Synthetic lethal gene repair for cancer therapy

Abstract

Provided herein are methods and compositions for inhibiting the growth of a cancer cell or treating a cancer, wherein the cancer cell or cancer expresses a mutant protein tyrosine phosphatase receptor type F (PTPRF) or a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein.

Description

SYNTHETIC LETHAL GENE REPAIR FOR CANCER THERAPYCROSS-REFERENCE TO RELATED APPLICATION[00011 This application claims the benefit under 35 U.S.C § 119(e) of U.S. provisional application No. 63 / 514740, filed on July 20, 2023, the entire contents of which are incorporated herein by reference.BACKGROUND

[0002] Synthetic lethality describes a genetic interaction in which simultaneous mutations in two genes lead to synergistic cell death compared to individual mutations in the same genes. The concept of synthetic lethality is useful for discovering anti-cancer drug leads with high therapeutic indices: one can search for small molecules that are only lethal in the presence of a specific oncogenic mutation.SUMMARY OF THE DISCLOSURE

[0003] In one aspect, provided herein is a method of inhibiting the growth of a cancer cell expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) or a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, the method comprising, or alternatively consisting essentially of, or yet further consisting of contacting the cancer cell with a PTPRD inhibitor and / or a PTPRF inhibitor.J0004] In one aspect, provided herein is a method of inhibiting the growth of a cancer cell expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) protein, the method comprising, or alternatively consisting essentially of, or yet further consisting of contacting the cancer cell with a PTPRD inhibitor.

[0005] In one aspect, provided herein is a method of inhibiting the growth of a cancer cell expressing a mutant PTPRF protein, the method comprising, or alternatively consisting essentially of, or yet further consisting of contacting the cancer cell with a PTPRD inhibitor.

[0006] In some embodiments of the above methods, the PTPRD or the PTPRF inhibitor comprises, or alternatively consists essentially of, or yet further consists of illudalic-acid-7-BIA or an analog of illudalic-acid-7-BIA, that is optionally combined with a pharmaceutically acceptable salt of each thereof.(0007] In some embodiments, the method further comprises, or alternatively consists essentially of, or yet further consists of contacting the cancer cell with a second anti-cancer agent that is different from the PTPRD inhibitor or the PTPRF inhibitor.

[0008] In some embodiments, the second anti-cancer agent is a chemotherapeutic agent or an immunotherapy.

[0009] In some embodiments, the second agent is contacted prior to, concurrently or subsequent to the PTPRD or the PTPRF inhibitor.

[0010] In some embodiments, the method further comprises, or alternatively consists essentially of, or yet further consists of determining the cancer cell growth inhibition subsequent to the contacting of the cancer cell with the PTPRD or the PTPRF inhibitor.

[0011] In some embodiments, the cancer cell is a primary cancer cell, a metastatic cancer cell, or an immortalized cancer cell, that can be isolated from a patient or alternatively it is a cultured cell line.

[0012] In some embodiments, the cancer cell is a mammalian cancer cell.

[0013] In some embodiments, the mammalian cancer cell is selected from the group of a canine cancer cell, a feline cancer cell or a human cancer cell.

[0014] In some embodiments, the cancer cell is a liver cancer cell, optionally a hepatocellular cancer cell or a liver cholangiocarcinoma cell line, that is further optionally a mammalian liver cancer cell, yet further optionally a mammalian canine cancer cell, a feline cancer cell or a human cancer cell.

[0015] In some embodiments, the cancer cell has a PTPRF and / or PTPRD loss of function mutant or has a copy loss of PTPRF and / or PTPRD.

[0016] In each of the above embodiments, the contacting is in vitro or in vivo.

[0017] In another aspect, provided is a method for one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of cancer in a subject suffering from a cancer, the cells of the cancer expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) or a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, the method comprising, or alternatively consisting essentially of, or yet further consisting of administering to subject an effective amount of an PTPRF inhibitor and / or PTPRD inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subject.

[0018] In another aspect, provided is a method for one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of a subject suffering from a cancer wherein the cells of the cancer express a mutant PTPRF, the method comprising, or alternatively consisting essentially of, or yet further consisting of administering to subject an effective amount of an PTPRD inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subject.

[0019] In another aspect, provided is a method for one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of cancer in a subject suffering from a cancer wherein the cells of the cancer express mutant PTPRD, the method comprising, or alternatively consisting essentially of, or yet further consisting of administering to subject an effective amount of an PTPRF inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subject.

[0020] In some embodiments, the PTPRD or the PTPRF inhibitor comprises, or alternatively consists essentially of, or yet further consists of illudalic-acid-7-BIA or an analog of illudalic- acid-7-BIA, that is optionally combined with a pharmaceutically acceptable salt of each thereof.

[0021] In some embodiments, the method further comprises, or alternatively consists essentially of, or yet further consists of administering to the subject a second anti-cancer agent.

[0022] In some embodiments, the second anti-cancer agent is a chemotherapeutic agent or an immunotherapy.

[0023] In some embodiments, the second agent is administered prior to, concurrently or subsequent to the PTPRD inhibitor and / or PTPRF inhibitor.

[0024] In some embodiments, the cancer is a primary cancer or a metastatic cancer, and the treatment is a first-line, second-line, third-line, fourth line, or fifth line therapy.

[0025] In some embodiments, the method further comprises, or alternatively consists essentially of, or yet further consists of determining the cancer cell growth inhibition subsequent to the administering the PTPRD or PTPRF inhibitor, optionally illudalic-acid-7- BIA or an analog of illudalic-acid-7-BIA, and / or the second anti-cancer agent.

[0026] In some embodiments, the subject is a mammal.

[0027] In some embodiments, the mammal is selected from the group of a canine, a feline or a human.

[0028] In some embodiments, the cancer is a liver cancer, optionally a hepatocellular cancer or a liver cholangio-carcinoma, that is a primary cancer or a metastatic cancer.

[0029] In some embodiments, the cancer expresses a loss of function of PTPRF mutant or PTPRD mutant or a copy loss PTPRF genotype or PTPRD genotype.

[0030] In some embodiments, the administration is selected from a first-line, second-line, third line, fourth-line or fifth-line therapy.

[0031] In some embodiments, the method further comprises, or alternatively consists essentially of, or yet further consists of determining whether the cancer expresses a mutant PTPRF or PTPRD protein, optionally a loss of function of PTPRF mutant or PTPRD mutant or a copy loss of PTPRF genotype or PTPRD genotype.

[0032] In some embodiments after administration, the subject experiences one or more endpoints selected from tumor response, reduction in tumor size, reduction in tumor burden,increase in overall survival, increase in progression free survival, inhibiting metastasis, improvement of quality of life, minimization of toxicity, or reduction of side-effects as compared to a subject not receiving the therapy.

[0033] In some embodiments of the above methods, the second agent is selected from the group of: 5-fluorouracil, pemetrexed, raltitrexed, nolatrexed, plevitrexed, GS7904L, capecitabine, methotrexate, pralatrexate, CT-900, NUC-3373, FOLFOX, FOLFOX4, FOLFIRI, MOF, deflexifol, or a combination of 5-FU with one or more selected from radiation, methyl-CCNU, leucovorin, oxaliplatin (such as cisplatin), irinotecan, mitomycin, cytarabine, and levamisole, anthracycline or other topoisomerase II inhibitor comprises daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, etoposide or teniposide.

[0034] In some embodiments, the second agent comprises, or alternatively consists essentially of, or yet further consists of one or more selected agents from monoclonal antibodies, optionally selected from monospecific antibodies, bispecific antibodies, multispecific antibodies and a bispecific immune cell engager, antibody-drug conjugates, CAR therapies optionally selected from a CAR NK therapy, a CAR T therapy, a CAR cytotoxic T therapy, a CAR gamma-delta T therapy, a CAR NK therapy, cell therapies, inhibitors or antagonists of an inhibitory immune checkpoint, activators or agonists of a stimulatory immune checkpoint optionally selected from an activating ligand, immune regulators, cancer vaccines, and a vector delivering each thereof to a subject optionally in an oncolytic virus therapy, and optionally wherein the monoclonal antibodies are selected from rituximab, blinatumomab, alemtuzumab, ibritumomab tiuxetan, bevacizumab, bevacizumab- awwb, cetuximab, panitumumab, ofatumumab, denosumab, pertuzumab, obinutuzumab, elotuzumab, ramucirumab, dinutuximab, daratumumab, trastuzumab, trastuzumab-dkst, nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, AMF 514 (MEDI0680), balstilimab, avelumab, durvalumab, atezolizumab, ipilimumab, tremelimumab, zalifrelimab, and AGEN1181, and further optionally wherein the antibody-drug conjugates are selected from moxetumomab pasudotox- tdfk, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, gemtuzumab ozogamicin, tagraxofusp-erzs, polatuzumab vedotin-piiq, enfortumab vedotin-ejfv,trastuzumab deruxtecan, and sacituzumab govitecan-hziy, and further optionally, a checkpoint inhibitor, optionally selected from GS4224, AMP -224, CA-327, CA-170, BMS- 1001, BMS-1166, peptide-57, M7824, MGD013, CX-072, UNP-12, NP-12, or a combination of two or more thereof, or an anti-PD-1 agent, an anti-PD-Ll agent, an anti-CTLA-4 agent, an anti-LAG-3 agent, an anti-TIM-3 agent, an anti-TIGIT agent, an anti-VISTA agent, an anti-B7-H3 agent, an anti-BTLA agent, an anti-ICOS agent, an anti-GITR agent, an anti-4- 1BB agent, an anti-OX40 agent, an anti-CD27 agent, an anti-CD28 agent, an anti-CD40 agent, and an anti-Siglec-15 agent.

[0035] In another aspect, provided herein is a method of inhibiting the growth of a liver cancer cell expressing a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, comprising contacting the cancer cell with illudalic-acid-7-BIA or an analog of illudalic-acid-7-BIA or a pharmaceutically acceptable salt of each thereof, optionally wherein the liver cancer is a hepatocellular carcinoma.

[0036] In another aspect, provided herein is a method of inhibiting the growth of a cancer cell in vivo or in vitro, comprising inhibiting expression of PTPRD and PTPRF in the cancer cell, wherein the genes encoding PTRPD or PTPRF are knocked down or inhibited with a chemical inhibitor.BRIEF DESCRIPTION OF THE FIGURES

[0037] FIGs. 1A-1B show that targeting PTPRD and PRPRF at the same time leads to increased apoptosis as compared to either gene targeted alone. (FIG. 1A) Double knockdown of PTPRD and PTPRF using CRISPRi leads to increased apoptosis at 48-, 72- and 96-hour time points (Gene A is PTPRD and Gene B is PTPRF). (FIG. IB) Knockdown of Gene A (PTPRD) has no effect on the viability of the cell line. Knockdown of Gene B (PTPRF) shows about 70% higher apoptosis than the scramble control. When both genes are knocked down at the same time, a 350% increase in apoptosis is observed, which is much higher than PTPRD+PTPRF (i.e., 0%+70% = 70%), demonstrating synthetic lethality. KD means gene knockdown and DKD means double gene knockdown.

[0038] FIG. 2 shows growth response curves of three separate liver hepatocarcinoma cell line clones where PTPRF has been knocked down and which were treated with a PTPRD inhibitor, Illudalic Acid-7-BIA. Top panels show the general growth curve, while the bottom panels zoom on the response curve. In the two clones with knockdown of PTPRF over 70% (the right two panels), there is a clear drug response curve. Compared to the other two clones with knockdown, a weaker response is observed in the clone with less knockdown (left panel). In the graphs, Compound 1 is Illudalic Acid-7-BIA.DETAILED DESCRIPTION

[0039] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation, the full bibliographic citations for some of which are found immediately preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this disclosure pertains.

[0040] As used herein, certain terms may have the following defined meanings. As used in the specification and claims, the singular form “a,” “an” and “the” include singular and plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a single cell as well as a plurality of cells, including mixtures thereof.

[0041] As used herein, the term “comprising” is intended to mean that the methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define methods, shall mean excluding other elements of any essential significance to the method. In the context of a composition or combination, “consisting essentially of’ a listing of active ingredients or agents that the recited elements are the only active agents in the composition or combination. “Consisting of’ shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods can include additional steps and components (comprising) or alternatively including steps of no significance (consisting essentially of) or alternatively, intending only the stated method steps (consisting of).

[0042] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term “about”. The term “about” also includes the exact value “X” in addition to minor increments of “X” such as “X + 0.1” or “X - 0.1.” It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0043] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an acronym for DNA loci that contain multiple, short, direct repetitions of base sequences. The prokaryotic CRISPR / Cas system has been adapted for use as gene editing (silencing, enhancing or changing specific genes) for use in eukaryotes (see, for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096): 816-21 (2012)). By transfecting a cell with elements including a Cas gene and specifically designed CRISPRs, nucleic acid sequences can be cut and modified at any desired location. Methods of preparing compositions for use in genome editing using the CRISPR / Cas systems are described in detail in US Pub. No. 2016 / 0340661, US Pub. No. 20160340662, US Pub. No. 2016 / 0354487, US Pub. No. 2016 / 0355796, US Pub. No. 20160355797, and WO 2014 / 018423, which are specifically incorporated by reference herein in their entireties.

[0044] CRISPR systems can also be used to downregulate or abrogate protein expression by inhibiting transcription (also designated CRISPRi) using dCas9 (a mutant version of the Cas9 enzyme that lacks endonuclease activity) fused to a repressor domain. As used herein, these systems are referred to as “CRISPRi” systems.

[0045] Receptor-type protein tyrosine phosphatase D (PTPRD) as reported by the NCBI database is a protein encoded by a gene that is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. This PTP contains an extracellular region, a single transmembrane segment and two tandem intracytoplasmic catalytic domains, and thus represents a receptor-type PTP. The extracellular region of this protein is composed of three Ig-like and eight fibronectin type III- like domains. Studies of the similar genes in chicken and fly suggest the role of this PTP is inpromoting neurite growth, and regulating neurons axon guidance. Multiple alternatively spliced transcript variants of this gene have been reported. A related pseudogene has been identified on chromosome 5. See, e.g., ncbi.nlm.nih.gov / gene / 5789, last accessed on July 16, 2021. The sequences of the gene and protein are known, and are reported in GeneCards, at genecards. org / cgi-bin / carddisp.pl?gene=PTPRD, last accessed on July 16, 2021. Paralogs for the gene are reported at genecards. org / cgi-bin / carddisp.pl?gene=PTPRD#paralogs, last accessed on July 16, 2021. PTPRD has a number of aliases including protein tyrosine phosphatase receptor type D.10046] Receptor-type protein tyrosine phosphatase F (PTPRF) as reported by the NCBI database is a protein encoded by a gene that is a member is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known to be signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. PTPRF has a number of aliases including protein tyrosine phosphatase receptor type F, leukocyte common antigen-related (LAR). See genecards.org / cgi- bin / carddisp.pl?gene=PTPRF, last accessed July 8, 2024. This PTP possesses an extracellular region, a single transmembrane region, and two tandem intracytoplasmic catalytic domains, and thus represents a receptor-type PTP. The extracellular region contains three Ig-like domains, and nine non-Ig like domains similar to that of neural-cell adhesion molecule. This PTP was shown to function in the regulation of epithelial cell-cell contacts at adherents junctions, as well as in the control of beta-catenin signaling. An increased expression level of this protein was found in the insulin-responsive tissue of obese, insulinresistant individuals, and may contribute to the pathogenesis of insulin resistance. Two alternatively spliced transcript variants of this gene, which encode distinct proteins, have been reported. See ncbi.nlm.nih.gov / gene?Db=gene&Cmd=DetailsSearch&Term=5792, last accessed on July 16, 2021. The sequences of the gene and protein are known, and are reported in GeneCards, at genecards. org / cgi-bin / carddisp.pl?gene=PTPRF, last accessed on July 16, 2021. Paralogs for the gene are reported at genecards.org / cgi- bin / carddisp.pl?gene=PTPRF#paralogs, last accessed on July 16, 2021.

[0047] A PTPRF inhibitor refers to a molecule or biological that inhibits the wild-type function of PTPRF. Non-limiting examples are PTPRF -targeted CRISPR or CRISPRisystems, PTPRF -targeted siRNA and a small molecule inhibitor of PTPRF enzymatic function, or a pharmaceutically acceptable salt of each thereof. Targeted CRISPR inhibition with a CRISPR or CRISPRi system can be gene knockdown or gene knockout.

[0048] A PTPRD inhibitor refers to a molecule or biological that inhibits the wild-type function of PTPRD. Non-limiting examples are PTPRD-targeted CRISPR or CRISPRi systems, PTPRD-targeted siRNA and 7-BIA, illudalic-acid-7-BIA, an analog of 7-BIA or a pharmaceutically acceptable salt of each thereof. Targeted CRISPR inhibition with a CRISPR or CRISPRi system can be gene knockdown or gene knockout.

[0049] Loss of function mutant intends a mutation in a gene that results in an altered gene product that lacks the molecular function of the wild-type gene. Loss of function can be determined using ALoFT (see nature. com / articles / s41467-017-00443-5 and nature. com / articles / s41586-020-2308-7, last accessed on July 16, 2021).

[0050] Copy loss mutation intends a mutation in the coding gene or polynucleotide that results in decreased gene or sequence fragment copies in the genome. Copy loss mutation can be determined using qPCR.

[0051] As used herein, 7-BIA (7-butoxy illudalic acid) is a receptor-type protein tyrosine phosphatase D (PTPRD) inhibitor. 7-BIA is a small molecule (chemical) inhibitor. As used herein, 7-BIA and illudalic-acid-7-BIA are used interchangeably. It is commercially available, for example from MedChemExpress (medchemexpress.com, last accessed on July 16, 2021) and TargetMol, catalog number T101910 (targetmol.com, last accessed on July 16, 2021), the chemical structure is shown below:

[0052] The term “chemotherapy” encompasses cancer therapies that employ chemical or biological agents or other therapies, such as radiation therapies, e.g., a small molecule drug or a large molecule, such as antibodies, CAR therapies, RNAi and gene therapies. Non-limiting examples of chemotherapies are provided below. Unless specifically excluded, when a specific therapy is recited, equivalents of the therapy are within the scope of this disclosure.

[0053] Bevacizumab (BV) is sold under the trade name Avastin® by Genentech. It is a humanized monoclonal antibody that binds to and inhibits the biologic activity of human vascular endothelial growth factor (VEGF). Biological equivalent antibodies are identified herein as modified antibodies which bind to the same epitope of the antigen, prevent the interaction of VEGF to its receptors (FltOl, KDR a.k.a. VEGFR2) and produce a substantially equivalent response, e.g., the blocking of endothelial cell proliferation and angiogenesis. Irinotecan and bevacizumab is also in the class of cancer drugs that inhibit angiogenesis (angiogenesis inhibitors).

[0054] Irinotecan (CPT-11) is sold under the trade name of Camptosar®. It is a semisynthetic analogue of the alkaloid camptothecin, which is activated by hydrolysis to SN-38 and targets topoisomerase I. Chemical equivalents are those that inhibit the interaction of topoisomerase I and DNA to form a catalytically active topoisomerase I-DNA complex. Chemical equivalents inhibit cell cycle progression at G2-M phase resulting in the disruption of cell proliferation. An equivalent of irinotecan is a composition that inhibits a topoisomerase. Non-limiting examples of an equivalent of irinotecan include topotecan, camptothecin and lamellarin D, etoposide, or doxorubicin.

[0055] Oxaliplatin (trans- / -diaminocyclohexane oxalatoplatinum; L-OHP; CAS No. 61825- 94-3) is sold under the trade name of Elotaxin. It is a platinum derivative that causes cell cytotoxicity. Oxaliplatin forms both inter- and intra-strand cross links in DNA, which prevent DNA replication and transcription, causing cell death. Non-limiting examples of an equivalent of oxaliplatin include carboplatin and cisplatin.

[0056] Topoisomerase inhibitors are agents designed to interfere with the action of topoisomerase enzymes (topoisomerase I and II), which are enzymes that control the changes in DNA structure by catalyzing the breaking and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle. In one aspect, topoisomerase inhibitors includeirinotecan, topotecan, camptothecin and lamellarin D, or compounds targeting topoisomerase IA. In another aspect, topoisomerase inhibitors include etoposide, doxorubicin or compounds targeting topoisomerase II.

[0057] Pyrimidine antimetabolite includes, without limitation, fluorouracil (5-FU), its equivalents and prodrugs. In one embodiment, a pyrimidine antimetabolite is a chemical that inhibits the use of a pyrimidine. The presence of antimetabolites can have toxic effects on cells, such as halting cell growth and cell division, so these compounds can be used as chemotherapy for cancer.

[0058] Fluorouracil (5-FU) belongs to the family of therapy drugs called pyrimidine based anti-metabolites. It is a pyrimidine analog, which is transformed into different cytotoxic metabolites that are then incorporated into DNA and RNA thereby inducing cell cycle arrest and apoptosis. Chemical equivalents are pyrimidine analogs which result in disruption of DNA replication. Chemical equivalents inhibit cell cycle progression at S phase resulting in the disruption of cell cycle and consequently apoptosis. Equivalents to 5-FU include prodrugs, analogs and derivative thereof such as 5’-deoxy-5-fluorouridine (doxifluroidine), 1- tetrahydrofuranyl-5-fluorouracil (ftorafur), Capecitabine (Xeloda), S-l (MBMS-247616, consisting of tegafur and two modulators, a 5-chloro-2,4-dihydroxypyridine and potassium oxonate), ralititrexed (tomudex), nolatrexed (Thymitaq, AG337), LY231514 and ZD9331, as described for example in Papamicheal (1999) The Oncologist 4:478-487.

[0059] “5-FU based adjuvant therapy” refers to 5-FU alone or alternatively the combination of 5-FU with other treatments, that include, but are not limited to radiation, methyl-CCNU, leucovorin, oxaliplatin, irinotecin, mitomycin, cytarabine, levamisole. Specific treatment adjuvant regimens are known in the art as FOLFOX, FOLFOX4, FOLFIRI, MOF (semustine (methyl-CCNU), vincrisine (Oncovin) and 5-FU). For a review of these therapies see Beaven and Goldberg (2006) Oncology 20(5):461-470. An example of such is an effective amount of 5-FU and Leucovorin. Other chemotherapeutics can be added, e.g., oxaliplatin or irinotecan.

[0060] Capecitabine is a prodrug of (5-FU) that is converted to its active form by the tumorspecific enzyme PynPase following a pathway of three enzymatic steps and two intermediary metabolites, 5'-deoxy-5-fluorocytidine (5'-DFCR) and 5'-deoxy-5-fluorouridine (5'-DFUR). Capecitabine is marketed by Roche under the trade name Xeloda®.[00611 The present therapy can be used in combination with other traditional therapies, nonlimiting examples of such include radiation, methyl-CCNU, leucovorin, oxaliplatin, irinotecin, mitomycin, cytarabine, levamisole. Specific treatment adjuvant regimens are known in the art as FOLFOX, FOLFOX4, FOLFOX6, FOLFIRI, MOF (semustine (methyl- CCNU), vincrisine (Oncovin) and 5-FU). For a review of these therapies see Beaven and Goldberg (2006) Oncology 20(5):461-470. An example of such is an effective amount of 5- FU and Leucovorin. Other chemotherapeutics can be added, e.g., oxaliplatin or irinotecan.

[0062] FOLFIRI is a chemotherapy regimen for treatment of colorectal cancer. It is made up of the following drugs: FOL - folinic acid (leucovorin), a vitamin B derivative used as a "rescue" drug for high doses of the drug methotrexate and that modulates / potentiates / reduces the side effects of fluorouracil; F - fluorouracil (5-FU), a pyrimidine analog and antimetabolite which incorporates into the DNA molecule and stops synthesis; and IRI - irinotecan (Camptosar), a topoisomerase inhibitor, which prevents DNA from uncoiling and duplicating.[ 00631 FOLFOX is a chemotherapy regimen for treatment of colorectal cancer. It is made up of the following drugs: FOL - folinic acid (leucovorin), F - fluorouracil (5-FU), and OX- oxaliplatin.

[0064] FOLFOXFIRI is a chemotherapy regimen for treatment of colorectal cancer. It is made up of the following drugs: FOL - folinic acid (leucovorin), F - fluorouracil (5-FU), OX- oxaliplatin and IRI - irinotecan (Camptosar).

[0065] An “effective amount” is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents disclosed herein for any particular subject depends upon a variety of factors including the activity of the specific compound employed, bioavailability of the compound, the route of administration, the age of the animal and its body weight, general health, sex, the diet of the animal, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form ofadministration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level commensurate with the concentrations found to be effective in vivo. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.10066] “Therapeutically effective amount” of a drug or an agent refers to an amount of the drug or the agent that is an amount sufficient to obtain a pharmacological response or alternatively, is an amount of the drug or agent that, when administered to a patient with a specified disorder or disease, is sufficient to have the intended effect, e.g., treatment, alleviation, amelioration, palliation or elimination of one or more manifestations of the specified disorder or disease in the patient. A therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations.

[0067] As used herein, “treating” or “treatment” of a disease in a subject refers to (1) preventing the symptoms or disease from occurring in a subject that is predisposed or does not yet display symptoms of the disease; (2) inhibiting the disease or arresting its development; or (3) ameliorating or causing regression of the disease or the symptoms of the disease. As understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For the purposes of this technology, beneficial or desired results can include one or more, but are not limited to, alleviation or amelioration of one or more symptoms, diminishment of extent of a condition (including a disease), stabilized (i.e., not worsening) state of a condition (including disease), delay or slowing of condition (including disease), progression, amelioration or palliation of the condition (including disease), states and remission (whether partial or total), whether detectable or undetectable. In one aspect, treatment excludes prophylaxis.

[0068] When the disease is cancer, the following clinical endpoints are non-limiting examples of treatment: (1) elimination of a cancer in a subject or in a tissue / organ of the subject or in a cancer loci; (2) reduction in tumor burden (such as number of cancer cells, number of cancer foci, number of cancer cells in a foci, size of a solid cancer, concentrate of a liquid cancer in the body fluid, and / or amount of cancer in the body); (3) stabilizing or delay or slowing or inhibition of cancer growth and / or development, including but not limitedto, cancer cell growth and / or division, size growth of a solid tumor or a cancer loci, cancer progression, and / or metastasis (such as time to form a new metastasis, number of total metastases, size of a metastasis, as well as variety of the tissues / organs to house metastatic cells); (4) less risk of having a cancer growth and / or development; (5) inducing an immune response of the patient to the cancer, such as higher number of tumor-infiltrating immune cell, higher number of activated immune cells, or higher number cancer cell expressing an immunotherapy target, or higher level of expression of an immunotherapy target in a cancer cell; (6) higher probability of survival and / or increased duration of survival, such as increased overall survival (OS, which may be shown as 1-year, 2-year, 5-year, 10-year, or 20-year survival rate), increased progression free survival (PFS), increased disease free survival (DFS), increased time to tumor recurrence (TTR) and increased time to tumor progression (TTP). In some embodiments, the subject after treatment experiences one or more endpoints selected from tumor response, reduction in tumor size, reduction in tumor burden, increase in overall survival, increase in progression free survival, inhibiting metastasis, improvement of quality of life, minimization of drug-related toxicity, and avoidance of side-effects (e.g., decreased treatment emergent adverse events). In some embodiments, improvement of quality of life includes resolution or improvement of cancer-specific symptoms, such as but not limited to fatigue, pain, nausea / vomiting, lack of appetite, and constipation; improvement or maintenance of psychological well-being (e.g., degree of irritability, depression, memory loss, tension, and anxiety); improvement or maintenance of social well-being (e.g., decreased requirement for assistance with eating, dressing, or using the restroom; improvement or maintenance of ability to perform normal leisure activities, hobbies, or social activities; improvement or maintenance of relationships with family). In some embodiments, improved patient quality of life that is measured qualitatively through patient narratives or quantitatively using validated quality of life tools known to those skilled in the art, or a combination thereof. Additional non-limiting examples of endpoints include reduced hospital admissions, reduced drug use to treat side effects, longer periods off-treatment, and earlier return to work or caring responsibilities. In one aspect, prevention or prophylaxis is excluded from treatment.

[0069] As used herein, immune cells are cells of the immune system, including but not limited to lymphocytes (such as, T-cells, B-cells, natural killer (NK) cells, and natural killer T (NKT) cells), myeloid-derived cells (such as granulocytes (basophils, eosinophils, neutrophils, mast cells), monocytes, macrophages, and dendritic cells (DC)). T cells are divided into two broad categories: CD8+ T cells or CD4+ T cells, based on which protein is present on the cell's surface. CD8+ T cells also are called cytotoxic T cells or cytotoxic lymphocytes (CTLs). The four major CD4+ T-cell subsets are TH1, TH2, TH17, and Treg, with "TH" referring to "T helper cell." T cells may also refer to gamma delta T cell.Dendritic cells (DC) are an important antigen-presenting cell (APC), and they also can develop from monocytes. In some embodiments, the immune cells refer to a killer cell, including but not limited to: a cytotoxic T cell, a gamma delta T cell, a NK cell and a NK-T cell. In one embodiment, the immune cell is a CD45+ cell.

[0070] The term “subject,” “host,” “individual,” and “patient” are as used interchangeably herein to refer to animals, typically mammalian animals. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. In some embodiments, a subject has or is diagnosed of having or is suspected of having a cancer.

[0071] In certain embodiments, the terms “disease” “disorder” and “condition” are used interchangeably herein, referring to a cancer, a status of being diagnosed with a cancer, or a status of being suspect of having a cancer. “Cancer”, which is also referred to herein as “tumor”, is a known medically as an uncontrolled division of abnormal cells in a part of the body, benign or malignant. In one embodiment, cancer refers to a malignant neoplasm, a broad group of diseases involving unregulated cell division and growth, and invasion to nearby parts of the body. Non-limiting examples of cancers include carcinomas, sarcomas, leukemia and lymphoma, e.g., colon cancer, colorectal cancer, rectal cancer, gastric cancer,esophageal cancer, head and neck cancer, breast cancer, brain cancer, lung cancer, stomach cancer, liver cancer, gall bladder cancer, or pancreatic cancer. In one embodiment, the term “cancer” refers to a solid tumor, which is an abnormal mass of tissue that usually does not contain cysts or liquid areas, including but not limited to, sarcomas, carcinomas, and certain lymphomas (such as Non-Hodgkin's lymphoma). In another embodiment, the term “cancer” refers to a liquid cancer, which is a cancer presenting in body fluids (such as, the blood and bone marrow), for example, leukemias (cancers of the blood) and certain lymphomas.

[0072] Additionally or alternatively, a cancer may refer to a local cancer (which is an invasive malignant cancer confined entirely to the organ or tissue where the cancer began), a metastatic cancer (referring to a cancer that spreads from its site of origin to another part of the body), a non-metastatic cancer, a primary cancer (a term used describing an initial cancer a subject experiences), a secondary cancer (referring to a metastasis from primary cancer or second cancer unrelated to the original cancer), an advanced cancer, an unresectable cancer, or a recurrent cancer. As used herein, an advanced cancer refers to a cancer that had progressed after receiving one or more of: the first line therapy, the second line therapy, or the third line therapy.

[0073] The term “contacting” means direct or indirect binding or interaction between two or more. A particular example of direct interaction is binding. A particular example of an indirect interaction is where one entity acts upon an intermediary molecule, which in turn acts upon the second referenced entity. Contacting as used herein includes in solution, in solid phase, in vitro, ex vivo, in a cell and in vivo. Contacting in vivo can be referred to as administering, or administration.

[0074] As used herein, the term “administration” and “administering” are used to mean introducing an agent into a subject. Routes of administration include, but are not limited to, oral (such as a tablet, capsule or suspension), topical, transdermal, intranasal, vaginal, rectal, subcutaneous intravenous, intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, intraocular, subconjunctival, sub-Tenon’s, intravitreal, retrobulbar, intracam eral, intratumoral, epidural and intrathecal.

[0075] An “immunotherapy agent” means a type of cancer treatment which uses a patient’s own immune system to fight cancer, including but not limited to a physical intervene, achemical substance, a biological molecule or particle, a cell, a tissue or organ, or any combinations thereof, enhancing or activating or initiating a patient's immune response against cancer. Non-limiting examples of immunotherapy agents include antibodies, immune regulators, checkpoint inhibitors, an antisense oligonucleotide (ASO), a RNA interference (RNAi), a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system, a viral vector, an anti-cancer cell therapy (e.g., transplanting an anti-cancer immune cell optionally amplified and / or activated in vivo, or administering an immune cell expressing a chimeric antigen receptor (CAR)), a CAR therapy, and cancer vaccines. As used herein, unless otherwise specified, an immunotherapy agent is not an inhibitor of thymidylate biosynthesis, or an anthracycline or other topoisomerase II inhibitor. As used herein, immune checkpoint refers to a regulator and / or modulator of the immune system (such as an immune response, an anti-tumor immune response, a nascent anti-tumor immune response, an antitumor immune cell response, an anti-tumor T cell response, and / or an antigen recognition of T cell receptor in the process of immune response). Their interaction activates either inhibitory or activating immune signaling pathways. Thus a checkpoint may contain one of the two signals: a stimulatory immune checkpoint that stimulates an immune response, and an inhibitory immune checkpoint inhibiting an immune response. In some embodiments, the immune checkpoint is crucial for self-tolerance, which prevents the immune system from attacking cells indiscriminately. However, some cancers can protect themselves from attack by stimulating immune checkpoint targets. In some embodiments, the immune checkpoints are present on T cells, antigen-presenting cells (APCs) and / or tumor cells.

[0076] One target of an immunotherapy agent is a tumor-specific antigen while the immunotherapy directs or enhances the immune system to recognize and attack tumor cells. Non-limiting examples of such agent includes a cancer vaccine presenting a tumor-specific antigen to the patient’s immune system, a monoclonal antibody or an antibody-drug conjugate specifically binding to a tumor-specific antigen, a bispecific antibody specifically binding to a tumor-specific antigen and an immune cell (such as a T-cell engager or a NK- cell engager), an immune cell (such as a killer cell) specifically binding to a tumor-specific antigen (such as a CAR-T cell, a CAR-NK cell, and a CAR-NKT cell), a polynucleotide (or a vector comprising the same) transfecting / transducing an immune cell to express an tumor-specific antibody of an antigen binding fragment thereof (such as a CAR), or a polynucleotide (or a vector comprising the same) transfecting / transducing a cancer cell to express an antigen or a marker which can be recognized by an immune cell.

[0077] Another exemplified target is an inhibitory immune checkpoint which suppresses the nascent anti-tumor immune response, such as A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CTLA-4 / B7-1 / B7-2, IDO, KIR, LAG3, N0X2, PD-1, PD-L1 and TIM-3, VISTA, SIGLEC7 (Sialic acid-binding immunoglobulin-type lectin 7, also designated as CD328) and SIGLEC9 (Sialic acid-binding immunoglobulin-type lectin 9, also designated as CD329). Non-limiting examples of such agent includes an antagonist or inhibitor of an inhibitory immune checkpoint, an agent reducing the expression and / or activity of an inhibitory immune checkpoint (such as via an antisense oligonucleotide (ASO), a RNA interference (RNAi), or a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) system), an antibody or an antibody-drug conjugate or a ligand specifically binding to and reducing (or inhibiting) the activity of an inhibitory immune checkpoint, an immune cell with reduced (or inhibited) an inhibitory immune checkpoint (and optionally specifically binding to a tumor-specific antigen, such as a CAR-T cell, a CAR-NK cell, and a CAR-NKT cell), and a polynucleotide (or a vector comprising the same) transfecting / transducing an immune cell or a cancer cell to reduce or inhibit an inhibitory immune checkpoint thereof. Reducing expression or activity of such inhibitory immune checkpoint enhances immune response of a patient to a cancer.

[0078] A further possible immunotherapy target is a stimulatory checkpoint molecule (including but not limited to 4-1BB, CD27, CD28, CD40, CD122, CD137, 0X40, GITR and ICOS), wherein the immunotherapy agent actives or enhances the anti-tumor immune response. Non-limiting examples of such agent includes an agonist of a stimulatory checkpoint, an agent increasing the expression and / or activity of a stimulating immune checkpoint, an antibody or an antibody-drug conjugate or a ligand specifically binding to and activating or enhancing the activity of a stimulating immune checkpoint, an immune cell with increased expression and / or activity of a stimulating immune checkpoint (and optionally specifically binding to a tumor-specific antigen, such as a CAR-T cell, a CAR-NK cell, and a CAR-NKT cell), and a polynucleotide (or a vector comprising the same)transfecting / transducing an immune cell or a cancer cell to express a stimulating immune checkpoint thereof.

[0079] Additional or alternative targets may be utilized by an immunotherapy agent, such as an immune regulating agent, including but not limited to, an agent activating an immune cell, an agent recruiting an immune cell to a cancer or a cancer cell, or an agent increasing immune cell infiltrated into a solid tumor and / or a cancer loci. Non-limiting examples of such agent is an immune regulator or a variant, a mutant, a fragment, an equivalent thereof.

[0080] In some embodiments, an immunotherapy agent utilizes one or more targets, such as a bispecific T cell engager, a bispecific NK cell engager, or a CAR cell therapy. In some embodiments, the immunotherapy agent targets one or more immune regulatory or effector cells.

[0081] As used herein, the term “antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits, rat, canine, donkey, mice, camelids (such as dromedaries, llamas, and alpacas), as well as non-mammalian species, such as shark immunoglobulins. Unless specifically noted otherwise, the term “antibody” includes intact immunoglobulins and “antibody fragments” or “antigen binding fragments” that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 103M'1greater, at least 104M-1greater or at least 105M'1greater than a binding constant for other molecules in a biological sample). The term “antibody” also includes genetically engineered forms such as chimeric antibodies (for example, murine or humanized non-primate antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Owen et al., Kuby Immunology, 7thEd., W.H. Freeman & Co., 2013; Murphy, Janeway ’s Immunobiology, 8thEd., Garland Science, 2014; Male et al., Immunology (Roitt), 8thEd., Saunders, 2012; Parham, The Immune System, 4thEd., Garland Science, 2014. The term “antibody” includes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule, such as the whole antibody and any antigen binding fragment or a single chain thereof. The terms “antibody,” “antibodies” and “immunoglobulin” also include immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fab', F(ab)2, Fv, scFv, dsFv, Fd fragments, dAb, VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies and kappa bodies; multispecific antibody fragments formed from antibody fragments and one or more isolated. Examples of such include, but are not limited to a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, at least one portion of a binding protein, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues. The antibodies can be polyclonal, monoclonal, multispecific (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

[0082] As used herein, the term “monoclonal antibody” refers to an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0083] In some embodiments, the antibody is a bispecific immune cell engager, referring to a bispecific monoclonal antibody that is capable of recognizing and specifically binding to a tumor antigen (such as CD19, EpCAM, MCSP, HER2, EGFR or CS-1) and an immune cell, and directing an immune cell to cancer cells, thereby treating a cancer. Non-limiting examples of such antibody include bispecific T cell engager, bispecific cytotoxic T lymphocytes (CTL) engager, and bispecific NK cell engager. In one embodiment, the engager is a fusion protein consisting of two single-chain variable fragments (scFvs) ofdifferent antibodies. Additionally or alternatively, the immune cell is a killer cell, including but not limited to: a cytotoxic T cell, a gamma delta T cell, a NK cell and a NK-T cell.

[0084] As used herein, the term “antigen binding domain” refers to any protein or polypeptide domain that can specifically bind to an antigen target.10085] The term “chimeric antigen receptor” (CAR), as used herein, refers to a fused protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a polypeptide different from a polypeptide from which the extracellular domain is derived, and at least one intracellular domain. The “chimeric antigen receptor (CAR)” is sometimes called a “chimeric receptor”, a “T-body”, or a “chimeric immune receptor (CIR).” The “extracellular domain capable of binding to an antigen” means any oligopeptide or polypeptide that can bind to a certain antigen. The “intracellular domain” or “intracellular signaling domain” means any oligopeptide or polypeptide known to function as a domain that transmits a signal to cause activation or inhibition of a biological process in a cell. In certain embodiments, the intracellular domain may comprise, alternatively consist essentially of, or yet further comprise one or more costimulatory signaling domains in addition to the primary signaling domain. The “transmembrane domain” means any oligopeptide or polypeptide known to span the cell membrane and that can function to link the extracellular and signaling domains. A chimeric antigen receptor may optionally comprise a “hinge domain” which serves as a linker between the extracellular and transmembrane domains.

[0086] As used herein, a CAR therapy may refer to administrating an immune cell expressing a CAR into a subject as well as contacting a vector expressing a CAR in an immune cell (such as in vivo).

[0087] As used herein, the term “NK cell,” also known as natural killer cell, refers to a type of lymphocyte that originates in the bone marrow and play a critical role in the innate immune system. NK cells provide rapid immune responses against viral-infected cells, tumor cells or other stressed cell, even in the absence of antibodies and major histocompatibility complex on the cell surfaces. NK cells for using in a cell therapy and / or a CAR therapy may either be isolated or obtained from a commercially available source. Non-limiting examples of commercial NK cell lines include lines NK-92 (ATCC® CRL-2407™), NK-92MI 1(ATCC® CRL-2408™). Further examples include but are not limited to NK lines HANK1, KHYG-1, NKL, NK-YS, NOI-90, and YT. Non-limiting exemplary sources for such commercially available cell lines include the American Type Culture Collection, or ATCC, (www.atcc.org / ) and the German Collection of Microorganisms and Cell Cultures (www.dsmz.de / ).[0088| As used herein, the term “T cell,” refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and are distinguished from other lymphocytes, such as B cells, by the presence of a T-cell receptor on the cell surface. T-cells for using in a cell therapy and / or a CAR therapy may either be isolated or obtained from a commercially available source. “T cell” includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells), cytotoxic T-cells (CD8+ cells), natural killer T- cells, T-regulatory cells (Treg) and gamma-delta T cells. A “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses. Non-limiting examples of commercially available T-cell lines include lines BCL2 (AAA) Jurkat (ATCC® CRL-2902™), BCL2 (S70A) Jurkat (ATCC® CRL- 2900™), BCL2 (S87A) Jurkat (ATCC® CRL-2901™), BCL2 Jurkat (ATCC® CRL- 2899™), Neo Jurkat (ATCC® CRL-2898™), TALL- 104 cytotoxic human T cell line (ATCC # CRL-11386). Further examples include but are not limited to mature T-cell lines, e.g., such as Deglis, EBT-8, HPB-MLp-W, HUT 78, HUT 102, Karpas 384, Ki 225, My-La, Se-Ax, SKW-3, SMZ-1 and T34; and immature T- cell lines, e.g., ALL-SIL, Bel3, CCRF-CEM, CML-T1, DND-41, DU.528, EU-9, HD-Mar, HPB-ALL, H-SB2, HT-1, JK-T1, Jurkat, Karpas 45, KE-37, KOPT-K1, K-Tl, L-KAW, Loucy, MAT, MOLT-1, MOLT 3, MOLT-4, MOLT 13, MOLT-16, MT-1, MT-ALL, P12 / Ichikawa, Peer, PER0117, PER-255, PF-382, PFI-285, RPMI-8402, ST-4, SUP-T1 to T14, TALL-1, TALL-101, TALL-103 / 2, TALL-104, TALL-105, TALL-106, TALL-107, TALL-197, IK-6, TLBR-1, -2, -3, and -4, CCRF-HSB-2 (CCL-120.1), J.RT3-T3.5 (ATCC TIB-153), J45.01 (ATCC CRL-1990), J.CaM1.6 (ATCC CRL-2063), RS4;11 (ATCC CRL-1873), CCRF-CEM (ATCC CRM-CCL-119); and cutaneous T-cell lymphoma lines, e.g., HuT78 (ATCC CRM-TIB-161), MJ[G11] (ATCC CRL-8294), HuT102 (ATCC TIB-162). Null leukemia cell lines, including but not limited to REH, NALL-1, KM-3, L92-221, are another commercially available source of immune cellsfor using in a CAR therapy, as are cell lines derived from other leukemias and lymphomas, such as K562 erythroleukemia, THP-1 monocytic leukemia, U937 lymphoma, HEL erythroleukemia, HL60 leukemia, HMC-1 leukemia, KG-1 leukemia, U266 myeloma. Nonlimiting exemplary sources for such commercially available cell lines include the American Type Culture Collection, or ATCC, (atcc.org / ) and the German Collection of Microorganisms and Cell Cultures (dsmz.de / ).(0089] As used herein, a “tumor-specific antigen” refers to an antigenic substance produced in tumor cells, capable of triggering an immune response in a subject. In some embodiments, such tumor-specific antigen is not expressed on or in a cell in the subject, which is not a cancer cell. In some embodiment, such tumor-specific antigen may still be expressed in or on some non-cancer cells. For example, a tumor-specific antigen may not be expressed on the cell surface of a non-cancer cell in the subject. In one embodiment, the tumor-specific antigen may be expressed in or on a non-cancer cell of the subject, but in a much lower level compared to a cancer cell. In another embodiment, the tumor-specific antigen may be expressed in or on a non-cancer cell of the subject which is not adjacent to a cancer or a cancer cell. Non-limiting examples of a tumor-specific antigen includes: Alphafetoprotein (AFP), Beta-2-microglobulin (B2M), Beta-human chorionic gonadotropin (Beta-hCG), Bladder Tumor Antigen (BTA), C-kit / CDl 17, CA15-3 / CA27.29, CA19-9, CA-125, CA 27.29, Calcitonin, Carcinoembryonic antigen (CEA), Chromogranin A (CgA), Cytokeratin fragment 21-1, Des-gamma-carboxy prothrombin (DCP), Estrogen receptor (ER) / progesterone receptor (PR), Epithelial tumor antigen (ETA), Fibrin / fibrinogen, Gastrin, HE4, overexpressed HER2 / neu, 5-HIAA, Lactate dehydrogenase, Melanoma-associated antigen (MAGE), MUC-1, Neuron-specific enolase (NSE), Nuclear matrix protein 22, Programmed death ligand 1 (PD-L1), Prostate-specific antigen (PSA), Prostatic Acid Phosphatase (PAP), Soluble mesothelin-related peptides (SMRP), Somatostatin receptor, Tyrosinase, Thyroglobulin, abnormal products of ras, p53, alpha folate receptor, 5T4, avP6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD25, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvni, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRoc, GD2, GD3, Glypican-3 (GPC3), HL A- A 1 +M AGE 1 ,HLA-A2+MAGE1, HL A- A3 +M AGE 1 , HLA-A1+NY-ES0-1, HL A-A2+NY-ES0- 1 , HLA-A3+NY-ES0- 1 , IL- 1 IRoc, IL-13Ra2, Lambda, Lewis- Y, Kappa, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, R0R1, SSX, Survivin, TAG72, TEMs, VEGFR2, and WT-1.100901 As used herein, a “vector” refers to a construct which is capable of delivering, and, in some embodiments expressing, a polynucleotide in to a cell. Non-limiting examples of delivery vectors include viral vectors, nucleic acid expression vectors (such as a plasmid), naked DNA, and certain eukaryotic cells (e.g., producer cells). In some embodiments, nucleic acids described by the disclosure are delivered via a viral vector. Examples of viral vectors include retroviral vectors (e.g., Maloney murine leukemia virus, MML-V), adenoviral vectors (e.g., AD 100), lentiviral vectors (e.g., HIV and FIV-based vectors), and herpesvirus vectors (e.g., HSV, HSV-1, HSV-2), as described by Chira et al. (Oncotarget, 2015, 6(31); 30673-30703). In some embodiments, nucleic acids described by the disclosure are delivered by an adeno-associated virus (AAV) vector (e.g., a recombinant AAV (rAAV) vector).100911 Expression plasmids expressing the peptide can be purchased from Origene (www.origene.com, No. SCI 16851, last accessed October 14, 2020, referencing NM_005218, last accessed October 14, 2020).

[0092] The phrase “first line” or “second line” or “third line” refers to the order of treatment received by a patient. First line therapy regimens are treatments given first, whereas second or third line therapy are given after the first line therapy or after the second line therapy, respectively. The National Cancer Institute defines first line therapy as “the first treatment for a disease or condition. In patients with cancer, primary treatment can be surgery, chemotherapy, radiation therapy, or a combination of these therapies. First line therapy is also referred to those skilled in the art as “primary therapy and primary treatment.” See National Cancer Institute website at cancer.gov. Typically, a patient is given a subsequent chemotherapy regimen because the patient did not show a positive clinical or sub-clinical response to the first line therapy or the first line therapy has stopped.

[0093] In one aspect, the term “equivalent” or “biological equivalent” of an antibody means the ability of the antibody to selectively bind its epitope protein or fragment thereof as measured by ELISA or other suitable methods. Biologically equivalent antibodies include,but are not limited to, those antibodies, peptides, antibody fragments, antibody variant, antibody derivative and antibody mimetics that bind to the same epitope as the reference antibody.

[0094] In one aspect, the term “equivalent” of “chemical equivalent” of a chemical means the ability of the chemical to selectively interact with its target protein, DNA, RNA or fragment thereof as measured by the inactivation of the target protein, incorporation of the chemical into the DNA or RNA or other suitable methods. Chemical equivalents include, but are not limited to, those agents with the same or similar biological activity and include, without limitation a pharmaceutically acceptable salt or mixtures thereof that interact with and / or inactivate the same target protein, DNA, or RNA as the reference chemical.

[0095] The term “allele,” which is used interchangeably herein with “allelic variant” refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions and insertions of nucleotides. An allele of a gene can also be a form of a gene containing a mutation.

[0096] As used herein, the term “determining the genotype of a cell or tissue sample” intends to identify the genotypes of polymorphic loci of interest in the cell or tissue sample. In one aspect, a polymorphic locus is a single nucleotide polymorphic (SNP) locus. If the allelic composition of a SNP locus is heterozygous, the genotype of the SNP locus will be identified as “X / Y” wherein X and Y are two different nucleotides. If the allelic composition of a SNP locus is heterozygous, the genotype of the SNP locus will be identified as “X / X” wherein X identifies the nucleotide that is present at both alleles.

[0097] The term “genetic marker” refers to an allelic variant of a polymorphic region of a gene of interest and / or the expression level of a gene of interest.[0098[ The term “polymorphism” refers to the coexistence of more than one form of a gene or portion thereof. A portion of a gene of which there are at least two different forms, i.e.,two different nucleotide sequences, is referred to as a “polymorphic region of a gene.” A polymorphic region can be a single nucleotide, the identity of which differs in different alleles.

[0099] The term “genotype” refers to the specific allelic composition of an entire cell or a certain gene and in some aspects a specific polymorphism associated with that gene, whereas the term “phenotype” refers to the detectable outward manifestations of a specific genotype.

[0100] The term “encode” as it is applied to polynucleotides refers to a polynucleotide which is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce the mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0101] The term “isolated” as used herein refers to molecules or biological or cellular materials being substantially free from other materials. In one aspect, the term “isolated” refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source. The term “isolated” also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Moreover, an “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. The term “isolated” is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.

[0102] The term “treating” as used herein is intended to encompass curing as well as ameliorating at least one symptom of the condition or disease. For example, in the case of cancer, a response to treatment includes a reduction in cachexia, increase in survival time, elongation in time to tumor progression, reduction in tumor mass, reduction in tumor burden and / or a prolongation in time to tumor metastasis, time to tumor recurrence, tumor response,complete response, partial response, stable disease, progressive disease, progression free survival, overall survival, each as measured by standards set by the National Cancer Institute and the U.S. Food and Drug Administration for the approval of new drugs. In one aspect, treatment excludes prevention or prophylaxis.10.1.031 The term “clinical outcome”, “clinical parameter”, “clinical response”, or “clinical endpoint” refers to any clinical observation or measurement relating to a patient’s reaction to a therapy. Non-limiting examples of clinical outcomes include tumor response (TR), overall survival (OS), progression free survival (PFS), disease free survival, time to tumor recurrence (TTR), time to tumor progression (TTP), relative risk (RR), toxicity or side effect.101041 The term “suitable for a therapy” or “suitably treated with a therapy” shall mean that the patient is likely to exhibit one or more desirable clinical outcomes as compared to patients having the same disease and receiving the same therapy but possessing a different characteristic that is under consideration for the purpose of the comparison. In one aspect, the characteristic under consideration is a genetic polymorphism or a somatic mutation. In another aspect, the characteristic under consideration is expression level of a gene or a polypeptide. In one aspect, a more desirable clinical outcome is relatively higher likelihood of or relatively better tumor response such as tumor load reduction. In another aspect, a more desirable clinical outcome is relatively longer overall survival. In yet another aspect, a more desirable clinical outcome is relatively longer progression free survival or time to tumor progression. In yet another aspect, a more desirable clinical outcome is relatively longer disease free survival. In further another aspect, a more desirable clinical outcome is relative reduction or delay in tumor recurrence. In another aspect, a more desirable clinical outcome is relatively decreased metastasis. In another aspect, a more desirable clinical outcome is relatively lower relative risk. In yet another aspect, a more desirable clinical outcome is relatively reduced toxicity or side effects. In some embodiments, more than one clinical outcomes are considered simultaneously. In one such aspect, a patient possessing a characteristic, such as a genotype of a genetic polymorphism, can exhibit more than one more desirable clinical outcomes as compared to patients having the same disease and receiving the same therapy but not possessing the characteristic. As defined herein, the patient is considered suitable for the therapy. In another such aspect, a patient possessing acharacteristic can exhibit one or more desirable clinical outcome but simultaneously exhibit one or more less desirable clinical outcome. The clinical outcomes will then be considered collectively, and a decision as to whether the patient is suitable for the therapy will be made accordingly, taking into account the patient’s specific situation and the relevance of the clinical outcomes. In some embodiments, progression free survival or overall survival is weighted more heavily than tumor response in a collective decision making.

[0105] A “tumor response” (TR) refers to a tumor’s response to therapy. A “complete response” (CR) to a therapy refers to the clinical status of a patient with evaluable but non- measurable disease, whose tumor and all evidence of disease have disappeared following administration of the therapy. In this context, a “partial response” (PR) refers to a response that is anything less than a complete response. “Stable disease” (SD) indicates that the patient is stable following the therapy. “Progressive disease” (PD) indicates that the tumor has grown (i.e. become larger) or spread (i.e. metastasized to another tissue or organ) or the overall cancer has gotten worse following the therapy. For example, tumor growth of more than 20 percent since the start of therapy typically indicates progressive disease. “Nonresponse” (NR) to a therapy refers to status of a patient whose tumor or evidence of disease has remained constant or has progressed.

[0106] “Overall Survival” (OS) refers to the length of time of a cancer patient remaining alive following a cancer therapy.101071 “Progression free survival” (PFS) or “Time to Tumor Progression” (TTP) refers to the length of time following a therapy, during which the tumor in a cancer patient does not grow. Progression-free survival includes the amount of time a patient has experienced a complete response, partial response or stable disease.10108] “Disease free survival” refers to the length of time following a therapy, during which a cancer patient survives with no signs of the cancer or tumor.

[0109] “ Time to Tumor Recurrence (TTR)” refers to the length of time, following a cancer therapy such as surgical resection or chemotherapy, until the tumor has reappeared (come back). The tumor may come back to the same place as the original (primary) tumor or to another place in the body.[01101 “Relative Risk” (RR), in statistics and mathematical epidemiology, refers to the risk of an event (or of developing a disease) relative to exposure. Relative risk is a ratio of the probability of the event occurring in the exposed group versus a non-exposed group.

[0111] The term “determining” or “identifying” is to associate or affiliate a patient closely to a group or population of patients who likely experience the same or a similar clinical response to a therapy.

[0012] The term “selecting” a patient for a therapy refers to making an indication that the selected patient is suitable for the therapy. Such an indication can be made in writing by, for instance, a handwritten prescription or a computerized report making the corresponding prescription or recommendation.

[0113] When a genetic marker or polymorphism “is used as a basis” for identifying or selecting a patient for a treatment described herein, the genetic marker or polymorphism is measured before and / or during treatment, and the values obtained are used by a clinician in assessing any of the following: (a) probable or likely suitability of an individual to initially receive treatment s); (b) probable or likely unsuitability of an individual to initially receive treatment(s); (c) responsiveness to treatment; (d) probable or likely suitability of an individual to continue to receive treatment(s); (e) probable or likely unsuitability of an individual to continue to receive treatment(s); (f) adjusting dosage; (g) predicting likelihood of clinical benefits; or (h) toxicity. As would be well understood by one in the art, measurement of the genetic marker or polymorphism in a clinical setting is a clear indication that this parameter was used as a basis for initiating, continuing, adjusting and / or ceasing administration of the treatments described herein.

[0114] “Having the same cancer” is used when comparing one patient to another or alternatively, one patient population to another patient population. For example, the two patients or patient population will each have or be suffering from colon cancer.

[0115] A “normal cell corresponding to the tumor tissue type” refers to a normal cell from a same tissue type as the tumor tissue. A non-limiting examples is a normal lung cell from a patient having lung tumor, or a normal colon cell from a patient having colon tumor.

[0116] The term “amplification” or “amplify” as used herein means one or more methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification can be exponential or linear. A target nucleic acid can be either DNA or RNA. The sequences amplified in this manner form an “amplicon.” While the exemplary methods described hereinafter relate to amplification using the polymerase chain reaction (“PCR”), numerous other methods are known in the art for amplification of nucleic acids (e.g., isothermal methods, rolling circle methods, etc.). The skilled artisan will understand that these other methods can be used either in place of, or together with, PCR methods.[01171 The term “complement” as used herein means the complementary sequence to a nucleic acid according to standard Watson / Crick base pairing rules. A complement sequence can also be a sequence of RNA complementary to the DNA sequence or its complement sequence and can also be a cDNA. The term “substantially complementary” as used herein means that two sequences hybridize under stringent hybridization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length. In particular, substantially complementary sequences comprise a contiguous sequence of bases that do not hybridize to a target or marker sequence, positioned 3' or 5' to a contiguous sequence of bases that hybridize under stringent hybridization conditions to a target or marker sequence.[01 IS] As used herein, the term “hybridize” or “specifically hybridize” refers to a process where two complementary nucleic acid strands anneal to each other under appropriately stringent conditions. Hybridizations are typically conducted with probe-length nucleic acid molecules. Nucleic acid hybridization techniques are well known in the art. Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions such that sequences having at least a desired level of complementarity will stably hybridize, while those having lower complementarity will not. For examples of hybridization conditions and parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus, N.J.[01191 “Primer” as used herein refers to an oligonucleotide that is capable of acting as a point of initiation of synthesis when placed under conditions in which primer extension is initiated (e.g., primer extension associated with an application such as PCR). The primer is complementary to a target nucleotide sequence, and it hybridizes to a substantially complementary sequence in the target and leads to addition of nucleotides to the 3 '-end of the primer in the presence of a DNA or RNA polymerase. The 3 '-nucleotide of the primer should generally be complementary to the target sequence at a corresponding nucleotide position for optimal expression and amplification. An oligonucleotide “primer” can occur naturally, as in a purified restriction digest or can be produced synthetically. The term "primer" as used herein includes all forms of primers that can be synthesized including, peptide nucleic acid primers, locked nucleic acid primers, phosphorothioate modified primers, labeled primers, and the like.

[0120] Primers are typically between about 5 and about 100 nucleotides in length, such as between about 15 and about 60 nucleotides in length, such as between about 20 and about 50 nucleotides in length, such as between about 25 and about 40 nucleotides in length. In some embodiments, primers can be at least 8, at least 12, at least 16, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 nucleotides in length. An optimal length for a particular primer application can be readily determined in the manner described in H. Erlich, PCR Technology. Principles and Application for DNA Amplification (1989).

[0121] “Probe” as used herein refers to nucleic acid that interacts with a target nucleic acid via hybridization. A probe can be fully complementary to a target nucleic acid sequence or partially complementary. The level of complementarity will depend on many factors based, in general, on the function of the probe. A probe or probes can be used, for example to detect the presence or absence of a mutation in a nucleic acid sequence by virtue of the sequence characteristics of the target. Probes can be labeled or unlabeled, or modified in any of a number of ways well known in the art. A probe can specifically hybridize to a target nucleic acid.

[0122] Probes can be DNA, RNA or a RNA / DNA hybrid. Probes can be oligonucleotides, artificial chromosomes, fragmented artificial chromosome, genomic nucleic acid, fragmentedgenomic nucleic acid, RNA, recombinant nucleic acid, fragmented recombinant nucleic acid, peptide nucleic acid (PNA), locked nucleic acid, oligomer of cyclic heterocycles, or conjugates of nucleic acid. Probes can comprise modified nucleobases, modified sugar moieties, and modified internucleotide linkages. A probe can be fully complementary to a target nucleic acid sequence or partially complementary. A probe can be used to detect the presence or absence of a target nucleic acid. Probes are typically at least about 10, 15, 21, 25, 30, 35, 40, 50, 60, 75, 100 nucleotides or more in length.

[0123] “Detecting” as used herein refers to determining the presence of a nucleic acid or marker of interest in a sample or the presence of a protein or marker of interest in a sample. Detection does not require the method to provide 100% sensitivity and / or 100% specificity.

[0124] “Detectable label” as used herein refers to a molecule or a compound or a group of molecules or a group of compounds used to identify a nucleic acid or protein of interest. In some cases, the detectable label can be detected directly. In other cases, the detectable label can be a part of a binding pair, which can then be subsequently detected. Signals from the detectable label can be detected by various means and will depend on the nature of the detectable label. Detectable labels can be isotopes, fluorescent moieties, colored substances, and the like. Examples of means to detect detectable label include but are not limited to spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means.

[0125] “TaqMan® PCR detection system” as used herein refers to a method for real time PCR. In this method, a TaqMan® probe which hybridizes to the nucleic acid segment amplified is included in the PCR reaction mix. The TaqMan® probe comprises a donor and a quencher fluorophore on either end of the probe and in close enough proximity to each other so that the fluorescence of the donor is taken up by the quencher. However, when the probe hybridizes to the amplified segment, the 5'-exonuclease activity of the Taq polymerase cleaves the probe thereby allowing the donor fluorophore to emit fluorescence which can be detected.

[0126] As used herein, the term “sample” or “test sample” refers to any liquid or solid material containing nucleic acids. In suitable embodiments, a test sample is obtained from abiological source (i.e., a "biological sample"), such as cells in culture or a tissue sample from an animal, preferably, a human. In an exemplary embodiment, the sample is a biopsy sample.

[0127] “Target nucleic acid” as used herein refers to segments of a chromosome, a complete gene with or without intergenic sequence, segments or portions a gene with or without intergenic sequence, or sequence of nucleic acids to which probes or primers are designed. Target nucleic acids can include wild type sequences, nucleic acid sequences containing mutations, deletions or duplications, tandem repeat regions, a gene of interest, a region of a gene of interest or any upstream or downstream region thereof. Target nucleic acids can represent alternative sequences or alleles of a particular gene. Target nucleic acids can be derived from genomic DNA, cDNA, or RNA. As used herein, target nucleic acid can be native DNA or a PCR-amplified product.

[0128] As used herein the term “stringency” is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds, under which nucleic acid hybridizations are conducted. With high stringency conditions, nucleic acid base pairing will occur only between nucleic acids that have sufficiently long segments with a high frequency of complementary base sequences. Exemplary hybridization conditions are as follows. High stringency generally refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018 M NaCl at 65°C. High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5*Denhardf s solution, 5*SSC (saline sodium citrate) 0.2% SDS (sodium dodecyl sulfate) at 42°C., followed by washing in 0.1 x SSC, and 0.1% SDS at 65°C. Moderate stringency refers to conditions equivalent to hybridization in 50% formamide, 5*Denhard s solution, 5* SSC, 0.2% SDS at 42°C, followed by washing in 0.2* SSC, 0.2% SDS, at 65°C. Low stringency refers to conditions equivalent to hybridization in 10% formamide, 5*Denhard s solution, 6*SSC, 0.2% SDS, followed by washing in l x SSC, 0.2% SDS, at 50°C.

[0129] As used herein the term “substantially identical” refers to a polypeptide or nucleic acid exhibiting at least 50%, 75%, 85%, 90%, 95%, or even 99% identity to a reference amino acid or nucleic acid sequence over the region of comparison. For polypeptides, the length of comparison sequences will generally be at least 20, 30, 40, or 50 amino acids or more, or the full length of the polypeptide. For nucleic acids, the length of comparisonsequences will generally be at least 10, 15, 20, 25, 30, 40, 50, 75, or 100 nucleotides or more, or the full length of the nucleic acid.Descriptive Embodiments

[0130] The disclosure further provides diagnostic, prognostic and therapeutic methods, which are based, at least in part, on determination of the identity of a genotype of interest identified herein.[01311 For example, information obtained using the diagnostic assays described herein is useful for determining if a subject is suitable for cancer treatment of a given type. Based on the prognostic information, a doctor can recommend a therapeutic protocol, useful for reducing the malignant mass or tumor in the patient or treat cancer in the individual.[01321 A patient’s likely clinical outcome following a clinical procedure such as a therapy or surgery can be expressed in relative terms. For example, a patient having a particular genotype or expression level can experience relatively longer overall survival than a patient or patients not having the genotype or expression level. The patient having the particular genotype or expression level, alternatively, can be considered as likely to survive. Similarly, a patient having a particular genotype or expression level can experience relatively longer progression free survival, or time to tumor progression, than a patient or patients not having the genotype or expression level. The patient having the particular genotype or expression level, alternatively, can be considered as not likely to suffer tumor progression. Further, a patient having a particular genotype or expression level can experience relatively shorter time to tumor recurrence than a patient or patients not having the genotype or expression level. The patient having the particular genotype or expression level, alternatively, can be considered as not likely to suffer tumor recurrence. Yet in another example, a patient having a particular genotype or expression level can experience relatively more complete response or partial response than a patient or patients not having the genotype or expression level. The patient having the particular genotype or expression level, alternatively, can be considered as likely to respond. Accordingly, a patient that is likely to survive, or not likely to suffer tumor progression, or not likely to suffer tumor recurrence, or likely to respond following a clinical procedure is considered suitable for the clinical procedure.

[0133] It is to be understood that information obtained using the diagnostic assays described herein can be used alone or in combination with other information, such as, but not limited to, genotypes or expression levels of other genes, clinical chemical parameters, histopathological parameters, or age, gender and weight of the subject. When used alone, the information obtained using the diagnostic assays described herein is useful in determining or identifying the clinical outcome of a treatment, selecting a patient for a treatment, or treating a patient, etc. When used in combination with other information, on the other hand, the information obtained using the diagnostic assays described herein is useful in aiding in the determination or identification of clinical outcome of a treatment, aiding in the selection of a patient for a treatment, or aiding in the treatment of a patient. In a particular aspect, the genotypes or expression levels of one or more genes as disclosed herein are used in a panel of genes, each of which contributes to the final diagnosis, prognosis or treatment.

[0134] The methods are useful in the assistance of an animal, a mammal or yet further a human patient. For the purpose of illustration only, a mammal includes but is not limited to a human, a simian, a feline, a canine, a murine, a bovine, an equine, a porcine or an ovine subject.Modes for Carrying Out the DisclosureTherapeutic Methods

[0135] In vivo and in vitro use

[0136] Provided herein is a method of inhibiting the growth of a cancer cell expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) or a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, comprising, or consisting essentially of, or yet further consisting of, contacting the cancer cell with a PTPRD inhibitor and / or a PTPRF inhibitor.

[0137] Also provided is a method of inhibiting the growth of a cancer cell expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) protein, comprising, or consisting essentially of, or yet further consisting of, contacting the cancer cell with a PTPRD inhibitor.

[0138] Yet further provided is a method of inhibiting the growth of a cancer cell expressing a mutant PTPRD protein, comprising, or consisting essentially of, or yet further consisting of, contacting the cancer cell with a PTPRF inhibitor.

[0139] In one aspect of the above methods, the PTPRD inhibitor or the PTPRF inhibitor comprises illudalic-acid-7-BIA or an analog of illudalic-acid-7-BIA or a pharmaceutically acceptable salt of each thereof. See FIG. 2.

[0140] In another aspect of the above methods, the PTPRD inhibitor or the PTPRF inhibitor comprises a CRISPR or CRISPRi system which targets the genes encoding PTPRD and / or PTPRF.

[0141] In a further aspect, the methods further comprise, or consist essentially of, or yet further consist of, contacting the cancer cell with a second anti-cancer agent. Examples of such agents are provided herein.

[0142] In one aspect, the second anti-cancer agent is a chemotherapeutic agent or an immunotherapy that may be contacted prior to, concurrently or subsequent to the PTPRD or PTPRF inhibitor.

[0143] The cancer cell can be any animal cell, typically a mammalian cell. Non-limiting examples of mammalian cells include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. The cell can be from a mammal at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. In some embodiments, a subject has or is diagnosed of having or is suspected of having a cancer. In some aspects, the cancer cell is a primary cancer cell, a metastatic cancer cell, or an immortalized cancer cell. The cell can be mammalian, such as a feline, canine or human cell. In a further aspect, the cancer cell is a liver cancer cell, optionally a hepatocellular cancer cell or a liver cholangio-carcinoma cell line. The cell can be isolated from a biopsy or a cultured cell line, e.g., a commercially available cell line.

[0144] In further aspects, the methods further comprise, or consist essentially of, or yet further consist of determining the cancer cell growth inhibition subsequent to the contacting of the cancer cell with the PTPRD inhibitor or PTPRF inhibitor.

[0145] In a further aspect of these methods, the cancer cell has a loss of function PTPRF mutant or PTPRD mutant or copy loss of PTPRF genotype or PTPRD genotype.

[0146] The methods can be practiced in vivo or in vitro.

[0147] When practiced in vitro, the methods provide an assay to determine personalized therapy for the subject or patient. They also can be used for assaying for possible combination therapies.

[0018] When practiced in vivo, they can be used as a therapy for an animal patient or human patient. The in vivo administration can also be used to assay for personalized therapies for the subject or possible combination therapies.

[0149] In a further aspect, provided herein is a method of inhibiting the growth of a liver cancer cell expressing a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, comprising contacting the cancer cell with illudalic-acid-7-BIA or an analog of illudalic-acid-7-BIA or a pharmaceutically acceptable salt of each thereof, optionally wherein the liver cancer is a hepatocellular carcinoma. The illudalic-acid-7-BIA inhibits PTPRF. In some embodiments, the contacting is in vivo or in vitro. See FIG. 2.

[0150] In a further embodiment, provided herein are methods of inhibiting the growth of a cancer cell in vivo or in vitro, comprising knocking out or knocking down PTPRD expression in the cancer cell, and inhibiting PTPRF with a chemical inhibitor in the cancer cell.

[0151] In a further embodiment, provided herein are methods of inhibiting the growth of a cancer cell in vivo or in vitro, comprising knocking out or knocking down PTPRF expression in the cancer cell, and inhibiting PTPRD with a chemical inhibitor in the cancer cell.]0152] In a further embodiment, provided herein are methods of inhibiting the growth of a cancer cell in vivo or inv vitro, comprising inhibiting knocking down or knocking out both the genes encoding PTRPD and PTPR. See FIGS. 1A and IB. In a further embodiment,provided herein are methods of inhibiting the growth of a cancer cell in vivo or inv vitro, comprising inhibiting both PTPRD and PTPRF in the cancer cell with a chemical inhibitor.

[0153] In one aspect, CRISPRi (CRISPR interference) is performed to knock down the function of PTPRF and / or PTPRD.

[0154] In another aspect, CRISPR is performed to knock out the function of PTPRF and / or PTPRD.101551 Therapeutic use in a subject

[0156] Also provided is a method for one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of cancer in a subject suffering from a cancer cell expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) or a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, comprising, or consisting essentially of, or yet further consisting of administering to subject an effective amount of an PTPRF inhibitor and / or PTPRD inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subject.

[0157] Further provided is a method for one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of a subject suffering from a cancer expressing a mutant PTPRF, comprising, or consisting essentially of, or yet further consisting of administering to subject an effective amount of a PTPRD inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subject.

[0158] Still further provided is a method for one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of cancer in a subject suffering from a cancer expressing mutant PTPRD comprising, or consisting essentially of, or yet further consisting of administering to subject an effective amount of a PTPRF inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subject.

[0159] In one aspect of the methods, the PTPRD or the PTPRF inhibitor comprises illudalic- acid-7-BIA or an analog of illudalic-acid-7-BIA or a pharmaceutically acceptable salt of each thereof.

[0160] In a further aspect of the methods, a second anti-cancer agent is administered, such as for example, a chemotherapeutic agent or an immunotherapy that can be contacted prior to, subsequent to, or concurrently with the PTPRD inhibitor or the PTPRF inhibitor. Nonlimiting examples include 5-fluorouracil, pemetrexed, raltitrexed, nolatrexed, plevitrexed, GS7904L, capecitabine, methotrexate, pralatrexate, CT-900, NUC-3373, FOLFOX, FOLFOX4, FOLFIRI, MOF, deflexifol, or a combination of 5-FU with one or more selected from radiation, methyl-CCNU, leucovorin, oxaliplatin (such as cisplatin), irinotecan, mitomycin, cytarabine, and levamisole, anthracycline or other topoisomerase II inhibitor comprises daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, etoposide or teniposide. The second agent can also comprises one or more selected from monoclonal antibodies, optionally selected from monospecific antibodies, bispecific antibodies, multispecific antibodies and a bispecific immune cell engager, antibody-drug conjugates, CAR therapies optionally selected from a CAR NK therapy, a CAR T therapy, a CAR cytotoxic T therapy, a CAR gamma-delta T therapy, a CAR NK therapy, cell therapies, inhibitors or antagonists of an inhibitory immune checkpoint, activators or agonists of a stimulatory immune checkpoint optionally selected from an activating ligand, immune regulators, cancer vaccines, and a vector delivering each thereof to a subject optionally in an oncolytic virus therapy, and optionally wherein the monoclonal antibodies are selected from rituximab, blinatumomab, alemtuzumab, ibritumomab tiuxetan, bevacizumab, bevacizumab- awwb, cetuximab, panitumumab, ofatumumab, denosumab, pertuzumab, obinutuzumab, elotuzumab, ramucirumab, dinutuximab, daratumumab, trastuzumab, trastuzumab-dkst, nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, AMF 514 (MEDI0680), balstilimab, avelumab, durvalumab, atezolizumab, ipilimumab, tremelimumab, zalifrelimab, and AGEN1181, and further optionally wherein the antibody-drug conjugates are selected from moxetumomab pasudotox- tdfk, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, gemtuzumab ozogamicin, tagraxofusp-erzs, polatuzumab vedotin-piiq, enfortumab vedotin-ejfv,trastuzumab deruxtecan, and sacituzumab govitecan-hziy, and further optionally, a checkpoint inhibitor, optionally selected from GS4224, AMP -224, CA-327, CA-170, BMS- 1001, BMS-1166, peptide-57, M7824, MGD013, CX-072, UNP-12, NP-12, or a combination of two or more thereof, or an anti-PD-1 agent, an anti-PD-Ll agent, an anti-CTLA-4 agent, an anti-LAG-3 agent, an anti-TIM-3 agent, an anti-TIGIT agent, an anti-VISTA agent, an anti-B7-H3 agent, an anti-BTLA agent, an anti-ICOS agent, an anti-GITR agent, an anti-4- 1BB agent, an anti-OX40 agent, an anti-CD27 agent, an anti-CD28 agent, an anti-CD40 agent, and an anti-Siglec-15 agent.10.1611 The cancer being treated by these methods can be a primary cancer or a metastatic cancer. The cancers being treated include for example, a liver cancer, optionally a hepatocellular cancer or a liver cholangio-carcinoma. In another aspect, the cancer expresses a loss of function PTPRF or PTPRD mutant or a copy loss of PTPRF or PTPRD. The administration of the therapy can be selected from a first-line, second-line, third line, fourthline or fifth-line therapy. The cancer cell can be a primary cancer cell isolated from a patient or a culture cell line, e.g., a commercially available cell line.

[0162] The methods can further comprise, or consist essentially of, or yet further consist of, determining the cancer cell growth inhibition subsequent to the contacting of the cancer cell with the PTPRD or the PTPRF inhibitor and / or the second agent.

[0163] The subject can be any animal, typically a mammal. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. In some embodiments, a subject has or is diagnosed of having or is suspected of having a cancer.

[0014] The methods can further comprise, or consist essentially of, or yet further consist of, determining whether the cancer expresses a mutant PTPRF protein or mutant PTPRD protein,optionally a loss of function PTPRF mutant or PTPRD mutant or a copy loss of PTPRF genotype or PTPRD genotype.

[0165] In the methods of this disclosure, after administration the subject experiences one or more endpoints selected from tumor response, reduction in tumor size, reduction in tumor burden, increase in overall survival, increase in progression free survival, inhibiting metastasis, improvement of quality of life, minimization of toxicity, avoidance of side-effects or a reduction of side effects as compared to a subject not receiving the therapy.

[0016] In a further aspect, the effective amount is a therapeutically effective amount. In a yet further aspect, the agent is administered in a pharmaceutically acceptable carrier. The contacting can be any one of the groups of: a first line, second line, third line, a fourth line, or a fifth line therapy.

[0167] The subject can be any animal, typically a mammal. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. In some embodiments, a subject has or is diagnosed of having or is suspected of having a cancer.

[0168] In one aspect, one or more cancer cells, tumors or lymph nodes comprising the cells have been removed from the subject prior to the administration of the agent.

[0169] The cancer or tumor that is the target of these methods can be a primary cancer, metastatic or recurrent cancer.

[0170] In a further aspect, the methods further comprise, consist essentially of, or yet further consist of, administering a second agent to the subject. In the aspects of these methods, after administration of the agent or the second agent, the subject experiences one or more endpoints selected from tumor response, reduction in tumor size, reduction in tumor burden,increase in overall survival, increase in progression free survival, inhibiting metastasis, improvement of quality of life, minimization of toxicity, and avoidance of side-effects.

[0171] Non-limiting examples of the second agent is selected from the group of: 5- fluorouracil, pemetrexed, raltitrexed, nolatrexed, plevitrexed, GS7904L, capecitabine, methotrexate, pralatrexate, CT-900, NUC-3373, FOLFOX, FOLFOX4, FOLFIRI, MOF, deflexifol, or a combination of 5-FU with one or more selected from radiation, methyl- CCNU, leucovorin, oxaliplatin (such as cisplatin), irinotecan, mitomycin, cytarabine, and levamisole.

[0172] In one aspect, the second agent comprises an inhibitor of folate-mediated one-carbon metabolism. In another aspect, the second agent comprises anthracycline or other topoisomerase II inhibitor comprises daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, etoposide and teniposide.

[0173] In a yet further aspect, the second agent comprises one or more selected from monoclonal antibodies, optionally selected from monospecific antibodies, bispecific antibodies, multispecific antibodies and a bispecific immune cell engager, antibody-drug conjugates, CAR therapies optionally selected from a CAR NK therapy, a CAR T therapy, a CAR cytotoxic T therapy, a CAR gamma-delta T therapy, a CAR NK therapy, cell therapies, inhibitors or antagonists of an inhibitory immune checkpoint, activators or agonists of a stimulatory immune checkpoint optionally selected from an activating ligand, immune regulators, cancer vaccines, and a vector delivering each thereof to a subject optionally in an oncolytic virus therapy. Non-limiting examples of monoclonal antibodies are selected from rituximab, blinatumomab, alemtuzumab, ibritumomab tiuxetan, bevacizumab, bevacizumab- awwb, cetuximab, panitumumab, ofatumumab, denosumab, pertuzumab, obinutuzumab, elotuzumab, ramucirumab, dinutuximab, daratumumab, trastuzumab, trastuzumab-dkst, nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, AMF 514 (MEDI0680), balstilimab, avelumab, durvalumab, atezolizumab, ipilimumab, tremelimumab, zalifrelimab, and AGEN1181. Non-limiting examples of antibody-drug conjugates are selected from moxetumomab pasudotox-tdfk, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, gemtuzumabozogamicin, tagraxofusp-erzs, polatuzumab vedotin-piiq, enfortumab vedotin-ejfv, trastuzumab deruxtecan, and sacituzumab govitecan-hziy.

[0174] In one aspect, the CAR cell therapy is CAR T-cell therapy selected from tisagenlecleucel and axicabtagene ciloleucel. Non-limiting examples of immune regulators are selected from interleukins, aldesleukin, interferon alfa-2a / 2b, pexidartinib, erythropoietin, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), thalidomide, lenalidomide, pomalidomide, and imiquimod.

[0175] In one aspect, the cancer vaccines are selected from CG live (THERACYS®) and sipuleucel-T (PROVENGE®).

[0176] In one aspect, the oncolytic virus therapy is selected from oncorine (H101) and talimogene laherparepvec (IMLYGIC®).

[0177] In another aspect, the second agent comprises a checkpoint inhibitor. Non-limiting examples of such include GS4224, AMP-224, CA-327, CA-170, BMS-1001, BMS-1166, peptide-57, M7824, MGD013, CX-072, UNP-12, NP-12, or a combination of two or more thereof.

[0178] Additional checkpoint inhibitors comprises one or more selected from an anti-PD-1 agent, an anti-PD-Ll agent, an anti-CTLA-4 agent, an anti -LAG-3 agent, an anti-TIM-3 agent, an anti-TIGIT agent, an anti-VISTA agent, an anti-B7-H3 agent, an anti-BTLA agent, an anti-ICOS agent, an anti-GITR agent, an anti-4-lBB agent, an anti-OX40 agent, an anti- CD27 agent, an anti-CD28 agent, an anti-CD40 agent, and an anti-Siglec-15 agent. In a further aspect, the checkpoint inhibitor comprises an anti-PDl agent or an anti-PD-Ll agent. In one aspect, the anti-PDl agent comprises an anti-PDl antibody or an antigen binding fragment thereof. In a further aspect, the anti-PDl antibody comprises nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, AMF 514, or a combination of two or more thereof. In another aspect, the anti- PD-Ll agent comprises an anti-PD-Ll antibody or an antigen binding fragment thereof. In a further aspect, the anti-PD-Ll antibody comprises avelumab, durvalumab, atezolizumab, envafolimab, or a combination of two or more thereof.Prognostic and Diagnostic Methods

[0179] Provided herein is a method for determining a better response to a therapy comprising administration of a PTPRF inhibitor or a PTPRD inhibitor in a subject in need thereof, e.g., suffering from a cancer, comprising determining whether the subject’s cancer expresses a mutant PTPRF protein or mutant PTPRD protein, wherein the mutation in PTPRF or PTPRD protein results in a non-functional PTPRF protein or PTPRD protein. In some embodiments, the mutation is a loss of function mutation. In some embodiments, the loss of function mutation is due to an early stop codon, a frameshift mutation or a mutation in the PTP catalytical domain (e.g., a point mutation or a deletion) that impairs or abolishes the enzymatic (protein phosphatase) activity. Subjects who have a mutant PTPRF protein can be treated with a PTPRD inhibitor. Subjects who have a mutant PTPRD protein can be treated with a PTPRF inhibitor. Subjects who do not have the mutant protein can be treated with a different therapy or therapies.

[0180] The subject can be any animal, typically a mammal. Any suitable mammal can be treated by a method described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, and the like), domestic animals (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs) and experimental animals (e.g., mouse, rat, rabbit, guinea pig). In some embodiments, a mammal is a human. A mammal can be any age or at any stage of development (e.g., an adult, teen, child, infant, or a mammal in utero). A mammal can be male or female. In some embodiments, a subject is a human. In some embodiments, a subject has or is diagnosed of having or is suspected of having a cancer.

[0181] In a further aspect, a second anti-cancer agent is administered to the subject, prior to, concurrently or subsequent to the PTPRF inhibitor or PTPRD inhibitor, non-limiting examples of such are described herein and known in the art.

[0182] Any suitable method for identifying the genotype or phenotype in the patient sample can be used and the disclosures described herein are not to be limited to these methods. For the purpose of illustration only, the genotype is determined by a method comprising, or alternatively consisting essentially of, or yet further consisting of, sequencing, hybridization, nucleic acid amplification, including polymerase chain reaction (PCR), real-time PCR,reverse transcriptase PCR (RT-PCR), nested PCR, ligase chain reaction, or PCR-RFLP, or microarray. These methods as well as equivalents or alternatives thereto are described herein.

[0183] Information obtained using the diagnostic assays described herein is useful for determining if a subject will likely, more likely, or less likely to respond to cancer treatment of a given type. Based on the prognostic information, a doctor can recommend a therapeutic protocol, useful for treating reducing the malignant mass or tumor in the patient or treat cancer in the individual.

[0184] In addition, knowledge of the identity of a particular allele in an individual (the gene profile) allows customization of therapy for a particular disease to the individual’s genetic profile, the goal of “pharmacogenomics”. For example, an individual’s genetic profile can enable a doctor: 1) to more effectively prescribe a drug that will address the molecular basis of the disease or condition; 2) to better determine the appropriate dosage of a particular drug and 3) to identify novel targets for drug development. The identity of the genotype or expression patterns of individual patients can then be compared to the genotype or expression profile of the disease to determine the appropriate drug and dose to administer to the patient.{0185] The ability to target populations expected to show the highest clinical benefit, based on the normal or disease genetic profile, can enable: 1) the repositioning of marketed drugs with disappointing market results; 2) the rescue of drug candidates whose clinical development has been discontinued as a result of safety or efficacy limitations, which are patient subgroup-specific; and 3) an accelerated and less costly development for drug candidates and more optimal drug labeling.

[0186] Also provided is a method of identifying inhibition of tumor growth or tumor cell proliferation by pretreatment with an agent that knocks-down function of protein tyrosine phosphatase receptor type F (PTPRF) in tumor cells, comprising contacting the tumor cells with a range of varying concentration of one or more candidate agents that inhibit the function of protein tyrosine phosphate receptor D (PTPRD) and determining the range of varying concentration that inhibit the growth of a tumor.

[0187] Also provided is a method of identifying inhibition of tumor growth or tumor cell proliferation by pretreatment with an agent that knocks-down function of protein tyrosine phosphatase receptor type F (PTPRF) in tumor cells, comprising contacting the tumor cells with a range of varying concentration of one or more candidate agents that inhibit the function of protein tyrosine phosphate receptor F (PTPRF) and determining the range of varying concentration that inhibit the growth of a tumor.

[0188] Also provided is a method of identifying inhibition of tumor growth or tumor cell proliferation by pretreatment with an agent that knocks-down function of protein tyrosine phosphatase receptor type D (PTPRD) in tumor cells, comprising contacting the tumor cells with a range of varying concentration of one or more candidate agents that inhibit the function of protein tyrosine phosphate receptor D (PTPRD) and determining the range of varying concentration that inhibit the growth of a tumor.

[0189] In one aspect, the inhibition of tumor growth is determined for concentrations of more than candidate agent. In another aspect, the inhibition of tumor growth is determined for concentrations of two candidate agents. In a yet further aspect, the two PTPRD inhibitors are provided in a ratio of about 1 : 1 to about 1 : 10, or alternatively from about 1 : 1 to about 1 :9, or alternatively from about 1 : 1 to about 1 :8, or alternatively from about 1 : 1 to about 1 :7, or alternatively from about 1 : 1 to about 1 :6, or alternatively from about 1 : 1 to about 1 :5, or alternatively from about 1 : 1 to about 1 :4, or alternatively from about 1 : 1 to about 1 :3, or alternatively from about 1 : 1 to about 1 :2, or alternatively from about 1 : 1 to each other. In a further aspect, the candidate agent comprises illudalic-acid-7-BIA or an analogue of illudalic- acid-7-BIA.

[0010] In one aspect, CRISPRi (CRISPR interference) is performed to knock down the function of PTPRF and / or PTPRD.[0191 [ In another aspect, CRISPR is performed to knock out the function of PTPRF and / or PTPRD.

[0192] The tumor or cancer cells are liver cancer cells (obtained from a biopsy or cultured from the biopsy), e.g., liver hepatocellular carcinoma cells or liver Cholangio-carcinomacells, or they can be commercial or established cell lines obtained for example, from the American Type Culture Collection (ATCC)

[0193] The methods can be practiced in vivo or in vitro.Biological Sample Collection and Preparation

[0194] Methods of obtaining test samples are known to those of skill in the art and include, but are not limited to, aspirations, tissue sections, swabs, drawing of blood or other fluids, surgical or needle biopsies. These methods can be used to isolate subject samples prior to analyzing for the genotype or phenotype.

[0195] In some aspects, the biological sample is a tissue or a cell sample. Suitable patient samples in the methods include, but are not limited to, blood, plasma, serum, a biopsy tissue, fine needle biopsy sample, amniotic fluid, plasma, pleural fluid, saliva, semen, serum, tissue or tissue homogenates, frozen or paraffin sections of tissue or combinations thereof. In some aspects, the biological sample comprises, or alternatively consisting essentially of, or yet further consisting of, at least one of a tumor cell, a normal cell adjacent to a tumor, a normal cell corresponding to the tumor tissue type, a blood cell, a peripheral blood lymphocyte, or combinations thereof. In some aspects, the biological sample is an original sample recently isolated from the patient, a fixed tissue, a frozen tissue, a resection tissue, or a microdissected tissue. In some aspects, the biological samples are processed, such as by sectioning of tissues, fractionation, purification, nucleic acid isolation, or cellular organelle separation.

[0196] In some embodiments, nucleic acid (DNA or RNA) is isolated from the sample according to any methods known to those of skill in the art. In some aspects, genomic DNA is isolated from the biological sample. In some aspects, RNA is isolated from the biological sample. In some aspects, cDNA is generated from mRNA in the sample. In some embodiments, the nucleic acid is not isolated from the biological sample (e.g., the polymorphism is detected directly from the biological sample).Compositions and Modes of Administration[01971 The agents or drugs can be administered as a composition. A “composition” typically intends a combination of the active agent and another carrier, e.g., compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder,stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like and include pharmaceutically acceptable carriers. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates.

[0198] Various delivery systems are known and can be used to administer a chemotherapeutic agent of the disclosure, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, receptor-mediated endocytosis. See e.g., Wu and Wu (1987) J. Biol. Chem. 262:4429-4432 for construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of delivery include but are not limited to intra-arterial, intra-muscular, intravenous, intranasal and oral routes. In a specific embodiment, it can be desirable to administer the pharmaceutical compositions of the disclosure locally to the area in need of treatment; this can be achieved by, for example, and not by way of limitation, local infusion during surgery, by injection or by means of a catheter.

[0019] The agents identified herein as effective for their intended purpose can be administered to subjects or individuals identified by the methods herein as suitable for the therapy. Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the agent.

[0200] Methods of administering pharmaceutical compositions are well known to those of ordinary skill in the art and include, but are not limited to, oral, microinjection, intravenous or parenteral administration. The compositions are intended for topical, oral, or local administration as well as intravenously, subcutaneously, or intramuscularly. Administration can be effected continuously or intermittently throughout the course of the treatment.Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the cancer being treated and the patient and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.Kits{0201 ] Kits or panel for use in detecting the mutant PTPRF and / or PTPRD in patient or subject biological samples are provided. In some embodiments, a kit comprises, or consists essentially of, or yet further consists of at least one reagent necessary to perform the assay.For example, the kit can comprise an enzyme, a buffer or any other necessary reagent (e.g. PCR reagents and buffers). For example, in some aspects, a kit contains, in an amount sufficient for at least one assay, any of the hybridization assay probes, amplification primers, and / or antibodies suitable for detection in a packaging material. Alternatively or in addition, the kit comprises a PTPRF inhibitor and / or PTPRD inhibitor and instructions for use.

[0202] The various components of the kit can be provided in a variety of forms. For example, in some aspects, the required enzymes, the nucleotide triphosphates, the probes, primers, and / or antibodies are be provided as a lyophilized reagent. These lyophilized reagents can be pre-mixed before lyophilization so that when reconstituted they form a complete mixture with the proper ratio of each of the components ready for use in the assay. In addition, the kits can contain a reconstitution reagent for reconstituting the lyophilized reagents of the kit. In exemplary kits for amplifying target nucleic acid derived from a colorectal cancer patients, the enzymes, nucleotide triphosphates and required cofactors for the enzymes are provided as a single lyophilized reagent that, when reconstituted, forms a proper reagent for use in the present amplification methods.

[0203] Typically, the kits will also include instructions recorded in a tangible form (e.g., contained on paper or an electronic medium) for using the packaged probes, primers, and / or antibodies in a detection assay for determining the presence or amount of the polymorphism of interest in a test sample.

[0204] In some aspects, the kits further comprise a solid support for anchoring the nucleic acid of interest on the solid support. The target nucleic acid can be anchored to the solid support directly or indirectly through a capture probe anchored to the solid support and capable of hybridizing to the nucleic acid of interest. Examples of such solid support include but are not limited to beads, microparticles (for example, gold and other nano particles), microarray, microwells, multiwell plates. The solid surfaces can comprise a first member of a binding pair and the capture probe or the target nucleic acid can comprise a second member of the binding pair. Binding of the binding pair members will anchor the capture probe or the target nucleic acid to the solid surface. Examples of such binding pairs include but are not limited to biotin / streptavidin, hormone / receptor, ligand / receptor, and antigen / antibody.

[0205] In one aspect, the kit further comprises, or consists essentially of, or yet further consists of an effective amount of the therapy.

[0206] The test samples used in the diagnostic kits include cells, protein or membrane extracts of cells, or biological fluids such as sputum, blood, serum, plasma, or urine. The test samples can also be a tumor cell, a normal cell adjacent to a tumor, a normal cell corresponding to the tumor tissue type, a blood cell, a peripheral blood lymphocyte, or combinations thereof. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are known in the art and can be readily adapted in order to obtain a sample which is compatible with the system utilized.

[0207] The kits can include all or some of the positive controls, negative controls, reagents, primers, sequencing markers, probes and antibodies described herein for determining the subject’s genotype in the polymorphic region of the gene of interest or target region.102081 As amenable, these suggested kit components can be packaged in a manner customary for use by those of skill in the art. For example, these suggested kit components can be provided in solution or as a liquid dispersion or the like.

[0209] Typical packaging materials would include solid matrices such as glass, plastic, paper, foil, micro-particles and the like, capable of holding within fixed limits hybridization assay probes, and / or amplification primers. Thus, for example, the packaging materials can include glass vials used to contain sub-milligram (e.g., picogram or nanogram) quantities of a contemplated probe, primer, or antibodies or they can be microtiter plate wells to which probes, primers, or antibodies have been operatively affixed, i.e., linked so as to be capable of participating in an amplification and / or detection methods.

[0210] The instructions will typically indicate the reagents and / or concentrations of reagents and at least one assay method parameter which might be, for example, the relative amounts of reagents to use per amount of sample. In addition, such specifics as maintenance, time periods, temperature, and buffer conditions can also be included.[02111 The diagnostic systems contemplate kits having any of the hybridization assay probes, amplification primers, or antibodies described herein, whether provided individually or in one of the combinations described above, for use in determining the presence or amount of a polymorphism of interest.102121 The disclosure now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the disclosure.EXAMPLESExample 1: Targeting PTPRD and PTPRF together results in synthetic lethality10213] CRISPRi was used to inhibit either the PTPRD or the PTPRF gene individually, or in tandem, to check for the canonical signs of Synthetic Lethality. In FIG 1A, there is a statistically higher cell death in the double knockdown than a matched double scramble CRISPRi cell line at each time point after the 24-hour time point. This shows that double knockdown of PTPRD and PTPRF can kill cells. In FIG. IB, the synthetic lethality of the gene pair is demonstrated. In a non-synthetic lethal situation concerning a non-interacting gene pair, the effects of knocking down each gene is expected to be additive. In contrast, in a synthetic lethal scenario, a synergistic effect is expected. In FIG. IB, the knockdown of GeneA (PTPRD) has no effect on the viability of the cell line. The knockout of Gene B (PTPRF) shows 70% higher apoptosis than the scramble control. When both genes are knocked down at the same time, a synergistic, rather than additive, increase in apoptosis (-350%) is observed, demonstrating that PTPRD and PTPRF make an interacting gene pair which shows synthetic lethality. The combined effect of both PTPRD and PTPRF inhibition was 5 times greater than inhibition of PTPRF alone, which had moderate inhibitory potential.Example 2: Inhibiting PTPRD is effective in killing cells where PTPRF is knocked down

[0214] PTPRF was knocked down using CRISPRi in 3 separate clones of a hepatocellular carcinoma cell line. Knockdown of each line was quantified for PTPRF (shown as Gene A in FIG. 2) via qPCR, values of which are shown in the legend. Next, each clone was treatedwith a PTPRD inhibitor, Illudalic Acid-7-BIA, with a reported IC50 of l-3uM. In this way, both PTPRF and PTPRD were inhibited in the hematocellular carcinoma cell line.

[0215] In the two clones with knockdown of PTPRF over 70% (right two panels), there is a clear drug response curve, and the ECso is between 3-4pM depending on the clones. On the other hand, the clone with lower PTPRF knockdown (leftmost panels) show a weaker response, but still a response nevertheless. That is, even a partial knockdown of a gene can be sufficient for synthetic lethality. As a comparison, when a wild type version of this cell line was treated with the PTPRD inhibitor; there is no effect on viability at any dose from 500nM to 50uM. This shows that the drug has no effect on a cell line with wild type functional PTPRF, but has an effect in viability at the expected concentration, on a PTPRF knocked-down background. Thus, the relative level of gene knockdown may affect the level of synthetic lethality. These results indicate that a PTPRD inhibitor would work better in a PTPRF null patient population (showing PTPRF loss of function or PTPRF copy loss). Similarly, a PTPRF inhibitor is expected to work in a PTPRD null patient population (showing PTPRD loss of function or PTPRD copy loss).

[0216] Copy loss of PTPRF is observed in several tumors including liver cholangiocarcinomas (15%), and hepatocellular carcinomas (5%).

[0217] The synthetic lethality observed between PTPRF and PTPRD in this disclosure is surprising because there are 21 paralogs in the PTP family, which were largely thought to be redundant with each other in their cellular functions.Embodiments

[0218] Additional exemplary embodiments of the disclosure provided herein include:

[0219] 1. A method of inhibiting the growth of a cancer cell expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) or a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, comprising contacting the cancer cell with a PTPRD inhibitor or a PTPRF inhibitor, wherein the growth inhibition of the cancer cell with thePTPRD inhibitor or the PTPRF inhibitor has a greater effect on viability than the expression of the mutant protein alone.

[0220] 2. A method of embodiment 1, wherein the cancer cell expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) protein is contacted with a PTPRD inhibitor.

[0221] 3. A method of embodiment 1, wherein the cancer cell expressing a mutant PTPRD protein is contacted with a PTPRF inhibitor.

[0222] 4. The method of any preceding embodiment, wherein the PTPRD inhibitor or the PTPRF inhibitor comprises illudalic-acid-7-BIA or an analog of illudalic-acid-7-BIA or a pharmaceutically acceptable salt of each thereof.(0223] 5. The method of any preceding embodiment, further comprising contacting the cancer cell with a second anti-cancer agent.

[0224] 6. The method of embodiment 5, wherein the second anti-cancer agent is a chemotherapeutic agent or an immunotherapy.

[0225] 7. The method of embodiment 5, wherein the second agent is contacted prior to, concurrently or subsequent to the PTPRD inhibitor or the PTPRF inhibitor.

[0226] 8. The method of any preceding embodiment, wherein the cancer cell is a primary cancer cell, a metastatic cancer cell, or an immortalized cancer cell.

[0227] 9. The method of any preceding embodiment, further comprising determining the cancer cell growth inhibition subsequent to the contacting of the cancer cell with the PTPRD inhibitor or the PTPRF inhibitor.

[0228] 10. The method of any preceding embodiment, wherein the cancer cell is a mammalian cancer cell.

[0229] 11. The method of embodiment 10, wherein the mammalian cancer cell is selected from the group of a canine cancer cell, a feline cancer cell or a human cancer cell.

[0230] 12. The method of any preceding embodiment wherein the cancer cell is a liver cancer cell, optionally a hepatocellular cancer cell or a liver cholangiocarcinoma cell line.(0231 ] 13. The method of any preceding embodiment, wherein the cancer cell has loss of function mutant PTPRF protein or PTPRD protein or copy loss of PTPRF genotype or PTPRD genotype.

[0232] 14. The method of any preceding embodiment, wherein the contacting is in vitro or in vivo.[ 02331 15. A method for one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of cancer in a subject suffering from a cancer cell expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) or a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, comprising administering to subject an effective amount of a PTPRF inhibitor or PTPRD inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subject.

[0234] 16. A method of embodiment 15, wherein the subject suffering from a cancer expressing a mutant PTPRF is administered an effective amount of an PTPRD inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subject.10235] 17. A method of embodiment 15, wherein the subject suffering from a cancer expressing mutant PTPRD is administered an effective amount of an PTPRF inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subject.

[0236] 18. The method of any of embodiment 15-17, wherein the PTPRD or the PTPRF inhibitor comprises illudalic-acid-7-BIA or an analog of illudalic-acid-7-BIA or a pharmaceutically acceptable salt of each thereof.

[0237] 19. The method of any of embodiment 15-18, further comprising administering to the subject a second anti-cancer agent.

[0238] 20. The method of embodiment 19, wherein the second anti-cancer agent is a chemotherapeutic agent or an immunotherapy.(0239] 21. The method of embodiment 19, wherein the second agent is administered prior to, concurrently or subsequent to the PTPRD inhibitor or PTPRF inhibitor.(0240] 22. The method of any of embodiment 15-21, wherein the cancer is a primary cancer or a metastatic cancer.

[0241] 23. The method of any of embodiment 15-22, further comprising determining the cancer cell growth inhibition subsequent to the contacting of the cancer cell with the PTPRD inhibitor or PTPRF inhibitor.

[0242] 24. The method of any of embodiment 15-23, wherein the subject is a mammal.

[0243] 25. The method of embodiment 24, wherein the mammal is selected from the group of a canine, a feline or a human.

[0244] 26. The method of any of embodiment 15-25 wherein the cancer is a liver cancer, optionally a hepatocellular cancer or a liver cholangio-carcinoma.(0245] 27. The method of any of embodiment 15-26, wherein the cancer expresses a loss of function PTPRF mutant or PTPRD mutant or a copy loss of PTPRF genotype or PTPRD genotype.|0246| 28. The method of any of embodiment 15-27, wherein the administration is selected from a first-line, second-line, third line, fourth-line or fifth-line therapy.

[0247] 29. The method of any of embodiment 15-28, further comprising determining whether the cancer expresses a mutant PTPRF protein or a mutant PTPRD protein, optionally a loss of function PTPRF mutant or PTPRD mutant or a copy loss of PTPRF genotype or PTPRD genotype.

[0248] 30. The method of any one of embodiment 15-29, wherein after administration the subject experiences one or more endpoints selected from tumor response, reduction in tumorsize, reduction in tumor burden, increase in overall survival, increase in progression free survival, inhibiting metastasis, improvement of quality of life, minimization of toxicity, avoidance of side-effects or a reduction of side-effects.

[0249] 31. The method of any one of embodiment 19 or 21-30, wherein the second agent is selected from the group of: 5-fluorouracil, pemetrexed, raltitrexed, nolatrexed, plevitrexed, GS7904L, capecitabine, methotrexate, pralatrexate, CT-900, NUC-3373, FOLFOX, FOLFOX4, FOLFIRI, MOF, deflexifol, or a combination of 5-FU with one or more selected from radiation, methyl-CCNU, leucovorin, oxaliplatin (such as cisplatin), irinotecan, mitomycin, cytarabine, and levamisole, anthracycline or other topoisomerase II inhibitor comprises daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, etoposide or teniposide.

[0250] 32. The method of any one of embodiment 19-30, wherein the second agent comprises one or more selected from monoclonal antibodies, optionally selected from monospecific antibodies, bispecific antibodies, multispecific antibodies and a bispecific immune cell engager, antibody-drug conjugates, CAR therapies optionally selected from a CAR NK therapy, a CAR T therapy, a CAR cytotoxic T therapy, a CAR gamma-delta T therapy, a CAR NK therapy, cell therapies, inhibitors or antagonists of an inhibitory immune checkpoint, activators or agonists of a stimulatory immune checkpoint optionally selected from an activating ligand, immune regulators, cancer vaccines, and a vector delivering each thereof to a subject optionally in an oncolytic virus therapy, and optionally wherein the monoclonal antibodies are selected from rituximab, blinatumomab, alemtuzumab, ibritumomab tiuxetan, bevacizumab, bevacizumab-awwb, cetuximab, panitumumab, ofatumumab, denosumab, pertuzumab, obinutuzumab, elotuzumab, ramucirumab, dinutuximab, daratumumab, trastuzumab, trastuzumab -dkst, nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, AMF 514 (MEDI0680), balstilimab, avelumab, durvalumab, atezolizumab, ipilimumab, tremelimumab, zalifrelimab, and AGEN1181, and further optionally wherein the antibody-drug conjugates are selected from moxetumomab pasudotox-tdfk, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, gemtuzumab ozogamicin, tagraxofusp-erzs, polatuzumab vedotin-piiq, enfortumab vedotin-ejfv, trastuzumab deruxtecan, and sacituzumab govitecan-hziy, and further optionally, a checkpoint inhibitor, optionally selected from GS4224, AMP- 224, CA-327, CA-170, BMS-1001, BMS-1166, peptide-57, M7824, MGD013, CX-072, UNP-12, NP-12, or a combination of two or more thereof, or an anti-PD-1 agent, an anti-PD- L1 agent, an anti-CTLA-4 agent, an anti -LAG-3 agent, an anti-TIM-3 agent, an anti-TIGIT agent, an anti-VISTA agent, an anti-B7-H3 agent, an anti-BTLA agent, an anti-ICOS agent, an anti-GITR agent, an anti-4- IBB agent, an anti-OX40 agent, an anti-CD27 agent, an anti- CD28 agent, an anti-CD40 agent, and an anti-Siglec-15 agent.

[0251] 33. A method of inhibiting the growth of a liver cancer cell expressing a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, comprising contacting the cancer cell with illudalic-acid-7-BIA or an analog of illudalic-acid-7-BIA or a pharmaceutically acceptable salt of each thereof, optionally wherein the liver cancer is a hepatocellular carcinoma.

[0252] 34. A method of inhibiting the growth of a cancer cell in vivo or in vitro, comprising inhibiting expression of PTPRD and PTPRF in the cancer cell, wherein the genes encoding PTRPD and / or PTPRF are knocked down or PTPRD and / or PTPRF are inhibited with a chemical inhibitor.

[0253] 35. The method of embodiment 34, wherein the genes encoding PTRPD and PTPRF are knocked down.

[0254] 36. The method of embodiment 34, wherein PTPRD and PTPRF are inhibited with a chemical inhibitor.Equivalents

[0255] Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the disclosure embodied therein herein disclosed can be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this disclosure. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure.

[0256] The disclosure has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the disclosure with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0257] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0258] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting the growth of a cancer cell expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) or a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, comprising contacting the cancer cell with a PTPRD inhibitor or a PTPRF inhibitor, wherein the contacting is in vitro or in vivo, optionally wherein the cancer cell is a primary cancer cell, a metastatic cancer cell, or an immortalized cancer cell, and optionally wherein the cancer cell has loss of function mutant PTPRF protein or PTPRD protein or copy loss of PTPRF genotype or PTPRD genotype, and further optionally wherein the growth inhibition of the cancer cell with the PTPRD inhibitor or the PTPRF inhibitor has a greater effect on viability than the expression of the mutant protein alone.

2. The method of claim 1, wherein the cancer cell expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) protein is contacted with a PTPRD inhibitor.

3. The method of claim 1, wherein the cancer cell expressing a mutant PTPRF protein is contacted with a PTPRD inhibitor.

4. The method of any of claims 1-3, wherein the PTPRD inhibitor or the PTPRF inhibitor comprises illudalic-acid-7-BIA or an analog of illudalic-acid-7-BIA or a pharmaceutically acceptable salt of each thereof.

5. The method of any of claims 1-4, further comprising contacting the cancer cell with a second anti-cancer agent, optionally wherein the second anti-cancer agent is a chemotherapeutic agent or an immunotherapy, and further optionally wherein the second agent is contacted prior to, concurrently or subsequent to the PTPRD inhibitor or the PTPRF inhibitor.

6. The method of any of claims 1-5, further comprising determining the cancer cell growth inhibition subsequent to the contacting of the cancer cell with the PTPRD inhibitor or the PTPRF inhibitor.

7. The method of any of claims 1-6, wherein the cancer cell is a mammalian cancer cell, optionally wherein the mammalian cancer cell is selected from the group of a canine cancer cell, a feline cancer cell or a human cancer cell.

8. The method of any of claims 1-7 wherein the cancer cell is a liver cancer cell, optionally a hepatocellular cancer cell or a liver cholangiocarcinoma cell line.

9. A method for one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of cancer in a subject suffering from a cancer cell expressing a mutant protein tyrosine phosphatase receptor type F (PTPRF) or a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, comprising administering to subject an effective amount of a PTPRF inhibitor or a PTPRD inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subject, wherein the administration is selected from a first-line, second-line, third line, fourthline or fifth-line therapy, optionally determining whether the cancer expresses a mutant PTPRF protein or PTPRD protein, optionally wherein the cancer expresses a loss of function PTPRF mutant or PTPRD mutant or a copy loss PTPRF genotype or PTPRD genotype optionally wherein the cancer is a primary or a metastatic cancer, and optionally further comprising administering to the subject a second anti-cancer agent, wherein the second agent is administered prior to, concurrently or subsequent to the PTPRD inhibitor or PTPRF inhibitor, optionally wherein the second anti-cancer agent is a chemotherapeutic agent or an immunotherapy.

10. The method of claim 9, wherein the subject suffering from a cancer expressing a mutant PTPRF is administered an effective amount of an PTPRD inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subject.

11. The method of claim 9, wherein the subject suffering from a cancer expressing mutant PTPRD is administered an effective amount of an PTPRF inhibitor, thereby achieving one or more of inhibiting the growth of, metastasis, recurrence, or promoting treatment of the cancer in the subj ect.

12. The method of any of claims 9-11, wherein the PTPRD inhibitor or the PTPRF inhibitor comprises illudalic-acid-7-BIA or an analog of illudalic-acid-7-BIA or a pharmaceutically acceptable salt of each thereof.

13. The method of any of claims 9-12, further comprising determining the cancer cell growth inhibition subsequent to the contacting of the cancer cell with the PTPRD inhibitor or PTPRF inhibitor.

14. The method of any of claims 9-13, wherein the subject is a mammal, optionally wherein the mammal is selected from the group of a canine, a feline or a human.

15. The method of any of claims 9-14, wherein the cancer is a liver cancer, optionally a hepatocellular cancer or a liver cholangio-carcinoma.

16. The method of any one of claims 9-15, wherein after administration the subject experiences one or more endpoints selected from tumor response, reduction in tumor size, reduction in tumor burden, increase in overall survival, increase in progression free survival, inhibiting metastasis, improvement of quality of life, minimization of toxicity, avoidance of side-effects or a reduction of side-effects.

17. The method of any one of claims 9-16, wherein the second agent is selected from the group of: 5-fluorouracil, pemetrexed, raltitrexed, nolatrexed, plevitrexed, GS7904L, capecitabine, methotrexate, pralatrexate, CT-900, NUC-3373, FOLFOX, FOLFOX4, FOLFIRI, MOF, deflexifol, or a combination of 5-FU with one or more selected from radiation, methyl-CCNU, leucovorin, oxaliplatin (such as cisplatin), irinotecan, mitomycin, cytarabine, and levamisole, anthracycline or other topoisomerase II inhibitor comprises daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, etoposide or teniposide.

18. The method of any one of claims 9-16, wherein the second agent comprises one or more selected from monoclonal antibodies, optionally selected from monospecific antibodies, bispecific antibodies, multispecific antibodies and a bispecific immune cell engager, antibody-drug conjugates, CAR therapies optionally selected from a CAR NK therapy, a CAR T therapy, a CAR cytotoxic T therapy, a CAR gamma-delta T therapy, a CAR NK therapy, cell therapies, inhibitors or antagonists of an inhibitory immune checkpoint,activators or agonists of a stimulatory immune checkpoint optionally selected from an activating ligand, immune regulators, cancer vaccines, and a vector delivering each thereof to a subject optionally in an oncolytic virus therapy, and optionally wherein the monoclonal antibodies are selected from rituximab, blinatumomab, alemtuzumab, ibritumomab tiuxetan, bevacizumab, bevacizumab-awwb, cetuximab, panitumumab, ofatumumab, denosumab, pertuzumab, obinutuzumab, elotuzumab, ramucirumab, dinutuximab, daratumumab, trastuzumab, trastuzumab-dkst, nivolumab, pembrolizumab, cemiplimab, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, AMF 514 (MEDI0680), balstilimab, avelumab, durvalumab, atezolizumab, ipilimumab, tremelimumab, zalifrelimab, and AGEN1 181, and further optionally wherein the antibody-drug conjugates are selected from moxetumomab pasudotox-tdfk, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, gemtuzumab ozogamicin, tagraxofusp-erzs, polatuzumab vedotin-piiq, enfortumab vedotin-ejfv, trastuzumab deruxtecan, and sacituzumab govitecan-hziy, and further optionally, a checkpoint inhibitor, optionally selected from GS4224, AMP -224, CA- 327, CA-170, BMS-1001, BMS-1166, peptide-57, M7824, MGD013, CX-072, UNP-12, NP- 12, or a combination of two or more thereof, or an anti-PD-1 agent, an anti-PD-Ll agent, an anti-CTLA-4 agent, an anti-LAG-3 agent, an anti-TIM-3 agent, an anti-TIGIT agent, an anti- VISTA agent, an anti-B7-H3 agent, an anti-BTLA agent, an anti-ICOS agent, an anti-GITR agent, an anti -4- IBB agent, an anti-OX40 agent, an anti-CD27 agent, an anti-CD28 agent, an anti-CD40 agent, and an anti-Siglec-15 agent.

19. A method of inhibiting the growth of a liver cancer cell expressing a mutant protein tyrosine phosphatase receptor type D (PTPRD) protein, comprising contacting the cancer cell with illudalic-acid-7-BIA or an analog of illudalic-acid-7-BIA or a pharmaceutically acceptable salt of each thereof, optionally wherein the liver cancer is a hepatocellular carcinoma.

20. A method of inhibiting the growth of a cancer cell in vivo or in vitro, comprising inhibiting expression of PTPRD and PTPRF in the cancer cell, wherein the genes encoding PTRPD and PTPRF are knocked down or inhibited with a chemical inhibitor.

Images