JP2002535258A - Homing pro-apoptotic conjugates and methods of using homing pro-apoptotic conjugates - Google Patents

Abstract

(57) SUMMARY The present invention provides homing pro-apoptotic conjugates. The homing pro-apoptotic conjugate comprises a tumor homing molecule that selectively homes to a selected mammalian cell type or tissue conjugated to an antimicrobial peptide, wherein the conjugate is bound by the mammalian cell type or tissue. It is selectively internalized and exhibits high toxicity to the mammalian cell type or tissue, wherein the antimicrobial peptide has low mammalian cytotoxicity when not bound to the tumor homing molecule Having. The homing pro-apoptotic conjugate of the present invention can be, for example, CNGRC-GG- _D (KLAKLAK) ₂ or ACCDRGDCFC-GG- _D (KLAKLAK) ₂ . The conjugates of the invention are useful, for example, for treating patients with tumors having angiogenic vasculature.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This study is based on the National Cancer Center
ute) (USA) grants CA74238, CA28896 and CA3
0199, as well as a grant from the Department of Defense DAMD 17-98-1-8
581. The United States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention [0002] The present invention relates generally to the fields of cancer biology and drug delivery, and more particularly, to the selective targeting of antimicrobial peptides.

BACKGROUND INFORMATION [0003] Developments that have continued over the past quarter century have resulted in considerable improvements in the ability of physicians to diagnose cancer in patients. Unfortunately, methods of treating cancer have not kept pace with methods of diagnosing the disease. Thus, although mortality from various cancers has been decreasing due to physicians' ability to detect disease early, the ability to treat patients presenting with more advanced disease has progressed minimally. I haven't.

[0004] A major obstacle to progress in treating cancer is the relative lack of factors that can selectively target cancer while normal tissues are relentless. For example, radiation therapy and surgery, which are generally localized procedures, can cause significant damage to normal tissue in the area of treatment, scarring, and, in severe cases, normal tissue function. Causes loss. Generally, systemically administered chemotherapy can cause considerable damage to organs such as bone marrow, mucosa, skin and small intestine, which undergo rapid cell turnover and continuous cell division. As a result, undesirable side effects (eg, nausea, hair loss and decreased blood cell count) occur as a result of systemic treatment of cancer patients with chemotherapeutic agents. Such unwanted side effects often limit the amount of treatment that can be administered. Due to these shortcomings in treatment, cancer remains a major cause of patient morbidity and mortality.

[0005] Tumors are characterized in part by relatively high levels of active angiogenesis that result in the continuous formation of new blood vessels to support tumor survival, growth, and metastasis. The angiogenic blood vessels required for tumor survival and growth are distinguishable from the mature vasculature. One of the characteristic features of the angiogenic vasculature is that unique endothelial cell surface markers are expressed. Thus, the blood vessels in a tumor provide a potential target for targeting a therapeutic agent to the tumor, thereby reducing the likelihood that the agent will kill sensitive normal tissue. The targeting of anti-cancer therapeutics to angiogenic vasculature depends on the identification of compounds that selectively home to angiogenic vasculature.

[0006] Strong antibacterial activity has been observed for a class of peptides, including naturally occurring peptides (eg, melittin, gramicidin, magainin, defensin, and cecropin). Naturally occurring antimicrobial peptides and related synthetic antimicrobial sequences generally have an equal number of polar and non-polar residues in the amphipathic domain, and a sufficient number of basic residues, Neutral p
H gives the peptide an overall positive charge. The biological activity of amphipathic α-helical peptides against Gram-positive bacteria can result from the ability of these peptides to form ion channels through the membrane bilayer. Many antimicrobial peptides selectively bind bacteria while maintaining low mammalian cytotoxicity, with apparent differential sensitivity of bacterial cells due to membrane differences between bacterial and mammalian cells. Inhibits and kills. As shown herein, these antimicrobial peptides are conferred selective cytotoxic activity on certain eukaryotic cell types, such as endothelial cells of angiogenic vessels that support tumor growth. obtain.

[0007] There is a need for new anti-cancer therapeutics that are selectively targeted to the angiogenic vasculature. The present invention provides a homing pro-apoptotic peptide that binds an antimicrobial peptide to a tumor homing compound to produce a conjugate that has selective toxicity to the angiogenic vasculature.
This need is met by providing c peptides). Related advantages are provided as well.

SUMMARY OF THE INVENTION The present invention provides a pro-apoptotic conjugate that can selectively home to tissue. The conjugate comprises a tumor homing molecule that selectively homes to a selected mammalian cell type or tissue conjugated to an antimicrobial peptide, wherein the conjugate selectively binds to the mammalian cell type or tissue. Internalized and exhibits high toxicity to the mammalian cell type or tissue, and wherein the antimicrobial peptide has low mammalian cytotoxicity when not bound to the tumor homing molecule. In one embodiment, a homing pro-apoptotic conjugate of the invention exhibits selective toxicity on angiogenic endothelial cells.

[0009] In another embodiment, a homing pro-apoptotic conjugate of the invention comprises an antimicrobial peptide having an amphipathic α-helical structure. The antimicrobial peptide portion of the homing pro-apoptotic conjugate is, for example, the sequence (KLAKLAK) ₂ (SEQ ID NO: 20).
0); (KLAKKKA) ₂ (SEQ ID NO: 201); (KAAKKKA) ₂ (SEQ ID NO: 202); or (KLGKKLG) ₃ (SEQ ID NO: 203). In a preferred embodiment, the antimicrobial peptide comprises the sequence _D (KLAKLAK) ₂ .

[0010] The invention further provides a homing pro-apoptotic conjugate, wherein the tumor homing molecule is a tumor homing peptide. Such a tumor homing peptide may comprise the amino acid sequence NGR and may be, for example, the peptide CNGRC (SEQ ID NO: 8), NGRAHA (SEQ ID NO: 6), or CNGRCVSGCAGRC (SEQ ID NO: 3). Tumor homing peptides useful in the conjugates of the invention may also include the amino acid sequence RGD, and include, for example, the peptide CDCRGDCF
C (SEQ ID NO: 1). In a preferred embodiment, the homing pro-apoptotic conjugate of the invention has the sequence CNGRC-GG- _D (KLAKLAK) ₂ or A
It has CDCRGDCFC-GG- _D (KLAKLAK) ₂ .

[0011] The invention further provides a method of directing an antimicrobial peptide in vivo to a tumor having angiogenic vasculature. This method is practiced by administering a homing pro-apoptotic conjugate of the invention to a subject comprising a tumor having angiogenic vasculature. In such a method of the invention, the antimicrobial peptide can be, for example, the sequence _D (
(KLAKLAK) ₂ . In a preferred embodiment, the homing pro-apoptotic conjugate administered to the subject has the sequence CNGRC-GG- _D (KLAKLA
K) ₂ or ACCDRGDCFC-GG- _D (KLAKLAK) ₂ .

The present invention further provides a method of inducing selective toxicity in a tumor having an angiogenic vasculature in vivo. This method is practiced by administering a homing pro-apoptotic conjugate of the invention to a subject comprising a tumor having angiogenic vasculature. In such a method of the invention, the antimicrobial peptide is, for example, the sequence _D
(KLAKLAK) ₂ . In a preferred embodiment, the homing pro-apoptotic conjugate has the sequence CNGRC-GG- _D (KLAKLAK) ₂ or ACD
It has CRGDCFC-GG- _D (KLAKLAK) ₂ .

[0013] Also provided herein are methods of treating a patient having a tumor with angiogenic vasculature. In such a method of treatment, the homing pro-apoptotic conjugate of the invention is administered to a patient and is selectively toxic to a tumor. The antimicrobial peptide portion can include, for example, the sequence _D (KLAKLAK) ₂ . In a preferred embodiment, the homing pro-apoptotic conjugate has the sequence CNGRC-GG
_−D (KLAKLAK) ₂ or ACCDRGDCFC-GG- _D (KLAKLA
K) having ₂ .

[0014] The invention further comprises a prostate homing peptide conjugated to an antimicrobial peptide.
Provided is a chimeric prostate homing pro-apoptotic peptide, which is selectively internalized by prostate tissue and exhibits high toxicity to prostate tissue, while the antimicrobial peptide binds to the prostate homing peptide. Otherwise, it has low mammalian cytotoxicity. In the chimeric peptide of the present invention, the prostate homing peptide portion has, for example, the sequence SMSIA
RL (SEQ ID NO: 207) or a functionally equivalent sequence, and the antimicrobial peptide portion may have an amphipathic α-helix structure, for example, the following sequence: (KLAKLAK) ₂ (SEQ ID NO: 200), (KLACKLA) ₂ (
(SEQ ID NO: 201), (KAAKKAA) ₂ (SEQ ID NO: 202), or (KLG
KKLG) ₃ (SEQ ID NO: 203). In a preferred embodiment, the antimicrobial peptide moiety comprises the sequence _D (KLAKLAK) ₂ . An exemplary prostate homing pro-apoptotic peptide is referred to herein as SMSIARL-GG- _D (KLAKLA
K) Provided as ₂ .

Further, the present invention provides a method for inducing selective toxicity in vivo in prostate cancer. The method comprises administering to a subject having prostate cancer a chimeric prostate homing pro-apoptotic peptide comprising a prostate homing peptide conjugated to an antimicrobial peptide, wherein the chimeric peptide is selectively internalized by prostate tissue. And show high toxicity to prostate tissue, but the antimicrobial peptide has low mammalian cytotoxicity when not bound to the prostate homing peptide. Methods for inducing selective toxicity in prostate cancer in vivo can be performed, for example, using a prostate homing peptide comprising the sequence SMSIARL (SEQ ID NO: 207) or a functionally equivalent sequence. The antimicrobial peptide can include, for example, the sequence _D (KLAKLAK) ₂ . In a preferred embodiment, the chimeric prostate homing pro-apoptotic peptide has the sequence SMSI
ARL-GG- _D (KLAKLAK) ₂ .

DETAILED DESCRIPTION OF THE INVENTION Antimicrobial peptides (also known as lytic or channel-forming peptides)
Is a broad spectrum antimicrobial agent. These peptides typically disrupt bacterial cell membranes, causing cell lysis and death. More than 100 antimicrobial peptides occur naturally. In addition, analogs have been described by Javadpour et al.
Med. Chem. 39: 3107-3113 (1996); and Blon.
Delle and Houghten, Biochem. 31: 12688-12
694 (1992), each of which is newly synthesized, as described herein. Some antimicrobial peptides, such as melittin, are not selective and damage normal mammalian cells with minimal bactericidal concentrations, while others are bacterial cell selective. For example, naturally occurring magainins and cecropin exhibit substantial bactericidal activity at concentrations that are not lethal to normal mammalian cells.

Antimicrobial peptides often contain a cationic amino acid, which is attracted to the head group of the anionic phospholipid, causing preferential disruption of the negatively charged membrane. Once electrostatically bound, this amphipathic helix can distort the lipid matrix, resulting in loss of membrane barrier function (Epend, The Am
phipathic Helix, CRC Press: Boca Raton
(1993); Lugtenberg and van Alphen, Bioch.
im. Biophys. Acta, 737: 51-115 (1983), each of which is incorporated herein by reference. Prokaryotic cytoplasmic membranes maintain large transmembrane potentials and have high volumes of anionic phospholipids. In contrast,
The outer leaflets of eukaryotic plasma membranes generally have low or no membrane potential and are almost exclusively composed of zwitterionic phospholipids. Thus, due to different membrane compositions, antimicrobial peptides can selectively disrupt prokaryotic membranes when compared to eukaryotic membranes.

The present invention provides that an antimicrobial peptide sequence can bind to a tumor homing molecule to form a homing pro-apoptotic conjugate, which is generally non-toxic outside of eukaryotic cells. However, it relates to the surprising discovery that when targeted and internalized in eukaryotic cells, it promotes cell death following disruption of the mitochondrial membrane. Homing pro-apoptotic conjugate (
For example, HPP-1 (including the antimicrobial peptide _D (KLAKLAK) ₂ conjugated to the cyclic tumor homing molecule CNGRC (SEQ ID NO: 8)) can have selective toxicity in vivo to angiogenic endothelial cells, Therefore, it may be useful as a new kind of anti-cancer therapeutic agent.

Accordingly, the present invention provides a homing pro-apoptotic conjugate, wherein the homing pro-apoptotic conjugate is a tumor homing molecule that selectively homes to a selected mammalian cell type or tissue bound to an antimicrobial peptide. The conjugate is selectively internalized to a mammalian cell type or tissue and exhibits high toxicity to the mammalian cell type or tissue, and the antimicrobial peptide binds to the tumor homing molecule If not, it has low mammalian cytotoxicity. For example, a homing pro-apoptotic conjugate of the invention can be selective toxic to angiogenic endothelial cells, and methods of inducing selective toxicity in vivo, for example, in tumors with angiogenic vasculature May be useful.

As disclosed herein, the synthetic antimicrobial peptide _D (KLAKLAK) ₂ , which has selective toxicity to bacteria compared to eukaryotic cells, has a pronounced mitochondrial swelling at a concentration of 10 μM. Was induced (FIG. 2a). This concentration is significantly less than that required to kill eukaryotic cells, indicating that _D (KLAKLAK) ₂ preferentially destroys mitochondrial membranes as compared to eukaryotic membranes. Shown (see Example I). Furthermore, _D (KLAKLAK) ₂ is a characteristic caspase 3
While activated mitochondria-dependent cell-free apoptosis as measured by processing (FIG. 2b), the non-alpha helix-forming peptide, DLSLARLATARLAI (SEQ ID NO: 204), Did not activate. These results indicate that antimicrobial peptides such as _D (KLAKLAK) ₂ can disrupt mitochondrial membranes, which, like bacterial membranes, have high volumes of anionic phospholipids, Reflects the common ancestry of mitochondria (Epand, supra, 1993; Lugtenberg)
And van Alphen, supra, 1983; Matsuzaki et al., Bio.
chemistry 34: 6521-6526 (1995); Hovius et al., FEBS Lett. 330: 71-76 (1993); and Baltc.
heffsky and Balteffeffsky, Mitochondria
and Microsomes (Lee et al.), Addison-Wesley:
Reading, MA (1981), each of which is incorporated herein by reference.

As further disclosed herein, the antimicrobial peptide _D (KLAKLAK
₂ ) Cyclic tumor homing peptide CNGR via glycinylglycine bridge
C (SEQ ID NO: 8) and coupled to the peptide CNGRC-GG- _D (KLAKLA
K) ₂ (designated "HPP-1"). As disclosed herein, HPP-1 was tested in a tissue culture model of angiogenesis by assaying for cord formation. This cord formation is a migrating form, usually indicated by a change in endothelial cell morphology from a "cobblestone" shape to a cell chain or cord. Normal human skin microvascular cells (DMEC) at 60 μM HPP-1
In the proliferating state (FIG. 3c) or the cord-like array forming state (FIG. 3d),
It resulted in a decrease in percent survival over time, but the non-targeted _D (KLAKLA
K) Treatment with ₂ peptides resulted in negligible loss of viability (see Example II). Further, as shown in Table 1, HPP
LC ₅₀ of DMEC in the treated propagation or moving at -1 were lower order than the LC ₅₀ of angiogenesis inhibitory DMEC maintained at 100% confluency in monolayer. This indicates preferential killing by HPP-1 under angiogenic conditions. The results disclosed herein further indicate that the mitochondria of DMEC treated with _D (KLAKLAK) ₂ for 24 hours remained morphologically normal while C
NGRC-GG- _D (KLAKLAK) ₂ or ACCDRGDCFC-GG- _D
Mitochondria of DMEC treated with (KLAKLAK) ₂ showed changes in mitochondrial morphology before showing classical morphological indicators of apoptosis, including nuclear enrichment and fragmentation.

As shown in Example III, this HPP-1 peptide CNGRC-GG- _D (KLAKLAK)_TwoIs also active in vivo. 4a and 4b
As disclosed, gnus carrying human MDA-MD-435 breast carcinoma xenografts
Dogs were treated with HPP-1. Tumor volume compared to control group
In this group of HPP-1 treated animals, on average one order of magnitude smaller and the survival rate was longer.
Was. In addition, some of the HPP-1 treated mice had control mice.
Lived several months longer than This prevents both growth and metastasis of the primary tumor.
Indicates that it was harmed. Disruption of tumor architecture and extensive cell death can lead to tumor histopathology
Was evident on biological analysis, with about 50% apoptotic cell death. HP
P-1 is also effective against tumors derived from the human melanoma cell line C8161;
ACCDRGDCFC-GG-_D(KLAKLAK)_TwoIs MDA-MD-4
It was effective against 35 breast cancer tumors. In short, these results indicate that the tumor Homin
Homing pro-apoptotic marker based on peptide sequence and antimicrobial peptide sequence
Peptides can be non-toxic outside of eukaryotic cells, but targeted eukaryotic targets
Cellular death following disruption of mitochondrial membrane when internalized into cells
It can be promoted. Homing pro-apoptotic peptides such as HPP-1
Tide has selective toxicity to angiogenic endothelial cells, and as an anticancer therapeutic
It can be of particular value.

[0023] Further results disclosed herein indicate that retinal neovascularization can be selectively inhibited by the homing pro-apoptotic conjugates of the present invention. In particular, the homing pro-apoptotic conjugate CDCRGDCFC-GG- _D (KLAK
The number of retinal neovessels in mice treated with (LAK) ₂ was reduced to 30-40% of control levels (see Figure 5). Thus, a homing pro-apoptotic conjugate of the invention can include a tumor homing molecule or can include another homing molecule that selectively homes to a selected mammalian cell type or tissue.

[0024] The homing pro-apoptotic conjugates of the present invention are characterized by being highly toxic to the mammalian cell type being internalized. As used herein, the term "very toxic" means that the conjugate is relatively effective in causing cell death of the selected cell type or tissue. One of skill in the art can use one of a variety of well-known assays for cell viability to
Understand that toxicity can be analyzed. Generally, the term highly toxic is used to refer to conjugates that have a half-maximal killing concentration (LC ₅₀ ) of less than about 100 μM, preferably less than about 50 μM. For example, as disclosed herein,
Homing proapoptotic conjugate HPP-1, for DMEM cells and KS1767 cells DMEC cells and cord-like sequences formed during in angiogenesis growth, respectively, were characterized by LC ₅₀ that 51,34 and 42. further,
Prolonged survival of tumor-bearing mice treated with the homing pro-apoptotic conjugate of the present invention indicates that this selective toxicity can be reproduced in vivo.

As used herein, the term “antimicrobial peptide” refers to a naturally occurring or synthetic peptide that has antimicrobial activity, where the antimicrobial activity is the growth of one or more microorganisms The ability to kill or delay. The antimicrobial peptide can, for example, kill or slow the growth of bacteria, including gram-positive or gram-negative bacteria, or one or more strains of fungi or protozoa. Thus, antimicrobial peptides are, for example, Escherichia coli, Pseudomo
nas aeruginosa or Staphylococcus aureu
may have, for example, bacteriostatic or bactericidal activity against one or more of the strains. While not wishing to be bound by the following, antimicrobial peptides may have biological activity due to the ability to form ion channels through the membrane bilayer as a result of self-aggregation.

Antimicrobial peptides are typically very basic and can have a linear or cyclic structure. As discussed further below, antimicrobial peptides can have an amphipathic α-helix structure (US Pat. No. 5,789,542; Javapadou).
et al., supra, 1996; Blondelle and Houghten, supra, 1
992). Antimicrobial peptides are also described, for example, in Mancheno et al.
. Peptide Res. 51: 142-148 (1998).

The antimicrobial peptide can be a naturally occurring peptide or can be a synthetic peptide. Naturally occurring antimicrobial peptides are isolated from biological sources (eg, bacteria, insects, amphibians and mammals) and are thought to present inducible defense proteins that can protect the host organism from bacterial infection. . Naturally occurring antimicrobial peptides include gramicidin, magainin, melittin, defensin, and cecropin (eg, Maloy and Kari, Biopo).
lymers 37: 105-122 (1995); Alvarez-Brav.
o et al., Biochem. J. 302: 535-538 (1994); Bessa.
Ile et al., FEBS 274: 151-155 (1990); and Blon.
Dell and Houghter, Annual Reports in M
edinical Chemistry, edited by Bristol, pages 159-168, Academic Press, San Diego (each of which is incorporated herein by reference). As discussed further below, the antimicrobial peptide can also be an analog of the natural peptide, in particular, an analog that retains or enhances amphipathicity.

Antimicrobial peptides incorporated into the homing pro-apoptotic conjugates of the present invention have low mammalian cytotoxicity when not conjugated to tumor homing molecules.
Mammalian cytotoxicity can be easily assessed using conventional assays. For example, mammalian cytotoxicity can be assayed by lysis of human erythrocytes in vitro, as described in Javapadour et al., Supra, 1996. An antimicrobial peptide having "low mammalian cytotoxicity" is not soluble in human erythrocytes,
Alternatively, for lytic activity, a concentration higher than 100 μM, preferably 200 μM
, 300 μM, 500 μM or 1000 μM.

In a preferred embodiment, the present invention also provides a homing pro-apoptotic conjugate wherein the antimicrobial peptide moiety promotes mitochondrial membrane disruption when internalized by a eukaryotic cell.
Conjugate). In particular, such antimicrobial peptides preferentially destroy mitochondrial membranes as compared to eukaryotic membranes. Mitochondrial membranes have a high content of negatively charged phospholipids, similar to bacterial membranes, but in contrast to eukaryotic plasma membranes. Antimicrobial peptides can be assayed for activity in disrupting mitochondrial membranes, for example, using a mitochondrial swelling assay (as described in Example I) or another assay well known in the art. As disclosed herein, for example, _D (KLAKLAK) ₂ causes significant mitochondrial swelling by 1
Induction was performed at a concentration of 0 μM. This concentration is significantly lower than the concentration required to kill eukaryotic cells. For example, 50 μM, 40 μM, 30 μM, 20 μM
Antimicrobial peptides that induce significant mitochondrial swelling below 10 μM are considered peptides that promote mitochondrial membrane disruption.

The present invention also provides a homing pro-apoptotic conjugate, wherein the tumor homing molecule is linked to an antimicrobial peptide having an amphipathic α-helix structure.
In the homing pro-apoptotic conjugate of the present invention, the antimicrobial peptide moiety may be, for example, the sequence (KLAKLAK) ₂ , (SEQ ID NO: 200); (KLAKLA) ₂ (
SEQ ID NO: 201); (KAAKKAA) ₂ (SEQ ID NO: 202); or (KLG
KKLG) ₃ (SEQ ID NO: 203), especially the sequence _D (KLAKLAK) ₂ .
The homing pro-apoptotic conjugate of the present invention may be, for example, the sequence CNGRC-GG
_−D (KLAKLAK) ₂ or ACCDRGDCFC-GG- _D (KLAKLA
K) may have ₂ .

Antimicrobial peptides generally have a random coil conformation in dilute aqueous solutions, but high levels of helix are induced by helix-promoting solvents and amphiphilic media (eg, micelles, synthetic bilayers or cell membranes). obtain. α-helix structures are well known in the art, and an ideal α-helix has a translocation of 3.6 residues per turn and 1.5 ° per residue (5.4 per turn).
Å; Creighton, Proteins: Structures and
Molecular Properties, W.M. H. Freeman, New
York (1984)). In the amphipathic α-helix structure, polar and non-polar amino acid residues are:
Aligned into an amphipathic helix, this helix is such that, when the peptide is viewed along the helix axis, hydrophobic amino acid residues are preferentially present on one side and hydrophilic residues predominate on its opposite side Is an alpha helix that exists.

Antimicrobial peptides with widely varying sequences have been isolated, which share an amphipathic α-helix structure as a common feature (Saberwal et al., Bioch).
im. Biophys. Acta 1197: 109-131 (1994)).
Analogs of the native peptide with amino acid substitutions predicted to enhance amphipathicity and helix properties typically have increased antimicrobial activity. In general, analogs with increased antibacterial activity also have increased cytotoxicity against mammalian cells (Malloy et al., Biopolymers 37: 105-122 (1
995)).

As used herein with reference to an antimicrobial peptide, the term “amphiphilic α-helical structure” refers to a hydrophilic surface, including some polar residues, and non-polar residues at physiological pH. Α-helix having a hydrophobic surface. Polar residues are, for example,
A non-polar residue may be, for example, a leucine residue or an alanine residue, while being a lysine residue or an arginine residue. Antimicrobial peptides having an amphipathic α-helical structure generally have sufficient numbers of polar and non-polar residues in the amphipathic domain and an overall positive charge on the peptide at neutral pH. With a large number of basic residues (Saberwal et al., Biochim. Biophy.
s. Acta 1197: 109-131 (1994), which is incorporated herein by reference). One skilled in the art will appreciate that helix-promoting amino acids (eg, leucine and alanine) can be advantageously included in the antimicrobial peptides of the invention (see, eg, Creighton, supra, 1984).

Various antimicrobial peptides having an amphipathic α-helical structure are well known in the art. Such peptides include synthetic, minimalist peptides based on the heptad building block scheme, where the repeating heptads are composed of repeating trimers with one additional residue. . As such a synthetic antimicrobial peptide, for example, a compound represented by the general formula [(X ₁ X ₂ X ₂ )]
(X ₁ X ₂ X ₂ ) X ₁ ] _n (SEQ ID NO: 205) or [(X ₁ X ₂ X ₂ ) X ₁ (X ₁ X ₂ X ₂ )] _n (SEQ ID NO: 206) Where X ₁ is a polar residue,
X ₂ is a non-polar residue; and n is 2 or 3 (see Javapadour et al., Supra, 1996). (KLAKLAK) ₂ (SEQ ID NO: 200);
(KLAKKLA) ₂ (SEQ ID NO: 201); (KAAKKKA) ₂ (SEQ ID NO: 20)
2); and (KLGKKLG) ₃ (SEQ ID NO: 203) are examples of synthetic antimicrobial peptides having an amphipathic α-helix structure. Similar synthetic antimicrobial peptides having an amphipathic α-helical structure are also described, for example, by McLaughlin and Bec.
It is known in the art, as described in US Pat. No. 5,789,542 to ker.

Helicality can be readily determined by one skilled in the art, for example, using circular dichroism spectroscopy. The percentage of α-helices can be found, for example, in Javapadour et al., supra, 1996 (also see McLean et al., Biochemistry 30: 31-37 (1991), which is incorporated herein by reference). It can be determined after measuring the molar ellipticity as described at 222 nm. An amphipathic α-helical antimicrobial peptide of the invention can have, for example, at least about 20% when assayed in an amphipathic medium (eg, 25 mM SDS).
Spiral. One skilled in the art will recognize that such antimicrobial peptides having an amphipathic α-helical structure may have, for example, at least about 25%, 30%, 35% or 40% helix when assayed in 25 mM SDS. To understand the. Antimicrobial peptides having an α-helical structure, for example, when assayed in 25 mM SDS, have a 25% to 90% helix; 25% to 60% helix;
25% to 40% helix; 30% to 90% helix; 30% to 60% helix; 30% to 50% helix; 40% to 90% It may have a helix or a helix between 40% and 60%. Amphipathicity can be easily determined, for example, using the helix wheel representation of the peptide (eg, Bl
ondelle and Houghten, supra, 1994).

An exemplary homing pro-apoptotic conjugate of the invention, CNGRC-GG
The structure of _-D (KLAKLAK) ₂ is shown in FIG. As can be seen in FIG. 1, the homing domain CNGRC (SEQ ID NO: 8) is a disulfide-bonded cyclic structure and is linked via a glycinylglycine bridge to the membrane disruption domain _D (KLAKLAKKLAKLAK). D-amino acids in the membrane disrupting antimicrobial portion of the conjugate may be useful in conferring increased stability to the conjugate in vivo. Furthermore, the membrane disruption _D (KLAKLAKKLAKLAK) part forms an amphipathic helix. In particular, lysine residues are aligned on one face of the helix (shown as dark, shaded areas of the helix), while non-polar leucine and alanine residues are opposed to this helix (lightly shaded). (Marked with).

The homing pro-apoptotic conjugate of the present invention can be a chimeric peptide, wherein the tumor homing molecule is a tumor homing peptide. The homing pro-apoptotic chimeric peptide of the present invention can have various lengths from about 18 amino acids to about 50 amino acids or more. The chimeric peptide of the present invention can have, for example, about 20 to about 50 amino acids, preferably 20 to 40 amino acids, more preferably 20 to 30 amino acids. Such chimeric peptides are, for example, 40, 35, 30, 27, 2,
It may have an upper limit of 5 or 21 amino acids. The chimeric peptides of the present invention can be linear or cyclic. In a preferred embodiment, the homing pro-apoptotic chimeric peptide of the present invention includes a cyclic tumor homing peptide moiety.

[0038] The homing pro-apoptotic chimeric peptide of the present invention can also be a peptidomimetic. As used herein, the term “peptidomimetic” is used broadly to mean a peptide-like molecule that has substantially the activity of the corresponding peptide. Peptidomimetics include chemically modified peptides, peptide-like molecules containing non-naturally occurring amino acids, peptoids, and the like, which have selective homing activity and high toxicity of the peptide that induced the peptidomimetic (eg, “Bur
ger's Medicinal Chemistry and Drug D
discovery ", 5th edition, volumes 1 to 3 (edited by ME Wolff; Wil)
eye Interscience 1995), which is incorporated herein by reference). For example, D amino acids can be advantageously included in the antimicrobial peptide portion of the chimeric peptides of the invention (see Examples I and II). Peptidomimetics offer various advantages over peptides, including increased stability during passage through the gastrointestinal tract, and therefore can be advantageously used as oral therapeutics.

In the homing pro-apoptotic conjugates of the present invention, a “binding domain” may be used to link the tumor homing peptide and the antimicrobial peptide, and may, for example, impart overall flexibility to the conjugate. The binding domain can be, for example, a glycinyl glycine linker, an alanine yl alanine linker or other linker incorporating glycine, alanine or other amino acids. The use of a glycinylglycine binding domain is described in Example II.

The vasculature within the tumor generally undergoes active angiogenesis, resulting in the continuous formation of new blood vessels that support the growing tumor. Such angiogenic blood vessels include endothelial cell surface markers (α _v β ₃ integrin (Brooks,
Cell 79: 1157-1164 (1994); WO 95/14714 (
International application filing date Nov. 22, 1994)) and receptors for angiogenic growth factors (Mustonen and Alitalo, J. Cell Bi).
ol. 129: 895-898 (1995); Lappi, Semin. Can
cer Biol. 6: 279-288 (1995)). Furthermore, tumor vasculature is histologically distinguishable from other blood vessels in that the tumor vasculature is fenestrated (Folkman,
Nature Med. 1: 27-31 (1995); Rak et al., Antica.
ncer Drugs 6: 3-18 (1995)). Thus, the unique characteristics of the tumor vasculature make it a particularly attractive target for anti-cancer therapeutics.

As disclosed herein, tumor homing molecules can bind to the endothelial lining of tumor microvessels. The vasculature within the tumor is probably unique due to continuous neovascularization, which results in the formation of new blood vessels required for tumor growth. The unique properties of angiogenic neovasculature within tumors are reflected in the presence of specific markers on endothelial and perivascular cells (Folkman, Nature
Biotechnol. 15: 510 (1997); Risau, FASEB.
J. 9: 926-933 (1995); Brooks et al., Supra, 1994);
These markers appear to be targeted by the disclosed tumor homing molecules.

The ability of tumor homing molecules to target blood vessels within a tumor offers significant advantages over systemic treatment methods or methods that directly target tumor cells. For example, tumor cells depend on the vascular supply for survival, and the endothelial lining of the blood vessels is readily accessible to circulating probes. Conversely, in order to reach solid tumor cells, the therapeutic agent is potentially a dense fibrous stroma with a long diffusion distance, tightly packed tumor cells, and high interstitial pressure to prevent extravasation (Burrows and Thorpe, Pharmacol. Ther. 64:
155-174 (1994)).

Furthermore, if the tumor vasculature is targeted, killing of all target cells may not be required. Because partial exposure of the endothelium can lead to the formation of occlusive thrombi that stop blood flow through the affected tumor vessels (Burrows and Thorpe, supra, 1994). Furthermore, unlike direct tumor targeting, there is an endogenous amplification mechanism in tumor vasculature targeting. A single capillary loop has 100
Up to one tumor cell can be nourished, each of which is critically dependent on the blood supply (Denekamp, Cancer Metast. Rev. 9: 267).
-282 (1990); Folkman, supra, 1997).

As shown above and exemplified herein, tumor homing molecules that are selective for angiogenic endothelial cells of the tumor vasculature are those in which the pro-apoptotic antimicrobial peptide binds to normal and healthy organs or tissues. While reducing the likelihood of having toxic effects,
It may be particularly useful for directing pro-apoptotic antimicrobial peptides to the tumor vasculature. Thus, in one embodiment, the present invention provides a homing pro-apoptotic conjugate comprising a tumor homing molecule that homes to an angiogenic endothelial cell linked to an antimicrobial peptide, wherein the conjugate comprises When selectively internalized by angiogenic endothelial cells and exhibits high toxicity to angiogenic endothelial cells, and when not linked to a tumor homing molecule, antimicrobial peptides have low mammalian cytotoxicity.

As used herein, the term “selective toxicity” means cell death of a selected cell type or tissue that is enhanced as compared to a control cell type or tissue. Generally, selective toxicity is at least twice as high as that of a selected cell type or tissue (eg, angiogenesis) as compared to a control cell type or tissue (eg, angiostatic endothelial cells). Endothelial cells). Thus, as used herein, the term selective toxicity refers to the specific toxicity by which cell death essentially occurs only in the selected cell type or tissue, as well as the selected cell type or tissue. And toxicity that occurs in a limited number of cell types or tissues. One of skill in the art further understands that the term selective toxicity refers to cell death caused by all mechanisms, including apoptotic and necrotic cell death. Accordingly, the homing pro-apoptotic conjugates of the present invention, which exhibit selective toxicity for angiogenic endothelial cells, have enhanced angiogenic endothelial cells compared to angiostatic endothelial cells or other types of surrounding cells. This results in cell death of the cell.

As disclosed herein, an identified tumor homing molecule converts a desired antimicrobial peptide linked to a homing molecule into a selected cell type (eg, an angiogenic endothelial cell). Useful for targeting. After being internalized by angiogenic endothelial cells in the tumor vasculature, the antimicrobial peptide is toxic to endothelial cells, thereby limiting blood supply to the tumor and inhibiting tumor growth.

Tumor homing molecules useful in homing pro-apoptotic conjugates of the invention include, for example, NGR motifs (eg, CNGRC (SEQ ID NO: 8); NGRA
HA (SEQ ID NO: 6) or CNGRCVSGCAGRC (SEQ ID NO: 3)). Tumor homing molecules useful in the present invention also include RGD
Can include a motif and can be, for example, a CDCRGDCFC (SEQ ID NO: 1) or a GSL motif (eg, the peptide CGSLVRC (SEQ ID NO: 5)
). Additional tumor homing molecules can be identified by screening libraries of molecules by in vivo panning as described in further detail below (also, Examples IV-VIII; published on April 22, 1997). US Patent No. 5,622,699; and Pasqualini
And Ruoslahti, Nature 380: 364-366 (1996).
), Each of which is incorporated herein by reference).

As used herein, the term “tumor homing molecule” refers to an organic chemical molecule (eg, a drug; a nucleic acid molecule; a peptide) that selectively homes in vivo to a selected cell type or tissue. Or peptidomimetic or protein). By "selectively home" is meant that in vivo, a tumor homing molecule preferentially binds to a selected cell type or tissue as compared to a control cell type, tissue or organ, and Generally, it is characterized by at least two times more localization in the selected cell type or tissue as compared to a control cell type or tissue. Tumor homing molecules useful in the present invention can be, for example, molecules that preferentially bind to endothelial cells of the angiogenic vasculature as compared to other cell types or angiostatic vasculature.

[0049] Tumor homing molecules were identified using in vivo panning as follows. Panning in vivo against breast cancer, melanoma and Kaposi's sarcoma identified phages expressing various peptides that selectively home to tumors (see Tables 2, 3 and 4, respectively). Thing). Due to the large size of the phage (900 nm-1000 nm) and the short time that the phage can circulate (3-5 minutes), a significant number of phages exit the circulatory system, especially in the brain and kidney. It seems. Histostaining studies have shown that the identified tumor homing molecules primarily home to and bind to endothelial cell surface markers that appear to be expressed in an organ-specific manner. These results indicate that in vivo panning can be used to identify and analyze endothelial cell specificity. Such an analysis is not possible with endothelial cells in culture.
This is because cultured cells tend to lose tissue-specific differences (P
auli and Lee, Lab. Invest. 58: 379-387 (198
8)).

The conditions under which the in vivo panning was performed identified a tumor homing peptide that primarily binds to endothelial cell markers, but the specific presence of phage expressing the tumor homing peptide was also observed in tumor parenchyma, especially in the administration of this peptide. Later observed late (Example VII). These results demonstrate that phage expressing the peptide can pass through the blood vessels in the tumor, presumably due to the fenestration properties of the blood vessels, and that the in vivo panning method is not compatible with the target molecule expressed by the tumor cells. And that it may be useful for identifying target molecules expressed by endothelial cells.

The phage peptide display library is available from Smith and Scott
(Supra, 1993; see also Koivunen et al., Biotechnology).
13: 265-270 (1995); Koivunen et al., Meth. Enzy
mol. 245: 346-369 (1994b), each of which is incorporated herein by reference). Oligonucleotides encoding peptides having a substantially randomized amino acid sequence were synthesized based on the "NNK" codon. Where "N" is
A, T, C or G, and “K” is G or T. "NNK" encodes 32 triplets encoding 20 amino acids and an amber stop codon (Scott and Smith (supra) 1990). In some libraries, at least one codon encoding cysteine was also included in each oligonucleotide so that cyclic peptides could be formed through disulfide bonds (Example IV). This oligonucleotide was inserted in frame with the sequence encoding gene III protein (gIII) in Vector Fuse 5, resulting in the expression of a peptide-gIII fusion protein. Following expression, this fusion protein was expressed on the surface of phage containing vectors (Koivunen et al. (Supra) 1994b; Smith and Scott).
t (supra) 1993).

Following the in vivo panning method, the phages were isolated based on their ability to selectively home to human breast carcinomas, murine melanomas or human caposi's sarcomas displaying only slightly different peptide sequences ( See Tables 2, 3 and 4, respectively). One of screening, peptides that selectively bind to alpha _V-containing integrins has been demonstrated previously (Koivunen et al., Supra 1995
WO 95/14714), the arginine-glycine-aspartate (RGD) integrin recognition sequence (Ruoslahti, Ann. Rev.);
. Cell Level. Biol. 12: 697 (1996)). Most sequences of the remaining tumor homing peptides did not show significant similarity to known ligands for endothelial cell receptors. But,
One of the tumor homing peptides is asparagine-glycine-arginine (NG
R) Contains motifs. This motif is a weak integrin binding motif similar to the motif present in integrin binding peptides (July 1, 1996).
Ruoslahti et al., U.S. Patent No. 5,536,814, issued June 6, incorporated herein by reference; also, Koivunen et al., Supra, 1994.
a)). Other screens showed many NGR-containing peptides (see Table 2). Despite the weak integrin binding ability of NGR peptides, integrin receptors cannot be target molecules recognized by the NGR tumor homing peptides exemplified herein. As used herein, the term "integrin" means a heterodimeric cell surface adhesion receptor.

The peptides expressed by phage homing to breast tumors include the peptides CGRECPRLCQSSC (SEQ ID NO: 2) and CNGRCVSGCAGRC
(SEQ ID NO: 3; see Table 2; Example V). Similarly, peptide CD
Tumor homing peptides, including CRGDCFC (SEQ ID NO: 1) and CGSLVRC (SEQ ID NO: 5), were identified from two other phage libraries administered to breast tumor bearing mice (Table 2). Some of these motifs and novel motifs have also been isolated by screening against mouse melanoma and human Kaposi's sarcoma (see Tables 3 and 4). These results demonstrated that tumor homing molecules could be identified using in vivo panning methods.

The three major tumor homing motifs identified may be particularly useful in homing pro-apoptotic conjugates of the invention. The tumor homing molecule part
Using a homing pro-apoptotic conjugate containing an NGR, RGD or GSL motif, the linked antimicrobial peptide can target endothelial cells of the angiogenic vasculature.

[0055] In one embodiment, the invention provides a homing pro-apoptotic conjugate comprising a tumor homing peptide comprising the sequence NGR linked to an antimicrobial peptide. In such a homing pro-apoptotic conjugate of the invention, the tumor homing peptide can be, for example, CNGRC (SEQ ID NO: 8); NGRAHA (SEQ ID NO: 6) or CNGRCVSGCAGRC (SEQ ID NO: 3). In a preferred embodiment, the homing pro-apoptotic conjugate has the sequence CNGR
C-GG- _D (KLAKLAK) ₂ .

In another embodiment, the present invention provides a homing pro-apoptotic conjugate comprising a tumor homing peptide comprising the sequence RGD linked to an antimicrobial peptide. In such a homing pro-apoptotic conjugate, the tumor homing peptide can be, for example, CDCRGDCFC (SEQ ID NO: 1). In a preferred embodiment, the homing pro-apoptotic conjugate has the sequence ACCDRG
DCFC-GG- _D (KLAKLAK) ₂ .

The present invention further provides a homing pro-apoptotic conjugate comprising a tumor homing peptide comprising the sequence GSL linked to an antimicrobial peptide. In such a homing pro-apoptotic conjugate, the tumor homing peptide can be, for example, CGSLVRC (SEQ ID NO: 5).

As discussed above, one motif is the peptide structure CDCRG
Includes DCFC (SEQ ID NO: 1) sequence embedded in RGD (Ruoslahti, supra 1996), the peptide structure is known to selectively bind to alpha _V integrins (Koivunen et al. (Supra) 1995; WO 95/1
4714). α _V β ₃ integrin and α _V β ₅ integrin are markers for angiogenic vessels (Brooks et al., supra) 1994; Friedlande
r et al., Science 270: 1500 (1995)), peptide CDCRG.
Phages expressing DCFC (SEQ ID NO: 1) were investigated for tumor targeting,
And, as disclosed herein, the tumors homed in a highly selective manner (see Example VI). In addition, homing by the CDCRGDCFC (SEQ ID NO: 1) phage was inhibited by co-administration of free CDCRGDCFC (SEQ ID NO: 1) peptide.

Another breast tumor homing peptide has the sequence CNGRCVSGCAGRC (SEQ ID NO: 3), which contains an NGR motif previously shown to have weak integrin binding activity (Koivunen et al., J. Biol). Chem.
268: 20205-20210 (1993); Koivunen et al., Supra.
994a; WO 95/14714). Since the NGR-containing peptide was identified, two additional peptides (NGRAHA, a linear peptide (SEQ ID NO: 6))
And the cyclic peptide CVLNGRMEC (SEQ ID NO: 7), each of which contains an NGR motif, was investigated for tumor homing. C
Similar to phage expressing NGRCVSGCAGRC (SEQ ID NO: 3), NGR
Phage expressing AHA (SEQ ID NO: 6) or CVLNGRMEC (SEQ ID NO: 7) homed to the tumor. In addition, tumor homing was independent of tumor type or species, as this phage accumulated selectively in human breast carcinomas and in tumors of mouse melanoma-bearing and human Kaposi's sarcoma xenograft-bearing mice .

Various peptides, including RGD-containing peptides and NGR-containing peptides, were generally bound to tumor vessels. This smallest cyclic NGR peptide, CNGRC (
SEQ ID NO: 8) was synthesized based on the CNGRCVSGCAGRC (SEQ ID NO: 3) sequence. The CNGRC (SEQ ID NO: 8) peptide is CNGRCVSGCAGRC
Phage accumulation in breast carcinoma xenografts was inhibited when co-injected with phage expressing either (SEQ ID NO: 3), NGRAHA (SEQ ID NO: 6) or CVLNGRMEC (SEQ ID NO: 7). However, even when a dose that is up to 10 times higher than that of the peptide that inhibited the homing of NGR phage, CNGRC (
SEQ ID NO: 8) peptide did not inhibit homing of phage expressing CDCRGDCFC (SEQ ID NO: 1) peptide. By comparison, the required amount is CNGRC
The CDCRGDCFC (SEQ ID NO: 1) peptide partially inhibited the homing of NGR phage, although 5-10 times more than the amount of peptide (SEQ ID NO: 8). These results indicate that the NGR and RGD peptides bind to different receptor sites in the tumor vasculature.

A third motif, GSL (glycine-serine-leucine), was also identified according to the in vivo panning method in mammary carcinoma, melanoma or kaposi sarcoma-bearing mice. CGSLVRC which is a GSL peptide
Homing of phage expressing (SEQ ID NO: 5) was inhibited by co-administration of free CGSLVRC (SEQ ID NO: 5) peptide. RGD peptide and NGR
Like the peptide, the phage expressing the GSL peptide also bound to tumor blood vessels. As disclosed herein, in view of the identification of conserved RGD, NGR and GSL motifs present in tumor homing peptides, peptides containing such motifs may be identified as tumor homing peptides
In particular, it is recognized that it may be useful to form homing pro-apoptotic conjugates that can selectively deliver antimicrobial peptides to tumors.

Various peptide libraries containing up to 13 amino acids have been constructed, and the NGR peptide CNGRCVSGCAGRC (SEQ ID NO: 3) was obtained as a result of an in vivo panning strategy on breast tumors. This NGR peptide obtained by screening a random peptide library was a tumor homing peptide. In addition, the peptide library has the formula CXXX
NGRXX (SEQ ID NO: 13) or CXXCNGRRCX (SEQ ID NO: 14), each of which is biased against the NGR sequence, and when used for in vivo panning methods against breast tumors, many NGRs The peptide was obtained (see Table 2).

These results indicate that the tumor homing molecules of the present invention may include the amino acid sequence RGD or NGR or GSL. Such tumor homing molecules are
Small peptides such as 5 amino acids (eg, CNGRC (SEQ ID NO: 8))
It is. Such tumor homing peptides can also be at least 13 amino acids long (the largest peptides exemplified herein), but also up to 20 amino acids long, or up to 30 amino acids long, if desired. Or 50-1
It can be up to 00 amino acids long. Advantageously, the tumor homing peptides of the invention are produced by chemical synthesis.

Immunohistochemical analysis was performed by circulating for about 4 minutes followed by comparing tissue staining for phage perfused through the mouse heart, or using tissues analyzed 24 hours after phage injection. It was implemented. 24 hours after administration,
There are essentially no circulating phage and therefore no perfusion is required (Pasqua
lini et al. (supra) 1997). Strong phage staining was observed in the tumor vasculature, but 4 ng from administration of CNGRCVSGCAGRC (SEQ ID NO: 3) phage.
After a minute, nothing was observed in the normal endothelium in the investigated samples (Example VII). By comparison, the staining of the tumor was intense at 24 hours, and it appeared that the blood vessels had spread outwardly into the tumor parenchyma. NGRAHA (SEQ ID NO: 6) phage and CVLNGRMEC (SEQ ID NO: 7) phage showed similar staining patterns (Example VII). In contrast, control organs and tissues showed little or no immunostaining, confirming the specificity of the NGR motif in tumor blood vessels. However, the spleen and liver captured the phage as expected. Because, regardless of the presence of peptide expression by the phage, the general properties of phage particles are taken up by the reticulocellular system (Pasqu
alini et al. (supra) 1997).

[0065] Immunostaining was also observed after administration of phage expressing the GSL motif-containing peptide, CLSGSLSC (SEQ ID NO: 4), and in this case, like phages of the NGR peptide, was found to be intravascular within melanoma tumors. (See below; see also Examples VII and VIII). Similarly, immunostaining after administration of phage expressing the RGD motif-containing peptide CDCRGDCFC (SEQ ID NO: 1) to breast tumor-bearing mice is located in blood vessels within the tumor,
It was not observed in kidney or various other non-tumor tissues (see Examples VI and VII; see also Pasqualini et al., Supra, 1997). These results demonstrate that the various tumor homing peptides generally home to tumor vasculature.

The general applicability of the in vivo panning method to identify tumor homing molecules was investigated by injecting phage expressing various populations of peptides into mice bearing syngeneic melanomas ( Example VIII). The B16 mouse melanoma model was selected for these studies. Because the tumor that forms is
It is highly vascularized and the biology of this tumor line is well characterized (Miner et al., Cancer Res. 42: 4631-46).
38 (1982)). Moreover, since the B16 melanoma cells are cells of mouse origin, species differences between the host and the tumor cell donor may include, for example,
It does not affect the distribution of phage to tumors as compared to the distribution of phage to normal organs. As disclosed herein, in vivo panning methods on B16 melanoma cells have revealed tumor homing peptides, including, for example, the GSL moiety-containing peptide CLSGSLSC (SEQ ID NO: 4; see also Table 3), And immunohistochemical staining of tumors and other organs using anti-phage antibodies was performed by CLSGS
LSC (SEQ ID NO: 4) expressing phage demonstrated immunostaining in melanoma but essentially no staining in skin, kidney or other control organs (Example VIII). The staining pattern generally followed blood vessels within the melanoma, but was not strictly restricted to blood vessels.

Although in vivo panning methods have been performed in mice, tumor homing molecules (eg, peptides containing an NGR, RGD, or GSL motif) may also potentially target human vasculature. NGR phage bound to blood vessels in transplanted human breast tumors, but not to blood vessels in normal tissue. This indicates that this motif may be particularly useful for tumor targeting in patients. CDCRGDCFC (SEQ ID NO: 1) peptide is selectively expressed in tumor blood vessels of human patients, human alpha _V integrin (Koi
vunen et al. (supra) 1995) (Max et al., Int. J. Canc.)
er 71: 320 (1997); Max et al., Int. J. Cancer 72
: 706 (1997)). The use of a homing pro-apoptotic conjugate in which CDCRGDCFC (SEQ ID NO: 1) is linked to an antimicrobial peptide
It offers the further advantage of being able to target the tumor cells themselves. This is because breast carcinoma cells can express, for example, α _v β ₃ integrin (Pasqual
ini et al., supra, 1997). In fact, many human tumors express this integrin. This is particularly true for certain tumors such as malignant melanoma (Albelda et al., Ca
ncres. 50: 6757-6764 (1990); Danen et al., I.
nt. J. Cancer 61: 491-496 (1995); Felding.
-Habermann et al. Clin. Invest. 89: 2018-20
22 (1992); Sanders et al., Cold Spring Harb. S
ymp. Quant. Biol. 58: 233-240 (1992); Mitj.
ans et al. Cell. Sci. 108: 3067-3078 (1995)).
May be involved in the progression of Unlike the CDCRGDCFC (SEQ ID NO: 1) peptide,
The NGR peptide does not appear to bind to MDA-MD-435 breast carcinoma cells.
However, the NGR peptide can deliver a therapeutically effective amount of doxorubicin to breast tumors, which means that even if the tumor homing molecule homes only to the tumor vasculature (ie, not directly to the tumor cells), Such vasculature targeting
Indicates that it is not enough to give the effect of the moiety linked to the molecule.

Since α _V β ₃ integrin is expressed by endothelial cells in the angiogenic vasculature, experiments have shown that tumor vasculature undergoing angiogenesis is disclosed herein. Such methods were used to determine if they could be targeted in vivo. Peptide CDCR known to bind to α _v β ₃ integrin
Phage expressing GDCFC (SEQ ID NO: 1; Koivunen et al., Supra, 1995) were injected into mice bearing tumors formed from human breast carcinoma cells, mouse melanoma cells or human caposi sarcoma cells (Example VII). checking).
The CDCRGDCFC (SEQ ID NO: 1) phage selectively homed to each of the tumors, whereas such homing did not occur with control phage. For example, in mice bearing tumors formed by transplantation of human breast carcinoma cells, 20- to 80-fold more CDCRGDCFC (SEQ ID NO: 1) phage accumulate in tumors than non-selective control phages. Was done.

[0069] Tissue staining for phage revealed that CDCRGDCFC (
SEQ ID NO: 1) showed accumulation of phage, whereas no staining was observed in brain, kidney or other control organs. The specificity of tumor homing by the CDCRGDCFC (SEQ ID NO: 1) phage was demonstrated by competition experiments. In this experiment, co-injection with the free CDCRGDCFC (SEQ ID NO: 1) peptide greatly reduced tumor homing of RGD phage, whereas co-injection with non-RGD containing control peptide did not Homing was not affected (Example VI). These results indicate that the α _V β ₃ target molecule is expressed on the luminal surface of endothelial cells in tumors, and that peptides that bind to α _V- containing integrins undergo this integrin, and thus angiogenesis It demonstrates that it can selectively bind to the vasculature.

The results of these studies indicate that tumor homing molecules can be identified by in vivo panning methods and, in some cases, tumor homing molecules can home to vascular tissue and tumor parenchyma within the tumor. This may be due to the fenestrated nature of the blood vessels, which would allow rapid phage egress from the circulatory system. Due to the ability of such tumor homing molecules to home to tumors, they are useful for targeting linked antimicrobial peptides to tumors. Accordingly, the invention provides conjugates comprising a tumor homing molecule linked to a moiety, such conjugates being useful for targeting the moiety to tumor cells.

The ability of a molecule that homes to a particular tumor to selectively home to another tumor of the same or similar histological type may be due to, for example, human tumors growing in nude mice or for these experiments. Can be determined using mouse tumors in syngeneic mice. For example, various human breast cancer cell lines (breast carcinoma of MDA-MB-435 (Price et al., Cancer Res. 50: 717-721 (1990))
), SKBR-1-II and SK-BR-3 (Fogg et al., J. Natl. C
canceler Inst. 59: 221-226 (1975)), and a mouse breast tumor line (EMT6 (Rosen et al., Int. J. Cancer 57:70).
6-714 (1994)) and C3-L5 (Lala and Parhar, I).
nt. J. Cancer 54: 677-684 (1993)) is readily available and is commonly used as a model for human breast cancer. For example, using such a breast tumor model, information about the specificity of the breast tumor homing molecules identified for a variety of breast tumors can be obtained, and homing to a wide variety of different breast tumors, or the most convenient Molecules that provide a good specificity profile can be identified. Further, such an analysis may yield new information (eg, about tumor stroma). This is because stromal cell gene expression (similar to endothelial cell gene expression) can be altered by the tumor so that it cannot be regenerated in vitro.

The selective homing of molecules (eg, peptides or proteins) to tumors results from the specific recognition by peptides of specific cell target molecules (eg, cell surface receptors present on cells in the tumor). obtain. Homing selectivity is 1
Depending on the specific target molecule expressed on only one or several different cell types,
As a result, this molecule homes primarily to tumors. As discussed above,
The identified tumor homing peptide is capable of recognizing, at least in part, endothelial cell surface markers in blood vessels present in the tumor. However, most cell types, especially particular cell types unique to organs or tissues, can express unique target molecules. Thus, in vivo panning can be used to identify molecules that selectively home to particular types of tumor cells (eg, breast cancer cells); specific homing is demonstrated by performing appropriate competition experiments. obtain.

As used herein, the term “tumor” refers, at least in part, to a mass of cells characterized by comprising angiogenic vasculature. the term"
"Tumor" is used extensively to include tumor parenchymal cells as well as supporting stroma, including angiogenic blood vessels that infiltrate the tumor parenchymal cell mass. Tumors are generally malignant tumors (ie, "cancers"), but tumors can also be non-malignant. However, neovascularization is associated with tumors. The terms “normal” tissue or “non-tumor” tissue are used to refer to tissue that is not “tumor”. As disclosed herein, a tumor homing molecule can be identified based on the ability of the molecule to home to a tumor but not to the corresponding non-tumor tissue.

As used herein, the term “corresponding” when used in reference to a tumor or tissue, or both, refers to two or more tumors, or two or more tissues, or a tissue or tissue. It means that the tumors are of the same histological type. One of skill in the art will appreciate that the histological type of a tissue can be determined by the function of the cells containing the tissue.
on)). Thus, those skilled in the art will recognize, for example, that the non-tumor tissue corresponding to breast tumor is normal breast tissue, while the non-tumor tissue corresponding to melanoma is skin, which includes melanocytes. . Further, for the purposes of the present invention, a tumor homing molecule can specifically bind to a target molecule expressed by the vasculature in a tumor, which generally includes blood vessels that undergo neovascularization;
In this case, it will be appreciated that the tissue corresponding to the tumor includes non-tumor tissue including blood vessels that do not undergo active angiogenesis.

[0075] Tumor homing molecules useful in the present invention can be identified by screening libraries of molecules by in vivo panning as disclosed herein, and US Pat. No. 5,622,699 ( Published April 22, 1997); and Pasqualini and Ruoslahti, Natur
e 380: 364-366 (1996), each of which is incorporated herein by reference. As used herein, the term "library" refers to a collection of molecules. Libraries can contain several or many different molecules, varying from about 10 molecules to billions or more.
If desired, the molecule can be attached to a tag, which can facilitate recovery or identification of the molecule.

As used herein, the term “molecule” refers to a polymeric or non-polymeric organic chemical (eg, a drug); a nucleic acid molecule (eg, RNA, cDNA or oligonucleotide); a peptide ( A variant or modified peptide or peptide-like molecule as referred to herein as a peptidomimetic, which mimics the activity of the peptide); or a protein (eg, an antibody or growth factor receptor or a derivative thereof). It is used broadly to mean a fragment (eg, an Fv fragment, Fd fragment or Fab fragment of an antibody that contains a binding domain). For convenience, the term "peptide" is used extensively herein to mean peptides, proteins, fragments of proteins, and the like. The molecule can also be a non-naturally occurring molecule, which is not naturally occurring but is produced as a result of an in vitro method, or a naturally occurring molecule (eg, a protein expressed from a cDNA library). Or fragments thereof).

[0077] Tumor homing molecules can also be peptidomimetics. As used herein, the term “peptidomimetic” is used broadly to mean a peptidomimetic molecule that has the binding activity of a tumor homing peptide. With respect to the tumor homing peptides of the present invention, peptidomimetics (which are chemically modified peptides,
Including peptide-like molecules containing non-naturally occurring amino acids, peptoids, etc.)
This peptidomimetic has the binding activity of the tumor homing peptide from which it is derived (see, for example, Burger's Medicinal Chemistry and
Drug Discovery, supra, 1995).

[0078] Methods for identifying peptidomimetics are well known in the art and include, for example, screening a database containing a library of potential peptidomimetics. For example, Cambridge Structural
Database contains a collection of more than 300,000 compounds with known crystal structures (Allen et al., Acta Crystallogr. Section B, 35: 2331 (1979)). The depository for this structure is that the new crystal structure has been determined and the appropriate shape (eg, the same shape as the tumor homing molecule, as well as potential geometric and chemical It is frequently updated if it can be screened for compounds with (complementarity). If the crystal structure of the tumor homing peptide or the target molecule that binds the tumor homing molecule is not available, the structure may be, for example,
The program CONCORD (Rusinko et al., J. Chem. Inf. Com.
put. Sci. 29: 251 (1989)). Available Chemicals Director, another database
y (Molecular Design Limited, Informati
ons Systems (San Leandro CA) contains about 100,000 compounds that are commercially available and can also be searched to identify potential peptidomimetics of tumor homing molecules.

Methods for preparing libraries containing diverse populations of various types of molecules (eg, peptides, peptoids and peptidomimetics) are well known in the art, and various libraries are commercially available. (See, eg, Ecker and Crooke, Biotechnology 13: 351-360 (199).
5), and Blondelle et al., Trends Anal. Chem. 14
: 83-92 (1995), and references cited therein, each of which is incorporated herein by reference; also, Goodma
n and Ro, Peptidomimetics for Drug Design
gn, "Burger's Medicinal Chemistry and
Drug Discovery ", Vol. 1 (edited by ME Wolff; John
Wiley & Sons 1995), pp. 803-861, and Gor.
don et al. Med. Chem. 37: 1385-1401 (1994), each of which is incorporated herein by reference). Where the molecule is a peptide, protein or fragment thereof, the molecule may be produced directly in vitro, or may be expressed from a nucleic acid, which may be produced in vitro. Synthetic peptide and nucleic acid chemistry methods are well known in the art.

A library of molecules can also be produced, for example, by constructing a cDNA expression library from mRNA collected from a cell, tissue, organ or organism of interest. Methods for producing such libraries are well known in the art (eg, Sambrook et al., Molecular Cloni).
ng: A laboratory manual (Cold Spring H
arbor Laboratory Press 1989, which is incorporated herein by reference). Preferably, the peptide encoded by the cDNA is expressed on the surface of a cell or virus containing the cDNA. For example, the cDNA can be cloned into a phage vector, such as fuse 5 (Example IV), wherein upon expression, the encoded peptide is expressed as a fusion protein on the surface of the phage.

Further, the library of molecules can include a library of nucleic acid molecules, which can be DNA or RNA, or analogs thereof. For example,
Nucleic acid molecules that bind to cell surface receptors are well known (eg, O'Conn).
ell et al., Proc. Natl. Acad. Sci. , USA 93: 5883.
-5887 (1996); Tuerk and Gold, Science 249.
: 505-510 (1990); Gold et al., Ann. Rev .. Biochem
. 64: 763-797 (1995), each of which is incorporated herein by reference). Thus, a library of nucleic acid molecules can be administered to a subject with a tumor, and subsequently, tumor homing molecules can be identified by in vivo panning. If desired, the nucleic acid molecule can be, for example, a nucleic acid analog that is less susceptible to attack by nucleases (eg, J
elinek et al., Biochemistry 34: 11363-11372 (
1995); Latham et al., Nucl. Acids. Res. 22: 2817
-2822 (1994); Tam et al., Nucl. Acids. Res. 22: 9
77-986 (1994); Reed et al., Cancer Res. 59: 656
5-6570 (1990), each of which is incorporated herein by reference.) As shown herein, in vivo panning is a method for identifying tumor homing molecules. This tumor homing molecule can be used to link to an antimicrobial peptide to form a homing pro-apoptotic conjugate of the invention. In vivo panning involves administering the library to a subject, collecting a sample of the tumor, and identifying a tumor homing molecule. The presence of a tumor homing molecule can be identified using various methods well known in the art. Generally, the presence of a tumor homing molecule in a tumor is identified based on one or more properties common to the molecules present in the library, and then the structure of the particular tumor homing molecule is identified. For example, sensitive detection methods such as mass spectrometry, either alone or in combination with methods such as gas chromatography, can be used to identify tumor homing molecules in tumors. Therefore, if the library
When containing a wide variety of molecules, generally based on the structure of organic molecules such as drugs, tumor homing molecules can be identified by determining the presence of a parent peak for a particular molecule.

If desired, the tumor can be collected and then processed using a method such as HPLC. Such methods provide, for example, a molecule-enriched fraction having a defined range of molecular weights or polar or non-polar properties, depending on the general properties of the molecules comprising the library. obtain. The conditions for HPLC depend on the chemistry of the particular molecule and are well known to those skilled in the art. Similarly, methods for large-scale removal of potential interfering cellular material (eg, DNA, RNA, proteins, lipids or carbohydrates) are enriched in fractions containing organic molecules using, for example, selective extraction methods. As well as methods for converting, it is well known in the art. The library is a collection of diverse organic chemical molecules, each of which is linked to a specific oligonucleotide tag such that the specific molecule is identified by determining the oligonucleotide sequence using the polymerase chain reaction (PCR). ), The genomic DNA is
To reduce the potential of background PCR reactions, they can be removed from collected tumor samples. In addition, libraries can include diverse populations of molecules, such as organic chemical molecules, each linked to a common shared tag. Based on the presence and properties of the shared tag, molecules of a library that selectively home to a tumor can be substantially isolated from a sample of the tumor. These and other methods enrich a collected tumor sample for a particular tumor homing molecule, thereby removing potential contaminants from the collected sample and increasing the selectivity of detection of the molecule. It may be useful to increase.

[0083] The evidence provided herein indicates that a sufficient number of tumor homing molecules home to tumors during in vivo panning, so that the molecules can be rapidly identified. For example, various independent phages expressing the same peptide have been identified in tumors formed from transplanted human breast cancer cells (Table 2), mouse melanoma cells (Table 3) or human Kaposi's sarcoma cells (Table 4). Was.

A substantial fraction of the identified tumor homing molecules have the same structure,
The peptide insert of only a few isolated phages was determined. However, it should be recognized that hundreds of thousands to millions of phages expressing organ homing peptides were recovered after in vivo panning for organ homing molecules (
See, for example, US Pat. No. 5,622,699; Pasqualini and Ruo.
slahti, supra, 1996). These results indicate that specific tumor homing molecules are present in substantial numbers in tumors after in vivo homing, thereby increasing the ease with which the molecules can be identified.

The ease of identification of tumor homing molecules, especially untagged molecules, depends on various factors, including potentially contaminating background cellular material. Thus, if the tumor homing molecule is an untagged peptide,
Many must home to tumors to identify peptides specific to a background of cellular proteins. In contrast, a smaller number of untagged organic chemical homing molecules (eg, drugs) are identifiable.
This is because such molecules are usually not present in the body, or are only present in small numbers. In such cases, sensitive methods such as mass spectrometry can be used to identify tumor homing molecules. One skilled in the art will recognize that the method of identifying a molecule will depend in part on the chemistry of the particular molecule.

[0086] Where the tumor homing molecule is, or is tagged with, a nucleic acid molecule, assays such as PCR are particularly useful for identifying the presence of the molecule.
Because, in principle, PCR can detect the presence of a single nucleic acid molecule (eg, Erlich, PCR Technology: Principle).
s and Applications for DNA Amplicica
Tion (Stockton Press 1989), which are incorporated herein by reference). Preliminary studies show that 10 ng of approximately 600
After intravenous injection of the 0 base pair plasmid into mice and circulation for 2 minutes, the plasmid was demonstrated to be detectable by PCR in lung samples.
These results indicate that the nucleic acid molecule is sufficiently stable when administered in the circulation, so that in vivo panning can be used to identify nucleic acid molecules that selectively home to tumors .

[0087] The molecules of the library are tagged, which may facilitate recovery or identification of the molecules. As used herein, the term “tag” refers to a physical, chemical, or biological moiety (eg, a plastic microbead, oligonucleotide, or oligonucleotide, respectively,) that is linked to a molecule of a library. Bacteriophage). Methods for tagging molecules are well known in the art (H
ermanson, Bioconjugate Technologies (Aca
demic Press 1996), which is incorporated herein by reference.

A tag, which can be a shared tag or a specific tag, can be useful for identifying the presence or structure of a tumor homing molecule in a library. As used herein, the term "shared tag" refers to a physical, chemical or biological portion common to each molecule of a library. For example, biotin can be a shared tag that is linked to each molecule in the library. A shared tag can be useful for identifying the presence of a molecule of a library in a sample, and can also be useful for substantially isolating the molecule from a sample. For example, if the shared tag is biotin, the biotin-tagged molecules in the library can be substantially isolated by binding to streptavidin or their presence binds labeled streptavidin Can be identified. Where the library is a phage display library, phage expressing the peptide are another example of a shared tag. This is because each peptide in the library is linked to a phage.
Further, a peptide such as a hemagglutinin antigen may be a covalent tag linked to each molecule in the library, thus, to substantially isolate library molecules from a selected tumor sample. Allows the use of antibodies specific for the hemagglutinin antigen.

A covalent tag can be a nucleic acid molecule, which is useful for identifying the presence of a library molecule in a sample, or for substantially isolating a library molecule from a sample. obtain. For example, each of the molecules of the library can be linked to the same selected nucleotide sequence, making up a shared tag. The affinity column containing the nucleotide sequence that is complementary to the shared tag then hybridizes the molecules of the library containing the shared tag, and thus can be used to substantially isolate the molecule from a tumor sample. . A nucleotide sequence complementary to a portion of the shared nucleotide sequence tag can also be used as a PCR primer so that the presence of the molecule containing the shared tag can be identified in the sample by PCR.

[0090] The tag can also be a specific tag. As used herein, the term "specific tag" refers to a physical, chemical, or biological tag that is linked to a specific molecule in a library and is unique to that specific molecule. It is. Specific tags are
It is particularly useful if it is easily identifiable. A nucleotide sequence that is unique to a particular molecule in the library is an example of a specific tag. For example, methods of synthesizing peptides tagged with a unique nucleotide sequence provide a library of molecules, each containing a specific tag, such that the identity of the peptide is known when determining the nucleotide sequence. (Brenner and Lerner,
Proc. Natl. Acad. Sci. , USA 89: 5381-5383.
(1992), which is incorporated herein by reference).
The use of nucleotide sequences as specific tags for peptides or other types of molecules provides a convenient means for identifying the presence of a molecule in a sample. This is because extremely sensitive methods (eg, PCR) can be used to determine the nucleotide sequence of a specific tag, thereby identifying the sequence of the molecule linked to it. Similarly, a nucleic acid sequence encoding a peptide expressed on a phage is another example of a specific tag. This is because sequencing of the specific tag identifies the amino acid sequence of the expressed peptide.

The presence of a shared or specific tag may provide a means for identifying or recovering a tumor homing molecule after in vivo panning. Further, the combination of a shared tag and a specific tag can be particularly useful for identifying tumor homing molecules. For example, a library of peptides can be prepared such that each is linked to a specific nucleotide sequence tag (eg, Brenner and Lern).
er, supra, 1992). Here, each specific nucleotide sequence tag incorporates a shared tag (eg, biotin) therein. Upon homing to a tumor, the particular tumor homing peptide can be substantially isolated from a sample of the tumor based on the shared tag, and the specific peptide is identified, for example, by PCR of the specific tag. (See Erlich, supra, 1989).

[0092] The tag may also function as a support. As used herein, the term "support" refers to a tag having a defined surface to which a molecule can be attached. Generally, tags useful as supports are shared tags. For example, the support may be a biological tag (eg, a virus or virus-like particle (eg, bacteriophage (“phage”)); a bacterium (eg, E. coli); or a eukaryotic cell (eg, yeast, insect Cells or mammalian cells)); or, alternatively, a physical tag (eg, liposomes or microbeads, which can be composed of plastic, agarose, gelatin, or other biological or inert material)) Can be If desired, a shared tag useful as a support can have a specific tag attached to it. Therefore, the phage display library
For example, it may be considered to consist of a phage, which is a covalent tag that is also a support, and a nucleic acid sequence encoding the peptide to be expressed, which nucleic acid sequence is a specific tag.

Generally, the support has a diameter in its shortest dimension of less than about 10 μm to about 50 μm. This allows the support to be able to pass through the capillary bed present in the subject relatively unobstructed and occlude the circulation. In addition, the support can be non-toxic (so that it does not interfere with the normal expression of cell surface molecules or the normal physiology of the subject) and can be biodegradable (especially in selected tumors). If the subject used for in vivo panning is not killed to collect

When the molecule is linked to a support, the tagged molecule is positioned such that portions of the molecule that are likely to be able to interact with the target molecule in cells in the subject can participate in the interaction. As described above, comprising molecules attached to the surface of the support. For example, if a tumor homing molecule appears to be a ligand for a growth factor receptor, the binding portion of the molecule attached to the support will allow it to interact with the growth factor receptor on cells in the tumor. Is positioned. If desired, a suitable spacer molecule is positioned between the molecule and the support such that the potential tumor homing molecule does not interfere with its ability to interact with the target molecule. The spacer molecule also has a reactive group, which provides a convenient and efficient means of linking the molecule to a support.
And, if desired, a tag (which may facilitate recovery or identification of the molecule) (see Hermanson, supra, 1996).

As exemplified herein, a peptide that could home to a selected tumor (eg, breast cancer or melanoma) was expressed as the N-terminus of the fusion protein. Here, the C-terminus consisted of a phage coat protein. Upon expression of this fusion protein, the C-terminal coat protein ligated the fusion protein to the surface of the phage so that the N-terminal peptide was in a position that interacted with the target molecule in the tumor. Thus, a molecule with a shared tag was formed by ligation of the peptide to the phage. Here, the phage provides a biological support, the peptide molecules are linked as a fusion protein, and the phage coding portion of the fusion protein is
The nucleic acid encoding the peptide, acting as a spacer molecule, provided a specific tag that allowed identification of the tumor homing peptide.

The term “in vivo panning” as used herein, when used in relation to the identification of tumor homing molecules, administers a library to a subject and selectively homes to a tumor in the subject. Means a method of screening a library (see US Pat. No. 5,622,699). The term "administering to a subject," when used in reference to a library of molecules or a portion of such a library, in its broadest sense, refers to the ability of a library to be administered to a subject (generally a vertebrate, In particular, it is used to mean delivered to a tumor in a mammal (eg, a human).

The library can be administered to a subject, for example, by injecting the library into the circulation of the subject such that the molecule passes through a tumor; after a suitable period of time,
Circulation is terminated by sacrificing the subject or by removing a sample of the tumor (Example IV; US Pat. No. 5,622,699; Pasqua).
lini and Ruoslahti, supra, 1996). Alternatively, a cannula can be inserted into a blood vessel in a subject, such that the library can be administered by perfusion for an appropriate period of time, after which the library can be removed from circulation by the cannula Or, the subject may be sacrificed to collect the tumor, or the tumor may be sampled to terminate circulation. Similarly, libraries can be shunted through one or a few organs, including tumors, by cannulation of appropriate blood vessels in a subject. It is recognized that the library can also be administered to a detached perfused tumor. Such panning in detached perfused tumors can be useful for identifying molecules that bind to tumor molecules, and can be used as an initial screening of a library if desired.

The use of in vivo panning to identify tumor homing molecules involves screening phage peptide display libraries in tumor-bearing mice and identifying specific peptides that selectively homed to breast or melanoma tumors Is exemplified herein (Example IV). However, protein receptor molecules (eg, including antibodies or antigen-binding fragments of antibodies (eg, Fv, Fd, or Fab fragments)); hormone receptors (
For example, phage libraries displaying growth factor receptors); or cell adhesion receptors (eg, integrins or selectins) can also be used for practicing the present invention. Variants of such molecules can be obtained by well-known methods (eg, random mutagenesis, site-directed mutagenesis, or codon-based mutagenesis (Hu
See US Pat. No. 5,264,563, issued Nov. 23, 1993, which is incorporated herein by reference. If desired, the peptide can be chemically modified after expression of the phage, but before administration to the subject. Thus, various types of phage display libraries can be screened by in vivo panning.

[0099] Phage display technology provides a means for expressing diverse populations of random or selectively randomized peptides. Various methods of phage display and methods for generating diverse populations of peptides are well known in the art. See, for example, Ladner et al. (US Pat. No. 5,223,409, issued Jun. 29, 1993, which is incorporated herein by reference).
Describe a method for preparing a diverse population of binding domains on the surface of a phage. In particular, Ladner et al. Describe phage vectors useful for generating phage display libraries, as well as methods for selecting potential binding domains, and randomly or selectively mutated binding domains. Describes a method for generating.

Similarly, Smith and Scott (Meth. Emzymol. 217:
228-257 (1993); Scott and Smith, Science
ce 249: 386-390 (1990), each of which is incorporated herein by reference) describes a method of generating a phage peptide display library comprising vectors, and expressed. Methods for diversifying the population of peptides are described (see also Huse, WO 91/07141 and WO 91/07149, which are incorporated herein by reference); and Example IV checking). Phage display technology is particularly powerful, for example, when used with codon-based mutagenesis methods, which can be used to generate random peptides, or randomly biased or desired biased peptides. (Huse, US Pat. No. 5,264,563, supra, 1993). These or other well-known methods can be used to generate a phage display library. Phage display libraries can be subjected to in vivo panning to identify tumor homing molecules useful in homing pro-apoptotic conjugates of the invention.

In addition to screening phage display libraries, in vivo panning can be used to screen various other types of libraries, including, for example, RNA or DNA libraries or chemical libraries. . If desired, the tumor homing molecule can be tagged. This may facilitate recovery of the molecule from the tumor or identification of the molecule in the tumor.
For example, if a library of organic molecules, each containing a shared tag, is screened, the tag can be a moiety such as biotin. This moiety can be directly attached to the molecule or can be linked to a support containing the molecule. Biotin provides a useful covalent tag for recovering molecules from selected tumor samples using an avidin or streptavidin affinity matrix. Further, the molecule or a support containing the molecule can be linked to a hapten, such as 4-ethoxy-methylene-2-phenyl-2-oxazolin-5-one (phOx). This hapten is used as an anti-p coupled to magnetic beads as a means of recovering molecules.
can be bound by a hOx antibody. Methods for purifying biotin or phOx labeled conjugates are known in the art, and materials for performing these procedures are commercially available (eg, Invitrogen, La Jo
11la CA; and Promega Corp. Madison WI).
If a phage library is to be screened, this phage is
V can be recovered using methods such as those disclosed in US Pat.

[0102] In vivo panning provides a method for directly identifying tumor homing molecules that can selectively home to tumors. As used herein, the term “homing” or “selectively home” means that a particular molecule binds relatively specifically to a target molecule present in a tumor after administration to a subject. Means to do. Generally, tumor homing molecules are characterized, in part, by exhibiting at least a 2-fold (2 ×) greater specific binding to a tumor as compared to a control organ or tissue.

[0103] It should be appreciated that in some cases, the molecule may be non-specifically localized to an organ or tissue, including a tumor. For example, in vivo panning of a phage display library can produce high background in organs that contain significant components of the retinal endothelial system (RES), such as liver and spleen. Thus, for example, when a tumor is present in the liver, non-specific binding of molecules due to uptake by the RES may make identification of tumor homing molecules more difficult.

However, selective homing of tumor homing molecules can be distinguished from non-specific binding by detecting differences in the ability of different individual phages to home to tumors. For example, selective homing involves combining a putative tumor homing molecule (eg, a peptide expressed on a phage) with a large excess of uninfected phage or an approximately 5-fold excess of phage expressing a non-selective peptide and subjecting the subject to Inject the mixture,
It can then be identified by collecting a sample of the tumor. In the latter case, for example, if the number of injected phage expressing the tumor homing peptide is low enough not to saturate the target molecule, more than about 20% of the phage in the tumor will The decision to express the homing molecule is definitive evidence that the peptide expressed by the phage is a specific tumor homing molecule. In addition, non-specific localization can be distinguished from selective homing, for example, by performing competition experiments using phage that express the putative tumor homing peptide in combination with an excess of “free” peptide (implementation). Example VII).

In addition, various methods can be used to prevent non-specific binding of molecules to organs containing components of the RES. For example, molecules that selectively home to tumors present in organs containing components of the RES will first block the RES using, for example, polystyrene latex particles or dextran sulfate (Kalin et al., Nucl. M.).
ed. Biol. 20: 171-174 (1993); Illum et al. Ph
arm. Sci. 75: 16-22 (1986); Takeya et al. Gen
. Microbiol. 100: 373-379 (1977), each of which is incorporated herein by reference), and then can be obtained by administering the library to a subject. For example, pre-administration of Dextran Sulfate 500 or polystyrene microspheres prior to administration of a test substance can be achieved by Kupf
It has been used to block non-specific uptake of test substances by fer cells, which are RES components of the liver (Illum et al., supra, 1986).
Similarly, non-specific uptake of drugs by RES can be attributed to carbon particles or silica
keya et al., supra, 1977) or gelatin colloids (Kalin et al., supra,
1993). Accordingly, various agents useful for blocking non-specific uptake by the RES are known and commonly used.

Non-specific binding of phage to RES or other sites can also be prevented, for example, by co-injecting a mouse with a specific phage display library together with the same phage rendered non-infectious ( Smith et al., Supra, 1990, 1.
993). In addition, tumor homing peptides in organs containing RES components can be identified by preparing phage display libraries using phage that exhibit low background binding to specific organs. For example, M
errill et al. (Proc. Natl. Acad. Sci., USA 93: 3.
188-3192 (1996), which is incorporated herein by reference)
Selected λ phage that were not taken up by the RES and, as a result, remained in circulation for an extended period of time. For example, filamentous phage variants can be selected using similar methods.

[0107] Selective homing of a tumor homing molecule can be demonstrated by determining the specificity of a tumor homing molecule for a tumor relative to a control organ or tissue. Selective homing can also be demonstrated by showing that molecules that home to a tumor as identified by one round of in vivo panning are enriched for tumor homing molecules in a subsequent round of in vivo panning. For example, phage-expressed peptides that selectively home to melanoma tumors were isolated by in vivo panning and then subjected to a further round of in vivo panning. After the second round of screening, phage recovered from the tumor showed a 3-fold enrichment in homing to the tumor compared to the brain. Phage recovered from tumors after the third round of screening showed an average of 10-fold enrichment in homing to tumors compared to brain. Selective homing can also be demonstrated by showing that molecules that home to selected tumors as identified by one round of in vivo panning are enriched for tumor homing molecules in subsequent rounds of in vivo panning. .

[0108] Tumor homing molecules can be identified by in vivo panning, for example, using a mouse containing the implanted tumor. Such a transplanted tumor, for example,
It can be a human tumor transplanted into an immunodeficient mouse, such as a nude mouse, or a mouse tumor that is maintained in tissue culture or by passage in mice. Due to the conserved nature of cellular receptors, and the conserved nature of ligands that bind to specific receptors, angiogenic vasculature and histologically similar tumor cells in various species are known to be tumor homing molecules. Are expected to be able to share a common cell surface marker useful as a target molecule. Therefore, those skilled in the art
For example, tumor homing molecules identified using in vivo panning in mice with defined histological types of mouse tumors (e.g., melanoma) may also have the corresponding target molecule in tumors in humans or other species. Recognize binding. Similarly, tumors that grow in laboratory animals require concomitant neovascularization, just as is required for tumors that grow in humans or other species. Thus, a tumor homing molecule that binds to a target molecule present in the vasculature in a tumor grown in a mouse may also bind to a corresponding target molecule in the vasculature of a tumor in a human or other mammalian subject. It is. The general ability of tumor homing molecules, identified by homing to human capsular sarcoma or to mouse melanoma, for example, by homing to human breast tumors, indicates that the target molecule is shared by many tumors. In fact, the results disclosed herein are:
Demonstrates that the target molecule is expressed in the neovasculature, which is the host tissue (see Example VII).

Tumor homing molecules identified using in vivo panning in experimental animals (eg, mice), for example, demonstrate that the molecules can also specifically bind to tumor samples obtained from patients This can easily be tested for the ability to bind to the corresponding tumor in a human subject. For example, CDCRGDC
FC (SEQ ID NO: 1) phage and NGR peptide have been shown to bind to blood vessels in microscopic sections of human tumors, while little or no binding occurs in blood vessels of non-tumor tissues. Thus, conventional methods can be used to confirm that tumor homing molecules identified using in vivo panning in laboratory animals can also bind to target molecules in human tumors.

The steps of administering the library to a subject, collecting a selected tumor and identifying a tumor homing molecule that homes to the tumor include a single round of in vivo panning. Although not required, one or more additional rounds of in vivo panning are generally performed. If an additional round of in vivo panning is performed, molecules recovered from the tumor in the previous round were administered to the subject, and this subject was used in a previous round where only a portion of the tumor was collected Can be the same subject.

By performing a second round of in vivo panning, the relative binding selectivity of the molecules recovered from the first round can be determined by administering the identified molecules to a subject, collecting a tumor, And determining whether more phage is recovered from the tumor after the second round of screening as compared to the phage recovered after the first round. Although not required, a control organ or tissue can also be harvested, and molecules recovered from the tumor can be compared to molecules recovered from the control organ. Ideally, no molecules will be recovered from the control organ or tissue after the second or subsequent round of in vivo panning. However, in general, the population of molecules is also present in a control organ or tissue. In this case, the ratio of the molecule in the selected tumor compared to the control organ (selected molecule: control molecule) can be determined. For example, phage that home to melanoma after the first round of in vivo panning showed a 3-fold enrichment when homing to selected tumors compared to control organs (brain) after two additional rounds of panning (
Example VIII).

[0112] Additional rounds of in vivo panning can be used to determine whether a particular molecule homes only to a selected tumor, or also in one or more normal organs or tissues of a subject. A target on the tumor that is expressed or sufficiently similar to the target molecule for the tumor may be recognized. Tumor homing molecules are also unlikely to home to the corresponding normal tissue. This is because in vivo panning methods select only those molecules that home to the selected tumor. If a tumor homing molecule is also directed to home to one or more normal organs or tissues in addition to the tumor, the organ or tissue is considered to constitute a selected tissue or family of organs. Using the method of in vivo panning,
Molecules that home only to selected tumors can be distinguished from molecules that also home to one or more selected organs or tissues. Such identification is accomplished by collecting various organs or tissues during subsequent rounds of in vivo panning.
Promoted.

The term “control organ or tissue” is used to mean an organ or tissue other than a tumor for which identification of a tumor homing molecule is desired. A control organ or tissue is characterized when the tumor homing molecule does not selectively home to the control organ. Control organs or tissues can be collected, for example, to identify nonspecific binding of the molecule or to determine the homing selectivity of the molecule. In addition, non-specific binding can be identified, for example, by administering a control molecule, which does not home to a tumor, but is known not to be chemically similar to a potential tumor homing molecule. Alternatively, where the administered molecule is bound to a support, administration of the support alone can also be used to identify non-specific binding. For example,
Phage expressing the gene III protein alone but without the peptide fusion protein can be studied by in vivo panning to determine the level of non-specific binding of the phage support.

As disclosed herein, specific homing of tumor homing molecules is
It can easily be identified by examining selected tumor tissues as compared to corresponding non-tumor tissues as well as control organs or control tissues. For example, immunohistological analysis can be performed on tumor tissue and corresponding non-tumor tissue using antibodies specific for the phage used to display the tumor homing peptide (see Example VII). See). Alternatively, an antibody that is specific for a shared tag that is expressed with the peptide, such as a FLAG epitope, can be used, such as a commercially available detection system.

In general, a library of molecules, including random diverse populations or selectively randomized molecules of interest, is prepared and then administered to a subject. At a selected time after dosing, the subject is sacrificed and the tumor is harvested so that the molecules present in the tumor can be identified (see Example IV). If desired, one or more control organs or tissues or portions of control organs or tissues can be sampled. For example, mice bearing breast cancer or melanoma tumors are injected using a phage peptide display library, and after about 1-5 minutes, the mice are anesthetized and frozen in liquid nitrogen, or Perfusion through the heart to terminate the circulation of the tumor, the tumor and one or more control organs are collected from each phage present in the tumor, and the control organs are collected and represented. Peptides that selectively homed to specific tumors were identified (see Examples IV, V and VIII).

In the examples provided, animals were sacrificed and selected tumors and control organs or tissues were collected. However, it is recognized that only a portion of the tumor is collected to recover the support containing the tumor homing molecule, and similarly, only a portion of the organ or tissue need be collected as a control. Should. Thus, for example, a portion of a tumor may be collected by biopsy, such that a molecule such as a peptide expressed by a phage can be administered to the same subject two or more times as desired. If the molecule to be administered a second time to the same subject is, for example, tagged or bound to a support, the tag or support should be such that it does not interfere with subsequent rounds of screening. It should be non-toxic and biodegradable.

In vitro screening of phage libraries has previously been used to identify peptides that bind to antibodies or cell surface receptors (Smi
th and Scott, supra, 1993). For example, in vitro screening of phage peptide display libraries has been described by integrin adhesion receptors (Koivunen et al., J. Cell Biol. 124: 373-380 (1).
994a). This is incorporated herein by reference) and the human urokinase receptor (Goodson et al., Proc. Natl. Acad. Sci.
USA 91: 7129-7133 (1994)) and have been used to identify novel peptides. However, such in vitro studies have determined whether a peptide capable of specifically binding a selected receptor in vitro also binds the receptor in vivo, or whether the peptide or the receptor is a specific organ in the body. Does not provide insight into whether it is unique or not. In addition, in vitro methods are performed using defined and well-characterized target molecules in artificial systems. For example, Goodso
n et al., supra, 1994, utilized cells expressing a recombinant urokinase receptor. However, such in vitro methods require prior knowledge of the target molecule,
And any information is limited in that little is available when it comes to in vivo availability.

In vitro panning on cells in culture has also been used to identify molecules that can specifically bind to receptors expressed by cells (Bar
ry et al., Nature Med. 2: 299-305 (1996), which is incorporated herein by reference. However, cell surface molecules expressed by cells in vivo often change when the cells are grown in culture. Thus, in vitro panning methods using cells in culture are also limited in that there is no guarantee that the molecules identified due to their binding to cells in culture will have the same ability in vivo. Furthermore, it is not possible to use in vitro panning to identify molecules that home only to the tumor cells used in the screening but not to other cell types.

In contrast, in vivo panning does not require prior knowledge or the availability of target molecules, and identifies molecules that bind to cell surface target molecules that are expressed in vivo. Also, since "non-targeted" tissues are present during screening, the possibility of isolating tumor homing molecules that lack homing specificity is
Greatly reduced. In addition, any molecules that may be particularly susceptible to degradation in the circulation in vivo are not recovered, eg, in obtaining a tumor homing molecule by in vivo panning due to metabolic activity. Thus, in vivo panning
Identifying tumor homing molecules that selectively home in vivo to target molecules present in the tumor offers significant advantages over previous methods.

Although the mechanism by which the disclosed method of in vivo panning works is not well defined, one possibility is that molecules such as peptides expressed on phage will be recognized and implicated in blood vessels in tumors. Along with the target molecule present on the endothelial cells. Evidence is, for example, that vascular tissue in different organs is different from another,
And show that such differences may be involved in regulating cellular trafficking in the body. For example, lymphocytes may have specific “addresses” by endothelial cells in their tissues.
homing to lymph nodes or other lymphoid tissues, due in part to the expression of "address" molecules (Salmi et al., Proc. Natl. Acad. Sci.,
USA 89: 11436-11440 (1992); Springer, Ce.
11 76: 30-314 (1994)). Similarly, various leukocytes may recognize sites of inflammation due in part to the expression of endothelial cell markers induced by inflammatory signals (Butcher and Picker, Science 272:
60-66 (1996); Springer, supra, 1994).
Thus, the endothelial cell marker is selectively bound by the tumor homing molecule,
And provides a potential target that can be used to direct the bound antimicrobial peptide to the tumor.

Additional components can be included as part of the homing pro-apoptotic conjugate, if desired. For example, in some cases it may be desirable to utilize an oligopeptide spacer between the tumor homing molecule and the antimicrobial peptide. Such spacers are described, for example, in Fitzpatrick and Garn.
ett, Anticancer Drug Des. 10: 1-9 (1995)
Are well known in the art, as described in

The homing pro-apoptotic chimeric peptides of the present invention can be readily synthesized in the required amounts using conventional methods of solid state peptide synthesis. The chimeric peptides of the present invention can also be obtained from commercial suppliers (eg, AnaSpec, Inc .; San.
Jose, CA). If the antimicrobial peptide is to be conjugated to a non-peptide tumor homing molecule, the antimicrobial peptide moiety can be independently synthesized using well-known methods, or can be obtained from a commercial supplier.

Some methods that can be used to attach antimicrobial peptides to tumor homing molecules are known in the art, which depend on the chemical characteristics of the molecule. For example, methods for coupling haptens to carrier proteins, as commonly used in the field of applied immunology (eg, Harlow and Lane, supra, 1
988; Hermanson, supra, 1996).

[0124] Premade antimicrobial peptides can also be conjugated to tumor homing peptides using, for example, carbodiimide conjugates (Baumi).
nger and Wilchek, Meth. Enzymol. 70: 151-1
59 (1980). This is incorporated herein by reference). Carbodiimides include a group of compounds having the general formula R—NＣCＮN—R ′, wherein R and R ′ may be aliphatic or aromatic; And used for the synthesis of peptide bonds. The preparative procedure is simple, relatively fast and performed under mild conditions. The carbodiimide compound attacks the carboxylic acid group and changes the carboxylic acid group into a reactive site for a free amino group. Carbodiimide conjugates have been used to conjugate various compounds to carriers for the production of antibodies.

The water-soluble carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), may be useful for conjugating antimicrobial peptides to tumor homing molecules. Such conjugates include an amino group (e.g.,
Requires the presence of an antimicrobial peptide), and a carboxyl group, which can be provided by a tumor homing molecule.

In addition to using carbodiimides for the direct formation of peptide bonds, EDC also provides active esters (eg, N-hydroxysuccinimide (NH
S) ester). The NHS ester, which binds only to the amino group, can then be used to induce the formation of an amide bond with one amino group of doxorubicin. EDC and NHS in combination
Is commonly used for conjugation to increase the yield of conjugate formation (Bauminger and Wilchek, supra, 1980).

Other methods for conjugating antimicrobial peptides to tumor homing molecules can also be used. For example, sodium periodate oxidation followed by reductive alkylation of the appropriate reactants can be used, as well as glutaraldehyde crosslinking.
However, despite the choice of method for producing the conjugates of the present invention, it must be determined that the tumor homing molecule retains its targeting ability and that the antimicrobial peptide maintains its antimicrobial activity. Be recognized. Methods known in the art can confirm the activity of a homing pro-apoptotic conjugate of the invention.

The yield of antimicrobial peptide / tumor homing molecule conjugate formed is determined using conventional methods. For example, HPLC or capillary electrophoresis or other quantitative or qualitative methods can be used (eg, Liu et al., J. C.
Chromatogr. 735: 357-366 (1996); Rose et al.
Chromatogr. 425: 419-412 (1988). Each of these is incorporated herein by reference; for example, also see Example VII.
I). In particular, those skilled in the art will recognize that the choice of method for determining the yield of a conjugate reaction will depend in part on the physical or chemical characteristics of the particular antimicrobial peptide and tumor homing molecule. After conjugation, the reaction product is desalted to remove any free peptide or molecule.

The present invention also provides a method of directing an antimicrobial peptide in vivo against a tumor having angiogenic vasculature. The method is performed by administering a homing pro-apoptotic conjugate of the invention to a subject containing a tumor having angiogenic vasculature. In a method of the invention for directing an antimicrobial peptide in vivo against a tumor having angiogenic vasculature, the antimicrobial peptide may be, for example,
Sequence _D (KLAKLAK) ₂ may be included. A particularly useful conjugate that can be administered to a subject containing a tumor with angiogenic vasculature is CNGRC-GG- _D (KLA
KLAK) ₂ and ACCDRGDCFC-GG- _D (KLAKLAK) ₂ .

The invention further provides a method of inducing selective toxicity in vivo in a tumor having angiogenic vasculature. The method is performed by administering a homing pro-apoptotic conjugate of the invention to a subject containing a tumor having angiogenic vasculature. Antimicrobial peptides useful in inducing selective toxicity in the methods of the invention can be, for example, peptides comprising the sequence _D (KLAKLAK) ₂ . A particularly useful conjugate that can be administered to induce in vivo selective toxicity in tumors with angiogenic vasculature is CNGRC-GG- _D (KLAKLA
K) ₂ and ACCDRGDCFC-GG- _D (KLAKLAK) ₂ .

[0131] Also provided herein are methods of treating a patient having a tumor with angiogenic vasculature. In such a method of treatment, a homing pro-apoptotic conjugate of the invention is administered to a patient and is selectively toxic to a tumor.
The antimicrobial peptide moiety can include, for example, the sequence _D (KLAKLAK) ₂ . In a preferred embodiment, the homing pro-apoptotic conjugate has the sequence CNGRC-G
G- _D (KLAKLAK) ₂ and ACCDRGDCFC-GG- _D (KLAKL
AK) ₂ .

When administered to a subject, a homing pro-apoptotic conjugate of the invention can be administered, for example, as a pharmaceutical composition comprising the conjugate and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions (eg, water or physiologically buffered saline) or other solvents or vehicles (eg, glycols, glycerol, oils (eg, Olive oil or injectable organic esters).

Pharmaceutically acceptable carriers can include, for example, physiologically acceptable compounds that act to stabilize or increase the absorption of the conjugate. Such physiologically acceptable compounds include, for example, carbohydrates (eg, glucose, sucrose or dextran); antioxidants (eg, ascorbic acid or glutathione); chelating agents; low molecular weight proteins; Stabilizers or excipients are included. One skilled in the art understands that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the compound. Pharmaceutical compositions may also include agents such as cancer therapeutics.

One skilled in the art will appreciate that the homing pro-apoptotic conjugates of the present invention can be administered to a subject as a pharmaceutical composition by various routes, eg, orally or parenterally (eg, intravenously). It is known. Pharmaceutical compositions containing the conjugate can be administered by injection or intubation. Pharmaceutical compositions can also be tumor homing molecules, into which antimicrobial peptides can be incorporated, associated with liposomes or other polymer matrices (Gregoridis, Liposome Tec).
hnology, Volume 1 (CRC Press, Boca Raton, FL
1984), incorporated herein by reference). For example, liposomes composed of phospholipids or other lipids are non-toxic, physiologically acceptable, and metabolizable carriers that are relatively simple to make and administer.

For the therapeutic methods disclosed herein, an effective amount of a homing pro-apoptotic conjugate should be administered to a subject. As used herein, the term "effective amount" means the amount of a conjugate that produces a desired effect. Effective amounts often depend on the particular antimicrobial peptide bound to the tumor homing molecule. An effective amount of a homing pro-apoptotic conjugate in which a tumor homing molecule is linked to a particular antimicrobial peptide can be determined using methods well known to those skilled in the art.

The route of administration of the homing pro-apoptotic conjugate will depend, in part, on the chemical structure of the molecule. For example, peptides are not useful, especially when administered orally. This is because peptides can be degraded in the gastrointestinal tract. But,
To make the peptide less sensitive to degradation by endogenous proteases,
Or by chemically modifying the peptide to be better absorbed through the gastrointestinal tract (
This includes the incorporation of D amino acids), but is well known (eg, Blond
Elle et al., supra, 1995; Ecker and Croke, supra, 1995.
Goodman and Ro, supra, 1995). Such modifications can be made on tumor homing peptides identified by in vivo panning, and on antimicrobial peptides. In addition, a library of peptidomimetics, which may contain D amino acids, other non-naturally occurring amino acids, or chemically modified amino acids; or may be organic molecules that mimic the structure of peptides; or Methods of preparing peptoids (such as vinylogous peptoids) are known in the art and can be used to identify tumor homing molecules that are stable for oral administration.

[0137] Tumor homing peptides can have a linear or cyclic structure. Some peptides contain a cysteine residue to allow cyclization of the peptide. In particular,
Peptides containing at least two cysteine residues spontaneously cyclize. In addition, such cyclic peptides may also be active when present in a linear form (see, eg, Koivunen et al., Supra, 1993). For example, NGRAHA (SEQ ID NO: 6), a linear peptide, was also useful as a tumor homing molecule (see Table 2). Thus, in some cases, disclosed herein or otherwise identified as a tumor homing peptide,
One or more cysteine residues in the tumor homing peptide can be deleted without significantly affecting the tumor homing activity of the peptide. Methods for determining the need for cysteine residues or amino acid residues N- or C-terminal to cysteine residues for the tumor homing activity of the peptides of the invention are conventional and well known in the art.

As further disclosed herein, some, but not all, tumor homing molecules can also home to angiogenic vasculature that is not contained within a tumor. For example, tumor homing molecules containing either the RGD motif or the GSL motif specifically homed to retinal neovasculature (Smith et al., In
vest. Opththamol. Vis. Sci. 35: 101 to 111 (1
994) (incorporated herein by reference), whereas tumor homing peptides containing the NGR motif did not substantially accumulate in this angiogenic vasculature. These results indicate that tumor vasculature expresses target molecules that are not substantially expressed in other types of angiogenic vasculature. The methods disclosed herein can be used to distinguish tumor homing peptides from peptides that home to non-tumor angiogenic vasculature. One of skill in the art will understand to administer a conjugate having a tumor homing peptide that preferentially homes to the tumor vasculature to treat the tumor.

The present invention provides chimeric prostate homing pro-apoptotic peptides. This
It can be used, for example, in the treatment of benign prostate hyperplasia or prostate cancer. This specification
As disclosed herein, the SMSIARL peptide (SEQ ID NO: 207) contains all
When administered locally, it selectively localizes to prostate tissue, specifically the prostate vasculature.
(See Examples IX.B and IX.E). In addition, prostate Homin
Gupeptide SMSIARL (SEQ ID NO: 207) binds to prostate tissue (eg,
(Eg, biotin or phage). Honcho
As further disclosed in the text, apoptosis is induced by SMSIARL-GG.
−_D(KLAKLAK)_TwoInduction in mouse prostate by systemic administration of chimeric peptide
There was no evidence that apoptosis was observed in non-prostate tissue (FIG. 7).
And Example IX. C). The results disclosed herein also
SMSIARL-GG-_D(KLAKLAK)_TwoAdministration of the chimeric peptide is
MP mice (Gingrich et al., Cancer Res. 56: 4096-4).
Prostate cancer under the influence of a transgene as described in
) Can be extended. FIG. 8 shows the SMSIARL-GG- _D (KLAKLAK)_TwoMice treated with vehicle alone,_D(KLAKLAK
)_TwoTreatment with peptide alone or SMSIARL peptide (SEQ ID NO: 207) alone
This indicates that the mice survived longer than the mice in which they were placed. Based on these results, the present invention
Is a chimeric prostate homing pro-apoptotic peptide, and further described below.
The present invention provides a method of using this peptide for the treatment of patients with prostate cancer.
Offer.

Accordingly, the present invention provides a chimeric prostate homing pro-apoptotic peptide comprising a prostate homing peptide linked to an antimicrobial peptide. Here, the chimeric peptide is selectively internalized into prostate tissue and shows high toxicity to that tissue, while the antimicrobial peptide has low mammalian cytotoxicity if not linked to the prostate homing peptide . In the chimeric peptide of the present invention, the prostate homing peptide portion has, for example, the sequence SMSIARL.
(SEQ ID NO: 207) or a functionally equivalent sequence, and the antimicrobial peptide portion comprises the sequence (KLAKLAK) ₂ (SEQ ID NO: 200), (KLAKLA) ₂ (SEQ ID NO: 201), (KAAKKAA) ₂ (SEQ ID NO: 202) or (KLGK
KLG) ₃ (SEQ ID NO: 203). In a preferred embodiment, the antimicrobial peptide moiety comprises the sequence _D (KLAKLAK) ₂ . An exemplary prostate homing pro-apoptotic peptide is SMSIARL-
Provided herein as GG- _D (KLAKLAK) ₂ .

The invention further provides a method of directing an antimicrobial peptide in vivo against prostate cancer. The method includes administering a chimeric prostate homing pro-apoptotic peptide comprising a prostate homing peptide linked to an antimicrobial peptide. Here, the chimeric peptide is selectively internalized into prostate tissue and shows high toxicity to that tissue, while the antimicrobial peptide has low mammalian cytotoxicity when not linked to the prostate homing peptide. In the method of the present invention, the prostate homing peptide has, for example, the sequence SMSIARL
(SEQ ID NO: 207) or a functionally equivalent sequence, and the antimicrobial peptide can include a sequence such as _D (KLAKLAK) ₂ . In a preferred embodiment,
The chimeric prostate homing pro-apoptotic peptide has the sequence SMSIARL-G
G- _D (KLAKLAK) ₂ .

A method for inducing selective toxicity in prostate cancer in vivo is also provided by the present invention. The method comprises administering a chimeric prostate homing pro-apoptotic peptide to a subject having prostate cancer. Here, the chimeric peptide comprises a prostate homing peptide linked to an antimicrobial peptide, which is selectively internalized in prostate tissue and exhibits high toxicity to that tissue, while the antimicrobial peptide comprises When not linked to a prostate homing peptide, it has low mammalian cytotoxicity. Methods for inducing selective toxicity in vivo in prostate cancer are described, for example, in the sequence SMSIARL (SEQ ID NO: 20).
7) Or, using prostate homing peptides containing functionally equivalent sequences. The antimicrobial peptide can include, for example, the sequence _D (KLAKLAK) ₂ . In a preferred embodiment, the chimeric prostate homing pro-apoptotic peptide comprises the sequence SMSIARL-GG- _D (KLAKLAK) ₂ .

In addition, the present invention provides for administering to a patient having prostate cancer a chimeric prostate homing pro-apoptotic peptide of the invention, whereby the chimeric peptide is selectively toxic to tumors. A method for treating a patient is provided. The chimeric peptide comprises a prostate homing peptide linked to an antimicrobial peptide, and the chimeric peptide is selectively internalized into prostate tissue and exhibits high toxicity to that tissue, while the antimicrobial peptide When not linked to a homing peptide, it has low mammalian cytotoxicity.
The prostate homing peptide portion can include, for example, the sequence SMSIARL (SEQ ID NO: 207) or a functionally equivalent sequence, and the antimicrobial peptide portion can include, for example, the sequence _D (KLAKLAK) ₂ . In a preferred embodiment for the treatment of a patient having a prostate tumor, the chimeric peptide has the sequence SMSIARL-GG
-Contains _D (KLAKLAK) ₂ .

[0144] As used herein, the term "prostate homing peptide" refers to a peptide that selectively homes to prostate tissue in vivo relative to control tissue (eg, brain). Such peptides are generally characterized by at least a 2-fold greater localization to prostate tissue as compared to a control cell type or tissue. Prostate homing peptides can selectively home, for example, to prostate vasculature as compared to other cell types or other vasculature (see Example IX).

The chimeric peptides of the present invention are selectively delivered to the prostate by the selective homing activity of the prostate homing peptide moiety. Various prostate homing peptides are useful in the present invention. As the peptide, injection of X ₇ libraries to mice (Table 7) and U.S. Patent SMSIARL identified by subsequent in vivo panning stated No. 5,622,699 (SEQ ID NO: 207) and VSFLEYR (SEQ ID NO: 222 ). The prostate homing peptides SMSIARL (SEQ ID NO: 21) and VSFLEYR (SEQ ID NO: 22) showed 34-fold and 17-fold enrichment, respectively, in the prostate compared to brain.

In one embodiment, the invention relates to the sequence SMSIARL (SEQ ID NO: 207)
Or it depends on a prostate homing peptide containing a functionally equivalent sequence. Array S
With respect to MSIARL (SEQ ID NO: 207), the term "functionally equivalent sequence"
As used herein, as shown in FIG. 9 for the sequence SMSIARL (SEQ ID NO: 207), in that it selectively binds to the endothelium of prostate blood vessels, and in that this sequence binds selectively to the same receptor. An array that functions similarly.

It is understood that the chimeric prostate homing pro-apoptotic peptides of the present invention can be used to induce selective toxicity in various prostate disorders.
Such disorders include benign nodular prostatic hyperplasia, as well as primary or secondary cancers, including clinically apparent and asymptomatic cancers. Cancers to be treated with the chimeric peptides of the present invention include prostate carcinomas, such as adenocarcinoma.

The following examples are intended to illustrate but not limit the invention.

Example I Characterization of _D (KLAKLAK) ₂ This example demonstrates that _D (KLAKLAK) ₂ preferentially disrupts mitochondrial membranes and induces mitochondria-dependent apoptosis. I do.

[0150] (KLAKLAK) ₂ and shows a synthetic 14-mer KLAKLAKKLAKLAK
(SEQ ID NO: 200) was selected. Because it kills bacteria at concentrations two orders of magnitude lower than those needed to kill eukaryotic cells (Javadpo).
ur et al. Med. Chem. 39: 3107-3113 (1996)). All the D-enantiomers, _D (KLAKLAK) _2, were used to avoid protease degradation (Bassallle et al., FEBS Lett. 274: 15).
1-155 (1990); Wade et al., Proc. Natl. Acad. Sci
87: 4761-4765 (1990)).

( _D (KLAKLAK) ₂ preferentially disrupts mitochondrial membranes) The ability of _D (KLAKLAK) ₂ to preferentially disrupt mitochondrial membranes across eukaryotic plasma membranes is demonstrated by mitochondrial swelling assays. And apoptosis were evaluated in mitochondria-dependent cell-free systems and by cytotoxicity assays.

A mitochondrial swelling assay was performed as follows. Briefly, rat liver mitochondria were prepared as described by Ellerby et al. Neurosci. 17: 6165
６１6178 (1997). The peptide was analyzed by HPLC for 9
(DLSLARLATARLAI (SEQ ID NO: 204)
), Coast Scientific, Inc. , San Diego, CA
All other peptides are AnaSpec, Inc .; ). Mitochondria 10 μM
_D (KLAKLAK) ₂ , 10 μM DLSLARLATARLAI negative control peptide (SEQ ID NO: 204), or 200 μM Ca ⁺² (as a positive control) were treated. The peptide was added to mitochondria in the cuvette. Swelling was quantified by measuring the absorbance at 520 nm.

As shown in FIG. 2a, 10 μM _D (KLAKLAK) ₂ induced marked mitochondrial swelling. Mild swelling was observed at a concentration of 3 μM. This is two orders of magnitude higher than the concentration required to kill eukaryotic cells (about 300 μM), as measured by the lethal concentration required to kill 50% of the cell monolayer (LC ₅₀ ; Table 1). Low. Non-α helix forming peptide DLSLARLATARLAI used as negative control
(SEQ ID NO: 204) did not induce mitochondrial swelling. These results demonstrate that _D (KLAKLAK) ₂ preferentially disrupts mitochondrial membranes as compared to eukaryotic plasma membranes.

[0154]

[Table 1] _(D (KLAKLAK) ₂ induces the mitochondrial-dependent apoptosis) _D (KLAKLAK) ₂ peptide in a cell-free system configured in normal mitochondria suspended in normal cytosolic extracts, mitochondrial-dependent apoptosis Were assayed for their ability to activate (Ellerby et al., J. Neuro).
osci. 17: 6165-6178 (1997)). Apoptosis was measured by the characteristic caspase 3 which processes the active protease from the inactive pro-form (Alnemri et al., Cell 87: 171 (1996)).

The cell-free apoptosis assay was performed essentially as follows. Cell-free systems were reconstituted as described in Ellerby et al., Supra, 1997. For mitochondrial-dependent reactions, rat liver mitochondria were converted to dermal microvascular endothelial cells (dermal microvessel endothelial cells).
ell) in normal (non-apoptotic) cytosolic extract prepared from After adding the peptide at a concentration of 100 μM and incubating at 30 ° C. or 37 ° C. for 2 hours, mitochondria were removed by centrifugation,
The supernatant was then purified on a 12% gel (Biorad; Hercules, CA) by S
Analyzed by DS / PAGE immunoblotting. Protein is converted to PVDF membrane (
Biorad) and anti-caspase 3 antibody (Santa Cruz)
Biotechnology; Santa Cruz, CA) followed by ECL detection (Amersham; Arlington High).
ts, IL).

Characteristic caspase 3 treatment was measured in dermal microvascular endothelial cell lysates as described in Ellerby et al., Supra, 1997. Briefly, an aliquot of cell lysate (1 μl lysate solution, 8-15 mg / ml) was transferred to 100 μM N-acetyl-Asp-Glu-Val-Asp-pNA (DEVO-pNA; BioMol).
100 μl, 100 mM HEPES, 10% sucrose, 0.1% CHAP
S, 1 mM DTT, pH 7.0). The hydrolysis of DEVD-pNA was monitored spectrophotometrically (400 nm) at 25 ° C.

As shown in FIG. 2b, lane 4, characteristic caspase 3 processing active protease was observed in the presence of mitochondria and _D (KLAKLAK) ₂ . Non-alpha helix forming peptide DLSLARLATA used as negative control
RLAI (SEQ ID NO: 204) was inactive and non-lethal to eukaryotic cells when tested in a cell-free system (FIG. 2b; see also Ellerby et al., Supra, 1997). .

Taken together, these results indicate that _D (KLAKLAK) ₂ preferentially disrupts mitochondrial membranes and activates mitochondrial-dependent apoptosis compared to eukaryotic plasma membranes. Show.

Example II Characterization of HPP-1 This example demonstrates that CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1) inhibits angiogenesis in a tissue culture model. Demonstrate.

A chimera containing a homing domain linked to an antimicrobial peptide via a glycinylglycine bridge was prepared. As described above, the peptide was converted to AnaSpec, Inc.
Was synthesized commercially at> 90% purity by HPLC.

The homing domain is a cyclic (disulfide bond between cysteines) CNGRC
Peptide (SEQ ID NO: 8, see FIG. 1) or bicyclic ACCDRGDCFC
Peptides (SEQ ID NO: 16), both of which have tumor homing properties (Pasqualini et al., Nature Biotech. 15:
542-546 (1997); Arap et al., Science 279: 377-.
380 (1998)) and can be internalized (Arap et al., Supra.
1998; Hart et al. Biol. chem. 269: 12468-124
74 (1994); Bretscher et al., EMBO. J. 8: 1341-13
48 (1989)). The homing domain was synthesized from all L-amino acids due to the putative chimeric properties of the homing domain-receptor interaction. Glycinylglycine crosslinks were used to link the homing and antimicrobial domains, confer flexibility on the peptide, and minimize potential steric interactions.

Survival of skin microvascular endothelial cells treated with HPP-1 The efficacy and specificity of HPP-1 was determined by Goto et al., Lab. Invest. 6
9: 508-517 (1993) as assessed in a tissue culture model of angiogenesis. During angiogenesis, capillary endothelial cells proliferate and migrate (Risau, Nature 386: 671-674 (1997); Ze).
tter, Ann. Rev .. Med. 49: 407-424 (1998)). Cord formation is a form of migration, which is performed in vitro from the usual "cobblestone"
It is represented by a change in the morphology of the endothelial cells into the cell chain or cord-like arrangement shown in FIG.

The effects of HPP-1 were assayed on normal human dermal microvascular endothelial cells (DMEC) under angiogenic conditions of proliferation and cord formation. In addition, the effect of HPP-1 was assayed under monolayer angiostatic conditions maintained at 100% confluency (as described below).

Briefly, dermal microvascular endothelial cells (DMEC) were purchased from CADMEC Growth
h Media ^™ (Cell Applications, Inc., San
(Deigo, CA). The skin microvascular endothelial cells are then
Three experimental conditions: growth (500 ng / ml human recombinant vascular endothelial growth factor (
VEGF; 30% confluency in growth medium supplemented with Pharmingen); non-proliferation (100% confluency in medium formulated to maintain monolayers); and cord formation (60% confluency (required for induction) in media formulated to induce cord formation). KS1767 and MDA-MB-435 cells were purchased from Arap et al.
Cultured as described in Hernier et al., Supra, 1994, supra.

The percent viability and LC ₅₀ (Table 1) were determined by the morphology of apoptosis.
llerby et al. Neurosci. 17: 6165-6178 (1997)
) Was determined. For percent viability assays, skin microvascular endothelial cells were isolated from 60 μM HPP-1 or control peptide _D (KL
(AKLAK) ₂ . At the indicated time points, cell culture media was aspirated from the adherent cells and the cells were gently washed once with PBS at 37 ° C. Subsequently, a 20-fold dilution of the dye mixture (100 μg / ml acridine orange and 100 μg / ml ethidium bromide) in PBS was gently pipetted into the cells and they were observed on an inverted microscope (Nikon TE 300). .
Cells with nuclei exhibiting chromatin margination and aggregation and / or nuclear fragmentation (early / middle apoptosis) or cells with lost plasma membrane (late apoptosis) are scored as not viable. did. At a given time point in each experiment, at least 500 cells were scored. Percent survival was calculated relative to the untreated control. Monolayer, proliferation (60%
The confluency) and LC ₅₀ for the cord-like array formed was scored at 72 hours.

As shown in FIG. 3, treatment of skin microvascular endothelial cells with 60 mM HPP-1 under conditions of proliferation or cord formation compared to untreated controls.
A reduction in percent viability over time occurred (see FIGS. 3c and 3d, respectively). In contrast, treatment with the non-targeting peptide _D (KLAKLAK) ₂ as a negative control resulted in a slight decrease in viability. In addition, the LC ₅₀ for proliferating or migrating dermal microvascular endothelial cells treated with HPP-1 was determined for angiostatic dermal microvascular endothelial cells maintained in a monolayer of 100% confluency. Of the order of magnitude smaller than the LC _{50 of} Table 1 (Table 1). These results demonstrate preferential killing by HPP-1 under angiogenic conditions compared to angiostatic conditions.

Various controls were also assayed for their effect on viability of skin microvascular endothelial cells. LC ₅₀ for non-targeting control _D (KLAKLAK) ₂ in angiogenesis conditions was similar to LC ₅₀ for HPP-1 in angiogenesis inhibition conditions. In addition, unbound _D (KLAKLAK) ₂ and CNGRC (SEQ ID NO: 8)
), The non-targeted form of CARAC-GG- _D (KLAKLAK) ₂ , and the scrambled form of CGRNC-GG- _D (KLAKLAK) ₂ are represented by _D (KLA
Similar results were obtained for (KLAL) ₂ . In addition, an alternative prototype AC
DCRGDCFC-GG- _D (KLAKLAK) ₂ is CNGRC-GG- _D (K
(LAKLAK) ₂ produced similar results as the prototype for (HPP-1;
Data not shown).

Mitochondrial morphology of cutaneous microvascular endothelial cells treated with HPP-1 The mitochondrial morphology of cutaneous microvascular endothelial cells was determined using 60 μM HPP-1, A
CDCRGDCFC-GG- _D (KLAKLAK) ₂ or untargeted _D (KLAK
In the proliferating cells after treatment with (LAK) ₂ , the following evaluation was performed. Skin microvascular endothelial cells were treated with peptides for 24 and 72 hours followed by 30 minutes at 37 ° C.
Mitochondrial staining MitoTracker Red ^™ (Mo
cyclic Probes, Inc. , Eugene, OR) and 500
Staining was performed by incubation with nM nuclear staining DAPI (Molecular Probes, Inc.). Subsequently, mitochondria were visualized using fluorescence microscopy (× 100) under an inverted microscope (Nikon) using a triple wavelength filter set.

The mitochondria of cutaneous microvascular endothelial cells treated with _D (KLAKLAK) ₂ for 24 hours remained morphologically normal, while mitochondria treated with each prototype had altered mitochondria. The form was shown. In particular, the altered mitochondrial morphology is associated with the CNGR before the cells round up.
C-GG- _D (KLAKLAK) ₂ or ACCDRGDCFC-GG- _D (KL
(AKLAK) ₂ was apparent in about 80% of the cells. Finally,
Cutaneous microvascular endothelial cells treated with CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1) are a classic morphological indicator of apoptosis, including nuclear aggregation and fragmentation as observed at 72 hours (Ellerby et al., Supra, 1997).
). Apoptotic cell death was confirmed by an assay for caspase activity (FIG. 3b; see Ellerby et al., Supra, 1997).

Survival of KS1767 and MDA-MB-435 Cells Treated with HPP-1 The chimeric HPP-1 peptide was expressed in KS1767 cells (from Kaposi's sarcoma), as well as in proliferating or migrating skin microspheres. Toxic to vascular endothelial cells (Table 1; Hernier et al., AIDS 8: 575-581 (1994)). In contrast, HPP-1 was approximately 1 order of magnitude less toxic to MDA-MB-435 human breast cancer cells (Table 1). KS1767 cells (cells of endothelial cell origin)
Is similar to angiogenic endothelial cells and binds the CNGRC peptide (SEQ ID NO: 8), but MDA-MB-435 cells do not (Samaniego et al.,
Amer. J. Path. 152: 1433-1443 (1998); Arap.
Et al., Supra, 1998).

Thus, these results demonstrate that HPP-1 induces mitochondrial swelling and apoptosis in skin microvascular endothelial cells.

Example III In Vivo Activity of Homing Proapoptotic Peptides This example demonstrates that CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1)
It demonstrates inhibiting tumor growth and extending survival of tumor-bearing animals. This example further demonstrates that CDCRGDCFC-GG- _D (KLAKLAK) ₂ inhibits retinal neovascularization.

A. In Vivo Activity of CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1) The activity of HPP-1 was determined using nude mice with human MDA-MD-435 breast cancer xenografts. The test was performed in vivo as follows. In short, MDA-
Tumor xenografts from MB-435 and C8161 were prepared as described in Arap et al., Supra.
998 in female nude mice at 2 months of age (Ja
ckson Labs; Bar Harbor, ME). Mice were treated with 2,2,2-tribromoethanol (Aldrich; Milwau) prepared in distilled water.
After anesthesia with a mixture of (kee, WI) and 2-methyl-butanol (Aldrich), the peptides were administered intravenously via the tail vein at a dose of 250 μg / week / mouse in a volume of 200 μg. Three-dimensional measurement of tumor
Performed by calipers under anesthesia and used to calculate tumor volume (P
asqualini et al., supra, 1996).

As shown in FIG. 4a, the tumor volume was similar to the control (non-targeted CARAC-
GG- _D (KLAKLAK) ₂ and unbound _D (KLAKLAK) ₂ and CNG
On the order of magnitude smaller than in the RC (mixture of SEQ ID NO: 8 peptides). As further shown in FIG. 4b, the survival in the HPP-1 treated group was longer than in the control group. Some of the HPP-1 treated mice lived longer than control mice for several months. It indicated that both primary tumor growth and metastasis were inhibited by HPP-1. Histopathological analysis revealed overt destruction of tumor structure and extensive cell death in the tumor, demonstrating that this cell death was approximately equally apoptotic and necrotic. In a similar experiment, HPP-1 also produced the human melanoma cell line C8161.
(Welch et al., Int. J. Cance).
r 47: 227-237 (1991)).

These results indicate that the homing pro-apoptotic conjugate (eg, CNGRC-
GG- _D (KLAKLAK) ₂ (HPP-1)) has been shown to have potent antitumor activity in vivo.

B. In Vivo Activity of CDCRGDCFC-GG- _D (KLAKLAK) ₂ Retinal angiogenesis was oxygen-induced in neonatal mice. Subsequently, mice were treated with a single 13 μg intravenous dose (1 animal per group) of vehicle; CDC
RGDCFC-GG- _D (KLAKLAK) ₂ ; or unconjugated CDCRGDC
Treated with a control mixture of FC (SEQ ID NO: 1) and _D (KLAKLAK) ₂ . Four days later, the number of retinal neovessels was determined in each treatment.

The results shown in FIG. 5 are for mice treated with vehicle alone (column 1; black bar).
Or the homing pro-apoptotic peptide CDCRGDCFC-G compared to mice treated with the untargeted pro-apoptotic peptide (column 3; gray bars).
_G-D (KLAKLAK) mice treated with _2; in (column 2 twill pattern bars), demonstrating that the number of retinal neovascular decreased. In particular, the angiogenic response in mice treated with the homing pro-apoptotic peptide CDCRGDCFC-GG- _D (KLAKLAK) ₂ was only 30-40% of the response observed in control mice.

These results indicate that homing pro-apoptotic peptides (eg, CDCRG
Show that DCFC-GG- _D (KLAKLAK) ₂ ) can selectively inhibit angiogenic responses (eg, retinal neovascularization).

Example IV In Vivo Panning This example demonstrates methods for preparing phage libraries and in vivo panning using in vivo panning to identify phage that express tumor homing peptides. Demonstrate methods for screening.

A. Preparation of phage library The phage display library was prepared according to Koivunen et al. (Supra, 1995).
Constructed using the fuse 5 vector as described by Koivunen et al., Supra, 1994b). CX ₅ C (SEQ ID NO: 9), C
Libraries encoding peptides named X ₆ C (SEQ ID NO: 10), CX ₇ C (SEQ ID NO: 11), and CX ₃ CX ₃ CX ₃ C (SEQ ID NO: 12) were prepared. here"
“C” indicates cysteine and “X _N ” indicates a predetermined number of individually selected amino acids. If at least two cysteine residues are present in the peptide, these libraries may represent a cyclic peptide. In addition, a library containing no defined cysteine residues was also constructed. Such libraries are mainly
While producing linear peptides, cyclic peptides can also occur due to random possibilities.

A biased library based on the sequence CXXXNGRXX (SEQ ID NO: 13) was also constructed. Further, in some cases, CXXXNGRXX (SEQ ID NO: 13)
) Library contains cysteine residues adjacent to the NGR sequence (ie, CXX
CNGRCX) incorporation was further biased (SEQ ID NO: 14; see Table 2).

A library containing defined cysteine residues was generated using an oligonucleotide constructed so that "C" is encoded by codon TGT and "X _N " is encoded by NNK. . Where "N" is a mixture of equal molar concentrations of A, C, G and T, and "K" is a mixture of equal molar concentrations of G and T. Thus, the peptide represented by CX ₅ C (SEQ ID NO: 9) can be represented by an oligonucleotide having the sequence TGT (NNK) ₅ TGT (SEQ ID NO: 14). Oligonucleotides were subjected to 3 cycles of PCR
Duplexed by amplification, purified, and fused to the gene I in the Fuse 5 vector.
Ligation to nucleic acid encoding II protein. As a result, upon expression, this peptide is present at the N-terminus of the gene III protein as a fusion protein.

This vector was transfected into MC1061 cells by electroporation. Bacteria were cultured for 24 hours in the presence of 20 μg / ml tetracycline, and phage were then collected from the supernatant by double precipitation with polyethylene glycol. Each library contained approximately 5 × 10 ^{9 to} 5 × 10 ¹⁴ transduction units (TU; individual recombinant phage).

B. In Vivo Panning of Phage Tumors were implanted into mice as described in Examples V and VI below. 1
A mixture of the phage library containing TU of × 10 ⁹ to 1 × 10 ¹⁴ was prepared by adding 200 μl of TU.
diluted in 1 DMEM and anesthetized (AVERTIN (0.015 ml / g);
U.S. Patent No. 5,622,699; Pasqualini and Ruoslah.
ti, supra, 1996) were injected into the tail vein of mice. 1-4
After a minute, the mice were quenched in liquid nitrogen. To recover phage, cadaver was partially thawed at room temperature for 1 hour, tumor and control organs were collected, weighed, and then 1 ml of DMEM-PI (protease inhibitor (PI
); Phenylmethylsulfonylfluoride (PMSF; 1 mM), aprotinin (20 μg / ml), leupeptin (1 μg / ml in DMEM).

Alternatively, following introduction of the library into mice, cycling the library
It is terminated by perfusion through the heart. Briefly, mice are anesthetized with AVERTIN, then the heart is exposed and 10 cc via a 0.5 mm cannula.
A 0.4 mm needle connected to a syringe was inserted into the left ventricle. An incision was made in the right atrium and 5-10 ml of DMEM was slowly administered and allowed to perfuse the entire body for about 5-10 minutes. Perfusion effectiveness was monitored directly by histological analysis.

The tumor and organ samples were washed three times with ice-cold DMEM-PI containing 1% bovine serum albumin (BSA) and then washed with 1 ml of K91-kan bacteria.
Incubated directly for hours. 1 containing 0.2 μg / ml tetracycline
0 ml of NZY medium (NZY / tet) was added to the bacterial culture. The mixture is incubated for 1 hour in a shaker at 37 ° C., then 10 μl or 1 μl
Aliquots of 00 μl were plated on agar plates (tet / agar) containing 12.5 μg / ml tetracycline.

[0187] Individual colonies containing phage recovered from the tumor were removed for 16 hours with 5 ml of N
Grow in ZY / tet. Bacterial cultures obtained from individual colonies were pooled and phage purified and re-injected into mice for a second in vivo panning as described above. Usually, a third panning was performed again. Phage DNA was purified from individual bacterial colonies obtained from the final round of in vivo panning and D encoding the peptide expressed by the selected phage.
The NA sequence was determined (see Koivunen et al., Supra, 1994b).

Example V Identification of Tumor Homing Peptides by In Vivo Panning on Breast Tumors This example demonstrates that in vivo panning was performed on breast tumors to identify tumor homing peptides that home to various tumors. Demonstrate what we can do.

Human 435 breast cancer cells (Price et al., Cancer Res. 50: 717-
721 (1990)) were inoculated into the fat pads of the breasts of nude mice. When the tumors reach approximately 1 cm in diameter, either phage targeting experiments (in which phage expressing a specific peptide were administered to tumor-bearing mice) or in vivo panning were performed. went.

Breast tumor-bearing mice were injected with 1 × 10 ⁹ phage expressing a library of CX ₃ CX ₃ CX ₃ C (SEQ ID NO: 12) peptides. Here, X ₃ represents three groups of randomly selected random amino acids. Circulate the phage for 4 minutes,
The mice were then anesthetized, quenched in liquid nitrogen during anesthesia, and the tumors were removed. Phages were isolated from tumors and subjected to two additional rounds of in vivo panning.

[0191]

[Table 2] Following the third round of panning, the phage was quantified and the peptide sequence expressed by the cloned phage was determined. The cloned phage expresses a variety of different peptides, including the peptides shown in Table 2. Similarly, CX ₇ C (SEQ ID NO: 11) and CX ₅ C (SEQ ID NO: 9) libraries were screened and breast tumor homing peptides were identified (Table 2). These results indicate that in vivo panning on breast tumors can identify tumor homing molecules.

Example VI In Vivo Targeting of Tumors of Phage Expressing RGD (an an RGD) Peptide to Tumors Human 435 breast carcinoma cells were inoculated into the breast fat pad of nude mice. When the tumors reached approximately 1 cm in diameter, phage expressing a particular RGD-containing peptide were administered to tumor-bearing mice. Similar results to those discussed below were also obtained in nude mice bearing tumors formed by implantation of human melanoma C8161 cells or by implantation of mouse B16 melanoma cells.

1 × 10 ⁹ phage or control (no insert) phage expressing an RGD-containing peptide (CDCRGDCFC (SEQ ID NO: 1; see Koivunen et al., Supra, 1995)) were injected into mice intravenously ( iv) and allowed to circulate for 4 minutes. The mouse is then snap frozen.
Or perfused through the heart under anesthesia, and removed various organs, including tumors, brain, and kidneys, and quantified the phage present in the organs (US Pat. No. 5,622,699; Pasqualini and Ruoslahti, supra, 1996).

When compared to brain and kidney, phage expressing the CDCRGDCFC (SEQ ID NO: 1) peptide (about 2-3 fold) were detected in breast tumors,
Figure 4 shows that the GDCFC (SEQ ID NO: 1; RGD phage) peptide results in the selective homing of phage to breast tumors. In a parallel study, unselected phage expressing various diverse peptides were injected into tumor-bearing mice,
And various organs were tested for the presence of phage. When compared to this tumor, there was considerably more phage in the kidney and to a lesser extent in the brain. Therefore, phages expressing RGD 80 times more than unselected phages were concentrated in this tumor. These results indicate that phage expressing the RGD-containing peptide home to tumors, presumably due to the expression of α _v β ₃ integrin on blood vessels that form in tumors.

The specificity of the breast tumor homing peptide was demonstrated by competition experiments in which 500 μg of the free peptide (ACCDRGDCFCCG (SEQ ID NO: 1)
6); see Pasqualini et al., Supra, 1997) and phage expressing the tumor homing peptide reduced the amount of phage in the tumor by about 10-fold, while the inactive control peptide (GRGESP) Co-injection with (SEQ ID NO: 17) had essentially no effect, indicating that phage displaying peptides capable of binding to integrins expressed on the vasculature of angiogenesis were It has been demonstrated that they can selectively home to organs or tissues (eg, tumors containing such vasculature) in vivo.

Example VII Immunohistological Analysis of Tumor Homing Peptides This example provides a method for identifying the localization of tumor homing molecules by immunohistological studies.

The localization of tumor homing peptide expressing phage was determined by tissue obtained either 5 minutes or 24 hours after administration of phage “peptide-phage” expressing tumor homing peptide to tumor-bearing mice. Sections were identified by immunochemical methods. For samples obtained 5 minutes after administration of peptide-phage, mice were perfused with DMEM and various organs including tumors were removed.
And fixed in Bouin's solution. For the sample obtained at 24 hours, no peptide-phage remained in circulation and therefore no perfusion was required. Histological sections were prepared and reacted with anti-M13 (phage) antibodies (Pharmac
ia Biotech; US Patent No. 5,622,699; Pasqualin
i and Ruoslahti, supra, 1996). Visualization of the conjugated anti-M13 antibody was performed according to the manufacturer's instructions, using a peroxidase-conjugated secondary antibody (
Sigma; St. Louis MO).

As discussed in Example VI, phage expressing tumor homing peptide (
CDCRGDCFC (SEQ ID NO: 1; “RGD phage”) was administered intravenously to mice bearing breast tumors. In addition, RGD phage was administered to mice bearing mouse melanoma or human Kaposi's sarcoma. Phage circulation was terminated, and mice were sacrificed as described above, and tumor samples and skin samples adjacent to the tumor, brain, kidney, lung and liver were collected. Immunohistochemical staining for phage showed accumulation of RGD phage in blood vessels present in breast tumors and melanomas and Kaposi's sarcoma, but little or no staining was observed in control organs.

A similar experiment identified a phage expressing the tumor homing peptide (CNGRCVSGCAGRC (SEQ ID NO: 3; “NGR phage”)) identified by in vivo panning on tumors formed by MDA-MB-435 breast carcinoma.
Was carried out using In these experiments, NGR phage or control phage that do not express the peptide were administered to mice bearing MDA-MB-435 breast carcinoma or tumors formed by human SLK Kaposi's sarcoma xenografts, and the mice were then treated as above And tumor and control organs (including brain, lymph nodes, kidneys, pancreas, uterus, mammary fat pad, lung, intestine, skin, skeletal muscle, heart and renal goblet, bladder and ureter epithelium) Was recovered. Histological samples were prepared and tested by immunostaining as described above.

In samples obtained from mice sacrificed 4 minutes after administration of NGR phage, immunostaining of both breast tumor and Kaposi's sarcoma vasculature was observed. Very little or no staining was observed in the endothelium of these tumors in mice receiving control phage without insert. In samples obtained from mice sacrificed 24 hours after administration of NRG phage, staining of the tumor samples appeared to have spread to the breast parenchyma and Kaposi's sarcoma parenchyma outside the vessels. In addition, little or no staining was observed in samples prepared from these tumors in mice receiving control phage without insert. In addition, little or no staining was observed in various control organs in mice that received NGR phage.

In other experiments, similar results were obtained with the NGR tumor homing peptide (NGRA
Obtained after administration of phage expressing HA (SEQ ID NO: 6) or CVLNGRMEC (SEQ ID NO: 7) to tumor-bearing mice. Also, as discussed below, similar results were obtained using phage expressing the GSL tumor homing peptide (CLSGSLSC (SEQ ID NO: 4)) identified by in vivo panning of melanoma (Examples below). VIII).

These results indicate that the tumor homing peptide selectively homed to the tumor, particularly to the vasculature in the tumor, and that the tumor homing peptide identified, for example, by in vivo panning against breast carcinoma, And selectively home to other tumors, including melanoma. Furthermore, these results demonstrate that immunohistochemical analysis provides a convenient assay for identifying the localization of phage expressing tumor homing peptides.

Example VIII Identification of Tumor Homing Peptides by In Vivo Panning on Melanoma Tumors The general applicability of the in vivo panning method to identify tumor homing peptides was demonstrated in transplanted mouse melanoma tumors. Were tested by performing in vivo panning.

Mice bearing melanoma were transformed with B16B15 producing highly vascularized tumors
b Produced by implantation of mouse melanoma cells. B16B15b mouse melanoma cells were injected subcutaneously into the mammary fat pad of nude mice (2 months old) and tumors were allowed to grow to a diameter of about 1 cm. In vivo panning was performed as described above. About 1 × 10 ¹² transducing units of phage expressing a CX ₅ C (SEQ ID NO: 9), CX ₆ C (SEQ ID NO: 10) or CX ₇ C (SEQ ID NO: 11) library
It was injected intravenously and circulated for 4 minutes. Mice were then snap frozen in liquid nitrogen or perfused through the heart under anesthesia to remove tumor tissue and brain (control organ) and phage isolated as described above. Three in vitro pannings were performed.

[0205]

[Table 3] The amino acid sequence was determined for inserts in 89 cloned phage recovered from B16B15b tumors. The peptides expressed by these phages have two dominant sequences, CLSGSLSC (SEQ ID NO: 4; 52% of sequenced clones) and WGTGLC (SEQ ID NO: 18; 25 of clones).
%; See Table 3). Reinfection of phage expressing one selected peptide resulted in an approximately 3-fold enrichment of phage that homed to tumors compared to brain.

The localization of phage expressing the tumor homing peptide in mouse organs was also examined by immunohistochemical staining of tumors and various other tissues (see Example VII). In these experiments, 1 × 10 ⁹ pfu of control (no insert) phage or phage expressing the tumor homing peptide (CLSGSLSC (SEQ ID NO: 4)) were injected intravenously into tumor-bearing mice and
Circulated for minutes.

Immunostaining was evident in melanomas obtained from mice injected with phage expressing the CLSGSLSC (SEQ ID NO: 4) tumor homing peptide. Although melanoma staining is generally localized to blood vessels within the tumor, some staining was also present in the tumor parenchyma. Essentially, no staining was observed in tumors obtained from mice injected with control phage without insert, or in skin or kidney samples obtained from mice injected with any phage. Was. However, immunostaining was detected in the liver sinusoids and spleen, indicating that phage can be non-specifically captured in organs containing RES.

Using a similar method, in vivo panning was performed in mice bearing SLK human Kaposi's sarcoma. Tumor homing peptides have been identified. This is disclosed in Table 4. Together, these results demonstrate that the in vivo panning method is generally applicable to methods for screening phage libraries to identify phage that express tumor homing peptides.

[0209]

[Table 4] Example IX Characterization of Chimeric Peptide Consisting of Prostate Homing Peptide and _D (KLAKLAK) ₂ This example demonstrates that the chimeric peptide SMSIARL-GG- _D (KLAKLAK) ₂ was selected in prostate tissue after systemic administration. Demonstrate that it can induce apoptosis and prolong the survival of animals with experimental prostate cancer.

[0210] The X ₇ library (A. Isolation of prostate homing peptides), were injected into mice, and as described in WO99 / forty-six thousand two hundred eighty-four, preferentially found sequences in prostate when compared to brain Was isolated. Prostate homing peptide SMSIARL (SEQ ID NO: 207) and VSFL
EYR (SEQ ID NO: 222), in the prostate when compared to the brain,
It showed 34- and 17-fold abundance. Additional prostate homing sequences identified by in vivo panning are shown in Table 5.

[0211]

[Table 5] B. Prostate Homing of Prostate Homing Peptide Biotin Conjugates In vivo screening of a heptapeptide phage library identified prostate homing peptides that were 35-fold more concentrated in the prostate than various other tissues. This phage displays the peptide SMSIARL (SEQ ID NO: 207). Co-injection of the synthetic SMSIARL peptide (SEQ ID NO: 207) was performed by SMS
Prostate-selective homing of IARL (SEQ ID NO: 207) -bearing phage was inhibited. In addition, antibody staining of tissue sections showed that after intravenous injection of phage into mice, the SMSIARL (SEQ ID NO: 207) phage was localized to prostate tissue but not to other tissues. Control phage also did not accumulate in the prostate. The SMSIARL (SEQ ID NO: 207) phage also homes to rat prostate tissue.

As shown in FIG. 6, the biotin-conjugated SMSIARL (SEQ ID NO: 207) synthetic peptide was shown to home to the prostate. Briefly, 1 mg of biotin-conjugated prostate homing peptide SMSIARL (SEQ ID NO: 207)
Alternatively, the mice were injected intravenously with the biotin-labeled control peptide CARAC (SEQ ID NO: 208), and the mice were sacrificed 10 minutes later. Prostate and other tissues were collected, excised, and processed for staining with avidin-peroxidase. Biotin staining was found predominantly in the lumen of the gland rather than the vasculature 10 minutes after systemic injection of the conjugate. These results indicate that the SMSIARL (SEQ ID NO: 207) peptide as well as other moieties that bind to the SMSIARL peptide (eg, phage or biotin) migrate to the prostate epithelium and then to the glandular lumen.

C. Induction of Prostate-Selective Apoptosis The SMSIARL peptide (SEQ ID NO: 207) was replaced by the pro-apoptotic peptide (SEQ ID NO: 207).
The ability to deliver pro-apoptotic peptide ( _D (KLAKLAK) ₂ ) to the prostate was analyzed. Briefly, SMSIARL-GG- _D (K
(LAKLAK) ₂ chimeric peptide or control substance in a single dose of 250
Administered in μg peptide / mouse and performed TUNEL staining on tissues obtained 24 hours later. As shown in FIG. 7, mice injected with the SMSIARL-GG- _D (KLAKLAK) ₂ chimera showed increased apoptosis in their prostate, and particularly in the capillary endothelium and basal muscle epithelial cells of the glands of the prostate. . There was no evidence of increased apoptosis in other tissues such as testis, kidney and brain. SMSIARL (SEQ ID NO: 207) and _D (KLAKLAK) ₂ 2
No apoptosis was observed in negative control mice treated with 50 μg of the unconjugated mixture.

_D. SMSIARL-GG- _D (KLAKLAK) ₂ Treatment of TRAMP Mice TRAMP mice develop prostate cancer under the influence of a foreign gene as described by Gingrich et al. (Supra, 1996). . SMSIARL-GG- _D (
(KLAKLAK) ₂ chimeric peptide was assayed for its ability to suppress the development of cancer in TRAMP mice. Treatment was for 12-week-old mice (10 per group)
, And mice received the SMSIARL-GG- _D (KLAKLAK) ₂ peptide or control peptide at 250 μg / dose biweekly for a total of 10 doses. Four mice without visible tumors were killed within minutes after injection, followed by SM
SIALL-GG- _D (KLAKLAK) was excluded from the _two groups. As shown in FIG.
SMSIARL-GG- _D (KLAKLAK) ₂ treated mice were treated with control (which is the vehicle, _D (KLAKLAK) ₂ peptide alone or SM
Survived longer than mice treated with the SIARL peptide (SEQ ID NO: 207) alone. Thus, targeted pro-apoptotic SMSIA over a period of months
Treatment of TRAMP mice with the RL-GG- _D (KLAKLAK) ₂ compound
This resulted in a distinct increase in survival of treated mice compared to control mice.

(E. Prostate Homing SMSIARL (SEQ ID NO: 207) phage binds to human prostate vasculature) Human prostate tissue sections, including both normal and cancerous tissues, were prepared using 10 ⁹ TU S
MSIARL phage (SEQ ID NO: 207) was covered and phage binding was detected with anti-phage antibodies and peroxidase staining. As shown in FIG.
SIARL (SEQ ID NO: 207) phage binds to the endothelium of human prostate blood vessels (
See panels a and b). No staining of the endothelium was seen with phage without peptide insert (panel c). Furthermore, when the soluble SMSIARL peptide (SEQ ID NO: 207) is included at 0.3 mg / ml in the shroud, the SMSIA
RL (SEQ ID NO: 207) phage staining was inhibited. The results shown in FIG. 9 indicate that at least some tumors carry the homing peptide receptor. In addition, the peptide (SEQ ID NO: 207) can bind to cancer vasculature in the prostate, while blood vessels in some other human tissues can
(SEQ ID NO: 207) Not stained by phage.

All journal articles, references and patent citations provided above are hereby incorporated by reference herein, whether in parentheses or otherwise set forth previously. Incorporated.

Although the invention has been described with reference to the above examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

[Brief description of the drawings]

FIG. 1 shows a computer generated model and amino acid sequence of CNGRC-GG- _D (KLAKLAK) ₂ (referred to as “HPP-1”). Upper panel: CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1) is composed of a homing domain and a membrane disruption domain linked by a binding domain. Lower panel: amino acid sequence of "HPP-1", corresponding to the structure shown in the upper panel.

FIG. 2 shows mitochondrial swelling and mitochondria-dependent apoptosis in the presence of _D (KLAKLAK) ₂ . a. _In the presence of _D (KLAKLAK) ₂ or Ca ²⁺ (positive control)
The mitochondrial swelling curve (optical absorption spectrum) is shown. b. Caspase 3 showing the 32 kDa pro-form and the 8 kDa and 20 kDa process forms in the presence of _D (KLAKLAK) ₂ or DLSLARLATARLAI (SEQ ID NO: 204) in the presence or absence of mitochondria
Immunoblots for cleavage. A representative experiment is shown. The results were reproduced in three independent experiments.

FIG. 3 shows mitochondrial swelling and apoptosis in skin microvascular endothelial cells treated with CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1). a. Skin microvascular endothelial cell cord array formation, bar scale = 250 μm.

FIG. 3 shows mitochondrial swelling and apoptosis in skin microvascular endothelial cells treated with CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1). b. Treated with CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1),
DEVD-pNA hydrolysis (caspase activation) in proliferating skin microvascular endothelial cells.

FIG. 3 shows mitochondrial swelling and apoptosis in skin microvascular endothelial cells treated with CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1). c. Viability of proliferating skin microvascular endothelial cells treated with HPP-1 over time (
Viability of proliferating skin microvascular endothelial cells over time treated with black bars) or control peptide _D (KLAKLAK) ₂ (grey bars). (T-test, P <0.05).

FIG. 3 shows mitochondrial swelling and apoptosis in skin microvascular endothelial cells treated with CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1). d. Survival rate of cord-forming skin microvascular endothelial cells treated with HPP-1 over time (black bars) or proliferating skin microvascular endothelial cells treated with control peptide _D (KLAKLAK) ₂ (grey bars) Survival over time. (T-test, P <0.05
).

FIG. 3E shows mitochondrial swelling and apoptosis in skin microvascular endothelial cells treated with CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1).

FIG. 3 shows mitochondrial swelling and apoptosis in skin microvascular endothelial cells treated with CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1).

FIG. 3 shows mitochondrial swelling and apoptosis in skin microvascular endothelial cells treated with CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1).

FIG. 4 shows the effect of HPP-1 treatment on nude mice bearing human MDA-MB-435-derived breast carcinoma xenografts. a. Tumor volume of HPP-1 treated tumors when compared to control CARAC-GG- _D (KLAKLAK) ₂ treated tumors. The difference in tumor volume between day 1 and day 50 is shown (t-test, P = 0.027). b. Survival of nude mice bearing breast carcinoma xenografts from human MDA-MB-435 treated with HPP-1 or control peptide ( _D (KLAKL
AK) ₂ and CNGRC (SEQ ID NO: 8)) treated human MDA-MB
Kap showing survival of nude mice bearing mammary carcinoma xenografts from -435
Lan-Meier survival plot. Each group consisted of 13 animals. (Log-Rank test, P <0.05).

FIG. 5 shows CDC on oxygen-induced neovascularization of the retina in neonatal mice.
The effect of RGDCFC-GG- _D (KLAKLAK) ₂ is shown. The number of new blood vessels in the retina was determined by treatment with the vehicle (solid bar); CDCRGDCFC-GG- _D
2 ) treatment with ₂ (stripe bars); and unbound CDCRGDCFC (SEQ ID NO: 1)
) And _D (KLAKLAK) ₂ treated with a control mixture (twill bars).

FIG. 6 shows the accumulation of biotin conjugate of intravenously injected prostate homing peptide in prostate tissue. a. Biotin-labeled prostate homing peptide SMSI
Avidin-peroxidase staining of prostate sections from mice injected with ARL (SEQ ID NO: 207). b. Avidin-peroxidase staining of prostate sections from mice injected with the biotin-labeled control peptide CARAC (SEQ ID NO: 208).

FIG. 7 shows SMSIARL-GG- _D (KLAKL) in normal mouse prostate.
₂ shows apoptosis induced by AK) ₂ . a. SMSIARL-GG- _D (KL
AKNE) TUNE of prostate tissue from mice treated with ₂ chimeric peptides
L staining. b. Higher magnification field similar to TUNEL staining in a. c. TUNEL staining of negative control mice treated with 250 μg of a mixture of unbound SMSIARL (SEQ ID NO: 207) and _D (KLAKLAK) ₂ .

FIG. 8 shows SMSIARL-GG- _D (KLAKLAK) ₂ , vehicle only, _D (
KLAKLAK) ₂ peptide alone or SMSIARL peptide (SEQ ID NO: 2)
07) shows the survival of TRAMP mice treated only with 07).

FIG. 9 shows binding of prostate homing SMSIARL (SEQ ID NO: 207) phage to human prostate vasculature. a and b. Peroxidase staining of human prostate tissue sections, including both normal and cancerous tissues, overlaid with 10 ⁹ TU SMSIARL phage (SEQ ID NO: 207) and detected using anti-phage antibodies. a is an overview (× 20), while b shows details from panel a at a higher magnification (× 40). c. Using phage lacking the peptide insert,
Peroxidase staining as in panel a. d. Soluble S contained in the overlay
Peroxidase staining as in a using the MSIARL peptide (SEQ ID NO: 207).

──────────────────────────────────────────────────続 き Continuation of front page (72) Inventor Pascarini, Renata United States of America California 92075, Solana Beach, ES. Sierra Avenue No. 29 707 (72) Inventor Luosrati, Elkki Eye. United States California 92067, Rancho Santa Fe, P. Oh. Box 1054 F-term (reference) 4C084 AA02 AA07 AA14 BA01 BA08 BA18 BA19 BA41 CA59 CA62 MA66 NA05 NA14 ZA812 ZB262 4H045 AA10 AA30 BA17 BA18 EA28 EA51 FA20

Claims (35)

[Claims] 1. A homing pro-apoptotic conjugate, wherein the homing pro-apoptotic conjugate comprises a tumor homing molecule selectively homing to a selected mammalian cell type or tissue, conjugated to an antimicrobial peptide. The conjugate is selectively internalized by the mammalian cell type or tissue and exhibits high toxicity to the mammalian cell type or tissue, and the antimicrobial peptide is not bound to the tumor homing molecule In some cases, a homing pro-apoptotic conjugate having low mammalian cytotoxicity. 2. The conjugate of claim 1, wherein said conjugate exhibits selective toxicity to angiogenic endothelial cells. 3. The conjugate according to claim 1, wherein the antimicrobial peptide has an amphipathic α-helix structure. 4. The conjugate according to claim 1, wherein the antimicrobial peptide comprises: (KLAKLAK) ₂ (SEQ ID NO: 200); (KLAKLA) ₂ (SEQ ID NO: 201); (KAAKKAA) ₂ (Sequence Conjugate comprising a sequence selected from the group consisting of: (KLGKKLG) ₃ (SEQ ID NO: 203). 5. The conjugate according to claim 4, wherein said antimicrobial peptide comprises the sequence _D (KLAKLAK) ₂ . 6. The conjugate of claim 1, wherein said conjugate is a chimeric peptide and said tumor homing molecule is a tumor homing peptide. 7. The conjugate according to claim 6, wherein the conjugate is a chimeric peptide having at most 27 amino acids. 8. The conjugate according to claim 7, wherein the conjugate is a chimeric peptide having at most 21 amino acids. 9. The conjugate of claim 6, wherein said tumor homing peptide comprises the amino acid sequence NGR. 10. The conjugate of claim 9, wherein said tumor homing peptide is from the group consisting of: CNGRC (SEQ ID NO: 8); NGRAHA (SEQ ID NO: 6); and CNGRCVSGCAGRC (SEQ ID NO: 3) A conjugate that is a peptide of choice. 11. The conjugate of claim 6, wherein said tumor homing peptide comprises the amino acid sequence RGD. 12. The conjugate of claim 11, wherein said tumor homing peptide is CDCRGDCFC (SEQ ID NO: 1). 13. A homing pro-apoptotic conjugate comprising a sequence selected from the group consisting of: CNGRC-GG- _D (KLAKLAK) ₂ and ACCDRGDCFC-GG- _D (KLAKLAK) _2. body. 14. The homing pro-apoptotic conjugate according to claim 13, wherein the homing is selected from the group consisting of: CNGRC-GG- _D (KLAKLAK) ₂ and ACCDRGDCFC-GG- _D (KLAKLAK) _2. Pro-apoptotic conjugate. 15. A method of directing an antimicrobial peptide in vivo to a tumor having an angiogenic vasculature, wherein the subject comprises a tumor having an angiogenic vasculature. Administering an apoptotic conjugate,
Method. 16. The method of claim 15, wherein the antimicrobial peptide comprises:
A method comprising the sequence _D (KLAKLAK) ₂ . 17. The method of claim 16, wherein the homing pro-apoptotic conjugate is selected from the group consisting of: CNGRC-GG- _D (KLAKLAK) ₂ and ACCDRGDCFC-GG- _D (KLAKLAK) _2. Will be the way. 18. A method for inducing selective toxicity in a tumor having an angiogenic vasculature in vivo, wherein the subject comprises a tumor having an angiogenic vasculature. Administering a pro-apoptotic conjugate. 19. The method of claim 18, wherein said antimicrobial peptide comprises:
A method comprising the sequence _D (KLAKLAK) ₂ . 20. The method of claim 19, wherein the homing pro-apoptotic conjugate is selected from the group consisting of: CNGRC-GG- _D (KLAKLAK) ₂ and ACCDRGDCFC-GG- _D (KLAKLAK) _2. Will be the way. 21. A method of treating a patient having a tumor having angiogenic vasculature, comprising the step of administering to the patient a homing pro-apoptotic conjugate of claim 1, thereby comprising: Wherein the conjugate is selectively toxic to the tumor. 22. The method of claim 21, wherein said antimicrobial peptide comprises:
A method comprising the sequence _D (KLAKLAK) ₂ . 23. The method of claim 22, wherein the homing pro-apoptotic conjugate is selected from the group consisting of: CNGRC-GG- _D (KLAKLAK) ₂ and ACCDRGDCFC-GG- _D (KLAKLAK) _2. Will be the way. 24. A chimeric prostate homing pro-apoptotic peptide, wherein the chimeric prostate homing pro-apoptotic peptide comprises a prostate homing peptide bound to an antimicrobial peptide, wherein the chimeric peptide is selectively internalized by prostate tissue; And exhibit high toxicity to the prostate tissue, and the antimicrobial peptide, when not bound to the prostate homing peptide,
A chimeric prostate homing pro-apoptotic peptide with low mammalian cytotoxicity. 25. The chimeric peptide according to claim 24, wherein the prostate homing peptide comprises the sequence SMSIARL (SEQ ID NO: 207) or a functionally equivalent sequence. 26. The chimeric peptide according to claim 24, wherein the antimicrobial peptide has an amphipathic α-helix structure. 27. The chimeric peptide according to claim 24, wherein the antimicrobial peptide comprises: (KLAKLAK) ₂ (SEQ ID NO: 200); (KLAKLA) ₂ (SEQ ID NO: 201); (KAAKKAA) ₂ (SEQ A chimeric peptide comprising a sequence selected from the group consisting of: (KLGKKLG) ₃ (SEQ ID NO: 203). 28. The chimeric peptide according to claim 24, wherein the antimicrobial peptide comprises the sequence _D (KLAKLAK) ₂ . 29. The chimeric peptide according to claim 28, which has the sequence SMS
A chimeric peptide comprising IARL-GG- _D (KLAKLAK) ₂ . 30. The chimeric peptide according to claim 29, wherein the sequence is SMS.
A chimeric peptide consisting of IARL-GG- _D (KLAKLAK) ₂ . 31. A method for inducing selective toxicity of prostate cancer in vivo, comprising the step of administering the chimeric peptide of claim 24 to a subject having prostate cancer. 32. The method of claim 31, wherein the prostate homing peptide comprises the sequence SMSIARL (SEQ ID NO: 207) or a functionally equivalent sequence. 33. The method of claim 31, wherein the antimicrobial peptide comprises:
A method comprising the sequence _D (KLAKLAK) ₂ . 34. The method of claim 33, wherein said chimeric peptide comprises the sequence SMSIARL-GG- _D (KLAKLAK) ₂ . 35. The method of claim 34, wherein said chimeric peptide is SMSIARL-GG- _D (KLAKLAK) ₂ .