WO2007016542A2 - Her-2 blocking bifunctional targeted peptides - Google Patents

Abstract

Provided herein are bispecific peptides comprising a targeting peptide and a proapoptotic peptide. The targeting peptide may be a Her-2 targeting peptide.

Description

HER-2 BLOCKING BIFUNCTIONAL TARGETED PEPTIDES

Related Applications

This application claims the benefit of U.S. provisional application no. 60/704,486, filed August 1, 2005, the contents of which is incorporated herein by reference in its entirety.

Background

Normal tissue homeostasis is achieved by an intricate balance between the rate of cell proliferation and cell death. Disruption of this balance either by increasing the rate of cell proliferation or decreasing the rate of cell death can result in the abnormal growth of cells and is thought to be a major event in the development of cancer, as well as other cell proliferative disorders such as restenosis.

The effects of cancer are catastrophic, causing over half a million deaths per year in the United States alone. Conventional strategies for the treatment of cancer include chemotherapy, radiotherapy, surgery or combinations thereof, however further advances in these strategies are limited by lack of specificity and excessive toxicity to normal tissues. In addition, certain cancers are refractory to treatments such as chemotherapy, and some of these strategies such as surgery are not always viable alternatives. For example, non-small- cell lung cancer (NSCLC), which includes squamous cell carcinoma, adenocarcinoma and large-cell carcinoma, accounts for 75-80% of all lung cancers (American Cancer Society, 1993). Current multimodality therapeutic strategies applied to regionally advanced NSCLC are minimally effective with the overall cure rate being only about 10% (Belani et al. (1993) Semin. Oncol. 20:302 and Roth et al. (1994) Lung Cancer 11 Suppl. 3:S25).

Cancer is now understood to be the result of multiple genetic changes (Goyette et al. (1992) MoI. Cell Biol. 12:1387) and it is well established that many cancers are caused, at least in part, by genetic alterations that result in either the over expression of one or more genes, or the expression of abnormal or mutant gene or genes. For example, the expression of oncogenes is known to play a role in the development of cancer. Oncogenes are defined as genetically altered genes whose mutated expression product or level of expression somehow disrupts normal cellular function or control (Spandidos et al. (1989) Anticancer Res. 9:821). These types of mutations are believed to have effects on the malignant growth of cells derived from practically every tissue. Oncogenes also include tumor suppressor genes, such as p53 and p53-like genes, whose lack of expression results in uncontrolled cell proliferation.

The Neu/HER-2/erbB2 proto-oncogene is a member of the epidermal growth factor (EGF) receptor family. Neu is a rodent gene and Her-2 is its human homologue. This group of receptor tyrosine kinases plays an essential role during growth and differentiation of many tissues. In addition, overexpression of these proteins is associated with several types of human cancers (reviewed in Hynes et al. (1994) Biochim Biophys Acta 1198(2-3), 165-84; Kim et al. (1999) Exp Cell Res 253(1), 78-87; and Hung et al. (1999) Seminars in Oncology 26 (4 Suppl 12), 51-9). The Neu/Her-2 gene is amplified and the protein overexpressed in 20-30% of human breast carcinomas, and this event correlates with poor prognosis. This observation strongly suggests that Neu/Her-2 plays a direct role in the development of breast tumors. Furthermore, targeted expression of constitutively active Neu to the mouse mammary gland results in induction of multifocal mammary tumors in females harboring the transgene (Muller et al. (1988) Cell 54(1), 105-15; Andrechek et al.(2000) PNAS 97(7), 3444-9). In addition, Neu antisense treatment not only affects proliferation but also activates apoptotic pathways in Neu-overexpressing cells, demonstrating that this proto-oncogene is involved in both proliferation and survival (Roh et al. (2000) Cancer Res 60(3), 560-5).

The fact that repression of Neu tyrosine kinase suppresses the malignant phenotype of cells overexpressing the oncogene together with its extracellular accessibility have made Neu an excellent target for tumor therapy. For that reason, efforts have been directed towards the development of reagents that directly interfere with Neu activity. Firstly, small molecules that inhibit the tyrosine kinase activity of Neu are being developed. Tyrphostins are an example of this category of compounds (Levitzki et al. (1995) Science 267(5205), 1782-8). Inhibitory activity towards a vast array of protein tyrosine kinases in the nanomolar and micromolar range has been reported for several tyrphostins in vitro, but only a few work on intact cells. More recently AG- 1478, a quinazoline inhibitor of the EGF receptor tyrosine kinase, has been shown to suppress tumorigenesis in MMTV/Neu + TGFα bigenic mice. This compound induces the sequestration of Neu in inactive Neu/EGF receptor heterodimers (Lenferink et al. (2000) PNAS 97(17), 9609-14). AG1478 and PDl 53035 both bind to the ATP binding site on the EGF receptor, acting as competitive inhibitors. These compounds have a cytostatic effect on cells, i.e. cells will continue to proliferate upon removal of these drugs (Albanell et al. (1999) Journal of Mammary Gland Biology & Neoplasia 4(4), 337-51). One of the major drawbacks of these types of inhibitors is that, in general, they affect more than one tyrosine kinase due to the high degree of homology within certain conserved domains of this class of enzymes (Levitzki et al. (1995) Science 267(5205), 1782-8). Thus, the major challenge at the moment lies in the improvement of selectivity for several of these promising compounds, hi addition, the antiproliferative effect of natural tyrosine kinase inhibitors, and a new class of irreversible inhibitors of Neu are currently being tested on Neu-overexpressing cancer cells (Hung et al. (1999) Seminars in Oncology 26(4 Suppl 12), 51-9; Arteaga et al. (1997) J Biol Chem 272(37), 23247-54; and Fry et al.(1998) Proc Natl Acad Sci U S A 95(20), 12022-7). Another approach to inhibiting Neu is the use of neutralizing monoclonal antibodies directed against the extracellular domain of Neu. Herceptin™, the humanized recombinant version of 4D5, has shown in general an antiproliferative effect on Neu-overexpressing cells, and have proven successful when used in combination with cytotoxic chemotherapeutic agents for the treatment of Neu-overexpressing breast cancers (Hudziak et al.(1989) MoI Cell Biol 9(3), 1165-72; Carter et al. (1992) Proc Natl Acad Sci U S A 89(10), 4285-9; Lewis et al.(1993) Cancer Immunol Imrnunother 37(4), 255-63; Pegram et al. (1999) Oncogene 18(13), 2241-51; and Pegram et al. (1998) Breast Cancer Research & ^• Treatment 52(1-3), 65-77). Although treatment of cells expressing high levels of Neu with neutralizing antibodies results in a decrease in receptor phosphorylation and Neu protein level, not all cells are growth inhibited (Lane et al. (2000) MoI Cell Biol 20(9), 3210-23). The outcome from clinical studies performed on patients with tumors overexpressing Neu treated with Herceptin™ is that not all respond to the antibody treatment (Cobleigh et al. (1999) Journal of Clinical Oncology 17(9), 2639-48). These results correlate with the observations at the cellular level, and suggest that other pathways might be contributing to the uncontrolled growth of Neu-overexpressing tumor cells.

Summary

Provided herein are bispecific peptides comprising, e.g., a targeting peptide and a pro-apoptotic peptide. The targeting peptide may be a Her-2 targeting petpide, e.g., a peptide comprising an amino acid sequence that is at least about 80%, 90% identical to

YCDGFYACYMDV. The targeting peptide may also comprise or consist essentially of the amino acid sequence YCDGFYACYMDV. The pro-apoptotic peptide may comprise an amino acid sequence that is at least about 80% or 90% identical to (KLAKLAK)2. The pro-apoptotic peptide may also comprise or consist essentially of the amino acid sequence (KLAKLAK)2. A bispecific peptide may comprise a Her-2 targeting peptide comprising an amino acid sequence consisting essentially of YCDGF Y AC YMD V and a pro-apoptotic peptide comprising an amino acid sequence consisting essentially of (KLAKLAK)2. The targeting peptide may be located N-terminally or C-terminally relative to the pro-apoptotic peptide.

A bispecific peptide may further comprise a linker, which may comprise from 1 to 20 amino acids; from 1 to 10 amino acids; from 1 to 5 amino acids. A linker may comprise 2 amino acids, e.g., GG. The linker may be a cleavable linker. Also provided herein are peptidomimetics of a bispecific peptide described herein.

A bispecific peptide may also be further attached to a molecule, such as a label, e.g., that can be detected. A molecule may be a molecule that stabilizes the bispecific peptide, e.g., a polymer. A molecule may be biodegradable.

Further provided are pharmaceutical compositions and kits, e.g., comprising a bispecific peptide described herein. Nucleic acids encoding a bispecific peptide are also described.

A bispecific peptide may be used for the preparation of a medicament for treating or preventing a disease characterized by the undesirable presence of Her-2 containing cells in a subject. It may also be used in a method for treating or preventing a disease characterized by the undesirable presence of Her-2 containing cells in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a bispecific peptide. The disease may be cancer, e.g., carcinoma, such as breast carcinoma. A subject may further be treated with another treatment, e.g., chemotherapy.

Brief description of the drawings

Fig. 1. Schematic representation and primary sequence of the BHAP peptide. Cysteines form an intra-molecular disulfide bridge.

Detailed description Definitions

For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below. "Abnormal growth of cells" means cell growth independent of normal regulatory mechanisms (e.g., loss of contact inhibition), including abnormal growth resulting form expression of an oncogene. An abnormal growth of cells can be a slower growth or a faster growth. The term "analog" of a compound refers to a compound having a substantial structural similarity to a particular compound and having essentially the same biological activity as the compound.

An "anti-neoplastic agent" refers to a chemotherapeutic agent effective against cancer. "c-erbB" refers to a cellular gene which encodes the epidermal growth factor receptor (EGFr). The c-erbB gene is a member of the tyrosine-specific protein kinase family to which many proto-oncogenes belong. The erbB gene is an oncogene form of c- erbB which is present in the avian erythroblastosis virus and which encodes a protein which lacks essentially all of the extracellular domain which renders it constitutively active. "c-erbB-2", which is referred to herein interchangeably as "erbB2", "HER-2",

"Neu", "Neu^τ" or "neu oncogene"), encodes a pi 85 tumor antigen which is a growth factor receptor having extracellular, transmembrane, and intracellular domains. This protein differs from its normal counterpart, the neu proto-oncogene or epidermal growth factor receptor (EGFr) in a single amino acid (point mutation) located in the transmembrane domain, causing the receptor to become constitutively active, i.e., active in the absence of ligand.

The term "antiproliferative" therapeutic or compound refers to a compound or therapeutic which inhibits cell proliferation.

The term "cytostatic" when referring to the activity of a compound means that the compound causes the cell to cell cycle arrest, but it does not kill the cell. Thus, removal of the drug from the environment of the cell may result in the regain of cell proliferation.

The term "unwanted cell proliferation" refers to cell proliferation that is undesirable. Unwanted cell proliferation can refer to cells which are proliferating normally and to cells which are proliferating abnormally, such as cancerous cells. For example, a wart is a tissue in which unwanted epithelial cell proliferation is occurring.

The terms "excessive cell proliferation," used interchangeably herein with "hyper- proliferation" of cells refers to cells, which divide more often than their normal or wild-type counterpart or which are not sensitive to normal mechanisms of growth control. For example, cells are excessively proliferating when they double in less than 24 hours if their normal counterparts double in 24 hours. Excessive proliferation can be detected by simple counting of the cells, with or without specific dyes, or by detecting DNA replication or transcription, such as by measuring incorporation of a labeled molecule or atom into DNA or RNA.

"Inhibiting cell proliferation" refers to decreasing the rate of cell division, by arresting or slowing down the cell cycle. The term refers to complete blockage of cell proliferation, i.e., cell cycle arrest, as well as to a lengthening of the cell cycle. For example, the period of a cell cycle can be increased by about 10%, about 20%, about 30, 40, 50, or 100%. The duration of the cell cycle can also be augmented by a factor of two, three, 4, 5, 10 or more.

"Normalizing cell proliferation" refers to reducing the rate of cell proliferation of a cell that proliferates excessively relative to that of its normal or wild-type counterpart, or increasing the rate of cell proliferation of a cell that proliferates poorly relative to its normal or wild-type counterpart.

"Modulating cell differentiation" refers to the stimulation or inhibition of cell differentiation.

The term "oncogene" refers to a gene which is associated with certain forms of cancer. Oncogenes can be of viral origin or of cellular origin. An oncogene is a gene encoding a mutated form of a normal protein or is a normal gene which is expressed at an abnormal level, e.g., over-expressed. Over-expression can be caused by a mutation in a transcriptional regulatory element, e.g., the promoter, or by chromosomal rearrangement resulting in subjecting the gene to an unrelated transcriptional regulatory element. The normal cellular counterpart of an oncogene is referred to as "proto-oncogene." Proto- oncogenes generally encode proteins which are involved in regulating cell growth, and are often growth factor receptors. Numerous different oncogenes have been implicated in tumorigenesis. Tumor suppressor genes, e.g., p53 or p53-like genes are also encompassed by the term "proto-oncogene." Thus, a mutated tumor suppressor gene which encodes a mutated tumor suppressor protein or which is expressed at an abnormal level, in particular an abnormally low level, is referred to herein as "oncogene." The terms "oncogene protein" refer to a protein encoded by an oncogene.

"Suppression of an oncogenic phenotype" of a cell refers to a reduction in the transforming, tumorigenic or metastatic potential of the cell. The term "transformed cell" refers to a cell which was converted to a state of unrestrained growth, i.e., they have acquired the ability to grow through an indefinite number of divisions in culture. Transformed cells may be characterized by such terms as neoplastic, anaplastic and/or hyperplastic, with respect to their loss of growth control. Transformed cells include cancer cells, such as cells over-expressing a proto-oncogene or expressing a mutated form of an proto-oncogene (i.e., an oncogene). Transformed cells also include cells infected by a microorgarnism, e.g., viruses. Exemplary viruses are retroviruses.

The term "proliferative disorder" refers to any disease/disorder of a tissue marked by unwanted or aberrant proliferation of at least some cells in the tissue. Such diseases include cancer, as well as benign diseases or disorders, such as warts or other benign tumors.

Throughout this application, the term "proliferative skin disorder" refers to any disease/disorder of the skin marked by unwanted or aberrant proliferation of cutaneous tissue. These conditions are typically characterized by epidermal cell proliferation or incomplete cell differentiation, and include, for example, X-linked ichthyosis, psoriasis, atopic dermatitis, allergic contact dermatitis, epidermolytic hyperkeratosis, and seborrheic dermatitis. For example, epidermodysplasia is a form of faulty development of the epidermis. Another example is "epidermolysis", which refers to a loosened state of the epidermis with formation of blebs and bullae either spontaneously or at the site of trauma. The term "carcinoma" refers to a malignant new growth made up of epithelial cells tending to infiltrate surrounding tissues and to give rise to metastases. Exemplary carcinomas include: "basal cell carcinoma", which is an epithelial tumor of the skin that, while seldom metastasizing, has potentialities for local invasion and destruction; "squamous cell carcinoma", which refers to carcinomas arising from squamous epithelium and having cuboid cells; "carcinosarcoma", which include malignant tumors composed of carcinomatous and sarcomatous tissues; "adenocystic carcinoma", carcinoma marked by cylinders or bands of hyaline or mucinous stroma separated or surrounded by nests or cords of small epithelial cells, occurring in the mammary and salivary glands, and mucous glands of the respiratory tract; "epidermoid carcinoma", which refers to cancerous cells which tend to differentiate in the same way as those of the epidermis; i.e., they tend to form prickle cells and undergo cornification; "nasopharyngeal carcinoma", which refers to a malignant tumor arising in the epithelial lining of the space behind the nose; and "renal cell carcinoma", which pertains to carcinoma of the renal parenchyma composed of tubular cells in varying arrangements. Another carcinomatous epithelial growth is "papillomas", which refers to benign tumors derived from epithelium and having a papillomavirus as a causative agent; and "epidermoidomas", which refers to a cerebral or meningeal tumor formed by inclusion of ectodermal elements at the time of closure of the neural groove.

As used herein, the term "psoriasis" refers to a hyperproliferative skin disorder which alters the skin's regulatory mechanisms. In particular, lesions are formed which involve primary and secondary alterations in epidermal proliferation, inflammatory responses of the skin, and an expression of regulatory molecules such as lymphokines and inflammatory factors. Psoriatic skin is morphologically characterized by an increased turnover of epidermal cells, thickened epidermis, abnormal keratinization, inflammatory cell infiltrates into the dermis layer and polymorphonuclear leukocyte infiltration into the epidermis layer resulting in an increase in the basal cell cycle. Additionally, hyperkeratotic and parakeratotic cells are present. The term "keratosis" refers to proliferative sldn disorder characterized by hyperplasia of the horny layer of the epidermis. Exemplary keratotic disorders include keratosis follicularis, keratosis palmaris et plantaris, keratosis pharyngea, keratosis pilaris, and actinic keratosis.

As used herein, "immortalized cells" refers to cells which have been altered via chemical and/or recombinant means such that the cells have the ability to grow through an indefinite number of divisions in culture.

A "patient" or "subject" can mean either a human or non-human animal , e.g., an ovine, bovine, porcine, equine, bird, canine, feline or non-human primate.

The term "cosmetic preparation" refers to a form of a pharmaceutical preparation which is formulated for topical administration.

An "effective amount" of a compound of the invention, with respect to the subject method of treatment, refers to an amount of a compound of the invention in a preparation which, when applied as part of a desired dosage regimen brings about a change in the rate of cell proliferation and/or the state of differentiation of a cell or cell Idling so as to produce a result according to clinically acceptable standards for the disorder to be treated or the cosmetic purpose.

The "growth state" of a cell refers to the rate of proliferation of the cell and the state of differentiation of the cell. "Treating" a subject refers to curing or improving at least one symptom of the disease. For example, treating cancer in a subject includes reducing or maintaining tumor load or curing the subject.

A targeting peptide may be a Her-2 targeting peptide, such as a human Her-2 targeting peptide. A targeting peptide may comprise an amino acid sequence that is functionally similar to YCDGFY ACYMDV (SEQ ID NO: 1), e.g., an amino acid sequence that comprises or consists essentially of SEQ E) NO: 1. Peptides that differ from SEQ ID NO: 1 by the addition, substitution or deletion of one or more amino acids (e.g., 1, 2, 3, 4 or 5) may also be used provided that they still home to a target cell. Targeting peptides are further described in U.S. 20030148932.

A pro-apoptotic peptide may comprise an amino acid sequence that is functionally similar to (KLAKLAK)2, i.e., KLAKLAKKLAKLAK (SEQ ID NO: 2), e.g., an amino acid sequence that comprises or consists essentially of SEQ ID NO: 2. Peptides that differ from SEQ ID NO: 2 by the addition, substitution or deletion of one or more amino acids (e.g., 1, 2, 3, 4 or 5) may also be used provided that they still home to a target cell. Targeting peptides are further described in U.S. 20010046498.

Peptides may also comprise synthetic amino acids, D amino acids, L amino acids or a combination thereof.

The subunits of a bispecific peptide may be covalently or non-covalently linked. When covalently linked, the two units may be directly linked or linked through a linker. The linker may comprise from 1 to 30, 1 to 20, 1 to 10, 1-5 or 1-2 amino acids.

A large number of linking groups for linking the two peptidic units of the bispecific peptide are known in the art and are amenable to the present invention. For example, the linking group can be an enzymatically degradable linker, such as the Gly-Phe-Leu-Gly linker described in WO 03/086382. Certain oligopeptide linkers offer the advantage that they can be cleaved by preselected cellular enzymes, for instance, those enzymes found in lysosomes of cancerous cells or proliferating endothelial cells. The number of amino acids in the oligopeptide can vary. For example, the oligopeptide linker may contain from 1 to about 5 amino acids, or the oligopeptide linker may contain from about 4 to about 10 amino acids. In certain instances, the oligopeptide linker contains about 3 or 4 amino acids.

The linking group may also be a pH sensitive linker. For example, an acid-labile linking group would degrade when placed in a medium having a pH of less than about 7, whereas an base-labile linking group would degrade when placed in a medium having a pH of greater than about 7. In certain instances, it may be advantageous for the pH sensitive linker to be stable at or near neutral pH in order to simplify handling of the peptide conjugate. In these situations, an acid-labile linking group that degrades only when the pH is less than about 6 may be preferred. Likewise, a base-labile linking group that degrades only when the pH is greater than about 8 may be preferred. Examples of acid-labile linking groups include linkers that have an ester, amide, or hydrazone group because these groups undergo hydrolysis under acidic conditions. Similarly, linking groups comprising cis- aconityl may be used as a pH-sensitive linker because they undergo degradation under acidic conditions. Representative pH-sensitive linkers amenable to the present invention are also described in F. Kratz et al. Crit. Rev. Ther. Drug Carrier Syst. 16 (1999) 245-288; A. Al-Shamkhani et al. Int. J. Pharm. 122 (1995) 107-119; K. Ulbrich Materials Structure 10 (2003) 3-5; and T. Etrych. et al. Macromol. Biosci 2 (2002) 43-52.

The linking group may also be a non-degradable linker, i.e., the linker is not pH sensitive, nor is it susceptable to enzymatic degradation. Representative examples of non- degradable linkers include an alkyl group, aryl group, alkenyl group, glycine-glycine group, and various oligopeptides.

A bispecific peptide may also comprise a linker sequence with a thrombin cleavage site. An exemplary nucleotide sequence encoding such a site has the following nucleotide sequence: 5' tct aga ggt ggt eta gtg ccg cgc ggc age ggt tec ccc ggg ttg cag 3', which encodes a peptide having the amino acid sequence: Ser Arg GIy GIy Leu VaI Pro Arg GIy Ser GIy Ser Pro GIy Leu GIn.

Generally, the bispecific peptides of the invention can be used to normalize, e.g., inhibit or block the proliferation of cells and/or modulate the differentiation of cells and/or induce apoptosis. The peptides can also be used to kill target cells, e.g., by inducing apoptosis in the cells. The cells whose proliferation or differentiation is to be normalized or which are to be killed are referred herein as "target cells." A target cell can essentially be any cell whose proliferation is to be inhibited or its differentiation modulated or which is to be killed. For example, target cells can be cells which are defective, e.g., non-responsive to, normal cell proliferation control mechanisms. In one embodiment, target cells are transformed cells, e.g., cells which express a mutated form of a molecule, e.g., a protein, or which over-express a molecule, e.g., a protein. As further discussed herein, a target cell can be a cell which is defective in its response to a growth factor, e.g., EGF. Thus, preferred target cells are those containing a defect in a growth factor receptor or signal transduction molecule relaying the information to the nucleus. Exemplary target cells are those which express an activated form of a proto-oncogene, e.g., the Neu oncogene. Target cells may express or overexpress the Neu/Her-2 protein. In one embodiment, the invention provides methods for treating cancer, e.g., cancers that are caused by, or associated with, expression of an oncogene or over-expression of a proto-oncogene, e.g., the Neu proto-oncogene. An exemplary cancer that can be treated is breast cancer, in particular, forms of breast cancers which are associated with an over- expression of the Neu proto-oncogene. Amplification and/or overexpression of the human erbB2 gene correlates with a poor prognosis in breast and ovarian cancers, in particular, carcinomas. Slamon et al., Science 235:177-82 (1987); Slamon et al., Science 244:707-12 (1989). Overexpression of erbB2 has been correlated with other carcinomas including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon and bladder. Accordingly, in U.S. Pat. No. 4,968,603, Slamon et al. describe and claim various diagnostic assays for determining erbB2 gene amplification or expression in tumor cells. Slamon et al. discovered that the presence of multiple copies of the erbB2 oncogene in tumor cells indicates that the disease is more likely to spread beyond the primary tumor site, and that the disease may therefore require more aggressive treatment than might otherwise be indicated by other diagnostic factors. Slamon et al. conclude that the erbB2 gene amplification test, together with the determination of lymph node status, provides greatly improved prognostic utility.

In view of the similarity of the Neu proto-oncogene with other members of the family of EGF receptors, the bispecific peptides of the invention are likely to inhibit the proliferation of cells transformed with other members of the EGF receptor family, such as Neu-erb2-related genes. Accordingly, the bispecific peptides of the invention can be used for treating diseases, in particular, malignancies, that are associated with the presence of such related genes. The effectiveness of the bispecific peptides in treating these other diseases can be tested using appropriate cell lines or animal models.

For example, the Neu proto-oncogene is related to erbBl, a 170 IdDa protein, which has been causally implicated in human malignancy. In particular, increased expression of this gene has been observed in more aggressive carcinomas of the breast, bladder, lung, and stomach. ErbB gene amplification or overexpression, or a combination of both, has been demonstrated in squamous cell carcinomas and glioblastomas (Libermann, T. A., Nusbaum, H. R., Razon, N., Kris, R., Lax, L, Soreq, H., Whittle, N., Waterfield, M.D., Ullrich, A. & Schlessinger, J., 1985, Nature 313:144-147). Accordingly, the bispecific peptides are believed to be useful for treating these malignancies.

A further related gene of the Neu proto-oncogene is erbB3 (or HER3), which encodes the ErbB-3 receptor (pl80.sup.HER3). This receptor has been described, e.g., in U.S. Pat. Nos. 5,183,884 and 5,480,968; Kraus et al., PNAS USA 86:9193-97 (1989); EP Patent Application No. 444,961Al; Kraus et al., PNAS USA 90:2900-04 (1993). Kraus et al. (1989) discovered that markedly elevated levels of erbB3 mRNA were present in certain human mammary tumor cell lines indicating that erbB3, like erbBl and erbB2, may play a role in human malignancies. Also, Kraus et al. (1993) showed that EGF-dependent activation of the ErbB-3 catalytic domain of a chimeric EGFR/ErbB-3 receptor resulted in a proliferative response in transfected NIH-3T3 cells. Furthermore, these researchers demonstrated that some human mammary tumor cell lines display a significant elevation of steady-state ErbB-3 receptor tyrosine phosphorylation, further implicating this receptor in human malignancies. The role of erbB3 in cancer has been explored by others, and this gene has been found to be overexpressed in breast (Lemoine et al., Br. J. Cancer 66:11 loll [1992]), gastrointestinal (Poller et al., J. Pathol. 168:275-80 [1992]; Rajkumer et al., J. Pathol. 170:271-78 [1993]; Sanidas et al., Int. J. Cancer 54:935-40 [1993]), and pancreatic cancers (Lemoine et al., J. Pathol. 168:269-73 [1992], and Friess et al., Clinical Cancer Research 1:1413-20 [1995]). Yet another member of the class I subfamily of growth factor receptor protein tyrosine kinases (EGF receptor family) has been further extended to include the ErbB-4 (HER4) receptor, which is the product of the erbB4 (HER4) gene. See EP Patent Application No. 599,274; Plowman et al., PNAS USA 90:1746-50 (1993); and Plowman et al., Nature 366:473-75 (1993). Plowman et al. found that increased erbB4 expression closely correlated with certain carcinomas of epithelial origin, including breast adenocarcinomas. Diagnostic methods for detection of human neoplastic conditions (especially breast cancers) that evaluate erbB4 expression are described in EP Patent Application No. 599,274.

The therapeutic methods of the invention generally comprises administering to a subject in need thereof, a pharmaceutically or therapeutically effective amount of a bispecific peptide. The bispecific peptides of the invention can be administered in a "growth inhibitory amount," i.e., an amount of the bispecific peptide which is pharmaceutically effective to inhibit or decrease proliferation of target cells. The bispecific peptides can also be administered in a "differentiation modulating amount", e.g., "differentiation-inducing amount" or "differentiation-inhibiting amount," which is an amount of the bispecific peptide which is pharmaceutically effective to modulate differentiation of target cells. The bispecific peptides of the invention can also be administered in a "cell death inducing amount," which is an amount of a bispecific peptide which is pharmaceutically effective to induce cell death of target cells. The bispecific peptides of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, in a pharmaceutical composition, according to standard pharmaceutical practice. The bispecific peptides can be administered orally or parenterally, including intravenously, intramuscularly, intraperitoneally, subcutaneously, rectally and topically. In a preferred embodiment, one or more bispecific peptides are injected directly into a tumor of the subject to be treated.

Toxicity and therapeutic efficacy of the bispecific peptides can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD_5O (the dose lethal to 50% of the population) and the ED₅Q (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅o/EDso. Reagents which exhibit large therapeutic indices are preferred. While reagents that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such reagents to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such reagents lies preferably within a range of circulating concentrations that include the ED_5O with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any reagent used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC5₀ (i.e., the concentration of the test bispecific peptide which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. For example, some bispecific peptides of the invention may be effective at concentrations of 1OnM, 10OnM, or lμM. Based on these numbers, it is possible to derive an appropriate dosage for administration to subjects.

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystallme cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a water soluble taste masking material such as hydroxypropylmethyl-cellulose or hydroxypropylcellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate may be employed. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the form of an oil- in- water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring agents, preservatives and antioxidants.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant. The pharmaceutical compositions may be in the form of a sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.

The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the active ingredient is dissolved in the oily phase. For example, the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.

The injectable solutions or microemulsions may be introduced into a patient's blood- stream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the instant bispecific peptide. In order to maintain such a constant concentration, a continuous intravenous delivery device may be utilized. An example of such a device is the Deltec CADD-PLUS™ model 5400 intravenous pump. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Bispecific peptides of the invention may also be administered in the form of a suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol. For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the bispecific peptide of the invention are employed. For purposes of this application, topical application shall include mouth washes and gargles.

The bispecific peptides for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specific amounts, as well as any product which results, directly or indirectly, from combination of the specific ingredients in the specified amounts.

The bispecific peptides identified by the instant method may also be co- administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated. For example, the instant bispecific peptides may be useful in combination with known anti-cancer and cytotoxic agents.

Drugs can be co-administered to a subject being treated with a bispecific peptide of the invention include antineoplastic agents selected from vinca alkaloids, epipodophyllotoxins, anthracycline antibiotics, actinomycin D, plicamycin, puromycin, gramicidin D, taxol, colchicine, cytochalasin B, emetine, maytansine, or amsacrine.

Classes of compounds that can be used as the chemotherapeutic agent (antineoplastic agent) include: alkylating agents, antimetabolites, natural products and their derivatives, hormones and steroids (including synthetic analogs), and synthetics. Examples of bispecific peptides within these classes are given below. Alkylating agents (including nitrogen mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas and triazenes): Uracil mustard, Chlormethine, Cyclophosphamide (Cytoxan™), Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylene-melamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, Dacarbazine, and Temozolomide. Antimnetabolites (including folic acid antagonists, pyrimidine analogs, purine analogs and adenosine deaminase inhibitors): Methotrexate, 5-Fluorouracil, Floxuridine, Cytarabine, 6- Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, Pentostatine, and Gemcitabine. Natural products and their derivatives (including vinca alkaloids, antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins): Vinblastine, Vincristine, Vindesine, Bleomycin, Dactinomycin, Daunorabicin, Doxorabicin, Epirubicin, Idarubicin, paclitaxel (paclitaxel is commercially available as Taxol.RTM. and is described in more detail below in the subsection entitled "Microtubule Affecting Agents"), Mithramycin, Deoxycoformycin, Mitomycin-C, L- Asparaginase, Interferons (especially IFN-a), Etoposide, and Teniposide.

Hormones and steroids (including synthetic analogs): I7.alpha.-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Tamoxifen, Methylprednisolone, Methyltestosterone, Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, Zoladex. Synthetics (including inorganic complexes such as platinum coordination complexes): Cisplatin, Carboplatin, Hydroxyurea, Amsacrine, Procarbazine, Mitotane, Mitoxantrone, Levamisole, and Hexamethylmelamine. Methods for the safe and effective administration of most of these chemotherapeutic agents are known to those skilled in the art. In addition, their administration is described in the standard literature. For example, the administration of many of the chemotherapeutic agents is described in the "Physicians' Desk Reference" (PDR), e.g., 1996 edition (Medical Economics Company, Montvale, NJ. 07645-1742, USA). If formulated as a fixed dose, such combination products employ the combinations of this invention within the dosage range described below and the other pharmaceutically active agent(s) within its approved dosage range. Combinations of the instant invention may also be used sequentially with known pharmaceutically acceptable agent(s) when a multiple combination formulation is inappropriate. Radiation therapy, including x-rays or gamma rays which are delivered from either an externally applied beam or by implantation of tiny radioactive sources, may also be used in combination with a bispefic peptide to treat cancer.

When a composition according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, and response of the individual patient, as well as the severity of the patient's symptoms.

All the essential materials and reagents required for administering the compounds of the invention may be assembled together in a kit. When the components of the kit are provided in one or more liquid solutions, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred.

For in vivo use, as discussed below, the compound may be provided in combination with one or more other drugs, e.g., chemo- or radiotherapeutic agent. These normally will be a separate formulation, but may be formulated into a single pharmaceutically acceptable composition. The container means may itself be geared for administration, such as an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to a target area of the body, or even applied to and mixed with the other components of the kit. The compositions of these kits also may be provided in dried or lyophilized forms.

When reagents or components are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means. The kits of the invention may also include an instruction sheet defining administration of the agent and, e.g., explaining how the agent will decrease proliferation of cells.

The kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained. Irrespective of the number or type of containers, the kits of the invention also may comprise, or be packaged with a separate instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal. Such an instrument may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle. Other instrumentation includes devices that permit the reading or monitoring of compound levels or reactions in vitro. The present description is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, GenBank accession numbers, issued patents, published patent applications as cited throughout this application) are hereby expressly incorporated by reference.

Examples

Example 1 : A bifunctional targeted peptide that blocks HER-2 tyrosine kinase and disables mitochondrial function in HER-2-positive carcinoma cells The figures corresponding to this example may be found in Fantin et al. (2005) Cancer Res. 65:6891.

HER-2-overexpression is observed in a large number of cancer patients and correlates with poor disease outcome. We have engineered a bifunctional peptide, BHAP, to simultaneously neutralize HER-2 and destabilize mitochondrial membranes. The chimeric peptide is biologically active and capable of selectively triggering apoptosis of HER-2 overexpressing cancer cells in culture, even those previously described as HerceptinTM resistant. Furthermore, BHAP slows down growth of HER-2-overexpressing human mammary xenografts established in SCID mice. BHAP is the first bifunctional peptide with this dual mechanism of action. Because BHAP shows anti-carcinoma activity both in vitro and in vivo, it may represent a novel and useful HER-2-targeted peptide entity with potential therapeutic applications. Furthermore, the BHAP paradigm suggests that development of tailored peptides with anti-carcinoma activity is a reality. The approach taken to create BHAP can be extended to the development of hybrid peptides against a number of overexpressed cellular receptors implicated in the development and progression of cancer.

We consider that BHAP indeed offers a new direction for drug development for the treatment of breast cancer. To date unfortunately there are only two therapeutic alternatives widely used for the treatment of metastatic breast cancer: chemotherapeutic agents such as placlitaxel and the HER-targeting antibody HerceptinTM. Both agents show significant benefits yet limited clinical success. Clinical studies have shown that the objective response rate of HerceptinTM monotherapy is 12 to 34% for a median of 9 months (Cobleigh, MA,et.al.; J Clin Oncol 1999; 17:2639-48). However the majority of patients develop Herceptin resistance by one year, a phenomenon that still remains poorly understood Albanell J, and Baselga J; J Natl Cancer Institute 2001; 93:1830-1832). Our work shows that BHAP targets HER-2 overexpressing MDA-MB-453 and MDA-MB-361 human breast cancer cell lines, which have been shown to be Herceptin resistant. Abstract The HER-2 oncoprotein is commonly overexpressed in a variety of human malignancies and has become an attractive anti-tumor target. A number of strategies to inhibit the HER-2 receptor tyrosine kinase are currently the focus of intensive preclinical and clinical research. In the present study we have engineered a bifunctional peptide, BHAP, that consists of two modular domains: a HER-2 targeting/neutralizing domain and a mitochondriotoxic, pro-apoptotic domain. The chimeric peptide is biologically active and capable of selectively triggering apoptosis of HER-2 overexpressing cancer cells in culture, even those previously described as Herceptin™ resistant. Furthermore, BHAP slows down growth of HER-2-overexpressing human mammary xenografts established in SCE) mice. This approach can be extended to the development of tailored targeted chimeric peptides against a number of overexpressed cellular receptors implicated in the development and progression of cancer. Significance HER-2-overexpression is observed in a large number of cancer patients and correlates with poor disease outcome. Most anti-HER-targeted therapies to date are aimed to neutralize HER-2 function by monoclonal anti-HER-2 -based strategies as well as by small molecules tyrosine kinase inhibitors. We have developed a new class of HER-2- targeted molecule that exploits the accessibility of this transmembrane protein to directly neutralize receptor activity and to act as a selective gate for toxin delivery to mitochondria. BHAP is the first bifunctional peptide with a dual mechanism of action. The hybrid peptide shows anti-carcinoma activity both in vitro and in vivo. Because BHAP is active against a number of HER-2-overexpressing human breast cancer cell lines, including those resistant to anti-HER-2 antibody treatment, it may represent a novel and useful HER-2-targeted peptide entity with potential therapeutic applications. Furthermore, the approach taken in this work can be extended to the development of hybrid peptides against a number of cellular receptors implicated in the development and progression of cancer. Introduction

Conventional chemotherapy and tumor-targeted therapy are two complementary approaches currently employed for the treatment of cancer. Most chemotherapeutic agents are aimed to preferentially affect tumor cells due to their faster rate of cell proliferation. However, despite the unquestionable benefits of systemic traditional anti-cancer therapies, treatment related toxicities normally arise from death of rapidly dividing normal cells. As a consequence of the identification of essential molecular players and pathways involved in tumorigenesis in recent years, tumor selective targets are beginning to emerge. HER-2, also known as erbB-2/neu, is among those genes frequently altered in human cancers (1, 2). The HER-2 proto-oncogene belongs to the epidermal growth factor receptor family that includes three other members: ErbB-1/HER-l/EGFR, ErbB-3/HER-3 and ErbB-4/HER-4 (3). This group of receptor tyrosine kinases, through homodimerization and heterodimerization, activates a number of signaling pathways that mediate physiological responses during embryo development and in adult tissues. Various lines of evidence indicate that their overexpression/ dysregulation leads to malignant transformation and tumorigenesis (4). In particular, overexpression of HER-2, often as a consequence of gene amplification, is observed in 20-30% of breast cancers and correlates with poor prognosis (5, 6). The elevated levels of HER-2 detected in many breast tumors (up to 100 fold higher than in normal mammary tissue) and the accessibility of the receptor from the extracellular space makes HER-2 a suitable candidate for the development of targeted therapies. Herceptin™, the humanized recombinant version of a mouse monoclonal antibody against HER-2, is currently used for the treatment of HER-2 positive breast cancers (7). In preclinical studies Herceptin™ has been shown to neutralize HER-2 activity as well as elicit antibody- dependent cell cytotoxicity (8).

Preclinical and clinical data indicates that effective HER-2 receptor neutralization in HER-2 positive tumors does not always translate into inhibition of tumor growth. The mechanisms by which many HER-2 positive tumors escape anti-HER-2-directed therapy are still not fully defined. Growth factor receptor signaling redundancy created by the activity of the IGF-I receptor and EGF receptor members may help to explain, in part, resistance to HER-2 neutralization (3, 9, 10). More recently, resistance to Herceptin™ has been associated with decreased levels of p27^klpl and PTEN deficiency (11, 12). Thus, it has been proposed that combination targeted therapies may be required to bypass resistance to anti-HER-2 monotherapy (13). hi the present work we asked whether it would be possible to overcome these barriers by engineering a "bifunctional peptide" capable to neutralize HER-2 and to deliver a toxin to disable mitochondria function in tumors overexpressing HER-2. To this end we created a hybrid peptide, BHAP (for bifunctional, HER-2 blocking and apoptosis-inducing peptide), composed of the previously described anti-HER-2 peptide (AHNP) fused to a mitochondriotoxic, pro-apoptotic peptide (PAP). The anti-HER-2/neu peptidomimetic (AHNP) is an exocyclic peptide designed to fit the CDR3 loop of Herceptin (14, 15). Previous work has established that AHNP binds HER-2 selectively and inhibits anchorage independent growth of HER-2-overexpressing cells. AHNP exhibits cytostatic effect towards HER-2-overexpessing tumors in vivo, similar to that of Herceptin. The mitochondriotoxic PAP domain of sequence (KLAKLAK)₂ is a synthetic peptide originally developed to enhance the activity of a natural anti-microbial peptide (16). This class of polypeptides that preferentially permeabilizes bacterial membranes rich in anionic phospholipids, have also been shown to affect mitochondria function in vitro (17, 18). The PAP selected for this study cannot efficiently permeate across eukaryotic plasma membranes and consequently exhibits low mammalian cell cytotoxicity. However, when coupled to selective targeting domains, (KLAKLAK)₂ is internalized by cells, induces mitochondrial damage and triggers apoptosis(19). This approach has been successfully employed to target angiogenic endothelial cells and the vasculature of white fat (19, 20). It has been proposed that the cationic and amphipatic nature of PAPs drives the alignment of the positively charged peptide surface with the negatively charged mitochondrial membrane, affecting its electro-elastic properties and compromising the organelle's biological function (21). A unique feature of BHAP is that the anti-HER-2 peptide domain serves both to selectively target the toxin and to simultaneously neutralize HER-2 in cells overexpressing the protein.

BHAP is the first hybrid peptide of its class, intentionally designed to perturb two essential cellular functions such as growth factor receptor signaling and mitochondria activity. Our results indicate that the engineered peptide is selectively internalized by human breast cancer cells through HER-2-mediated endocytosis and induces apoptosis in vitro and in vivo, and that HER-2 overexpression is sufficient to render tumor cells sensitive to the fusion peptide. BHAP was effective against the HER-2-overexpressing human breast cancer cell lines tested, even those like MDA-MB -453 and MDA-MB-361 that have been previously described as Herceptin™ resistant (22), illustrating the potential therapeutic application of BHAP. At the molecular level, the chimeric peptide is capable of inactivating HER-2 as well as inducing mitochondrial damage, yet the anti-tumor property of BHAP primarily correlates with its mitochondriotoxic effect, explaining the spectrum of tumor cell responses. In addition, to increase the avidity of the peptide for HER-2 we have created tetramers through streptavidin-binding of bio tin-labeled BHAP with improved in vitro efficacy.

Material and Methods:

Reagents: Linear peptides were synthesized as carboxy-terminus amides and purified to 90-95% in the W.M, Keck Facility (Yale University, CT). The anti-HER-2 peptide, AHNP, of sequence YCDGFY ACYMDV (from amino- to carboxy-terminus), and the PAP of sequence KLAKLAKKLAKLAK were linked through a di-glycine linker to produce BHAP of sequence (AHNP)-GG-(PAP). The PAP domain was synthesized using D-amino acids to minimize proteolytic degradation (43). BHAP derivatives with fluorescein- and biotin-labeled carboxy-terminus were also synthesized. AHNP and BHAP were cyclized by air oxidation as described (15). Cyclized peptides were lyophilized and subjected to MALDI-MS to determine their purity. Anti-phospho-HER-2, anti-HER-2 and anti-Src were purchased from Upstate Biotechnology Inc. Antibodies against MAP kinase and phospho-MAP kinase, PKB and phospho-PKB, PLC-γl and phospho-PLC-γl were obtained from Cell Signaling. Anti-Src [pY418] was purchased from Biosource International. Unless specified, reagents used for flow cytometry were obtained from BD Biosciences. Cell culture and generation of stable cell lines: Human breast cancer cell lines

SKBR-3, BT474, MDA-MB-453 (MDA-453) , MDA-MB-361 (MDA-361), MDA-MB-231 (MDA-231), MCF-7 and human mammary epithelial MCF-IOA cells were obtained from the ATCC and grown in Dulbecco's modified Eagle's medium (DMEM), 10 % FBS at 37 ⁰C/ 5% CO₂. The HER-2 cDNA was subcloned into pcDNA-3 (Stratagene) as follows. Apal-digested pcDNA-3 was filled in with T4 DNA polymerase, and subsequently digested with Xhol. The Sall/Dral fragment containing full length HER-2 cDNA was excised from ρBR322/HER-2 (44) and was ligated into the linearized pcDNA-3. Transfection of MDA- MB-231 cells was done using Fugene Reagent according to manufacturer's protocol (Roche). Finally, stable clones were selected in medium containing G418 at 0.5 mg/mL. Cell lysate preparation and immunoblotting: Cells were washed with PBS and lysed in buffer (40 niM HEPES, 150 mM NaCl₅ 10 mM sodium pyrophosphate, 2% Nonidet P- 40, 10 mM NaF, 2 mM EDTA₅ 5 μM Na₃VO₄.) containing Complete™ protease inhibitor cocktail (Roche). Insoluble material was removed by centrifugation. Protein concentration was determined on the supernatants using Bradford reagent (Bio-Rad). Protein lysates were resolved by SDS-PAGE and transferred to ktrmobilon-P membranes (Millipore) in

Towbin's transfer buffer (25 mM Tris, 190 mM glycine, 20% methanol, 0.005% SDS). Membranes were blocked with 1.5 % BSA in TBST (150 mM NaCl, 20 mM Tris-HCl pH 7.4, 0.3% Tween 2O)₅ and incubated with primary antibodies (1 μg/mL) and horseradish peroxidase-conjugated secondary antibodies in TBST/ 0.2% BSA. Membranes were subjected to chemiluminescence detection (Pierce).

Effect of fusion peptide on cell proliferation: To monitor cell proliferation a few modifications were introduced to a previously described BrdU incorporation-based assay (30). Cells (5,000/40 μL) were seeded in a white 384-well plate (Costar). Untreated or peptide-treated cells were incubated at 37 °C with 5% CO2 for 24 h. BrdU was added to a final concentration of 20 μM to the cells 14 h prior to fixation. Detection of BrdU incorporation was done with 0.5 μg/mL of mouse anti-BrdU antibody (PharMingen) and 1 :5,000 dilution of HRP -conjugated anti-mouse IgG, followed by addition of the enhanced chemiluminescence reagent (Pierce). Chemiluminescent signal from each plate was detected by autoradiograph and luminometer (Lmax; Molecular Devices) to quantify results.

Detection of apoptosis by flow cytometry : Untreated and peptide-treated human breast cancer cells were fixed and subjected to TUNEL assay with ApoBrdU™ following manufacturer's procedure. Peptide detection was done with anti-fiuorescein conjugated to AlexaFluor 488 (Molecular Probes). Analysis of labeled cells was done using the FACSCalibur and Cell quest software (Becton-Dickinson).

Detection of cytochrome c release: Mitochondria were isolated from SKBR-3 and MDA-MB-231 cells by differential centrifugation in ice-cold mito buffer (0.25 M sucrose, 1OmM Tris-HCl (pH 7.4), O.lmM EGTA) as described (45). In vitro release of cytochrome c from mitochondria was performed as follows (46). Mitochondria (50 μg) were left untreated or were treated for 1 hr with BHAP or AHNP (lOμM and 50 μM) in 0.1 niL mito buffer, and pelleted at 14,000 rpm for 5 min. Quantification of cytochrome c in supernatant fraction was done by anti-cytochrome c ELISA assay following manufacturer's procedure (R&D Systems). Release of cytochrome c from mitochondria to the cytosol from untreated or SKBR-3 and MD A-MB-231 cells treated as indicated was done as previously described (47). The mitochondria- and cytosol- containing fractions were analyzed by Western blot. Immunodetection was performed with mouse monoclonal anti-cytochrome c antibody (PharMingen) and anti-manganese superoxide dismutase (anti-MnSOD, Stressgen). Immuno fluorescent localization of cytochrome c and peptide: Untreated or peptide- treated cells were fixed with ice-cold 3.7% paraformaldehyde at RT, permeabilized with ice-cold 0.01% (v/v) Nonidet P-40 for 20 min and incubated with 0.5% BSA/PBS for 15 min. Fluorescein-labeled peptide was detected with rabbit anti-fluorescein- AlexaFluor 488 (Molecular Probes) and cytochrome c with monoclonal anti-cytochrome c antibody (PharMingen) and TRITC-conjugated anti-mouse secondary antibody (Jackson

ImmunoResearch) in PBSTB. Cells were photographed under Axioskop microscope using a Spot Camera (Diagnostic Instruments). Detection of peptide internalization and mitochondrial accumulation by electron- microscopy: Untreated or peptide-treated cells were harvested and fixed for 4 h at room temperature in 0.1 M sodium phosphate buffer (pH 7.4) containing 4% paraformaldehyde. Ultrathin cryosections were then stained with anti-fluorescein antibody followed by protein A conjugated to lOnm gold particles as previously described (48). The grids were examined using a JEOL 1200EX transmission electron microscope.

Determination of body distribution and toxicity of the fusion peptide: Mice were treated with fusion peptide at doses ranging from 1-50 mg/kg. Liver, heart, kidney, lung, spleen, skeletal muscle and blood were collected from mice 24 h post injection, Evidence of toxicity was determined by examination of tissue sections. Concentration of peptide in plasma was determined by LC/MS (Waters-MicroMass) against peptide standards. Plasma samples were processed with Montage albumin depleting kit and concentrated with ZipTip containing Cl 8 reverse phase media (Millipore). Peptide was eluted in 50% acetonitrile/ 0.1% formic acid solution and analyzed. Peptide accumulation in tumors was analyzed by flow cytometry. Cell suspensions were prepared from tumors dissected from vehicle or BHAP^F— treated mice 1 hr post-injection. Samples were fixed and co-incubated with anti- HER-2-APC conjugate and anti-fluorescein- AlexaFluor 488 conjugate. Cells gated on HER-2, which represent the human carcinoma cell population within the tumor cell samples, were analyzed for the presence of labeled peptide using the FACSCalibur (Becton-Dickinson).

In vivo efficacy in xenograft models: Human breast cancer cells were resuspended to 5-7 xlO⁶ cells/ 100 μL in PBS/20% matrigel (BD Biosciences), and injected into mammary gland fat pads of 6-8 week old CB.17-SCID female mice (Taconic Farms). To support growth of BT474 tumors, mice were implanted with 17β-estradiol pellets (Innovative Research) (49). Tumors were measured with a caliper and tumor volume calculated using the formula: volume (mm³) = width² x length/2. Treatment began when tumors reached a volume of > 200 mm³, Fusion and control peptides as well as vehicle were administered as described in the text. Mice were monitored weekly for weigh loss and tumor progression. AU animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School.

Results BHAP inhibits growth of HER-2-overexpressing human breast cancer cells The effect of BHAP (Fig. 1) on cell growth was tested on a panel of human breast cancer cell lines using a cell-based proliferation assay.

Whole cell lysates were prepared from a panel of human breast cancer cell lines and, for comparison, from immortalized non-transformed mammary epithelial MCF-IOA cells. HER-2 protein levels were assessed by Western blot. The growth inhibitory effect of BHAP was evaluated by cytoblot assay. Human breast cancer cell lines were treated with BHAP or control peptides for 24 hr, pulsed with BrdU for 15 hr and subjected to immunodetection of incorporated BrdU. The inhibitory concentration 50 (IC₅o) for the cell panel tested were determined by quantification of chemilumiscenet signals. Cells with diverse (high, medium and normal) HER-2 expression profile were selected for this purpose. The panel included SKBR-3, BT474, and Herceptin™-resistant MDA-361 and MDA-453 cells. MDA-231 breast cancer cells, like the non-transformed mammary epithelial MCF-IOA cells, express low (normal) HER-2 protein levels and were included in experimental group for comparison. The effect of BHAP on cell proliferation was monitored by BrdU-incorporation. A representative cytoblot assay for some of the cell lines tested is shown. Chemiluminescent signals were quantified and IC₅₀ determined for BHAP responder cells among the panel examined. BHAP treatment resulted in a dose- dependent inhibition of cellular proliferation, and the degree of inhibition correlated with the level of HER-2 overexpression. Unlike AHNP-induced growth inhibitory effect that is limited to cells with the highest HER-2 protein level, BHAP blocked proliferation of all

HER-2-overexpressing cell lines tested within the concentration range of the assay. Former studies have established that the low toxicity of the pro-apoptotic peptide (PAP) against mammalian cells is a consequence of its inability to traverse the plasma membrane. Indeed, cells incubated with PAP alone were unaffected by the treatment. The fusion of the pro- apoptotic domain to AHNP improved its potency and expanded its spectrum of action towards MDA-453 and MDA-361, while retaining its selectivity for cells with higher than normal levels of HER-2. Furthermore, MDA-231 cells that were resistant to BHAP treatment due to low levels of HER-2 expression became sensitive to the peptide upon HER-2-overexpression. Altogether, this data implies that HER-2-overexpression is necessary and sufficient to confer sensitivity to BHAP.

BHAP is internalized by receptor mediated endocvtosis and localizes to mitochondria

The results from the cytoblot assay confirmed that PAP alone is not toxic to HER-2 -overexpressing human cells unless PAP is coupled to the HER-2 targeting peptide. In control studies BHAP, but not AHNP, was detectable by liquid chromatography- mass spectrometry analysis in samples prepared from mitochondria of peptide-treated SKBR-3 and BT474 cells (data not shown). To examine the internalization of BHAP we used a fluorescein-labeled form of this peptide, BHAP^F. In the proliferation assay the activity of the labeled peptide was identical to that of BHAP. Cells treated with BHAP^F were analyzed by immunofluorescence microscopy, utilizing anti-fluorescein-Alexa Fluor 488 conjugates to amplify the signal. A punctate fluorescent signal spreading throughout the cytoplasm was observed when HER-2-overexpressing cells were exposed to peptide for 30 minutes. Preincubation of cells with anti-HER-2 antibodies prevented internalization of BHAP. To assess whether BHAP entered by receptor-mediated endocytosis, cells were treated with receptor internalization inhibitors prior to incubation with the labeled peptide (23). Pretreatment with 5 μM phenylarsenoxide for 30 minutes or 450 mM sucrose for 5 minutes effectively blocked internalization of the peptide, and a weak fluorescent signal remained restricted to the external periphery of the cells. These results are consistent with BHAP entering the cells through HER-2 -mediated endocytosis.

Immunofluorescence microscopy did not provide sufficient detail to conclude the fate of the peptide once endocytosed. For that reason the internalization of BHAP^F was monitored by immuno-gold electron microscopy. Ultrathin cryosections prepared from cells treated BHAP^F for 3 hr were subjected to anti-fluorescein immunodetection followed by protein A-gold.

HER-2-overexpressing breast cancer cells were left untreated or treated with the peptide and fixed in glutaraldehyde/ paraformaldehyde-containing buffer at various times thereafter. Samples fixed upon 3-5 hr of incubation showed gold particles localizing to the plasma membrane, sub-compartments of the endocytic pathway such as vesicles, endosome and multi-vesicular bodies (MVB) (24), and to a lesser extent to mitochondria. Analysis of samples subjected to 8-16 hr of incubation showed extensive labeling of mitochondria, with gold particles frequently decorating areas of mitochondria fusion/fission.

Electron microscopy analysis of glutaraldehyde-fixed samples, that better preserves membranes, reveals loss of internal organization and collapse of inner mitochondrial membrane following BHAP treatment (24 hr). On the other hand, no significant immunogold labeling or alterations of mitochondria morphology were detected in low HER-2-expressing MDA-231 cells. Thus, in sensitive HER-2-overexpressing cells an increase in peptide accumulation correlates with destabilization of mitochondria membranes.

HER-2 and downstream signaling; are differentially affected by BHAP in responsive cell lines Like Herceptin™, the peptidomimetic AHNP exerts a growth inhibitory effect on some HER-2-overexpresing cell lines (14). The anti-tumor effect that follows Herceptin™ treatment has been attributed to antibody-mediated cell cytoxicity elicited by the recombinant antibody, and to neutralization of HER-2 activity (8). In preclinical studies inhibition of tumor cell proliferation by Herceptin™ has been associated with a variety of effects including receptor internalization and endocytic degradation, as well as direct blockage of HER-2 activity and downstream signaling. Therefore, we examined the effect BHAP, and AHNP for comparison, on HER-2 and on three major signal transduction pathways activated by this tyrosine kinase receptor.

Whole cell lysates were prepared from human breast cancer cell lines left untreated or treated for 3 hr with BHAP and AHNP for a comparison. Aliquots containing equivalent amounts of protein were subjected to immunoblot analysis using antibodies against HER-2, PLCγl, PKB, MAPK and Src as well as antibodies against the phosphorylated forms of the proteins.

Treatment with both peptides for 3 hr resulted in a decrease in HER-2 protein levels (approximately 40-50 %) and a uniform dramatic tyrosine dephosphorylation in all the cell lines tested. The phosphorylation state of downstream signaling partners PKB/Akt, MAPK, PLC -γl and Src was also affected by AHNP and BHAP, to varying degrees in the different cell lines. Despite the comparable effect of BHAP and AHNP on HER-2-mediated cell signaling, their antiproliferative effect on the tumor panel analyzed is different. While BHAP inhibited growth of all selected cell lines with higher than normal levels of HER-2, AHNP was not active against MDA-453 and MDA-361 cell lines. Downregulation of HER-2 signaling may result from BHAP treatment and may contribute to its anti-carcinoma effect, however, the peptide-mediated cytotoxicity is most likely a consequence of the mitochondrial damage. Of note, the effect of BHAP and AHNP treatment on the pathways studied was more pronounced in SKBR-3 and BT474 cells. Both peptides were also effective in inducing donwregulation of HER-2 levels as well as dephosphorylation of the protein in HER-2-overexpressing MDA-453, MDA-361 and the low HER-2 expressing-MDA-231 cells. However, they were only capable of marginally interfering with signaling downstream of HER-2 in these three carcinoma cells. These results could potentially be explained in terms of the relative expression levels of other EGFR family members and their ligands, as well as of other co-receptors and downstream partners, and the relative contribution of these factors to sustain growth promoting signaling pathways in these cell lines (3, 9, 10, 25). As previously suggested, MDA-361 and MDA-453 may represent examples of HER-2-overexpressing cell lines that do not solely rely on HER-2 signaling to sustain their proliferation. Growth factor receptor signaling redundancy in cancer cells may be among the underlying causes of the limited efficacy of receptor-neutralization strategies. Thus, fusion of the toxic PAP domain to the HER-2 neutralizing AHNP may then represent an effective way to bypass this form of resistance. BHAP affects mitochondria in vitro and induces apoptosis in HER-2-overexpressing cells

The pro-apoptotic domain of our fusion peptide (KLAKLAK)₂ belongs to a class of compounds that induces apoptosis via a direct effect on mitochondria. A key event following mitochondria permeabilization during the course of the apoptotic response is the release to the cytosol of pro-apoptotic proteins, such as cytochrome c, normally stored in the intermembrane space (26, 27). This event is in turn followed by the activation of cysteine aspartyl proteases (caspases) as well as endonucleases that execute the cleavage of specific protein substrates and of genomic DNA. PAP has been previously shown to induce release of cytochrome c and loss of mitochondrial potential from mitochondria in vitro (19). To determine if BHAP behaved similarly, cytochrome c release from isolated mitochondria in response to the fusion peptide was quantified by ELISA assay.

Mitochondria (50 μg) isolated from SKBR-3 (gray bars) and MDA-231 (black bars) cells were incubated for 1 hr in 100 μL of mito buffer containing BHAP or control peptides and pelleted. Quantification of cytochrome c release was done by ELISA. SKBR-3 and MDA-231 cells were left untreated or treated with BHAP. Mitochondria- and cytosol- containing fractions were prepared as described under "Material and Methods" and subjected to anti-cytochrome c Western blot. Loadings were normalized to cell number. A significant redistribution of cytochrome c from the mitochondria pellet to the supernatant was observed upon both PAP and BHAP treatment. In contrast, incubation of mitochondria in AHNP-containing buffer did not result in cytochrome c release. These results indicate that AHNP has no effect on mitochondria membrane unless fused to PAP. Death of HER-2-overexpressing cells in response to BHAP treatment exhibits the typical hallmarks of apoptotic demise, i.e., cell shrinkage and nuclear condensation partially blocked by the pan-caspase inhibitor z-VAD-fmk (data not shown). We also examined the localization of cytochrome c following BHAP treatment of tumor cell lines. Treatment of sensitive cells with BHAP for 8 hr resulted in release of cytochrome c from mitochondria to the cytosol as determined by anti-cytochrome c Western blot analysis. Quantification of the signals is consistent with a 60 % reduction of cytochrome c in the mitochondria fraction of BHAP-treated SKBR-3 cells. On the other hand, no release of other mitochondrial proteins such as manganese superoxide dismutase (MnSOD) was detectable in the same samples. These results were further confirmed by immunofluoresence analysis of untreated cells or cells exposed to fluorescein-labeled BHAP followed by simultaneous detection of the peptide and cytochrome c. Moreover, following peptide treatment oligonucleosomal DNA fragmentation was only detectable by TUNEL assay in cells overexpressing HER-2. Unlike the non-selective mitochondriotoxic effect of PAP and BHAP on in vitro isolated mitochondria, BHAP merely triggered cytochrome c release from mitochondria of tumor cells in a HER-2-dependent manner. These results along with the electron microscopy studies are consistent with the notion that following selective HER- 2-mediated endocytosis of BHAP, peptide molecules reach the mitochondria and selectively trigger apoptosis in HER-2-overexpressing breast cancer cells. BHAP inhibits tumor growth in vivo The anti-carcinoma effect of BHAP observed in vitro prompted us to test whether this fusion peptide exhibited efficacy in vivo. In preliminary studies, mice were injected intraperitoneally with BHAP at various doses. At high doses, i.e. 50 mg/kg, out of five mice treated with BHAP at 50 mg/kg one died approximately 24 hr post-injection. Histologic examination of tissues collected at necropsy showed signs of nephrotoxicity at this dose. No indication of rhabdomyalysis or hemolysis was detectable among samples from the treated group. Peptide accumulation in tubules and collecting ducts in the kidneys during excretion are most likely responsible for the toxic effect. Lower doses (10mg/kg) were well tolerated. No toxic effects were detectable upon a comprehensive histologic examination of tissue sections. Blood samples were collected from mice tail veins at various times points post-injection and peptide levels in plasma were determined by liquid chromatography-mass spectrometry. A peak in plasma (17 μM) was detected 30 minutes after injection. The effect of BHAP on tumor growth was assessed in three tumor xenograft models. Tumors were initiated in CB.17-SCID mice by injection of HER-2-overexpressing human breast cancer MDA-453 and BT474 as well as MDA-231 cells into the fat pads of the fourth mammary glands. BHAP accumulation in tumors following intraperitoneal injection of fluorescein-labeled fusion peptide was examined by flow cytometry.

Tumors from HER-2-overexpressing BT474 and MDA-453 (Herceptin™ resistant) cells were initiated in CB.17-SCID mice as described in the text. Cell suspensions prepared from tumors dissected from mice treated with vehicle or BHAP^F 1 hr post injection, were subjected to flow cytometry. BT474 (black) and MDA-453 (grey) samples were gated for HER- 2 positive cells and analyzed for the presence of labeled peptide as indicated under "Material and Methods". When indicated BHAP and control peptides were administered by intraperitoneal injection. Tumor volumes were calculated as indicated under "Material and Methods".

Analysis of cell suspensions prepared from tumors 1 hr post-injection showed that 35-75 % of HER-2 positive BT474 and MDA-453 cells accumulated BHAP. BHAP was not detectable in MDA-213 -derived tumors (data not shown). Thus, BHAP selectively homes to HER-2-overexpressing carcinoma cells. Peptides were administered to mice bearing established tumors as follows: when tumor volume reached 200-300 mm³, mice were treated with lOmg/kg daily every other day, for a total of three doses as indicated. Progression of tumor growth was monitored thereafter. For a comparison, mice were treated with AHNP and PAP alone as well. Administration of BHAP resulted in inhibition of growth of MDA-453- and BT474-derived tumors. In contrast, AHNP was only effective against BT474-derived xenografts. BHAP showed no anti-tumor effect on mice bearing MDA-231 initiated tumors. Thus, the selectivity of BHAP for HER-2-overexpressing tumor cells exhibited in vitro was also recapitulated in vivo. No body weigh loss in excess of 5.5% was observed. Histologic examination of sections prepared from MDA-453 and BT474-derived tumors excised 24 hr from the final BHAP dose revealed areas with condensed cells and extensive cell death. Apoptotic (TUNEL positive) cells were detected by flow cytometry in samples from BHAP-treated MDA-453 and BT474 xenografts (data not shown). The overall 4.3 and 3.8 fold decrease in tumor burden in BHAP-treated mice bearing BT474 and MDA-453 carcinoma xenografts respectively demonstrates that the peptide effectively slowed down tumor growth. The mouse study also highlights the importance of the dual targeting effect. In the case of BT474, the two-fold increase in the growth inhibitory activity of BHAP vs. AHNP suggests that addition of the toxin further improves the outcome of the HER-2 targeted approach.

Development of BHAP tetramers with a lower ICsn: Streptavidin has been widely used as an adaptor molecule to produce oligomers from a variety of proteins and peptides. Streptavidin is a tetramer with unique affinity for biotin (Kd= 4 x 10^"14 M) (28). We exploited the stability of the streptavidin: 4 biotin complex to test whether oligomerization of biotinylated-BHAP (BHAP^B) could improve its activity. BHAP ^B and streptavidin (SAv) were incubated for 10 minutes. Samples were resolved in a reversed polarity native gel (pH 3.9; β-alanine/HAc buffer) at 300V (4°C) and stained with Coomassie blue. BHAP^B tetramer is the predominant species at 4:1 ratio. The BHAP tetramer can be visualized by Coomassie Blue staining of samples resolved by non-denaturing polyacrilamide gel electrophoresis. The effect of the BHAP - streptavidin oligomer on the proliferation of breast cancer cells was compared to that of monomelic BHAP. For this experiment we included MCF-7 cells that express intermediate (lower than MDA-453 and higher than MDA-231) levels of HER-2 in comparison to the rest of the panel. Like the monomer, the BHAP tetramer exhibited selectivity for the cells overexpressing HER-2. The IC₅₀ of the oligomer for responsive cell lines ranged from 100- 800 nM, a 19 to 80 fold decrease when compared to BHAP monomer. Thus, this approach resulted in a significant improvement of the efficacy of BHAP. against the HER-2 positive breast cancer cells tested. Discussion In the present study we have linked two functional domains to produce a novel chimeric peptide BHAP. BHAP was designed as a "bifunctional peptide" both to block the HER-2 protein function and to affect mitochondria in target HER-2-overexpressing carcinoma cells. The choice of a mitochondriotoxic peptide over a mitochondiotoxic small molecule was based on the simplicity of peptide chemistry for conjugation purposes.

Mitochondria are unique organelles in that they play a central role in a plethora of biological functions essential for cell survival (29). A number of laboratories, including ours (30), have focused efforts to develop and to characterize novel small molecules and peptides that selectively obliterate mitochondria function in tumor cells by directly targeting the organelle as a means to interfere with tumor progression (31, 32). The attractive perspective of this strategy lies in the potential to circumvent resistance to apoptosis that normally arises as a consequence of accumulation of mutations in apoptosis signaling intermediates upstream of mitochondria in a variety of cancers (33-35). It is well established that the selectivity of a toxin for tumor cell mitochondria over normal cell mitochondria can be dictated by an inherent differential trait of the organelle between the normal and transformed cellular states. Alternatively, a promiscuous and/or plasma membrane impermeable mitochondria toxin could be turned into a selective anti-carcinoma reagent through targeted delivery. Our data shows that indeed it is feasible to target the plasma membrane impermeable mitochondriotoxic (KLAKLAK)₂ peptide to cancer cells overexpressing the HER-2 tyrosine kinase receptor.

Characterization of the BHAP -mediated effects at the cellular level shows that the fusion peptide is selectively endocytosed by cells with high levels of HER-2. As shown by electron microscopy, once internalized the peptide traffics through the endocytic compartment. Although still not fully understood at the molecular level, the ability of polycationic and amphipatic "membrane active" peptides like (KLAKLAK)₂ to escape the acidic endosome environment has been previously described (36, 37). Our results indicate that BHAP translocates endosomal/lysosomal membranes and reaches the mitochondria. The organelle's functional and structural integrity is compromised as a consequence of the PAP mitochondrial membrane-disrupting ability, triggering apoptosis in the target cells. Besides the mitochondrial effects, BHAP directly affects HER-2. However, despite this direct and general effect of BHAP on HER-2 tyrosine phosphorylation among the HER-2-overexpressing cell panel, the phosphorylation state of downstream signaling partners was significantly affected only in a subset. Regardless of the limited effect of the fusion peptide on signaling in MDA-453 and MDA-361, their proliferation is still significantly halted following treatment, rnactivation of proliferation promoting signaling pathways in response to BHAP cannot account for the comparable IC_5O of the HER-2 positive cancer cell panel tested. Therefore, HER-2 neutralization may contribute only in some cases to the growth inhibitory effect of BHAP on HER-2-overexpressing cells. But it is likely that the direct disrupting effect on mitochondria membrane is the general underlying mechanism of action of the fusion peptide.

We have also begun to explore the use of oligomeric forms of BHAP as a means to increase the avidity of the chimeric peptide for HER-2. For instance, tetramers with higher ligand avidity have been successfully produced from biotinylated class I MHC monomers (38, 39). Likewise, we have produced tetrameric BHAP by streptavidin binding of biotinylated monomers. The "BHAP tetramer" exhibits 19-80 fold lower IC₅₀ (per mol of monomer) than BHAP against the panel of cell lines overexpressing HER-2. Further studies will determine the efficacy of the oligomeric form of BHAP in vivo. In addition to the effects observed in vitro, the fusion peptide exhibits anti-tumor effect in HER-2 positive human breast carcinoma xenografts. The fact that HER-2- overexpression is sufficient to render cancer cells sensitive to fusion peptide treatment has important therapeutic implications. Our work shows that BHAP is effective against the Herceptin™-resistant MDA-453 and MDA-361 cancer cell lines. Thus, the mitochondrial injury imposed by a reagent like BHAP may help to overcome the barrier encountered by therapies focused on HER-2 neutralization or by an insufficient antibody-mediated cell cytotoxicity elicited by HER-2 directed antibodies. The benefits of BHAP treatment in two xenograft transplant models clearly provide proof-of concept in vivo. The results illustrate that overexpression of HER-2 can be used as a selective entry gate for the designed peptide- based toxin. Under the dose regimen chosen for the in vivo experiments, BHAP did not completely eradicate tumors. In the future we will test different BHAP dose schedules as well as BHAP tetramer in xenograft models.

Finally, the BHAP paradigm suggests that the development of tailored peptides with anti-carcinoma activity is feasible. Like HER-2 a number of receptors overexpressed in an array of solid tumor types have already been defined. In the future it may be possible to develop personalized medicines by creating peptide libraries that will enable customized treatment of tumors according to their molecular profile. Stable and high affinity peptides against a number of cell surface targets continue to emerge by phage display methodology and by protein-based peptide library screening or, like AHNP, by molecular modeling after receptor-directed antibodies. Recent advances in conjugation technology may allow to reintroduce extremely potent cytotoxic small molecules that have been found to be too toxic for therapeutic purposes. To date, a number of antibody-immunoconjugates have been developed (40-42). In comparison to peptides, antibodies are in general more stable molecules and display exquisite affinity for their cognate antigens. However, peptides still exhibit some attractive features. The small molecular mass of a peptide increases the molar ratio of toxin to targeting agent, hi addition, stable peptides with reasonable dissociation constants exhibit superior ability to diffuse across tissues, improving tumor exposure to the drug. In comparison to recombinant antibodies, the more cost effective large-scale production of peptides may provide a viable alternative to produce peptide-based prodrug conjugates. This approach would minimize bystander toxicities providing an effective alternative to increase the therapeutic index of cytotoxic compounds for the treatment of cancer. References

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Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

Claims:

1. A bispecific peptide comprising a targeting peptide and a pro-apoptotic peptide.

2. The bispecific peptide of claim I₃ wherein the targeting peptide is a Her-2 targeting petpide.

3. The bispecific peptide of claim 2, wherein the Her2 targeting peptide comprises an amino acid sequence that is at least about 80% identical to YCDGFYACYMDV.

4. The bispecific peptide of claim 3, wherein the Her2 targeting peptide comprises an amino acid sequence that is at least about 90% identical to YCDGFYACYMDV.

5. The bispecific peptide of claim 4, wherein the Her2 targeting peptide comprises the amino acid sequence YCDGFYACYMDV.

6. The bispecific peptide of claim 2, wherein the Her-2 targeting peptide comprises an amino acid sequence consisting essentially of YCDGFYACYMDV.

7. The bispecific peptide of any one of the above claims, wherein the pro-apoptotic peptide comprises an amino acid sequence that is at least about 80% identical to (KLAKLAK)2.

8. The bispecific peptide of any one of the above claims, wherein the pro-apoptotic peptide comprises an amino acid sequence that is at least about 90% identical to (KLAKLAK)2.

9. The bispecific peptide of any one of the above claims, wherein the pro-apoptotic peptide comprises the amino acid sequence (KLAKLAK)2.

10. The bispecific peptide of any one of the above claims, wherein the pro-apoptotic peptide comprises an amino acid sequence consisting essentially of (KLAKLAK)2.

11. The bispecific peptide of any one of the above claims, wherein the Her-2 targeting peptide comprises an amino acid sequence consisting essentially of YCDGFYACYMDV and the pro-apoptotic peptide comprises an amino acid sequence consisting essentially of (KLAKLAK)2.

12. The bispecific peptide of any one of the above claims, further comprising a linker.

13. The bispecific peptide of claim 12, wherein the linker comprises from 1 to 20 amino acids.

14. The bispecific peptide of claim 13, wherein the linker comprises from 1 to 10 amino acids.

15. The bispecific peptide of claim 14, wherein the linker comprises from 1 to 5 amino acids.

16. The bispecific peptide of claim 15, wherein the linker comprises 2 amino acids.

17. The bispecific peptide of claim 16, wherein the linker comprises the amino acid sequence GG.

18. The bispecific peptide of any one of claims 12-17, wherein the linker is a cleavable linker.

19. The bispecific peptide of any one of the above claims, wherein the targeting peptide is located N-terminally relative to the pro-apoptotic peptide.

20. The bispecific peptide of any one of the above claims, wherein the targeting peptide is located C-terminally relative to the pro-apoptotic peptide.

21. A peptidomimetic of the bispecific peptide of any of the above claims.

22. The bispecific peptide of any one the above claims, further attached to a molecule.

23. The bispecific peptide of claim 22, wherein the molecule is a label that can be detected.

24. The bispecific peptide of claim 22, wherein the molecule is a molecule that stabilizes the bispecific peptide.

25. The bispecific peptide of claim 24, wherein the molecule is a polymer.

26. The bispecific peptide of claim 23, wherein the molecule is biodegradable.

27. A pharmaceutical composition comprising a bispecific peptide of any of the above claims.

28. A kit comprising a bispecific peptide of any of the above claims.

29. A nucleic acid encoding a bispecific peptide of any of the above claims.

30. Use of a bispecific peptide of any of the above claims for the preparation of a medicament for treating or preventing a disease characterized by the undesirable presence of Her-2 containing cells in a subject.

31. The use of claim 30, wherein the disease is a cancer.

32. The use of claim 31, wherein the disease is a carcinoma.

33. The use of claim 32, wherein the disease is breast carcinoma.

34. The use of any one of claims 30-33, further comprising providing to the subject another treatment for the disease.

35. The use of claim 34, wherein the other treatment is chemotherapy.