JP2003520808A - Chimeric prostate homing peptide with pro-apoptotic activity - Google Patents

Abstract

The present invention is directed to a chimeric prostate homing pro-apoptotic peptide comprising a prostate homing peptide linked to an antimicrobial peptide, wherein the chimeric peptide is selectively internalized by prostate tissue, and It provides a chimeric peptide that is highly toxic to tissues and has low mammalian cytotoxicity when the antimicrobial peptide is not linked to the prostate homing peptide. In a preferred embodiment, the chimeric prostate homing pro-apoptotic peptide comprises the sequence SMSIARL-GG- _D (KLAKLAK) ₂ . Methods for using such chimeric peptides to treat patients with prostate cancer are also provided.

Description

Detailed Description of the Invention

This study is based on the National Cancer Institute (USA).
) Grants CA74238, CA28896 and CA30199, and the Department of Defense grant DAMD17-98-1-8581. The US Government has certain rights in this invention.

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to the field of cancer biology and drug delivery, and more specifically to the selective targeting of antimicrobial peptides to the prostate. To do.

Background Information Prostate adenocarcinoma has become the most frequent cancer diagnosed in American men in recent years,
For the first time exceeded the frequency of lung cancer. An estimated 30,000 American men with this malignancy die in a year. In addition, with the highest frequency reported in the United States and Scandinavian countries, and the moderately high frequency seen throughout Europe, prostate cancer affects men worldwide. Prostate cancer is a disease of elderly men, and 75% of all patients with this disease are 60-80 years old. At the age of 50 years, the estimated lifetime chance of developing clinically apparent prostate cancer is approximately 10% for American men. However, anatomical studies have shown that the true frequency of prostate cancer is actually significantly higher than indicated by clinical evidence.

[0004] A major obstacle to advances in cancer (eg, prostate cancer) targeting is the relative lack of drugs that can selectively target cancer but do not act on normal tissues. For example, radiation therapy and surgery, which is generally local treatment, can cause substantial damage to normal tissue in the treated area, causing scarring and, in severe cases, of normal tissue. Cause loss of function. Chemotherapy, which is generally administered systemically, can cause substantial damage to organs such as the bone marrow, mucous membranes, skin and small intestine, which result in rapid cell turnover and continuity. Cause specific cell division. As a result, undesired side effects, such as nausea, hair loss and cytopenia, occur as a result of systemically targeting cancer patients with chemotherapeutic agents. Such undesired side effects often result in limitations on the amount of treatment that can be administered. Because of these shortcomings of treatment, cancer remains the cause of morbidity and mortality in patients.

A potent antibacterial activity is found in naturally occurring peptides (eg melittin, gramicidin, magainin, defensins and cecropins (ce).
have been observed for a class of peptides including cropin)). Naturally occurring antimicrobial peptides, and related synthetic antimicrobial sequences, generally have the same number of polar and non-polar residues within an amphipathic domain and a sufficient number of basic residues, It gives the peptide an overall positive charge at neutral pH. The biological activity of amphipathic α-helix peptides against Gram-negative bacteria is due to their ability to form ion channels through the bilayer of the membrane. Many antimicrobial peptides selectively inhibit and kill bacteria by virtue of the specific sensitivity of the bacterial cells, apparently due to differences in the membranes of the bacteria and mammalian cells, but retain low mammalian cytotoxicity . As shown herein, these antimicrobial peptides can be endowed with selective cytotoxic activity against specific eukaryotic cell types, such as prostate endothelial cells.

Accordingly, there is a need for new anti-cancer therapeutics that can selectively target the prostate. The present invention provides a prostate-homing pro-apoptotic peptide that combines an antimicrobial peptide with a prostate homing peptide to produce a conjugate with selective toxicity to the prostate. Meet the need. Relevant advantages are still provided.

SUMMARY OF THE INVENTION The present invention provides a chimeric prostate-homing proapoptotic peptide comprising a prostate-homing peptide linked to an antimicrobial peptide, wherein the chimeric peptide is selectively selected by prostate tissue. Although internalized and highly toxic there, the antimicrobial peptide has low mammalian cytotoxicity when not bound to the prostate-homing peptide. In the chimeric peptide of the present invention,
The 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 an amphipathic α-helical structure such as the sequence (KLAKLAK) ₂ (SEQ ID NO: 200). ), (KL
AKKLA) ₂ (SEQ ID NO: 201), (KAAKKAA) ₂ (SEQ ID NO: 202)
Or (KLGKKLG) ₃ (SEQ ID NO: 203)). In a preferred embodiment, the antimicrobial peptide portion comprises the sequence _D (KLAKLAK) ₂ . Exemplary prostate-homing pro-apoptotic peptides are described herein as SMSIA.
It is provided as RL-GG- _D (KLAKLAK) ₂ . The invention further provides a method for in vivo antimicrobial peptides against prostate cancer. This method
Administering a chimeric prostate-homing proapoptotic peptide comprising a prostate-homing peptide linked to an antimicrobial peptide, wherein the chimeric peptide is selectively internalized by prostate tissue, where it exhibits high toxicity. However, antimicrobial peptides have poor mammalian cell properties when not bound to prostate-homing peptides. In the methods of the invention, the prostate homing peptide can include, for example, an antimicrobial peptide that can include the sequence SMSIARL (SEQ ID NO: 207) or a functionally equivalent sequence, and a sequence such as _D (KLAKLAK) ₂ . In a preferred embodiment, the chimeric prostate-homing proapoptotic peptide comprises the sequence SMSIARL-GG- _D (KLAKLAK) ₂ .

The present invention also provides a method of inducing selective toxicity in prostate cancer in vivo. The method comprises administering to a subject having prostate cancer a chimeric prostate-homing pro-apoptotic peptide comprising a prostate-homing peptide linked to an antimicrobial polypeptide, wherein the chimeric peptide is induced by prostate tissue. If selectively internalized and highly toxic there, but not bound to the prostate homing peptide, the antimicrobial peptide has low mammalian toxicity. Methods of inducing selective toxicity in prostate cancer in vivo include, for example:
It can be performed in vivo with a prostate-homing peptide comprising the sequence SMSIARL (SEQ ID NO: 207) or a functionally equivalent sequence. Examples of antibacterial peptides include the sequence _D (KLAKLAK) ₂ . In a preferred embodiment,
As chimeric prostate-homing pro-apoptotic peptide the sequence SMSIA
RL-GG- _D (KLAKLAK) ₂ may be mentioned.

[0009]   Further, the present invention provides a chimeric prostate-homing proapoptotic peptide of the invention.
The peptide is administered to the patient so that the chimeric peptide is selective for tumors.
Methods of treating patients with prostate cancer by being toxic are provided. Texture
Lapeptides include prostate-homing peptides linked to antimicrobial peptides,
The chimeric peptide is then selectively internalized by prostate tissue and
Although highly toxic here, the antimicrobial peptide is bound to the prostate-homing peptide.
If not, it has low mammalian cytotoxicity. Prostate-Homing Peptide
The portion may be, for example, the sequence SMSIARL (SEQ ID NO: 207) or a functionally equivalent
Sequence, and the antimicrobial peptide portion can be, for example, a sequence_D(KLAKLAK) _Two Can be included. In a preferred embodiment, the chimeric peptide has the sequence SMSIA.
RL-GG-_D(KLAKLA)_Twoincluding.

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

Antibacterial peptides often contain cationic amino acids that are attracted to the head groups of anionic phospholipids, resulting in preferential destruction of negatively charged membranes. Once electrostatically bound, this amphipathic helix can distort its lipid matrix, resulting in loss of membrane barrier function (Epand, The Am.
phipathic Helix, CRC Press: Boca Raton
(1993); Lutenberg and van Alphan, 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. Therefore, due to the different membrane composition, antimicrobial peptides are compared to eukaryotic membranes,
It can destroy prokaryotic membranes selectively.

The present invention provides that an antimicrobial peptide sequence may bind to a tumor homing molecule to produce a homing pro-apoptotic conjugate, which homing pro-apoptotic conjugate is generally located outside of a eukaryotic cell. Is non-toxic, but relates to the surprising finding that it promotes disruption of mitochondrial membranes and subsequent cell death when targeted and internalized in eukaryotic cells. Homing pro-apoptotic conjugates (eg HPP-1 (which is a cyclic tumor homing molecule CN
Including antimicrobial peptide _D (KLAKLAK) ₂ bound to GRC (SEQ ID NO: 8))
) May have in vivo selective toxicity to angiogenic endothelial cells and may therefore be useful as a new class of anti-cancer therapeutic agents.

Accordingly, the present invention provides a homing pro-apoptotic conjugate, which homing pro-apoptotic conjugate is a tumor that selectively homes to a selected mammalian cell type or tissue conjugated to an antimicrobial peptide. A homing molecule, the conjugate is selectively internalized by the mammalian cell type or tissue and is highly toxic to the mammalian cell type or tissue, and the antimicrobial peptide binds the tumor homing molecule. If it is not bound to a molecule, it has low mammalian cytotoxicity. For example, the homing pro-apoptotic conjugates of the invention may exhibit selective toxicity to angiogenic endothelial cells, and induce selective toxicity in vivo, eg, in tumors with angiogenic vasculature. Can be useful in the method.

As disclosed herein, the synthetic antimicrobial peptide _D (KLAKLAK) ₂ , which has selective toxicity to bacteria as compared to eukaryotic cells, has a marked mitochondrial swelling at a concentration of 10 μM. Were induced (Fig. 2a). This concentration is significantly lower than that required to kill eukaryotic cells, indicating that _D (KLAKLAK) ₂ preferentially disrupts mitochondrial membranes as compared to eukaryotic membranes. Shown (see Example I). Furthermore, _D (KLAKLAK) ₂ activated mitochondrial-dependent cell-free apoptosis as measured by the characteristic caspase-3 processing (FIG. 2b), but non-α-helix formation. The peptide DLSLARLATARLAI (SEQ ID NO: 204) did not activate this apoptosis. 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, which , Reflects a common ancestor of bacteria and mitochondria (Epand, supra, 1993; L
ugtenberg and van Alphan, supra, 1983; Matsu.
zaki et al., Biochemistry 34: 6521-6526 (1995).
); Hovius et al., FEBS Lett. 330: 71-76 (1993);
And Baltcheffsky and Baltcheffsky, Mito
chondria and Microsomes (Lee et al.), Addiso
n-Wesley: Reading, MA (1981), each of which is incorporated herein by reference).

[0015]   This antimicrobial peptide, as disclosed further herein._D(KLAKLAK
)_TwoTo the cyclic tumor homing peptide CNGR via the glycinylglycine bridge
Conjugated to C (SEQ ID NO: 8), the peptide CNGRC-GG-_D(KLAKL
AK)_Two(Referred to as "HPP-1"). As disclosed herein
In addition, HPP-1 was tested for angiogenesis by assaying cord formation.
Of the tissue culture model. This cord formation is usually “cobblestone” to cellular.
It is a migratory form indicated by changes in endothelial cell morphology into chains or cords. 60μ
By treatment of normal human dermal microvascular cells (DMEC) with MHPP-1,
In the proliferative state (Fig. 3c) or the cord formation state (Fig. 3d), the survival percentage is increased with time.
Leading to a decrease in_D(KLAKLAK)_TwoTreatment with peptides
In some cases, there was only a negligible loss of viability (see Example II).
Teru). Further, as shown in Table 1, during proliferation treated with HPP-1
Or LC of DMEC on the move_FiftyIs a single layer with 100% confluency
LC of maintained angiostatic DMEC_FiftyThan
It was a low order. This is due to the superiority of HPP-1 under angiogenic conditions.
Indicates proactive killing. The results disclosed herein further include_D(KLAKLAK) _Two Mitochondria of DMEC treated for 24 h remained morphologically normal
However, CNGRC-GG-_D(KLAKLAK)_TwoOr ACDCRGDCF
C-GG-_D(KLAKLAK)_TwoThe mitochondria of DMEC treated with
Before showing classical morphological indicators of apoptosis, including nuclear condensation and fragmentation
, Showed changes in mitochondrial morphology.

[0016]   As shown in Example III, this HPP-1 peptide CNGRC-GG- _D (KLAKLAK)_TwoAlso has activity in vivo. Figures 4a and 4b
As disclosed in U.S.A.
Mouse mice were treated with HPP-1. Tumor volume compared to control group
, One order smaller on average and survival in this HPP-1 treated group of animals
Was long. Furthermore, some of these HPP-1 treated mice were
He lived a few months longer than Lumaus. This means that both primary tumor growth and metastasis
Indicates that the one was inhibited. Destruction of tumor architecture and widespread cell death can result in
Evident on tissue pathological analysis, with about 50% apoptotic cell death
. HPP-1 is also effective against tumors derived from the human melanoma cell line C8161
, And ACDCRGDCFC-GG-_D(KLAKLAK)_TwoIs MDA-
It was effective against MD-435 breast cancer tumors. In short, these results are
Homing pro-apopt based on homing peptide sequences and antimicrobial peptide sequences
Proteolytic peptides can be non-toxic to the outside of eukaryotic cells, but
Destruction and destruction of mitochondrial membranes when internalized into nucleolar target cells
And that it can promote cell death. Homing prop like HPP-1
B.The apoptotic peptide has selective toxicity to angiogenic endothelial cells.
Therefore, it may have particular value as an anticancer therapeutic agent.

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

The homing pro-apoptotic conjugates of the invention are characterized by being highly toxic to internalized mammalian cell types. As used herein, the term "highly toxic" means that the conjugate is relatively effective in causing cell death of the selected cell type or tissue. Those of skill in the art will appreciate that toxicity can be analyzed using one of a variety of well-known assays for cell viability. Generally, this term (highly toxic) refers to a half maximal killing concentration (LC ₅₀ ) of less than about 100 μM, preferably about 50 μM.
Used to refer to a conjugate that is less than. For example, as disclosed herein, the homing pro-apoptotic conjugate HPP-1 is associated with D in angiogenic proliferation.
For MEC cells and cord forming DMEM cells and KS1767 cells,
It was characterized by an LC _{50 of} 51, 34 and 42, respectively. Furthermore, it is shown that the extended survival of tumor-bearing mice treated with the homing pro-apoptotic conjugates of the invention allows this selective toxicity to be reproduced in vivo.

As used herein, the term “antimicrobial peptide” means a naturally occurring or synthetic peptide having antimicrobial activity, which antimicrobial activity is the growth of one or more microorganisms. The ability to kill or delay. The antimicrobial peptide can kill or delay the growth of, for example, bacteria, including Gram-positive or Gram-negative bacteria, or one or more strains of fungi or protozoa. Therefore, antibacterial peptides are, for example, Escherichia coli, Pseudomo
nas aeruginosa or Staphylococcus aureu
For example, it may have bacteriostatic or bactericidal activity against one or more strains of S. Although not wishing to be bound by the following, antimicrobial peptides can have biological activity due to their ability to form ion channels through the membrane bilayer as a result of self-aggregation.

The antimicrobial peptide is typically highly basic and may have a linear or cyclic structure. As discussed further below, the antimicrobial peptide may have an amphipathic alpha-helical structure (US Pat. No. 5,789,542; Javadpou).
r et al., supra, 1996; Blondelle and Houghten, supra, 1
992). Antimicrobial peptides are also described, for example, in Mancheno et al., J.
． Peptide Res. 51: 142-148 (1998), may be a β chain / sheet forming peptide.

The antimicrobial peptide can be a naturally occurring peptide or it can be a synthetic peptide. Naturally occurring antimicrobial peptides are believed to be isolated from biological sources (eg, bacteria, insects, amphibians and mammals) and present inducible protective proteins that may protect the host organism from bacterial infections. . Naturally occurring antimicrobial peptides include gramicidin, magainin, melittin, defensin, and cecropine (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
Delle and Houghten, Annual Reports in M
edicinal Chemistry, Bristol, eds., 159-168, Academic Press, San Diego, each of which is incorporated herein by reference). As discussed further below, the antimicrobial peptide may also be an analog of the natural peptide, particularly one that possesses or enhances amphipathicity.

The antimicrobial peptides incorporated into the homing pro-apoptotic conjugates of the invention have low mammalian cytotoxicity when not bound to tumor homing molecules. Mammalian cytotoxicity can be readily assessed using conventional assays. For example, mammalian cytotoxicity can be assayed in vitro by lysis of human erythrocytes, as described by Javadour et al., Supra, 1996. Antimicrobial peptides with "low mammalian cytotoxicity" are not soluble in human erythrocytes or, due to their lytic activity, concentrations above 100 μM, preferably 200 μM.
Concentrations higher than M, 300 μM, 500 μM or 1000 μM are required.

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

The present invention also provides a homing pro-apoptotic conjugate in which a tumor homing molecule is bound to an antimicrobial peptide having an amphipathic α-helix structure.
In the pro-apoptotic conjugate of the present invention, the antimicrobial peptide portion has, for example, the sequence (KLAKKL) ₂ (SEQ ID NO: 200); (KLAKKKA) ₂ (SEQ ID NO: 201); (KAAKKKA) ₂ (SEQ ID NO: 202); or (KLGKK
LG) ₃ (SEQ ID NO: 203), and in particular the sequence _D (KLAKLAK) ₂ . Homing pro-apoptotic conjugates of the invention may have, for example, the sequence CNGR.
C-GG- _D (KLAKLAK) ₂ or ACDCRGDCFC-GG- _D (K
LAKLAK) ₂ .

[0025] Antimicrobial peptides generally have random coil conformations in dilute aqueous solutions, but still high levels of helicity are associated with helix-promoting solvents and amphipathic media (eg micelles, synthetic bilayers or cell membranes). Can be induced by. α
The -helix structure is well known in the art, and the ideal α-helix has 3.6 residues per turn and 1.5 Å translation per residue.
Tion) (5.4Å per turn; C
reighton, Proteins: Structure and Mole
Clarular Properties, W.W. H. Freeman, New Yor
k (1984)). In the amphipathic α-helix structure, polar and non-polar amino acid residues are aligned into the amphipathic helix, and when the peptide is observed along the helix axis, the helix is The hydrophobic amino acid residues are predominantly on one side and the hydrophilic residues are predominantly on the opposite side, α
It is a helix.

A widely varying sequence of antimicrobial peptides was isolated, which shared an amphipathic α-helix structure as a common feature (Saberwal et al., Biochim.
Biophys. Acta 1197: 109-131 (1994)). Analogs of native peptides containing amino acid substitutions that are expected to enhance amphipathicity and helicity typically have increased antibacterial activity. In general, analogs with increased antibacterial activity also have increased cytotoxicity on mammalian cells (Maloy et al., Biopolymers 37: 105-122 (19).
95)).

As used herein with reference to antimicrobial peptides, the term “amphipathic α-helix structure” refers to a hydrophilic surface containing some polar residues at physiological pH, .Alpha.-helix with a hydrophobic face containing non-polar residues. The polar residue can be, for example, a lysine residue or an arginine residue, while the non-polar residue can be, for example, a leucine residue or an alanine residue. An antimicrobial peptide having an amphipathic α-helix structure generally has the same number of polar and non-polar residues in its amphipathic domain and has an overall positive charge at neutral pH. Have a sufficient number of basic residues (Saberwall et al., Biochim).
． Biophys. Acta 1197: 109-131 (1994), incorporated herein by reference. Those skilled in the art will appreciate that helix-promoting amino acids (eg,
It is understood that leucine and alanine) may be advantageously included in the antimicrobial peptides of the invention (see, eg, Creighton, supra, 1984).

[0028]   Various antimicrobial peptides having an amphipathic α-helix structure are well known in the art.
Is. As such a peptide, each 7-group (heptad)
Based on a 7-tuple building block scheme, consisting of individual trimers containing residues
In addition, synthetic minimal peptides can be mentioned. Examples of such synthetic antimicrobial peptides include
For example, the general formula [(X₁X_TwoX_Two) (X₁X_TwoX_Two) X₁]_n(SEQ ID NO: 205)
Or [(X_lX_TwoX_Two) X₁(X₁X_TwoX_Two)]_n(SEQ ID NO: 206) (X₁ Is a polar residue, X_TwoIs a non-polar residue and n is 2 or 3
) Peptide (see Javadpour et al., Supra, 1996).
Be done. (KLAKLAK)_Two(SEQ ID NO: 200); (KLAKKLA)_Two(Array
No. 201); (KAAKKAA)_Two(SEQ ID NO: 202); (KLGKKLG) _Three (SEQ ID NO: 203) is a synthetic antibacterial peptide having an amphipathic α-helix structure.
It is an example of the code. Similar synthetic amphipathic antibacterial peptide with amphiphilic α-helix structure
Ptides are also known in the art, eg, McLaughlin and B
No. 5,789,542 to Ecker.

Spirality can be readily determined by one of ordinary skill in the art using, for example, circular dichroism spectroscopy. The percent α-helix can be determined after measuring the molar ellipticity at 222 nm, as described, for example, in Javadpour et al., supra, 1996 (also McLean et al., Biochemistry 30: 31-3).
7 (1991) (incorporated herein by reference)). The amphipathic α-helix antimicrobial peptide of the present invention is an amphipathic medium (eg, 25 mM).
When assayed in SDS), for example, it may have at least about 20% helicity. Such an antimicrobial peptide having an amphipathic α-helix structure is
When assayed in MSDS, for example, at least about 25%, 30%,
Those skilled in the art will understand that they can have a helicity of 35%, or 40%. Antimicrobial peptides having an α-helix structure are 25% to 90% helical; 25% to 60% helical; 25% to 50% helical; 25% to 40% when assayed in 25 mM SDS. 30% to 90% helical; 30% to 60% helical; 30% to 50% helical; 40% to 90% helical or 40% to 60% helical. You can The amphipathic property is, for example, the helical wheel of the peptide (
can be easily determined using a physical wheel display (eg,
See Blondelle and Houghten, supra, 1994).

CNGRC-G, an exemplary homing pro-apoptotic conjugate of the invention
The structure of G- _D (KLAKLAK) ₂ is shown in FIG. As can be observed in FIG.
Homing domain CNGRC (SEQ ID NO: 8) is a disulfide-bonded cyclic structure and is linked to membrane disruption domain _D (KLAKLAKKLAKLAK) via a glycinylglycine bridge. D-amino acids in the membrane-disrupting antimicrobial portion of this conjugate may be useful in conferring increased stability to the conjugate in vivo. Furthermore, the membrane-disrupted _D (KLAKLAKKLAKLAK) portion forms an amphipathic helix. In particular, lysine residues are aligned on one face of the helix (shown as the dark, shaded region of the helix), while nonpolar leucine and alanine residues are opposite to this helix (lighter Shaded)
Aligned on the surface.

Homing pro-apoptotic conjugates of the invention can be chimeric peptides, where the tumor homing molecule is a tumor homing peptide. Homing pro-apoptotic chimeric peptides of the invention can have various lengths from about 18 amino acids to about 50 amino acids or more. The chimeric peptide of the present invention may 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 include, for example, 40, 35, 30, 27, 25
Or it may have an upper limit of 21 amino acids. The chimeric peptide of the present invention is
It can be linear or cyclic. In a preferred embodiment, the homing pro-apoptotic chimeric peptides of the invention include cyclic tumor homing peptide moieties.

The homing pro-apoptotic chimeric peptides of the present invention can also be peptidomimetics. As used herein, the term “peptidomimetic” is used broadly to mean a peptidomimetic 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 for the peptides from which the peptidomimetic is derived (eg, "
Burger's Medicinal Chemistry and Dru
g Discovery, 5th Edition, Volumes 1 to 3 (ME Wolff, edited;
Wiley Interscience 1995), which is incorporated herein by reference). For example, the D amino acid may be advantageously included in the antimicrobial peptide portion of the chimeric peptides of the invention (see Example I and Example II). Peptidomimetics offer various advantages over peptides, including increased stability during transit through the gastrointestinal tract, and can therefore be advantageously used as oral therapeutic agents.

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

The vasculature within a tumor generally undergoes active angiogenesis, resulting in the continuous formation of new blood vessels that support the growing tumor. Such angiogenic blood vessels are characterized by an endothelial cell surface marker (α _v β ₃ integrin (Brooks) with a unique angiogenic vasculature.
, Cell 79: 1157-1164 (1994); WO 95/14714.
(International filing date November 22, 1994)) and receptors for angiogenic growth factors (Mustonen and Alitaro, J. Cell B).
iol. 129: 895-898 (1995); Lappi, Semin. Ca
ncer Biol. 6: 279-288 (1995)), which is distinguishable from the mature vasculature. Furthermore, the 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., Antic.
ancer Drugs 6: 3-18 (1995)). Thus, the unique features of 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 continued 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 tumors offers considerable advantages over systemic treatment methods or methods that target tumor cells directly. For example, tumor cells rely on the vascular supply for survival, and the endothelial lining of blood vessels is readily accessible to circulating probes. Conversely, in order to reach solid tumor cells, the therapeutic agent must be a dense fibrous stroma with potentially long diffusion distances, tightly packed tumor cells, and high interstitial pressure that prevents extravasation. (Burrows and Thorpe, Pharmacol. Ther. 64:
155-174 (1994)).

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

As described above and exemplified herein, a tumor homing molecule that is selective for vasculogenic viable endothelial cells of tumor blood vessels is a pro-apoptotic antimicrobial peptide that binds to normal, healthy organs or tissues. It may be particularly useful for directing pro-apoptotic antimicrobial peptides to tumor blood vessels, while remaining less likely to have toxic effects. Thus, in one embodiment, the invention provides a homing pro-apoptotic conjugate. The conjugate comprises a tumor homing molecule linked to an antimicrobial peptide that selectively homes to angiogenic endothelial cells, wherein the conjugate is selectively internalized by angiogenic endothelial cells. , Shows high toxicity to it, and the antimicrobial peptide is less toxic to mammalian cells when not linked to tumor homing molecules.

As used herein, the term “selective toxicity”
By “xicity)” is meant enhanced cell death in a selected cell type or tissue as compared to a control cell type or tissue. In general, selective toxicity is
Characterized by cell death of at least twice as much in a selected cell type or tissue (eg, angiogenic endothelial cells) as compared to a control cell type or tissue (eg, angiogenic endothelial cells) Attached. Thus, the term selective toxicity, as used herein, refers to a particular toxicity, whereby cell death essentially occurs only in the selected cell type or tissue, and the selected cell type or tissue. In addition, it includes toxicity that occurs in a limited number of cell types or tissues.
The person skilled in the art further understands that the term selective toxicity refers to cell death caused by all mechanisms including apoptotic cell death and necrotic cell death. Thus, homing pro-apoptotic conjugates of the present invention that exhibit selective toxicity to angiogenic endothelial cells may be compared to vascularized angiostatic endothelial cells or other types of surrounding cells. It results in increased cell death of the forming endothelial cells.

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

Tumor homing molecules useful in the homing pro-apoptotic conjugates of the invention include, for example, NGR motifs (eg, CNGRC (SEQ ID NO: 8); NGR).
It can be a peptide containing AHA (SEQ ID NO: 6), or CNGRCVSGCAGRC (SEQ ID NO: 3)). Tumor homing molecules useful in the present invention may also include an RGD motif, and include, for example, CDCRGDCFC (SEQ ID NO: 1).
Or may include a GSL motif (eg, peptide CGSLVRC (SEQ ID NO: 5)). Additional tumor homing molecules can be identified by screening a library of molecules by in vivo panning as described in more detail below (see also, Examples IV-VIII; 19).
US Patent No. 5,622,699, published April 22, 1997; and Pasqua
lini and Ruoslahti, Nature 380: 364-366 (
1996) (each of which is incorporated herein by reference)).

The term “tumor homing molecule” as used herein is an organic compound such as a drug; a nucleic acid molecule; a peptide or peptidomimetic or protein (selective in vivo against a selected cell type or tissue). To home). "Selectively home" means that, in vivo, the tumor homing molecule binds preferentially to a selected cell type or tissue as compared to a control cell type, tissue or organ, and It is meant to be generally characterized by at least a 2-fold greater localization in the selected cell type or tissue as compared to the 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 angiogenic blood vessels as compared to other cell types or angiogenic blood vessels.

Tumor homing molecules were identified using in vivo panning as follows. In vivo panning 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). Large phage size (900-1000 nm) and short time for phage to circulate (3-
It is unlikely that a significant number of phages have left the circulatory system (especially brain and kidney) due to (5 min). Tissue staining studies have shown that the identified tumor homing molecules home and bind primarily to endothelial cell surface markers, which 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 their tissue-specific differences (P
auli and Lee, Lab. Invest. 58: 379-387 (198)
8)).

Although the conditions under which in vivo panning was performed identified tumor-homing peptides that generally bind to endothelial cell markers, the specific presence of phage expressing tumor-homing peptides also revealed that in tumor parenchyma, In particular, it was observed at a later time point after administration of the peptide (Example VII). These results demonstrate that peptide-expressing phage can cross blood vessels in tumors, presumably due to the fenestrated nature of blood vessels, and in vivo panning methods target tumor cell-expressed targets. We show that it can be useful for identifying molecules and target molecules expressed by endothelial cells.

[0045] A peptide display library of phage was constructed essentially by Smith and Scott (supra, 1993; see also: Koivunen et al., B.
iotechnology 13: 265-270 (1995); Koivun
en et al. , Meth. Enzymol. 245: 346-369 (1994b)
(Each incorporated herein by reference)).
Oligonucleotides encoding peptides having a substantially random amino acid sequence were synthesized based on the "NNK" codon. Here, “N” means A, T,
C or G, and "K" is G or T. "NNK" is 20
Encodes 32 triplets, encoding the amino acids of and the amber stop codon (Scott and Smith, supra, 1990). In some libraries, at least one codon encoding cysteine may also be included in each oligonucleotide, resulting in disulfide bond formation of a cyclic peptide (Example IV). This oligonucleotide was inserted in frame with the sequence encoding gene III protein (gIII) into vector fuse5, thereby expressing the peptide-gIII fusion protein. After expression,
The fusion protein was expressed on the surface of phage containing this vector (Koivu
nen et al., supra, 1994b; Smith and Scott, supra, 1993).
.

After in vivo panning, phage isolated based on their ability to selectively home to human breast cancer, mouse melanoma, or human Kaposi's sarcoma had only a few peptide sequence differences (Table 1 and Table 2, respectively). See 2, 3, and 4). The peptide sequence represented by one of the screens, in the case of peptides previously demonstrated to selectively bind to α _v containing integrins (Koivunen et al., Supra, 1995; WO 95/14714), arginine- Glycine-aspartic acid (RGD) integrin recognition sequence (Ruosla
hti, Ann. Rev. Cell. Devel. Biol. 12: 697 (1
996)). Most of the remaining tumor homing peptide sequences
It showed no significant similarity to known ligands for endothelial cell receptors.
However, one of the tumor homing molecules is asparagine-glycine-arginine (
NGR) motif was included. This motif is a weak integrin-binding motif similar to that present in integrin-binding peptides (Ruoslaht).
i et al., U.S. Patent No. 5,536,814 (published July 16, 1996), which is incorporated herein by reference; also see Koivunen et al., supra, 1994a. . Other screens showed many NGR-containing peptides (see Table 2). Despite the weak integrin binding capacity of the NGR peptide, the integrin receptor need not be the target molecule recognized by the NGR tumor homing peptides exemplified herein.
As used herein, the term "integrin" means a heterodimeric cell surface adhesion receptor.

Peptides expressed by phage homed to breast tumors are the peptides CGRECPRLCQSSC (SEQ ID NO: 2) and the peptide CNGRCVSG.
CAGRC (SEQ ID NO: 3; Table 2; see Example V) was included. Similarly, tumor homing peptides, including peptide CDCRGDCFC (SEQ ID NO: 1) and peptide 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, were also isolated by screening against mouse melanoma and human Kaposi's sarcoma (see Tables 3 and 4).
These results demonstrated that tumor homing molecules can be identified using in vivo panning.

The three major tumor homing motifs identified may be particularly useful in homing pro-apoptotic conjugates of the invention. Homing pro-apoptotic conjugates in which a portion of the tumor homing molecule contains an NGR motif, RGD motif, or GSL motif can be used to target antimicrobial peptides bound to endothelial cells of angiogenic blood vessels.

In one embodiment, the present invention provides a homing pro-apoptotic conjugate. This conjugate comprises a tumor homing peptide containing the sequence NGR linked to an antimicrobial peptide. In such homing pro-apoptotic conjugates of the invention, the tumor homing peptide is, for example, CNGRC (SEQ ID NO: 8); NG.
It can be RAHA (SEQ ID NO: 6) or CNGRCVSGCAGRC (SEQ ID NO: 3). In a preferred embodiment, the homing pro-apoptotic conjugate is
It comprises the sequence CNGRC-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 ACDCRGD.
CFC-GG- _D (KLAKLAK) ₂ is included.

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 known to selectively bind to α _v integrin (Koivunen et al., Supra, 1995; WO 95/1.
4714), which contained the sequence RGD embedded in the peptide structure CDCRGDCFC (SEQ ID NO: 1) (Ruoslahti, supra, 1996). The α _v β ₃ and α _v β ₅ integrins are markers for angiogenic blood vessels (Brooks et al., supra, 1994; Friedlander et al., Science 270: 1500 (
1995)), so phage expressing the peptide CDCRGDCFC (SEQ ID NO: 1) were tested for tumor targets and homed to tumors in a very selective manner (Example VI, as disclosed herein). See). Furthermore, CDC
Homing by RGDCFC (SEQ ID NO: 1) phage was performed with free CDCRGD
It was inhibited by co-administration of CFC (SEQ ID NO: 1) peptide.

Another breast tumor homing peptide had the sequence CNGRCVSGCAGRC (SEQ ID NO: 3). This sequence contains the NGR motif previously shown to have weak integrin binding activity (Koivunen et al., J. Biol.
Chem. 268: 20205-20210 (1993); Koivunen et al. , Supra, 1994a; WO 95/14714). Since NGR-containing peptides were identified, two additional peptides, the linear peptide NGRAHA (SEQ ID NO: 6) and the cyclic peptide CVLNGRMEC (SEQ ID NO: 7), each containing an NRG motif, were tested for tumor homing. did. Similarly, CN
Phage expressing GRCVSGCAGRC (SEQ ID NO: 3), NGRAHA (SEQ ID NO: 6) or CVLNGRMEC (SEQ ID NO: 7) homed to tumors. Moreover, since tumor homing is tumor type or species independent, this phage selectively accumulated in human breast cancer, as well as in tumors of mice bearing mouse melanoma and human Kaposi's sarcoma xenografts.

Various peptides, including RGD-containing peptides and NGR-containing peptides,
Generally bound to tumor vessels. The minimal 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 was labeled with CNGRCVSGCAGRC (
Co-injection with phage expressing either SEQ ID NO: 3), NGRAHA (SEQ ID NO: 6) or CVLNGRMEC (SEQ ID NO: 7) suppressed accumulation of this phage in breast cancer xenografts. However, even when administered up to 10 times higher than the peptide that suppresses homing of NGR phage, CNG
The RC (SEQ ID NO: 8) peptide did not suppress homing of phage expressing the CDCRGDCFC (SEQ ID NO: 1) peptide. Relative to CDCRGDCF
The C (SEQ ID NO: 1) peptide partially suppressed homing of NGR phages, but required a high amount up to 5 to 10 times higher than that of the CNGRC peptide (SEQ ID NO: 8). These results indicate that NGR and RGD peptides bind to different receptor sites in the tumor vasculature.

A third motif (GSL (glycine-serine-leucine)) was also identified in mouse-bearing breast cancer, malignant melanoma, or Kaposi's sarcoma by an in vivo panning method using: Homing of phage expressing the GSL peptide (CGSLVRC (SEQ ID NO: 5)) was inhibited by co-injection of free CGSLVRC (SEQ ID NO: 5) peptide. Like the RGD and NGR peptides,
Phage expressing the GSL peptide also bound to tumor blood vessels. Given the identification of conserved RGD, NGR and GSL motifs present in tumor homing peptides, peptides containing such motifs may be useful as tumor homing peptides, as disclosed herein. It is recognized that, in particular, it may be useful for forming homing pro-apoptotic peptides that can selectively deliver antimicrobial peptides to tumors.

Various peptide libraries containing up to 13 amino acids were constructed and N
The GR peptide (CNGRCVSGCAGRC (SEQ ID NO: 3)) was obtained as a result of an in vivo panning procedure on breast tumors. The NGR peptide, which was obtained by screening a random peptide library,
It was a tumor homing peptide. In addition, the peptide library was labeled with the formula CXX.
When constructed on the basis of XNGRXX (SEQ ID NO: 13) or CXXCNGRCX (SEQ ID NO: 14), each of which is biased to NGR sequences, and when breast tumors were used for in vivo panning methods, a large number of NGR peptides were obtained. (Table 2
checking).

These results indicate that the tumor homing molecule of the invention may comprise 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))
Can be. Such tumor homing peptides can also be at least 13 amino acids in length, which is the largest peptide exemplified herein, as well as, if desired, 23 amino acids or less in length. , Or 30 amino acids or less, or 50-100 amino acids in length. Conveniently, the tumor homing peptides of the invention are produced by chemical synthesis.

Immunohistochemical analysis was performed by circulating tissue for approximately 4 minutes and comparing tissue staining for phage perfused through the heart of mice or by using tissue analyzed 24 hours after injection of phage. Twenty-four hours after administration, the phage are essentially free of circulation and do not require perfusion (Pasqualini et al.,
Ibid, 1997). In the samples tested 4 minutes after administration of the CNGRCVSGCAGRC (SEQ ID NO: 3) phage, intense phage staining was observed in tumor vessels except normal endothelium (Example VII). In comparison, tumor staining is
It was strong at 24 hours and appeared to extend outside the blood vessels in the parenchyma of the tumor. The NGRAHA (SEQ ID NO: 6) and CVLNGRMEC (SEQ ID NO: 7) phages showed similar staining patterns (Example VII). On the other hand, control organs and tissues showed little or no immunostaining, confirming the specificity of the NGR motif for tumor vessels. As expected, the spleen and liver captured the phage, but their uptake by the reticuloendothelial system was unrelated to the presence of peptide expression by this phage, due to the general nature of phage particles (Pa
squalini et al., supra, 1997).

After administration of the phage expressing the GSL motif containing the peptide (CLSGSLSC (SEQ ID NO: 4)), immunostaining was also observed, which staining was similar to that of the NGR peptide in blood vessels (in this case, in melanoma tumors). ) (See below; see also Example VII and Example VIII). Similarly, peptides (CDC
Immunostaining for breast tumors carried by mice following administration of phage expressing RGD motifs, including RGDCFC (SEQ ID NO: 1), was localized to blood vessels in the tumor but in brain, kidney or various other non-tumor Not observed in tissues (see also Example VI and Example VII; also Pasqualini et al., Supra, 1997). These results demonstrate that various tumor homing peptides generally home to the tumor vasculature.

A general application of the in vivo panning method to identify tumor homing molecules was tested by injecting mice expressing syngeneic melanoma with phages expressing different populations of peptides. Example VIII). The B16 mouse melanoma model was selected for these studies. Because the tumor is a highly angiogenic form and the biology of this tumor line has been fully characterized (Miner et al., Cancer Res. 42: 4631-4638 (1).
982)). Furthermore, due to the mouse origin of B16 melanoma cells, species differences between host and tumor cell donors may be different, for example, in the distribution of phage in tumors compared to the distribution of phage in normal organs. Does not affect As disclosed herein, in vivo panning methods on B16 melanoma cells reveal tumor-homing peptides (eg, containing a GSL portion that includes the peptide CLSGSLSC (SEQ ID NO: 4; see also Table 3)). And immunohistochemical staining of tumors and other organs with anti-phage antibodies showed that CLSGSLSC (SEQ ID NO: 4) expressing phage showed essentially no staining in skin, kidney or other control organs. It was demonstrated to show immunostaining in melanoma (Example VIII). This staining pattern generally tracks blood vessels in melanoma, but is not strictly limited to blood vessels.

Although the in vivo panning method was performed in mice, it appears that tumor homing molecules (eg, peptides containing NGR, RGD or GSL motifs) can also target human vasculature. NGR phage do not bind to blood vessels in normal tissue but to blood vessels in transplanted human breast tumors. This indicates that this motif may be particularly useful for targeting tumors in patients. CDCRG
The DCFC (SEQ ID NO: 1) peptide is human α _V -integrin (Koivune).
n et al., supra, 1995), this peptide is selectively expressed in tumor blood vessels of human patients (Max et al., Int. J. Cancer 71: 320 (19).
97); Max et al., Int. J. Cancer 72: 706 (1997)).
The use of homing pro-apoptotic conjugates containing CDCRGDCFC (SEQ ID NO: 1) that binds antimicrobial peptides has been demonstrated, for example, by breast cancer cells capable of expressing integrin α _V β ₃ (Pasqualini et al., Supra, 1997). Due to
It provides the additional advantage that the antimicrobial peptide can target the tumor cells themselves. In fact, many human tumors express this integrin, which may be associated with the progression of certain tumors (eg malignant melanoma) (Albelda et al., Ca.
ncer Res. 50: 6577-1676 (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. Ouant. Biol. 58: 233-240 (1992); Mitj.
ans et al. Cell. Sci. 108: 3067-3078 (1995)).
. Unlike the CDCRGDCFC (SEQ ID NO: 1) peptide, the NGR peptide is
It does not appear to bind to MDA-MD-435 breast tumor cells. However, the NGR peptide was able to deliver a therapeutically effective amount of doxorubicin to breast tumors. This means that even if the tumor homing molecule homes only to the tumor vasculature (ie, not directly to the tumor cells), targeting such vasculature is not linked to the moiety linked to the molecule. It is not enough to give the effect of.

Since α _v β ₃ integrin is expressed by endothelial cells in the angiogenic vasculature, experiments were performed to disclose the vasculature of angiogenic tumors. Such methods were used to determine if they could be targeted in vivo. Peptides known to bind to α _V β ₃ integrin (CDCRG
DCFC (see SEQ ID NO: 1; Koivunen et al., Supra, 1995)
) Was injected into mice bearing tumors formed from human breast cancer cells, mouse melanoma cells or human Kaposi's sarcoma cells (Example VII).
The CDCRGDCFC (SEQ ID NO: 1) phage selectively homed to their tumors, respectively, whereas such homing did not occur with control phage. For example, in a mouse bearing a tumor formed by transplantation of human breast cancer cells, 20 to 80 times or more CDCRGDCFC (SEQ ID NO: 1) phage accumulated in the tumor as compared with the non-selective control phage.

Tissue staining for phage showed 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 CDCRGDCFC (SEQ ID NO: 1) phage was demonstrated by co-injection of free CDCRGDCFC (SEQ ID NO: 1) peptide, whereas co-injection of non-RGD containing control peptide decreased tumor homing of RGD phage. It was demonstrated by competition experiments, which did not affect homing of RGD phage (see 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 can selectively bind to this integrin, resulting in vascular Demonstrate that it can bind to the neovasculature.

The results of these studies indicate that tumor homing molecules can be identified by in vivo panning methods and, in some cases, probably due to the fenestration of blood vessels that allows for rapid clearance of phage from the circulatory system. Indicate that tumor homing molecules can home to vascular tissue in tumors and parenchyma of tumors. Due to the ability of such tumor homing molecules to home to tumors, these molecules are
It is useful for targeting tumor bound antimicrobial peptides. Therefore, the present invention provides
Providing a conjugate comprising a tumor homing molecule attached to a moiety, such moiety comprising:
Useful for targeting individual parts to tumor cells.

The ability of the molecule to home to a particular tumor that selectively homes to another tumor of the same or similar histological type was determined for these experiments, eg, human tumors in nude mice. It can be determined using growth or mouse tumor growth in syngeneic mice. For example, various human breast cancer cell lines (including DA-MB-435 breast cancer (including Price et al., Cancer Res. 50: 717-721 (1990)), SKBR-1-II and SK-BR-3 (Fohh et al. , J. Natl
． Cancer Inst. 59: 221-226 (1975)), as well as a mouse mammary tumor line (EMT6 (Rosen et al., Int. J. Cancer 57:
706-714 (1994)) and C3-L5 (Lala and Par).
har, Int. J. Cancer 54: 677-684 (1993))
It is readily available and is commonly used as a model for human breast tumors. Such breast tumor models can be used, for example, to obtain information about the specificity of breast tumor homing molecules identified for various breast tumors, and to home to a wide variety of breast tumors or at the most convenient location. Molecules that provide a specificity profile can be identified. Moreover, such analysis may yield new information (eg, about tumor stroma). Because stromal cell gene expression (
Endothelial cells) can be modified by this tumor so that they cannot replicate in vitro.

Selective homing of molecules (eg peptides or proteins) to tumors is due to specific recognition by peptides of specific cell targeting molecules (eg cell surface receptors present on cells in the tumor). obtain. Homing selectivity is 1
Depends on specific target molecules expressed on only one or on several different cell types,
As a result, this molecule primarily homes to tumors. As discussed above,
The identified tumor homing peptides can, at least in part, recognize endothelial cell surface markers in blood vessels present in the tumor. However, most cell types, especially particular cell types that are unique to an organ or tissue, can express unique target molecules. Therefore, in vivo panning can be used to identify molecules that selectively home to specific types of tumor cells (eg, breast cancer cells); specific homing is demonstrated by performing appropriate competition experiments. obtain.

As used herein, the term “tumor” means a mass of cells characterized, at least in part, by including angiogenic vasculature. the term"
"Tumor" is used broadly 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 term “normal” tissue or “non-tumor” tissue is used to refer to tissue that is not “tumor”. As disclosed herein, tumor homing molecules can be identified based on their ability to home to tumor but not to corresponding non-tumor tissue.

As used herein, the term “corresponding” when used in reference to tumors or tissues or both, includes two or more tumors, or two or more tissues, or tissues and It means that the tumors are of the same histological type.
Those skilled in the art will appreciate that the histological type of a tissue is a function of cells containing the tissue.
n) is recognized. Thus, for example, non-tumor tissue corresponding to a breast tumor is normal breast tissue, while non-tumor tissue corresponding to melanoma is skin (
It contains melanocytes). Further, for the purposes of the present invention, a tumor homing molecule may 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 is recognized that the tissue corresponding to the tumor includes non-tumor tissue containing blood vessels that do not undergo active angiogenesis.

Tumor homing molecules useful in the present invention can be identified by screening a library of molecules by in vivo panning as disclosed herein, and US Pat. No. 5,622,699 ( (Published April 22, 1997); and Pasqualini and Ruoslahti, Nature.
e 380: 364-366 (1996), each of which is incorporated herein by reference. As used herein, the term "library" means a collection of molecules. A library may contain several or many different molecules, varying from about 10 molecules to billions or more of molecules.
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” includes polymeric or non-polymeric organic chemicals (eg, drugs); nucleic acid molecules (eg, RNA, cDNA or oligonucleotides); peptides ( A variant or modified peptide or peptide-like molecule, referred to herein as a peptidomimetic, which mimics the activity of the peptide); or a protein (eg, an antibody or growth factor receptor or their 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 broadly 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 in vitro methods, or a naturally occurring molecule (eg, a protein expressed from a cDNA library). Or a fragment thereof).

Tumor homing molecules can also be peptidomimetics. As used herein, the term "peptidomimetic" is used broadly to mean a peptidomimetic molecule having the binding activity of a tumor homing peptide. With respect to the tumor homing peptides of the present invention, peptidomimetics, which include chemically modified peptides,
Peptide-like molecules containing non-naturally occurring amino acids, including peptoids)
It has the binding activity of the tumor-homing peptide from which this peptidomimetic is derived (eg, "Burger's Medicinal Chemistry and
Drug Discovery ", supra, 1995).

Methods for identifying peptidomimetics are well known in the art and include, for example, screening databases containing libraries 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)). A depository for this structure is that a new crystal structure has been determined and appropriate shape (eg, the same shape as the tumor-homing molecule, as well as potential geometric and chemical for the target molecule bound by the tumor-homing peptide). Frequently updated if compounds with (complementarity) can be screened. If the crystal structure of the tumor-homing peptide, or target molecule that binds the tumor-homing molecule, is not available, the structure is, for example,
Program CONCORD (Rusinko et al., J. Chem. Inf. Com
put. Sci. 29: 251 (1989)). Another Database, Available Chemicals Director
y (Molecular Design Limited, Informationi)
ons Systems; San Leandro CA) contains approximately 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 different types of molecules (eg, peptides, peptoids and peptidomimetics) are well known in the art, and various libraries are commercially available. (Eg, Ecker and Crooke, Biotechnology 13: 351-360 (199
5), and Blondelle et al., Trends Anal. Chem. 14
: 83-92 (1995), as well as the references cited therein, each of which is incorporated herein by reference; also Goodma.
n and Ro, Peptidomimetics for Drug Desi
gn, “Burger's Medicinal Chemistry and
"Drug Discovery", Volume 1 (ME Wolff ed .; 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). If the molecule is a peptide, protein or fragment thereof, the molecule can be produced directly in vitro or can be expressed from a nucleic acid, which can be produced in vitro. Methods of synthetic peptide and nucleic acid chemistry 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 this 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), where upon expression the encoded peptide is expressed as a fusion protein on the surface of the phage.

In addition, 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); Turk 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 having a tumor, and subsequently tumor homing molecules can be identified by in vivo panning. If desired, the nucleic acid molecule can be a nucleic acid analog, eg, less susceptible to attack by a nuclease (eg, J.
Elinek et al., Biochemistry 34: 11363-11372 (
1995); Ratham 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 used to identify tumor homing molecules. This tumor homing molecule can be used to link antimicrobial peptides to form the homing pro-apoptotic conjugates of the invention. In vivo panning involves administering a library to a subject, collecting a sample of tumor, and identifying tumor homing molecules. The presence of tumor homing molecules can be identified using various methods well known in the art. Typically,
The presence of tumor homing molecules in tumors is identified based on one or more properties in common with 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 can be used to identify tumor homing molecules in tumors, either alone or in combination with methods such as gas chromatography. Thus, if the library contains diverse 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 the particular molecule.

If desired, tumors can be harvested and then processed using methods such as HPLC. Such methods provide, for example, a fraction enriched in molecules having a defined range of molecular weights or polar or non-polar properties, etc., depending on the general properties of the molecule containing the library. obtain. 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, eg, using selective extraction methods. As well as methods for achieving this, are well known in the art. The library comprises a variety of organic chemical molecules, each of which is linked to a specific oligonucleotide tag such that the specific molecule is identified by using the polymerase chain reaction (PCR) to determine the oligonucleotide sequence. Genomic DNA,
It can be removed from collected tumor samples to reduce the potential for background PCR reactions. In addition, the library can include a diverse population of molecules, such as organic chemical molecules, each linked to a common covalent tag. Based on the presence and properties of the shared tag, the molecules of the library that selectively home to tumors can be substantially isolated from the tumor sample. These and other methods
It may be useful for enriching a collected sample of tumors for a particular tumor homing molecule, thereby removing potential contaminants from the collected sample and increasing the sensitivity of detection of the molecule .

The evidence provided herein shows that a sufficient number of tumor homing molecules home to tumors during in vivo panning so that the molecules can be readily 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). It was

A substantial fraction of the identified tumor homing molecules had the same structure, but only a few peptide inserts of the isolated phage were determined. However, it should be appreciated that hundreds of thousands to millions of phage expressing organ homing peptides were recovered after in vivo panning for organ homing molecules (eg, US Pat. No. 5,622,699). Issue; Pasqualini and Ruosla
hti, 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 molecule can be identified.

The ease of identifying tumor homing molecules, particularly those that are not tagged, depends on a variety of factors, including the presence of potentially contaminating background cellular material.
Therefore, if the tumor homing molecule is an untagged peptide, to identify a particular peptide against the background of cellular proteins,
The greater number must home to the tumor. In contrast, such molecules are normally absent in the body or only in small numbers in the body, so that a very small number of untagged organic chemical homing molecules such as drugs are identifiable. . In such cases, sensitive methods such as mass spectrometry can be used to identify tumor homing molecules. Those skilled in the art will recognize that the method of identifying a molecule depends, in part, on the chemistry of the particular molecule.

When the tumor homing molecule is or is tagged with a nucleic acid molecule,
In principle, PCR can detect the presence of a single nucleic acid molecule, so assays such as PCR can be particularly useful for identifying the presence of a molecule (eg, Erl).
ich, PCR Technology: Principles and Ap
applications for DNA Amplification (Sto
Cckton Press 1989), which is incorporated herein by reference). Preliminary studies have shown that after intravenous injection of 10 ng of approximately 6000 bp plasmid into mice and 2 minutes in circulation, the plasmid was blocked with P in lung samples.
It was proved to be detectable by CR. These results indicate that nucleic acid molecules are sufficiently stable when administered in circulation so that in vivo panning can be used to identify nucleic acid molecules that selectively home to tumors.

The molecules of the library can be tagged, which can facilitate recovery or identification of the molecules. As used herein, the term “tag” refers to a physical, chemical, or biological molecule, such as a plastic microbead, oligonucleotide, or bacteriophage, that is linked to a molecule of the library. Means a part. Methods of tagging molecules are well known in the art (Hermanson, Bi.
oconjugate Technologies (Academic Press)
1996), which is incorporated herein by reference).

Tags that may be shared or specific tags may be useful for identifying the presence or structure of tumor homing molecules in the library. As used herein, the term "shared tag" means the physical, chemical, or biological moiety that is common to each molecule in the library. For example, biotin can be a shared tag linked to each molecule in the library. The shared tag may be useful for identifying the presence of the molecule of the library in the sample, and may also be useful for substantially isolating the molecule from the sample.
For example, if the shared tag is biotin, the biotin-tagged molecule in the library can be substantially isolated by binding to streptavidin, or its presence can be associated with labeled streptavidin. Can be identified by the binding of When the library is a phage display library, each peptide in the library is linked to a phage, so phage expressing the peptide are another example of a shared tag. In addition, a peptide such as a hemagglutinin antigen can be a shared tag linked to each molecule in the library, thereby substantially isolating the molecule of the library from a selected tumor sample. To enable the use of antibodies specific for the hemagglutinin antigen.

A shared tag is also a nucleic acid sequence that may be useful for identifying the presence of a molecule of the library in a sample, or for substantially isolating a molecule of the library from a sample. obtain. For example, each of the molecules of the library can be linked to the same selected nucleotide sequence that constitutes its shared tag.
An affinity column containing a nucleotide sequence complementary to the shared tag can then be used to hybridize molecules of the library containing the shared tag, thus substantially removing the molecule from the tumor sample. Let go. Nucleotide sequences that are complementary to a portion of the shared nucleotide sequence tag also have a P sequence such that the presence of the molecule containing the shared tag can be identified in the sample by PCR.
It can be used as a CR primer.

The tag can also be a specific tag. The term “specific tag” as used herein refers to a physical, chemical, or biological molecule that is linked to and is unique to a particular molecule in a library. Means a tag. Specific tags are particularly useful if they are easily identifiable. Nucleotide sequences that are unique to a particular molecule in the library are examples of specific tags. For example, a method of synthesizing peptides tagged with unique nucleotide sequences provides a library of molecules each containing a specific tag such that the identity of the peptides is known in determining each nucleotide sequence. Yes (Brenner and Lerne
r, Proc. Natl. Acad. Sci. , USA 89: 5381-53.
83 (1992), which is incorporated herein by reference). Highly sensitive methods such as PCR can be used to determine the nucleotide sequence of a specific tag and identify the sequence of the molecule linked thereby, so that it is specific for peptides or other types of molecules. The use of nucleotide sequences as unique tags provides a simple means to identify the presence of molecules in a sample. Similarly, nucleic acid sequences encoding peptides expressed in phage are another example of specific tags, as sequencing of the specific tag identifies the amino acid sequence of the expressed peptide.

The presence of shared or specific tags may provide a means for identifying or recovering tumor-homing molecules of the invention after in vivo panning. Furthermore, the combination of shared tags and specific tags may 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, Brenne.
r and Lerner, supra, 1992), where each specific nucleotide sequence tag has incorporated therein a shared tag such as biotin. Upon tumor homing, specific tumor homing peptides can be substantially isolated from a sample of tumor based on a shared tag, and specific peptides can be identified by, for example, PCR of specific tags. (See Erlich, supra, 1989).

The tag may also serve as a support. As used herein, the term "
By "support" is meant a tag having a defined surface to which molecules can be attached. Generally, tags that are useful as supports are shared tags. For example, the support may be a virus or virus-like particle (“phage”) such as a bacteriophage;
Bacteria such as E. coli; or biological tags such as eukaryotic cells such as yeast, insect, or mammalian cells; or physical tags such as liposomes or microbeads, Can be composed of plastic, agarose, gelatin, or other biological or inert substances. If desired, a shared tag useful as a support can have a specific tag attached thereto. Thus, the phage display library used in the exemplified method comprises a phage (which is a shared tag that is also a support), and a nucleic acid sequence encoding the expressed peptide (which nucleic acid sequence is a specific tag). Can be considered to consist of.

In general, the support is about 10 μm in its shortest dimension so that the support can pass relatively unimpeded through the capillary beds present in the subject and may not block the circulation. It should have a diameter of less than about 50 μm. Moreover, the support may be non-toxic, so that it does not perturb the normal expression of cell surface molecules or the normal physiological state of the subject, and in particular the subject used for in vivo panning collects selected tumors. It is biodegradable if not sacrificed.

When the molecule is linked to a support, the tagged molecule comprises the molecule attached to the surface of the support, so that it is suspected that it may interact with the target molecule in the cell in the subject. A portion of a molecule is arranged so that it can participate in the interaction. For example, if a tumor homing molecule is suspected to be a ligand for a growth factor receptor, the binding portion of the molecule attached to the support is positioned so that it can interact with the growth factor receptor on cells in the tumor. If desired, a suitable spacer molecule can be placed between the molecule and the support so that the potential tumor homing molecule's ability to interact with the target molecule is not hindered. The spacer molecule can also include a reactive group, which provides a convenient and efficient means of linking the molecule to the support, and can, if desired, include a tag that can facilitate recovery or identification of the molecule. (Her
Manson, supra, 1996).

As exemplified herein, a peptide suspected of being able to home to a selected tumor such as breast cancer or melanoma is expressed as the N-terminus of the fusion protein, where the C-terminus is the phage. Made of coat protein. Upon expression of the fusion protein, the C-terminal coat protein linked the fusion protein to the surface of the phage such that the N-terminal peptide was in a position in the tumor to interact with the target molecule. Thus, a molecule with a shared tag is formed by the ligation of a peptide to a phage, where the phage provides the biological support and the peptide molecule is ligated as a fusion protein, the phage coding portion of the fusion protein. Acts as a spacer molecule, and the nucleic acid encoding the peptide provided a specific tag that allowed the identification of tumor homing peptides.

As used herein, the term “in vivo panning”, as used with respect to identifying tumor homing molecules, refers to the step of administering a library to a subject, and selecting for tumors in the subject. It refers to a method of screening a library by identifying molecules that home positively (US Pat. No. 5,6.
22, 699). The term "administering to a subject" when used in reference to a library of molecules or a portion of such a library refers to a subject in which the library is a vertebrate in general, and in particular mammals such as humans. It is used in its broadest sense to mean delivered to the tumor in the body.

The library can be administered to a subject by, for example, injecting the library into the circulation of the subject so that the molecule penetrates the tumor; after an appropriate time, the circulation will kill the subject. Or by removing a sample of the tumor (Example IV; US Pat. No. 5,622,699; Pasqual).
See also ini and Ruoslahti, supra, 1996). Alternatively,
The cannula may be inserted into the blood vessel of the subject such that the library is administered by perfusion for an appropriate time and then the library may be removed from the circulation through the cannula to terminate the circulation or The body can be sacrificed to collect the tumor, or the tumor can be sampled. Similarly, the library contains tumors by cannulation of appropriate blood vessels in the subject.
It can be shunted through one or several organs. It is recognized that the library can also be administered to isolated perfused tumors. Such panning in isolated perfused tumors can be useful to identify molecules that bind to the tumor and, if desired, can be used as an initial screen of the library.

Use of In Vivo Panning to Identify Tumor-Homing Molecules Screens Phage Peptide Display Libraries in Tumor-Bearing Mice and Identifies Specific Peptides that Selectively Home to Breast or Melanoma Tumors By way of example (Example IV). However, protein receptor molecules (including, for example, antibodies or antigen-binding fragments of antibodies (eg, Fv, Fd, or Fab fragments)); hormone receptors (
Phage libraries displaying, for example, growth factor receptors); or cell adhesion receptors such as integrins or selectins can also be used for the practice of the invention. Variants of such molecules are well known in the art (eg, random mutagenesis, site-directed mutagenesis, or codon-based mutagenesis (Hu
Se, 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 prior to administration to the subject. Thus, various types of phage display libraries can be screened by in vivo panning.

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

Similarly, Smith and Scott (Meth. Emzymol. 217:
228-257 (1993); also Scott and Smith, Scien.
Ce 249: 386-390 (1990, each of which is hereby incorporated by reference), for methods of generating a phage peptide display library containing the vector and expressed. Methods for diversifying populations of peptides have also been described (see also Huse, WO91 / 07141 and WO91 / 07149, which are incorporated herein by reference); and Example IV. checking). Phage display technology is particularly powerful when used with, for example, 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 methods or other well known methods can be used to generate phage display libraries. The phage display library can be subjected to in vivo panning to identify tumor homing molecules useful in the homing proapoptotic conjugates of the invention.

In addition to screening phage display libraries, in vivo panning can be used to screen a variety of 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 molecules from tumors or identification of molecules in tumors.
For example, when a library of organic molecules, each containing a shared tag, is screened, the tag can be a biotin-like moiety. This moiety can be directly attached to the molecule or linked to a support containing the molecule. Biotin provides a covalent tag that is useful for recovering molecules from selected tumor samples using avidin or streptavidin affinity matrices. Additionally, 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 anti-p linked to magnetic beads as a means of recovering molecules.
It can be bound by hOx antibodies. 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.
lla CA; and Promega Corp. , Madison WI).
When a phage library is screened, this phage is used in Example I.
It can be recovered using methods such as those disclosed in V.

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 homing" means that a particular molecule binds to a target molecule present in a tumor relatively specifically after administration to a subject. Means to do. In general, tumor homing molecules are characterized, in part, by showing at least 2-fold (2x) greater specific binding to tumors as compared to control organs or tissues.

It should be appreciated that in some cases, the molecule may be non-specifically localized to organs or tissues, including tumors. For example, in vivo panning of phage display libraries can result in high background in organs containing prominent 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 the molecule due to uptake by RES can 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 phage 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 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 a tumor homing peptide is low enough not to saturate the target molecule, then more than about 20% of the phage in the tumor will be a putative tumor. The determination to express a homing molecule is the conclusive evidence that the peptide expressed by the phage is a specific tumor homing molecule. Furthermore, non-specific localization can be distinguished from selective homing, for example, by performing competition experiments with phage that express the putative tumor homing peptide in combination with an excess of "free" peptide. Example VII).

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

Non-specific binding of phage to RES or other sites can also be prevented, for example, by co-injecting a specific phage display library with the same phage that has been rendered non-infective ( Smith et al., Supra, 1990, 1.
993). Moreover, tumor-homing peptides in organs containing RES components can be identified by preparing phage display libraries with phage that show 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).
Were not taken up by RES, and consequently selected λ-type phages that remained in circulation for long periods of time. For example, filamentous phage variants can be selected using similar methods.

Selective homing of tumor homing molecules can be demonstrated by determining the specificity of tumor homing molecules for tumors relative to control organs or tissues. Selective homing can also be demonstrated by showing that molecules that home to tumors as identified by one round of in vivo panning are enriched for tumor homing molecules in subsequent rounds 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, the phage recovered from the tumor showed a 3-fold enrichment in tumor homing compared to the brain. Phage recovered from tumors after the third round of screening showed an average of 10-fold enrichment in tumor homing 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. .

Tumor homing molecules can be identified by in vivo panning, for example, using mice containing transplanted tumors. Such transplanted tumors include, for example:
It can be a human tumor transplanted into an immunodeficient mouse such as a nude mouse, or a mouse tumor 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 associated with tumor homing molecules. It is expected that they may share common cell surface markers useful as target molecules. 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 (eg, melanoma) are also associated with corresponding target molecules in tumors in humans or other species. Recognize to combine. Similarly, tumors that grow in experimental animals require associated neoangiogenesis, just as is required for tumors that grow in humans or other species. Therefore, tumor-homing molecules that bind to target molecules present in the vasculature in tumors grown in mice may also bind to corresponding target molecules in the vasculature of tumors in human or other mammalian subjects. Is. The general ability of tumor homing molecules to bind to both human Kaposi's sarcoma or to mouse melanoma, identified by homing to human breast tumors, for example, indicates that the target molecule is shared by many tumors. In fact, the results disclosed herein are
Demonstrate 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) demonstrate, for example, that the molecules can also specifically bind to a sample of tumor obtained from a patient. This can be easily tested for the ability to bind the corresponding tumor in human subjects. For example, CDCRGDC
FC (SEQ ID NO: 1) phage and NGR peptides have been shown to bind blood vessels in microscopic sections of human tumors, whereas in blood vessels of non-tumor tissue little or no binding occurs. Thus, conventional methods can be used to confirm that tumor homing molecules identified using in vivo panning in experimental animals can also bind to target molecules in human tumors.

Administering the library to a subject, collecting selected tumors and identifying tumor homing molecules that home to the tumors involves a single 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 was performed, the molecule recovered from the tumor in the previous round was administered to the subject, which was used in the previous round in which only a portion of the tumor was collected. It 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 is determined by administering the identified molecule to a subject, collecting the tumor, And by comparing the phage recovered after the first round with more phage recovered from the tumor after the second round of screening. Although not necessarily, control organs or tissues can also be collected, and molecules recovered from the tumor can be compared to molecules recovered from this control organ. Ideally, no molecule is 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 control organs or tissues. In this case, the ratio of molecules in selected tumors compared to control organs (selected molecules: control molecules) can be determined. For example, phage that home to melanoma after the first round of in vivo panning showed a 3-fold enrichment in homing to selected tumors compared to control organs (brain) after two additional rounds of panning. (Example VIII).

An additional round of in vivo panning may 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. It may recognize a target on the tumor that is expressed or is sufficiently similar to the target molecule for the tumor. Tumor homing molecules are also unlikely to home to the corresponding normal tissue. This is because the method of in vivo panning selects 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, then that organ or tissue is considered to constitute a selected tissue or family of organs. Using the method of in vivo panning,
Molecules that home to only selected tumors can be identified as 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.
Be promoted.

The term “control organ or tissue” is used to mean a non-tumor organ or tissue in which identification of tumor homing molecules is desired. Control organs or tissues are characterized when tumor-homing molecules do not selectively home to control organs. Control organs or tissues can be collected, for example, to identify non-specific binding of the molecule or to determine 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 the tumor but is known not to be chemically similar to potential tumor homing molecules. Alternatively, if the administered molecule is bound to a support, administration of support alone can also be used to identify non-specific binding. For example,
Phage expressing the gene III protein alone but no peptide fusion protein can be studied by in vivo panning to determine the level of non-specific binding of the phage support.

Specific homing of tumor homing molecules, as disclosed herein, includes:
It can be readily identified by examining selected tumor tissues as compared to the 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 tumor homing peptides (see Example VII). See). Alternatively, an antibody that is specific for the shared tag expressed with the peptide, such as the FLAG epitope, can be used, as in commercially available detection systems.

In general, a library of molecules, which comprises a random diverse population or selectively randomized molecules of interest, is then administered to a subject. At a selected time post-administration, the subject is sacrificed and the tumor harvested so that the molecule present in the tumor can be identified (see Example IV). If desired, one or more control organs or control tissues or portions of control organs or control tissues may be sampled. For example, mice with breast cancer or melanoma tumors are injected with the phage peptide display library, then about 1-5 minutes later, the mice are anesthetized and frozen in liquid nitrogen or, preferably, phage. Perfusion through the heart to terminate the circulation of the tumor, the tumor and one or more control organs are collected from each of the phage present in the tumor, and the control organs are harvested and representative We identified peptides that selectively home to common tumors (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 needs to be harvested in order to harvest the support containing the tumor homing molecule, and likewise only a portion of the organ or tissue needs to be harvested as a control. Should be. Thus, for example, a portion of a tumor can be harvested by biopsy, so that molecules such as peptides expressed by phage can be administered to the same subject more than once, as desired. If the molecule to be administered a second time to the same subject is, for example, tagged or bound to a support, this tag or support will 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 performed using 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)), which has been used to identify novel peptides that specifically bind. However, such in vitro studies show whether a peptide capable of specifically binding to a selected receptor in vitro also binds that receptor in vivo, or whether the peptide or its 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 carried out in artificial systems using well-defined and well-characterized target molecules. For example, Goodso
n et al., supra, 1994 utilized cells expressing recombinant urokinase receptor. However, such in vitro methods require prior knowledge of the target molecule,
And any information is limited in that it is rarely available in the case of 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 the 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 are often altered when the cells are grown in culture. Therefore, in vitro panning methods using cells in culture are also limited in that there is no guarantee that the identified molecule will have the same potency in vivo due to its binding to cells in culture. Furthermore, it is not possible to use in vitro panning to identify molecules that home only to tumor cells used in the screen, but not to other cell types.

In contrast, in vivo panning requires no prior knowledge or availability of the target molecule and identifies molecules that bind to cell surface target molecules expressed in vivo. Also, because "non-targeted" tissue is present during screening, the possibility of isolating tumor homing molecules lacking homing specificity is
Greatly reduced. Moreover, any molecule that may be particularly susceptible to degradation in the circulation in vivo, eg, in obtaining a tumor homing molecule by in vivo panning due to metabolic activity is not recovered. Therefore, in vivo panning
The identification of tumor homing molecules that selectively home in vivo to target molecules present in tumors provides significant advantages over previous methods.

Although the mechanism by which the disclosed methods of in vivo panning operate is not well defined, one possibility is that molecules such as peptides expressed on phage are recognized and vascularized in tumors. Along with binding to target molecules present on endothelial cells. Evidence is, for example, that vascular tissue in various organs is different from another,
And that such differences may be involved in the regulation of cellular transport in the body. For example, lymphocytes are specific “addresses ()” by endothelial cells in their tissues.
due to the expression of "address" molecules (Salmi et al., Proc. Natl. Acad. Sci.,
USA 89: 11436-11440 (1992); Springer, Ce.
ll 76: 30-314 (1994)). Similarly, various leukocytes may recognize sites of inflammation, in part due to the expression of endothelial cell markers induced by inflammatory signals (Butcher and Picker, Science 272:
60-66 (1996); Springer, supra, 1994).
Thus, endothelial cell markers are selectively bound by tumor homing molecules,
It then 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, for example, Fitzpatrick and Gar.
nett, Anticancer Drug Des. 10: 1-9 (1995
) Are well known in the art.

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. Chimeric peptides of the present invention may also be obtained from commercial suppliers (eg AnaSpec, Inc .; Sa
n Jose, CA). Where the antimicrobial peptide is to be attached to a non-peptide tumor homing molecule, the antimicrobial peptide moieties can be independently synthesized using well known methods or obtained from commercial suppliers.

Several methods that can be used to attach antimicrobial peptides to tumor homing molecules are known in the art, depending on the chemical characteristics of the molecule. For example, methods of attaching a hapten to a carrier protein, such as are conventionally used in the field of applied immunology (eg, Harlow and Lane, supra, 1).
988; Hermanson, supra, 1996).

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). Which is hereby incorporated by reference). Carbodiimides include a group of compounds having the general formula R-N = C = N-R ', where R and R'may be aliphatic or aromatic. And used for the synthesis of peptide bonds. The preparative procedure is simple, relatively fast, and carried out under mild conditions. The carbodiimide compound attacks the carboxylic acid group, converting 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 conjugation requires the presence of an amino group, which can be provided, for example, by the antimicrobial peptide, and a carboxyl group, which can be provided by the tumor homing molecule.

In addition to using carbodiimides for the direct formation of peptide bonds, EDC also employs active esters such as N-hydroxysuccinimide (NH
S) ester) can be prepared. 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. The combined use of EDC and NHS 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 can glutaraldehyde cross-linking.
However, despite the choice of the method of producing the conjugates of the invention, the decision must be made that the tumor homing molecule retains its targeting capacity and that the antimicrobial peptide retain its antimicrobial activity. Be recognized. Methods known in the art can confirm the activity of the homing pro-apoptotic conjugates 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 qualitative or quantitative 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; also see Example VIII). In particular, one of ordinary skill in the art will recognize that the choice of method for determining the yield of the 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 antimicrobial peptides in vivo against tumors having angiogenic vasculature. This method is practiced by administering the homing pro-apoptotic conjugates of the invention to a subject containing a tumor having an angiogenic vasculature. In the method of the invention for directing an antimicrobial peptide in vivo against a tumor having an angiogenic vasculature, the antimicrobial peptide may be, for example:
The sequence _D (KLAKLAK) ₂ may be included. Particularly useful conjugates that can be administered to a subject containing a tumor having angiogenic vasculature are CNGRC-GG- _D (KL.
AKLAK) ₂ and ACDCRGDCFC-GG- _D (KLAKLAK) ₂ .

The present invention further provides methods of inducing selective toxicity in vivo in tumors having angiogenic vasculature. This method is practiced by administering the homing pro-apoptotic conjugates of the invention to a subject containing a tumor having an angiogenic vasculature. An antimicrobial peptide useful in inducing selective toxicity in the methods of the invention can be, for example, a peptide containing the sequence _D (KLAKLAK) ₂ . A particularly useful conjugate that can be administered to induce selective toxicity in vivo in tumors with angiogenic vasculature is CNGRC-GG- _D (KLAKL.
AK) ₂ and ACDCRGDCFC-GG- _D (KLAKLAK) ₂ .

Also provided herein is a method of treating a patient having a tumor having an angiogenic vasculature. In such methods of treatment, the homing pro-apoptotic conjugates of the invention are administered to a patient and are selectively toxic to tumors.
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 and ACDCRGDCFC-GG- _D (KLA
KLAK) ₂ .

When administered to a subject, the homing pro-apoptotic conjugates 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, glycerols, oils (eg Olive oil or injectable organic ester).

Pharmaceutically acceptable carriers can include, for example, physiologically acceptable compounds that act to stabilize or increase 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; or other Stabilizers or excipients are included. Those of skill in the art understand that the choice of pharmaceutically acceptable carrier, including physiologically acceptable compounds, depends, for example, on the route of administration of the compound. The pharmaceutical composition may also include agents such as cancer therapeutic agents.

One of skill in the art will appreciate that the homing pro-apoptotic conjugates of the invention may be administered to a subject as pharmaceutical compositions by a variety of routes, eg, orally or parenterally (eg, intravenously). Is known. The pharmaceutical composition containing the conjugate may be administered by injection or intubation. The pharmaceutical composition can also be a tumor-homing molecule, into which an antimicrobial peptide can be incorporated, bound to liposomes or other polymer matrices (Gregoriadis, Liposome Tec).
hology, Volume 1 (CRC Press, Boca Raton, FL
1984), which is 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 methods of treatment disclosed herein, an effective amount of homing pro-apoptotic conjugate must be administered to the subject. As used herein, the term "effective amount" means the amount of conjugate that produces the desired effect. Effective amounts often depend on the particular antimicrobial peptide attached to the tumor homing molecule. Effective amounts of homing pro-apoptotic conjugates in which the tumor homing molecule is linked to a particular antimicrobial peptide can be determined using methods well known to those of skill in the art.

The route of administration of homing pro-apoptotic conjugates depends, in part, on the chemical structure of the molecule. For example, peptides are not useful, especially when administered orally. This is because this peptide can be degraded in the digestive tract. However, methods of chemically modifying the peptide to make it less susceptible to degradation by endogenous proteases or to be more absorbed through the gastrointestinal tract, including the incorporation of D amino acids, have been described by Well known (eg Blo
ndelle et al., supra, 1995; Ecker and Crooke, supra, 19
95; Goodman and Ro, supra, 1995). Such modifications can be performed on tumor-homing peptides identified by in vivo panning, and antimicrobial peptides. Furthermore, a library of peptidomimetics (
It may include D amino acids, other non-naturally occurring amino acids, or chemically modified amino acids; or it may be an organic molecule that mimics the structure of a peptide; or such as a vinyl analog peptoid. Methods of preparing peptoids) are known in the art and can be used to identify tumor-homing molecules that are stable for oral administration.

Tumor homing peptides can have a linear or cyclic structure. Some peptides contained a cysteine residue, allowing cyclization of the peptide. In particular,
Peptides containing at least two cysteine residues cyclize spontaneously. Furthermore, such cyclic peptides may also be active when present in linear form (see, eg, Koivunen et al., Supra, 1993). For example, the linear peptide NGRAHA (SEQ ID NO: 6) was also useful as a tumor homing molecule (see Table 2). Thus, in some cases, disclosed herein, 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 this peptide. Methods for determining the need for cysteine residues or amino acid residues N-terminal or C-terminal to cysteine residues for 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 may also home to angiogenic vasculature that is not contained within the tumor. For example, tumor homing molecules containing either the RGD motif or the GSL motif specifically homed to the retinal neovasculature (Smith et al., In.
vest. Opththamol. Vis. Sci. 35: 101-111 (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. The person skilled in the art will understand that, preferably, for treating a tumor, a conjugate having a tumor homing peptide, which selectively homes to the tumor vasculature, is administered.

The present invention provides chimeric prostate homing pro-apoptotic peptides.
It can be used, for example, in the treatment of benign prostatic hyperplasia or prostate cancer. As disclosed herein, the SMSIARL peptide (SEQ ID NO: 207) is
When administered systemically, it can be selectively localized to prostate tissue, particularly the prostate vasculature (see Examples IX.B and IX.E). In addition, the prostate homing peptide SMSIARL (SEQ ID NO: 207) is a linking moiety to prostate tissue (
For example, it can be used to selectively deliver biotin or phage). As further disclosed herein, apoptosis is associated with SMSIARL-G.
Systemic administration of the G- _D (KLAKLAK) ₂ chimeric peptide was induced in mouse prostate; there was no evidence of apoptosis observed in non-prostate tissues (see Figure 7 and Example IX.C). The results disclosed herein also demonstrate that administration of SMSIARL-GG- _D (KLAKLAK) ₂ chimeric peptide
TRAMP mouse (Gingrich et al., Cancer Res. 56: 409.
6-4102 (1996), which develops prostate cancer under the influence of the transgene). FIG. 8 shows the SMSIARL-
_{GG-D (KLAKLAK)} mice treated with _2, vehicle _alone, D (KLA
KLAK) ₂ peptide alone, or SMSIARL peptide (SEQ ID NO: 207)
Shown to have survived longer than mice treated alone. Based on these results, the present invention provides chimeric prostate homing pro-apoptotic peptide and methods of using this peptide for the treatment of patients with prostate cancer, as described further below.

[0134]   Accordingly, the present invention includes a prostate homing peptide linked to an antimicrobial peptide.
A chimeric prostate homing pro-apoptotic peptide is provided. Where
Chimeric peptide is selectively internalized in prostate tissue, and its
While highly toxic to tissues, antibacterial peptides have been shown to interfere with prostate homing peptides.
When not linked to Tide, it has low mammalian cytotoxicity. Chimera of the present invention
In peptide, the prostate homing peptide moiety has, for example, the sequence SMSIAR
L (SEQ ID NO: 207) or a functionally equivalent sequence, and an antimicrobial peptide
The part is the sequence (KLAKLAK)_Two(SEQ ID NO: 200), (KLAKKLA)_Two (SEQ ID NO: 201), (KAAKKAA)_Two(SEQ ID NO: 202), or (KL
GKKLG)_ThreeMay have an amphipathic α-helix structure such as (SEQ ID NO: 203)
. In a preferred embodiment, the antimicrobial peptide moiety is a sequence_D(KLAKLAK) _Two including. An exemplary prostate homing pro-apoptotic peptide is SMSI
ARL-GG-_D(KLAKLAK)_TwoAs provided herein.

The invention further provides methods for directing in vivo antimicrobial peptides against prostate cancer. The method comprises administering a chimeric prostate homing proapoptotic peptide comprising a prostatic homing peptide linked to an antimicrobial peptide, wherein the chimeric peptide is selectively internalized by prostatic tissue, and the tissue The antimicrobial peptides have low mammalian cytotoxicity when not linked to the prostate homing peptide. In the methods of the invention, the prostate homing peptide can include, 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- containing _{D (KLAKLAK) 2.}

Also provided by the present invention are methods of inducing selective toxicity in vivo in prostate cancer. The method comprises administering to a subject containing prostate cancer a chimeric prostate homing pro-apoptotic peptide comprising a prostatic homing peptide linked to an antimicrobial peptide, wherein the chimeric peptide is selected by prostate tissue. Internalized and highly toxic to this tissue, while the antimicrobial peptide is not linked to the prostate homing peptide:
Has low mammalian cytotoxicity. Methods of inducing selective toxicity in vivo in prostate cancer can be carried out, for example, with 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 proapoptotic peptide has the sequence SMSIAR.
L-GG- _D (KLAKLAK) ₂ .

[0137]   Furthermore, the present invention provides that a patient with prostate cancer is provided with this patient's chimeric progenitor.
Method of treating by administering gland homing pro-apoptotic peptide
The chimeric peptide is selectively toxic to tumors
. This chimeric peptide is a prostate homing peptide linked to an antimicrobial peptide.
And the chimeric peptide is selectively internalized by prostate tissue.
, And highly toxic to this tissue, while the antimicrobial peptide
It has low mammalian cytotoxicity when not linked to the minging peptide. Front
The gland homing peptide portion has, for example, the sequence SMSIARL (SEQ ID NO: 207).
Or may include a functionally equivalent sequence, and the antimicrobial peptide moiety may be, for example, a sequence _D (KLAKLAK)_TwoCan be included. For treating patients with prostate tumors
In a preferred embodiment, the chimeric peptide has the sequence SMSIARL-GG-._D (KLAKLAK)_Twoincluding.

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

The chimeric peptides of the present invention are selectively delivered to the prostate due to the selective homing activity of the prostate homing peptide moieties. A variety of prostate homing peptides are useful in the present invention, which includes SMSIARL (SEQ ID NO: 207).
And wherein VSFLEYR (SEQ ID NO: 222), these are identified by injection into mice of _{X 7} library (Table 7), and subsequent in vivo panning (e.g., as described in U.S. Patent No. 5,622,699) Was done. The prostate homing peptides, SMSIARL (SEQ ID NO: 21) and VSFLEYR (SEQ ID NO: 22), are 34-fold and 1 in prostate, respectively, when compared to the brain.
It showed a 7-fold enrichment.

In one embodiment, the invention features the sequence SMSIARL (SEQ ID NO: 207).
Alternatively, it relies on a prostate homing peptide containing a functionally equivalent sequence. the term"
A "functionally equivalent sequence", as used herein with respect to the sequence SMSIARL (SEQ ID NO: 207), refers to the sequence SMSIARL (SEQ ID NO: 207) in FIG.
As shown in Fig. 3, a sequence is shown that selectively binds to the endothelium of prostate blood vessels and functions similarly in that this sequence selectively binds to the same receptor.

The chimeric prostate homing pro-apoptotic peptides of the invention can be used to induce selective toxicity in various prostate disorders. Such disorders include benign nodular prostate hyperplasia and primary and secondary cancers including clinically apparent and subclinical cancers. Cancers treated with the chimeric peptides of the invention include prostate carcinomas such as adenocarcinomas.

The following working receptors are intended to be exemplary, but not limiting of the invention.

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

[0144]   Synthetic 14-mer KLAKLAKKLAKLAK (SEQ ID NO: 200) ((KL
(AKLAK)_TwoIs called). Because it kills eukaryotic cells
Kills bacteria at a concentration that is two orders of magnitude lower than that required to damage
(Javadpool et al., J. Med. Chem. 39: 3107).
-3113 (1996)). All D-enantiomers,_D(KLAKLAK) _Two , To avoid degradation by proteases (Bessalle et al., F.
EBS Lett. 274: 151-155 (1990); Wade et al., Pro.
c. Natl. Acad. Sci. 87: 4761-4765 (1990)).

[0145] _{(D (KLAKLAK) 2} preferentially disrupt mitochondrial membranes) collapses preferentially mitochondrial membrane than eukaryotic plasma membranes _D (KLAKL
The capacity of AK) ₂ was assessed by the mitochondrial swelling assay and the cell-free system of mitochondrial-dependent apoptosis, and by the cytotoxicity assay.

The mitochondrial swelling assay was performed as follows. Briefly, rat liver mitochondria were prepared as described in Ellerby et al. Neurosci. 17: 6
Prepared as described in 165-6178 (1997). Peptide, HP
Synthesized by LC to a purity higher than 90% (DLSLLARLATARLAI
(SEQ ID NO: 204) Coast Scientific, Inc. , San D
IEGO, CA; all other peptides are described in AnaSpec, Inc. ). Mitochondria were treated with _D (KLAKLAK) ₂ at a concentration of 10 μM and DL at a concentration of 10 μM.
SLARLATARAI negative control peptide (SEQ ID NO: 204
), Or with Ca ⁺² at a concentration of 200 μM as a positive control. Peptide was added to the mitochondria in the cuvette and swelled to 520
It was quantified by measuring the visible absorbance at nm.

As shown in FIG. 2a, 10 μM _D (KLAKLAK) ₂ induced significant mitochondrial swelling. When measured at the lethal concentration required to kill 50% of cell monolayers (LC ₅₀ ; Table 1), a mild swelling was evident at a concentration of 3 μM, killing eukaryotic cells. Required concentration (about 300 μM
) Was two orders of magnitude lower than The non-α-helix forming peptide DLSLARLATARLAI (SEQ ID NO: 2 used as negative control)
04) did not induce mitochondrial swelling. These results are _D (KLA
We demonstrate that KLAK) ₂ preferentially disrupts the mitochondrial membrane compared to the eukaryotic plasma membrane.

[0148]

[Table 1] ( _D (KLAKLAK) ₂ induces mitochondrial-dependent apoptosis) _D (KLAKLAK) ₂ peptide is mitochondrial-dependent in a cell-free system composed of normal mitochondria suspended in normal cytosolic extract. Assayed for the ability to activate sexual apoptosis (Ellerby et al., J. Am.
Neurosci. 17: 6165-6178 (1997)). Apoptosis was measured by the characteristic caspase-3 processing from an inactive proform to an active protease (Alnemri et al., Cell 87:
171 (1996)).

The cell-free apoptosis assay was performed essentially as follows. The cell-free system was reconstructed as described by Ellerby et al. (Supra, 1997), and for mitochondrial-dependent reactions, rat liver mitochondria were prepared from normal (non-apoptotic) skin microvascular endothelial cells. Suspended in cytosolic extract. 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, and the supernatant was
Analyzed by SDS / PAGE immunoblotting on a% gel (Biorad; Hercules, CA). PVDF membrane (Biorad)
And the anti-caspase-3 antibody (Santa Cruz Biotechnol
Ogy; Santa Cruz, CA) followed by E
CL detection (Amersham; Arlington Heights, IL) was performed.

The characteristic caspase-3 processing was characterized by Ellerby et al. (Supra, 1997).
) In skin microvascular endothelial cell lysates as described in (1). Briefly, 100 μl aliquots of cell lysate (1 μl lysate, 8-15 mg / ml)
MN-Acetyl-Asp-Glu-Val-Asp-pNA (DEVD-pN
A; BioMol; 100 μl, 100 mM HEPES, 10% sucrose, 0.1% CHAPS, 1 mM DTT, pH 7.0). DEV
The hydrolysis of D-pNA was monitored with a spectrophotometer (400 nm) at 25 ° C.

As shown in FIG. 2b, lane 4, the active protease, a characteristic caspase processing, was observed in the presence of mitochondria and _D (KLAKLAK) ₂ . The non-α-helix forming peptide DLSLARLATARLAI (SEQ ID NO: 204) used as a negative control was inactive when tested in a cell-free system and was not lethal to eukaryotes (FIG. 2b;
See also Ellerby et al., Supra, 1997).

In summary, these results indicate that _D (KLAKLAK) ₂ preferentially disrupts mitochondrial membranes and activates mitochondrial-dependent apoptosis when 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. To do.

A chimera containing a homing domain linked to an antimicrobial peptide via a glycinylglycine bridge was prepared. As above, the peptide was labeled with AnaSpec, I
Commercially synthesized to> 90% purity by HPLC by nc.

This homing domain is cyclic (disulfide bond between cysteines) CNG.
RC peptide (SEQ ID NO: 8; see Figure 1), or dual cyclic ACDCRG
DCFC peptide (SEQ ID NO: 16), both of which have tumor-homing properties (Pasqualini et al., Nature Biotech. 1).
5: 542-546 (1997); Arap et al., Science 279: 37.
7-380 (1998)) and can be absorbed (Arap et al., Supra, 1998.
Hart et al., J .; Biol. Chem. 269: 12468-12474 (1
994); Brettcher et al., EMBO. J. 8: 1341-1348 (1
989)). Due to the putative chiral nature of the homing domain-receptor interaction, this homing domain was synthesized from all L-amino acids. This glycinylglycine bridge was used to couple the homing and antimicrobial domains to give flexibility to the peptide and minimize potential steric interactions.

Viability of dermal 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) and evaluated in a tissue culture model of angiogenesis. During angiogenesis, capillary endothelial cells proliferate and migrate (
Risau, Nature 386: 671-674 (1997); Zette.
r, Ann. Rev. Med. 49: 407-424 (1998)). Cord formation is a form of migration, which is represented by the change in endothelial cell morphology in vitro from normal “cobblestones” to chains or cords of cells, as shown in FIG. 3 ( See also Goto et al., Supra, 1993).

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

Briefly, dermal microvascular endothelial cells (DMEC) were isolated from CADMEC Gro.
wth Media ^™ (Cell Applications, Inc .; S
an Diego, CA). Then, the dermal microvascular endothelial cells were added to 3
Cultured under one experimental condition: Growth, 30% confluency in growth medium supplemented with 500 ng / ml human recombinant vascular endothelial growth factor (VEGF; Pharmingen); no growth, formulated to maintain monolayers. 100% in conditioned medium
, And 60% confluency in the medium formulated to induce cord formation (required for induction). KS1767 and MDA-MB-
435 cells from Arap et al., Supra, 1998; Hernier et al., Supra, 199.
Cultured as described in 4.

Percent viability and LC ₅₀ (Table 1) were calculated according to Ellerby et al. Ne
urosci. 17: 6165- 6178 (1997), by morphology of apoptosis. For percent viability assay, dermal microvascular endothelial cells were treated with 60 μM HPP-1 or control peptide _D (
KLAKLAK) ₂ . At the indicated time points, cell culture medium was aspirated from adherent cells and the cells were gently washed once with 37 ° C PBS. Subsequently, the dye mixture in PBS (100 μg / ml acridine orange and 10
A 20-fold dilution of 0 μg / ml ethidium bromide) was pipetted slowly onto the cells, which was viewed under an inverted microscope (Nikon TE 300). Chromatin marginal orientation and condensation, and / or nuclear fragmentation (early / intermediate apoptosis)
Cells with nuclei or immunocompromised plasma membranes (late apoptosis) were scored as non-viable. In each experiment, at least 500 until a given time
Individual cells were recorded. The percent viability was calculated relative to untreated controls. The LC ₅₀ for monolayer, proliferation (60% confluency), and cord formation was recorded at 72 hours.

As shown in FIG. 3, treatment of dermal microvascular endothelial cells with 60 mM HPP-1 reduced percent viability over time under conditions of proliferation or cord formation compared to untreated controls. (See FIGS. 3c and 3d, respectively). In contrast, non-targeting peptide _D (KLAK as a negative control
Treatment with LAK) ₂ led to a negligible loss in viability. Furthermore, HP
Was treated with P-1, LC ₅₀ for dermal microvascular endothelial cells growing or move, LC about 100% confluency angiogenesis inhibitory dermal micro endothelial cells maintained in monolayer at Sea The order of magnitude was lower than ₅₀ (
Table 1). These results demonstrate preferential killing by HPP-1 under angiogenic conditions compared to angiostatic conditions.

[0161]   Various controls were also used to determine the effect on dermal microvascular endothelial cell viability.
Assayed. Non-targeted control under angiogenic conditions_D(KLAKLAK) _Two About LC_FiftyIs the LC for HPP-1 under vascular static conditions_FiftyWhen
It was similar. Furthermore, unbound_D(KLAKLAK)_TwoAnd CNGRC (sequence
No. 8) mixture, non-targeted form CARAC-GG-_D(KLAKLAK)_Two , And the scrambled form of CGRNC-GG-_D(
(KLAKLAK)_TwoIs_D(KLAKLAK)_TwoAnd gave similar results. further
, An alternative prototype ACDCRGDCFC-GG-_D(KLAKLAK)_TwoIs
, CNGRC-GG-_D(KLAKLAK)_Two(HPP-1; data not shown)
The results were similar to those for.

Mitochondrial Morphology of HPP-1 Treated Dermal Microvascular Endothelial Cells The mitochondrial morphology of dermal microvascular endothelial cells was determined as follows: 60 μM H
It was evaluated in proliferating cells after treatment with PP-1, ACDCRGDCFC-GG- _D (KLAKLAK) ₂ or non-targeted _D (KLAKLAK) ₂ . Dermal microvascular endothelial cells were stained with 100 nM mitochondria MitoTracker
Red ^™ (Molecular Probes, Inc., Eugene, O.
R) and 500 nM nuclear stain DAPI (Molecular Probes,
Inc. ) For 30 minutes at 37 ° C. and stained 24 and 72 hours after peptide treatment. The mitochondria were subsequently visualized using a fluorescence microscope (100x) under an inverted microscope with a triple wavelength filter set (Nikon).

While the mitochondria of dermal microvascular endothelial cells treated with _D (KLAKLAK) ₂ for 24 hours remained morphologically normal, the mitochondria treated with each prototype showed altered mitochondrial morphology. Indicated. In particular, the altered mitochondrial morphology can be confused by CNGR prior to round-up of cells.
C-GG- _D (KLAKLAK) ₂ or ACDCRGDCFC-GG- _D (K
It was apparent in about 80% of cells treated with LAKLAK) ₂ . Ultimately, CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1) treated dermal microvascular endothelial cells showed a classical morphological indicator of apoptosis, as seen at 72 hours ( Including nuclear condensation and fragmentation) (Ellerby et al., Supra, 1997). Apoptotic cell death was confirmed by assaying for caspase activity (see Figure 3b; Ellerby et al., Supra, 1997).

Viability of KS1767 and MDA-MB-435 Cells Treated with HPP-1 The chimeric HPP-1 peptide was conjugated to KS1767 cells as well as to proliferating or migrating dermal microvascular endothelial cells. (This is from Kaposi's sarcoma) (Table 1; Hernier et al., AIDS 8: 575-581 (1
994)). In contrast, HPP-1 is only about an order of magnitude larger than MDA-MB-43.
It was of low toxicity to 5 human breast cancer cells (Table 1). KS1767 cells (this is
Endothelial origin) has something in common with angiogenic endothelial cells and binds to the CNGRC peptide (SEQ ID NO: 8), whereas MDA-MB-435 cells do not (S.
amaniego et al., Amer. J. Path. 152: 1433-1443 (
1998); Arap et al., Supra, 1998).

In summary, these results demonstrate that HPP-1 induces mitochondrial swelling and apoptosis in dermal microvascular endothelial cells.

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

A. CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1) In Vivo Activity HPP-1 activity was determined in nude mice bearing human MDA-MD-435 breast cancer xenografts as follows. And tested in vivo. In short, MDA-M
B-435 and C8161-induced tumor xenografts were prepared according to Arap et al., Supra, 19
98, established in 2-month-old female nude mice (Jackso.
n Labs; Bar Harbor, ME). 2,2,2 prepared in distilled water
-After anesthetizing the mice with a mixture of tribromoethanol (Aldrich; Milwaukee, WI) and 2-methyl-butanol (Aldrich), the peptide was passed through the tail vein in a volume of 200 [mu] l, dose of 250 [mu] g / week / mouse. Administered slowly intravenously. Three-dimensional measurement of tumors was performed with calipers under anesthesia and used to calculate tumor volume (Pasquali.
ni et al., supra, 1996).

As shown in FIG. 4a, tumor volume was controlled by the control (which was non-targeted C
ARAC-GG- _D (KLAKLAK) ₂ as well as unbound _D (KLAKLAK)
₂ ) and CNGRC (which is a mixture of CNGRC (SEQ ID NO: 8) peptides) was an order of magnitude smaller on average. As further shown in Figure 4b, survival was longer in the HPP-1 treated group than in the control group. Some HPP-1 treated mice lived several months longer than control mice, indicating that both primary tumor growth and metastasis were inhibited by HPP-1. Histopathological analysis reveals overt tumor structure destruction and extensive cell death in the tumor, demonstrating that this cell death is approximately equally apoptotic and necrotic. In a similar experiment, HPP-1 also induced human melanoma cell line C
Effective against 8161-derived tumors (Welch et al., Int. J. Canc.
er 47: 227-237 (1991)).

These results show that homing pro-apoptotic conjugates (eg CNGRC)
-GG- _D (KLAKLAK) ₂ (HPP-1)) has potent antitumor activity in vivo.

B. In Vivo Activity of CDCRGDCFC-GG- _D (KLAKLAK) ₂ Retinal angiogenesis was oxygen-induced in neonatal mice. Then,
Mice were given a single 13 μg intravenous dose (1 animal per group) of vehicle; CD
CRGDCFC-GG- _D (KLAKLAK) ₂ ; or unconjugated CDCRG
It was treated with a control mixture of DCFC (SEQ ID NO: 1) and _D (KLAKLAK) ₂ . Four days later, retinal neovascular numbers were determined for each treatment.

[0171]   The results shown in FIG. 5 indicate that mice treated with vehicle alone (column 1; black bars).
Or mice treated with non-targeted pro-apoptotic peptides (column 3; Aya)
Homing pro-apoptotic peptide CDCRGD compared to the patterned bar)
CFC-GG-_D(KLAKLAK)_TwoTreated with (column 2; striped)
), A decrease in the number of retinal neovasculature was demonstrated. Especially homing
Proapoptotic peptide CDCRGDCFC-GG-_D(KLAKLAK) _Two The angiogenic response in mice treated with is observed in control mice.
Only 30-40% of the observed responses.

These results show that homing pro-apoptotic peptides (eg CDCR
It is shown that GDCFC-GG- _D (KLAKLAK) ₂ ) can selectively inhibit an angiogenic response (eg, retinal neovascularization).

Example IV In Vivo Panning This example uses in vivo panning to identify a method for preparing a phage library and phage expressing tumor homing peptides. Demonstrate a method for screening.

A. Phage Library Preparation Phage display libraries were prepared according to Koivunen et al. (Supra, 1995).
Constructed using the Fuse 5 vector as described by Koivunen et al., Supra, 1994b). Prepare libraries encoding peptides named CX ₅ C (SEQ ID NO: 9), CX ₆ C (SEQ ID NO: 10), CX ₇ C (SEQ ID NO: 11), CX ₃ CX ₃ CX ₃ C (SEQ ID NO: 12) did. Here, "C" indicates cysteine and "X _N " indicates a predetermined number of individually selected amino acids. These libraries may represent cyclic peptides if at least two cysteine residues are present in the peptide. In addition, a library without defined cysteine residues was also constructed. Such libraries primarily result in the production of linear peptides, but cyclic peptides can also occur due to random potential.

A biased library based on the sequence CXXXNGRXX (SEQ ID NO: 13) was also constructed. Furthermore, in some cases, CXXXXXNRXX (SEQ ID NO: 13
) Library was further biased by the incorporation of cysteine residues flanking the NGR sequence (ie, CXXCNGRCX (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 the codon TGT and "X _N " is encoded by NNK. did. Here "N" is a mixture of equal molar concentrations of A, C, G and T, and here "K" is a mixture of equal molar concentrations of G and T. Thus, the peptide represented by CX ₅ C (SEQ ID NO: 9) may be represented by an oligonucleotide having the sequence TGT (NNK) ₅ TGT (SEQ ID NO: 14). Oligonucleotide for 3 cycles of PC
This peptide was ligated to the nucleic acid encoding the gene III protein in the fuse 5 vector such that it was double-stranded by R amplification, purified, and, upon expression, as a fusion protein, at the N-terminus of the gene III protein. .

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

B. In Vivo Panning of Phage Tumors were transplanted into mice as described in Examples V and VI below. 1
Mix a mixture of phage libraries containing 200 × 10 ⁹ to 1 × 10 ¹⁴ TUs with 200
Dilute in μl DMEM and anesthetize (AVERTIN (0.015 ml / g)
US Patent No. 5,622,699; Pasqualini and Ruosla;
hti, supra, 1996). 1 to
After 4 minutes, mice were snap frozen in liquid nitrogen. To recover the phage, the carcasses were partially thawed at room temperature for 1 hour, the tumor and control organs were collected and weighed, then 1 ml DMEM-PI (protease inhibitor (PI); phenylmethyl). DMEM containing sulfonyl fluoride (PMSF; 1 mM), aprotinin (20 μg / ml), leupeptin (1 μg / ml))
Powdered in.

Alternatively, following the introduction of the library into the mouse, cycling the library
It is terminated by perfusion through the heart. Briefly, mice were anesthetized with AVERTIN, then the heart was exposed and 10 cc via a 0.5 mm cannula.
A 0.4 mm needle connected to the 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 the whole body was perfused for about 5-10 minutes. The efficiency of perfusion was directly monitored by histological analysis.

Tumor and organ samples were washed 3 times with ice-cold DMEM-PI containing 1% bovine serum albumin (BSA) and then directly incubated with 1 ml K91-kan bacteria for 1 hour. 10 ml of NZY medium (NZY / tet) containing 0.2 μg / ml tetracycline was added to the bacterial culture. The mixture was incubated for 1 hour on a shaker at 37 ° C., then 10 μl or 100 μl aliquots were plated on agar plates (tet / agar) containing 12.5 μg / ml tetracycline.

Individual colonies containing phage recovered from the tumor were treated with 5 ml N 4 for 16 hours.
Grow in ZY / tet. Bacterial cultures obtained from individual colonies were pooled and phage were purified and reinjected into mice as above for a second round of in vivo panning. Usually a third panning was also done. Phage DNA was purified from individual bacterial colonies obtained from the final round of in vivo panning and D encoding a 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 Against 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 that you can be broken.

Human 435 breast cancer cells (Price et al., Cancer Res. 50: 717-.
721 (1990)) was inoculated into the fat pad of the breast of nude mice. If the tumors reach approximately 1 cm in diameter, either perform phage targeting experiments (where phage expressing specific peptides were administered to tumor-bearing mice) or perform in vivo panning. 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. Where X ₃ represents three groups of independently selected random amino acids. Phage were circulated for 4 minutes, then mice were anesthetized, snap frozen in liquid nitrogen during anesthesia and tumors removed. Phages were isolated from tumors and subjected to two additional rounds of in vivo panning.

[0185]

[Table 2] Following the third round of panning, the phage were quantified and the peptide sequences expressed by the cloned phage determined. The cloned phage express 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 RGD (an an RGD) Peptide-Expressing Phage to Tumors Human 435 breast cancer cells were inoculated into the breast fat pad of nude mice. The tumor
Phage expressing specific RGD-containing peptides were administered to tumor-bearing mice when they reached approximately 1 cm in diameter. Results similar 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.

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

Phage expressing the CDCRGDCFC (SEQ ID NO: 1) peptide (about 2-3 times more) were detected in breast tumors when compared to the brain and kidney, which was the CDCR.
It is shown that the GDCFC (SEQ ID NO: 1; RGD phage) peptide causes selective homing of phage to breast tumors. In a parallel study, unselected phage expressing a wide variety of peptides were injected into tumor-bearing mice,
Various organs were then tested for the presence of phage. There were significantly more phage in the kidney and to a lesser extent in the brain when compared to the tumor. Therefore, 80% more RGD-expressing phage were concentrated in this tumor than non-selected phage. These results indicate that phage expressing RGD-containing peptides home to tumors, probably due to the expression of α _V β ₃ integrin on blood vessels forming in the tumor.

The specificity of the breast tumor homing peptide was demonstrated by competition experiments, in which 500 μg of the free peptide (ACCDRGDCFCG (SEQ ID NO: 1
6); Pasqualini et al., Supra, 1997) and co-injection with tumor-homing peptide expressing phage reduced the amount of phage in the tumor by approximately 10-fold, but with an inactive control peptide (GRGESP). Co-injection with (SEQ ID NO: 17) had essentially no effect.These results indicate that phage displaying peptides capable of binding to integrins expressed on the angiogenic vasculature were in vivo. Demonstrate that it can selectively home to organs or tissues, such as tumors containing such vasculature.

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

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

As discussed in Example VI, phage expressing tumor homing peptides (
CDCRGDCFC (SEQ ID NO: 1; “RGD phage”) was administered intravenously to mice bearing breast tumors. In addition, RGD phage was administered to mice carrying mouse melanoma or human Kaposi's sarcoma. Phage circulation was terminated and mice were sacrificed as above and tumor samples and skin samples adjacent to 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 melanoma and Kaposi's sarcoma, while little or no staining was observed in control organs.

A similar experiment was performed with tumor-homing peptide-expressing phage (CNGRCVSGCAGRC (SEQ ID NO: 3; “NGR phage”)) identified by in vivo panning on tumors formed by MDA-MB-435 breast cancer. Carried out. In these experiments, NGR phage or control phage that did not express the peptide were administered to mice bearing tumors formed by MDA-MB-435 breast cancer or human SLK Kaposi's sarcoma xenograft, and then the mice were as described above. Sacrifice and control tumors and control organs including brain, lymph nodes, kidneys, pancreas, uterus, breast fat pads, lungs, intestines, skin, skeletal muscle, heart and kidney calyx, bladder and ureteral epithelium Collected. Histological samples were prepared and examined by immunostaining as described above.

Immunostaining of the vasculature of both breast tumors and Kaposi's sarcoma was observed in samples obtained from mice sacrificed 4 minutes after administration of NGR phage. Very little or no staining was observed on the endothelium of these tumors in mice that received control phage without inserts. In samples obtained from mice sacrificed 24 hours after administration of NRG phage, staining of tumor samples appeared to have spread to mammary tumor and Kaposi's sarcoma parenchyma outside the vessels. Moreover, little or no staining was observed in the samples prepared from these tumors in mice that received control phage without inserts. Furthermore, 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 identified by in vivo panning of melanoma (CLSGSLSC (SEQ ID NO: 4)) (Examples below). VIII).

These results show that tumor-homing peptides selectively home to tumors, particularly the vasculature in tumors, and that tumor-homing peptides identified by, for example, in vivo panning against breast cancer also show caposarcoma and We demonstrate that it selectively homes 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 for Melanoma Tumors The general applicability of the in vivo panning method for identifying tumor homing peptides was demonstrated in transplanted mouse melanoma tumors. Were tested by performing in vivo panning on.

Melanoma-bearing mice produce highly vascularized tumors B16B
It was prepared by transplantation of 15b mouse melanoma cells. B16B15b mouse melanoma cells were injected subcutaneously into the mammary fat pad of nude mice (2 months old) and the tumors were allowed to grow to approximately 1 cm in diameter. In vivo panning was performed as described above. CX ₅ C (SEQ ID NO: 9), CX ₆ C (SEQ ID NO: 10) or CX ₇ C
About 1 × 10 ¹² transducing units of phage expressing the (SEQ ID NO: 11) library were injected intravenously and circulated for 4 minutes. Mice were then snap frozen in liquid nitrogen or perfused transcardially under anesthesia to remove tumor tissue and brain (control organs) and phage isolated as described above. In vitro panning was performed 3 times.

[0199]

[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 the sequenced clones) and WGTGLC (SEQ ID NO: 18; 25 of the clones).
%; See Table 3). Reinfection of phage expressing one selected peptide resulted in approximately 3-fold enrichment of tumor-homed phage compared to brain.

The localization of phage expressing tumor homing peptides 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 tumor-homing peptide (phage expressing CLSGSLSC (SEQ ID NO: 4) was injected intravenously into tumor-bearing mice, and 4
Circulate for minutes.

Immunostaining was evident in melanoma obtained from mice injected with phage expressing the CLSGSLSC (SEQ ID NO: 4) tumor homing peptide. Melanoma staining is generally localized to blood vessels within the tumor, although some staining was also present in the tumor parenchyma. In essence, 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 either phage. There wasn't. However, immunostaining was detected in liver sinusoids and spleen, indicating that the phage could be nonspecifically captured by organs containing RES.

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

[0203]

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

[0204] 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 peptides SMSIARL (SEQ ID NO: 207) and VSFL
EYR (SEQ ID NO: 222), in the prostate, respectively, when compared to the brain,
It showed 34-fold and 17-fold richness. Additional prostate homing sequences identified by in vivo panning are shown in Table 5.

[0205]

[Table 5] B. Prostate Homing Peptide Homing Peptide Biotin Conjugates In vivo screening of heptapeptide phage libraries was used to identify 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 synthetic SMSIARL peptide (SEQ ID NO: 207)
Inhibited prostate-selective homing of IARL (SEQ ID NO: 207) -carrying phage. In addition, antibody staining of tissue sections showed that SMSIARL (SEQ ID NO: 207) phage was localized to prostate tissue but not to other tissues after intravenous injection of phage into mice. 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 biotin-labeled control peptide CARAC (SEQ ID NO: 208) was injected intravenously into mice, which were sacrificed 10 minutes later. Prostate and other tissues were harvested, 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 show 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.

[0207]   (C. Induction of prostate-selective apoptosis)   The SMSIARL peptide (SEQ ID NO: 207) is a proproapoptotic pep
Cide (pro-apoptotic peptide) (_D(KLAKLAK) _Two ) Was delivered to the prostate. Simply put, SMSIARL-GG
−_D(KLAKLAK)_TwoSingle dose of chimeric peptide or control substance
Of TUN in 250 μg peptide / mouse and tissue obtained 24 hours later
EL staining was performed. As shown in FIG. 7, SMSIARL-GG-_D(KLAK
LAK)_TwoMice injected with the chimera show that their prostate, and in particular the prostate
It showed increased apoptosis in capillary endothelium and basal myoepithelial cells. Testis,
There is no evidence of increased apoptosis in other tissues such as kidney and brain
It was SMSIARL (SEQ ID NO: 207) and_D(KLAKLAK)_TwoOf 250
In negative control mice treated with μg of unconjugated mixture,
No apoptosis was observed.

_D. TRAMP Mouse SMSIARL-GG- _D (KLAKLAK) ₂ Treatment TRAMP mice develop prostate cancer under the influence of transgenes as described in Gingrich et al. (Supra, 1996). . SMSIARL-GG- _D (
The KLAKLAK) ₂ chimeric peptide was assayed for its ability to suppress the development of cancer in TRAMP mice. 12-week-old mice (10 per group)
Starting at 2, mice received SMSIARL-GG- _D (KLAKLAK) ₂ peptide or control peptide biweekly at 250 μg / dose (total 10 doses). Four mice without visible tumors were killed within minutes after injection and
SMSIARL-GG- _D (KLAKLAK) ₂ group excluded. As shown in FIG. 8, mice treated with SMSIARL-GG- _D (KLAKLAK) _{2 were} treated with control (which is the vehicle, _D (KLAKLAK) ₂ peptide alone,
Alternatively, it survived longer than mice treated with the SMSIARL peptide (SEQ ID NO: 207) alone. Therefore, TRAM with targeted pro-pro-apoptotic SMSIARL-GG- _D (KLAKLAK) ₂ compounds over a period of several months
Treatment of P mice caused a clear 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 containing both normal and cancerous tissue were treated with 10 ⁹ TU S.
MSIARL phage (SEQ ID NO: 207) were overlaid and phage binding was detected with anti-phage antibody 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 endothelium was seen with phage without peptide insert (panel c). Furthermore, when the soluble SMSIARL peptide (SEQ ID NO: 207) was included at 0.3 mg / ml in the cover, SMSIA
RL (SEQ ID NO: 207) phage staining was inhibited. The results shown in FIG. 9 indicate that at least some tumors carry homing peptide receptors. In addition, the peptide (SEQ ID NO: 207) may bind to cancer vessels within the prostate, while blood vessels in some other human tissues are associated with SMSIARL.
(SEQ ID NO: 207) Not stained by phage.

All scientific articles, references, and patent citations provided above, whether parenthesized or otherwise, are incorporated herein by reference.

Although the present 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 claims that follow.

[Sequence list]

[Brief description of drawings]

FIG. 1 is a computer-generated model and CNGRC referred to as “HPP-1”.
1 shows the amino acid sequence of -GG- _D (KLAKLAK) ₂ . Top panel: CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1)
It consists of a homing domain and a membrane-disrupting domain linked by a coupling domain. Lower panel: Amino acid sequence of "HPP-1" which matches the structure shown in the upper panel.

FIG. 2 shows mitochondrial swelling and mitochondria-dependent apoptosis in the presence of _D (KLAKLAK) ₂ . a. The mitochondrial swelling curve (absorption spectrum) in the presence of _D (KLAKLAK) ₂ or Ca ⁺² (positive control) is shown. b. _D (KLAKLAK) ₂ or D in the presence or absence of mitochondria
Caspase-3 cleaved immunoblot showing the pre-form of 32 kDa and the processed forms of 8 and 20 kDa in the presence of LSLARLATARLAI (SEQ ID NO: 204). A representative experiment is shown. The results were reproduced in 3 independent experiments.

FIG. 3 is a cutaneous microvascular endothelial cell treated with CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1).
3) shows mitochondrial swelling and apoptosis in the cell. a. Cutaneous microvascular endothelial cell cord formation, scale bar = 250 μm. b. Hydrolysis of DEVD-pNA (Caspase activation) in proliferating skin microvascular endothelial cells treated with CNGRC-GG- _D (KLAKLAK) ₂ (HPP-1). c. HPP-1 (black bar) or control peptide _D (KLAKLA
K) Viability of proliferating skin microvascular endothelial cells treated for a long time with ₂ (grey bar).
(T-test, P <0.05). d. HPP-1 (black bar) or control peptide _D (KLAKLA
K) Viability of chondrogenic skin microvascular endothelial cells treated for a long time with ₂ (grey bar). (T-test, P <0.05).

FIG. 4 shows the effect of HPP-1 treatment on nude mice bearing human MDA-MB-435-derived breast cancer xenografts. a. Tumor volume of HPP-1 treated tumors when compared to control CARAC-GG- _D (KLAKLAK) ₂ treated tumors. Differences in tumor volume between day 1 and day 50 are shown (t-test, P = 0.027). b. HPP-1 or control peptide ( _D (KLAKLAK) ₂ and CN
Kaplan-Meie showing the survival of nude mice bearing xenografts of human MDA-MB-435-derived breast cancer treated with GRC (mixture of SEQ ID NO: 8).
r Survival plot. Each group consisted of 13 animals. (Log-Rank test, P <0.05)

FIG. 5: Oxygen-induced retinal neovascularization (oxigen-i) in neonatal mice.
The effect of CDCRGDCFC-GG- _D (KLAKLAK) _{2 on} the reduced final neovascularization is shown. The retinal neovascular number is vehicle (black bar); CDCRGDCFC-GG- _D (KLAKLAK) ₂ (hatched bar); and unbound CDCRGDCFC (SEQ ID NO: 1) and _D (KLA).
Treatment of KLAK) _{2 with} a control mixture (hatch bar) is shown.

FIG. 6 shows the accumulation of biotin conjugates of prostate-homing peptide injected intravenously into prostate tissue. a. Biotin-labeled prostate homing peptide, SM
Avidin-peroxidase staining of prostate sections from mice injected with SIARL (SEQ ID NO: 207). b. Biotin-labeled control peptide CARAC
Avidin-veroxidase staining of prostate sections from mice injected with (SEQ ID NO: 208). b. Avidin-veroxidase staining of prostate sections from mice injected with biotin-labeled control peptide CARAC (SEQ ID NO: 208).

FIG. 7 shows SMSIARL-GG- _D (KLAKLA) in normal mouse prostate.
K) ₂ shows apoptosis induced by. a. T of prostate tissue from mice treated with SMSTARL-GG- _D (KLAKLAK) ₂ chimeric peptide
UNEL staining. b. a. Greater expansion of the same area as that of. c. TUNEL staining of negative control mice treated with 250 μg of an unconjugated mixture of SMSIARL (SEQ ID NO: 207) and _D (KLAKLAK) ₂ .

FIG. 8 shows survival of TRAMP mice treated with SMSIARL-GG- _D (KLAKLAK) ₂ , vehicle alone, _D (KLAKLAK) ₂ peptide alone, or SMSIARL peptide (SEQ ID NO: 207).

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 overlaid with 10 ⁹ TU SMSIARL phage (SEQ ID NO: 207) containing both normal and cancerous tissue and detected with anti-phage antibody. a is the appearance (x20), while b shows the details from panel a with a larger magnification (x40). c. Peroxidase staining as in panel a with phage lacking peptide insertion. d. Soluble SMSIA included in overlay
Peroxidase staining as in a with the RL peptide (SEQ ID NO: 207).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 43/00 105 C07K 7/06 C07K 7/06 7/08 7/08 14/00 14/00 A61K 37 / 02 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL , SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG BR, BY, BZ, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL , IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72 ) Inventor Arap, Wadich United States Texas 77025, Huston, Buffalo Speedway No. 328 7171 (72) Inventor Bredesen, Dale E. United States California 94949, Novato, Raccoon Drive Number 1 Sea 19 (72) Inventor Elby, H.C. Michael USA California 94947, Novato, Mayers Court 6F Term (reference) 4C084 AA02 AA07 BA01 BA08 BA17 BA18 BA19 BA41 DA27 DA41 NA14 ZA81 ZB26 ZB35 4H045 AA10 AA30 BA14 BA16 BA17 BA18 DA83

Claims (22)

[Claims] 1. A chimeric prostate homing proproapoptotic peptide comprising a prostatic homing peptide linked to an antimicrobial peptide, said chimeric peptide being selectively internalized by prostatic tissue and to said prostatic tissue. Chimeric peptide that exhibits high toxicity and has low mammalian cytotoxicity when the antimicrobial peptide is not linked to the prostate homing peptide. 2. The chimeric peptide of claim 1, wherein the prostate homing peptide comprises the sequence SMSIARL (SEQ ID NO: 207), or a functionally equivalent sequence. 3. The antimicrobial peptide has an amphipathic α-helix structure,
The chimeric peptide according to claim 1. 4. The chimeric peptide according to claim 1, wherein the antimicrobial peptide is: (KLAKLAK) ₂ (SEQ ID NO: 200); (KLAKKKA) ₂ (SEQ ID NO: 201); (KAAKKAA) ₂ (SEQ ID NO: 202); and (KLGKKLG) ₃ (SEQ ID NO: 203), a chimeric peptide comprising a sequence selected from the group consisting of: 5. The chimeric peptide of claim 1, wherein the antimicrobial peptide comprises the sequence _D (KLAKLAK) ₂ . 6. A chimeric peptide according to claim 5, comprising the sequence SMSIARL-GG- _D (KLAKLAK) ₂ . 7. Chimeric peptide according to claim 6, consisting of the sequence SMSIARL-GG- _D (KLAKLAK) ₂ . 8. A method of directing an antimicrobial peptide to prostate cancer in vivo, said method comprising the step of administering the chimeric peptide of claim 1. 9. The method of claim 8, wherein the prostate homing peptide comprises the sequence SMSIARL (SEQ ID NO: 207), or a functionally equivalent sequence. 10. The antimicrobial peptide comprises the sequence _D (KLAKLAK) ₂ .
The method of claim 8. 11. The chimeric peptide has the sequence SMSIARL-GG- _D (K
11. The method of claim 10, comprising LAKLAK) ₂ . 12. The chimeric peptide is SMSIARL-GG- _D (KLA).
The method of claim 11, wherein the method is KLAK) ₂ . 13. A method of inducing selective toxicity in prostate cancer in vivo, said method comprising the step of administering a chimeric peptide of claim 1 to a subject having prostate cancer. 14. The method of claim 13, wherein the prostate homing peptide comprises the sequence SMSIARL (SEQ ID NO: 207), or a functionally equivalent sequence. 15. The antimicrobial peptide comprises the sequence _D (KLAKLAK) ₂ .
The method according to claim 13. 16. The chimeric peptide has the sequence SMSIARL-GG- _D (K
16. The method of claim 15, comprising LAKLAK) ₂ . 17. The chimeric peptide is SMSIARL-GG- _D (KLA).
17. The method of claim 16, which is KLAK) ₂ . 18. A method of treating a patient having prostate cancer, said method comprising the step of administering to said patient the chimeric peptide of claim 1, whereby said chimeric peptide is present in said tumor. A method which is selective toxicity to. 19. The method of claim 18, wherein the prostate homing peptide comprises the sequence SMSIARL (SEQ ID NO: 207), or a functionally equivalent sequence. 20. The antimicrobial peptide comprises the sequence _D (KLAKLAK) ₂ .
The method according to claim 18. 21. The chimeric peptide has the sequence SMSIARL-GG- _D (K
21. The method of claim 20, comprising LAKLAK) ₂ . 22. The chimeric peptide is SMSIARL-GG- _D (KLA).
22. The method of claim 21, which is KLAK) ₂ .