WO2021185719A1

WO2021185719A1 - Rgd-containing peptide and use thereof for cancer treatment

Info

Publication number: WO2021185719A1
Application number: PCT/EP2021/056451
Authority: WO
Inventors: Christian Auclair
Original assignee: Ac Bioscience Sa
Priority date: 2020-03-15
Filing date: 2021-03-15
Publication date: 2021-09-23

Abstract

The invention relates to an RGD-containing peptide and use thereof for treating cancer and/or cancer metastases.

Description

RGD-CONTAINING PEPTIDE AND USE THEREOF FOR CANCER TREATMENT

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Cancer is a generic name for a wide range of cellular malignancies characterized by unregulated growth, lack of differentiation, and the ability to invade local tissues and metastasize. These neoplastic malignancies affect, with various degrees of prevalence, every tissue and organ in the body.

A number of therapeutic agents have been developed over the past few decades for the treatment of various types of cancer. The most commonly used types of anticancer agents include: DNA- alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate, a folate antagonist, and 5-fluorouracil, a pyrimidine antagonist), microtubule disrupters (e.g., vincristine, vinblastine) or microtubule stabilizers (e.g. paclitaxel), topoisomerase inhibitors (e.g., doxorubicin, daunomycin), alkylating agents (e.g. cisplatin), and hormone therapy (e.g., tamoxifen, flutamide, gene targeted therapies, such as protein-tyrosine kinase inhibitors (e.g. imatinib; the EGFR kinase inhibitor. More recently, checkpoint inhibitors such as anti-CTLA4 and anti-PDl/PDLl are increasingly used for cancer treatment.

An ideal anti-neoplastic drug would selectively kill cancer cells, with a high therapeutic index relative to its toxicity towards non-malignant cells. It would also retain its efficacy against malignant cells, even after prolonged exposure to the drug. Unfortunately, few of the current chemotherapies possess such an ideal profile and most possess low therapeutic indexes. Furthermore, cancerous cells exposed to slightly sub-lethal concentrations of a chemotherapeutic agent will very often develop resistance to such an agent, and quite often cross-resistance to several other antineoplastic agents as well. Additionally, for any given cancer type one frequently cannot predict which patient is likely to respond to a treatment, even with targeted therapies, such as mutated EGFR or c-kit kinase inhibitors, thus necessitating considerable trials in order to find the most effective therapy. Thus, there is a need for more efficacious treatment for neoplasia and other proliferative disorders, and for more effective means for determining which cancers will specifically respond to the treatment which in turn requires the identification of specific molecular target(s).

Integrins are a family of cell surface receptors that attach cells to the matrix and mediate mechanical and chemical signals from it. These signals regulate the activities of cytoplasmic kinases, growth factor receptors, and ion channels and control the organization of the intracellular actin cytoskeleton. Many integrin signals converge on cell cycle regulation, directing cells to live or die, to proliferate, or to exit the cell cycle and differentiate. Accordingly, integrins regulates a diverse array of cellular functions crucial to the initiation, progression and metastasis of solid cancers. The importance of integrins in several cell types that affect tumor progression has made them pertinent targets for cancer therapy. Integrin antagonists, including the anb3 and anb5 inhibitor cilengitide, have shown encouraging activity in Phase II clinical trials and cilengitide is currently being tested in a Phase III trial in patients with glioblastoma.

Integrins have been shown to be a dominant factor in enhancing cancer aggressiveness. High expression of integrin av and b3 has been shown to closely associate with bone metastasis and tissue remodeling in prostate cancer. Besides, integrin av and b3 overexpression in prostate cancer tightly relates to angiogenesis and tumor metastasis potentials. Moreover, integrin av and b3 facilitates bone remodeling that results in bone metastasis by mediating adhesion and migration via vitronectin. In esophageal carcinomas, the integrin a6 has been shown to link with tumor invasion. For pancreatic cancer, integrins a2b1, a5b1 and anb5 have been shown to mediate cancer cell adhesion to the ECM components fibronectin, laminin, collagen, and vitronectin, respectively. These integrin engagements are involved in cell proliferation and essential for cancer invasion. It is evident that integrins may represent candidate targets to interfere with cancer growth and progression. Disrupting integrin-ligand interactions has the potential to interfere with key survival and proliferation signals that support cancer growth. Importantly, integrin inhibition may simultaneously target key aspects of tumor cell behavior as well as crucial features of the tumor microenvironment such as angiogenesis or functions of cancer-associated fibroblasts that support cancer growth or invasion.

Early studies showed that blocking a large subset of integrins, mainly a5b1 and anb3 by using RGD peptides (arginine-glycine-aspartic acid) can interfere with tumor cell invasion in vitro and metastasis in mouse models (Humphries et al., Science, 1986: 233, 470). Subsequently, various synthetic peptides containing the RGD sequence or other integrin binding sequences, nonpeptide RGD mimetics, and disintegrins (integrin-binding proteins isolated from viper snake venoms) have been demonstrated to be able to block experimental tumor cell metastasis in animal model systems (Curley et al., Cellular and Molecular Life Sciences, 1999: 56, 427). Interestingly, similar approaches could at the same time inhibit tumor angiogenesis (Brooks et al., Journal of Clinical Investigation, 1995: 96, 1815). Based on these initial promising findings, a variety of RGD-related peptides, peptides covering alternative integrin recognition motifs, nonpeptide mimetics, and humanized integrin-directed antibodies have been developed. These are in various stages of (pre) clinical testing or already on the market for a variety of diseases. In the context of cancer treatment, at present strategies for targeting bΐ integrins or av integrins have entered phase I, phase II, and even phase III clinical trials (Brooks et al., Journal of Clinical Investigation, 1995: 96, 1815).

Despite many attempts for developing integrin antagonists as antitumor agents, few molecules if any, including the most promising cilengitid, have demonstrated a major clinical benefit. Clinical trials with cilengitide in patients with either lung cancer or metastatic melanoma or recurrent or metastatic head and neck tumors, showed little effect on overall survival, whereas preliminary data in advanced glioblastoma seemed to suggest an effect on overall survival. The multicentre, randomized, open-label phase III CENTRIC trial investigated the potential benefit of the combination of cilengitide with standard treatment in patients newly diagnosed with glioblastoma with methylated MGMT promoter. However, results of this study have not shown cilengitide to be better in terms of progression-free survival or overall survival than the standard treatment. Thus, Stupp and colleagues suggested not pursuing the testing of cilengitide in further anticancer clinical trials.

In addition, the RGD peptides are generally not suitable for in vivo use because of their short half life in the body.

Among antitumor integrins antagonists subjected to clinical trials, AMEP (Anti angiogenic MEtargidin Peptide) is the disintegrin domain of human metargidin (also known as ADAM- 15). It is an anti-cancer agent in experimental models with proven anti -angiogenic properties by binding of anb3 and a5b! integrins on endothelial cells (Bosnjak et al, Gene Ther. 2015; 22:578; Trochon-Joseph et al. Cancer Res. 2004;6 4:2062). Furthermore, intramuscular gene electrotransfer of plasmid AMEP has been shown to inhibit tumor growth in murine models. In 2015, it has been reported the results from a first-in-man study investigating intratumoral gene electrotransfer of plasmid AMEP into cutaneous melanoma. Plasmid AMEP was well tolerated and measurements of treated lesions suggested local anti -turn or efficacy (Spanggaard et al., Hum Gene Ther Clin Dev. 2013; 24:99). In this study, AMEP mRNA was found in treated lesions; however, AMEP protein could not be detected with available methodology. In 2017, it has been reported the results from a phase I study investigating gene electrotransfer to muscle tissue using plasmid AMEP in patients with advanced solid tumors. No objective responses were observed in this study (Spanggaard et al., Oncologica Acta 56, 2017).

AMEP was a promising molecule, however the length of the peptide and the complexity of the structure (90 amino acids and 10 cysteines) does not allow the synthesis and the use of the native peptide as therapeutic agent. This requires the use of a plasmid encoding the AMEP peptide introduced in the patient’s muscles by electrotransfer process. The short lifetime of the plasmid AMEP and the low expression level explain the lack of efficacy of this strategy.

Therefore, there is still a need to develop therapeutic agents which retain the positive properties of RGD-containing peptides for longer periods of time than is normally currently the case, which allow easier synthesis and easier manipulation.

SUMMARY OF THE INVENTION

An aspect of the present invention provides an RGD-containing peptide comprising RGD sequence, PHK sequence and a spacer between the RGD and the PHK sequences, wherein the RGD-containing peptide has a length of 40 to 60 amino acids, and wherein the spacer consists of 14 to 20 amino acids.

Another aspect of the present invention provides a use of the RGD-containing peptide of the invention for inhibiting RGD-binding integrins.

Another aspect of the present invention provides a pharmaceutical composition comprising the RGD-containing peptide of the invention and at least one pharmaceutically acceptable carrier, excipient or diluent. Another aspect of the present invention provides an RGD-containing peptide of the invention for use in a method of inhibiting RGD-binding integrins activity in a subject.

Another aspect of the invention provides an RGD-containing peptide of the invention for use in a method for treating cancer, wherein cancer cells express RGD-binding integrins.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows comparative effect of peptide AMEP (42 amino acids) referred as WT AMEP and an optimized chimeric peptide containing RGD and PHK sequences referred as phkAMEP on the proliferation of vascular endothelial cells (HUVEC, ATCC CRL-1730). Proliferation was measured according to conventional procedure. Peptide concentrations were expressed in pg/ml.

DETAILED DESCRIPTION OF THE INVENTION

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components. Also as used in the specification and claims, the language "comprising" can include analogous embodiments described in terms of "consisting of “ and/or "consisting essentially of’. As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

As used in the specification and claims, the term "and/or" used in a phrase such as "A and/or B" herein is intended to include "A and B", "A or B", "A", and "B".

The terms "peptide" or “polypeptide” are used interchangeably herein to refer to a peptide or a polypeptide (i.e. a polymer of amino acid residues joined together by an amide bond between adjacent amino acid residues) that is typically no more than 60, 70, 80, 90 or 100 amino acids in length. In some embodiments, peptides are at least about 40, 50, 60 or 70 amino acid residues long. The terms “peptide” or "polypeptide" encompass native or artificial proteins, protein fragments and polypeptide analogues of a protein sequence. Therefore, "peptides" of the invention are distinguishable over full-length proteins and other polypeptides.

As used herein the term "therapeutically effective amount" is defined as an amount of the agent that will inhibit proliferation, metastasis, and/or invasion of the cancer. For example, it may decrease, reduce, inhibit or otherwise abrogate the growth of a cancer cell, induce apoptosis, inhibit angiogenesis of a tumour cell, inhibit metastasis, or induce cytotoxicity in cells. Thus, an effective amount is an amount sufficient to detectably and repeatedly ameliorate, reduce, minimize or limit the extent of the disease or its symptoms. More rigorous definitions may apply, including elimination, eradication or cure of disease.

"Invasiveness" is the degree to which an organism such as cancer cell is able to spread through the body from one region such as the original site of tumour.

"Metastasis" is the process by which cancer spreads from the place at which it first arose as a primary tumour to distant locations in the body. Metastasis depends on the cancer cells acquiring two separate abilities: increased motility and invasiveness. Cells that metastasize are basically of the same kind as those in the original tumour. If a cancer arises in the lung and metastasizes to the liver, the cancer cells in the liver are lung cancer cells. However, the cells have acquired increased motility and the ability to invade another organ.

As used herein the term "pharmaceutically acceptable excipients, diluents and/or carriers" means that the compositions or components thereof so described are suitable for suitable for administration to subject (patient) body without undue toxicity, incompatibility, instability, irritability, allergic response, and the like.

The terms "patient," "subject," "individual," and "host" are used interchangeably herein to refer to a living animal, including a human and a non-human animal. The subject may, for example, be an organism possessing cancer cells that express integrins and capable of responding to inhibition of these integrins. The subject may be a mammal, such as a human or non-human mammal, for example, farm animals, such as horses, cows, pigs, family pets, such as dogs or cats, and other animals including non-human primates, rats, and mice. The term "subject" does not preclude individuals that are entirely normal with respect to a disease, or normal in all respects.

The term "treatment" refers to a therapeutic or preventative measure. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

An aspect of the invention provides an RGD-containing peptide that comprises RGD sequence, PHK sequence and a spacer between the RGD and the PHK sequences, wherein the RGD- containing peptide has a length of 40 to 60 amino acids, and wherein the spacer consists of 14 to 20 amino acids. The presence of the PHK sequence can increase the binding constant of the RGD sequence to recognize RGD ligands.

In some embodiments of the invention, the RGD-containing peptide according to formula I comprises an N-terminal sequence, RGD sequence, PHK sequence, a spacer between the RGD and the PHK sequences and C-terminal sequence, wherein the RGD-containing peptide has a length of 40 to 60 amino acids, and wherein the spacer consists of 14 to 20 amino acids.

Formula I

In some other embodiments of the invention, the RGD-containing peptide according to formula I consists of an N-terminal sequence, RGD sequence, PHK sequence, a spacer between the RGD and the PHK sequences and C-terminal sequence, wherein the RGD-containing peptide has a length of 40 to 60 amino acids, and wherein the spacer consists of 14 to 20 amino acids.

Formula I

In the context of the present invention, the RGD sequence is arginine-glycine-aspartic acid, and the PHK sequence is proline-histidine-lysine.

In some embodiments, the RGD-containing peptide of the invention has a length of 40 to 50 amino acids, 42 to 50 amino acids, 45 to 50 amino acids, 40 to 51 amino acids, 42 to 51 amino acids, 45 to 51 amino acids, 42 to 55 amino acids, 45 to 55 amino acids, 42 to 60 amino acids, 45 to 60 amino acids, 50 to 60 amino acids. In some other embodiments, the RGD-containing peptide of the invention has a length of at least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids. In some other embodiments, the RGD-containing peptide of the invention has a length of maximum 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acids.

In some embodiments of the RGD-containing peptide of the invention, the spacer consists of 14, 15, 16, 17, 18, 19 or 20 amino acids. In other embodiment of the RGD-containing peptide of the invention, the spacer consists of 14 to 19 amino acids, 14 to 18 amino acids, 14 to 17 amino acids, 14 to 16 amino acids, 14 to 15 amino acids, 15 to 20 amino acids, 15 to 19 amino acids, 15 to 18 amino acids, 15 to 17 amino acids, 15 to 16 amino acids, 16 to 20 amino acids, 16 to 19 amino acids, 16 to 18 amino acids, 16 to 17 amino acids, 17 to 20 amino acids, 17 to 19 amino acids, 17 to 18 amino acids, 18 to 20 amino acids, 18 to 19 amino acids, 19 to 20 amino acids. The spacer can have any suitable amino acid sequence that allows correct RGD- containing peptide folding, 3D structure and ideal 3D positioning of the RGD sequence in respect to the PHK sequence. In a preferred embodiment of the RGD-containing peptide of the invention, the spacer is CDLPEFCPGDSSQC (SEQ ID NO: 1). In another preferred embodiment of the RGD-containing peptide of the invention, the spacer is CDLPEFCPGDSSQCPPD (SEQ ID NO: 2). In another preferred embodiment of the RGD- containing peptide of the invention, the spacer is CDLPEFCPGDSSQCPPDV (SEQ ID NO: 3). In a preferred embodiment of the RGD-containing peptide of the invention, the spacer is selected from the group comprising CDLPEFCPGDSSQC (SEQ ID NO: 1), CDLPEFCPGDSSQCPPD (SEQ ID NO: 2), CDLPEFCPGDSSQCPPDV (SEQ ID NO: 3). In some embodiments of the RGD-containing peptide of the invention, the spacer can be a polymer or a PEG-polymer. The length of the polymer or the PEG should correspond to the length of the spacer expressed in amino acids, as mentioned above.

In other preferred embodiments, the polymer is selected from the group comprising DSG, DSS, BS3, TSAT (trifunctional), BS(PEG)5, BS(PEG)9, DSP, DTSSP, DST, BSOCOES, EGS, Sulfo-EGS, DMA, DMP, DMS, DTBP, DFDNB, BMOE, BMB, BMH, TMEA (trifunctional), BM(PEG)2, BM(PEG)3, DTME, AMAS, BMPS, GMBS and Sulfo-GMBS, MBS and Sulfo- MBS, SMCC and Sulfo-SMCC, EMCS and Sulfo-EMCS, SMPB and Sulfo-SMPB, SMPH, LC-SMCC, Sulfo-KMUS, SM(PEG)2, SM(PEG)4, SM(PEG)6, SM(PEG)8, SM(PEG)12, SM(PEG)24, SPDP or SPDP, LC-SPDP and Sulfo-LC-SPDP, SMPT, PEG4-SPDP, PEG12- SPDP, SIA, SBAP, SIAB, Sulfo-SIAB, ANB-NOS, Sulfo-SANPAH, ATFB, SDA and Sulfo- SDA, LC-SDA and Sulfo-LC-SDA, SDAD and Sulfo-SDAD, DCC, EDC or ED AC, BMPH, EMCH, MPBH, KMUH, PDPH, PMPI, SPB.

In some preferred embodiments, the RGD-containing peptide of the invention is selected from the group comprising:

QNCQLRPSGWQCRPTRGDCDLPEFCPGDSSQCPHKVSLGD (SEQ ID NO: 4) QNCQLRPSGWQCRPTRGDCDLPEFCPGDSSQCPPDPHKVSLG (SEQ ID NO: 5) CASDGPCCQNCQLRPSGWQCRPTRGDCDLPEFCPGDSSQCPPDPHKVSLG (SEQ ID NO: 6)

CASDGPCCQNCQLRPSGWQCRPTRGDCDLPEFCPGDSSQCPPDVPHKVSLG (SEQ ID NO: 7)

In some embodiments of the RGD-containing peptide of the invention, the N-terminal sequence consists of 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids. In some embodiments the N- terminal sequence consists of 15 to 23 amino acids, 12 to 30 amino acids, 16 to 20 amino acids. The N-terminal sequence can have any suitable amino acid sequence that allows correct RGD- containing peptide folding, 3D structure and/or ideal 3D positioning of the RGD sequence in respect to the PHK sequence. In some other embodiments, the N-terminal sequence is QNCQLRPSGWQCRPT (SEQ ID NO: 8) or CASDGPCCQNCQLRPSGWQCRPT (SEQ ID NO: 9). In some embodiments of the RGD-containing peptide of the invention, the C-terminal sequence consists of 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In some embodiments the C-terminal sequence consists of 3 to 10 amino acids, 4 to 7 amino acids, 4 to 5 amino acids. The C-terminal sequence can have any suitable amino acid sequence that allows correct RGD-containing peptide folding, 3D structure and/or ideal 3D positioning of the RGD sequence in respect to the PHK sequence. In some other embodiments, the C-terminal sequence is VSLGD (SEQ ID NO: 10) or VSLG (SEQ ID NO: 11) or VSLGDGE (SEQ ID NO: 12). In some other embodiments, the C-terminal sequence is selected from the group comprising VSLGD (SEQ ID NO: 10), VSLG (SEQ ID NO: 11), VSLGDGE (SEQ ID NO: 12). In some other embodiments, the C-terminal sequence is selected from the group comprising VSLGD (SEQ ID NO: 10), VSLG (SEQ ID NO: 11).

In other preferred embodiments, the RGD-containing peptide of the invention is either a linear peptide or a cyclized peptide.

In some embodiments, the RGD-containing peptide of the invention is conjugated to an agent that increases the accumulation of the peptide in a cell. Such an agent can be a compound which induces receptor mediated endocytosis such as for example the membrane transferrin receptor mediated endocytosis of transferrin conjugated to therapeutic drugs (Qian Z. M. et al., "Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway" Pharmacological Reviews, 54, 561, 2002) or a cell membrane permeable carrier which can, be selected e. g. among the group of fatty acids such as decanoic acid, myristic acid and stearic acid, which have already been used for intracellular delivery of peptide inhibitors of protein kinase C (Ioannides C.G. et al., "Inhibition of IL-2 receptor induction and IL-2 production in the human leukemic cell line Jurkat by a novel peptide inhibitor of protein kinase C" Cell Immunol., 131, 242, 1990) and protein-tyrosine phosphatase (Kole H.K. et al., "A peptide- based protein-tyrosine phosphatase inhibitor specifically enhances insulin receptor function in intact cells" J. Biol. Chem. 271, 14302, 1996) or among peptides. Preferably, cell membrane permeable carriers are used, more preferably a cell membrane permeable carrier peptide is used. In case the cell membrane permeable carrier is a peptide then it will preferably be a positively charged amino acid rich peptide. Preferably such positively charged amino acid rich peptide is an arginine rich peptide. It has been shown in Futaki et al. (Futaki S. et al., "Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery" J. Biol. Chem., 276, 5836, 2001), that the number of arginine residues in a cell membrane permeable carrier peptide has a significant influence on the method of internalization and that there seems to be an optimal number of arginine residues for the internalization, preferably they contain more than 6 arginines, more preferably they contain 9 arginines (R9). Usually arginine rich peptides are selected from the group comprising the HIV- TAT 48-57 peptide, the FHV-coat 35 -49 peptide, the HTLV-II Rex 4- 16 peptide and the BMV gag 7-25 peptide. Preferably, the arginine rich peptide is HIV-TAT 48-57 peptide.

Since an inherent problem with native peptides (in L-form) is degradation by natural proteases, the RGD-containing peptide of the invention peptide, as well as the cell membrane permeable peptide of the invention, may be prepared to include D-forms and/or "retro-inverso isomers" of the peptide.

In this case, retro-inverso isomers of fragments and variants of the RGD-containing peptide, as well as of the cell membrane permeable peptide, of the invention are prepared. Protecting the peptide from natural proteolysis should therefore increase the effectiveness of the RGD- containing peptide of the invention. A higher biological activity is predicted for the retro- inverso containing peptide when compared to the non-retro-inverso containing analogue owing to protection from degradation by native proteinases. Furthermore the retro-inverso containing peptides have been shown to exhibit an increased stability and lower immunogenicity (Sela M. and Zisman E., "Different roles of D-amino acids in immune phenomena" FASEB J. 11, 449, 1997). Retro-inverso peptides are prepared for peptides of known sequence as described for example in Sela and Zisman, (1997).

By "retro-inverso isomer" is meant an isomer of a linear peptide in which the direction of the sequence is reversed and the chirality of each amino acid residue is inverted; thus, there can be no end-group complementarity.

Also encompassed by the present invention are modifications of the RGD-containing peptide of the invention (which do not normally alter primary sequence), including in vivo or in vitro chemical derivitization of peptides, e. g, acetylation or carboxylation. Also included are modifications of glycosylation, e. g., those made by modifying the glycosylation patterns of a peptide during its synthesis and processing or in further processing steps, e. g, by exposing the peptide to enzymes which affect glycosylation e. g. mammalian glycosylating or deglycosylating enzymes. Also included are sequences which have phosphorylated amino acid residues, e. g. phosphotyrosine, phosphoserine, or phosphothreonine.

The invention also includes analogues in which one or more peptide bonds have been replaced with an alternative type of covalent bond (a "peptide mimetic") which is not susceptible to cleavage by peptidases. Where proteolytic degradation of the peptides following injection into the subject is a problem, replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic will make the resulting peptide more stable and thus more useful as an active substance. Such mimetics, and methods of incorporating them into peptides, are well known in the art.

Also useful are amino-terminal blocking groups such as t-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyl oxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4,- dinitrophenyl. Blocking the charged amino-and carboxy -termini of the peptides would have the additional benefit of enhancing passage of the RGD-containing peptide of the invention through the hydrophobic cellular membrane and into the cell.

The peptides of the invention may be prepared by classical methods known in the art. These standard methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis, and recombinant DNA technology. A preferred method for peptide synthesis is solid phase synthesis. Solid phase peptide synthesis procedures are well-known in the art.

The RGD-containing peptide of the invention is an RGD-binding integrin inhibitor.

Integrins are a family of integral cytoplasmic membrane proteins that mediate cell interactions with other cells and with the extracellular matrix. Approximately one third of the members of the integrin family directly bind to a specific amino acid motif, arginine-glycine-aspartate (RGD), that is contained within the sequence of their cognate protein ligands. It has been established in the art that peptides containing the RGD sequence, and synthetic small molecule compounds that mimic the RGD sequence, are capable of binding to these integrin receptors with varying degrees of specificity, and thereby inhibit the binding to normal physiologic ligands. Many human diseases are characterized by either or both of two common contributing pathological mechanisms: angiogenesis and fibrosis. Different subsets of the RGD-binding integrins have predominant roles in driving these dual processes, so that simultaneous antagonism of angiogenesis and fibrosis requires agents capable of binding potently to several target integrins. This contrasts with agents designed specifically for binding to a single integrin which may be less effective in some applications due to their more restricted mechanism of action. Integrins which have been shown to have a role in promoting angiogenesis include anb3, anb5, and a5b1.

Antagonists of RGD-binding integrins should be useful for treatment of human diseases having angiogenesis or fibrosis as a principal part of their pathology. In particular, the important role of a5b1 in angiogenesis is supported by numerous studies. Because a5b1 expression is not confined to the endothelium, it has other functional roles in addition to angiogenesis. It is expressed to varying degrees in many cell types including fibroblasts, hematopoietic and immune cells, smooth muscle cells, epithelial cells, and tumour cells. Expression on tumour cells has been implicated in the progression of tumour growth and metastasis.

Eight members of the integrin superfamily recognize the tripeptide motif Arg-Gly-Asp (RGD) within extracelluar matrix (ECM) proteins. These integrins constitute an important subfamily and play a major role in cancer progression and metastasis via their tumour biological functions.

Another aspect of the invention provides a use of the RGD-containing peptide of the invention for inhibiting RGD-binding integrins.

Another aspect of the invention provides a method for inhibiting RGD-binding integrins, wherein the method comprises using the RGD-containing peptide of the invention.

Another aspect of the invention provides a method of inhibiting RGD-binding integrins activity in a biological sample comprising contacting said biologic sample with the RGD-containing peptide of the invention.

Another aspect of the invention provides a pharmaceutical composition comprising the RGD- containing peptide of the invention and at least one pharmaceutically acceptable carrier, excipient and/or diluent. Certain pharmaceutical compositions of the invention are single unit dosage forms suitable for oral or mucosal (such as nasal, sublingual, vaginal, buccal, or rectal) administration to a subject. Examples of dosage forms include, but are not limited to tablets, caplets, capsules, such as soft elastic gelatine capsules, cachets, troches, lozenges, dispersions, suppositories, powders, solutions, aerosols (such as nasal sprays or inhalers), liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (such as aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs.

The formulation of the pharmaceutical composition of the invention should suit the mode of administration. For example, oral administration requires enteric coatings to protect the RGD- containing peptide of the invention from degradation within the gastrointestinal tract. Similarly, a formulation may contain ingredients that facilitate delivery of the RGD-containing peptide of the invention to the site of action. For example, the RGD-containing peptide of the invention may be administered in liposomal formulations, in order to protect them from degradative enzymes, facilitate transport in circulatory system, and effect delivery across cell membranes to intracellular sites.

The pharmaceutical compositions of the invention suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (such as chewable tablets), caplets, capsules, and liquids (such as flavoured syrups). Such dosage forms contain predetermined amounts of the RGD-containing peptide of the invention, and may be prepared by methods of pharmacy well known to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences.

Typical oral dosage forms are prepared by combining the RGD-containing peptide of the invention in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration.

Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by conventional methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the RGD-containing peptide of the invention with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary. Disintegrants may be incorporated in solid dosage forms to facility rapid dissolution. Lubricants may also be incorporated to facilitate the manufacture of dosage forms (such as tablets). For example pharmaceutically acceptable excipients particularly suitable for use in conjunction with tablets include, for example, inert diluents, such as celluloses, calcium or sodium carbonate, lactose, calcium or sodium phosphate; disintegrating agents, such as cross- linked povidone, maize starch, or alginic acid; binding agents, such as povidone, starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc.

Other pharmaceutical compositions of the invention are transdermal and topical dosage forms administered to a patient. Such dosage forms are selected from the group comprising ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to a person skilled in the art (see for example Remington's Pharmaceutical Sciences); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985). Transdermal dosage forms include "reservoir type" or "matrix type" patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of the compounds of the invention.

Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal, topical, and mucosal dosage forms are well known to a person skilled in the art, and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied.

The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve delivery of one or more RGD-containing peptides of the invention. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates may also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients so as to improve delivery. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery-enhancing or penetration-enhancing agent. Different salts, hydrates or solvates of the active ingredients can be used to further adjust the properties of the resulting composition.

Other pharmaceutical compositions of the invention are parenteral dosage forms administered to subjects (patients) by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are specifically sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to a person skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

The composition, shape, and type of a dosage form of the pharmaceutical composition of the invention will vary depending on its use. For example, a dosage form used in the acute treatment of a disease, such as cancer, may contain larger amounts of one or more of the RGD-containing peptides of the invention than a dosage form used in the chronic treatment of the same disease.

Another aspect of the invention provides a method of inhibiting RGD-binding integrins activity in a subject comprising administering to said subject the RGD-containing peptide of the invention or the pharmaceutical composition of the invention.

An embodiment of the invention provides an RGD-containing peptide of the invention or a pharmaceutical composition of the invention for use in a method of inhibiting RGD-binding integrins activity in a subject. Another aspect of the present invention provides a method for treating cancer, wherein cancer cells express RGD-binding integrins, comprising administering to a subject in need thereof a therapeutically effective amount of the RGD-containing peptide of the invention or the pharmaceutical composition of the invention.

An embodiment of the invention provides an RGD-containing peptide of the invention or a pharmaceutical composition of the invention for use in a method for treating cancer, wherein cancer cells express RGD-binding integrins.

In the context of the present invention, “treating cancer” refers to inhibiting proliferation, metastasis, and/or invasion of the cancer cells.

In some embodiments of the method for treating cancer of the invention, RGD-binding integrins are selected from the group consisting of a5b1, anbΐ, a8b1, anb3, anb5, anbό, a6b4, a4b1 and anb8. In some preferred embodiments of the method for treating cancer of the invention, RGD- binding integrins are anb3 and/or a5b1.

In some embodiments of the method for treating cancer of the invention, cancers comprising cancer cells express RGD-binding integrins are selected from the group comprising, but not limited to, melanoma, NSCLC, and pancreatic cancer.

In some embodiments of the method for treating cancer of the invention, the method further comprises other anticancer treatments selected from the group comprising chemotherapy, radiotherapy and/or immunotherapy. In some embodiments, the other anticancer treatments are carried out simultaneously or sequentially with the administration of the RGD-containing peptide of the invention or the pharmaceutical composition of the invention.

In one embodiment of the method of the invention, the RGD-containing peptide of the invention is administered at the same time as the chemotherapy, radiotherapy and/or immunotherapy treatment. In another embodiment of the method of the invention, the RGD-containing peptide of the invention is administered prior to chemotherapy, radiotherapy and/or immunotherapy treatment. In another embodiment of the method of the invention, the RGD-containing peptide of the invention is administered after the chemotherapy, radiotherapy or immunotherapy treatment. In some embodiments of the method for treating cancer of the invention, the method further comprises administering to said subject one or more other anti-cancer agents. In some embodiments, the one or more other anti-cancer agents are administered simultaneously or sequentially with the RGD-containing peptide of the invention or the pharmaceutical composition of the invention.

In an embodiment, the present invention provides a method for treating cancer, wherein cancer cells express RGD-binding integrins, comprising administering simultaneously or sequentially to a subject in need thereof a therapeutically effective amount of the RGD-containing peptide of the invention and one or more other anti-cancer agents.

In one embodiment of the method of the invention, the RGD-containing peptide of the invention is administered at the same time as the administration of the one or more other anti-cancer agents. In another embodiment of the method of the invention, the RGD-containing peptide of the invention is administered prior to the administration of the one or more other anti-cancer agents. In another embodiment of the method of the invention, the RGD-containing peptide of the invention is administered after the administration of the one or more other anti-cancer agents.

In some embodiments of the method for treating cancer, the one or more other anti-cancer agents are selected from the group comprising a kinase inhibitor, a cytotoxic agent, a checkpoint inhibitor.

In some preferred embodiments, the kinase inhibitor is known in the art and is selected from the group comprising c-KIT inhibitor, EGFR inhibitor, VEGF inhibitor, c-MET inhibitor, B- RAF inhibitor, MEK inhibitor.

In some preferred embodiments, the cytotoxic agent is known in the art and is selected from the group comprising alkylating agents, topoisomerase inhibitors, tubulin dynamics modulators, antimetabolites.

In some preferred embodiments, the checkpoint inhibitor is known in the art and is selected from the group comprising CTLA4 inhibitors, PD1 inhibitors, PDL1 inhibitors. In the methods for treating cancer of the present invention, the one or more other anti-cancer agents or treatments can be co-administered simultaneously or sequentially with the RGD- containing peptide of the invention, as judged to be appropriate by the administering physician, in combination with any additional circumstances pertaining to the individual patient.

In some embodiments, the simultaneous or sequential co-administration of the RGD-containing peptide of the invention and the one or more other anti-cancer agent provides a synergistical effect.

Physicians skilled in the art would be able to dose the RGD-containing peptide of the invention, as well as any contemplated combination therapy, designing both dosage amounts and dosage frequency.

The effective amount refers to an amount of the composition that is capable of producing a medically desirable result in a treated subject. Take cancer treatment as an example, compared to an untreated subject, the desirable result comprises the inhibition of proliferation, metastasis, and/or invasion, and may decrease of tumor mass, growth rate, metastasis, alleviation of symptoms, extension of life, and/or improvement of life quality. The exact dosage for administration depends on the types, extent or symptom of the disease, as well as the health conditions, age, sex, weight, or drug toleration of the subject to be administered. The amount for administration also varies with the extent, severity, and type of tumor. One skilled in the art can decide the suitable dosage for administration according the foregoing or other factors.

The treatments may include various "unit doses". Unit dose is defined as containing a predetermined-quantity of the therapeutic composition calculated to produce the desired responses in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, and the particular route and formulation, are within the skill of those in the clinical arts. Also of import is the subject to be treated, in particular, the state of the subject and the protection desired. A unit dose need not be administered as a single injection but may comprise continuous infusion over a set period of time.

Generally, the RGD-containing peptide of the invention is administered to a subject at a dose ranging from about 1 pg/kg to about 20 mg/kg, about 1 pg/kg to about 10 mg/kg, about 1 pg/kg to about 1 mg/kg, about 10 pg/kg to about 100 pg/kg, about 10 pg/kg to about 1 mg/kg, about 100 pg/kg to about 1 mg/kg, about 100 pg/kg to about 10 mg/kg, or about 200 pg/kg to about 1 mg/kg. For example, the RGD-containing peptide of the invention can be administered at a dose ranging from about 80 pg to about 1600 mg, about 80 pg to about 800 mg, about 80 pg to about 80 mg, about 800 pg to about 8 mg, about 800 pg to about 80 mg, about 8 mg to about 80 mg, about 8 mg to about 800 mg, or about 16 mg to about 80 mg. In another example, the RGD-containing peptide of the invention can be administered at a dose ranging from about 50 pg to about 1000 mg, about 50 pg to about 500 mg, about 50 pg to about 50 mg, about 500 pg to about 5 mg, about 500 pg to about 50 mg, about 5 mg to about 50 mg, or about 5 mg to about 500 mg, or about 10 mg to about 50 mg.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practising the present invention and are not intended to limit the application and the scope of the invention.

EXAMPLES

The biological property of the synthesized peptides has been assessed by the inhibition of proliferation of the human umbilical vein endothelial cells (HUVEC). This cell line express both av and bΐ integrins subunits which are the natural targets of ADAM 15 disintegrin domain (Nath and al. 1999, J Cell Sci. 112 :579).

Figure 1 shows that the short c-terminal fragment of AMEP (42 AA) containing the RGD sequence has no anti-proliferative effect on HUVEC cells. In contrast the optimized AMEP peptide derivative displaying both RGD and PHK sequence displays a significant anti proliferative effect with an IC50 close to 5 pg/ml.

Claims

1. An RGD-containing peptide according to formula I consisting of an N-terminal sequence, RGD sequence, PHK sequence, a spacer between the RGD and the PHK sequences and C-terminal sequence, wherein the RGD-containing peptide has a length of 40 to 60 amino acids, and wherein the spacer consists of 14 to 20 amino acids.

Formula I

2. The RGD-containing peptide of claim 1, wherein the spacer is selected from the group comprising CDLPEFCPGDSSQC (SEQ ID NO: 1), CDLPEFCPGDSSQCPPD (SEQ ID NO: 2), CDLPEF CPGD S SQCPPD V (SEQ ID NO: 3).

3. The RGD-containing peptide of any one of claims 1-2, wherein the N-terminal sequence is QNCQLRPSGWQCRPT (SEQ ID NO: 8) or CASDGPCCQNCQLRPSGWQCRPT (SEQ ID NO: 9).

4. The RGD-containing peptide of any one of claims 1-3, wherein the C-terminal sequence is selected from the group comprising VSLGD (SEQ ID NO: 10), VSLG (SEQ ID NO: 11), VSLGDGE (SEQ ID NO: 12).

5. The RGD-containing peptide of any one of claims 1-4, selected from the group comprising:

QNCQLRPSGWQCRPTRGDCDLPEFCPGDSSQCPHKVSLGD (SEQ ID NO: 4)

QN C QLRP S GW Q CRPTRGDCDLPEF CPGD S S QCPPDPHK V SLG ( SEQ ID NO: 5) CASDGPCCQNCQLRPSGWQCRPTRGDCDLPEFCPGDSSQCPPDPHKVSLG (SEQ ID NO: 6)

CASDGPCCQNCQLRPSGWQCRPTRGDCDLPEFCPGDSSQCPPDVPHKVSLG (SEQ ID NO: 7)

6. A use of the RGD-containing peptide of any one of claims 1-5 for inhibiting RGD- binding integrins.

7. A pharmaceutical composition comprising the RGD-containing peptide of any one of claims 1-5 and at least one pharmaceutically acceptable carrier, excipient or diluent.

8. An RGD-containing peptide of any one of claims 1-5 for use in a method of inhibiting RGD-binding integrins activity in a subject.

9. An RGD-containing peptide of any one of claims 1-5 for use in a method for treating cancer, wherein cancer cells express RGD-binding integrins.

10. The RGD-containing peptide for use according to claim 9, wherein RGD-binding integrins are selected from the group consisting of a5b1, anbΐ, a8b1, anb3, anb5, anbό, a6b4, a4b1 and anb8.

11. The RGD-containing peptide for use according to claim 9, wherein RGD-binding integrins are anb3 and/or a5b1.

12. The RGD-containing peptide for use according to any one of claims 9-11, wherein the method further comprises other anticancer treatments selected from the group comprising chemotherapy, radiotherapy and/or immunotherapy.

13. The RGD-containing peptide for use according to claim 12, wherein the other anticancer treatments are carried out simultaneously or sequentially with the administration of the RGD- containing peptide of any one of claims 1-5.

14. The RGD-containing peptide for use according to any one of clams 9-11, wherein the method further comprises administering to said subject one or more other anti-cancer agents.

15. The RGD-containing peptide for use according to claim 14, wherein the one or more other anti-cancer agents are administered simultaneously or sequentially with the RGD- containing peptide of any one of claims 1-5.

16. The RGD-containing peptide for use according to any one of claims 14-15, wherein the one or more other anti-cancer agents are selected from the group comprising a kinase inhibitor, a cytotoxic agent, a checkpoint inhibitor.

17. The RGD-containing peptide for use according to claim 16, wherein the kinase inhibitor is selected from the group comprising c-KIT inhibitor, EGFR inhibitor, VEGF inhibitor, c-MET inhibitor, B-RAF inhibitor, MEK inhibitor.

18. The RGD-containing peptide for use according to claim 16, wherein the cytotoxic agent is selected from the group comprising alkylating agents, topoisomerase inhibitors, tubulin dynamics modulators, antimetabolites.

19. The RGD-containing peptide for use according to claim 16, wherein the checkpoint inhibitor is selected from the group comprising CTLA4 inhibitors, PD1 inhibitors, PDL1 inhibitors.