WO2021009761A1 - Combination therapy for the treatment of cancer - Google Patents

Abstract

A method of treating a solid tumor in a subject in need thereof is provided. The method comprising, administering to the subject a therapeutically effective amount of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator, wherein said CD4 antagonist is not a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof, thereby treating the solid tumor in the subject.

Description

COMBINATION THERAPY FOR THE TREATMENT OF CANCER

RELATED APPLICATION/S

This application claims the benefit of priority from U.S. Provisional Patent Application No. 62/875,522 filed July 18, 2019, incorporated by reference in its entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 82998SequenceListing.txt, created on 15 July 2020, comprising 39,749 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to a combination therapy for the treatment of cancer.

Cancer is the second leading cause of death in the U.S. A. The estimates for 2014 are that approximately 585,000 people will die of cancer and 1.6 million new cases will be diagnosed (American Cancer Society, Cancer Facts & Figures 2014).

For early stage cancers, surgical removal is a very effective treatment.

However, for more advanced cases and non-solid hematological malignancies, standard, non-specific cancer treatments such as chemotherapy and radiotherapy are typically used. These treatments affect many healthy cells and result in elevated toxicity and effective in only a minor percentage of treated individuals. Moreover, even individuals that initially respond to therapy are at risk for relapses, and often develop resistance.

Additional background art includes:

U.S. 20190125867

W02017/009842

W02017/009843

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of treating a solid tumor in a subject in need thereof, the method comprising, administering to the subject a therapeutically effective amount of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator, wherein the CD4 antagonist is not a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof, thereby treating the solid tumor in the subject.

According to an aspect of some embodiments of the present invention there is provided a therapeutically effective amount of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator for use in treating solid tumor in a subject in need thereof, wherein the CD4 antagonist is not a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof.

According to an aspect of some embodiments of the present invention there is provided an article of manufacture for use in treating cancer comprising a therapeutically effective amount of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator, wherein the CD4 antagonist is not a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof.

According to some embodiments of the invention, the CD4 antagonist is an antibody.

According to some embodiments of the invention, the CD4 antagonist is a small molecule.

According to some embodiments of the invention, the solid tumor is pancreatic cancer.

According to some embodiments of the invention, the CD4 antagonist, chemotherapy and checkpoint regulator cause a decrease in tumor growth.

According to some embodiments of the invention, the CD4 antagonist, chemotherapy and checkpoint regulator cause a synergistic increase in activation state of CD8+ T cells infiltrating the solid tumor.

According to some embodiments of the invention, the CD4 antagonist, chemotherapy and checkpoint regulator cause a synergistic increase in size of CD8+ T cells infiltrating the solid tumor.

According to some embodiments of the invention, the CD4 antagonist, chemotherapy and checkpoint regulator cause a decrease in Tregs infiltrating the solid tumor.

According to some embodiments of the invention, the Tregs comprise FOXP3+CD4+ T cells.

According to some embodiments of the invention, the immune checkpoint regulator is an immune checkpoint inhibitor.

According to some embodiments of the invention, the immune checkpoint inhibitor is anti

PD-1.

According to some embodiments of the invention, the chemotherapy comprises ILF.

According to some embodiments of the invention, the CD4 antagonist, chemotherapy and checkpoint regulator are administered concomitantly. According to some embodiments of the invention, the CD4 antagonist, chemotherapy and checkpoint regulator are administered sequentially.

According to some embodiments of the invention, the CD4 antagonist, chemotherapy and checkpoint regulator are in a co-formulation.

According to some embodiments of the invention, the CD4 antagonist, chemotherapy and checkpoint regulator are in separate formulations.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

Fig. 1 is a bar graph showing a decrease in tumor volume due to combination of chemotherapy with BL8040 and anti PD-1.

Fig. 2 shows an increase in CD8+ size (a marker for T cell activation) in tumors treated with combination of chemotherapy with BL8040 and anti PD-1.

Fig. 3 shows that tumors treated with combination of chemotherapy with BL8040 and anti PD-1 have similar amounts of CD8 cells and increase number of CD8+CD69 activated T cells.;

Fig. 4 shows that tumors treated with combination of chemotherapy BL8040 and anti PD- 1 have significantly reduced amounts of CD4.

Fig. 5 shows that tumors treated with combination of chemotherapy, BL8040 and anti PD- 1 have significantly reduced amounts of CD4+FOXP3+ cells (100% reduction) and moderate reduction in total CD45+ cells (20-30%). DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a combination therapy for the treatment of cancer.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Chemotherapy, immune checkpoint modulation and target therapy are only a few of treatment modalities that are currently available today for rebutting cancer.

The present inventor has now surprisingly uncovered that CD4 inhibition combined with chemotherapy and immune checkpoint modulation can act synergistically in activating CD8+ T cells.

Specifically, a combination of chemotherapy [Irrinotecan, Leucovorin and Fluorouracil (ILF)] with the peptide set forth in SEQ ID NO: 1 (also termed as BL8040) and anti PD-1 did not reduce or change the number of CD8+ T cells accumulating in the tumors. However, it did change the activation state of CD8+ T cells, increasing their size and the percentage of CD8+CD69+ cells within the tumor. The combination significantly reduced the number of CD4+ T cells and CD4+ FOXP3+ cells accumulating in the tumors changing dramatically the ratio between activating CD8+ T killer cells and regulatory CD4+ T cells.

It is therefore suggested that the ability of BL8040 to inhibit trafficking of CD4+ T and CD4+FOXP3+ cells allow a better activation of the immune cells contributing to the effect of chemotherapy and immunotherapy on tumor survival.

The ability of the peptide set forth in SEQ ID NO: 1 to inhibit trafficking of regulatory T cells into the tumor, suggests that any CD4 antagonist or regulator thereof can be used to achieve the same synergy as that displayed with the peptide in combination with the chemotherapy and checkpoint regulator.

Thus, according to an aspect of the invention there is provided a method of treating a solid tumor in a subject in need thereof, the method comprising, administering to the subject a therapeutically effective amount of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator, wherein the CD4 antagonist is not a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof, thereby treating the solid tumor in the subject.

According to another aspect of the invention there is provided a therapeutically effective amount of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator for use in treating solid tumor in a subject in need thereof, wherein the CD4 antagonist is not a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof.

According to another aspect of the invention there is provided an article of manufacture for use in treating cancer comprising a therapeutically effective amount of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator, wherein the CD4 antagonist is not a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof.

A CD4 antagonist, a chemotherapy and an immune checkpoint regulator may be referred to as“the combination” however, this does not necessitate that they are comprised in a single formulation/composition because separate formulations (e.g., 2 or 3) are also contemplated herein.

The terms “treating” or "treatment" refers to inhibiting, preventing or arresting the development of a pathology (e.g. cancer) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

As used herein the phrase "subject in need thereof" refers to a mammalian male or female subject (e.g., human being) who is diagnosed with cancer. Veterinary uses are also contemplated. The subject may be of any gender or at any age including neonatal, infant, juvenile, adolescent, adult and elderly adult. The subject may have been treated with one or two of the components of the combination comprising of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator and is now subjected to treatment with the rest of the combination.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

According to one embodiment the cancer is a primary tumor.

According to another embodiment the cancer is a cancer metastasis.

According to a specific embodiment, the cancer is a solid tumor.

Examples of cancer include but are not limited to, carcinoma, adenoma, or sarcoma.

Cancers to which the present invention is applied are solid cancers including various cancers except blood cancers (malignant lymphoma, leukemia, multiple myeloma). Typical specific examples include epithelial solid cancers such as lung cancer, breast cancer, gastric cancer, liver cancer, colon cancer, tongue cancer, thyroid cancer, renal cancer, prostate cancer, uterine cancer, cervical cancer, ovarian cancer. However, cancers are not limited as long as they are solid cancers, and the examples also include other solid cancers not belonging to epithelial solid cancers, such as melanoma and glioma. Other examples of the solid cancer include, but not limited to, at least one epithelial solid cancer selected from the group consisting of colon cancer, lung cancer, pancreatic cancer, renal cancer, and breast cancer, or at least one epithelial solid cancer selected from the group consisting of colon cancer, lung cancer, pancreatic cancer, and renal cancer, or at least one epithelial solid cancer selected from the group consisting of colon cancer, lung cancer, and breast cancer, or at least one solid cancer selected from melanoma and glioma. The term "treatment of solid cancer" includes both suppression of cancer growth and prolongation of life of cancer patients. The term "treatment of solid cancer" also includes treatment of primary cancer. For example, the therapeutic agent of the present invention may be applied to a patient who developed primary cancer which is different from the primary cancer the patient had first developed, for the purpose of treatment of the second or subsequent primary cancers.

According to specific embodiments the cancer is selected from the group consisting of lung cancer, glioma, colon cancer, ovarian cancer, renal cancer, melanoma cancer, hepatocellular cancer, gastric or stomach cancer, glioblastoma, cervical cancer, bladder cancer, breast cancer, colorectal cancer, prostate cancer, thyroid cancer, head and neck and pancreatic cancer.

According to specific embodiments, the cancer is selected from the group consisting of lung cancer, glioma, colon cancer and pancreatic cancer.

According to a specific embodiment, the solid tumor is pancreatic cancer.

According to a specific embodiment, the pancreatic cancer is a metastatic pancreatic adenocarcinoma or metastatic ductal adenocarcinoma.

As used herein “pancreatic ductal adenocarcinoma” (PDAC) is a type of exocrine pancreatic cancer. It develops from cells lining small tubes in the pancreas called ducts (duct cells in the diagram above). These carry the digestive juices, which contain enzymes, into the main pancreatic duct and then on into the duodenum (first part of the small intestine). PDAC can grow anywhere in the pancreas, although it is most often found in the head of the pancreas.

As used herein,“a therapeutically effective amount of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator” refers to an increase in the ratio between CD8+ and CD4+ cells i.e., number of CD8+ cell divided by number of CD4+ cells in the tumor edge and parenchyma, thereby increasing the activity of CD 8+ T cells in the tumor.

As used herein“an increase” refers to an increase of at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 100 %, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold or at least 10 fold increase in the ratio of the combination as compared to an absence thereof (control), where otherwise the conditions are identical and where each number represents a different embodiment.

CD8+ T cells activity can be measured according to methods which are well known in the art. According to a specific embodiment, the measure is the size of the CD8+ T cells, which increases with CD8+ T cells activity.

Other non-limiting markers of CD8+ T cells activation include, but are not limited to CD69, CD137, CD44 or CD154. Downregulation of markers can also be followed, e.g., CD62L. In any case, FACS can be a reliable assay for determining T cell activation, although other methods can also be used based on protein/mRNA gene expression, e.g., immunohistochemistry or PCR.

According to a specific embodiment, the marker for CD8+ T cell activation is CD69.

According to a specific embodiment, there is an increase of at least 15 % in the CD69+ CD8 + T cells (of the total CD8+ cells) in the tumor following treatment with the combination (see e.g., Figure 3 right panel).

According to a specific embodiment, there is an increase of at least 10 % in the ratio of CD8+ to CD4+ cells in the tumor following treatment with the combination.

According to an additional or an alternative embodiment, the combination causes a decrease in tumor growth.

As used herein“a decrease” refers to a decrease of at least 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 %, 95 %, 100 %, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold or at least 10 fold in the presence of the combination as compared to an absence thereof (control), where otherwise the conditions are identical and where each number represents a different embodiment.

There are numerous measures of tumor growth. According to a specific embodiment, tumor growth is measured by weight (such as in test animals indicative of size), or using imaging methods e.g., ultrasound, MRI, CT, x-ray and the like.

Thus, according to a specific embodiment, the combination causes a synergistic increase in activation state of CD8+ T cells infiltrating the solid tumor.

According to a specific embodiment, the combination causes a synergistic increase in size of CD8+ T cells infiltrating said solid tumor.

As shown in the Examples section which follows, treatment with the combination causes a decrease in regulatory T cells as evidenced by the complete disappearance of FOXP3+CD4+ T cells (see Figure 5).

As used herein“regulatory T cells” or“Tregs” refers to the subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FOXP3, and CD25 and are thought to be derived from the same lineage as naive CD4 cells. TGFP may be used to to differentiate from naive CD4+ cells and is important in maintaining Treg homeostasis. According to a specific embodiment, the combination causes a decrease in Tregs infiltrating said solid tumor.

According to a specific embodiment, the Tregs comprise FOXP3+CD4+ T cells. Hence according to a specific embodiment, the FOXP3+CD4+ T cells subpopulation decreases in the tumor following treatment with the combination, preferably to a level where they are hardly detectable by methods which are well known in the art e.g., immunohistochemistry.

According to a specific embodiment, the combination does not affect the level of CD8 T cells in the solid tumor (see e.g., Figure 3 left panel).

In support of this observation it was demonstrated that human CD4+ cells migrate better in response to the CXCR4 ligand SDF-1 than CD8+ cells Sawada et al. J Exp Med. 1998 May 4;187(9):1439-49.

As used herein a“CD4 antagonist” refers to an agent which causes down regulation is the regulatory/suppressive activity of CD4+ T cells.

According to a specific embodiment, the agent prevents trafficking of the cells to the tumor.

According to another specific embodiment, the agent downregulates CD4+ T cells signaling which results in the immune suppression.

According to another specific embodiment, the agent kills CD4+ T cells.

According to another specific embodiment, the agent inhibits CD4+ T cell migration.

According to a specific embodiment, the CD4 antagonist is a direct inhibitor of CD4 (i.e., binds CD4 and inhibits its activity.

According to another specific embodiment, the CD4 antagonist is an indirect inhibitor of CD4. Such are known in the art such as inhibitors of the chemokine receptors CCR6, CCR4, CCR5, CCR2 or CCR3, that regulate CD4 cell migration.

Inhibitors for such targets are well known in the art.

Some are provided infra, as adapted from the literature.

For instance:

CCR2- the small molecules BMS-741672 or AZ-889.

CCR4- Mogamulizumab is a humanized clinically approved antibody.

A CCR5 antagonist, includes the clinically approved CCR5 inhibitor Maraviroc.

Other antibodies, small molecules or others e.g., natural ligands, expression inhibitors etc. were known to the skilled artisan as inhibitors of such receptors at the date of filing.

According to a specific embodiment the CD4 antagonist is non-cytotoxic.

According to a specific embodiment the CD4 antagonist is cytotoxic. Numerous other anti CD4 antibodies have been described in the art and they can be divided for instance by their mode of binding e.g., those recognizing the D1 domain (e.g. Q4120, 6H10, 2D5, and 2F2), the D2 domain (e.g., mAb Mu5A8, Leu3A, OKT4A, F91-55, and M-T441) and the D3 and D4 domains (e.g., mAb OKT4 and L120). Many more such antibodies are available to the skilled artisan.

According to a specific embodiment, the anti-CD4 antibody is 15A7 or TNX-355 (Ibalizumab).

The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof (that are capable of binding to an epitope of an antigen). The relevance of the terms is for CD4 antagonists as well as for immune checkpoint regulators in the context of the invention.

As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

According to a specific embodiment, the antibody fragments include, but are not limited to, single chain, Fab, Fab’ and F(ab')2 fragments, Fd, Fcab, Fv, dsFv, scFvs, diabodies, minibodies, nanobodies, Fab expression library or single domain molecules such as VH and VL that are capable of binding to an epitope of the antigen in an HLA restricted manner.

Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as“light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as“heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv Fv (scFv), a disulfide- stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’)2, or antibody fragments comprising the Fc region of an antibody.

As used herein, the terms "complementarity-determining region" or "CDR" are used interchangeably to refer to the antigen binding regions found within the variable region of the heavy and light chain polypeptides. Generally, antibodies comprise three CDRs in each of the VH (CDR HI or HI; CDR H2 or H2; and CDR H3 or H3) and three in each of the VL (CDR LI or LI; CDR L2 or L2; and CDR L3 or L3).

The identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by Kabat et al. (See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989.), a compromise between Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see, Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268; and world wide web site www(dot)bioinf-org(dot)uk/abs), available complex crystal structures as defined by the contact definition (see MacCallum et al., J. Mol. Biol. 262:732-745, 1996) and the "conformational definition" (see, e.g., Makabe et al., Journal of Biological Chemistry, 283: 1156- 1166, 2008).

As used herein, the“variable regions” and "CDRs" may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches.

Functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows:

(i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;

(ii) single chain Fv (“scFv”), a genetically engineered single chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

(iii) disulfide-stabilized Fv (“dsFv”), a genetically engineered antibody including the variable region of the light chain and the variable region of the heavy chain, linked by a genetically engineered disulfide bond.

(iv) Fab, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CHI domains thereof;

(v) Fab’, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);

(vi) F(ab’)2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments held together by two disulfide bonds);

(vii) Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen; and (viii) Fcab, a fragment of an antibody molecule containing the Fc portion of an antibody developed as an antigen-binding domain by introducing antigen-binding ability into the Fc region of the antibody.

Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

Exemplary methods for generating antibodies employ induction of in-vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi D.R. et ah, 1989. Proc. Natl. Acad. Sci. U. S. A. 86:3833-3837; Winter G. et ah, 1991. Nature 349:293-299) or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique (Kohler G. et ah, 1975. Nature 256:495-497; Kozbor D. et ah, 1985. J. Immunol. Methods 81:31-42; Cote RJ. et ah, 1983. Proc. Natl. Acad. Sci. U. S. A. 80:2026-2030; Cole SP. et ah, 1984. Mol. Cell. Biol. 62:109-120).

In cases where target antigens are too small to elicit an adequate immunogenic response when generating antibodies in-vivo, such antigens (haptens) can be coupled to antigenically neutral carriers such as keyhole limpet hemocyanin (KLH) or serum albumin [e.g., bovine serum albumine (BSA)] carriers (see, for example, US. Pat. Nos. 5,189,178 and 5,239,078]. Coupling a hapten to a carrier can be effected using methods well known in the art. For example, direct coupling to amino groups can be effected and optionally followed by reduction of the imino linkage formed. Alternatively, the carrier can be coupled using condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents. Linker compounds can also be used to effect the coupling; both homobifunctional and heterobifunctional linkers are available from Pierce Chemical Company, Rockford, Ill. The resulting immunogenic complex can then be injected into suitable mammalian subjects such as mice, rabbits, and the like. Suitable protocols involve repeated injection of the immunogen in the presence of adjuvants according to a schedule which boosts production of antibodies in the serum. The titers of the immune serum can readily be measured using immunoassay procedures which are well known in the art.

The antisera obtained can be used directly or monoclonal antibodies may be obtained as described hereinabove.

Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light- heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

As described hereinabove, Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11: 1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a single complementarity determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

As mentioned, the antibody fragment may comprise a Fc region of an antibody termed “Fcab”. Such antibody fragments typically comprise the CH2-CH3 domains of an antibody. Fcabs are engineering to comprise at least one modification in a structural loop region of the antibody, i.e. in a CH3 region of the heavy chain. Such antibody fragments can be generated, for example, as follows: providing a nucleic acid encoding an antibody comprising at least one structural loop region (e.g. Fc region), modifying at least one nucleotide residue of the at least one structural loop regions, transferring the modified nucleic acid in an expression system, expressing the modified antibody, contacting the expressed modified antibody with an epitope, and determining whether the modified antibody binds to the epitope. See, for example, U.S. Patent Nos. 9,045,528 and 9,133,274 incorporated herein by reference in their entirety.

Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et ah, Nature, 321:522-525 (1986); Riechmann et ah, Nature, 332:323- 329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et ah, Nature, 321:522-525 (1986); Riechmann et ah, Nature 332:323-327 (1988); Verhoeyen et ah, Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et ah, J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boemer et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al., Nature 368: 856- 859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845- 51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

The cytotoxic activity of antibodies can comprise the ADCC activity and the complement- dependent cytotoxicity activity (CDC) activity. Increasing the cytotoxic activity of an antibody where necessary can be achieved such as by using an antibody-drug conjugate (ADC) concept.

Small molecules CD4 inhibitors can also be used. Other CD4 antagonists (inhibitors) can include soluble CD4 [e.g., Haim et al. PLoS Pathog. 2009 Apr; 5(4): el000360] or a nucleic acid agent which down-regulates gene expression.

As used herein“nucleic acid agent” (relevant here also to checkpoint regulation) refers to a nucleic acid agent, having a nucleic acid backbone, DNA, RNA, mimetics thereof or a combination of same that inhibits gene expression at the nucleic acid level. The nucleic acid agent may be encoded from a DNA molecule or provided to the cell per se.

Thus, the antagonist of some embodiments of the invention can be an RNA silencing agent. As used herein, the phrase "RNA silencing" refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi. As used herein, the term "RNA silencing agent" refers to an RNA which is capable of specifically inhibiting or "silencing" the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include non-coding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated. Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.

In one embodiment, the RNA silencing agent is capable of inducing RNA interference.

In another embodiment, the RNA silencing agent is capable of mediating translational repression.

According to another embodiment, the nucleic acid agent causes DNA editing using methods which are well known in the art.

The chemotherapeutic agent of the present invention can be, but not limited to, cytarabine (cytosine arabinoside, Ara-C, Cytosar-U), asprin, sulindac, curcumin, alkylating agents including: nitrogen mustards, such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); thylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphor amide (thiotepa), hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine ), 5-azacytidine, 2,2 difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2 '-deoxycoformycin (pento statin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophylotoxins such as etoposide and teniposide; antibiotics, such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin; enzymes such as L-asparaginase, cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, TNF-beta and GM-CSF, anti-angiogenic factors, such as angiostatin and endostatin, inhibitors of FGF or VEGF such as soluble forms of receptors for angiogenic factors, including soluble VGF/VEGF receptors, platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including Nmethylhydrazine (MIH) and procarbazine, adrenocortical suppressants such as mitotane (o,r' -DDD) and aminoglutethimide; hormones and antagonists including adrenocortico steroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; non-steroidal antiandrogens such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, anti-oxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors and receptor tyrosin kinase inhibitors such as imatinib mesylate (marketed as Gleevac or Glivac) and erlotinib (an EGF receptor inhibitor) now marketed as Tarveca; and anti-virals such as oseltamivir phosphate, Amphotericin B, and palivizumab.

In some embodiments the chemotherapeutic agent of the present invention is cytarabine (cytosine arabinoside, Ara-C, Cytosar-U), quizartinib (AC220), sorafenib (BAY 43-9006), lestaurtinib (CEP-701), midostaurin (PKC412), carboplatin, carmustine, chlorambucil, dacarbazine, ifosfamide, lomustine, mechlorethamine, procarbazine, pentostatin, (2'deoxycoformycin), etoposide, teniposide, topotecan, vinblastine, vincristine, paclitaxel, dexamethasone, methylprednisolone, prednisone, all-trans retinoic acid, arsenic trioxide, interferon- alpha, rituximab (Rituxan®), gemtuzumab ozogamicin, imatinib mesylate, Cytosar-U), melphalan, busulfan (Myleran®), thiotepa, bleomycin, platinum (cisplatin), cyclophosphamide, Cytoxan®)., daunorubicin, doxorubicin, idarubicin, mitoxantrone, 5-azacytidine, cladribine, fludarabine, hydroxyurea, 6-mercaptopurine, methotrexate, 6-thioguanine, or any combination thereof.

Examples include but are not limited to, gemtabicine, FOLFIRINOX, erlotinib, 5- fluorouracil, paclitaxel, nab-paclitaxel, docetaxel, capecitabine, oxaliplatin cisplatin, FOLFOXIRI, abraxane, an anti-CD40 antibody, oregovomab, Nelfinavir, cetuximab, tegafur, leucovorin, irinotecan and combinations thereof.

According to a specific embodiment the chemotherapy is irinotecan, a topoiseomarase inhibitor.

Irinotecan is converted by esterase enzymes into the more active metabolite, SN-38. The chemical name of irinotecan is (S)-4,l l-diethyl-3,4,12,14-tetrahydro-4-hydroxy-3,14-dioxolH- pyrano [4', 4'- : 6,7] -indolizino [ 1 ,2-b] quinolin-9-yl- [ 1 ,4'bipiperidine] - 1 '-carboxylate. Irinotecan hydrochloride trihydrate is also referred to by the name CPT-11 and by the trade name CAMPTOSAR®.

The topoisomerase inhibitor can be camptothecin conjugated to a biocompatible polymer such as a cyclodextrin or cyclodextrin analog (e.g., sulfonated cyclodextrins). For example, the topoisomerase inhibitor can be a cyclodextrin-containing polymer chemically bound to a camptothecin, irinotecan, SN-38 or other topoisomerase 1 inhibitor compound. A cyclodextrin- camptothecin conjugated topoisomerase 1 inhibitor can be administered at a pharmaceutically acceptable dose including 6, 12, or 18 mg/m2 weekly administration, or 12, 15 or 18 mg/m2 biweekly administration. Examples of camptothecin-cyclodextrin conjugate topoisomerase 1 inhibitors (e.g., the cyclodextrin-containing polymer conjugate with camptothecin designated "CRLX101"), and related intermediates for preparing the same, are disclosed, for example, in Greenwald et ah, Bioorg. Med. Chem., 1998, 6, 551-562, as well as United States Patent Application 2010/0247668, United States Patent Application 2011/0160159 and United States Patent Application 2011/0189092.

The topoisomerase inhibitor can also be a liposomal formulation of a topoisomerase inhibitor such as irinotecan, camptothecin or topotecan. Liposomal irinotecan (e.g., MM-398, also called "nal-IRI") is a highly stabilized liposomal formulation of irinotecan that provides for sustained exposure of irinotecan, and the active metabolite SN-38 in the tumor to a higher proportion of cells during the more sensitive S-phase of the cell cycle. MM-398 is a liposomal irinotecan that has shown promising preclinical and clinical activity in a range of cancer types, and was recently approved in the United States in combination with 5-FU/LV for patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy. Compared with free irinotecan, nal-IRI has an extended PK profile with prolonged local tumor exposure of MM-398 and SN-38. Since SN-38 is cleared more quickly from normal tissues than from tumor, it is hypothesized that delayed dosing of veliparib relative to MM-398 will allow for the expected window of maximum irinotecan-induced toxicity to pass in the absence of concurrent veliparib toxicity. However, the tumor levels of SN-38 are predicted to be sustained upon subsequent veliparib dosing, therefore maintaining the ability of both drugs to act on tumor tissue simultaneously and maintain synergy.

One suitable liposomal Topi inhibitor formulation is liposomal irinotecan available under the brand name ONIVYDE®. (irinotecan liposome injection) (Merrimack Pharmaceuticals, Inc. Cambridge, Mass.), previously designated "MM-398" prior to FDA approval, and liposomal irinotecan products that are bioequivalent to ONIVYDE. The ONIVYDE/MM-398 (irinotecan liposome injection) includes irinotecan as an irinotecan sucrosofate salt encapsulated in liposomes for intravenous use. The drug product liposome is a small unilamellar lipid bilayer vesicle, approximately 110 nm in diameter, which encapsulates an aqueous space which contains irinotecan in a gelated or precipitated state, as the sucrosofate salt. The liposome carriers are composed of l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 6.81 mg/mL; cholesterol, 2.22 mg/mL; and methoxy-terminated polyethylene glycol (MW 2000)- distearoylphosphatidylethanolamine (MPEG-2000-DSPE), 0.12 mg/mL. Each mL also contains 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES) as a buffer, 4.05 mg/mL; sodium chloride as isotonicity reagent, 8.42 mg/mL, ONIVYDE/MM-39S is believed to include about 80,000 molecules of irinotecan in a gelated or precipitated state as a sucrosofate salt, encapsulated in a liposome of about 100 nm in diameter.

Gemcitabine (e.g., Gemzar™) or Gemcitabine-based chemotherapy e.g., combined with other drugs such as albumin-bound paclitaxel (Abraxane), erlotinib (Tarceva), or capecitabine (Xeloda) or combined with radiation (this is called chemoradiation)

As used herein“leucovorin” refers to folinic acid that is typically administered in combination with Onivyde(R) and 5-FU.

5-FU is a thymidylate synthase (TS) inhibitor. Interrupting the action of this enzyme blocks synthesis of the pyrimidine thymidine, which is a nucleoside required for DNA replication. Thymidylate synthase methylates deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). Administration of 5-FU causes a scarcity in dTMP, so rapidly dividing cancerous cells undergo cell death via thymineless death. Calcium folinate provides an exogenous source of reduced folinates and hence stabilizes the 5-FU-TS complex, hence enhancing 5-FU's cytotoxicity. 5-FU is sold under the brand name Adrucil, among others.

According to a specific embodiment, the chemotherapy comprises a plurality of chemotherapeutic agents e.g., at least 2, or at least 3, e.g., 2, 3, 4, 5 chemotherapeutic agents).

According to a specific embodiment, the chemotherapy comprises irinotecan (e.g., liposome-encapsulated e.g., Onivyde^(R)), 5-FU and Leucovorin.

As used herein the term "immune-check point regulator" refers to a molecule that modulates the activity of one or more immune-check point proteins in an agonistic or antagonistic manner resulting in recruitment of an immune cell to elicit an immune activity against a cancer cell.

According to specific embodiments, the immune-check point regulator modulates the activity of a specific immune-check point protein with no cross reactivity with other immune- check point proteins. According to other specific embodiments, the immune-check point regulator modulates the activity of at least 2, at least 3, at least 4 immune-check point proteins.

According to specific embodiments the immune-check point regulator binds directly the immune-check point protein.

According to other specific embodiments, the immune-check point regulator indirectly binds the immune-check point protein through an intermediary molecule.

As used herein the term "activation" refers to the process of stimulating an immune cell (e.g. T cell, NK cell, B cell) that results in cellular proliferation, maturation, cytokine production and/or induction of regulatory or effector functions.

As used herein the term "immune-check point protein" refers to an antigen independent protein that modulates an immune cell response (i.e. activation or function). Immune-check point proteins can be either co- stimulatory proteins [i.e. positively regulating an immune cell activation or function by transmitting a co- stimulatory secondary signal resulting in activation of an immune cell] or inhibitory proteins (i.e. negatively regulating an immune cell activation or function by transmitting an inhibitory signal resulting in suppressing activity of an immune cell).

According to specific embodiments, the immune-check point protein regulates activation or function of a T cell. Numerous checkpoint proteins are known in the art and include, but not limited to, PD1, PDL-1, CTLA-4, CD80, LAG-3, TIM-3, KIR, IDO, 0X40, OX40L, CD 137 (4- 1BB), 4-1BBL, CD27, CD70, CD40, CD40L, GITR, CD28, CD86, and ICOS (CD278), ICOSL.

Methods of determining signaling of a stimulatory or inhibitory signal are well known in the art and include, but are not limited to, binding assay using e.g. BiaCore, HPLC or flow cytometry, enzymatic activity assays such as kinase activity assays, and expression of molecules involved in the signaling cascade using e.g. PCR, Western blot, immunoprecipitation and immunohistochemistry. Additionally or alternatively, determining transmission of a signal (co stimulatory or inhibitory) can be effected by evaluating immune cell activation or function. Methods of evaluating immune cell activation or function are well known in the art and include, but are not limited to, proliferation assays such as BRDU and thymidine incorporation, cytotoxicity assays such as chromium release, cytokine secretion assays such as intracellular cytokine staining ELISPOT and ELISA, expression of activation markers such as CD25, CD69 and CD69 using flow cytometry.

According to specific embodiments, determining the signaling activity is effected in-vitro or ex-vivo e.g. in a mixed lymphocyte reaction (MLR).

For the same culture conditions the signaling activity or the immune cell activation or function are generally expressed in comparison to the signaling, activation or function in a cell of the same species but not contacted with the immune-check point regulator or contacted with a vehicle control, also referred to as control.

Depending on the immune-check point protein (i.e. co-stimulatory or inhibitory) the immune-check point regulator can be an agonist or antagonist.

According to specific embodiment the immune-check point regulator is an antagonist.

As used herein the term“antagonist” refers to a molecule that prevents and/or inhibits the biological function and/or expression of an immune-check point protein.

According to specific embodiments, the antagonist prevents and/or inhibits the suppressive effect of an immune-check point protein on an immune cell (e.g. T cells).

According to specific embodiments, the antagonist prevents and/or inhibits signaling to an immune cell (e.g. T cell) by an immune-check point protein.

The molecule may be a reversible or an irreversible antagonist.

According to specific embodiments, the antagonist completely prevents the biological function (e.g. signal transduction) of the immune-check point protein.

According to other specific embodiments, the antagonist inhibits the biological function (e.g. signal transduction) of the immune-check point protein e.g., as detected by e.g. kinase activity, proliferation assay, cytotoxicity assay or cytokine secretion assay. The reduction may be by at least a 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % as compared to same in the absence of the antagonist.

As mentioned, depending on the immune-check point protein (i.e. co- stimulatory or inhibitory) the immune-check point regulator can be an agonist or antagonist. Thus, according to specific embodiments, the immune-check point regulator is an agonist.

As used herein the term“agonist” refers to a molecule that induces and/or increases the biological function and/or expression of an immune-check point protein.

According to specific embodiments, the agonist induces and/or increases the co stimulatory effect of an immune-check point protein on an immune cell (e.g. T cells).

According to specific embodiments, the agonist induces and/or increases signaling to an immune cell (e.g. T cell) by an immune-check point protein.

The agonist can be a naturally occurring activator or a functional derivative thereof; or non-naturally occurring activator.

According to specific embodiments, the agonist is a full agonist, that is, the effect of the agonist is equivalent to the effect of the naturally occurring activator (i.e. ligand). According to other specific embodiments, the agonist is a partial agonist, that is, the effect of the agonist is lower than the maximal effect of the naturally occurring activator (i.e. ligand). The effect of the agonist may be lower by at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 % at least 50 %, at least 60 %, at least 70 %, at least 80 % or at least 90 % as compared to the maximal effect of the naturally occurring activator.

According to yet other specific embodiments, the agonist is a super agonist, that is, the effect of the agonist is higher than the maximal effect of the naturally occurring activator (i.e. ligand). The effect of the agonist may be higher by at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 % at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 % or at least 2 fold, at least 4 fold, at least 5 fold or at least 10 fold as compared to the maximal effect of the naturally occurring activator.

According to specific embodiments, the agonist induces complete activation the biological function (e.g. signal transduction) of the immune-check point protein.

According to other specific embodiments, the agonist increases the biological function (e.g. signal transduction) of the immune-check point protein e.g., as detected by e.g. kinase activity, proliferation assay, cytotoxicity assay or cytokine secretion assay. The increase may be by at least a 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % as compared to same in the absence of the agonist.

According to specific embodiments, the agonist binds directly the immune-check point protein.

According to other specific embodiments, the agonist indirectly binds the immune-check point protein by acting through an intermediary molecule, for example the agonist binds to or modulates a molecule that in turn binds to or modulates the immune-check point protein.

Activating and/or increasing the biological function of an immune-check point protein can be effected at the protein level (e.g., antibodies, small molecules, peptides and the like) but may also be effected at the genomic level (e.g., activation of transcription via promoters, enhancers, regulatory elements) and/or the transcript level using a variety of molecules which promote transcription and/or translation (e.g., correct splicing, polyadenylation, activation of translation) of a co-stimulatory immune-check point protein.

According to a specific embodiment the immune-check point regulator (modulator) is selected from the group consisting of PD1 antagonist, PDL-1 antagonist, CTLA-4 antagonist, LAG-3 antagonist, TIM-3 antagonist, KIR antagonist, IDO antagonist, 0X40 agonist, CD 137 agonist, CD27 agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS agonist; More non-limiting examples are provided WO W02017/009842 and W02017/009843, each of which is incorporated by reference in its entirety.

According to a specific embodiment, the immune checkpoint regulator is an antibody.

According to a specific embodiment, the immune checkpoint regulator is a small molecule.

According to a specific embodiment, the immune checkpoint regulator is a peptide or soluble receptor or ligand.

Examples of immune checkpoint inhibitors include, but are not limited to, of cytotoxic T- lymphocyte antigen 4 (CTLA4), programmed death 1 (PD-1) or its ligands, lymphocyte activation gene-3 (LAG3), B7 homolog 3 (B7-H3), B7 homolog 4 (B7-H4), indoleamine (2,3)-dioxygenase (IDO), adenosine A2a receptor, neuritin, B- and T-lymphocyte attenuator (BTLA), killer immunoglobulin-like receptors (KIR), T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), inducible T cell costimulator (ICOS), CD27, CD28, CD40, CD244 (2B4), CD 160, GARP, 0X40, CD137 (4-1BB), CD25, VISTA, BTLA, TNFR25, CD57, CCR2, CCRS, CCR6, CD39, CD73, CD4, CD18, CD49b, CDld, CDS, CD21, TIMI, CD19, CD20, CD23, CD24, CD38, CD93, IgM, B220 (CD45R), CD317, CDl lb, Ly6G, ICAM-1, FAP, PDGFR, Podoplanin, and TIGIT.

Examples of clinically approved immune checkpoint inhibitors include, but are not limited to, Ipilimumab, (anti CTLA-4), Nivolimumab (anti PD-1) and Pembrolizumab (anti PD-1).

Anti-PDl antibodies suitable for use in the invention can be generated using methods well known in the art especially in light of the detailed description hereinabove. Alternatively, art recognized anti-PDl antibodies can be used. Examples of anti-PDl antibodies are disclosed for example in Topalian, et al. NEJM 2012, U.S. Patent Nos. 7,488,802; 8,008,449; 8,609,089; 6,808,710; 7,521,051; and 8168757, U.S. Patent Application Publication Nos. 20140227262; 20100151492; 20060210567; and 20060034826 and International Patent Application Publication Nos. WO2008156712; W02010089411; W02010036959; WO2011159877; WO2013/019906; WO2014159562; WO2011109789; WOOl/14557; W02004/004771; and W02004/056875, which are hereby incorporated by reference in their entirety.

Specific anti-PDl antibodies that can be used according to some embodiments of the present invention include, but are not limited to:

Nivolumab (also known as MDX1106, BMS-936558, ONO-4538), marketed by BMY as Opdivo, a fully human IgG4 antibody with the structure described in WHO Drug Information, Vol. 27, No. 1, pages 68-69 (2013) that binds to and blocks the activation of PD-1 by its ligands PD-L1 and PD-L2; Pidilizumab (also known as CT-011, hBAT, hBAT-1, produced by CureTech), a humanized monoclonal IgGl antibody that binds PD-1;

AMP-514 (also known as MEDI-0680, produced by AZY and Medlmmune), a humanized monoclonal IgG4 antibody that binds PD-1.

Humanized antibodies h409Al 1, h409A16 and h409A17, which are described in PCT Patent Application No. WO2008/156712;

According to a specific embodiment, the anti PD- 1 is Pembrolizumab (also known as MK- 3475, Keytmda, SCH 900475, produced by Merck). A humanized monoclonal IgG4 antibody with the structure described in WHO Drug Information, Vol. 27, No. 2, pages 161-162 (2013) that binds to and blocks the activation of PD1 by its ligands;

According to a specific embodiment, the chemotherapy comprises ILF, the CD4 antagonist is an antibody and the immune checkpoint regulator is anti-PD-1 (e.g., Pembrolizumab). According to a specific embodiment, the disease is pancreatic cancer.

The CD4 antagonist, chemotherapy and immune checkpoint regulator can be provided to the subject per se or as part of a pharmaceutical composition.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the CD4 antagonist and at least one of immune checkpoint regulator and/or the chemotherapy accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, intradermal, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

The CD4 antagonist, immune checkpoint regulator and/or the chemotherapy or the pharmaceutical composition comprising same can be administered in the same route or in separate routes.

According to a specific embodiment, The CD4 antagonist, immune checkpoint regulator and/or the chemotherapy or the pharmaceutical composition comprising same is administered subcutaneously.

According to another specific embodiment, the CD4 antagonist, immune checkpoint regulator and/or the chemotherapy of the invention or the pharmaceutical composition comprising same is administered intravenously.

According to a specific embodiment, the anti-cancer agent or the pharmaceutical composition comprising same is administered intravenously.

According to a specific embodiment, the anti-cancer agent or the pharmaceutical composition comprising same is administered via a subcutaneous route. Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.

Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use. The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Alternative embodiments include depots providing sustained release or prolonged duration of activity of the active ingredient in the subject, as are well known in the art.

Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, according to specific embodiments, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and the route of administration utilized.

The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et ah, 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).

Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved. The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

According an aspect of the present invention there is provided an article of manufacture identified for use in treating cancer, comprising a packaging material packaging the CD4 antagonist, the immune checkpoint regulator and/or the chemotherapy as described herein.

The CD4 antagonist, the immune checkpoint regulator and/or the chemotherapy and the agent may be packaged in the same container or in separate containers; each possibility represents a separate embodiment of the present invention.

According to specific embodiments, the CD4 antagonist, the immune checkpoint regulator and/or the chemotherapy are in separate containers.

According to specific embodiments, the CD4 antagonist, the immune checkpoint regulator and/or the chemotherapy are in separate formulations.

According to other specific embodiments, the CD4 antagonist, the immune checkpoint regulator and/or the chemotherapy are in a co-formulation.

According to a specific embodiment, the CD4 antagonist, the immune checkpoint regulator and/or the chemotherapy are co-administered.

According to a specific embodiment, the CD4 antagonist, immune checkpoint regulator and the chemotherapy are administered in a sequential manner.

According to a specific embodiment, at least one of or two of the CD4 antagonist immune checkpoint regulator and/or the chemotherapy are administered in a sequential manner. As mentioned the CD4 antagonist is not the peptide set forth in SEQ ID NO: 1 or analog or derivative or same as described in the following embodiments which are to be excluded from the scope of the claimed invention.

In various particular embodiments, the peptide analog or derivative has an amino acid sequence as set forth in the following formula (I) or a salt thereof:

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Ai-A₂-A₃-Cys-Tyr-A₄-A₅-A₆-A₇-As-A₉-Aio-Cys-An (I)

wherein:

Ai is an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue or a N-a- substituted derivative of these amino acids, or Ai is absent;

A2 represents an arginine or glutamic acid residue if Ai is present, or A2 represents an arginine or glutamic acid residue or a N-a-substituted derivative of these amino acids if Ai is absent;

A3 represents an aromatic amino acid residue;

A4, As and A9 each independently represents an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue;

Ab represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;

Ai represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;

As represents a tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;

A10 represents a citrulline, glutamic acid, arginine or lysine residue;

A11 represents an arginine, glutamic acid, lysine or citrulline residue wherein the C- terminal carboxyl may be derivatized;

and the cysteine residue of the 4-position or the 13-position can form a disulfide bond, and the amino acids can be of either L or D form.

Exemplary peptide analogs or derivatives according to formula (I) are peptides having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-72, as presented in Table 1 hereinbelow.

Table 1 - T-140 and currently preferred T-140 analogs

In another embodiment, the peptide analog consists essentially of an amino acid sequence as set forth in SEQ ID NO: 1. In another embodiment, the peptide used in the compositions and methods of the invention comprises an amino acid sequence as set forth in SEQ ID NO: 1. In another embodiment, the peptide is at least 60%, at least 70% or at least 80% homologous to SEQ ID NO: 1. In another embodiment, the peptide is at least 90% homologous to SEQ ID NO: 1. In another embodiment, the peptide is at least about 95% homologous to SEQ ID NO: 1. Each possibility represents a separate embodiment of the present invention.

In various other embodiments, the peptide is selected from SEQ ID NOs: 1-72, wherein each possibility represents a separate embodiment of the present invention.

It is expected that during the life of a patent maturing from this application many relevant CD4 antagonists, chemotherapies and/or immune checkpoint regulators will be developed and the scope of the terms is intended to include all such new technologies a priori.

As used herein the term“about” refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

The term“consisting of’ means“including and limited to”.

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases“ranging/ranges between” a first indicate number and a second indicate number and“ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Patent Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);“Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1- 317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

MATERIALS AND METHODS

Cell Lines and Cell Culture Conditions.

The Panc02 cell line is described in Partecke et al. Eur Surg Res. 2011;47(2):98-107.

Cells were cultured in RPMI-1640 medium, supplemented with 10 % (vol/vol) heat- inactivated FCS, 2 mM glutamine, 100 units/mL penicillin, and 100 pg/mL streptomycin. The cell cultures were maintained in a humidified atmosphere of 5 % (vol/vol) CO2 at 37 °C. Female C57B/6 5-wk old mice were purchased from Harlan Laboratories. All mice were kept in a specific pathogen-free facility. Mice were handled according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health. All experiments were approved by the Animal Care Committee of Hebrew University.

Tumor Models.

The mice were allowed to acclimate to the facility for at least 1 week before manipulation. Mice had free access to water and chow at all times.

S.c. tumors.

Tumor xenografts were established by s.c. injection of log-phase growth viable Panc02 cells, 2* 10⁶ (in 2000 pL PBS Panc02 cells. Mice were randomized immediately after the injection. 3 days after cell injection mice were treated as follows:

BL8040, 400 pg/mouse, SC injected in total 200 pi PBS at days 3,4 and 5;

Anti-PD-1, 100 pg/mouse, SC injected in total 200 pi PBS at day 3;

Irrinotecan, 0.2 mg/mouse, IV injected in total 200 pi PBS at day 3;

Leukovorin, 1.6 mg/mouse, SC injected in total 200 pi PBS at days 3, and 5;

Fluorouracil (5FU), 0.8 mg/mouse, SC injected in total 200 pi PBS at days 3, and 5.

Control animals were non-injected mice.

At day 7 mice were weighed and sacrificed, tumors were weighted, and paraffin embedded.

Immunohistochemical Staining.

Immunohistochemistry was done on 4- pm- thick formalin-fixed paraffin-embedded tissue sections by standard procedure. Staining was done using the following antibodies listed in Table 2:

Table 2

Statistical Analysis.

All data were subjected to statistical analysis using the Excel software package (Microsoft). A two-tailed Student t test was used to determine the difference between the groups. Differences were considered significant at P < 0.05. Data are given as mean ± SEM.

EXAMPLE 1

Decrease in pancreatic tumor volume due to combination of chemotherapy with BL8040 and anti PD-1

Syngeneic S.C. Panc02 tumors were treated with anti-PD-1, BL80/40 or chemotherapy (Irrinotecan, Leucovorin and Fluorouracil (ILF)) or combination of chemotherapy with BL8040 or combination of chemotherapy with BL8040 and anti PD- 1. Seven days after cell injection mice were sacrificed and tumors were weighed. The graph represents average tumor weight (mg) in each group ± SEM (n=8). Student T test (p) was calculated to compare ILF-treated group with combination of chemotherapy with BL8040 or combination of chemotherapy with BL8040 and anti PD-1.

As shown in Figure 1, the combination of chemotherapy with BL8040 and possibly anti PD1 was statistically significant in inhibiting tumor development, as determined by tumor weight.

EXAMPLE 2

T cell activation in tumors treated with combination of chemotherapy with BL8040 and anti PD-1.

Syngeneic S.C. Panc02 tumors were treated with anti-PD-1, BL80/40 or chemotherapy

(Irrinotecan, Leucovorin and Fluorouracil (ILF)) or combination of chemotherapy with BL8040 or combination of chemotherapy with BL8040 and anti PD-1. Seven days after cell injection mice were sacrificed and tumors were paraffin embedded and IHC-stained to detect CD8.

Pictures were taken at magnification of x20 and the size of the cells (35-56 cells per treatment) was evaluated using the ImageJ program. The P value was determined using Student 11 in CD8+ size (a marker for T cell activation) in tumors treated with combination of chemotherapy with BL8040 and anti PD- lest.

Staining shown in Figure 2 points to an increase in CD8+ size (a marker for T cell activation) in tumors treated with combination of chemotherapy with BL8040 and anti PD-1. EXAMPLE 3

Tumors treated with a combination of chemotherapy with BL8040 and anti PD-1 have similar amounts of CD8 cells and increase number of CD8+CD69 activated T cells

Syngeneic S.C. Panc02 tumors were treated with anti-PD-1, BL80/40 or chemotherapy (Irrinotecan, Leucovorin and Fluorouracil (ILF)) or combination of chemotherapy with BL8040 or combination of chemotherapy with BL8040 and anti PD- 1. Seven days after cell injection mice were sacrificed and tumors were paraffin embedded and IHC-stained to detect CD8 and CD69 positive cells. Pictures were taken at magnification of x20 and the number of cells (20-50 per field, in four fields was counted). The P value was determined using Student t test. Increase in the percentage of CD69+CD8+ cells was observed in the treatment combination of chemotherapy with BL8040 and anti PD-1 compare to control (45% vs 20% respectively).

As shown in Figure 3, tumors treated with a combination of chemotherapy with BL8040 and anti PD-1 have similar amounts of CD8 cells and increase number of CD8+CD69 activated T cells.

EXAMPLE 4

Tumors treated with combination of chemotherapy BL8040 and anti PD-1 have

significantly reduced amounts of CD4

Syngeneic S.C. Panc02 tumors were treated with anti-PD-1, BL80/40 or chemotherapy (Irrinotecan, Leucovorin and Fluorouracil (ILF)) or combination of chemotherapy with BL8040 or combination of chemotherapy with BL8040 and anti PD- 1. Seven days after cell injection mice were sacrificed and tumors were paraffin embedded and IHC-stained to detect CD4. Pictures were taken at magnification of x20 and the number of cells (up to 60 cells were counted per field, in four fields was counted). The P value was determined using Student t test. As can be seen in figure 4, tumors treated with combination of chemotherapy BL8040 and anti PD-1 have significantly reduced amounts of CD4 as compared to control, chemotherapy or BL8040 alone.

EXAMPLE 5

Tumors treated with combination of chemotherapy, BL8040 and anti PD-1 have significantly reduced amounts of regulatory T cells

Syngeneic S.C. Panc02 tumors were treated with anti-PD-1, BL80/40 or chemotherapy (Irrinotecan, Leucovorin and Fluorouracil (ILF)) or combination of chemotherapy with BL8040 or combination of chemotherapy with BL8040 and anti PD- 1. Seven days after cell injection mice were sacrificed and tumors were paraffin embedded and IHC-stained to detect CD4 and FOXP-3 double positive cells and CD45+ cells. Pictures were taken at magnification of x20 and the number of cells (up to 60 cells were counted per field, in four fields was counted). The P value was determined using Student t test. As shown in Figure 5, tumors treated with combination of chemotherapy, BL8040 and anti PD-1 have significantly reduced amounts of CD4+FOXP3+ cells (100% reduction) and moderate reduction in total CD45+ cells (20-30%) compared to control.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a solid tumor in a subject in need thereof, the method comprising, administering to the subject a therapeutically effective amount of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator, wherein said CD4 antagonist is not a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof, thereby treating the solid tumor in the subject.

2. A therapeutically effective amount of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator for use in treating solid tumor in a subject in need thereof, wherein said CD4 antagonist is not a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof.

3. An article of manufacture for use in treating cancer comprising a therapeutically effective amount of a CD4 antagonist, a chemotherapy and an immune checkpoint regulator, wherein said CD4 antagonist is not a peptide having an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or derivative thereof.

4. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-3, wherein said CD4 antagonist is an antibody.

5. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-3, wherein said CD4 antagonist is a small molecule.

6. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-5, wherein said solid tumor is pancreatic cancer.

7. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-6, wherein said CD4 antagonist, chemotherapy and checkpoint regulator cause a decrease in tumor growth.

8. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-7, wherein said CD4 antagonist, chemotherapy and checkpoint regulator cause a synergistic increase in activation state of CD8+ T cells infiltrating said solid tumor.

9. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-7, wherein said CD4 antagonist, chemotherapy and checkpoint regulator cause a synergistic increase in size of CD8+ T cells infiltrating said solid tumor.

10. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-9, wherein said CD4 antagonist, chemotherapy and checkpoint regulator cause a decrease in Tregs infiltrating said solid tumor.

11. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of claim 10, wherein said Tregs comprise FOXP3+CD4+ T cells.

12. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-9, wherein said immune checkpoint regulator is an immune checkpoint inhibitor.

13. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-9, wherein said immune checkpoint inhibitor is anti PD-1.

14. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-13, wherein said chemotherapy comprises ILF.

15. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-14, wherein said CD4 antagonist, chemotherapy and checkpoint regulator are administered concomitantly.

16. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-14, wherein said CD4 antagonist, chemotherapy and checkpoint regulator are administered sequentially.

17. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-14, wherein said CD4 antagonist, chemotherapy and checkpoint regulator are in a co-formulation.

18. The method or the CD4 antagonist, chemotherapy and checkpoint regulator or article of manufacture of any one of claims 1-14, wherein said CD4 antagonist, chemotherapy and checkpoint regulator are in separate formulations.