WO2004020457A2

WO2004020457A2 - Methods and compositions for the inhibition of dna repair protein xrcc-3

Info

Publication number: WO2004020457A2
Application number: PCT/US2003/027321
Authority: WO
Inventors: Philip Connell; Douglas Bishop; Ralph Weichselbaum
Original assignee: Philip Connell; Douglas Bishop; Ralph Weichselbaum
Priority date: 2002-08-30
Filing date: 2003-08-29
Publication date: 2004-03-11
Also published as: WO2004020457A3; AU2003268340A8; AU2003268340A1

Abstract

Methods and compositions comprising peptides derived from Rad51C.

Description

METHODS AND COMPOSITIONS FOR THE INHIBITION OF DNA

REPAIR PROTEIN XRCC-3

Field of the Invention

The present invention relates generally to the field of cancer cell growth. It concerns methods and compositions for the inhibition of DNA repair protein XRCC-3. More particularly, such methods and compositions involve the use of peptides derived from the N-terminal region of RAD51C.

Background of the Invention

The statistics relating to cancer deaths and new cancer occurrence are staggering. The American Cancer Society publishes these statistics annually and they can be found at www.cancer.org. The National Cancer Institute estimates that approximately 8.9 million Americans with a history of cancer were alive in 1997 and further estimated that a further 1,268,000 new cancer cases would be diagnosed in 2001. The overall costs for cancer in the year 2000 were estimated at $180.2 billion: $60 billion for direct medical costs (total of all health expenditures); $15 billion for indirect morbidity costs (cost of lost productivity due to illness); and, $105.2 billion for indirect mortality costs (cost of lost productivity due to premature death). Traditional methods for cancer treatment rely on surgery, radiation, gene therapy, cytotoxic chemotherapeutic drugs or combinations of two or more of these treatment strategies. Although the treatment of tumor cells with cytotoxic chemicals is well known in the art, presently, the therapeutic effect of cytotoxic anti- cancer drugs and therapies is limited by lack of specificity of treatment and excessive toxicity of these treatment regimens in normal tissues. In addition, certain cancers are refractory to treatments such as chemotherapy and radiation alone. Moreover, some of these strategies, such as surgery for example, are not always viable alternatives.

Once the diagnosis of cancer is established, the most urgent question is whether the disease is localized, or has metastasized to lymph nodes and distant organs. In nearly 50% of patients, surgical excision of primary neopjasia is ineffective, because metastasis has occurred by the time the tumor is large enough for resection (Sugarbaker, Curr. Prob. Cancer 3:1-59, 1979; Fidler and Balch, Curr. Probl. Surg. 24:137-208, 1987). Metastases can be located in different organs and in different regions of the same organ, making complete eradication by surgery, radiation, drugs, or biotherapy difficult. Furthermore, the organ microenvironment significantly influences the response of tumor cells to therapy (Fidler, J. Natl. Cancer Inst., 87: 1588, 1995), as well as the efficiency of anticancer drugs, which must be delivered to tumor foci in amounts sufficient to destroy cells without leading to undesirable side effects (Fidler and Poste, Semin. Oncol. 12:207-221, 1985). In addition, the treatment of metastatic cancer is greatly hindered due to the biological heterogeneity of cancer cells, and the rapid emergence of tumor cells that become resistant to most conventional anticancer agents (Fidler and Poste, Semin. Oncol. 12:207-221, 1985).

Platinum-based chemotherapeutic agents are standard treatments for many common epithelial malignancies, including ovarian, lung, uterine cervix, and head/neck carcinomas. In head and neck carcinomas, for example, these drugs have single agent response rates of 20-30% and remain standard components of multi-agent regimens for recurrent/metastatic disease. Platinum-based drugs are also commonly combined with radiotherapy in the treatment of numerous advanced primary tumors. However, despite advances in systemic therapies survival rates remains relatively poor for many patients with locally advanced primary tumors and recurrent/metastatic disease.

Therapy of cancers that are refractory to traditional therapeutic intervention therapy can be enhanced by increasing the drug sensitivity and responsiveness of the given cancer to the therapeutic agent. One promising strategy involves directly transferring a "chemosensitization" or "suicide" gene encoding a prodrug activation enzyme to malignant cells, in order to confer sensitivity to otherwise innocuous agents (Moolten, Cancer Gene Therapy 1 :279-287 (1994); Freeman et al., Semin. Oncol. 23:31-45 (1996); Deonarain et al, Gene Therapy 2: 235-244 (1995)). For example, tumor cells transduced with herpes simplex virus thymidine kinase (HSV-TK) acquire sensitivity to ganciclovir, a clinically proven agent originally designed for treatment of viral infections. Moolten and Wells, J. Natl. Cancer Inst. 82:297-300, 1990; Ezzeddine et al., New Biol. 3:608-614, 1991). Another example of gene-based chemosensitization is the use of the bacterial gene cytosine deaminase (CD), which has been shown to sensitize tumor cells to the antifungal agent 5-fluorocytosine as a result of its transformation to 5-flurouracil, a known cancer chemotherapeutic agent. (Caruso et al.. Proc. Natl. Acad. Sci. USA 90:7024-7028 (1993); Oldfield et al., Hum. Gene Ther. 4: 39 (1993); Culver. Clin. Chem. 40: 510 (1994); O'Malley, Jr. et al, Cancer Res. 56:1737-1741 (1996); Rainov et al, Cancer Gene Therapy 3:99-106 (1996). However, these gene-based chemosensitization techniques are somewhat limited by technological inabilities to deliver genes to a population of tumor cells with efficiency and reproducibility.

Another approach that has been to employ peptides or other small molecules that can serve to enhance the sensitivity of a tumor cell to therapeutic intervention. For example, U.S. Patent No. 6,025,365 (incorporated herein by reference in its entirety), describes the use of a protein kinase C, chelerythrine. This alkaloid was found to decrease the apoptotic threshold in tumor cells, especially radioresistant cells, following ionizing radiation. The induction of apoptosis by the combination of chelerythrine and ionizing radiation is preceded by activation of both neutral and acidic sphingomyelinases. This induction of apoptosis by the combination of chelerythrine and ionizing radiation treatment occurred at doses much lower than would be expected based on conventional cancer therapy treatments.

Thus, it is possible to sensitize cancer cells to treatment using a variety of sensitizing agents. However, given the ever increasing occurrence of new cancers, many of which are refractory to existing cancer therapy methods, additional methods and compositions for cancer therapy are needed.

Summary of the Invention

The present application provides a teaching of an isolated peptide derived from the N-terminal of region of RAD51C wherein the peptide inhibits the sub-nuclear assembly of Rad51. Preferably, the peptide inhibits homologous recombination repair (HRR). More preferably, the peptide inhibits HRR in mammalian cells. In certain aspects, the peptide is one which binds XRCC-3. Alternatively, the peptide is one which binds to one or more of the components of the Rad51 complex selected from the group consisting of XRCC-3, XRCC-2, Rad51B, Rad51C and Rad51D. In preferred embodiments, the peptide is an anti-tumor agent.

In specific embodiments, the peptide comprises between about the first 10 and about the first 30 amino acids of the N-terminus of Rad51C. In particularly preferred embodiments, the peptide is a dodecapeptide. In other preferred embodiments, the peptide comprises amino acids 14-25 of RAD51C and further comprises a second peptide sequence at the C-terminal. More specifically, the second peptide sequence at the C-terminal may be a C-terminal transduction domain. In preferred embodiments, the C-terminal transduction domain comprises a sequence of YARAAARQARA.

In particularly preferred embodiments, the peptide comprises a sequence selected from the group consisting of the sequences depicted in Figure 3, and a variant of any of the sequences depicted in Figure 3 wherein the variant inhibits the sub-nuclear assembly of Rad51. The peptide may have a sequence of

LVSFPLSPAVRV, or alternatively is a variant of this sequence that binds to XRCC- 3.

The present application contemplates methods and compositions of making the above peptides. Further the present application also provides a fusion protein comprising the N-terminal region of RAD51 C fused at the C-terminal to a C- terminal transduction domain, wherein the fusion protein inhibits the subnuclear assembly of Rad51. The invention further contemplates a composition comprising any of the peptides and/or fusion proteins described herein and a pharmaceutically acceptable carrier, excipient or diluent. The invention further contemplates a polynucleotide comprising a nucleotide sequence that encodes a peptide or a fusion peptide of the present invention. Expression vectors comprising such a polynucleotide also are within the scope of the present invention. Such an expression vector may be further characterized in that it may comprise the polynucleotide operably linked to a promoter to promote expression of the protein encoded by the polynucleotide in a host cell.

Recombinant host cells transformed or transfected with the polynucleotides described herein also form part of the invention. The recombinant host cell also may be ones which are transformed or transfected with an expression vector described herein. The present invention further comprises methods of using the compositions of the present invention. In exemplary embodiments, the present invention provides a method for inhibiting the growth of a cell comprising contacting the cell with a peptide derived from the N-terminal of region of RAD51C wherein the peptide inhibits the sub-nuclear assembly of Rad51 and a chemotherapeutic DNA damaging agent, wherein the dose of the peptide, when combined with the dose of the DNA damaging agent, is effective to inhibit growth of the cell. In preferred embodiments, the peptide increases the cytotoxicity of the chemotherapeutic agent against the cell. Any conventional methods of treating a subject using peptide compositions may be used to augment the methods of the present invention. In preferred embodiments, the peptide is contacted with the cell prior to contacting the cell with the DNA damaging agent. In alternative embodiments, the DNA damaging agent is contacted with the cell prior to contacting the cell with the peptide. In particularly preferred embodiments, the cell is a cancer cell. More particularly, the cancer cell is a bladder cancer cell, a blood cancer, a breast cancer cell, a lung cancer cell, a colon cancer cell, a prostate cancer cell, a liver cancer cell, a pancreatic cancer cell, a stomach cancer cell, a testicular cancer cell, a brain cancer cell, an ovarian cancer cell, a lymphatic cancer cell, a skin cancer cell, a brain cancer cell, a bone cancer cell, a soft tissue cancer cell.

In preferred aspects of the invention, the cell is located in a human subject. In more particular embodiments, the peptide is contacted with the cell by being administered by direct intratumoral injection. More particularly, the peptide is administered by injection into tumor vasculature. Any DNA damaging agent that is conventionally used to control the growth of a cell may be used in conjunction with the present invention. In certain embodiments, the DNA damaging agent is a chemotherapeutic agent selected from a group consisting of doxorubicin, daunorubicin, dactinomycin, mitoxantrone, cisplatin, procarbazine, mitomycin, carboplatin, bleomycin, etoposide, teniposide, mechlroethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, ifosfamide, melphalan, hexamethylmelamine, thiopeta, busulfan, camustine, lomustine, semustine, streptozocin, dacarbazine, adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D, hydrogen peroxide, nitrosurea, plicomycin, tamoxifen, taxol, transplatinum, vincristin, vinblastin and methotrexate.

In other embodiments, the DNA damaging agent is ionizing radiation therapy. In preferred embodiments, the cell is contacted with the peptide multiple times. In other aspects the cell is contacted with the DNA damaging agent multiple times. The invention may employ the use of tumor resection, before during or concurrently with the application of the peptide and/or DNA damaging agent. Of course, the peptide and the DNA damaging agent may be delivered both before and after the tumor resection. The dose of the peptide will vary depending on the size, age and other characteristics of the individual being treated and also may vary according to the stage of the cancer. Preferably the dose of the peptide is about 1 mg/kg to about 100 mg/kg. More preferably the dose of the peptide is about 1 mg/kg to about 4 mg/kg.

Also contemplated is a method of killing a tumor cell comprising contacting the tumor cell with a peptide derived from the N-terminal of region of RAD51C wherein the peptide inhibits the sub-nuclear assembly of Rad51 and a chemotherapeutic DNA damaging agent, wherein the dose of the peptide, when combined with the dose of the DNA damaging agent, is effective to kill the tumor cell. Preferably the peptide is a dodecapeptide. More preferably the peptide comprises a sequence selected from the group consisting of the sequences depicted in Figure 3 or a variant of one of the sequences in Figure 3, wherein the variant inhibits sub-nuclear assembly of Rad51. the present invention further contemplates a method of treating cancer in a mammal comprising administering a peptide derived from the N-terminal of region of RAD51C wherein the peptide inhibits the sub-nuclear assembly of Rad51 and a chemotherapeutic DNA damaging agent to the mammal, wherein the dose of the peptide, when combined with the dose of the DNA damaging agent, is effective to treat the cancer.

In addition, the invention contemplates a method of potentiating the effect of a chemotherapeutic DNA damaging agent on a tumor cell comprising contacting the tumor cell with isolated peptide derived from the N-terminal of region of RAD51C wherein the peptide inhibits the sub-nuclear assembly of Rad51 and further contacting the tumor cell with the DNA damaging agent.

Brief Description of the Drawings

Figure 1. Western blotting shows expression of XRCC3p in CHO cells transfected with the human XRCC3 gene. Nuclear extracts (18 μg/lane) were separated on a 4-20% SDS page, and blotted with primary anti-XRCC3 (1 : 1000) and secondary HRP-conjugated anti-rabbit IgG (1 : 1000). Cells lines by lane are: l=irslsf + pCB6, 2=irslsf + pXR3 + pCB6, 3=PC3, 4=BT-20. B) XRCC3-deficient CHO cells are extremely sensitive to cisplatin, and transfection with human XRCC3 results in platinum resistance. Cells were plated overnight, treated with varying doses of cisplatin for one hour, and then allowed to grow in peptide-containing media for 12 days. The resulting colonies were stained with crystal violet and counted.

Figure 2: XRCC3 defects make tumors sensitive to ionizing radiation and to treatment with cisplatin. Xenograft tumors were induced in the hind limbs of nude mice by injection with 5x106 - 5x107 cells. These cells consisted of XRCC3- deficient CHO cells (irslsf + ρCB6) or the XRCC3p-expressing line (irslsf + pXR3 + pCB6). Tumors were allowed to grow for 1-2 weeks (volumes of 250-500 mm3). A) For the cisplatin experiment mice received 5 daily intra-peritoneal injections with 10 μg cisplatin (or PBS control). B: In the ionizing radiation (IR) experiment, tumors were treated with 3 Gy/day for 5 consecutive days.

Figure 3: XRCC3-binding peptide sequences were aligned to an N- terminal region of Rad51C (amino acids 14-25). Amino acids represented in red represent sites of sequence identity.

Figure 4: Clonogenic survival assays were performed as follows: cells were plated overnight, treated with cisplatin for 1 hour, and then allowed to grow in peptide-containing media for 12 days. The resulting colonies were stained with crystal violet and counted. Figure 4A) XRCC3-expressing CHO cells (irslsf + pXR3 + pCB6) were treated with DMEM media containing 25 μM cisplatin (or media alone) followed by varied concentrations of peptide. Figure 4B) XRCC3-expressing CHO cells (irslsf + pXR3 + pCB6) were treated with varied concentrations of cisplatin followed by peptide (0.5 μM peptide in DMEM + 10% heat-inactivated FBS). Figure 4C) XRCC3 -deficient CHO cells (irslsf + pCB6) were treated with varied concentrations of cisplatin followed by peptide (0.5 μM peptide in DMEM + 10% heat-inactivated FBS). Figure 5. Rad51C(14-25)-PTD4 inhibits sub-nuclear assembly of Rad51 following DNA damage. Cells were treated with cisplatin or radiation, incubated with peptide for 3 hours, trypsinized, and fixed with PFA. Cells were then transferred to microscope slides and stained with primary anti-hsRad51 and secondary FITC-conjugated anti-rabbit IgG antibodies. Cells were examined via confocal microscopy and FITC-staining Rad51 foci were quantified for each cell. Each nucleus was then plotted in ascending order, based on number of Rad51 foci. Figure 5 A) XRCC3-expressing CHO cells (irslsf + ρXR3 + pCB6) were treated with cisplatin-(50 μM in serum-free DMEM) for one hour prior to incubation with peptide. Figure 5B) PC3 cells were irradiated with 9 Gy immediately prior to incubation with peptide.

Detailed Description of the Preferred Embodiments of the Invention

Traditional treatments such as radiotherapy and/or chemotherapy have limited ability to treat certain cancers. Moreover, certain cancers may become resistant to traditional cancer therapies. As such, recent developments have led to the identification of agents that act as chemosensitizing agents. The present invention, for the first time shows that use of peptides derived from the N-terminus of Rad51C are capable of sensitizing cancer cells against therapeutic intervention. Methods and compositions for exploiting this discovery are discussed in further detail herein below.

The therapeutic compositions and methods of the present invention are based on findings that involve elements of a large complex involved in mammalian homologous recombination repair pathway. In order to facilitate a more complete understanding of the invention, the present section provides details of the mechanisms of operation of this pathway and the components of the complex involved.

The eukaryotic Rad51 protein is related to the prokaryotic RecA protein, and is the key protein facilitating both mitotic and meiotic homologous recombination (Bianco et al., Front. Biosci., 3, D570-D603, 1998). The E. coli RecA protein is known to play a central role in the prokaryotic response to DNA damage. There are five Rad51 -related proteins (or paralogs) in human cells: XRCC2 (Liu et al, Mol. Cell, 1, 783-793, 1998; Cartwright et al. Nucleic Acids Res., 26, 3084-

3089, 1998; Johnson et al, Nature, 401, 397-399, 1999), XRCC3 (Liu et al, Mol Cell, 1, 783-793, 1998; Tebbs et al., Proc. Natl Acad. Sci. USA, 92, 6354-6358, 1995; Pierce et al, Genes Dev., 13, 2633-2638, 1999), Rad51B/Rad51Ll (7-9), Rad51C/Rad51L2 (10) and Rad51D/Rad51L3 (9,11,12). These Rad51 paralogs share 20-30% sequence identity with Rad51 and with each other, and probably arose by gene duplication, followed by the development of new functions (for review see 13- 15). The Rad51 paralogs were first implicated in homologous recombinational repair (HRR) on the basis of their sequence similarity to Rad51. In addition, there is now extensive evidence for an important role in HRR from analyses with mutations in hamster and chicken DT40 cell lines.

Genetic studies have shown that cells require Xrcc3 protein (Xrcc3p) for DNA repair via the homologous recombination repair (HRR) pathway. Cells lacking Xrcc3p are, consequently, 50-100 fold more sensitive to cross-linking chemotherapeutic drugs. Based on these data, phage display was used to develop dodecapeptides that directly bind Xrcc3 protein and inhibit HRR. Two consecutive rounds of phage display were performed and identified several Xrcc3p-binding peptide sequences. Several of these peptides displayed sequence similarity to Rad51C protein, a known binding partner of Xrcc3p. This portion of Rad51Cp (amino acids 14-25) was synthesized in fusion with a membrane transduction domain. The resulting peptide, Rad51C(14-25)-PTD4, was delivered to Chinese hamster ovary (CHO) cells and shown to be capable of inhibiting sub-nuclear assembly of the central HRR protein, Rad51 , following DNA damage. Colony forming assays showed that the peptide also sensitized cells to the lethal effects cisplatin. Identical experiments performed with a scrambled version of this peptide as a negative control showed no effect on Rad51 focus formation or sensitivity to cisplatin. Furthermore, CHO cells defective in XRCC3p expression were unaffected by the Rad51C(14-25)-PTD peptide. The activity of the peptide was also tested on three human breast tumor lines MCF7, BT20, and MDA MB-231. Thus, the present invention identifies peptides that inhibit HRR by disrupting, inhibiting or otherwise abrogating the sub-nuclear assembly of the Rad51 complex. These peptides potentiate the effects of DNA damaging agents and as such are useful in combination with DNA damaging agents for the therapy of cancer- related disorders.

A. Peptide Production The peptides of the present invention are peptides derived from the N- terminal of region of RAD51C which inhibit the subnuclear assembly of the Rad51 complex. Such peptides may be fusion proteins or peptides that comprise the above- discussed region of RAD51C as part of their sequence, or they may be labeled or otherwise modified peptides in which the labeling or modification may be used to facilitate the purification of the peptide, detection of the peptide itself or a detection of the interaction of the peptide with the Rad51 complex. Exemplary modifications are described in further detail herein below.

The present invention particularly contemplates the generation fusion proteins or fusion polypeptides, of the N-terminal region of the RAD51C described above or identified according to the present invention. This fusion polypeptide generally has all or a substantial portion of the N-terminal region of the RAD51C (e.g., this portion of the peptide may comprise any or all of the amino acid residues between about the first 10 and about the first 30 amino acids of the N-terminus of Rad51C), linked at the N- and/or C-terminus, to all or a portion of a second or third polypeptide. In particularly preferred embodiments, the peptide may comprises amino acids 14 to 25 of the N-terminal region of Rad51C. However, it should be understood that this is merely an exemplary embodiment of the peptides of the present invention, and any peptide that is derived from a portion of the Rad51 C peptide is envisioned as part of the invention so long as the peptide is one which disrupts the assembly of the Rad51 complex. It is contemplated that the fusion polypeptide may be produced by recombinant protein production or indeed by automated peptide synthesis as discussed elsewhere in the specification.

General principles for designing and making fusion proteins are well known to those of skill in the art. For example, fusions typically employ leader sequences from other species to permit the recombinant expression of a protein or peptide in a heterologous host. Another useful fusion includes the addition of an immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion polypeptide. Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification. The recombinant production of these fusions is described in further detail elsewhere in the specification. Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.

Other particular embodiments further contemplate a tagged sequence as a component of the fusion polypeptides of the present invention. There are various commercially available fusion protein expression systems that may be used to provide a tagged sequence in this context of the present invention. Particularly useful systems include but are not limited to the glutathione S-transferase (GST) system (Pharmacia, Piscataway, NJ), the maltose binding protein system (NEB, Beverley, MA), the FLAG system (D3I, New Haven, CT), and the 6xHis system (Qiagen, Chatsworth, CA). These systems are capable of producing recombinant polypeptides bearing only a small number of additional amino acids, which are unlikely to affect the biologically relevant activity of the recombinant fusion protein. For example, both the FLAG system and the 6xHis system add only short sequences, both of which are known to be poorly antigenic and which do not adversely affect folding of the polypeptide to its native conformation. In addition to providing fusion polypeptides as already described, the invention provides fusion proteins or peptide substrates that are further modified to incorporate, for example, a label or other detectable moiety.

Preferred peptide substrates will comprise internally quenched labels that result in increased detectability after cleavage of the peptide substrates. The peptides may be labeled using labels well known to those of skill in the art, e.g., biotin labels are particularly contemplated. The use of such labels is well known to those of skill in the art and is described in, e.g., U.S. No. Patent 3,817,837; U.S. Patent No. 3,850,752; U.S. Patent No. 3,996,345 and U.S. Patent No. 4,277,437. Other labels that will be useful include but are not limited to radioactive labels, fluorescent labels and chemiluminescent labels. U.S. Patents concerning use of such labels include for example U.S. Patent No. 3,817,837; U.S. Patent No. 3,850,752; U.S. Patent No. 3,939,350 and U.S. Patent No. 3,996,345. Any of the peptides of the present invention may comprise one, two, or more of any of these labels. The present invention provides peptide compositions for use in the inhibition of the formation of the Rad51 complex. Such peptides may be produced by conventional automated peptide synthesis methods or by recombinant expression.

The peptides of the invention can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., Pierce Chemical Co., (1984);Tam et al., J. Am. Chem. Soc, 105:6442, (1983); Merrifield, Science, 232: 341-347, (1986); and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1-284, (1979), each incorporated herein by reference. The novel isolated peptides of the present invention comprise the N-terminal region of RAD51C and inhibit the formation of the Rad51 complex and/or XRCC-3; these peptides can be readily synthesized and then screened for such inhibition and/or binding activity screening assays. In particularly preferred methods, the peptides of the present invention were synthesized by solid-phase technology employing a Model 433A from Applied Biosystems Inc. The purity of any given peptide substrate, generate through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art. In preferred embodiments, the authenticity is established by mass spectrometry as described in the examples.

As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides that comprise peptide sequences of the invention.

A variety of expression vector/host systems may be utilized to contain and express the peptide or fusion polypeptide coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g.. cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the peptide substrates or fusion polypeptides in bacteria, yeast and other invertebrates are described herein below.

Expression vectors for use in prokaryotic hosts generally comprise one or more phenotypic selectable marker genes. Such genes generally encode, e.g., a protein that confers antibiotic resistance or that supplies an auxotrophic requirement. A wide variety of such vectors are readily available from commercial sources. Examples include pSPORT vectors, pGEM vectors (Promega), pPROEX vectors (LTI, Bethesda, MD), Bluescript vectors (Stratagene), pET vectors (Novagen) and pQE vectors (Qiagen). The DNA sequence encoding the given peptide substrate or fusion polypeptide is amplified by PCR and cloned into such a vector, for example, pGEX 3X (Pharmacia, Piscataway, NJ) designed to produce a fusion protein comprising glutathione S transferase (GST), encoded by the vector, and a protein encoded by a DNA fragment inserted into the vector's cloning site. The primers for the PCR may be generated to include for example, an appropriate cleavage site. Treatment of the recombinant fusion protein with thrombin or factor Xa (Pharmacia, Piscataway, NJ) is expected to cleave the fusion protein, releasing the substrate or substrate containing polypeptide from the GST portion. The pGEX 3X/peptide construct is transformed into E. coli XL 1 Blue cells (Stratagene, La Jolla CA), and individual transformants are isolated and grown. Plasmid DNA from individual transformants is purified and partially sequenced using an automated sequencer to confirm the presence of the desired peptide or polypeptide encoding nucleic acid insert in the proper orientation.

While certain embodiments of the present invention oontemplate producing the peptides or polypeptides using synthetic peptide synthesizers and subsequent FPLC analysis and appropriate refolding of the cysteine double bonds, it is contemplated that recombinant protein production also may be used to produce the peptide compositions. For example, induction of the GST/substrate fusion protein is achieved by growing the transformed XL 1 Blue culture at 37°C in LB medium (supplemented with carbenicillin) to an optical density at wavelength 600 nm of 0.4, followed by further incubation for 4 hours in the presence of 0.5 mM Isopropyl β-D Thiogalactopyranoside (Sigma Chemical Co., St. Louis MO).

The GST fusion protein, expected to be produced as an insoluble inclusion body in the bacteria, may be purified as follows. Cells are harvested by centrifugation; washed in 0.15 M NaCI, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1 mg/ml lysozyme (Sigma Chemical Co.) for 15 minutes at room temperature. The lysate is cleared by sonication, and cell debris is pelleted by centrifugation for 10 minutes at 12,000 X g. The fusion protein containing pellet is resuspended in 50 mM Tris, pH 8, and 10 mM EDTA, layered over 50% glycerol, and centrifuged for 30 min. at 6000 X g. The pellet is resuspended in standard phosphate buffered saline solution (PBS) free of Mg++ and Ca++. The fusion protein is further purified by fractionating the resuspended pellet in a denaturing SDS polyacrylamide gel (Sambrook et al., supra). The gel is soaked in 0.4 M KCl to visualize the protein, which is excised and electroeluted in gel running buffer lacking SDS. If the GST/RAD51C-derived peptide fusion protein is produced in bacteria as a soluble protein, it may be purified using the GST Purification Module (Pharmacia Biotech).

The fusion protein may be subjected to thrombin digestion to cleave the GST from the peptide. The digestion reaction (20-40 μg fusion protein, 20 30 units human thrombin (4000 U/mg (Sigma) in 0.5 ml PBS) is incubated 16 48 hrs. at room temperature and loaded on a denaturing SDS PAGE gel to fractionate the reaction products. The gel is soaked in 0.4 M KCl to visualize the protein bands. The identity of the protein band corresponding to the expected molecular weight of the desired peptide product may be confirmed by partial amino acid sequence analysis using an automated sequencer (Applied Biosystems Model 473A, Foster City, CA).

Alternatively, the DNA sequence encoding the predicted peptide containing fusion polypeptide may be cloned into a plasmid containing a desired promoter and, optionally, a leader sequence (see, e.g., Better et al., Science, 240: 1041 43, 1988). The sequence of this construct may be confirmed by automated sequencing. The plasmid is then transformed into E. coli using standard procedures employing CaC12 incubation and heat shock treatment of the bacteria (Sambrook et al., supra). The transformed bacteria are grown in LB medium supplemented with carbenicillin, and production of the expressed protein is induced by growth in a suitable medium. If present, the leader sequence will effect secretion of the mature peptide and be cleaved during secretion.

The secreted recombinant protein is purified from the bacterial culture media by the method described herein throughout.

Similarly, yeast host cells from genera including Saccharomyces, Pichia, and Kluveromyces may be employed to generate the recombinant peptide. Preferred yeast hosts are S. cerevisiae and P. pastoris. Yeast vectors will often contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Vectors replicable in both yeast and E. coli (termed shuttle vectors) may also be used. In addition to the above-mentioned features of yeast vectors, a shuttle vector will also include sequences for replication and selection in E. coli. Direct secretion of polypeptides expressed in yeast hosts may be accomplished by the inclusion of nucleotide sequence encoding the yeast I-factor leader sequence at the 5' end of the substrate-encoding nucleotide sequence. Generally, a given substrate may be recombinantly expressed in yeast using a commercially available expression system, e.g., the Pichia Expression System (Invitrogen, San Diego, CA), following the manufacturer's instructions. This system also relies on the pre pro alpha sequence to direct secretion, but transcription of the insert is driven by the alcohol oxidase (AOX1) promoter upon induction by methanol. The secreted recombinant substrate is purified from the yeast growth medium by, e.g., the methods used to purify substrate from bacterial and mammalian cell supematants.

Alternatively, a synthetic DNA encoding the peptide of the invention may be cloned into the baculovirus expression vector pVL1393 (PharMingen, San Diego, CA; Luckow and Summers, Bio/Technology 6:47 (1988)). ,

In yet another alternative, the peptide-based therapeutic compositions of the present invention may be expressed in an insect system. Insect systems for protein expression are well known to those of skill in the art. In one such system. Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The substrate coding sequence is cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of substrate will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which the substrate is expressed (Smith et al, J Virol 46: 584, 1983; Engelhard EK et al, Proc Nat Acad Sci USA \ : 3224-7, 1994).

Mammalian host systems for the expression of recombinant proteins also are well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.

It is preferable that the transformed cells are used for long-term, high- yield protein production and as such stable expression is desirable. Once such cells are transformed with vectors that contain selectable markers along with the desired expression cassette, the cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The selectable marker is designed to confer resistance to selection and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell.

A number of selection systems may be used to recover the cells that have been transformed for recombinant protein production. Such selection systems include, but are not limited to, HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase genes, in tk-. hgprt- or aprt- cells, respectively. Also, anti-metabolite resistance can be used as the basis of selection for dhfr, that confers resistance to methotrexate; gpt, that confers resistance to mycophenolic acid; neo, that confers resistance to the aminoglycoside G418; als which confers resistance to chlorsulfuron; and hygro, that confers resistance to hygromycin. Additional selectable genes that may be useful include trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine. Markers that give a visual indication for identification of transformants include anthocyanins, D glucuronidase and its substrate, GUS, and luciferase and its substrate, luciferin.

In certain aspects of the present invention, it may be necessary to express the peptide substrates or the peptide substrate fusion proteins of the present invention. To achieve such expression, the present invention will employ vectors comprising polynucleotide molecules for encoding the peptide substrates or the fusion proteins of the present invention, as well as host cell transformed with such vectors. Such polynucleotide molecules may be joined to a vector, which generally includes a selectable marker and an origin of replication, for propagation in a host. These elements of the expression constructs used in the present invention are described in further detail herein below. The expression vectors include DNA encoding any of the given peptide or described above or below, operably linked to suitable transcriptional or translational regulatory sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, mRNA ribosomal binding sites, and appropriate sequences which control transcription and translation.

The terms "expression vector," "expression construct " or "expression cassette " are used interchangeably throughout this specification and are meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.

The choice of a suitable expression vector for expression of the peptides or polypeptides of the invention will of course depend upon the specific host cell to be used, and is within the skill of the ordinary artisan. Expression vectors for use in mammalian host cells may include transcriptional and translational control sequences derived from viral genomes. Commonly used promoter sequences and enhancer sequences which may be used in the present invention include, but are not limited to, those derived from human cytomegalovirus (CMV), Adenovirus 2, Polyoma virus, and Simian virus 40 (SV40). Methods for the construction of mammalian expression vectors are disclosed, for example, in Okayama and Berg (Mol. Cell Biol. 3:280 (1983)); Cosman et al. (Mol Immunol. 23:935 (1986)); Cosman et al. (Nature 312:768 (1984)); EP-A-0367566; and WO 91/18982. The expression construct will comprise a nucleic acid region that encodes the particular peptide of the present invention. Coding regions for use in constructing such expression vectors should encode at least from about amino acid 10 to about amino acid 30 of the N-terminal region of the RAD51C, although it is contemplated that larger polypeptides may be encoded as long as one the peptide generated comprises a from about amino acid 10 to about amino acid 30 of the RAD51C N-terminus.

In certain aspects of the present invention, the expression construct may further comprise a selectable marker that allows for the detection of the expression of the peptide or polypeptide. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, neomycin, puromycin, hygromycin, DHFR, zeocin and histidinol. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) (eukaryotic), β-galactosidase, luciferase, or chloramphenicol acetyltransferase (CAT) (prokaryotic) may be employed. Immunologic markers also can be employed. For example, epitope tags such as the FLAG system (IBI, New Haven, CT), HA and the 6xHis system (Qiagen, Chatsworth, CA) may be employed. Additionally, glutathione S-transferase (GST) system (Pharmacia, Piscataway, NJ), or the maltose binding protein system (NEB, Beverley, MA) also may be used. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art. Particularly prefeπed selectable markers that may be employed in the present invention are neomycin resistance or a GFP marker. Expression requires that appropriate signals be provided in the vectors. The present section includes a discussion of various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that may be used to drive expression of the nucleic acids of interest in host cells. Elements designed to optimize messenger RNA stability and translatability in host cells also are defined. The conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products also are provided, as is an element that links expression of the drug selection markers to expression of the mutant phenotype. In preferred embodiments, the nucleic acid encoding the given peptide or the nucleic acid encoding a selectable marker is under transcriptional control of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the DNA encoding the peptide substrate or the fusion polypeptide. Thus, a promoter nucleotide sequence is operably linked to a given DNA sequence if the promoter nucleotide sequence directs the transcription of the sequence. Similarly, the phrase "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins. At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

The particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of directing the expression of the nucleic acid in the targeted cell. Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include either a human or viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, β-actin, rat insulin promoter, the phosphoglycerol kinase promoter and glyceraldehyde-3-phosphate dehydrogenase promoter, all of which are promoters well known and readily available to those of skill in the art, can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized.

Selection of a promoter that is regulated in response to specific physiologic or synthetic signals can permit inducible expression of the gene product. Several inducible promoter systems are available for production of viral vectors. One such system is the ecdysone system (Invitrogen, Carlsbad, CA), which is designed to allow regulated expression of a gene of interest in mammalian cells. Another useful inducible system is the Tet-Off™ or Tet-On™ system (Clontech. Palo Alto, CA). In mammalian cells, the CMV immediate early promoter if often used to provide strong transcriptional activation. Modified versions of the CMV promoter that are less potent have also been used when reduced levels of expression of the transgene are desired. Retroviral promoters such as the LTRs from MLV or MMTV are contemplated to be useful in the present invention. Other viral promoters that may be used include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters such as from the El A, E2A, or MLP region, AAV LTR, cauliflower mosaic virus, HSV- TK, and avian sarcoma virus.

In some embodiments, regulatable promoters may prove useful. Such promoters include for example, those that are hormone or cytokine regulatable. Hormone regulatable promoters include MMTV, MT-1, ecdysone and RuBisco as well as other hormone regulated promoters such as those responsive to thyroid, pituitary and adrenal hormones.

Another regulatory element contemplated for use in the present invention is an enhancer. These are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization. Enhancers useful in the present invention are well known to those of skill in the art and will depend on the particular expression system being employed (Scharf D et al (1994) Results Probl Cell Differ 20: 125-62; Bittner et al (1987) Methods in Enzymol 153: 516-544). Where an expression construct employs a cDNA insert, one will typically desire to include a polyadenylation signal sequence to effect proper polyadenylation of the gene transcript. Any polyadenylation signal sequence recognized by cells of the selected transgenic animal species is suitable for the practice of the invention, such as human or bovine growth hormone and SV40 polyadenylation signals.

Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences. The termination region which is employed primarily will be one selected for convenience, since termination regions for the applications such as those contemplated by the present invention appear to be relatively interchangeable. The termination region may be native with the transcriptional initiation, may be native to the DNA sequence of interest, or may be derived for another source. In certain embodiments of the invention, the use of internal ribosome entry site (IRES) elements is contemplated to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5' methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, Nature, 334:320 325, 1988). IRES elements from two members of the picomavirus family (poliovirus and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988 supra), as well an IRES from a mammalian message (Macejak and Samow, Nature, 353:90 94, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker. B. Protein Purification

It will be desirable to purify the peptides of the present invention. Protein purification techniques are well known to those of skill in the art. These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the peptide or polypeptides of the invention from other proteins, the polypeptides or peptides of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion- exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography (FPLC) or even high performance liquid chromatography (HPLC). Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded polypeptide, protein or peptide. The term "purified polypeptide, protein or peptide" as used herein, is intended to refer to a composition, isolated from other components, wherein the polypeptide, protein or peptide is purified to any degree relative to its naturally- obtainable state. A purified polypeptide, protein or peptide therefore also refers to a polypeptide, protein or peptide, free from the environment in which it may naturally occur.

Generally, "purified" will refer to a polypeptide, protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the polypeptide, protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition. Various methods for quantifying the degree of purification of the polypeptide, protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "- fold purification number." The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed polypeptide, protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified polypeptide, protein or peptide.

There is no general requirement that the polypeptide, protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al, Biochem. Biophys. Res. Comm., 76:425, 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary. C. Therapeutic Applications of Peptides of the Invention

Tumor cell resistance to DNA damaging agents represents a major problem in clinical oncology. One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy. One way is by combining such traditional therapies with gene therapy. For example, the herpes simplex-thymidine kinase (HS-tA) gene, when delivered to brain tumors by a retroviral vector system, successfully induced susceptibility to the antiviral agent ganciclovir (Culver et al, 1992). One embodiment of the present invention, it is contemplated that the peptide compositions of the present invention may be administered in conjunction with chemo- or radiotherapeutic intervention, immunotherapy, or with any other therapy conventionally employed in the treatment of cancer.

To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of tumor cells, using the methods and compositions of the present invention, one would generally contact a "target" cell, a tumor or its vasculature with the peptide-based therapeutic compositions of the present invention and at least one other agent. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cancer by killing and/or inhibiting the proliferation of the cancer cells. This process may involve contacting the cells with the peptide-based composition and the agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the peptide-based therapeutic compositions of the present invention and the other includes the second agent.

Alternatively, the therapeutic treatment employing the peptide-based compositions described herein may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agent and peptide-based composition are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and the viral-based therapeutic would still be able to exert an advantageously combined effect. In such instances, it is contemplated that one would administer both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations. Repeated treatments with one or both agents is specifically contemplated. In specific embodiments, an anti-cancer therapy may be delivered which directly attacks the cancer cells in a manner to kill, inhibit or necrotize the cancer cell, in addition a therapeutic composition based on the peptide-based compositions of the present invention also is administered to the individual in amount effective to have an apoptotic, cell killing, or growth retarding effect. The peptide-based therapeutic compositions of the present invention may be administered following the other anti-cancer agent, before the other anti-cancer agent or indeed at the same time as the other anti-cancer agent.

Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell. Such agents and factors include radiation and waves that induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, also described as "chemotherapeutic agents," function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. Chemotherapeutic agents contemplated to be of use, include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide. The invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.

In treating cancer according to the invention, one would contact the tumor cells and/or the endothelia of the tumor vessels with an agent in addition to the therapeutic peptide-based therapeutic compositions of the present invention. This may be achieved by irradiating the localized tumor site with radiation such as X-rays, UV-light, *-rays or even microwaves. Alternatively, the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or cisplatin. Kinase inhibitors also are contemplated to be useful in combination therapies with the peptide-based therapeutic compositions of the present invention.

Agents that directly cross-link nucleic acids, specifically DNA. are envisaged to facilitate DNA damage leading to a synergistic, antineoplastic combination with the based therapy. Agents such as cisplatin, and other DNA alkylating agents may be used. Cisplatin has been widely used to treat cancer, with efficacious doses used in clinical applications of 20 mg/m^ for 5 days every three weeks for a total of three courses. Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.

Agents that damage DNA also include compounds that interfere with DNA replication, mitosis and chromosomal segregation. Such chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m^ at 21 day intervals for adriamycin, to 35-50 mg/m^ for etoposide intravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of nucleic acid precursors and subunits also lead to DNA damage. As such a number of nucleic acid precursors have been developed. Particularly useful are agents that have undergone extensive testing and are readily available. As such, agents such as 5-fluorouracil (5-FU), are preferentially used by neoplastic tissue, making this agent particularly useful for targeting to neoplastic cells. Although quite toxic, 5-FU, is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg/kg/day being commonly used.

By way of example the following is a list of chemotherapeutic agents and the cancers which have been shown to be managed by administration of such agents. Combinations of these chemotherapeutics with the peptide-based therapeutic compositions of the present invention may prove to be useful in amelioration of various neoplastic disorders. Examples of these compounds include adriamycin (also known as doxorubicin), VP-16 (also known as etoposide), and the like, daunorubicin (intercalates into DNA, blocks DNA-directed RNA polymerase and inhibits DNA synthesis); mitomycin (also known as mutamycin and or mitomycin-C) is an antibiotic isolated from the broth of Streptomyces caespitosus which has been shown to have antitumor activity; Actinomycin D also may be a useful drug to employ in combination with the peptides of the present invention because tumors which fail to respond to systemic treatment sometimes respond to local perfusion with dactinomycin which also is known to potentiate radiotherapy. It also is used in combination with primary surgery, radiotherapy, and other drugs, particularly vincristine and cyclophosphamide and has been found to be effective against Ewing's tumor, Kaposi's sarcoma, and soft-tissue sarcomas, choriocarcinoma, metastatic testicular carcinomas, Hodgkin's disease and non-Hodgkin's lymphomas.

Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus, is effective in the management of the following neoplasms either as a single agent or in proven combinations with other approved chemotherapeutic agents in squamous cell carcinoma such as head and neck (including mouth, tongue, tonsil, nasopharynx, oropharynx, sinus, palate, lip, buccal mucosa, gingiva, epiglottis, larynx), skin, penis, cervix, and vulva. It has also been used in the treatment of lymphomas and testicular carcinoma.

Cisplatin has been widely used to treat cancers such as metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other tumors and may be a useful combination with the peptide-based therapeutic compositions of the present invention. VP16 (etoposide) and is used primarily for treatment of testicular tumors, in combination with bleomycin and cisplatin, and in combination with cisplatin for small-cell carcinoma of the lung. It is also active against non-Hodgkin's lymphomas, acute nonlymphocytic leukemia, carcinoma of the breast, and Kaposi's sarcoma associated with acquired immunodeficiency syndrome (AIDS). Tumor Necrosis Factor [TNF; Cachectin] glycoprotein that kills some kinds of cancer cells, activates cytokine production, activates macrophages and endothelial cells, promotes the production of collagen and collagenases, is an inflammatory mediator and also a mediator of septic shock, and promotes catabolism, fever and sleep. TNF can be quite toxic when used alone in effective doses, so that the optimal regimens probably will use it in lower doses in combination with other drugs. Its immunosuppressive actions are potentiated by γ- interferon, so that the combination potentially is dangerous. A hybrid of TNF and interferon-α also has been found to possess anti-cancer activity.

Taxol an antimitotic agent original isolated from the bark of the ash tree, Taxus brevifolia, and its derivative paclitaxol have proven useful against breast cancer and may be used in the combination therapies of the present invention. Beneficial responses to vincristine have been reported in patients with a variety of other neoplasms, particularly Wilms' tumor, neuroblastoma, brain tumors, rhabdomyosarcoma, and carcinomas of the breast, bladder, and the male and female reproductive systems. Vinblastine also is indicated as a useful therapeutic in the same cancers as vincristine. The most frequent clinical use of vinblastine is with bleomycin and cisplatin in the curative therapy of metastatic testicular tumors. It is also active in Kaposi's sarcoma, neuroblastoma, and Letterer-Siwe disease (histiocytosis X), as well as in carcinoma of the breast and choriocarcinoma in women.

Melphalan also known as alkeran, L-phenylalanine mustard, phenylalanine mustard, L-PAM, or L-sarcolysin, is a phenylalanine derivative of nitrogen mustard. Melphalan is a bifunctional alkylating agent which is active against selective human neoplastic diseases. Melphalan is the active L-isomer of the D-isomer, known as medphalan, which is less active against certain animal tumors, and the dose needed to produce effects on chromosomes is larger than that required with the L-isomer. Melphalan is available in form suitable for oral administration and has been used to treat multiple myeloma. Available evidence suggests that about one third to one half of the patients with multiple myeloma show a favorable response to oral administration of the drug. Melphalan has been used in the treatment of epithelial ovarian carcinoma. Cyclophosphamide is stable in the gastrointestinal tract, tolerated well and effective by the oral and parental routes and does not cause local vesication, necrosis, phlebitis or even pain. Chlorambucil, a bifunctional alkylating agent of the nitrogen mustard type that has been found active against selected human neoplastic diseases. Chlorambucil is indicated in the treatment of chronic lymphatic (lymphocytic) leukemia, malignant lymphomas including lymphosarcoma, giant folhcular lymphoma and Hodgkin's disease. It is not curative in any of these disorders but may produce clinically useful palliation. Other factors that cause DNA damage and have been used extensively include what are commonly known as γ- rays, X-rays, and/or the directed delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also contemplated such as microwaves and UV-irradiation. It is most likely that all of these factors effect a broad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.

The skilled artisan is directed to "Remington's Pharmaceutical Sciences" 15th Edition, chapter 33, in particular pages 624-652. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.

The inventors propose that the regional delivery of peptide-based therapeutic compositions of the present invention to patients with therapy resistant cancers will be a very efficient method for counteracting the clinical disease.

Similarly, the chemo- or radiotherapy may be directed to a particular, affected region of the subject's body. Alternatively, systemic delivery of the peptide-based therapeutic compositions of the present invention and/or the agent may be appropriate in certain circumstances, for example, where extensive metastasis has occurred. In addition to combining the peptide-based therapeutic compositions of the present invention with chemo- and radiotherapies, it also is contemplated that combination with gene therapies will be advantageous. For example, targeting of peptide-based therapeutic compositions of the present invention and a tumor suppressor gene at the same time may produce an improved anti-cancer treatment. Any other tumor-related gene conceivably can be targeted in this manner, for example, p21, Rb, APC, DCC, NF-1, NF-2, BCRA2, pl6, FHIT, WT-1 , MEN-I, MEN-II, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf, erb, src.fms un, trk, ret, gsp, hst, bcl and abl. In addition to the anticancer therapeutics discussed above, it is contemplated that the peptide compositions of the invention may be combined with angiogenesis inhibitors, http://cancertrials.nci.nih.gov/news/angio is a website maintained by the National Institutes of Health which provides current information on the trials presently being conducted with anti-angiogenic agents. These agents include, for example, Marimastat (British Biotech, Annapolis MD; indicated for non-small cell lung, small cell lung and breast cancers); AG3340 (Agouron, LaJolla, CA; for glioblastoma multiforme); COL-3 (Collagenex, Newtown PA; for brain tumors); Neovastat (Aeterna, Quebec, Canada; for kidney and non-small cell lung cancer) BMS-275291 (Bristol-Myers Squibb, Wallingford CT; for metastatic non-small cell ling cancer); Thalidomide (Celgen; for melanoma, head and neck cancer, ovarian, metastatic prostate, and Kaposi's sarcoma; recurrent or metastatic colorectal cancer (with adjuvants); gynecologic sarcomas, liver cancer; multiple myeloma; CLL, recurrent or progressive brain cancer, multiple myeloma, non-small cell lung, nonmetastatic prostate, refractory multiple myeloma, and renal cancer);

Squalamine (Magainin Pharmaceuticals Plymouth Meeting, PA; non-small cell cancer and ovarian cancer); Endostatin (EntreMEd, Rockville, MD; for solid tumors); SU5416 (Sugen, San Francisco, CA; recurrent head and neck, advanced solid tumors, stage MB or IV breast cancer; recurrent or progressive brain (pediatric); Ovarian, AML; glioma, advanced malignancies, advanced colorectal, von-Hippel Lindau disease, advanced soft tissue; prostate cancer, colorectal cancer, metastatic melanoma, multiple myeloma, malignant mesothelioma: metastatic renal, advanced or recurrent head and neck, metastatic colorectal cancer); SU6668 (Sugen San Francisco, CA; advanced tumors); interferon-α; Anti-VEGF antibody (National Cancer Institute, Bethesda MD; Genentech San Franscisco, CA; refractory solid tumors; metastatic renal cell cancer, in untreated advanced colorectal); EMD121974 (Merck KCgaA, Darmstadt, Germany; HIV related Kaposi's Sarcoma, progressive or recurrent Anaplastic Glioma ); Interleukin 12 (Genetics Institute, Cambridge, MA; Kaposi's sarcoma) and IM862 (Cytran, Kirkland, WA; ovarian cancer, untreated metastatic cancers of colon and rectal origin and Kaposi's sarcoma). The parenthetical information following the agents indicates the cancers against which the agents are being used in these trials. It is contemplated that any of these disorders may be treated with the peptide-based compositions of the present invention either alone or in combination with the agents listed. In order to prepare the therapeutic peptide-based compositions for clinical use, it will be necessary to prepare the therapeutic compositions as pharmaceutical compositions, i.e., in a form appropriate for in vivo applications. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient. Aqueous compositions of the present invention comprise an effective amount of the peptide- based therapeutic composition, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.

The active compositions of the present invention include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. The pharmaceutical compositions may be introduced into the subject by any conventional method, e.g., by intravenous, intradermal, intramusclar, intramammary, intraperitoneal, intrathecal, retrobulbar, intravesicular, intrapulmonary (e.g., term release); by sublingual, nasal, anal, vaginal, or transdermal delivery, or by surgical implantation at a particular site. The treatment may consist of a single dose or a plurality of doses over a period of time.

The active compounds may be prepared for administration as solutions of free base or pharmacologically acceptable salts in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. For oral administration the compositions of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants. The compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups also can be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.

"Unit dose" is defined as a discrete amount of a therapeutic composition dispersed in a suitable carrier. Parenteral administration may be carried out with an initial bolus followed by continuous infusion to maintain therapeutic circulating levels of drug product. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient. The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the routes of administration. The optimal pharmaceutical formulation will be determined by one of skill in the art depending on the route of administration and the desired dosage. See for example Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publ. Co, Easton PA 18042) pp 1435-1712, incorporated herein by reference. Such formulations may influence the physical state, stability, rate of in vivo release and rate oϊin vivo clearance of the administered agents. Depending on the route of administration, a suitable dose may be calculated according to body weight, body surface areas or organ size. Further refinement of the calculations necessary to determine the appropriate treatment dose is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein as well as the pharmacokinetic data observed in animals or human clinical trials.

Appropriate dosages may be ascertained through the use of established assays for determining blood clotting levels in conjunction with relevant dose-response data. The final dosage regimen will be determined by the attending physician, considering factors that modify the action of drugs, e.g., the drug's specific activity, severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, further information will emerge regarding appropriate dosage levels and duration of treatment for specific diseases and conditions.

In gene therapy embodiments employing viral delivery of the peptide- based compositions, the unit dose may be calculated in terms of the dose of viral particles being administered. Viral doses include a particular number of virus particles or plaque forming units (pfu). For embodiments involving virus, particular unit doses include 10³, 10⁴, 10⁵, 10 ⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10^{1 1}, 10¹², 10 ¹³ or 10^4 pf_u. Particle doses may be somewhat higher (10 to 100-fold) due to the presence of infection defective particles.

It will be appreciated that the pharmaceutical compositions and treatment methods of the invention may be useful in fields of human medicine and veterinary medicine. Thus the subject to be treated may be a mammal, preferably human or other animal. For veterinary purposes, subjects include for example, farm animals including cows, sheep, pigs, horses and goats, companion animals such as dogs and cats, exotic and/or zoo animals, laboratory animals including mice rats, rabbits, guinea pigs and hamsters; and poultry such as chickens, turkey ducks and geese.

D. Examples

The present invention is described in more detail with reference to the following non-limiting examples which represent preferred embodiments of the invention. Those of skill in the art will understand that the techniques described in these examples represent techniques described by the inventors to function well in the practice of the invention, and as such constitute preferred modes for the practice thereof. However, it should be appreciated that those of skill in the art should in light of the present disclosure, appreciate that many changes can be made in the specific methods which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1: Materials & Methods

Cell lines and plasmids: The mammalian expression vector encoding cDNA for human XRCC3 (pXR3-10), a bacterial expression plasmid encoding 6xHis- tagged human XRCC3 (pET29-XR3), and CHO cell lines (AA8 and irslsf) were provided by Dr. Larry Thompson. The human breast carcinoma cell line BT-20 was provided by Dr. Suzanne Conzen. Both cell lines were maintained in Dulbeccos's modified eagle's medium (DMEM) (Gibco), 10% fetal bovine serum (FBS) (Intergen), and 100 units/ml penicillin and streptomycin (pen/strep) jn a CO₂ incubator at 37°C. The human prostate carcinoma cell line PC3 was purchased from ATCC, and was maintained in DMEM/F12 media (Gibco) containing 10% FBS and pen strep.

Cell transfection: The irslSF were stably transfected with human XRCC3 by lipofection. Briefly, irslSF cells were plated at 1x10^ cells/ well in a six well tissue culture plate in 75% DMEM 25% F-12 with 10% FBS. The cells were incubated at 37° in a CO₂ incubator until cells were 70% confluent. The cells were transfected with the pXR3-10 plasmid (2 ug) in OPTI-MEM I Reduced Serum Medium (Gibco BRL) with Lipofectin Reagent (Gibco BRL). Cells were co- transfected with a neomycin-resistant plasmid, pCB6 (0.23 ug). The neomycin- resistant plasmid was used as a vector control. After 2 hours, the transfection mixture was replaced with 75%DMEM/25% F-12 with 10% FBS. At 24 hours post transfection, the cells were harvested and transferred to 100 mm tissue culture dishes. The cells were selected with Genetacin (0.71 ug/ml, Sigma).

Antibodies: Rabbit polyclonal anti-hsRad51 antiserum was a gift of Akira Shinohara. The antibodies were purified from the serum using a HiTrap protein-A column (Amersham Pharmacia), followed by an affinity purification step.

Antiserum was also generated in rabbits (Convance Research Products) against His- tagged XRCC protein and purified with a HiTrap protein-A column (Pharmacia).

Other primary antibodies included anti-hsXRCC3 polyclonal (Novus Biologicals), and anti-hsHistone Hl(AE-4) monoclonal (Santa Cruz). Secondary antibodies included FITC-conjugated anti-rabbit IgG antibodies (Molecular Probes), HRP- conjugated anti-rabbit IgG (Santa Cruz), and HRP-conjugated anti-mouse IgG (Santa

Cruz).

Preparation of 6xHis-tagged XRCC3 protein: Electro-competent BL21(DE3)pLysS bacteria (Novagen) were electoporated with pET29-XR3, grown in LB (with 20ug/ml kanamycin and 20 ug/ml chloramphenicol) to an OD₅₄₀=0.3, and induced with 1 mM IPTG for 2 hours. Cells were centrifuged, resuspended in lysis buffer (8 M urea, 150 mM NaCI, 20 mM Tris pH-7.4), sonicated, and again centrifuged to pellet insoluble debris. To purify the 6xHis-tagged protein, the supernatent was incubated with nickel affinity resin (Clontech). The resin was subsequently washed three times with lysis buffer and once with lysis buffer containing 5 mM imidazole. Protein was eluted with lysis buffer containing 50 mM imidazole. In an attempt to promote partial protein refolding, the urea concentration was gently reduced by stepwise dialysis. The purified protein was poorly soluble at urea concentrations < 2 M, therefore it was stored in 2.5 M urea. Analysis on coomassie-stained SDS page showed a purity of > 95%.

Preparation of nuclear extracts and western blots: Nuclear extracts were prepared from CHO, PC3, and BT-20 cell lines as previously described (paper). Briefly, 1 X 10⁶ cells were scraped into 1.5 ml ice cold PBS and quickly microcentrifuged. The cell pellet was resuspended in 400 ul of buffer A ( 10 mM hepes pH-7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, and protease inhibitors), incubated on ice for 10 minutes, vortex ed for 10 seconds, and again microcentrifuged. Nuclei were resuspended in 100 ul of buffer C (20 mM hepes pH-7.9, 25% glycerol, 420 mM NaCI, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, and protease inhibitors), incubated on ice for 20 minutes, and microcentrifuged. The supernatant was collected, yielding 75-200 ug of protein per cell line. Nuclear extracts (20μg) were separated with a 4-20% gradient acrylamide SDS-PAGE, electroblotted onto PVDF membranes (Millipore), and blocked for 1 hr (TBS, 0.1 % tween-20, 5% dried milk). Blots were then incubated with the appropriate primary antibody at 1 : 1000 dilution for 1 hr, washed, incubated with a 1 : 1000 dilution of peroxidase-conjugated secondary antibody for 1 hr, and developed by enhanced chemo-luminescence (NEN Life Science Products). Phage display selection of XRCC3-binding peptides: Phage display was performed using the Ph.D.- 12 library (New England Biolabs). Our methods were similar to those described by Scott and Smith {Scott, 1990 #125} with some modifications. High-binding microtiter wells (Costar) were filled with lOOul of PBS and subsequently coated with 6xHis-XRCC3 protein by adding 2.5 ug of the partially renatured protein (stored in 2.5 M urea). Wells were blocked for 1 hour with 300 ul of blocking buffer (TBS, 0.1% tween-20, 5% dried milk) and washed five times with TBST. The coated wells were then incubated with phage (1 X 10^{1 1} pfu/well) for 30 minutes. Non-binding clones were washed away by 10 washes with TBST. The XRCC3-binding clones were then selectively competed off the coated wells using anti-XRCC3 polyclonal antibodies ( 100 ug/ml) in 100 ul TBST. Eluted phage were tittered in ER2738 bacteria, according to methods outlined in the Ph.D. -12 manual. For solid-phase amplification the eluted phage were infected into mid-log phase ER2738 bacteria, diluted into 10 ml of top agarose, and plated onto 150mm plates (6 x 10⁴ pfu plate). Overnight incubation at this plating density results in small plaques that only rarely overlap. On the following day, 10 ml of TBST were added to each plate. The amplified phage were harvested by incubation at 4° with gentle rocking and purified by PEG precipitation. These amplified phage served as the library for the second round of this 'biopanning' procedure. Each round of biopanning was performed using a control well coated with BSA. Enrichment is defined as the titer eluted from 6xHis-XRCC3 coated well divided by titer eluted from the BSA coated well.

Characterization of individual phage clones: After two rounds of biopanning, individual phage clones were randomly selected from plaques and amplified in ER2738 cells. Each clone was screened for binding to 6xHis-XRCC3 coated wells via phage ELISA, according to methods outlined in the Ph.D.- 12 manual. Clones with confirmed binding were chosen for further characterization by sequencing. Single stranded DNA templates were prepared by phenol/chloroform extraction of the amplified phage. These DNA templates were sequenced using the - 96 gill primer (New England Biolabs).

Preparation of synthetic peptides: Synthetic peptides were prepared by Genemed Sythesis, Inc (San Francisco). Each peptide was HPLC purified and its composition was confirmed on mass spectroscopy (by the manufacturer). The lyophilized peptides were stored at -80° C in sterile water, and concentrations were confirmed by UV spectroscopy.

Cytotoxicity assays: Cytotoxicity was determined by loss of colony- forming ability. Briefly, 800 cells were plated overnight in 100-mm cell culture dishes. On the following day, the cells were washed, treated with various concentrations cisplatin in serum-free DMEM medium for 1 hour, and again washed with serum free media. Finally, 10ml of peptide-containing media (DMEM, 10% heat-inactivated FBS, and 500nM peptide) was added to the cells and left on for the remainder of the experiment. After 10-12 days, the resulting colonies were fixed and stained with crystal violet. Plates were then imaged with a digital camera and colony number/plate were counted using NIH Image software. Colonies containing at least 50 cells were scored as survivors. Clonogenic survival rates were normalized to an appropriate set of control cells exposed to no drugs. Each treatment condition was performed in triplicate, and standard deviations were calculated. Rad51 focus formation assay: Rad51 focus formation was quantified as previously described (Doug's paper). Briefly, cells were plated overnight at 1x10^' cells/plate. Cells were then treated with a DNA-damaging agent (IR or cisplatin) and incubated for an additional 3 hrs. Cells were then trypsinized, fixed with 3% PFA, and cytospun onto glass microscope slides. The slides were dried, permeablized for 5 minutes (20 mM hepes pH-7.4, 0.5% triton, 50 mM NaCI, 3 mM MgCl₂, 300 mM sucrose), and blocked for 15 minutes (TBS with 1% BSA). Slides were then stained with anti-hsRad51 polyclonal antibody (1 :200 dilution) overnight at 4°, followed by incubation with FITC-conjugated anti -rabbit IgG antibodies (Molecular Probes). Nuclei were counterstained with DAPI. The nuclear foci of Rad51 were viewed via confocal microscopy and quantified with the Lab Spectrum software package.

Xenograft Studies with Cisplatin and Irradiation: Seven week-old female nude mice (Frederick Cancer research Institute, Frederick, MD) were housed in accordance with the University of Chicago's institutional guidelines. CHO tumor xenografts were induced in mice by subcutaneous injection of lxl 0⁷ cells (suspended in PBS) into the right hind limbs. Tumors were allowed to grow for one to two weeks to volumes of 200-400 mm³. Cisplatin (diluted in PBS) was administered via daily intraperitoneal injections. Ionizing radiation (IR) treatments consisted of five daily fractions of 3 Gy. Mice were irradiated using a GE Maxitron X-ray generator operating at 150 mV, 30 mA, with a 1 mm aluminum filter at a dose rate of

188cGy/min. For radiation treatments mice were immobilized in plexiglass restraining devices, such that their entire bodies were shielded with lead except for the tumor- bearing limbs. Tumors were directly measured with calipers, and volumes were calculated using the ellipsoid estimation formula (length x width x height 12). Standard error of the mean (SEM) was calculated for each data point.

EXAMPLE 2: Results

Establishment of cell lines: An isogenic pair of cell lines was established from an XRCC3-deficient CHO line. The irslsf cell line was co- transfected with an hsXRCC3-expressing plasmid (pXR3) and a gen^tacin-resistance plasmid (pCB6). Control cells were transfected with pCB6 only. Individual clones were selected based on genetacin resistance, and the resulting lines were subsequently tested for cisplatin resistance. Western blot analysis of nuclear extracts confirmed XRCC3 protein expression in the co-tranfected cells (irslsf + pXR3 + pCB6), but no detectable expression in the control cells (irslsf + pCB6). This expression level was relatively low compared to the human breast cancer cell line BT-20 and the human prostate cancer line PC3. (Figure la) Transfection with the XRCC3-containing plasmid resulted in cisplatin resistance, as previously demonstrated by Tebbs (Figure lb), (quote)

Tumor xenograft experiments: Cells that are deficient in HRR have well-known sensitivity to DNA-damaging therapies in cell culture-based experiments, however little is know about the significance of this DNA-repair pathway in solid tumor models. To confirm the potential of XRCC3 as therapeutic target, xenograft tumor experiments were thus performed. Xrcc3-defective and rescued CHO cells were injected subcutaneously into nude mice, and were allowed to form tumors. Pathological examination of the resulting tumors demonstrated that these tissues resembled fibrosarcomas histo logically. After appropriate tumor growth, the mice were treated with cisplatin intraperitoneally for 5 consecutive days or PBS as a control. All of the XRCC3-deficient tumors (irslsf + pCB6) completely resolved by day 14, and none re-grew by day 60. The XRCC3-rescued tumors (irslsf + pXR3 + pCB6), however, were essentially un-affected by this dose of cisplatin. (Figure 2a). Similar results, albeit less dramatic, were seen when ionizing radiation treatment was used in place of cisplatin. (Figure 2b)

Peptide development: XRCC3-binding peptides were selected from a phage display library, wherein each phage virion expresses five copies of a 12-amino acid random peptide. Phage were selected based of their ability to bind to XRCC3- coated microtiter wells. Two consecutive rounds of this affinity selection were performed. Using BSA-binding as a control, there was a xx-fold enrichment in 6xHis-XRCC3 binding after the first round of affinity selection and a yy-fold enrichment following the second round. The resulting phage clones were amplified, and their DNA were sequenced in order to determine the dodecapeptide sequences responsible for binding to 6xHis-XRCC3. Alignment of these peptide sequences revealed a consensus pattern that closely resembled an N-terminal (amino acids 14- 25) region of RAD51C protein. (Figure 3) Since RAD51C is a natural binding partner of XRCC3, we hypothesized that this 12 residue peptide (LVSFPLSPAVRV) represents the XRCC3-binding domain of RAD51C. This peptide sequence was synthetically constructed, connected by a poly-glycine linker to a C-terminal protein transduction domain (YARAAARQARA). This transduction sequence, PTD4, has been shown to cross lipid bilayers and result in intra-cellular drug delivery. This fusion peptide is hereafter be referred to as Rad51C(14-25)-PTD4. A second peptide was also synthesized, wherein the RAD51C-containing sequence was scrambled (SVVAPLLSRFVP). The resulting fusion peptide, referred to as RAD51C(scram'd 14-25)-PTD4, served as a negative control in all subsequent experiments.

Activity of peptides on cell survival: Colony forming assays were performed to assess the effect of RAD51 C( 14-25)-PTD4 on cellular survival. For these experiments cells were plated overnight and then treated with cisplatin for one hour. The cells were then incubated together with the peptide and allowed to form colonies. In the absence of cisplatin, neither RAD51C(14-25)-PTD4 nor the scrambled control peptide was toxic at concentrations up to 2 μM. However, when peptide treatments were combined with cisplatin, RAD5 lC(14-25)-PTD4 resulted in a significant sensitization at peptide concentrations as low as 125 nM. The scrambled control peptide resulted in no detectable cisplatin sensitization (Figure 4a). Treatment with the RAD51C(14-25)-PTD4 peptide lead to an approximate doubling of cisplatin cytotoxicity in XRCC3-expressing cells, however it had no measurable effect on XRCC3-deficient cells. (Figure 4b-4c).

Activity of peptides on HRR: The RAD51 C(l 4-25)-PTD4 peptide is believed to function by binding XRCC3 protein, and consequently inhibiting HRR. To support this hypothesized mechanism, Rad51 focus formation assays were performed. In this assay, cells are exposed to a DNA-damaging treatment and then stained to identify sites of sub-nuclear RAD51 assembly. CHO cells (irslsf + pXR3 + pCB6) were treated with cisplatin and then incubated with RAD51C(14-25)-PTD4. Examination of cells via confocal microscopy demonstrated that the RAD51C(14-25)- PTD4 peptide inhibited the formation Rad51 foci. As expected, the scrambled control peptide had no appreciable effect on HRR in any of these experiments. (Figure 5a) Similar experiments were performed using the cell lines, PC3 and BT-20. (Figure 5b- 5c)

Claims

What is Claimed Is:

I . An isolated peptide derived from the N-terminal of region of RAD51C wherein said peptide inhibits the sub-nuclear assembly of Rad51.

2. The isolated peptide of claim 1 , wherein said peptide inhibits homologous recombination repair.

3. The isolated peptide of claim 1, wherein said peptide binds XRCC-3.

4. The isolated peptide of claim 1, wherein said peptide binds to one or more of the components of the Rad51 complex selected from the group consisting of XRCC-3, XRCC-2, Rad51b, Rad51C and Rad51d.

5. The isolated peptide of claim 1, wherein said peptide is an anti- tumor agent.

6. The isolated peptide of claim 1, wherein said peptide is a dodecapeptide.

7. The isolated peptide of claim 1, wherein said peptide comprises between about the first 10 and about the first 30 amino acids of the N-terminus of Rad51C.

8. The isolated peptide of claim 1, wherein said peptide comprises amino acids 14-25 of RAD51C and further comprises a second peptide sequence at the C-terminal.

9. The isolated peptide of claim 8, wherein said second peptide sequence at the C-terminal is a C-terminal transduction domain.

10. The isolated peptide of claim 9, wherein said C-terminal transduction domain comprises a sequence of YARAAARQARA.

I I. The isolated peptide of claim 1, wherein said peptide comprises a sequence selected from the group consisting of the sequences depicted in Figure 3, and a variant of any of the sequences depicted in Figure 3 wherein said variant inhibits the sub-nuclear assembly of Rad51.

12. The isolated peptide of claim 1, wherein said peptide has a sequence of LVSFPLSPAVRV, or a variant thereof that binds to XRCC-3.

13. A fusion protein comprising the N-terminal region of RAD51C fused at the C-terminal to a C-terminal transduction domain, wherein said fusion protein inhibits the sub-nuclear assembly of Rad51.

14. A composition comprising a peptide or fusion protein of any of the preceding claims, and a pharmaceutically acceptable carrier, excipient or diluent.

15. A polynucleotide comprising a nucleotide sequence that encodes a peptide or a fusion peptide according to any one of claims 1 to 12.

16. An expression vector comprising a polynucleotide according claim 16.

17. The expression vector of claim 16, wherein said polynucleotide is operably linked to a promoter to promote expression of the protein encoded by the polynucleotide in a host cell.

18. A recombinant host cell transformed or transfected with a polynucleotide according to claim 15.

19. A recombinant host cell transformed or transfected with an expression vector of claim 16.

20. A method for inhibiting the growth of a cell comprising contacting said cell with a peptide derived from the N-terminal of region of RAD51C wherein said peptide inhibits the sub-nuclear assembly of Rad51 and a chemotherapeutic DNA damaging agent, wherein the dose of said peptide, when combined with the dose of said DNA damaging agent, is effective to inhibit growth of said cell.

21. The method of claim 20, wherein said peptide increases the cytotoxicity of said chemotherapeutic agent against said cell.

22. The method of claim 20, wherein said peptide is a peptide of any of claims 1 through 12 or a fusion protein of claim 13

23. The method of claim 20, wherein said peptide is contacted with said cell prior to contacting said cell with said DNA damaging agent.

24. The method of claim 20, wherein said DNA damaging agent is contacted with said cell prior to contacting said cell with said peptide.

25. The method of claim 20, wherein said cell is a cancer cell.

26. The method of claim 25, wherein said cancer cell is a bladder cancer cell, a blood cancer, a breast cancer cell, a lung cancer cell, a colon cancer cell, a prostate cancer cell, a liver cancer cell, a pancreatic cancer cell, a stomach cancer cell, a testicular cancer cell, a brain cancer cell, an ovarian cancer cell, a lymphatic cancer cell, a skin cancer cell, a brain cancer cell, a bone cancer cell, a soft tissue cancer cell.

27. The method of claim 1, wherein said cell is located in a human subject.

28. The method of claim 27, wherein said peptide is contacted with said cell by being administered by direct intratumoral injection.

29. The method of claim 27, wherein said peptide is administered by injection into tumor vasculature.

30. The method of claim 20, wherein said DNA damaging agent is a chemotherapeutic agent selected from a group consisting of doxorubicin, daunorubicin, dactinomycin, mitoxantrone, cisplatin, procarbazine, mitomycin, carboplatin, bleomycin, etoposide, teniposide, mechlroethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, ifosfamide, melphalan, hexamethylmelamine, thiopeta, busulfan, camustine, lomustine, semustine, streptozocin, dacarbazine, adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D, hydrogen peroxide, nitrosurea, plicomycin, tamoxifen, taxol, transplatinum, vincristin, vinblastin and methotrexate.

31. The method of claim 20, wherein said DNA damaging agent is ionizing radiation therapy.

32. The method of claim 20, wherein said cell is contacted with said peptide multiple times.

33. The method of claim 20, wherein said cell is contacted with said DNA damaging agent multiple times.

34. The method of claim 20, further comprising tumor resection.

35. The method of claim 34, wherein said tumor resection occurs prior to said contacting.

36. The method of claim 35, wherein said contacting comprises treating said resected tumor bed with said peptide and said DNA damaging agent.

37. The method of claim 34, wherein said tumor resection occurs after said contacting.

38. The method of claim 34, wherein said contacting occurs both before and after said tumor resection.

39. The method of claim 1 , wherein said dose of said peptide is about 1 mg/kg to about 100 mg/kg.

39. The method of claim 39, wherein said dose of said peptide is about 1 mg/kg to about 4 mg/kg.

40. A method of killing a tumor cell comprising contacting said tumor cell with a peptide derived from the N-terminal of region of RAD51C wherein said peptide inhibits the sub-nuclear assembly of Rad51 and a chemotherapeutic DNA damaging agent, wherein the dose of said peptide, when combined with the dose of said DNA damaging agent, is effective to kill said tumor cell.

41. The method of claim 40, wherein said peptide is a dodecapeptide.

42. The method of claim 40, wherein said peptide comprises a sequence selected from the group consisting of the sequences depicted in Figure 3.

43. A method of treating cancer in a mammal comprising administering a peptide derived from the N-terminal of region of RAD51C wherein said peptide inhibits the sub-nuclear assembly of Rad51 and a chemotherapeutic DNA damaging agent to said mammal, wherein the dose of said peptide, when combined with the dose of said DNA damaging agent, is effective to treat said cancer.

44. A method of potentiating the effect of a chemotherapeutic DNA damaging agent on a tumor cell comprising contacting said tumor cell with isolated peptide derived from the N-terminal of region of RAD51C wherein said peptide inhibits the sub-nuclear assembly of Rad51 and further contacting said tumor cell with said DNA damaging agent.