is added and incubated at room temperature for about 15-30 minutes. Reaction is 25 μl / well of 2M
Stop by adding H ₂ SO ₄ and read plate at 450 nm against control within 30 minutes.

Example 43: Fusion Protocol for Hybridoma Generation One week prior to the fusion step, P3X growth medium (1 × DMEM 0% (Gibco Catalog No. 11965-019), 5-10% fetal bovine serum, 1 X L-Glutamine (Biofluids Catalog No. 300), and 1X sodium pyruvate (Biofluides Cat No. 333)). Thaw fresh vials of P3X mouse myeloma cells into 1 well of a 6-well dish (thaw protocol (
(See below)), and the expansion of the cells was started in P3X growth medium.
The next day, if viability is good, transfer to a 100 mm dish. Cell densities should not exceed 10 ⁶ cells / ml or more. Moreover, the membranes of these cells should not look rough. By the day of the fusion procedure, there should be 6-8 dishes at 5-8 × 10 ⁵ cells / ml. It is a good idea to try some P3X cells in HAT medium. All cells die within about 4 days. Otherwise, P3X cells should be grown in P3X medium containing 15 ug / ml 8-azaguanine to remove revertants.

Four days before the fusion procedure, mice should be immunized with an intraperitoneal injection of approximately 10 μl of high purity protein.

One day prior to fusion, P3X cells were split and given fresh medium as the cells were healthy and needed to grow in exponential phase by the next day.

On the day of the fusion procedure, 50 ml P3X medium, PEG solution and HAT medium (1 × D
MEM 0%, 20% fetal bovine serum, 4% Hybridoma Supplem
ent-BM Conditioned HI (Boehringer-Mannhe
im), 1 × L-glutamine, 1 × NEAA, 1 × sodium pyruvate, 1 ×
HAT (Sigma Catalog No. H0262), 1 × 0.05M 2ME and 1 × penicillin-streptomycin) were placed in a 37 ° C. water bath. Prepare approximately 100 ml of cold DMEM 0% available.

All P3X plates are checked for possible contamination and cell health assessed. Resuspend cells from 4 plates or flasks and combine into 50 ml tubes. Centrifuge at 200 × G for 10 minutes. Aspirate the supernatant. 10
Resuspend each tube with ml DMEM 0% and pool. Viable cells are counted using trypan blue viability stain (viability should be greater than 90%). The total number of cells should be 2-4 × 10 ⁷ cells. If not enough cells, repeat this process with more plates. Leave cells at ambient room temperature (ART) until needed for further steps.

Prepare the hood, where the spleen is removed using the following: 70% EtOH,
Sterilization equipment (including sieve and suction rubber cup), 2 Petri dishes (with 10 ml DMEM 0%) and 15 ml centrifuge tubes (2
).

Mice are sacrificed and spleens are then harvested from carcass. Place the spleen in a Petri dish containing 0% DMEM. The other dish containing 10 ml DMEM 0% is sieved and covered with a plate lid. The spleen is transferred to a sieve using sterile forceps, and the spleen is shredded using a syringe with a needle so that the cells come out into the medium in pieces. The spleen is then gently sifted through using another suction rubber cup. Avoid grinding splenic organ tissue through a sieve. This is because this causes a large amount of fibroblast proliferation.

The sieve is removed and the spleen cell suspension is transferred to a 15 ml centrifuge tube.
The remaining cells are washed out of the dishes with 5 ml DMEM 0% and added to the tubes. Let the tube sit for 5 minutes and allow a large strip to sink to the bottom. The debris is then removed and the cell suspension is transferred to a second 15 ml tube. 200 x G cells
Centrifuge for 10 minutes. Aspirate s / n and resuspend spleen cells in 5 ml DMEM 0%. An additional 5 ml DMEM 0% is added and the total volume is transferred to a 50 ml tube.

Remove 10 μl of spleen cell suspension and add 500 μl trypan blue to it to count lymphocytes (Note: for normal spleen ensure that 10 ⁸
Are lymphocytes).

(Fusion) Add sufficient P3X cells to a 50 ml centrifuge tube containing spleen cells to give a lymphocyte to P3X cell ratio of 5: 1 (eg, 2 × 10 ⁸ for 10 ⁸ lymphocytes). ⁷ P3X cells are required). Total volume 45-5 with 0% DMEM
Bring to 0 ml and centrifuge at 200 × G for 10 minutes. Prepare a moving hood with a timer, warm PEG, warm P3X medium, and a beaker with water at approximately 38-40 ° C. Aspirate all of the supernatant from the P3X-lymphocyte pellet and tap the tube to break up the pellet. Place the tube in a small water bath. The fusion tube is kept in warm water with gentle infusion and 1 ml PEG is added dropwise over 1 minute. Then, shake occasionally and let stand for 1-2 minutes. Then, 1 ml of P3X medium is added dropwise for 1 minute. Then 3
ml P3X medium is added dropwise over 1 minute, and then 10 ml P3X medium is added to 1 ml.
Add dropwise over minutes.

Gently add P3X medium to a total volume of 45 ml. Leave the tube to stand for 10 minutes, then centrifuge at 200 × G for 10 minutes. Aspirate the supernatant,
The pellet is then gently resuspended in 5 ml or less of HAT medium. The cell suspension is transferred to a bottle containing 400 ml HAT medium and vortexed to mix.
Pour some cell suspension into a sterile reservoir.

Place cells in 96-well plates (200 μl / well) using a 12-channel pipette with a filtered tip. Place the plate in the incubator. Plates are monitored daily for hybridoma growth or contamination. Incubate the plate for 3 days. First feeding (
Medium exchange) was performed by aspirating approximately half of the medium in each well using an 8-position manifold, and 100-150 μl / well of HT.
Perform by about 7 days by replacing with medium. Feed approximately once a week before the initial screen serves to dilute any antibody produced by unfused lymphocytes. The lymphocytes were found to continue to produce antibodies in culture after 2 weeks. If many or all wells are filled with colonies in more than half of the wells and the color of the supernatant changes to orange / yellow, then within 2 weeks of fusion, ready for sampling for screening It is in.

Example 44: ELISA Screening of Mouse Hybridomas To screen mouse hybridomas, an ELISA plate (I
mmulon 2 "U" Bottom Microtiter Plate (Dynatech Ca
t. No. 011-010-3555)) 50 μl / well of antigen (2 μg /
ml PBS). Cover the ELISA plate with a plastic seal and incubate overnight at 4 ° C. After incubation, wash the plate 4 times with 200 μl / well PBS per wash. 3%
Block with BSA (200 μl / well) for 60 minutes at room temperature. Shake off blocking solution.

150 μl / well of hybridoma supernatant is added to Corning 96 well assay plates, then 50 μl of each supernatant is transferred from the Corning assay plates to the ELISA plates. Include culture medium and positive and negative mouse serum controls in the blank. Incubate for 1-2 hours at room temperature or overnight at 4 ° C. PBST (PBS containing 0.05% Tween) (25
Wash 4 times with 0 μl / well).

50 μl / well of biotinylated anti-mouse IgG H + L (0.1-0.3%
0.5 μg / ml in PBST containing BSA and 1% horse serum). Incubate at room temperature for 30-60 minutes. Plate 4 with PBST
Wash twice.

50 μl / well of ABC reagent (Vector Cat. No. PK-610)
0) is added to the plate and incubated for 30 minutes at room temperature. Wash plate 6 times with PBST.

Tetramethylbenzidine dihydrochloride (TMB) tablets (Sigma Ca
t. No. T-3405) EL by dissolving one in 5 ml of ddH ₂ O
Prepare a substrate for ISA detection. 5 ml of 0.1 M phosphate citrate buffer (25
． 7 ml 0.2 M sodium phosphate dibasic, 24.3 ml 0.1 M citric acid monohydrate, pH 5.0) are added. Add 2 μl of fresh 30% hydrogen peroxide, vortex and use immediately.

[0761]   After incubation and washing of the plate, add 100 μl of substrate solution
And incubate at room temperature for about 15-30 minutes. 25 μl / well of 2M H ₂ SO_FourThe reaction was stopped by adding
And read at 450 nm within 30 minutes.

Example 45: Relative Affinity Test of Monoclonal Antibodies Obtained from Culture Supernatant (A. Determination of Antigen Coating Concentration) About 1 ml of antigen (4 μg / ml in PBS) is prepared. Microdilution (micro
Dilution) Transfer to a tube. 0.5m in each of 9 microdilution tubes
Make 1 serial dilution by adding 1 l PBS, then starting from a 4 μg / ml tube and transferring 0.5 ml from tube to tube. Where 4, 2
Tubes containing 1, 0.5, 0.25, 0.125, 0.06, 0.03, 0.015 and 0.0075 μg / ml are obtained. Coat each 6 well of the plate with the above concentration (50 μl / well).

Cover plate and incubate overnight at 4 ° C. After incubation, the plates are washed 4 times with 200 μl / well PBS per wash. Block with 3% BSA (200 μl / well) for 60 minutes at room temperature. Shake off blocking solution.

The titer curve of mouse sera positive for the antigen is examined. Determine the serum dilution just above the titer curve. Add positive mouse sera of this dilution in PBS containing 0.1% BSA to rows 2 to columns 2-11 (50 μl / well). Columns 2-11 of rows E-G contain the negative control sera from the above dilutions. Incubate at 4 ° C overnight. After incubation, subtract the negative control serum value from the positive control serum value. Mean values (OD450) are plotted on a linear scale against antigen concentration. Less than maximum O. D. Determine the antigen coating concentration indicated. This is the coating concentration for use with the relative affinity assay.

(B. Boehringer Mannheim Biochemica K
it "Mouse-IgG ELISA" (Cat. No. 1333 151)
Measurement of Mouse IgG Concentration of Hybridoma Supernatant Sample Using Sample) The coating buffer concentrate is diluted 1/10 with ddH ₂ O. 10-20m
l is required. Obtain an aliquot of capture antibody. After coating the three tubes, thaw the buffer concentrate (blocking solution). Twelve wells are required for standards and 4-6 wells for each supernatant tested. Assuming a coating volume of 50 μl / well, calculate the required ml of diluted capture antibody. Dilute capture antibody in the following proportions: 25 μl capture Ab = X μl capture Ab 1 ml coating buffer ml coating buffer Nunc plates are coated with this solution and incubated for 30 minutes at room temperature on a shaker. After concentrated coating, the buffer is diluted 1/10 in ddH ₂ O. Plates in ELISA wash buffer (0.9% NaCl, 0.1% Tw
Wash with een 20) and block with 200 μl / well post-coating buffer (blocking solution) for 15 minutes at room temperature. Dilute IgG standards into post-coating buffer (blocking solution) to the following concentrations: 0.2, 0.1, 0.05, 0.025, 0.0125 and 0.00 in post-coating buffer.
625 μg / ml.

The supernatant is diluted in blocking buffer to a final concentration of 1/100 and 1/1000. After the blocking step, the plates are washed and 50 μl / well diluted IgG standard and diluted supernatant are added in duplicate. Incubate for 30 minutes at room temperature on a shaker.

Dilute the conjugate solution in post-coating buffer (blocking solution) according to the following ratio: 50 μl binding solution = X μl binding solution 1 ml blocking solution ml blocking solution Wash plate, Then add 50 μl / well of binding solution. Incubate the plate on a shaker for 30 minutes at room temperature.

One substrate tablet is dissolved in 5 ml of substrate buffer. Wash plate, and 5
Add 0 μl / well of substrate. Incubate the plate on a shaker for 30 minutes at room temperature and read at 405 nm.

C. Relative Affinity Assay Coat appropriate ELISA plates at a predetermined antigen concentration at 4 ° C. overnight. Block plate as above. PBS + 0.1% BSA 1 /
Make test supernatants of 3 serial dilutions.

Add 50 μl / well of the supernatant sample (containing undiluted sample) to the ELISA plate in duplicate or triplicate. The positive control consisted of several wells of positive control mouse serum at the same concentration used to determine the antigen coating concentration. Negative controls consist of several wells of dilution buffer. Cover plate and incubate at 4 ° C.

The IgG concentration of each supernatant is plotted against the mean value (OD450) in a 4-parameter curve fit. The curve of the supernatant to the left is the supernatant with the highest affinity.

Example 46 Ascites Production in Mice Hybridoma cells should be healthy and in the exponential growth phase for ascites production. Transfer cells to a 15 ml tube and count. 4x
Determine the volume containing 10 ⁶ cells, transfer the volume to a second tube, and centrifuge the cells. Pellet 0.9 ml HBSS (Hanks balanced salt solution)
Resuspend in and transfer to an Eppendorf tube.

Fill a 1 cc syringe with cell suspension and IP inject into mice as follows: 0.2 cc / mouse if the initial cell number is 4 × 10 ⁶ and initial cell number. Is 3 × 10 ⁶ , 0.3 cc / mouse. If the belly is very distended and feels slightly balloon-like to the touch (usually by day 9 or 10, but sometimes as late as day 14) It is time to “tap the mouse”.

A. Tapping Mice are retained in the left hand and an alcohol pad is used to wipe the abdominal area just above the left hind limb of the mice. A 19 gauge needle is inserted into the abdomen while retaining the mouse on a 15 ml centrifuge tube with an open mouth. Ascites should begin to drip into the centrifuge tube from the end of the needle. The average mouse should produce 3-6 ml of ascites.

B. Treatment and storage of ascites Ascites collected from each mouse in the group was pooled (all injected with the same hybridoma) and left at room temperature for 1-2 hours or at 37 ° C for 15- 15 hours. Place for 30 minutes. The ascites is then placed at 4 ° C overnight to allow clot formation. Centrifuge the solidified ascites for 10 minutes. Transfer liquid ascites to a 50 ml centrifuge tube and store at -20 ° C. Subsequent puncture can be added to the 50 ml tube. When all mice are sacrificed, pooled ascites is thawed, spun again, and aliquoted for long-term storage at -20 ° C or -70 ° C. Ascites
Titered by ELISA.

[0776] (Example 47: Mouse hybridoma and protocols for freezing and thawing myeloma) (A. frozen) cells to be frozen is healthy, is in the logarithmic growth phase, and from about 5 to 8 × 10 ⁵
There should be a concentration of cells / ml. Cells from 6 well plates or flasks are resuspended, transferred to 15 ml tubes and counted. The total number of cells is counted and divided into 1-3 × 10 ⁶ cells / vial to determine the number of frozen vials.

[0777]   Pellet cells at 200-300 x G for 5-10 minutes. Pellet the supernatant
Aspirated from, and sufficient chilled freezing medium (50% FBS in DMEM, 10%
DMSO; Origen DMSO Freeze Medium (IG
EN), Fisher Cat. No. IG-50-0715)
Achieve the desired number of cells / vial per ml (cell density is 5 x 10 ^Five ~ 1 x 10⁷Should be in the range of cells / vial). By cooling the cell suspension
Immediately transfer to cryovial (1 ml / vial) and place on ice
To do. Transfer vials to rate controlled freezer and place in -70 ° C freezer overnight. Long
Transfer vials to liquid nitrogen tanks after 24 hours for long term storage, or-
Transfer to a freezer at 130 ° C.

(B. Thaw) Add 10 ml of chilled medium (eg P3X medium) to a 15 ml tube. Remove frozen vials of frozen cells and keep on dry ice until ready for thawing. Thaw cells rapidly in a 37 ° C water bath. Hold the vial during thaw and continue to shake gently until only a small amount of ice remains in the vial. Do not warm the contents above 4 ° C. Wipe the outside of the cooling vial with alcohol. Transfer the cell suspension in the vial to 10 ml of chilled medium using a sterile Pasteur pipette without touching the edge of the chilled vial. 20
Spin at 0-300 x G for 5-10 minutes. Aspirate supernatant and 6 ml HT
Resuspend the pellet in cloning medium (supra). Transfer to one well of a 6-well dish. Viability is assessed after 24 hours. Viability should not be less than 50%.

Example 48: In Vitro Assay for Angiogenic Protein Activity The following assay is designed to design angiogenic protein activity (preferably VEGF).
-2 activity) is detected. For example, the extracellular domain of the Flt-4 receptor (SEQ ID NO: 27) (nucleotides encoding Galland et al., Genomics 13 (2): 475-478 (1992), which is incorporated herein by reference in its entirety). , Flk-1 transmembrane and intracellular domains (SEQ ID NO: 2
8) (Davis-Smith, which is incorporated herein by reference in its entirety).
Et al., EMBO J 15 (18): 4919-4927 (1996)) to produce a chimeric receptor. Therefore,
This chimeric receptor has amino acids 765 to 1356 of SEQ ID NO: 28, respectively.
SEQ ID NOs: fused to amino acids 1-775.

Alternatively, the chimeric receptor may be designed as outlined above, but which is hereby incorporated by reference in its entirety, Pacifici et al., JBC 269 (
3): 1571-1574 (1994), Flk-1
The erythropoietin receptor (EPOR) is replaced by the transmembrane and intracellular domains of erythropoietin receptor (EPOR) (see FIG. 1 for details).

The resulting DNA encoding the chimeric receptor is discussed, supra.
, Into a suitable mammalian, baculovirus, or bacterial expression vector (such as pC4, pCDNA3, or pA2). Mammalian host cells that can be used for expression of the chimeric receptor include NIH3T3 (supra), or the pre-B cell line BaF3 (Achen et al., PNAS 95 (2), incorporated herein by reference in its entirety). : 548-553
(1998)).

To test for activity, angiogenic proteins can be contacted with a cell line expressing the chimeric receptor or an extract thereof. Angiogenic proteins that bind to the chimeric receptor can then be detected by measuring any resulting signal transmitted by the chimeric receptor.

It will be appreciated that the present invention may be practiced other than as described in detail in the foregoing description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and are therefore within the scope of the appended claims.

Full disclosure of each document cited in the background of the invention, detailed description and examples, including patents, patent applications, academic literature, abstracts, laboratory manuals, books or other disclosures.
Are incorporated herein by reference. In addition, the sequence listing is incorporated by reference, and further, US patent application Ser. No. 08 / 207,412 (currently US Pat.
No. 817,485) and No. 08 / 803,926, which are incorporated herein by reference in their entirety.

[0785]

[Table 3] ATCC Deposit No. 97149, page 2 (Canada) The applicant is required to file a Canadian patent under the application or until the application is refused or abandoned and cannot be recovered or withdrawn until the Patent Office Commissioner ( Requesting that the Commissioner of Patents) permit the donation of a sample of the deposited biological material referred to in the application only to an independent expert nominated by the Commissioner, the applicant Prior to the completion of the regulatory preparations for the publication of, the fact must be notified in writing to the International Bureau.

(Norway) Applicants hereby agree that the application of samples is not subject to specialization in the art until the application has been published (by the Norwegian Patent Office) or has been decided by the Norwegian Patent Office without publication. Claim that it will only be done to the house. A request to this effect shall be made by the applicant to the Norwegian Patent Office before the time when the application is made publicly available under section 22 and section 33 (3) of the Norwegian Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Norwegian Patent Office (list of
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

(Australia) The Applicant hereby states that the provision of a sample of microorganisms is
Alternatively, before the abandonment, rejection or withdrawal of the application, a person who is a person skilled in the art who has no interest in the invention (skilled addre)
It is to be announced that it will only be performed for Ssee) (Australian Patent Law No. 3.25 (3)).

(Finland) The applicant hereby states that the application is (Patent and Control Committee (National
That samples will only be provided to experts in the art until published (by Board of Patents and Regulations) or until a decision by the National Patent and Legal Commission is made without publication. ,Claim.

(UK) Applicant hereby claims that the provision of a sample of microorganisms is made available only to professionals. A request to this effect must be made by the applicant to the International Bureau before the regulatory preparations for the international publication of the application are complete. ATCC Deposit No. 97149, page 3 (Denmark) The applicant hereby refuses to provide samples until the application has been published (by the Danish Patent Office) or has been decided by the Danish Patent Office without publication. Claim that it is done only to experts in the art. A request to this effect shall be filed by the applicant with the Danish Patent Office before the time when the application is made publicly available under Articles 22 and 33 (3) of the Danish Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Danish Patent Office (list of).
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

(Sweden) Applicants hereby advise that the application of a sample shall be prioritized in the art until the application has been published (by the Swedish Patent Office) or has been decided by the Swedish Patent Office without publication. Claim that it will only be done to the house. A request to this effect must be made by the applicant before the expiration of 16 months from the priority date.
It should be done for Temp (preferably PCT Applicant's
Form PCT / R described in Annex Z of Volume I of Guide
O / 134). If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert may be any person listed on the list of recognized experts prepared by the Swedish Patent Office or, in the individual case, any person approved by the applicant.

(Netherlands) Applicants hereby agree that until the issue date of the Dutch patent, or until the date when the application is rejected, withdrawn or lapped, the microorganism is specialized under the provisions of Patent Law 31F (1). Claim that it will only be done in the form of sample delivery to the house. A request for this shall be made by the applicant before the Dutch industrial property right before the date on which the application is made publicly available under Article 22C or Article 25 of the Dutch Patent Act, whichever is earlier. It shall be submitted to the Bureau.

[0792]

[Table 4] ATCC Deposit No. 75698, page 2 (Canada) The applicant is required to file a Canadian patent under the application, or until the application is refused or abandoned and cannot be recovered or withdrawn until the Commissioner of the Patent Office ( Requesting that the Commissioner of Patents) permit the donation of a sample of the deposited biological material referred to in the application only to an independent expert nominated by the Commissioner, the applicant Prior to the completion of the regulatory preparations for the publication of, the fact must be notified in writing to the International Bureau.

(Norway) Applicants hereby agree that the application of samples shall be deemed to be until the application has been published (by the Norwegian Patent Office) or determined by the Norwegian Patent Office without publication. Claim that it will only be done to the house. A request to this effect shall be made by the applicant to the Norwegian Patent Office before the time when the application is made publicly available under section 22 and section 33 (3) of the Norwegian Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Norwegian Patent Office (list of
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

(Australia) The Applicant hereby states that the provision of a sample of microorganisms is
Alternatively, before the abandonment, rejection or withdrawal of the application, a person who is a person skilled in the art who has no interest in the invention (skilled addre)
It is to be announced that it will only be performed for Ssee) (Australian Patent Law No. 3.25 (3)).

(Finland) Applicant hereby states that the application (Patent and Control Committee (National
That samples will only be provided to experts in the art until published (by Board of Patents and Regulations) or until a decision by the National Patent and Legal Commission is made without publication. ,Claim.

(UK) The applicant hereby claims that the provision of a sample of microorganisms is made available only to professionals. A request to this effect must be made by the applicant to the International Bureau before the regulatory preparations for the international publication of the application are complete. ATCC Deposit No. 75698, page 3 (Denmark) Applicants hereby submit samples until the application has been published (by the Danish Patent Office) or determined by the Danish Patent Office without publication. Claim that it is done only to experts in the art. A request to this effect shall be filed by the applicant with the Danish Patent Office before the time when the application is made publicly available under Articles 22 and 33 (3) of the Danish Patent Act. If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts prepared by the Danish Patent Office (list of).
any of those listed in "recognized experts", or
It can be any person approved by the applicant in the individual case.

(Sweden) Applicants hereby advise that the application of samples shall be prioritized in the art until the application has been published (by the Swedish Patent Office) or has been decided by the Swedish Patent Office without publication. Claim that it will only be done to the house. A request to this effect shall be filed by the applicant with the International Bureau not later than 16 months from the priority date (preferably PCT Applicant's Gui).
Form PCT / RO / 13 described in Annex Z of Volume I of de
4). If such a claim is made by the applicant, any claim for the provision of the sample by a third party shall indicate the expert used. The expert is a list of certified experts (lis) prepared by the Swedish Patent Office.
any of the persons listed in to of recognized experts),
Alternatively, it may be any person approved by the applicant in the individual case.

(Netherlands) Applicants hereby agree that until the issue date of the Dutch patent, or until the date when the application is rejected, withdrawn or lapped, the microorganism is specialized under the provisions of Patent Act 31F (1). Claim that it will only be done in the form of sample delivery to the house. A request for this shall be made by the applicant before the Dutch industrial property right before the date on which the application is made publicly available under Article 22C or Article 25 of the Dutch Patent Act, whichever is earlier. It shall be submitted to the Bureau.

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as covered by the claims.

[Sequence list]

[Brief description of drawings]

1A-1E show the full-length nucleotide sequence of VEGF2 (SEQ ID NO: 15) and the deduced amino acid sequence (SEQ ID NO: 16). This polypeptide contains about 419 amino acid residues, of which about 23 represent a leader sequence. Use the standard one-letter abbreviations for amino acids. Sequencing is model 373 automated DN
It was performed using an A sequencer (Applied Biosystems, Inc.). Sequencing accuracy is expected to be greater than 97%.

2A-2D show the nucleotide sequence (SEQ ID NO: 25) and deduced amino acid sequence (SEQ ID NO: 26) of the biologically active form of VEGF2. This polypeptide contains about 350 amino acid residues, of which the first about 24 amino acids represent the leader sequence.

3A-3C are PDGF-A (SEQ ID NO: 2), PDGF-B (SEQ ID NO: 4).
, PlGF (SEQ ID NO: 6), PlGF-2 (SEQ ID NO: 8), GDGF (SEQ ID NO: 10), VEGF (SEQ ID NO: 12), VEGF-A (SEQ ID NO: 14), VEGF
-2 (SEQ ID NO: 16), VEGF-3 (SEQ ID NO: 18), VEGF-D (SEQ ID NO: 20), VEGF-D1 (SEQ ID NO: 22), and VEGF-E (SEQ ID NO: 2)
FIG. 4 is a schematic diagram of amino acid sequence homology between 4). Boxed regions indicate the positions of conserved sequences and 8 conserved cysteine residues.

[Procedure amendment]

[Submission date] December 11, 2001 (2001.12.11)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] 0184

[Correction method] Change

[Contents of correction]

The angiogenic proteins of the present invention are all structurally related. It is particularly important that all eight cysteines are conserved within all members of the VEGF / PDGF family (see boxed regions in Figures 3A-3D). Furthermore, PDG
F / VEGF family signature, PXCVXXXRCXGCC (SEQ ID NO: 29
Is conserved in all angiogenic proteins disclosed herein (FIG. 3A).
~ 3D).

[Procedure Amendment 2]

[Document name to be amended] Statement

[Name of item to be corrected] 0187

[Correction method] Change

[Contents of correction]

The polynucleotide encoding each mature polypeptide of FIGS. 3A-3D may include: coding sequence of each mature polypeptide only; coding sequence of each mature polypeptide and additional coding sequences (eg, A leader or secretory sequence, or a proprotein sequence); the coding sequence (and optionally further coding sequence) and non-coding sequence of each mature polypeptide (
(For example, introns, or non-coding sequences 5'and / or 3'of the mature polypeptide coding sequence).

[Procedure 3]

[Document name to be amended] Statement

[Correction target item name] 0189

[Correction method] Change

[Contents of correction]

The present invention further relates to variants of the polynucleotides described herein above which encode fragments, analogs and derivatives of the polypeptides having the deduced amino acid sequences of Figures 3A-3D. The variant of the polynucleotide can be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

[Procedure amendment 4]

[Document name to be amended] Statement

[Correction target item name] 0190

[Correction method] Change

[Contents of correction]

Accordingly, the present invention thus encodes a polynucleotide encoding the same respective mature polypeptides shown in Figures 3A-3D, as well as fragments, derivatives or analogs of the polypeptides of Figures 3A-3D, thus Various polynucleotide variants. Such nucleotide variants include deletion variants, substitution variants, and addition or insertion variants.

[Procedure Amendment 5]

[Document name to be amended] Statement

[Name of item to be corrected] 0227

[Correction method] Change

[Contents of correction]

Angiogenic Polypeptides The present invention further relates to polypeptides having the deduced amino acid sequences of Figures 3A-3D, as well as fragments, analogs and derivatives of such polypeptides.

[Procedure correction 6]

[Document name to be amended] Statement

[Correction target item name] 0230

[Correction method] Change

[Contents of correction]

Fragments of angiogenic polypeptides and polynucleotides characterized by structural or functional domains are also preferred. Such fragments include α-helix and α-helix forming regions, β-sheet and β-sheet forming regions, turns and turn-forming regions, coils and coil-forming regions, hydrophilic regions, hydrophobic regions, α-amphipathic regions. , Β-amphipathic region, flexible region, surface forming region, substrate binding region and high antigenicity indicator region. Polypeptide fragments of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 contained in the conserved domain are specifically contemplated by the present invention (FIG. 3).
See A-3D). Moreover, polynucleotide fragments encoding these domains are also contemplated.

[Procedure Amendment 7]

[Document name to be amended] Statement

[Name of item to be corrected] 0367

[Correction method] Change

[Contents of correction]

Oligonucleotides that are complementary to the 5'end of the message (eg, 5'untranslated sequences up to and including the AUG start codon) should work most efficiently in inhibiting translation. is there. However, sequences complementary to the 3'untranslated sequences of mRNAs have likewise been shown to be effective in inhibiting translation of mRNAs. Generally, Wagner, R .; , 1994, Nature 372: 333-33.
See 5. Thus, oligonucleotides complementary to either the 5'or 3'untranslated noncoding regions of the angiogenic proteins shown in Figures 3A-3D inhibit translation of the endogenous mRNA encoding the angiogenic protein. It can be used for antisense approaches. Oligonucleotides complementary to the 5'untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region, although less efficient inhibitors of translation, can be used in accordance with the present invention. Whether designed to hybridize to the 5'region, 3'region or coding region of angiogenic mRNA, the antisense nucleic acid should be at least 6 nucleotides in length, and preferably 6 Is an oligonucleotide ranging from about 50 nucleotides in length. In certain aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
It is a nucleotide.

[Procedure Amendment 8]

[Document name to be amended] Statement

[Name of item to be corrected] Figure 3

[Correction method] Change

[Contents of correction]

3A-3D are PDGF-A (SEQ ID NO: 2), PDGF-B (SEQ ID NO: 4).
, PlGF (SEQ ID NO: 6), PlGF-2 (SEQ ID NO: 8), GDGF (SEQ ID NO: 10), VEGF (SEQ ID NO: 12), VEGF-A (SEQ ID NO: 14), VEGF
-2 (SEQ ID NO: 16), VEGF-3 (SEQ ID NO: 18), VEGF-D (SEQ ID NO: 20), VEGF-D1 (SEQ ID NO: 22), and VEGF-E (SEQ ID NO: 2)
FIG. 4 is a schematic diagram of amino acid sequence homology between 4). Boxed regions indicate the positions of conserved sequences and 8 conserved cysteine residues.

[Procedure Amendment 9]

[Document name to be corrected] Drawing

[Correction target item name] All drawings

[Correction method] Change

[Contents of correction]

[Figure 1]

[Fig. 2]

[Figure 3]

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) A61P 7/04 A61P 9/04 4C084 7/06 9/06 4H045 9/04 9/08 9/06 9 / 10 9/08 101 9/10 9/12 101 11/00 9/12 11/06 11/00 13/12 11/06 17/00 101 13/12 17/02 17/00 101 19/02 17/02 21/04 19/02 25/00 21/04 25/16 25/00 25/28 25/16 29/00 25/28 101 29/00 31/00 101 31/04 31/00 31/12 31/04 35/00 31/12 37/02 35/00 37/06 37/02 43/00 105 37/06 C07K 14/515 43/00 105 16/22 C07K 14/515 C12N 1/15 16/22 1/19 C12N 1/15 1/21 1/19 C12P 21/02 C 1/21 C12Q 1/02 5/10 1/68 C12P 21/02 G01N 33/15 Z C12Q 1/02 33/50 Z 1/68 33 / 53 DG01N 33/15 33/68 33/50 C12N 15/00 ZNAA 33/53 A61K 37/2 4 33/68 C12N 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT , SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, A, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA , UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Rosen, Craig A. United States Maryland 20882, Leightonsville, Rolling Hill Road 22400 (72) Inventor Ruben, Stephen M. United States Maryland 20832, Ornay, Heritage Hills Drive 18528 (72) Inventor Hu, Jin-shan United States California 94040, Mountain View, Number 420, Ortega Avenue 550 (72) Inventor Khao, Liang United States Maryland 20814, Bethesda, Suite 820 es, up Kusu Hill Road 5225 F-term (reference) 2G045 AA40 DA36 FB02 FB03 4B024 AA01 AA11 BA21 BA44 CA04 CA11 DA01 DA02 DA05 DA11 EA01 EA02 EA03 EA04 FA02 GA11 HA01 HA03 HA11 4B063 QA01 QA08 QA18 QA19 QQ05 QQ42 QQ52 QR08 QR33 QR42 QR55 QR59 QR62 QR74 QR80 QS05 4B064 AG13 CA01 CA19 CC24 DA01 DA13 4B065 AA01X AA57X AA87X AA93Y AB01 BA01 CA24 CA25 CA44 CA46 4C084 AA02 AA06 AA07 ZA ZA ZA 522 ZA 522 ZA 522 ZA 522 ZA 152 ZA 152 DA 02 DA 01 DA 27 DA 41 ZA452 ZA512 ZA532 ZA552 ZA592 ZA812 ZA892 ZA902 ZA942 ZA962 ZB072 ZB112 ZB152 ZB212 ZB262 ZB312 ZB332 ZB352 ZC352 ZC552 4H045 AA10 AA11 AA20 AA30 BA10 CA40 DA01 DA76 EA20 EA50 FA72 FA74

Claims (23)

[Claims] 1. An isolated nucleic acid molecule, which comprises: (a) a polynucleotide fragment of SEQ ID NO: X that is capable of hybridizing to SEQ ID NO: X; (b) SEQ ID NO: Y. (C) a polynucleotide encoding a polypeptide domain of SEQ ID NO: Y, which hybridizes to SEQ ID NO: X. (D) a polynucleotide that encodes the polypeptide epitope of SEQ ID NO: and is capable of hybridizing to SEQ ID NO: X; (e) a polynucleotide that encodes the polypeptide of SEQ ID NO: Organism that is a nucleotide and is capable of hybridizing to SEQ ID NO: X (F) a polynucleotide that is a variant of SEQ ID NO: X; (g) a polynucleotide that is an allelic variant of SEQ ID NO: X; (h) a species homologue of SEQ ID NO: Y (I) a polynucleotide capable of hybridizing to any one of the polynucleotides specified in (a) to (h) under stringent conditions, wherein the polynucleotide is Has a nucleotide sequence that is at least 95% identical to a sequence selected from the group consisting of a polynucleotide that does not hybridize under stringent conditions to a nucleic acid molecule that has a nucleotide sequence with only A residues or only T residues. An isolated nucleic acid molecule comprising a polynucleotide. 2. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence encoding a secreted protein. 3. The isolated of claim 1, wherein the polynucleotide fragment comprises a nucleotide sequence that encodes a sequence identified as SEQ ID NO: Y and is capable of hybridizing to SEQ ID NO: X. Nucleic acid molecule. 4. The isolated nucleic acid molecule of claim 1, wherein the polynucleotide fragment comprises the entire nucleotide sequence of SEQ ID NO: X, which sequence is hybridizable to SEQ ID NO: X. 5. The isolated nucleic acid molecule of claim 2, wherein the nucleotide sequence comprises consecutive nucleotide deletions from either the C-terminus or the N-terminus. 6. The isolated nucleic acid molecule of claim 3, wherein the nucleotide sequence comprises consecutive nucleotide deletions from either the C-terminus or the N-terminus. 7. A recombinant vector comprising the isolated nucleic acid molecule of claim 1. 8. A method of making a recombinant host cell comprising the isolated nucleic acid molecule of claim 1. 9. A recombinant host cell produced by the method of claim 8. 10. The recombinant host cell of claim 9, which comprises a vector sequence. 11. An isolated polypeptide comprising: (a) a polypeptide fragment of SEQ ID NO: Y; (b) a polypeptide fragment of SEQ ID NO: Y having biological activity; (c) a sequence. (D) polypeptide epitope of SEQ ID NO: (e) secreted form of SEQ ID Y; (f) full-length protein of SEQ ID Y; (g) variant of SEQ ID Y; A) an allelic variant of SEQ ID NO :; or (i) a species homolog of SEQ ID Y, comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of:
Isolated polypeptide. 12. The isolated polypeptide of claim 11, wherein the secreted form or the full-length protein comprises a contiguous amino acid deletion from either the C-terminus or the N-terminus. 13. An isolated antibody that specifically binds to the isolated polypeptide of claim 11. 14. Expressing the isolated polypeptide of claim 11.
Recombinant host cell. 15. A method for producing an isolated polypeptide, comprising: (a) culturing the recombinant host cell of claim 14 under conditions such that the polypeptide is expressed. A step; and (b) recovering the polypeptide. 16. A polypeptide produced by the method of claim 15. 17. A method for preventing, treating or ameliorating a medical condition, wherein a therapeutically effective amount of the polypeptide of claim 11 or the polynucleotide of claim 1 is administered to a mammalian subject. A method comprising the step of administering to. 18. A method of diagnosing a pathological condition or susceptibility to a pathological condition in a subject, comprising: (a) determining the presence or absence of a mutation in the polynucleotide of claim 1. And (b) diagnosing a pathological condition, or susceptibility to a pathological condition, based on the presence or absence of the mutation. 19. A method of diagnosing a pathological condition or susceptibility to a pathological condition in a subject, comprising: (a) the presence of expression of the polypeptide of claim 11 in a biological sample. Or a step of determining the amount; and (b) diagnosing a pathological condition or susceptibility to the pathological condition based on the presence or amount of expression of the polypeptide. 20. A method of identifying a binding partner for the polypeptide of claim 11, comprising: (a) contacting the polypeptide of claim 11 with a binding partner;
And (b) determining whether the binding partner results in the activity of the polypeptide. 21. A gene corresponding to the cDNA sequence of SEQ ID NO: Y. 22. A method of identifying activity in a biological assay, comprising:
Wherein the method comprises: (a) expressing SEQ ID NO: X in cells; (b) isolating the supernatant; (c) detecting activity in a biological assay; and (d) ) Identifying a protein in the supernatant that has the activity. 23. A product produced by the method of claim 20.