EP1417310A2 - Nouvelles sequences d'acides nucleiques et d'acides amines - Google Patents

Nouvelles sequences d'acides nucleiques et d'acides amines

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Publication number
EP1417310A2
EP1417310A2 EP02741129A EP02741129A EP1417310A2 EP 1417310 A2 EP1417310 A2 EP 1417310A2 EP 02741129 A EP02741129 A EP 02741129A EP 02741129 A EP02741129 A EP 02741129A EP 1417310 A2 EP1417310 A2 EP 1417310A2
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EP
European Patent Office
Prior art keywords
seq
nucleic acid
sequence
amino acid
acid sequence
Prior art date
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EP02741129A
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German (de)
English (en)
Inventor
Liat Mintz
Kinneret Savitzky
Sharon Engel
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Compugen Ltd
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Compugen Ltd
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Publication date
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Publication of EP1417310A2 publication Critical patent/EP1417310A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons

Definitions

  • the present invention concerns novel nucleic acid sequences, vectors and host cells containing them, amino acid sequences encoded by said sequences, and antibodies reactive with said amino acid sequences, as well as pharmaceutical compositions comprising any of the above.
  • the present invention further concerns methods for screening for candidate activator or deactivators utilizing said amino acid sequences.
  • VEGF Vascular endothelial growth factor
  • DNA sequencing suggests the existence of several molecular species of VEGF.
  • VEGFs are secreted proteins in contrast to other endothelial cell mitogens such as acidic or basic fibroblast growth factors and platelet-derived endothelial cell growth factors.
  • VEGF was found to augment human growth by inducing neovascularization.
  • neutralization of VEGF activity may have clinical application in inhibiting malignant cells-induced angiogenesis, decreasing blood supply to the cancerous tissue, leading eventually to its destruction.
  • VEGF has various other functions on endothelial cells, the most prominent of which is the induction of proliferation and differentiation. It was found to be capable of preventing serum starvation-induced apoptosis and this inhibition may represent a major aspect of the regulatory activity of VEGF on vascular endothelium. VEGF was also found to be involved in the development and the growth of ovarian corpus luteum (CL), since its development is dependent on the growth of new capillary vessels.
  • CL ovarian corpus luteum
  • Flt-1 receptors which inhibit vascular endothelial growth factor bioactivity, resulted in complete separation of corpus luteum angiogenesis in a rat model of hormonally induced ovulation, indicated that VEGF is essential for CL angiogenesis and may be involved in the control of fertility and treatment of ovarian disorders characterized by hypervascularity and hyperplasia.
  • the human VEGF gene has been recently assigned to chromosome 6p21.2.
  • VEGF may exist as one - of four different molecular species, having respectively, 121, 165, 189 and 206 amino acids (VEGFm, VEGF ⁇ 65 , BEGF 206 ).
  • VEGF165 lacks the residues encoded by exon 6, while VEGFm lacks
  • VEGF ⁇ has an insertion of 24 amino acids highly enriched in basic residues and VEGF206 has an additional insertion of 17 amino acids.
  • VEGF 16 5 is the predominant isoform secreted by a variety of normal and transformed cells. Transcripts encoding VEGF121 and VEGF 189 are detected in the majority of cells and tissues expressing the VEGF gene. In contrast, VEGF206 is
  • Native VEGF is a basic, heparin-binding, homodimeric glycoprotein of 45 kDA. These properties correspond to those of VEGF165.
  • VEGFm is an acidic polypeptide that fails to bind to heparin.
  • VEGF ⁇ 9 and VEGF200 are more basic and bind to heparin with greater affinity than VEGF165.
  • VEGFm is a freely soluble
  • VEGF165 is also secreted, although a significant fraction remains bound to the cell surface and the extracellular matrix (ECM).
  • ECM extracellular matrix
  • VEGF189 and VEGF206 are almost completely sequestered in the ECM, but may be released in a soluble form by heparin or heparinase. Also, these long forms may be released by plasmin following cleavage at the COOH terminus (Ferrara, N., European J. of Cancer,
  • Vascular endothelial growth factor variant (VEGFV) nucleic acid sequence - the sequences shown in SEQ ID NO: 1 or SEQ ID NO: 3, sequences having at least
  • This sequence is a sequence coding for a novel alternative splice variant of the native VEGF. While the known VEGF peptides include 206, 189, 105 or 121 amino acids, the novel VEGF variant peptide of the invention includes only 141 amino acids, 27 of which being in the signal peptide and 114 being present in the mature protein. According to the terminology used in the publication of Ferrara
  • SEQ ID NO: 3 is a sequence coding for the VEGF ⁇ 4 , containing 231 nucleotides of unique 3' untranslated region (UTR). Said 3' UTR, which starts at the nucleotide at position 409 and ends at the nucleotide at position 640 of SEQ ID NO: 3 is identified herein also as SEQ ID NO: 3
  • Vascular endothelial growth factor variant (VEGFV product) - also referred at times as the "VEGFV protein” or "VEGFV polypeptide” - is an amino acid sequence having the first 141 amino acids of the native VEGF. This naturally occurring sequence is the result of alternative splicing.
  • the amino acid sequence may be a peptide, a protein, as well as peptides or proteins having chemically modified amino acids (see below) such as a glycopeptide or glycoprotein.
  • An example of a VEGFV product is shown in SEQ ID NO: 2.
  • the term also includes analogues of said sequences in which one or more amino acids has been added, deleted, substituted (see below) or chemically modified (see below) as well as fragments of this sequence having at least 10 amino acid.
  • Nucleic acid sequence - a sequence composed of DNA nucleotides, RNA nucleotides or a combination of both types and may include natural nucleotides, chemically modified nucleotides and synthetic nucleotides.
  • Amino acid sequence - a sequence composed of any one of the 20 naturally appearing amino acids, amino acids which have been chemically modified (see below), or composed of synthetic amino acids.
  • Framents of VEGFV nucleic acid sequence a continuous portion, preferably of about 20 nucleic acid sequences of the VEGFV nucleic acid sequence.
  • the fragments encompassed by the invention are such that include a sequence that is not found in the native VEGF.
  • “Fragments of VEGFV product” a polypeptide which has an amino acid sequence which is the same as part of but not all of the amino acid sequence of the VEGF product.
  • Constant substitution refers to the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix.
  • Class I Cys
  • Class II Ser, Thr, Pro, Ala, Gly
  • Class III Asn, Asp, Gin, Glu
  • Class IV His, Arg, Lys
  • Class V He, Leu, Val, Met
  • Class VI Phe, Tyr, Trp
  • Non-conservative substitution refers to the substitution of an amino acid in one class with an amino acid from another class; for example, substitution of an Ala, a class II residue, with a class III residue such as Asp, Asn, Glu, or Gin.
  • “Chemically modified” - when referring to the protein product of the invention, means a product (protein) where at least one of its amino acid resides is modified either by natural processes, such as processing or other post-translational modifications, or by chemical modification techniques which are well known in the art.
  • modifications typical, but not exclusive examples include: acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristlyation, pegylation, prenylation, phosphorylation, ubiqutination, or any similar process.
  • chemically modified means where at least one of its nucleotides is modified either by methylations, acetylation, acylation, amidation, ADP-ribosylation, deamination, phosphorylation, substitution, insertion, deletion or any similar process.
  • Bioly active refers to the VEGFV product having structural, regulatory or biochemical functions of the naturally occurring VEGF product, for example the same effect on vascular endothelial cells, such as stimulating endothelial cells proliferation, cell differentiation, cell migration, cell survival, angiogenic activity or vascular permeability.
  • the term "Biologically active” refers to VEGFV product having altered levels of structural, regulatory or biochemical functions of the naturally occurring VEGF product as well. Altered levels means higher or lower function of the VEGFV product as compared to the naturally occurring VEGF product.
  • Immunologically active defines the capability of a natural, recombinant or synthetic VEGFV product, or any fragment thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • a biologically active fragment of VEGFV product denotes a fragment which retains some or all of the immunological properties of the VEGFV product, e.g can bind specific anti-VEGFV product antibodies or which can elicit an immune response which will generate such antibodies or cause proliferation of specific immune cells which produce VEGFV.
  • Optimal alignment is defined as an alignment giving the highest percent identity score. Such alignment can be performed using a variety of commercially available sequence analysis programs, such as the local alignment program
  • Mac Vector operated with " an open gap penalty of 10.0, an extended gap penalty of 0.1, and a BLOSUM similarity matrix. If a gap needs to be inserted into a first sequence to optimally align it with a second sequence, the percent identity is calculated using only the residues that are paired with a corresponding amino acid residue (i.e., the calculation does not consider residues in the second sequences that are in the "gap" of the first sequence).
  • Having at least X% identity refers to the percentage of residues that are identical in the two sequences when the sequences are optimally aligned.
  • 70% amino acid sequence identity means that 70% of the amino acids in two or more optimally aligned polypeptide sequences are identical.
  • isolated nucleic acid molecule having an VEGFV nucleic acid sequence is a nucleic acid molecule that includes the coding VEGFV nucleic acid sequence.
  • Said isolated nucleic acid molecule may include the VEGFV nucleic acid sequence as an independent insert; may include the VEGFV nucleic acid sequence fused to an additional coding sequences, encoding together a fusion protein in which the VEGFV coding sequence is the dominant coding sequence (for example, the additional coding sequence may code for a signal peptide); the VEGFV nucleic acid sequence may be in combination with non-coding sequences, e.g., introns or control elements, such as promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host; or may be a vector in which the VEGFV protein coding sequence is a heterologous.
  • Expression vector refers to vectors that have the ability to incorporate and express heterologous DNA fragments in a foreign cell. Many prokaryotic and eukaryotic expression vectors are known and commercially available. Selection of appropriate expression vectors is within the knowledge of those having skill in the art.
  • “Deletion " - is a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
  • “Insertion” or “addition” - is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring sequence.
  • substitution - replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively. As regards amino acid sequences the substitution may be conservative or non- conservative.
  • Antibody refers to IgG, IgM, IgD, IgA, and IgG antibody.
  • the definition includes polyclonal antibodies or monoclonal antibodies. This term refers to whole antibodies or fragments of the antibodies comprising the antigen-binding domain of the anti-VEGFV product antibodies, e.g. antibodies without the Fc portion, single chain antibodies, fragments consisting of essentially only the variable, antigen-binding domain of the antibody, etc.
  • Activator refers to a molecule which mimics the effect of the natural VEGFV product or at times even increases or prolongs the duration of the biological activity of said product, as compared to that induced by the natural product.
  • the mechanism may be by binding to the VEGFV receptor, by prolonging the lifetime of the VEGFV, by increasing the activity of the VEGFV on its target (vascular endothelial cells), by increasing the affinity of VEGFV to its receptor, etc.
  • Activators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which can bind to and activate the VEGFV product.
  • Deactivator or (“Inhibitor”) ' - refers to a molecule which modulates the activity of the VEGFV product in an opposite manner to that of the activator, by decreasing or shortening the duration of the biological activity of the VEGFV product. This may be done by blocking the binding of the VEGFV to its receptor (competitive or non-competitive inhibition), by causing rapid degradation of the VEGFV, etc.
  • Deactivators may be polypeptides, nucleic acids, carbohydrates, lipids, or derivatives thereof, or any other molecules which bind to and modulate the activity of said product.
  • Treating a disease refers to administering a therapeutic substance effective to ameliorate symptoms associated with a disease, to lessen the severity or cure the disease, or to prevent the disease from occurring.
  • ⁇ Detection refers to a method of detection of a disease. This term may refer to detection of a predisposition to a disease.
  • Probe - the VEGFV nucleic acid sequence, or a sequence complementary therewith, when used to detect presence of other similar sequences in a sample. The detection is carried out by identification of hybridization complexes between the probe and the assayed sequence.
  • the probe may be attached to a solid support or to a detectable label.
  • the present invention is based on the surprising finding that there exist in humans a novel variant of the VEGF protein, having 141 amino acid (114 amino acids of the mature protein without the signal peptide) than the known VEGF.
  • the nucleic sequences coding for this variant were identified as being from the same locus as the known VEGF and thus it was concluded that the variants are not encoded from a different gene than the known VEGF, but are the result of alternative splicing of the known VEGF.
  • the present invention provides a novel isolated nucleic acid molecule comprising or consisting of the coding sequence of SEQ ID NO: 1, fragments of said coding sequence having at least 20 nucleic acids, or a nucleic acid molecule comprising a sequence having at least 95%, preferably at least 98% identity to SEQ ID NO: 1
  • SEQ ID NO: l Also provided by the invention are novel sequences comprising or consisting of SEQ ID NO: 3 (the "joint").
  • SEQ ID NO: 3 is in fact a combination of SEQ ID NO: 1 and SEQ ID NO: 4, the two being joined between positions 408 and 409 of SEQ ID NO: 3.
  • SEQ ID NO: 3 includes a 3* untranslated region (3* UTR) sequence consisting of the 231 nucleotides between the positions numbers 409 and 640 of SEQ ID NO: 3.
  • SEQ ID NO: 4 is this 3' UTR, which is novel and is also an aspect of the invention.
  • fragments of SEQ ID NO: 3 or SEQ ID NO: 4 having at least 20 nucleic acids, or a nucleic acid molecule comprising a sequence having at least 70%, preferably 80%, more preferably 90% and most preferably 95% identity to SEQ ID NO: 3 or to SEQ ID NO: 4.
  • nucleic acid molecule comprising a sequence having at least 70%, preferably 80%, more preferably 90% and most preferably 95% identity to SEQ ID NO: 3 or to SEQ ID NO: 4.
  • Provided by the invention are also complete or partial sequences based on
  • SEQ ID NO: 3 that span the joint between SEQ ID NO: 1 and SEQ ID NO: 4.
  • sequences include a first part based on SEQ ID NO: 1 or at least the part thereof adjacent to the joint, and a second part based on SEQ ID NO: 4 or at least a part thereof adjacent to the joint.
  • provided by the invention are also sequences having at least 95% identity to said first part, and at least 70%, preferably 80%, more preferably 90% and most preferably 95% identity to said second part.
  • the present invention further provides a protein or polypeptide comprising or consisting of an amino acid sequence encoded by any of the above nucleic acid sequences, termed herein "VEGFV product", for example, an amino acid sequence having the sequence as depicted in SEQ ID NO: 2, as well as homologs of the amino acid sequences SEQ ID NO: 2 in which one or more of the amino acid residues has been substituted (by conservative or non-conservative substitution) added, deleted, or chemically modified.
  • VEGFV product an amino acid sequence having the sequence as depicted in SEQ ID NO: 2, as well as homologs of the amino acid sequences SEQ ID NO: 2 in which one or more of the amino acid residues has been substituted (by conservative or non-conservative substitution) added, deleted, or chemically modified.
  • the present invention further provides nucleic acid molecule comprising or consisting of a sequence which encodes the above amino acid sequences,
  • the present invention further provides expression vectors and cloning vectors comprising any of the above nucleic acid sequences, as well as host cells transfected by said vectors.
  • the present invention still further provides pharmaceutical compositions comprising, as an active ingredient, said nucleic acid molecules, said expression vectors, or said protein or polypeptide.
  • compositions are suitable for the treatment of diseases and pathological conditions, which can be ameliorated or cured by raising the level of the VEGFV product, for example when increased angiogenic effect is required as it is the case in several cardio-vascular diseases.
  • the present invention also provides a nucleic acid molecule comprising or consisting of a non-coding sequence which is complementary to that of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4, or complementary to any of the sequences defined above.
  • the complementary sequence may be a DNA sequence which hybridizes with the SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4 or hybridizes to a portion of that sequence having a length sufficient to inhibit the transcription of the complementary sequence.
  • the complementary sequence may be a DNA sequence which can be transcribed into an mRNA being an antisense to the mRNA transcribed SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4 or into an mRNA which is an antisense to a fragment of the mRNA transcribed from SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4 which has a length sufficient to hybridize with the
  • nucleic acids of the second aspect of the invention may be used for therapeutic or diagnostic applications for example for detection of the expression of
  • the proportion of expression of the VEGF variant of the present invention as compared to the known VEGF variants may be indicative to a variety of physiological or pathological conditions.
  • the present invention also provides expression vectors comprismg any one of the above defined complementary nucleic acid sequences and host cells transfected with said nucleic acid sequences or vectors, being complementary to those specified in the first aspect of the invention.
  • the invention also provides anti- VEGFV product antibodies, namely antibodies directed against the VEGFV product which specifically bind to said VEGFV product. Said antibodies are useful both for diagnostic and therapeutic purposes.
  • said antibody may be as an active ingredient in a pharmaceutical composition as will be explained below.
  • the present invention also provides pharmaceutical compositions comprising, as an active ingredient, the nucleic acid molecules, which comprise or consist of said complementary sequences, or of a vector comprising said complementary sequences.
  • the pharmaceutical composition thus provides pharmaceutical compositions comprising, as an active ingredient, said anti- VEGFV product antibodies.
  • the pharmaceutical compositions comprising said anti- VEGFV product antibodies or the nucleic acid molecule comprising said complementary sequence are suitable for the treatment of diseases and pathological conditions where a therapeutically beneficial effect may be achieved by neutralizing the VEGFV or decreasing the amount of the VEGFV product or blocking its binding to the receptor, for example, by the neutralizing effect of the antibodies, or by the decrease of the effect of the antisense mRNA in decreasing expression level of the
  • Decreasing the expression and function of the naturally occurring VEGF by administering to an individual the VEGFV of the invention may be required for the treatment of diseases and pathological conditions where, for example, one of biological activities of endothelial- cells should be inhibited, for example in curing eliminating or preventing the growth of solid tumors.
  • the present invention provides methods for detecting the level of the transcript (mRNA) of said VEGFV product in a body fluid sample, or in a specific tissue sample, for example by use of probes comprising or consisting of said coding sequences; as well as methods for detecting levels of expression of said product in tissue, e.g. by the use of antibodies capable of specifically reacting with the above amino acid sequences.
  • Detection of the level of the expression of the VEGF variant of the invention in particular as compared to that of the known VEGF variants may be indicative of a plurality of physiological or pathological conditions.
  • the method for detection of a nucleic acid sequence which encodes the VEGFV product in a biological sample, comprises:
  • the probe is part of a nucleic acid chip used for detection purposes, i.e. the probe is a part of an array of probes each present in a known location on a solid support.
  • the nucleic acid sequence used in the above method may be a DNA sequence, an RNA sequence, etc; it may be a coding sequence or a sequence complementary thereto (for respective detection of RNA transcripts or coding-DNA sequences).
  • quantization of the level of hybridization complexes and calibrating the quantified results it is possible also to detect the level of the transcript in the sample.
  • Methods for detecting mutations in the region coding for the VEGFV product are also provided, which may be methods carried-out in a binary fashion, namely merely detecting whether there is any mismatches between the normal
  • the present invention also concerns a method for detecting VEGFV product in a biological sample, comprising:
  • the invention also provides a method for identifying candidate compounds capable of binding to the VEGFV product and modulating its activity (being either activators or deactivators).
  • the method includes: 0) providing a protein or polypeptide comprising an amino acid sequence substantially as depicted in SEQ ID NO: 2, or a fragment of such a sequence;
  • VEGFV product to its native receptor, effect on the modulation in the effect of VEGFV on vascular endothelial cells, etc. Any modulator which changes such an activity has an intersecting potential.
  • the present invention also concerns compounds identified by the methods described above, which compound may either be an activator of the serotonin-receptor like product or a deactivator thereof.
  • Figs. 1 and 2 show a Northern blot analysis of RNA obtained from various tissues and tested with a probe specific for known VEGFs but lacking the VEGF variant of the invention (termed "VEGF”) (left), as well as a unique probe from the 3" UTR region of VEGF-114 (right).
  • VEGF vascular endothelial growth factor
  • Fig. 3 shows a Western blot analysis of Ac- VEGF- 114 infected insect cells using the VEGF (147) polyclonal antibody (Santa Cruz Biotechnology).
  • Example I VEGFV - nucleic acid sequence
  • the nucleic acid sequences of the invention include nucleic acid sequences which encode VEGFV product and fragments and analogs thereof.
  • the nucleic acid sequences may alternatively be sequences complementary to the above coding sequence, or to a region of said coding sequence or to the regulatory 3' untranslated region. The length of the complementary sequence is sufficient to avoid the expression of the coding sequence.
  • the nucleic acid sequences may be in the form of RNA or in the form of DNA, and include messenger RNA, synthetic RNA and DNA, cDNA, and genomic DNA.
  • the DNA may be double-stranded or single-stranded, and if single-stranded may be the coding strand or the non-coding (anti-sense, complementary) strand.
  • the nucleic acid sequences may also both include dNTPs, rNTPs as well as non naturally occurring sequences.
  • the sequence may also be a part of a hybrid between an amino acid sequence and a nucleic acid sequence.
  • a nucleic acid sequence encompassed by the present invention is also such with a degree of identity to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 4, as defined above.
  • the nucleic acid sequences may include the coding sequence by itself.
  • the coding region may be in combination with additional coding sequences, such as those coding for fusion protein or signal peptides, in combination with non-coding sequences, such as introns and control elements, promoter and terminator elements or 5' and/or 3' untranslated regions, effective for expression of the coding sequence in a suitable host, and/or in a vector or host environment in which the VEGFV nucleic acid sequence is introduced as a heterologous sequence.
  • the nucleic acid sequences of the present invention may also have the product coding sequence fused in-frame to a marker sequence which allows for purification of the VEGFV product.
  • the marker sequence may be, for example, a hexahistidine tag to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al. Cell 37:767 (1984)).
  • fragments also referred to herein as oligonucleotides, typically having at least 20 bases, preferably 20-30 bases corresponding to a region of the coding-sequence nucleic acid sequence.
  • the fragments may be used as probes, primers, and when complementary also as antisense agents, and the like, according to known methods.
  • the nucleic acid sequence may be substantially as depicted in SEQ ID NO: 1, in SEQ ID NO: 3 or in SEQ ID NO: 4, or fragments thereof or sequences having the above degree of identity to these sequences or fragments.
  • the sequence may be a sequence coding the amino acid sequence of SEQ ID NO: 2, or fragments or analogs of said amino acid sequence.
  • the nucleic acid sequences may be obtained by screening cDNA libraries using oligonucleotide probes which can hybridize to or PCR-amplify nucleic acid sequences which encode the VEGFV products disclosed above.
  • cDNA libraries prepared from a variety of tissues are commercially available and procedures for screening and isolating cDNA clones are well-known to those of skill in the art.
  • the nucleic acid sequences may be extended to obtain upstream and downstream sequences such as promoters, regulatory elements, and 5' and 3 1 untranslated regions (UTRs). Extension of the available transcript sequence may be performed by numerous methods known to those of skill in the art, such as PCR or primer extension (Sambrook et al, supra), or by the RACE method using, for example, the Marathon RACE kit (Clontech, Cat. # K1802-1).
  • genomic DNA is amplified in the presence of primer to a linker sequence and a primer specific to the known region.
  • the amplified sequences are subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR can be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T.
  • the primers may be designed using OLIGO(R) 4.06 Primer Analysis Software (1992; National Biosciences Inc, Madison, Minn.), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72°C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Capture PCR is a method for PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA. Capture PCR also requires multiple restriction enzyme digestions and ligations to place an engineered double-stranded sequence into a flanking part of the DNA molecule before PCR.
  • flanking sequences Another method which may be used to retrieve flanking sequences is that of Parker, J.D., et al, Nucleic Acids Res., 19:3055-60, (1991)). Additionally, one can use PCR, nested primers and PromoterFinderTM libraries to "walk in" genomic DNA (PromoterFinderTM; Clontech, Palo Alto, CA). This process avoids the need to screen libraries and is useful in finding intron/exon junctions. Preferred libraries for screening for full length cDNAs are ones that have been size-selected to include larger cDNAs. Also, random primed libraries are preferred in that they will contain more sequences which contain the 5' and upstream regions of genes.
  • a randomly primed library may be particularly useful if an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries are useful for extension into the 5' nontranslated regulatory region.
  • the nucleic acid sequences and oligonucleotides of the invention can also be prepared by solid-phase methods, according to known synthetic methods. Typically, fragments of up to about 100 bases are individually synthesized, then joined to form continuous sequences up to several hundred bases.
  • VEGFV nucleic acid sequences for the production of VEGFV products
  • nucleic acid sequences specified above may be used as recombinant DNA molecules that direct the expression of VEGFV products.
  • Codons preferred by a particular prokaryotic or eukaryotic host can be selected, for example, to increase the rate of VEGFV product expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
  • the nucleic acid sequences of the present invention can be engineered in order to alter a VEGFV product coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the product.
  • alterations may be introduced using techniques which are well known jn the art, e.g., site-directed mutagenesis, to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to produce splice variants, etc.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are also described in Sambrook, etal, (supra).
  • the present invention also relates to host cells which are genetically engineered with vectors of the invention, and the production of the product of the invention by recombinant techniques.
  • Host cells are genetically engineered (i.e., transduced, transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the expression of the VEGFV nucleic acid sequence.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art.
  • the nucleic acid sequences of the present invention may be included in any one of a variety of expression vectors for expressing a product.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and related sub-cloning procedures are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate transcription control sequence (promoter) to direct mRNA synthesis.
  • promoters include: LTR or SV40 promoter, the E.coli lac or trp promoter, the phage lambda PL promoter, and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation, and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coll
  • the vector containing the appropriate DNA sequence as described above, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • appropriate expression hosts include: bacterial cells, such as E.coli, Streptomyces, Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila and Spodoptera Sf9; animal cells such as CHO, COS, HEK
  • a number of expression vectors may be selected depending upon the use intended for the VEGFV product. For example, when large quantities of VEGFV product are needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified may be desirable.
  • Such vectors include, but are not limited to, multifunctional E.coli cloning and expression vectors such as Bluescript(R) (Stratagene), in which the VEGFV polypeptide coding sequence may be ligated into the vector in-frame with sequences for the amino-terminal Met and the subsequent 7 residues of beta-galactosidase so that a hybrid protein is produced; IN vectors (Van Heeke & Schuster J Biol Chem. 264:5503-5509, (1989)); pET vectors (Novagen, Madison WI); and the like.
  • Bluescript(R) Stratagene
  • IN vectors Van Heeke & Schuster J Biol Chem. 264:5503-5509, (1989)
  • pET vectors Novagen, Madison WI
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH.
  • PGH palladium phosphate
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH.
  • promoters such as the 35S and 19S promoters of CaMV (Brisson et al, Nature 310:511-514.
  • omega leader sequence from TMV (Takamatsu et al, EMBO J., 3:1671-1680, (1984); Broglie et al, Science 224:838-843, (1984)); or heat shock promoters (Winter J and
  • constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection.
  • pathogen-mediated transfection see Hobbs S. or Murry L.E. (1992) in McGraw Hill Yearbook of Science and Technology,
  • VEGFV product may also be expressed in an insect system.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the VEGFV product coding sequence may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of VEGFV coding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S.
  • a number of viral-based expression systems may be utilized.
  • a VEGFV product coding sequence may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing VEGFV protein in infected host cells (Logan and Shenk, Proc. Natl Acad. Sci. 81:3655-59, (1984).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • Specific initiation signals may also be required for efficient translation of a VEGFV protein coding sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where VEGFV product coding sequence, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed.
  • exogenous transcriptional control signals including the ATG initiation codon must be provided.
  • the initiation codon must be in the correct reading frame to ensure transcription of the entire insert.
  • Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf, D. et al, (1994) Results Probl Cell Differ., 20:125-62, (1994); Bittner et al., Methods in Enzymol 153:516-544, (1987)).
  • the present invention relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., and Battey, I. (1986) Basic Methods in Molecular Biology).
  • Cell-free translation systems can also be employed to produce polypeptides using RNAs derived from the DNA constructs of the present invention.
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the protein include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post-translational processing which cleaves a "pre-pro" form of the protein may also be important for correct insertion, folding and/or function.
  • Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc. have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
  • cell lines which stably express VEGFV product may be transformed using expression vectors which contain viral origins of replication or endogenous expression elements and a selectable marker gene.
  • cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M., et al, Cell 11:223-32, (1977)) and adenine phosphoribosyltransferase (Lowy I., et al, Cell 22:817-23, (1980)) genes which can be employed in tk- or aprt- cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler M., et al, Proc. Nail. Acad. Sci.
  • npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al, J. Mol Biol, 150:1-14, (1981)) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman S.C. and R.C. Mulligan, Proc. Nail. Acad. Sci.
  • Host cells transformed with a nucleotide sequence encoding VEGFV product may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the product produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing nucleic acid sequences encoding VEGFV product can be designed with signal sequences which direct secretion of VEGFV product through a prokaryotic or eukaryotic cell membrane.
  • VEGFV product may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle, Wash.).
  • the inclusion of a protease-cleavable polypeptide linker sequence between the purification domain and VEGFV protein is useful to facilitate purification.
  • One such expression vector provides for expression of a fusion protein compromising a VEGFV polypeptide fused to a polyhistidine region separated by an enterokinase cleavage site.
  • the histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography, as described in Porath, et al, Protein Expression and Purification, 3:263-281, (1992)) while the enterokinase cleavage site provides a means for isolating VEGFV polypeptide from the fusion protein.
  • pGEX vectors Promega, Madison, Wis.
  • GST glutathione S-transferase
  • such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to ligand-agarose beads (e.g., glutathione-agarose in the case of GST-fusions) followed by elution in the presence of free ligand.
  • ligand-agarose beads e.g., glutathione-agarose in the case of GST-fusions
  • the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.
  • Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, or other methods, which are well know to those skilled in the art.
  • the VEGFV products can be recovered and purified from recombinant cell cultures by any of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • the nucleic acid sequences of the present invention may be used for a variety of diagnostic purposes.
  • the nucleic acid sequences may be used to detect and quantitate expression of VEGFV in patient's cells, e.g. biopsied tissues, by detecting the presence of mRNA coding for VEGFV product.
  • the assay may be used to detect soluble VEGFV in the serum or blood. This assay typically involves obtaining total mRNA from the tissue or serum and contacting the mRNA with a nucleic acid probe.
  • the probe is a nucleic acid molecule of at least 20 nucleotides, preferably 20-30 nucleotides, capable of specifically hybridizing with a sequence included within the sequence of a nucleic acid molecule encoding VEGFV under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of VEGFV.
  • This assay can be used to distinguish between absence, presence, and excess expression of VEGFV product and to monitor levels of VEGFV expression during therapeutic intervention.
  • the invention also contemplates the use of the nucleic acid sequences as a diagnostic for diseases resulting from inherited defective VEGFV sequences, or diseases in which the purpose of the amount of the known VEGF to the novel
  • VEGF variant of the invention is altered. These sequences can be detected by comparing the sequences of the defective (i.e., mutant) VEGFV coding region with that of a normal coding region. Association of the sequence coding for mutant VEGFV product with abnormal VEGFV product activity may be verified.
  • sequences encoding mutant VEGFV products can be inserted into a suitable vector for expression in a functional assay system (e.g., colorimetric assay, complementation experiments in a VEGFV protein deficient strain of
  • mutant genes have been identified, one can then screen populations of interest for carriers of the mutant gene.
  • Nucleic acids used for diagnosis may be obtained from a patient's cells, including but not limited to such as from blood, urine, saliva, placenta, tissue biopsy and autopsy material.
  • Genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki, et ah, Nature 324:163-166, (1986)) prior to analysis.
  • RNA or cDNA may also be used for the same purpose.
  • PCR primers complementary to the nucleic acid of the present invention can be used to identify and analyze mutations in the gene of the present invention. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA of the invention or alternatively, radiolabeled antisense DNA sequences of the invention. Sequence changes at specific locations may also be revealed by nuclease protection assays, such RNase and SI protection or the chemical cleavage method (e.g. Cotton, et alProc. Natl Acad. Sci. USA,
  • Molecular beacons (Kostrikis L.G. et al, Science 279:1228-1229, (1998)), hairpin-shaped, single-stranded synthetic oligo- nucleotides containing probe sequences which are complementary to the nucleic acid of the present invention, may also be used to detect point mutations or other " sequence changes as well as monitor expression levels of VEGFV product. Such diagnostics would be particularly useful for prenatal testing.
  • Another method for detecting mutations uses two DNA probes which are designed to hybridize to adjacent regions of a target, with abutting bases, where the region of known or suspected mutation(s) is at or near the abutting bases.
  • the two probes may be joined at the abutting bases, e.g., in the presence of a ligase enzyme, but only if both probes are correctly base paired in the region of probe junction.
  • the presence or absence of mutations is then detectable by the presence or absence of ligated probe.
  • oligonucleotide array methods based on sequencing by hybridization (SBH), as described, for example, in U.S. Patent No. 5,547,839.
  • SBH sequencing by hybridization
  • the DNA target analyte is hybridized with an array of oligonucleotides formed on a microchip.
  • the sequence of the target can then be "read" from the pattern of target binding to the array.
  • RNA was obtained from the following tissues: putamen, temporal lobe, frontal lobe, occipital lobe, spinal cord, medulla, cerebral cortex, brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta lung and leukocytes and was hybridized either with an VEGF probe or with a probe from the unique 3' UTR region of the VEGF-V of the invention. The results are shown in Figs. 1 and 2. As can be seen the new VEGF-V probe of the invention shows, in general, similar hybridization patterns to those of native VEGF. D. Gene mapping utilizing nucleic acid sequences
  • the nucleic acid 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.
  • mapping of DNAs to chromosomes is an important first step in correlating those sequences with genes associated with disease.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 20-30 bp) from the VEGFV cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, which would complicate the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.
  • mapping of somatic cell hybrids or using instead radiation hybrids are rapid procedures for assigning a particular DNA to a particular chromosome.
  • sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
  • Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.
  • Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step.
  • FISH Fluorescence in situ hybridization
  • This technique can be used with cDNA as short as 50 or 60 bases.
  • Verma et al Human Chromosomes: a Manual of Basic Techniques, (1988) Pergamon Press, New York.
  • the OMIM gene map presents the cytogenetic map location of disease genes and other expressed genes.
  • the OMIM database provides information on diseases associated with the chromosomal location. Such associations include the results of linkage analysis mapped to this interval, and the correlation of translocations and other chromosomal aberrations in this area with the advent of polygenic diseases, such as cancer, in general and prostate cancer in particular.
  • Nucleic acid sequences of the invention may also be used for therapeutic purposes.
  • expression of VEGFV product may be modulated through antisense technology, which controls gene expression through hybridization of complementary nucleic acid sequences, i.e. antisense DNA or RNA, to the control, 5' or regulatory regions of the gene encoding VEGFV product.
  • antisense technology which controls gene expression through hybridization of complementary nucleic acid sequences, i.e. antisense DNA or RNA, to the control, 5' or regulatory regions of the gene encoding VEGFV product.
  • the 5' coding portion of the nucleic acid sequence which codes for the product of the present invention is used to design an antisense oligonucleotide of from about 10 to 40 base pairs in length. Oligonucleotides derived from the transcription start site, e.g.
  • An antisense DNA oligonucleotide is designed to be complementary to a region of the nucleic acid sequence involved in transcription (Lee et al, Nucl. Acids, Res., 6:3073, (1979); Cooney et al., Science 241:456, (1988); and Dervan et al, Science 251:1360, (1991)), thereby preventing transcription and the production of the VEGFV products.
  • An antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the VEGFV products (Okano J. Neurochem. 56:560,
  • the antisense constructs can be delivered to cells by procedures known in the art such that the antisense RNA or DNA may be expressed in vivo.
  • the antisense may be antisense mRNA or DNA sequence capable of coding such antisense mRNA.
  • the antisense mRNA or the DNA coding thereof can be complementary to the full sequence of nucleic acid sequences coding to the
  • VEGFV protein or to a fragment of such a sequence which is sufficient to inhibit production of a protein product.
  • expression of VEGFV product may be increased by providing coding sequences for coding for said product under the control of suitable control elements ending its expression in the desired host.
  • compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the formulation should suit the mode of administration.
  • Cells from a patient may be engineered with a nucleic acid sequence (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide.
  • DNA or RNA nucleic acid sequence
  • Such methods are well-known in the art.
  • cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered for expression of a polypeptide in vivo by procedures known in the art.
  • a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expression of the polypeptide in vivo.
  • the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.
  • Retroviruses from which the retroviral plasmid vectors mentioned above may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the retroviral plasmid vector is employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, psi-2, psi-AM, PA 12, T19-14X, VT-19-17-H2, psi-CRE, psi-CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller (Human Gene Therapy, Vol. 1, pg. 5-14, (1990)).
  • the vector may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
  • the producer cell line generates infectious retroviral vector particles which include the nucleic acid sequence(s) encoding the polypeptides.
  • retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vitro or in vivo.
  • the transduced eukaryotic cells will express the nucleic acid sequence(s) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic stem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, flbroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial epithelial cells.
  • the genes introduced into cells may be placed under the control of inducible promoters, such as the radiation-inducible Egr-1 promoter, (Maceri, H.J., et al, Cancer Res., 56(19):4311 (1996)), to stimulate VEGFV production or antisense inhibition in response to radiation, eg., radiation therapy for treating tumors.
  • inducible promoters such as the radiation-inducible Egr-1 promoter, (Maceri, H.J., et al, Cancer Res., 56(19):4311 (1996)
  • the substantially purified " VEGFV product of the invention has been defined above as the product coded from the nucleic acid sequence of the invention.
  • the amino acid sequence is an amino acid sequence having at least 70%, preferably at least 80% or 90% identity to the sequence identified as SEQ ID NO: 2.
  • the protein or polypeptide may be in mature and/or modified form, also as defined above. Also contemplated are protein fragments having at least 10 contiguous amino acid residues, preferably at least 10-20 residues, derived from the VEGFV product.
  • sequence variations are preferably those that are considered conserved substitutions, as defined above.
  • a protein with a sequence having at least 80% sequence identity with the protein identified as SEQ ID NO: 2, preferably by utilizing conserved substitutions as defined above is also part of the invention.
  • the protein has or contains the sequence identified SEQ ID NO: 2.
  • the VEGFV product may be (i) one in which one or more of the amino acid residues in a sequence listed above are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the VEGFV product is fused with another compound, such as a compound to increase the half-life of the protein (for example, polyethylene glycol (PEG)), or a moiety which serves as targeting means to direct the protein to its target tissue or target cell population (such as an antibody), or (iv) one in which additional amino acids are fused to the VEGFV product.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • the amino acid residues includes a substituent group
  • another compound such as a compound to increase the half-life of the protein (for example, polyethylene glycol (PEG)), or a moiety
  • 5 product may be produced by direct peptide synthesis using solid-phase techniques
  • In vitro peptide synthesis may be performed using manual techniques or by automation.
  • VEGFV product may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
  • the VEGF-V of the invention was cloned into the Back to Back
  • pFBHTal-VEGF Baculovirus Expression System 15 Baculovirus Expression System (Invitrogen). Briefly, the gene was inserted into the pFastBackHTal transfer vector (including a 6xHis tag on its 5'). The resulting transfer vector (pFBHTal-VEGF) was confirmed for the correct insertion by sequencing. pFBHTal-VEGF was then transformed into DHlOBac competent cells (containing the ACNPV viral genome in the form of bacmid
  • the medium contains two bands of size around 11KD and 15KD.
  • the 15 D band corresponds for the 6XHis-VEGF translation starting at the His tag AUG and the 11KD band corresponds for the VEGF translation starting from the VEGF gene AUG.
  • the VEGFV product of. the invention is generally useful in treating diseases and disorders which are characterized by a lower than normal level of VEGFV expression, and or diseases which can be cured or ameliorated by raising the level of the VEGFV product, even if the level is normal.
  • VEGFV products or fragments may be administered by any of a number of routes and methods designed to provide a consistent and predictable concentration of compound at the target organ or tissue.
  • the product-containing compositions may be administered alone or in combination with other agents, such as stabilizing compounds, and/or in combination with other pharmaceutical agents such as drugs or hormones.
  • VEGFV product-containing compositions may be administered by a number of routes including, but not limited to oral, intravenous, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means as well as by nasal application. VEGFV product-containing compositions may also be administered via liposomes. Such administration routes and appropriate formulations are generally known to those of skill in the art.
  • the product can be given via intravenous or intraperitoneal injection. Similarly, the product may be injected to other localized regions of the body. The product may also be administered via nasal insufflation. Enteral administration is also possible. For such administration, the product should be formulated into an appropriate capsule or elixir for oral administration, or into a suppository for rectal administration.
  • a therapeutic composition for use in the treatment method can include the product in a sterile injectable solution, the polypeptide in an oral delivery vehicle, the product in an aerosol suitable for nasal administration, or the product in a nebulized form, all prepared according to well known methods.
  • Such compositions comprise a therapeutically effective amount of the compound, and a pharmaceutically acceptable carrier or excipient.
  • a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the product of the invention may also be used to modulate angiogenesis, endothelial differentiation and proliferation, cell migration, as well as to modulate cell survival, apoptosis and vascular permeability either ex vivo or in vitro, for example, in cell cultures.
  • Example III Screening methods for activators and deactivators (inhibitors)
  • the present invention also includes an assay for identifying molecules, such as synthetic drugs, antibodies, peptides, or other molecules, which have a modulating effect on the activity of the VEGFV product, e.g. activators or deactivators of the VEGFV product of the present invention.
  • an assay comprises the steps of providing an VEGFV product encoded by the nucleic acid sequences of the present invention, contacting the VEGFV protein with one or more candidate molecules to determine the candidate molecules modulating effect on the activity of the VEGFV product, and selecting from the molecules a candidate's molecule capable of modulating VEGFV product physiological activity.
  • VEGFV product its catalytic or immunogenic fragments or oligopeptides thereof, can be used for screening therapeutic compounds in any of a variety of drug screening techniques.
  • the fragment employed in such a test may be free in solution, affixed to a solid support, borne on a cell membrane or located intracellularly.
  • the formation of binding complexes, between VEGFV product and the agent being tested, may be measured.
  • the activator or deactivator may work by serving as agonist or antagonist, respectively, of the
  • VEGFV receptor and their effect may be determined in connection with the receptor.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the receptor.
  • VEGFV product a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with the full VEGFV product or with fragments of VEGFV product and washed. Bound VEGFV product is then detected by methods well known in the art.
  • Substantially purified VEGFV product can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • Antibodies to the VEGFV product may also be used in screening assays according to methods well known in the art. For example, a "sandwich" assay may be performed, in which an anti- VEGFV antibody is affixed to a solid surface such as a microtiter plate and VEGFV product is added. Such an assay can be used to capture compounds which bind to the VEGFV product. Alternatively, such an assay may be used to measure the ability of compounds to influence with the binding of VEGFV product to the VEGFV receptor, and then select those compounds which effect the binding.
  • the purified VEGFV product is used to produce anti- VEGFV antibodies which have diagnostic and therapeutic uses related to the activity, distribution, and expression of the VEGFV product, in particular therapeutic applications in inhibiting the effect of the VEGFV on vascular endothelial cells.
  • Antibodies to VEGFV product may be generated by methods well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragments and fragments produced by an Fab expression library. Antibodies, i.e., those which inhibit dimer formation, are especially preferred for therapeutic use.
  • a fragment VEGFV product for antibody induction does not require biological activity but have to feature immunological activity; however, the protein fragment or oligopeptide must be antigenic.
  • Peptides used to induce specific antibodies may have an amino acid sequence consisting of at least five amino acids, preferably at least 10 amino acids of the sequences specified in SEQ ID NO: 2. Preferably they should mimic a portion of the amino acid sequence of the natural protein and may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of VEGFV protein amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Procedures well known in the art can be used for the production of antibodies to VEGFV product.
  • VEGFV product for the production of antibodies, various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with VEGFV product or any portion, fragment or oligopeptide which retains immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include but are not limited to Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corynebacterium parvum are potentially useful human adjuvants.
  • Monoclonal antibodies to VEGFV protein may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein (Nature 256:495-497, (1975)), the human B-cell hybridoma technique (Kosbor et al, Immunol. Today 4:72, (1983);
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al. (Proc. Natl. Acad. Sci. 86:3833-3837, 1989)), and Winter G and Milstein C, (Nature 349:293-299, (1991)).
  • Antibody fragments which contain specific binding sites for VEGFV protein may also be generated.
  • fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W.D. et al, Science 256:1275-1281, (1989)).
  • Antibodies which specifically bind VEGFV product are useful for the diagnosis of conditions or diseases characterized by over expression of VEGFV as well as for detection of diseases in which the proportion between the amount of the known VEGF and the novel VEGF variants of the invention is altered.
  • such antibodies may be used in assays to monitor patients being treated with VEGFV product, its activators, or its deactivators.
  • Diagnostic assays for VEGFV protein include methods utilizing the antibody and a label to detect VEGFV product in human body fluids or extracts of cells or tissues.
  • the products and antibodies of the present invention may be used with or without modification. Frequently, the proteins and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule.
  • a wide variety of reporter molecules are known in the art.
  • VEGFV product A variety of protocols for measuring VEGFV product, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS). As noted above, a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on VEGFV product is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, et al (supra). Such protocols provide a basis for diagnosing altered or abnormal levels of VEGFV product expression.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • Normal or standard values for VEGFV product expression are established by combining body fluids or cell extracts taken from normal subjects, preferably human, with antibody to VEGFV product under conditions suitable for complex formation which are well known in the art.
  • the amount of standard complex formation may be quantified by various methods, preferably by photometric methods. Then, standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by disease.
  • Deviation between standard and subject values establishes the presence of disease state.
  • the antibody assays are useful to determine the level of VEGFV present in a body fluid sample, in order to determine whether it is being overexpressed or underexpressed in the tissue, or as an indication of how VEGFV levels are responding to drug treatment.
  • the antibodies may have a therapeutical utility in blocking or decreasing the activity of the VEGFV product in pathological conditions where beneficial effect can be achieved by such a decrease.
  • the antibody employed is preferably a humanized monoclonal antibody, or a human MAb produced by known globulin-gene library methods.
  • the antibody is administered typically as a sterile solution by IV injection, although other parenteral routes may be suitable.
  • the antibody is administered in an amount between about 1-15 mg/kg body weight of the subject. Treatment is continued, e.g., with dosing every 1-7 days, until a therapeutic improvement is seen.

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Abstract

Nouvelles séquences d'acides nucléiques et d'acides aminés d'un nouveau variant du facteur de croissance endothéliale vasculaire (VEGF). La présente invention concerne encore des vecteurs d'expression et des cellules hôtes contenant ces séquences ainsi que des compositions pharmaceutiques et des méthodes de détection reposant sur l'utilisation desdites séquences.
EP02741129A 2001-06-20 2002-06-20 Nouvelles sequences d'acides nucleiques et d'acides amines Withdrawn EP1417310A2 (fr)

Applications Claiming Priority (3)

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US88450101A 2001-06-20 2001-06-20
WOPCT/US88/04050 2001-06-20
PCT/IL2002/000491 WO2002102848A2 (fr) 2001-06-20 2002-06-20 Nouvelles sequences d'acides nucleiques et d'acides amines

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EP1417310A2 true EP1417310A2 (fr) 2004-05-12

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CN106349389B (zh) * 2015-07-21 2019-11-15 科济生物医药（上海）有限公司 肿瘤特异性抗egfr抗体及其应用

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JP3591264B2 (ja) * 1997-12-24 2004-11-17 富士レビオ株式会社 Ｖｅｇｆ１２１特異的モノクローナル抗体及び測定方法
EP1053326A2 (fr) * 1998-02-06 2000-11-22 Collateral Therapeutics Variants du facteur angiogenique de croissance cellulaire endotheliale vasculaire vegf

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AU2002314491A1 (en) 2003-01-02
WO2002102848A3 (fr) 2004-03-04

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