Methods of selecting treatment regimen against solid tumors

Abstract

A method of selecting a treatment regimen for a subject diagnosed with a solid tumor is provided. The method comprising, determining in an in situ sample of the solid tumor of the subject a level of CXCL12, wherein: when the level is above a predetermined threshold selecting a treatment regimen which comprises a CXCR4 inhibitor; or when the level is below a predetermined threshold selecting a treatment regimen which does not comprise a CXCR4 inhibitor.

Description

[0001] METHODS OF SELECTING TREATMENT REGIMEN AGAINST SOLID TUMORS

[0002] RELATED APPLICATION

[0003] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 637,940 filed on 24 April 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] FIELD AND BACKGROUND OF THE INVENTION

[0005] The present invention, in some embodiments thereof, relates to methods of selecting treatment regimen against solid tumors.

[0006] CXCR4 is a G-protein coupled receptor that mediates the activity of CXC chemokines. To date, its only identified cognate ligand is CXCL12, also known as Stromal Cell-Derived Factor- 1 (SDF-1). CXCR4 plays an important role in mammalian development, mediating the migration and motility of tissue and hematopoietic stem and progenitor cells. Both CXCR4 and SDF-1 knock-out mice show embryonic lethality with essentially identical phenotypes involving tissue and vascular malformations, supporting the hypothesis that CXCR4 is the key receptor for the activity of SDF-1 (CXCR7 is a second known receptor of SDF-1). CXCR4 continues to be broadly expressed in the adult, with high levels detected on bone marrow stem and progenitor cells, various circulating lymphocytes (B-cells, activated T-cells), as well as endothelial precursor cells, and tissue macrophages and fibroblasts.

[0007] CXCR4 plays a pleiotropic role in human cancer. Its expression is upregulated in many tumor types, including cancers of the breast, lung, colon, pancreas, brain, prostate, ovary, as well as hematopoietic cancers. Some literature reports suggest that SDF-1 may act through CXCR4 as a growth and / or survival factor for some tumors. In models of metastatic cancer, CXCR4 positive tumors were shown to metastasize to distant sites, and this activity was inhibited by agents that silence the CXCR4 gene or antibodies that block CXCR4 or SDF-1. Consistent with this view, many common sites of metastasis in human cancer, such as bone marrow, lung, lymph node, and liver, express high levels of SDF-1. CXCR4 is expressed on stem cell-like or tumor initiating subpopulations of many tumors, and may mediate the ability of these cells to support the recurrence and metastatic spread of cancers. Furthermore, CXCR4 is expressed on endothelial precursor cells (EPCs), and its activity is required for incorporation of EPCs into functional vessels during angiogenesis. This may make a significant contribution to the vascularization and survival of tumors. CXCR4 signaling can also lead to induction of pro-angiogenic cytokines (e.g., VEGF), as well as integrins, adhesion molecules and matrix degrading enzymes that may mediate invasion by tumor cells. Lastly, CXCR4 expression is detected on tumor infiltrating lymphocytes and fibroblasts, as well as tumor associated macrophages. These cells tend to suppress immune recognition and attack on the tumor, and remodel the tumor microenvironment to encourage tumor growth and metastasis (see WO2017 / 021963).

[0008] The multiple roles of CXCR4 in tumor growth, vascularization, and metastasis, and its broad expression in many common tumor types, make this receptor an attractive target for therapeutic intervention using inhibitory agents.

[0009] 4F-benzoyl-TN14003 (also known as BKT140, hereinafter BL-8040), is a 14-residue bio stable synthetic peptide developed as a specific CXCR4 antagonist. It has been shown that BL- 8040 binds the CXCR4 receptor with high affinity and long receptor occupancy. BL-8040 was found to be toxic against several tumors such as myeloid leukemia, hematopoietic tumors and nonsmall cell lung cancer (International Patent Application No. IL2014 / 050939 and International Patent Application Publication No. W02013 / 16089556053 and W02008 / 075370).

[0010] W02020 / 065647 teaches the use of CXCR4 as a predictor of the clinical efficacy of CXCR4 antagonists, especially in non-solid tumors e.g., AML.

[0011] SUMMARY OF THE INVENTION

[0012] According to an aspect of some embodiments of the present invention there is provided a method of selecting a treatment regimen for a subject diagnosed with a solid tumor, the method comprising, determining in an in situ sample of the solid tumor of the subject a level of CXCL12, wherein: when the level is above a predetermined threshold selecting a treatment regimen which comprises a CXCR4 inhibitor; or when the level is below a predetermined threshold selecting a treatment regimen which does not comprise a CXCR4 inhibitor.

[0013] According to an aspect of some embodiments of the present invention there is provided a method of selecting a subject diagnosed with a solid tumor for treatment with a CXCR4 inhibitor, the method comprising, determining in an in situ sample of the solid tumor of the subject a level of CXCL12, wherein: when the level is above a predetermined threshold, selecting the subject for a treatment regimen which comprises a CXCR4 inhibitor; or when the level is below a predetermined threshold, selecting the subject for a treatment regimen which does not comprise a CXCR4 inhibitor. According to an aspect of some embodiments of the present invention there is provided a method of treating a subject diagnosed with a solid tumor, the method comprising, determining in an in situ sample of the solid tumor of the subject a level of CXCL12, wherein: when the level is above a predetermined threshold treating the subject with a treatment regimen which comprises a CXCR4 inhibitor; or when the level is below a predetermined threshold treating the subject with a treatment regimen which does not comprise a CXCR4 inhibitor.

[0014] According to an aspect of some embodiments of the present invention there is provided a CXCR4 inhibitor for use in treating a subject diagnosed with a solid tumor displaying an in situ level of CXCL12 above a predetermined threshold.

[0015] According to some embodiments of the invention, the determining further comprises determining a level of CXCR4 in the in situ sample.

[0016] According to some embodiments of the invention, the determining a level of CXCR4 in the in situ sample is on selected immune cells.

[0017] According to some embodiments of the invention, the determining comprises an immunoassay.

[0018] According to some embodiments of the invention, the immunoassay comprises immunohistochemistry (IHC).

[0019] According to some embodiments of the invention, the in situ comprises cancer cells and / or tumor microenvironment.

[0020] According to some embodiments of the invention, the tumor microenvironment comprises fibroblasts.

[0021] According to some embodiments of the invention, the solid tumor is pancreatic cancer.

[0022] According to some embodiments of the invention, the pancreatic cancer comprises metastatic pancreatic adenocarcinoma.

[0023] According to some embodiments of the invention, the CXCR4 inhibitor is a peptide, a small molecule, an antibody, a nucleic acid or a combination of same.

[0024] According to some embodiments of the invention, the CXCR4 inhibitor is a peptide.

[0025] According to some embodiments of the invention, the peptide is as set forth in SEQ ID NO: 1 or an analog of same.

[0026] According to some embodiments of the invention, the peptide of SEQ ID NO: 1 is motixafortide.

[0027] According to some embodiments of the invention, the peptide set forth in SEQ ID NO: 1 is administered subcutaneously. According to some embodiments of the invention, the peptide set forth in SEQ ID NO: 1 is administered at a dose of 0.5-5 mg / kg.

[0028] According to some embodiments of the invention, the peptide set forth in SEQ ID NO: 1 is administered at a dose of 0.5-2.5 mg / kg.

[0029] According to some embodiments of the invention, the peptide set forth in SEQ ID NO: 1 is administered at a dose of 0.75-1.5 mg / kg.

[0030] According to some embodiments of the invention, the peptide set forth in SEQ ID NO: 1 is administered intravenously.

[0031] According to some embodiments of the invention, the CXCR4 inhibitor is a small molecule.

[0032] According to some embodiments of the invention, the CXCR4 inhibitor is AMD3100.

[0033] According to some embodiments of the invention, the subject is naive (prior to first line treatment).

[0034] According to some embodiments of the invention, the subject is post first line treatment.

[0035] According to some embodiments of the invention, the treatment regimen comprises at least one of a chemotherapy, a targeted therapy and an immune modulator.

[0036] According to some embodiments of the invention, the immune modulator comprises a checkpoint modulator.

[0037] According to some embodiments of the invention, the checkpoint modulator is anti PD-1 or anti PD-L1.

[0038] 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.

[0039] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0040] The present invention, in some embodiments thereof, relates to methods of selecting treatment regimen against solid tumors.

[0041] 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.

[0042] Whilst conceiving embodiments of the invention, the present inventors harnessed the recently understood mechanism of action of motixafrtide, a CXCR4 inhibitor, towards the development of a biomarker for subject selection for anti cancer treatment using a CXCR4 inhibitor. It is suggested that assessing the level of CXCL12 at the tumor and tumor microenvironment (TME), would be predictive of treatment efficacy with a CXCR4 inhibitor and customize treatment.

[0043] Specifically, the tumor secretes CXCL12 thereby attracting tumor suppression cells such as MDSCs and Tregs. Motixafortide reduces attraction of suppressor cells to the TME by blocking CXCR4 (the receptor for CXCL12), thereby reducing the immunosuppression. In addition, cancer associated fibroblasts (CAFs) secrete CXCL12 and bind effector T cells to the tumor edge, preventing them from infiltrating into the tumor. Motixafortide prevents T cells to be blocked by CAFs at the tumor edge which leads to penetration of effector T cells into the tumor. Hence assessing CXCL12 levels at the tumor or TME is pivotal to selecting subjects for treatment with CXCR4 inhibitors, and Motixafortide in particular.

[0044] Thus, according to an aspect of the invention there is provided a method of selecting a treatment regimen for a subject diagnosed with a solid tumor, the method comprising, determining in an in situ sample of the solid tumor of the subject a level of CXCL12, wherein: when the level is above a predetermined threshold selecting a treatment regimen which comprises a CXCR4 inhibitor; or when the level is below a predetermined threshold selecting a treatment regimen which does not comprise a CXCR4 inhibitor.

[0045] Alternatively or additionally, there is provided a method of treating a subject diagnosed with a solid tumor, the method comprising, determining in an in situ sample of the solid tumor of the subject a level of CXCL12, wherein: when the level is above a predetermined threshold treating the subject with a treatment regimen which comprises a CXCR4 inhibitor; or when the level is below a predetermined threshold treating the subject with a treatment regimen which does not comprise a CXCR4 inhibitor.

[0046] Alternatively or additionally, there is provided a method of selecting a subject diagnosed with a solid tumor for treatment with a CXCR4 inhibitor, the method comprising, determining in an in situ sample of the solid tumor of the subject a level of CXCL12, wherein: when the level is above a predetermined threshold selecting the subject for a treatment regimen which comprises a CXCR4 inhibitor; or when the level is below a predetermined threshold selecting the subject for a treatment regimen which does not comprise a CXCR4 inhibitor.

[0047] Alternatively or additionally, there is provided a CXCR4 inhibitor for use in treating a subject diagnosed with a solid tumor displaying an in situ level of CXCL12 above a predetermined threshold.

[0048] As used herein “selecting” refers to informing the subject of decision of treatment or issuing a prescription.

[0049] As used herein, the term “subject” includes mammals, preferably human beings at any age diagnosed with a solid tumor / cancer.

[0050] According to a specific embodiment, the subject is diagnosed with metastatic pancreatic adenocarcinoma (as described below).

[0051] According to a specific embodiment the solid tumor is histologically confirmed (either previously or newly biopsied).

[0052] Solid tumors are heterotypic aggregates of many cell types, including cancer cells, cancer stem cells, connective-tissue cells, and immune cells. The collective cells in the vicinity of the tumor are referred to as the tumor microenvironment (TME).

[0053] The tumor microenvironment (TME) refers to the complex cellular and molecular landscape that surrounds a tumor. It includes various cell types, such as cancer cells, immune cells, fibroblasts and endothelial cells. Cancer-associated fibroblasts (CAFs) are activated fibroblasts found within the TME. They play roles in ECM remodeling, promoting tumor growth, angiogenesis, and metastasis.

[0054] Examples of solid tumors include, but are not limited to, pancreatic cancer, lung cancer, breast cancer, prostate cancer, colorectal cancer, colon cancer, hepatocellular carcinoma, ovarian cancer, prostate cancer, melanoma, brain tumors such as gliomas and meningiomas, liver cancer (hepatocellular carcinoma), kidney cancer (renal cell carcinoma), thyroid cancer, bladder cancer, stomach cancer (gastric cancer), esophageal cancer, sarcomas like osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, neuroblastoma, Wilms tumor (nephroblastoma), testicular cancer, endometrial cancer, cervical cancer, head and neck cancers, adrenal gland tumors such as adrenal cortical carcinoma and pheochromocytoma, mesothelioma, soft tissue sarcomas like leiomyosarcoma and liposarcoma, gastrointestinal stromal tumors (GIST), Merkel cell carcinoma, basal cell carcinoma, squamous cell carcinoma, germ cell tumor and cholangiocarcinoma. According to a specific embodiment, the tumor is selected from the group consisting of pancreatic cancer, ovarian cancer, prostate cancer, colon cancer and hepatocellular carcinoma.

[0055] According to a specific embodiment, the tumor is a cold tumor.

[0056] According to a specific embodiment, the tumor is a primary tumor.

[0057] According to a specific embodiment, the tumor is a metastatic tumor.

[0058] According to a specific embodiment, the tumor is pancreatic cancer.

[0059] According to a specific embodiment, the pancreatic cancer is a metastatic pancreatic adenocarcinoma.

[0060] As used herein “metastatic pancreatic adenocarcinoma” refers to stage lib to IV of the disease, when the tumor is present out of the pancreas i.e., lymph nodes or other distal locations.

[0061] 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.

[0062] According to a specific embodiment, the PDAC comprises intraductal papillary mucinous neoplasm.

[0063] In some embodiments, the cancer is recurrent e.g., recurrent pancreatic cancer.

[0064] In some embodiments, the cancer has reoccurred after remission.

[0065] In some embodiments, the individual has measurable disease (for example, according to RECIST criteria).

[0066] In some embodiments, the individual has one or more tumors (e.g., metastatic) measurable, for example, by CT scan (or MRI).

[0067] In some embodiments, the pancreatic cancer is unresectable pancreatic cancer. In some embodiments, the pancreatic cancer is a resectable pancreatic cancer.

[0068] In some embodiments, the pancreatic cancer is borderline resectable.

[0069] In some embodiments, the primary location of the pancreatic cancer is the head of the pancreas. In some embodiments, the primary location of the pancreatic cancer is the body of the pancreas. In some embodiments, the primary location of the pancreatic cancer is the tail of the pancreas.

[0070] As used herein “subject” refers to a human subject diagnosed with metastatic pancreatic adenocarcinoma.

[0071] In some embodiments, the subject is a female.

[0072] In some embodiments, the individual is a male. In some embodiments, the individual is under about 65 years old (such as under about any of 60, 55, 50, 45, or 40 years old).

[0073] In some embodiments, the subject is at least about 65 years old (such as at least about any of 70, 75, or 80 years old).

[0074] According to a specific embodiment, the subject is at least 18 years

[0075] When referring to pancreatic cancer, according to some embodiments, the subject has not been treated with any previous systemic chemotherapy for the pancreatic adenocarcinoma. Accordingly, treatment is first-line treatment.

[0076] According to another embodiment, treatment is first-line treatment against the pancreatic adenocarcinoma.

[0077] According to a specific embodiment, the treatment is post first-line treatment against the pancreatic adenocarcinoma.

[0078] Thus, methods, regimens, uses, described herein may also be used as a second line or third line therapy after the prior treatment for cancer has failed or has substantially failed, or the cancer is substantially refractory to the first line therapy. In some embodiments, the individual has received at least one line of therapy (e.g., chemotherapy, radiation or immunotherapy) for treating cancer prior to receiving the treatment described herein. In some embodiments, the patient has received 1 line of therapy or 2 lines of therapy (e.g., 1 line of chemotherapy or immunotherapy). Thus, the treatment described herein may be used as a second line therapy. The prior line of therapy described herein may be a prior line of chemotherapy or immunotherapy.

[0079] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical symptoms of the solid tumor.

[0080] According to a specific embodiment, the method is effected ex-vivo on a tissue biopsy which includes cancer cells and / or CAFs , though other modes of detection are also contemplated.

[0081] As used herein cells of the subject, refers to tumor cells and / or cells of the microenvironment such as comprised in a biological sample. For example, determining the level of CXCL12 on tumor cells and / or fibroblasts of the TME (i.e., CAFs) and optionally in immune cells in the sample. Immune cells may be subjected to analysis of CXCR4 couples with CD3 and / or CD8 for determining T cells and FoxP3 for determining Tregs. A multiplex assay is contemplated to refer to the use of a number of antibodies contacted with single biopsy, which will be differentiated by the mode of staining.

[0082] Methods of determining a level of CXCL12 and optionally CXCR4 are well known in the art. Thus a biopsy specimen may be obtained from the subject. Typically tissue sections are prepared on slides. The skilled artisan would elect a fixation method and typical processing to ensure preservation of antigenicity and morphology. Depending on the tissue type and fixation method, antigen retrieval may be done to unmask epitopes and improve antibody binding. Common methods include, but are not limited to, epitope retrieval (HIER) or enzymatic digestion. Blocking nonspecific sites is done on the tissue sections to reduce background staining. Blocking reagents such as serum, albumin (BSA), or appropriate blocking buffers can be used. The tissue sections are then incubated with one or more primary antibodies, targeting the antigens of interest e.g., CXCL12. When needed the skilled artisan would ensure that each primary antibody is validated for specificity and compatibility with a multiplex staining protocol. In addition the antibody concentrations and incubation conditions (e.g., temperature, duration) are optimized. Thereafter, the tissue sections are washed to remove unbound primary antibodies and reduce nonspecific background staining. The primary antibody may be chromogenically or fluorescently labeled, e.g., with a dye or distinct dyes when a number of antigens are detected, e.g., CXCR4 and CXCL12. When the primary antibody(s) is not labeled secondary antibodies conjugated to different fluorophores or enzyme labels to detect the bound primary antibodies are used. Measures are taken that each secondary antibody should be specific to the host species of the corresponding primary antibody and labeled with a distinct fluorophore or enzyme for multiplex detection. The antibody-antigen complexes are visualized using fluorescence microscopy or chromogenic detection methods. Optionally, counterstaining is performed with nuclear dyes (e.g., DAPI, Hoechst) or other markers to visualize specific cellular structures or compartments. The stained tissue sections are captured using an appropriate imaging system. Images are analyzed to quantify staining intensity, co-localization of markers, and other relevant parameters using image analysis software. Positive and negative controls are used as well as known reference samples.

[0083] Typically, the level of CXCL12 (and optionally CXCR4) is above a predetermined threshold.

[0084] Thus, “above a predetermined threshold” refers to a level in the same cells (e.g., cancer and / or TME) above that of non-responders to CXCR4 inhibition therapy (alone or combined e.g., CXCR4, anti PD-1 and a chemotherapy).

[0085] Examples of immunoassays include but are not limited to those which retain the tissue structure. These may be immunohistochemistry (IHC), immunofluorescent (IF) and multiplex IF or IHC. Multiplex assays are also contemplated herein combining the principles of IF or IHC with multiplexing capabilities to enable simultaneous detection of multiple protein targets within intact tissue sections. It preserves tissue structure while allowing for the spatial characterization of cell subsets.

[0086] In other embodiments the tissue structure is not maintained. These include, ELISA, mass cytometry (CyTOF) and in situ hybridization.

[0087] As used herein “CXCL12” also known as the stromal cell-derived factor 1 (SDF-1) refers to a chemokine protein that in humans is encoded by the CXCL12 gene on chromosome 10. : CXCL12, IRH, PBSF, SCYB 12, SDF1, TLSF, TP ARI, C-X-C motif chemokine ligand 12.

[0088] Antibodies for CXCL12 are well known in the art. Preferably used are those which provide a specific and sensitive result in any of the aforementioned immunoassays. Examples include, but are not limited to:

[0089] Abeam: Antibody Name: Anti-CXCL12 / SDF-1 antibody [EPR20472] (abl8919) Vendor: Abeam (www(dot)abcam(dot)com); Cell Signaling Technology (CST): Antibody Name: CXCL12 / SDF-1 (D8H4W) XP® Rabbit mAb (IHC Specific) (Cat# 3528) Vendor: Cell Signaling Technology (https: / / www(dot)cellsignal(dot)com); R&D Systems: Antibody Name: Human CXCL12 / SDF-1 alpha Antibody AF-310-NA Vendor: R&D Systems (https: / / www(dot)rndsystems(dot)com); Novus Biologicals: Antibody Name: CXCL12 / SDF-1 alpha Antibody (MAB310) Vendor: Novus Biologicals (https: / / www(dot)novusbio(dot)com); Proteintech provides polyclonal antibodies (i.e., 7402-1-AP) for IHC.

[0090] As used herein “CXCR4” also known as fusin or CD 184 is a protein that in humans is encoded by the CXCR4 gene.

[0091] According to a specific embodiment, the level of CXCR4 in the tissue biopsy is not measured.

[0092] Antibodies for CXCR4 are well known in the art. Preferably used are those which provide a specific and sensitive result in any of the aforementioned immunoassays. Examples include, but are not limited to:

[0093] Abeam: Antibody Name: Anti-CXCR4 antibody [EPR16824] (abl24824) Vendor: Abeam (www(dot)abcam(dot)com). Cell Signaling Technology (CST):

[0094] Antibody Name: CXCR4 (D1H8) Rabbit mAb (IHC Specific) (Cat# 97653), Vendor: Cell Signaling Technology (www(dot)cellsignal(dot)com); R&D Systems: Antibody Name: Human CXCR4 Antibody MAB 170, Vendor: R&D Systems (www(dot)rndsystems(dot)com); Novus Biologicals: Antibody Name: CXCR4 Antibody (12G5) (NBP2-42258), Vendor: Novus Biologicals (www(dot)novusbio(dot)com); Santa Cruz Biotechnology: Antibody Name: CXCR4 (12G5) Mouse mAb (sc-53534) Vendor: Santa Cruz Biotechnology (www(dot)scbt(dot)com).

[0095] According to a specific embodiment, other markers can be detected, such as tumor associated antigens, immune cells antigens such as CD8, CD3, FoxP3 and or any other antigen. As mentioned, when the level of CXCL12 is above a predetermined threshold the method contemplated selecting a treatment regimen or treating with a regimen a CXCR4 inhibitor; or when said level is below a predetermined threshold selecting a treatment regimen or treating with a treatment which does not comprise a CXCR4 inhibitor.

[0096] As used herein, the term "CXCR4 inhibitor" refers to molecules and compositions that interfere with or inhibit the biological activity of the CXCR4 receptor. Biological activity of the CXCR4 receptor can include, entry of the virus to the cell or replication of the virus in the cell (without being bound by theory). A CXCR4 inhibitor may also be interchanged herein with a “CXCR4 antagonist”.

[0097] The CXCR4 inhibitors can encompass numerous classes of chemical molecules, e.g., small organic or inorganic molecules, polysaccharides, biological macromolecules, e.g., peptides, proteins, peptide analogs and derivatives, peptidomimetics, antibodies, antibody fragments, nucleic acids, nucleic acid analogs and derivatives such as aptamers, an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues, naturally occurring or synthetic compositions.

[0098] Without wishing to be bound by a theory, a CXCR4 inhibitor can act by a number of different pathways. For example, a CXCR4 inhibitor can bind to a ligand bind site on the CXCR4 receptor and interfere with binding of the ligand to the CXCR4 receptor, bind to a nonligand binding site on the CXCR4 receptor and interfere with binding of the ligand to the CXCR4 receptor, bind with a CXCR4 receptor ligand and interfere with binding of the ligand to the CXCR4 receptor, or inhibit the expression of a polynucleotide (e.g., mRNA) expressing CXCR4

[0099] In some embodiments, a CXCR4 inhibitor inhibits the biological activity of the CXCR4 receptor by at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% relative to a control. In some embodiments, a CXCR4 inhibitor completely abrogates the biological activity of the CXCR4 receptor relative to a control. A control can comprise a sample that is not treated an inhibitor.

[0100] In some embodiments, a CXCR4 inhibitor is a nucleic acid. Exemplary CXCR4 nucleic acid inhibitors include, but are not limited to, antisense oligonucleotides, siRNAs, shRNAs, microRNAs, aptamers, ribozymes and decoy oligonucleotides. A CXCR4 nucleic acid inhibitor can inhibit the expression of a CXCR4 gene.

[0101] Exemplary anti CXCR4 siRNAs are described, for example, in U.S. Pat. App. Pub. No. 2007 / 0238868, No. 2009 / 0253772, content of both of which is incorporated herein by reference. Some exemplary CXCR4 antisense oligonucleotides are described, for example, in U.S. Pat. App. Pub. No. 2004 / 0209837, content of which is incorporated herein by reference.

[0102] In some embodiments, the CXCR4 inhibitor binds to CXCR4 or to CXCL12 (SDF- 1 alpha). In another embodiment, the CXCR4 inhibitor is an antibody or antibody fragment. In some embodiments, the CXCR4 inhibitor is a small molecule, for example, AMD-3100, ALX40-4C, T22, T140, Met-SDFlbeta, T134, or AMD-3465.

[0103] Exemplary CXCR4 inhibitors include, but are not limited to, 2,2'-bicyclam; 6,6'-bicyclam; the embodiments set forth in U.S. Pat. Nos. 5,021,409, and 6,001,826, and in particular 1,1'-[1,4- phenylene-bis(methylene)]-bis-l,4,8,l ltetraazacyclotetradecane, set forth in U.S. Pat. No. 5,583,131, and designated herein AMD3100. In some embodiments, a CXCR4 inhibitor can be N'- (1 Hbenzimidazol-2-yl methyl)-N'-(5,6,7,8-tetrahydroquinoline8-yl)-butane-l,4-diamine as described in U.S. Patent Publication No. 2003 / 0220341, CTCF-0214; CTCF-9908; CP-1221 (linear peptides, cyclic peptides, natural amino-acids, unnatural amino acids, and peptidomimetic compounds); 4F-benzoylTN24003; KRH-1120; KRH-1636; KRH-2731; polyphemusin analogue; ALX40-4C; or those described in WO 01 / 85196; WO 99 / 50461; WO 01 / 94420; WO 03 / 090512, each of which is incorporated by reference herein in its entirety.

[0104] In some embodiments, CXCR4 inhibitors include the T-140 analogs and antibodies described in US Patent Publication 2010 / 0055088, the cycle polyamines described in US Patent Publication 2009 / 0221683, and the compounds disclosed in US Patent Publication Nos. 2004 / 0209921, 2005 / 0059702, 2005 / 0043367, 2005 / 0277670, 2010 / 0178271, and 2003 / 0220341; U.S. Pat. Nos. 5,021,409, 6,001,826, 5,583,131, and Patent Publication WO 03 / 011277, each of which are incorporated herein by reference in their entirety.

[0105] CXCR4 inhibitors can also include, but are not limited to, polypeptides that specifically bind to CXCR4. Such inhibitors include T140 and derivatives of T140. Exemplary derivatives of T140 include, but are not limited to, TN14003, TC14012, and TE14011 as well as those found in Tamamura, H. et al. Org. Biomol. Chem. 1:3656-3662, 2003, which is incorporated by reference herein in its entirety.

[0106] According to specific embodiments, the CXCR4-antagonistic peptides of the present invention are for example, 4F-benzoyl-TN14003 (SEQ ID NO: 1) analogs and derivatives and are structurally and functionally related to the peptides disclosed in patent applications WO 2002 / 020561 and WO 2004 / 020462, also known as “T-140 analogs”, as detailed hereinbelow.

[0107] In various particular embodiments, the T-140 analog or derivative has an amino acid sequence as set forth in the following formula (I) or a salt thereof:

[0108] 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:

[0109] 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;

[0110] 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;

[0111] A3 represents an aromatic amino acid residue;

[0112] A4, As and A9 each independently represents an arginine, lysine, ornithine, citrulline, alanine or glutamic acid residue;

[0113] Ae represents a proline, glycine, ornithine, lysine, alanine, citrulline, arginine or glutamic acid residue;

[0114] A7 represents a proline, glycine, ornithine, lysine, alanine, citrulline or arginine residue;

[0115] As represents a tyrosine, phenylalanine, alanine, naphthylalanine, citrulline or glutamic acid residue;

[0116] A10 represents a citrulline, glutamic acid, arginine or lysine residue;

[0117] An 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.

[0118] Exemplary peptides 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.

[0119] Table 1 - T-140 and currently preferred T-140 analogs

[0120] According to a specific embodiment, in each one of SEQ ID NOS: 1-72, two cysteine residues are coupled in a disulfide bond.

[0121] In another embodiment, the analog or derivative has an amino acid sequence as set forth in SEQ ID NO:65 (H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH; TC14003).

[0122] In another embodiment, the peptide used in the compositions and methods of the invention 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.

[0123] In another embodiment, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOS: 1-4, 10, 46, 47, 51-56, 65, 66, 68, 70 and 71. In another embodiment, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOS: 4, 10, 46, 47, 68 and 70. In another embodiment, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOS: 1, 2, 51, 65 and 66. In another embodiment, the peptide has an amino acid sequence as set forth in any one of SEQ ID NOS:53-56.

[0124] In an embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO: 1. In another embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO:2. In another embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO:51. In another embodiment, the peptide has an amino acid sequence as set forth in SEQ ID NO:66.

[0125] According to a specific embodiment, the CXCR4 inhibitor is Motixafortide which can be under the brand name Aphexda. Another name of Motixafortide is BL804.

[0126] Other CXCR4 peptide inhibitors (antagonists) include but are not limited to LY2510924 (by Lilly Oncology), CTCE-9908 (Huang et al. 2009 Journal of Surgical Research 155:231-236), Fcl31 analogs and nanobodies as specified in the citations below (each of which is incorporated herein by reference in its entirety):

[0127] Tan NC, Yu P, Kwon Y-U, Kodadek T. High-throughput evaluation of relative cell permeability between peptoids and peptides. Bioorg Med Chem. 2008;16:5853-61.

[0128] Kwon Y-U, Kodadek T. Quantitative evaluation of the relative cell permeability of peptoids and peptides. J Am Chem Soc. 2007; 129: 1508.

[0129] Miller S, Simon R, Ng S, Zuckermann R, Kerr J, Moos W. Comparison of the proteolytic susceptibilities of homologous L-amino acid, D-amino acid, and N-substituted glycine peptide and peptoid oligomers. Drug Dev Res. 1995;35:20-32.

[0130] Yoshikawa Y, Kobayashi K, Oishi S, Fujii N, Furuya T. Molecular modeling study of cyclic pentapeptide CXCR4 antagonists: new insight into CXCR4-FC131 interactions. Bioorg Med Chem Lett. 2012;22:2146-50.

[0131] Jaahnichen S, Blanchetot C, Maussang D, Gonzalez-Pajuelo M, Chow KY, Bosch L, De Vrieze S, Serruys B, Ulrichts H, Vandevelde W. CXCR4 nanobodies (VHH -based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells. Proc Natl Acad Sci USA. 2010;107:20565-70.

[0132] According to a specific embodiment, the CXCR4 inhibitor is SEQ ID NO: 1, which is administered subcutaneously. According to a specific embodiment, the peptide set forth in SEQ ID NO: 1 is administered at a dose of 0.5-5 mg / kg.

[0133] According to a specific embodiment, the peptide set forth in SEQ ID NO: 1 is administered at a dose of 0.5-2.5 mg / kg.

[0134] According to a specific embodiment, the peptide set forth in SEQ ID NO: 1 is administered at a dose of 0.75-1.5 mg / kg.

[0135] According to a specific embodiment, the peptide set forth in SEQ ID NO: 1 is administered intravenously.

[0136] According to a specific embodiment, the CXCR4 inhibitor is a small molecule.

[0137] According to a specific embodiment, the treatment regimen comprises at least one of a chemotherapy, a targeted therapy and an immune modulator.

[0138] According to a specific embodiment, the immune modulator comprises a checkpoint modulator.

[0139] According to a specific embodiment, the checkpoint modulator is anti PD-1 or anti PD-L1.

[0140] Specific examples of other anti-cancer agents include, but are not limited to:

[0141] (i) a vaccine (e.g., IMCgplOO, Prophage G-100 & G-200, GV-1001, IMA-950, CV- 9201, CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAdl, GL-ONC1, ONCOS- 102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as GI-4000);

[0142] (ii) anti-cancer reactive mononuclear blood cells (MNBCs);

[0143] (iii) a cytokine capable of inducing activation and / or proliferation of a T cell;

[0144] (iv) an immune-check point modulator e.g., a 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;

[0145] More non-limiting examples are provided WO W02017 / 009842 and W02017 / 009843, each of which is incorporated by reference in its entirety.

[0146] According to a specific embodiment, the protocol for metastatic pancreatic cancer is provided in WO2020 / 148745 [i.e., SEQ ID NO: 1 and a chemotherapy comprising irinotecan, fluorouracil (5-FU) and leucovorin (LV)] and in W02020 / 079692 (i.e., SEQ ID NO: 1, an anti PD- 1 and a chemotherapy) each of which is incorporated herein by reference.

[0147] According to a specific embodiment, the anti PD1 is cemiplimab, zimberelimab or pembrolizumab. According to the embodiment, where the treatment does not comprise a CXCR4 inhibitor, any other treatment against cancer is contemplated, e.g., chemotherapy, radiotherapy and biological thereapy).

[0148] The subject mat be monitored for treatment efficacy such as by using imaging and molecular tools.

[0149] As used herein the term “about” refers to ± 10 %.

[0150] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0151] The term “consisting of’ means “including and limited to”.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0158] 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.

[0159] 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. 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 selecting a treatment regimen for a subject diagnosed with a solid tumor, the method comprising, determining in an in situ sample of said solid tumor of said subject a level of CXCL12, wherein: when said level is above a predetermined threshold selecting a treatment regimen which comprises a CXCR4 inhibitor; or when said level is below a predetermined threshold selecting a treatment regimen which does not comprise a CXCR4 inhibitor.

2. A method of selecting a subject diagnosed with a solid tumor for treatment with a CXCR4 inhibitor, the method comprising, determining in an in situ sample of the solid tumor of the subject a level of CXCL12, wherein: when said level is above a predetermined threshold, selecting the subject for a treatment regimen which comprises a CXCR4 inhibitor; or when said level is below a predetermined threshold, selecting the subject for a treatment regimen which does not comprise a CXCR4 inhibitor.

3. A method of treating a subject diagnosed with a solid tumor, the method comprising, determining in an in situ sample of said solid tumor of said subject a level of CXCL12, wherein: when said level is above a predetermined threshold treating the subject with a treatment regimen which comprises a CXCR4 inhibitor; or when said level is below a predetermined threshold treating the subject with a treatment regimen which does not comprise a CXCR4 inhibitor.

4. A CXCR4 inhibitor for use in treating a subject diagnosed with a solid tumor displaying an in situ level of CXCL12 above a predetermined threshold.

5. The method or inhibitor for use of any one of claims 1-4, wherein said determining further comprises determining a level of CXCR4 in said in situ sample.

6. The method or inhibitor for use of claim 5, wherein said determining a level of CXCR4 in said in situ sample is on selected immune cells.

7. The method or inhibitor for use of any one of claims 1-6, wherein said determining comprises an immunoassay.

8. The method or inhibitor for use of claim 7, wherein said immunoassay comprises immunohistochemistry (IHC).

9. The method or inhibitor for use of any one of claims 1-7, wherein said in situ comprises cancer cells and / or tumor microenvironment.

10. The method or inhibitor for use of claim 9, wherein said tumor microenvironment comprises fibroblasts.

11. The method or inhibitor for use of any one of claims 1-10, wherein said solid tumor is pancreatic cancer.

12. The method or inhibitor for use of claim 11, wherein said pancreatic cancer comprises metastatic pancreatic adenocarcinoma.

13. The method or inhibitor for use of any one of claims 1-11, wherein said CXCR4 inhibitor is a peptide, a small molecule, an antibody, a nucleic acid or a combination of same.

14. The method or inhibitor for use of any one of claims 1-13, wherein said CXCR4 inhibitor is a peptide.

15. The method or inhibitor for use of claim 14, wherein said peptide is as set forth in SEQ ID NO: 1 or an analog of same.

16. The method or inhibitor for use of claim 15, wherein said peptide of SEQ ID NO: 1 is motixafortide.

17. The method or inhibitor for use of claim 15 or 16, wherein said peptide set forth in SEQ ID NO: 1 is administered subcutaneously.

18. The method or inhibitor for use of any one of claims 15-17, wherein said peptide set forth in SEQ ID NO: 1 is administered at a dose of 0.5-5 mg / kg.

19. The method or inhibitor for use of any one of claims 15-18, wherein said peptide set forth in SEQ ID NO: 1 is administered at a dose of 0.5-2.5 mg / kg.

20. The method or inhibitor for use of any one of claims 15-19, wherein said peptide set forth in SEQ ID NO: 1 is administered at a dose of 0.75-1.5 mg / kg.

21. The method or inhibitor for use of claim 15 or 16, wherein said peptide set forth in SEQ ID NO: 1 is administered intravenously.

22. The method or inhibitor for use of any one of claims 1-13, wherein said CXCR4 inhibitor is a small molecule.

23. The method or CXCR4 inhibitor for use of any one of claims 1-13, wherein said CXCR4 inhibitor is AMD3100.

24. The method or CXCR4 inhibitor for use of any one of claims 1-23, wherein said subject is naive (prior to first line treatment).

25. The method or CXCR4 inhibitor for use of any one of claims 1-23, wherein said subject is post first line treatment.

26. The method or CXCR4 inhibitor for use of any one of claims 1-25, wherein said treatment regimen comprises at least one of a chemotherapy, a targeted therapy and an immune modulator.

27. The method or CXCR4 inhibitor for use of claim 26, wherein said immune modulator comprises a checkpoint modulator.

28. The method or CXCR4 inhibitor for use of claim 27, wherein said checkpoint modulator is anti PD-1 or anti PD-L1.

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