CD70 targeting in cancer with acquired resistance to targeted therapeutic agents

Abstract

Disclosed herein are CD70-targeted treatments for RAS, RET, and ALK mutant cancers. The treatments are effective for preventing RAS, RET, and ALK inhibitor resistance, as well as for treating cancers that have developed resistance to RAS, RET, and ALK inhibitors. The present disclosure provides a method for treating a RAS, RET, or ALK mutant cancer in a subject comprising administering a CD70-targeted therapy to the subject, thereby treating the RAS, RET, or ALK-mutant cancer in the subject.

Description

CD70 TARGETING IN CANCER WITH ACQUIRED RESISTANCE TO TARGETED THERAPEUTIC AGENTS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 610,941, filed December 15, 2023. The content of the prior application is considered part of and is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTIONFIELD OF THE INVENTION

[0002] The present disclosure relates generally to methods of treating cancer and more specifically to CD70-targeted treatments for KRAS, RET, and ALK mutant cancers.BACKGROUND INFORMATION

[0003] Tyrosine kinase inhibitors (TKIs) are effective for treating numerous cancers, including many KRAS, RET, and ALK mutant cancers. However, drug resistance is a major clinical challenge for tyrosine kinase inhibitor treatment. Resistance to TKIs not only diminishes the efficacy of TKI-based treatments, but often also coincides with broader phenotypic changes in the cancer that promote adaptability and persistence. The subpopulation of cancer cells with these traits is often referred to as “drug-tolerant persister cells” (DTPCs). While DTPC drug tolerance is often reversible, these cells often also exhibit reduced metabolic activity, low proliferation rates, and stem-like phenotypes that improve their tolerance to a broad range of cancer treatments and enhance their survival. Owing to these improved survival traits, it is hypothesized that DTPCs are a common cause of cancer relapse.SUMMARY OF THE INVENTION

[0004] The present disclosure is based on the seminal discovery that RAS, RET, and ALK inhibitor resistance increase CD70 expression, and accordingly that CD70 overexpression is a feature of drug tolerant persister cells (DTPCs) that survive initial RAS, RET, and ALK inhibitor treatments. Leveraging this discovery, the present disclosure provides CD70-targeted therapies that localize immune cells or toxic payloads to RAS, RET, and ALK mutant cancer.

[0005] In one aspect, the present disclosure provides a method for treating a RAS, RET, or ALK mutant cancer in a subject that includes administering a CD70-targeted therapy to the subject, thereby treating the RAS, RET, or ALK-mutant cancer in the subject.

[0006] In some embodiments, the subject was previously administered a RAS inhibitor, RET inhibitor, or ALK inhibitor or combination thereof for the cancer. In some embodiments, the cancer progressed during the treatment with the RAS inhibitor, the RET inhibitor, or the ALK inhibitor or the combination thereof. In some embodiments, the cancer is resistant to the RAS inhibitor, the RET inhibitor, or the ALK inhibitor or the combination thereof.

[0007] In some embodiments, CD70 is upregulated in the cancer. In some embodiments, the cancer has undergone an epithelial to mesenchymal transition. In some embodiments, the cancer expresses an EMT marker selected from CDH1, BIM, AXL, ZEB1, or ZEB2.

[0008] In some embodiments, the CD70-targeted therapy includes a CD70 antagonist, an antibody-drug conjugate, a chimeric antigen receptor (CAR) T cell, a CAR natural killer (NK) cell, a tyrosine kinase inhibitor therapy (TKI) or a combination thereof.

[0009] In some embodiments, the method further includes administering a RAS inhibitor, a RET inhibitor, an ALK inhibitor, or a combination thereof to the subject. In some embodiments, the RAS inhibitor is selected from adagrasib, sotorasib, MRTX1257, JNJ- 74699157, LY3499446, KRAS (G12C) inhibitor 6, ARS-853, BPI-421286, LY3537982, JDQ443, JAB-21000, RMC-6291, or GDC-6036; the RET inhibitor is selected from BLU6864, cabozantinib, dovitinib, foretinib, lenvatinib, ponatinib, pralsetinib, selpercatinib, sorafenib, sunitinib, or vandetanib; the ALK inhibitor is selected from alectinib, alkotinib, belizatinib, brigatinib, ceritinib, conteltinib, crizotinib, ensartinib, entrectinib, foritinib, lorlatinib, repotrectinib, or zotizalkib; or a combination thereof. In some embodiments, the CD70-targeted therapy is administered concurrently with and / or subsequently to the RAS inhibitor, the RET inhibitor, the ALK inhibitor, or the combination thereof.

[0010] In some embodiments, the RAS is KRAS. In some embodiments, the KRAS includes a mutation selected from G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61R, Q61Y, or a combination thereof; the KRAS is a KRAS fusion selected from an ALK fusion, a BRAF fusion, an FGFR2 fusion, an NRF1 fusion, an NTRK1 fusion, an NTRK3 fusion, a RAFI fusion, a ROS1 fusion, or a UBEL2L3 fusion; the RET is a RET fusion selected from a CCDC6 fusion, a CUX1 fusion, a KIAA1468 fusion, a KIF5B fusion, an NCOA4 fusion, or a TRIM33 fusion; the ALK is an ALK fusion selected from an EMIL4 fusion, a KIF5B fusion, a KLC1 fusion, a PTPN3 fusion, or a TGF fusion; or a combination thereof.

[0011] In some embodiments, the method further includes administering an additional therapy selected from chemotherapy, radiation, surgery, tyrosine kinase inhibitor therapy, or immunotherapy. In some embodiments, the additional therapy includes pembrolizumab, nivolumab, durvalumab, atezolizumab, carboplatin, pemetrexed, nab-paclitaxel, photofrin,cisplatin, docetaxel, gemcitabine, paclitaxel, vinorelbine, alectinib, lorlatinib, ceritinib, gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, brigatinib, cusatuzumab, vorsetuzumab, auristatin E (MMAE), duocarmycin, monomethyl auristatin F (MMAF) or pyrrolobenzodiazepine (PBD) or a combination thereof. In some embodiments, the method further includes administering an adjuvant, a neoadjuvant, or a combination thereof to the subject.

[0012] In some embodiments, the cancer is selected from bile duct cancer, bladder cancer, brain cancer, breast cancer, carcinoma, cervical cancer, colorectal cancer, endometrial cancer, epitheloid carcinoma of the bone, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, hepatocellular cancer, large cell lung carcinoma, leukemia, lung cancer, medulloblastoma, melanoma, ovarian cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, renal cell cancer, or thyroid cancer. In some embodiments, the cancer is non- small cell lung cancer (NSCLC). In some embodiments, the NSCLC is lung adenocarcinoma. In some embodiments, the subject is a non-smoker.

[0013] In some embodiments, the cancer includes a RAS, RET, or ALK activating mutation. In some embodiments, the cancer has not been tested for CD70 expression. In some embodiments, the cancer expresses CD70.

[0014] In some embodiments, the cancer is not an EGFR mutant cancer.

[0015] In another aspect, the present disclosure provides a method for treating cancer in a subject that includes administering to the subject a CD70-targeted therapy; and a RAS inhibitor, a RET inhibitor, or an ALK inhibitor, thereby treating the cancer in the subject.

[0016] In some embodiments, the CD70-targeted therapy is administered concurrently with the RAS inhibitor, the RET inhibitor, or the ALK inhibitor. In some embodiments, the CD70- targeted therapy blocks CD70 signaling. In some embodiments, the CD70-targeted therapy includes a CD70 antagonist or a CD27 antagonist.

[0017] In some embodiments, the cancer is not resistant to the RAS inhibitor, the RET inhibitor, or the ALK inhibitor; CD70 is not upregulated in the cancer; the cancer has not undergone an epithelial to mesenchymal transition (EMT); or a combination thereof.

[0018] In some embodiments, the RAS inhibitor is selected from adagrasib, sotorasib, MRTX1257, JNJ-74699157, LY3499446, KRAS (G12C) inhibitor 6, ARS-853, BPI-421286, LY3537982, JDQ443, JAB-21000, RMC-6291, or GDC-6036; the RET inhibitor is selected from BLU6864, cabozantinib, dovitinib, foretinib, lenvatinib, ponatinib, pralsetinib, selpercatinib, sorafenib, sunitinib, or vandetanib; the ALK inhibitor is selected from alectinib,alkotinib, belizatinib, brigatinib, ceritinib, conteltinib, crizotinib, ensartinib, entrectinib, foritinib, lorlatinib, repotrectinib, or zotizalkib; or a combination thereof.

[0019] In some embodiments, the RAS is KRAS. In some embodiments, the KRAS includes a mutation selected from G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61R, Q61Y, or a combination thereof; the KRAS is a KRAS fusion selected from an ALK fusion, a BRAF fusion, an FGFR2 fusion, an NRF1 fusion, an NTRK1 fusion, an NTRK3 fusion, a RAFI fusion, a ROS1 fusion, or a UBEL2L3 fusion; the RET is a RET fusion selected from a CCDC6 fusion, a CUX1 fusion, a KIAA1468 fusion, a KIF5B fusion, an NCOA4 fusion, or a TRIM33 fusion; the ALK is an ALK fusion selected from an EMIL4 fusion, a KIF5B fusion, a KLC1 fusion, a PTPN3 fusion, or a TGF fusion; or a combination thereof.

[0020] In some embodiments, the cancer is not an EGFR mutant cancer.

[0021] In a further aspect, the present disclosure provides a method of identifying a subject with cancer as a candidate for treatment with a CD70-targeted therapy that includes a) determining whether the cancer is resistant to a RAS inhibitor, a RET inhibitor, or an ALK inhibitor; and b) classifying the subject as a likely responder to the CD70-targeted therapy if the cancer is resistant the RAS inhibitor, the RET inhibitor, or the ALK inhibitor, thereby identifying subject as suitable for treatment with the CD70-targeted therapy; or c)c lassi tying the subject as an unlikely responder to the CD70-targeted therapy if the cancer is not resistant to the RAS inhibitor, the RET inhibitor, or the ALK inhibitor, thereby identifying subject as an unlikely responder and unsuitable for treatment with the CD70-targeted therapy, thereby identifying a subject with cancer as a candidate for a CD70-targeted treatment.

[0022] In some embodiments, determining includes in vivo tumor imaging, a solid tumor biopsy, a liquid tumor biopsy, a cancer biomarker panel, circulating tumor cell analysis, circulating tumor DNA analysis, circulating tumor RNA analysis, mRNA sequencing, DNA sequencing, protein expression measurements, in vitro inhibitor analysis, or a combination thereof.

[0023] In some embodiments, the cancer is not an EGFR mutant cancer.BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIGS. 1A-1C illustrate a series of plots of cancer cell expression data. FIG. 1A illustrates a plot showing endothelial to mesenchymal transition scores for KRAS mutant cancer cells prior to exposure to a KRAS G12C inhibitor, 24 hours after exposure to the inhibitor, 48 hours after exposure to the inhibitor, and following acquisition of resistance to the inhibitor. FIG. IB illustrates a plot showing CD70 expression data for KRAS mutant cancercells prior to exposure to a KRAS G12C inhibitor, 24 hours after exposure to the inhibitor, 48 hours after exposure to the inhibitor, and following acquisition of resistance to the inhibitor. FIG. 1C illustrates a plot that shows the percent of cancer cells from multiple KRAS inhibitor resistant and non-resistant clones that are CD70 positive.

[0025] FIGS. 2A-2B illustrate a series of bar graphs of mRNA expression in multiple KRAS mutant cancer cell lines prior to exposure to a KRAS inhibitor and following acquisition of resistance to the KRAS inhibitor. FIG. 2A illustrates graphs that show ZEB1 mRNA expression data for the cancer cell lines. FIG. 2B illustrates graphs that show CD70 mRNA expression data for the cancer cell lines.

[0026] FIGS. 3A-3D illustrate a set of graphs that show CD70 expression and CD70- targeting CAR T cell mediated lysis of a cancer cell line prior to and post-acquisition to KRAS inhibitor resistance. FIG. 3A illustrates a plot showing CD70 flow cytometry data. FIG. 3B illustrates a bar graph showing CD70 expression data. FIG. 3C illustrates a plot showing specific lysis values for non-KRAS inhibitor resistant cancer cells following exposure to CD70-targeting CAR T cells. FIG. 3D illustrates a plot showing specific lysis values for KRAS inhibitor resistant cancer cells following exposure to CD70-targeting CAR T cells.

[0027] FIGS. 4A-4D illustrate a set of graphs that show CD70 expression and CD70- targeting CAR T cell mediated lysis of a second cancer cell line prior to and post-acquisition to KRAS inhibitor resistance. FIG. 4A illustrates a plot showing CD70 flow cytometry data. FIG. 4B illustrate a bar graph showing CD70 expression data. FIG. 4C illustrates a plot showing specific lysis values for non-KRAS inhibitor resistant cancer cells following exposure to CD70-targeting CAR T cells. FIG. 4D illustrates a plot showing specific lysis values for KRAS inhibitor resistant cancer cells following exposure to CD70-targeting CAR T cells.

[0028] FIG. 5 illustrates a bar graph that shows relative cell viability upon treatment with 0 or 20 pg / ml of a CD70-targeting ADC for three KRAS inhibitor resistant cancer cell clones and one non-resistant cancer cell clone.

[0029] FIGS. 6A-6B illustrate a set of plots showing mRNA expression data for a RET fusion positive cancer cell line prior and subsequent to acquisition of resistance to a RET inhibitor. FIG. 6A illustrates a plot showing ZEB 1 mRNA expression. FIG. 6B illustrates a plot showing CD70 mRNA expression.

[0030] FIGS. 7A-7B illustrate a set of plots showing protein expression of CD70 on RET inhibitor resistant cells and CD70 targeting. FIG 7A illustrates a plot that shows CD70 expression on the surface of selpecatinib (RET inhibitor) resistant LC2ad (RET fusion positive)cancer cells. FIG 7B illustrates a graph that shows CD70 CAR mediated killing of LC2- selpercatinib resistant cells (CD70 positive).

[0031] FIG. 8 illustrates a plot showing CD70 RNA expression on H3122 cells treated with the ALK inhibitor crizotinib. Crizotinib treated DTPCs show increased expression of CD70.

[0032] FIGS. 9A-9D illustrate a set of plots showing gene expression of EMT related genes and CD70 in H2228 (ALK fusion positive) cells and H2228 cells with acquired resistance to the ALK inhibitor alectinib. H2228 alectinib resistant cells show an EMT gene expression signature with increased expression of vimentin (VIM) and AXL which are both markers of EMT. CD70 expression is increased in alectinib resistant cells. FIG. 9A illustrates a plot showing EMT scores for the H2228 cells. FIG. 9B illustrates a plot showing vimentin expression in the H2228 cells. FIG. 9C illustrates a plot showing AXL expression in the H2228 cells. FIG. 9D illustrates a plot showing CD70 expression in the H2228 cells.

[0033] FIGS. 10A-10E illustrate drug sensitivity curves of ALK fusion positive DFCI032 cells with or without acquired resistance to ALK inhibitors. FIG 10A illustrates a graph showing drug sensitivity curves of ALK fusion positive DFCI032 cells with or without acquired resistance to alectinib. FIG 10B illustrates a graph showing drug sensitivity curves of ALK fusion positive DFCI032 cells with or without acquired resistance to crizotinib. FIG 10C illustrates a graph showing drug sensitivity curves of ALK fusion positive DFCI032 cells with or without acquired resistance to brigatinib. FIG 10D illustrates a graph showing drug sensitivity curves of ALK fusion positive DFCI032 cells with or without acquired resistance to lorlatinib. FIG 10E illustrates a graph showing that ALK inhibitor resistant cells upregulate CD70 expression.DETAILED DESCRIPTION OF THE INVENTION

[0034] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0035] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and / or steps of the typedescribed herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0036] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0037] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0038] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0039] The present disclosure is based on the seminal discovery that RAS, RET, and ALK inhibitor resistance increases CD70 expression, and that CD70 overexpression is a feature of drug tolerant persister cells (DTPCs) that survive initial RAS, RET, and ALK inhibitor treatments. It is further demonstrated herein that this CD70 upregulation can be exploited to target RAS, RET, and ALK inhibitor resistant cells using antibody-drug conjugates (ADCs), chimeric antigen receptor (CAR) T cells, and other targeted therapies that direct cytotoxic payloads or oncolytic agents to drug resistant cells. As DTPCs are often a cause of cancer recalcitrance, these targeted therapies are particularly suitable for treating cancers with a high incidence of relapse.

[0040] Leveraging this discovery, in one aspect, the present disclosure provides a method for treating a RAS, RET, or ALK mutant cancer in a subject by administering a CD70-targeted therapy to the subject, thereby treating the RAS, RET, or ALK-mutant cancer in the subject. In some embodiments, the cancer is RAS or RET mutant cancer.

[0041] As used herein, the terms “RAS,” “RAS GTPase,” and “RAS protein” refer to any protein within the RAS family of GTPase proteins, as well as naturally occurring and engineered fusions, variants, and isoforms of these proteins. RAS family proteins activate numerous members of the MAP -kinase pathway. The RAS family encompasses more than 150 distinct proteins including, HRAS, KRAS, NRAS, DIRAS1 , DIRAS2, DIRAS3, ERAS, GEM, MRAS, NKIRAS1, NKIRAS2, and RRAS. However, in some embodiments disclosed herein, RAS specifically denotes KRAS. RAS mutations are common in many types of cancer, including but not limited to pancreatic cancer, colorectal cancer, lung cancer, non-small celllung cancer (NSCLC), appendiceal cancer, small-bowel cancer, biliary tract malignancies, endometrial cancer, cervical cancer, bladder cancer, liver cancer, myeloid leukemia, breast cancer, thyroid Cancer, Prostate adenocarcinoma, cutaneous melanoma, hepatocellular carcinoma, bladder urothelial, cervical squamous cell carcinoma, endometrial adenocarcinoma, ovarian serous cystadenocarcinoma, Uterine carcinosarcoma, breast invasive ductal carcinoma, breast invasive lobular carcinoma, papillary thyroid cancer, prostate adenocarcinoma, cholangiocarcinoma, esophageal adenocarcinoma, astrocytoma, and glioblastoma multiforme.

[0042] As used herein, the terms “RET” and “RET protein” refer to a receptor tyrosine kinase that is naturally encoded by the RET protooncogene, as well as natural and engineered fusions and variants thereof. RET activates numerous signaling pathways including PI3K / AKT, RAS / MAPK, JAK / STAT, and PKA / PKC. Aberrant RET activity often stems from oncogenic translocations that produce constitutively active RET fusion proteins, which can promote cancer pathogenesis through by enhancing cellular proliferation, migration, and differentiation. RET mutations are involved in various types of cancers, including but not limited to thyroid cancer, lung cancer, gastrointestinal cancer, breast cancer, pancreatic carcinoma, bladder carcinoma, ovarian carcinoma, and renal cell carcinoma, gallbladder carcinoma, gastric adenocarcinoma, gastric carcinoma, gastrointestinal stromal tumor, head and neck carcinoma, head and neck squamous cell carcinoma, hepatobiliary neoplasm, hepatoblastoma, hepatocellular carcinoma, lip and oral cavity carcinoma, lung carcinoma, malignant central nervous system neoplasm, malignant hepatobiliary neoplasm, malignant laryngeal neoplasm, malignant salivary gland neoplasm, malignant uterine neoplasm, multiple myeloma, nasal cavity and paranasal sinus carcinoma, nasopharyngeal carcinoma, neuroendocrine carcinoma, non-Hodgkin lymphoma, oropharyngeal carcinoma, osteosarcoma, poorly differentiated thyroid gland carcinoma, rhabdomyosarcoma, squamous cell lung carcinoma, urothelial carcinoma, and Wilms tumor.

[0043] “ALK” (also known as “CD246” and anaplastic lymphoma kinase) is a receptor tyrosine kinase in the insulin receptor superfamily. In humans, ALK is primarily expressed in the brain, central nervous system, and small intestines, where it promotes intercellular communication and neural development. Upon activation, ALK phosphorylates numerous receptor proteins to activate downstream MAPK-ERK, PI3K-AKT, PLCy, CRKL-C3G, and JAK-STAT signaling pathways. Genetic translocations and deletions that generate mutant or fusion ALK proteins have been implicated in numerous cancers including but not limited to lung cancer, neuroblastoma, renal cell carcinoma, breast cancer, colon cancer, renal medullary carcinoma, diffuse large b-cell lymphoma, glioblastoma, esophageal squamous cell carcinoma,diffuse large B-cell lymphoma, non-small cell lung cancer, esophageal squamous cell carcinoma, and anaplastic large cell lymphoma, and pulmonary sarcomatoid carcinoma.

[0044] As used herein, the terms “CD70” refer to a costimulatory molecule that helps activate cytotoxic T cells and regulate B cell activation. CD70 is expressed on the surface of activated lymphocytes and is the ligand for the CD27 receptor on B cells. CD70 is aberrantly expressed in various solid tumors, including brain, ovarian, lung, colon, and pancreatic cancer. High CD70 expression in cancer cells may inhibit the anti-tumor response.

[0045] The term “subject” as used herein refers to any individual or patient to which the disclosed methods are performed, to whom the disclosed compositions are administered, or from whom a biological material (e.g., a tissue sample, a cell, or a biofluid) is obtained. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be a non-human animal. Thus, other animals, including vertebrate such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, chickens, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0046] The term "treatment" is used interchangeably herein with the term "therapeutic method" or “therapy” and refers to 1) therapeutic treatments or measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic conditions or disorder, and / or 2) prophylactic / preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e. , those needing preventive measures).

[0047] The terms “administration of’ and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral, topical, or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrastemal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, nebulization, otic, buccal, conjunctival, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extraamniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarticular, intrabiliary, intrabronchial, intrabursal, intracartilaginous, intracaudal, intracavemous, intracavitary, intracerebroventricular, intracistemal, intracorneal, intracoronal, intracoronary, intracorpous cavemaosum, intradiscal, intraductal, intraduodenal, intradural, intraepidermal,intraesophageal, intragastric, intragingival, intrahippocampal, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intrapleural, intraprostatic, intrapulmonary, intrasinal, intrasynovial, intratendinous, intratesticular, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous bolus, intravenous drip, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasogastric, ophthalmic, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, retrobulbar, subconjunctival, sublingual, submucosal, topical, transmucosal, transplacental, transtympanic, ureteral, urethral, infraorbital, intraparenchymal, intraventricular, stereotactic administration subcuticular, or any combination thereof. A form of administration can be tailored for a particular cancer based on the properties and localization of the cancer and drug.

[0048] The terms “therapeutically effective amount”, “effective dose,” “therapeutically effective dose”, “effective amount,” or the like refer to that amount of the subject agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician. Generally, the response is either amelioration of symptoms in a patient or a desired biological outcome (e.g., treatment of the disease). Such amount should be sufficient to eliminate tumor cells. The effective amount can be determined as described herein.

[0049] In some embodiments, the subject was previously administered a RAS inhibitor, RET inhibitor, or ALK inhibitor or combination thereof for the cancer. While the CD70 signaling axis is separate from native RAS, RET, and ALK pathways, it was surprisingly shown herein that CD70 is upregulated during the acquisition of RAS, RET, and ALK inhibitor resistance. CD70 upregulation in RAS, RET, and ALK mutant cancers can be exploited to direct cytotoxic, immune activating, or other antineoplastic agents to the cancer.

[0050] In some embodiments, the cancer is resistant to the RAS inhibitor, the RET inhibitor, or the ALK inhibitor or the combination thereof. Alternatively, or in addition thereto, CD70 can be upregulated in the cancer. In further embodiments, the cancer has undergone an epithelial to mesenchymal transition. In some embodiments, the cancer expresses an EMT marker selected from CDH1, BIM, AXL, ZEB1, or ZEB2. As non-limiting examples, inhibitor resistance, CD70 upregulation, and EMT can be determined with in vivo tumor imaging (e.g., to detect DTPC or EMT biomarkers), solid tumor biopsies, liquid tumor biopsies, cancer biomarker panels, circulating tumor cell analyses, circulating tumor DNA analyses, circulating tumor RNA analyses, mRNA sequencing, DNA sequencing, protein expression measurements,in vitro inhibitor analyses, or combinations thereof. Inhibitor resistance can also be determined with a cell survival assay performed with an inhibitor of interest.

[0051] As used herein the term “resistance” or “treatment resistance” refers to the ability to resist drugs that are usually effective. Drug resistance may be present before treatment is given or may occur during or after treatment with the drug. In cancer treatment, there are many things that may cause resistance to anticancer drugs. For example, DNA changes or other genetic or molecular changes may change the way the drug gets into the cancer cells or the way the drug is broken down within the cancer cells. Drug resistance can lead to cancer treatment not working or to the cancer coming back.

[0052] In some embodiments, the cancer progressed during the treatment with the RAS inhibitor, the RET inhibitor, or the ALK inhibitor or the combination thereof. In such cases, the cancer progression may be taken as evidence of inhibitor resistance. For example, diminished efficacy of an initially effective RAS, RET, or ALK inhibitor may provide evidence for the development of inhibitor resistance in the cancer.

[0053] In some embodiments, the CD70-targeted therapy includes a CD70 antagonist, an antibody-drug conjugate, a chimeric antigen receptor (CAR) T cell, a CAR natural killer (NK) cell, a tyrosine kinase inhibitor therapy (TKI) or a combination thereof. In many embodiments, the CD70-targeted therapy directs a chemotherapeutic agent or species that otherwise causes or promotes cancer cell death, such as a T cell or NK cell, to the cancer. As CD70 will often be disproportionately expressed by the RAS , RET, or ALK mutant cancer, these CD70-targeted treatments can direct the chemotherapeutic agent or species to the cancer cells. Accordingly, in some embodiments, the therapy includes an antibody-drug conjugate, a chimeric antigen receptor (CAR) T cell, a CAR natural killer (NK) cell, a tyrosine kinase inhibitor therapy (TKI) or a combination thereof.

[0054] As used herein the term “CD70 antagonist” refers to a molecule, typically a monoclonal antibody, that blocks the interaction between the protein CD70 (Cluster of Differentiation 70) and its receptor, CD27, essentially preventing the activation signals that CD70 normally delivers to immune cells. CD70 antagonists are potential therapeutic targets for cancer treatment where CD70 is overexpressed and contributes to tumor growth and immune evasion.

[0055] As used herein the term “antibody-drug conjugate” or “ADC” refers to a substance made up of an antibody chemically linked to a drug. ADC may be a therapeutic agent that combines a monoclonal antibody (mAb) with a cytotoxic drug to treat cancer.

[0056] As used herein the term “CAR T cell” refers to a genetically engineered T cell, a type of immune cell, where a synthetic receptor called a CAR is added to its surface, allowing it to specifically recognize and kill cancer cells by binding to a unique antigen on a tumor cell surface. CAR T cell is a type of immunotherapy that uses genetically modified T cells to treat cancer.

[0057] As used herein the term “CAR natural killer (NK) cell” refers to a type of immunotherapy that uses genetically modified cells to treat cancer. CAR NK cells are derived from natural killer cells.

[0058] As used herein the term “tyrosine kinase inhibitor therapy” or “TKI therapy” refers to a type of targeted cancer treatment that works by blocking the activity of enzymes called tyrosine kinases, which are crucial for cell growth and signaling, effectively preventing cancer cells from proliferating. TKI therapy specifically targets tyrosine kinases, often found in elevated levels in cancer cells, and does not harm healthy cells broadly.

[0059] In some embodiments, the method further includes administering a RAS inhibitor, a RET inhibitor, an ALK inhibitor, or a combination thereof to the subject. In some embodiments, the CD70-targeted therapy is administered concurrently with and / or subsequently to the RAS inhibitor, the RET inhibitor, the ALK inhibitor, or the combination thereof. This can include continuing administration of the RAS, RET, and / or ALK inhibitor while administering the CD70-targeted therapy.

[0060] As used herein the term “RAS inhibitor” refers to drugs that target the RAS protein to treat cancer. RAS protein inhibitors block the activity of the RAS protein, which plays a critical role in cell growth and differentiation. RAS protein inhibitors can be small molecules that enter the cell and inhibit proteins or enzymes. RAS protein inhibitors can also bind to an intracellular chaperone protein, which then binds to the RAS protein to form a tri-complex that inhibits RAS-dependent signaling. As non-limiting examples, in some embodiments, the RAS inhibitor is selected from adagrasib (MRTX849), sotorasib (AMG510), MRTX1257, JNJ-74699157, LY3499446, KRAS (G12C) inhibitor 6, ARS-853, BPI-421286, LY3537982, JDQ443, JAB- 21000, RMC-6291, or GDC-6036, lonafamib (SCH-66336), deltarasin, and MRTX1133.

[0061] As used herein the term “RET inhibitor” refers to a type of targeted therapy that treats cancers caused by abnormalities in the RET proto-oncogene protein. RET inhibitors block the activity of the RET proto-oncogene, which is involved in cell growth. This inhibits the growth of tumors that have increased RET activity. RET inhibitors target the ATP binding pocket of the kinase domain, which is activated by RET mutations and fusions. In some embodiments,the RET inhibitor is selected from BLU6864, cabozantinib, dovitinib, foretinib, lenvatinib, ponatinib, pralsetinib, selpercatinib, sorafenib, sunitinib, or vandetanib.

[0062] As used herein the term “ALK inhibitor” refers to anti-cancer drugs that target and block the activity of the ALK protein. In some embodiments, the ALK inhibitor is selected from alectinib, alkotinib, belizatinib, brigatinib, ceritinib, conteltinib, crizotinib, ensartinib, entrectinib, foritinib, lorlatinib, repotrectinib, or zotizalkib.

[0063] As used herein the term “cancer treatment” refers to any treatment that prevents or stops, or slows, the growth of cancer. Examples of anti-cancer treatment include but are not limited to chemotherapy, radiation therapy, immunotherapy, resection surgery, hormone therapy, targeted therapy, and stem cell or bone marrow transplant.

[0064] In some embodiments, the method further includes administering an additional therapy. As non-limiting examples, the additional therapy can be selected from chemotherapy, radiation, surgery, tyrosine kinase inhibitor therapy, or immunotherapy.

[0065] By chemotherapy is meant a cancer treatment that uses drugs to kill cancer cells, stop or slow their growth. Chemotherapy can be used to cure cancer, reduce the chance of it returning, or ease symptoms and are generally known to those of skill in the art. Chemotherapy can be used in conjunction with other treatments, such as radiotherapy or surgery. Chemotherapy can be administered in many ways, including orally, intravenously, or topically. The treatment can take place at home, in a hospital, or at a day clinic for example. Examples of chemotherapy include but are not limited to, Actinomycin, Azacitidine, Azathioprine, Bleomycin, Bortezomib, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Epothilone, Etoposide, Fiuorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Imatinib, Irinotecan, Mechlorethamine, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Teniposide, Tioguanine, Topotecan, Valrubicin, Vinblastine, Vincristine, Vindesine, Vinorelbine, panitumamab, Erbitux (cetuximab), matuzumab, IMC-IIF 8, TheraCIM hR3, denosumab, Avastin (bevacizumab), Humira (adalimumab), Herceptin (trastuzumab), Remicade (infliximab), rituximab, Synagis (palivizumab), Mylotarg (gemtuzumab oxogamicin), Raptiva (efalizumab), Tysabri (natalizumab), Zenapax (dacliximab), NeutroSpec (Technetium (99mTc) fanolesomab), tocilizumab, ProstaScint (Indium-Ill labeled Capromab Pendetide), Bexxar (tositumomab), Zevalin (ibritumomab tiuxetan (IDEC-Y2B8) conjugated to yttrium 90), Xolair (omalizumab), MabThera (Rituximab), ReoPro (abciximab), MabCampath (alemtuzumab), Simulect (basiliximab), LeukoScan (sulesomab), CEA-Scan (arcitumomab), Verluma (nofetumomab),Panorex (Edrecolomab), alemtuzumab, CDP 870, natalizumab Gilotrif (afatinib), Lynparza (olaparib), Perjeta (pertuzumab), Otdivo (nivolumab), Bosulif (bosutinib), Cabometyx (cabozantinib), Ogivri (trastuzumab-dkst), Sutent (sunitinib malate), Adcetris (brentuximab vedotin), Alecensa (alectinib), Calquence (acalabrutinib), Yescarta (ciloleucel), Verzenio (abemaciclib), Keytruda (pembrolizumab), Aliqopa (copanlisib), Nerlynx (neratinib), Imfinzi (durvalumab), Darzalex (daratumumab), Tecentriq (atezolizumab), Tarceva (erlotinib), nitrogen mustards, alkylsulfonates, nitrosoureas, triazines, ethylenimines, platinum drugs, genotoxic agents, all-trans retinoic acid, arsenic trioxide, asparaginase, eribulin, ixabepilone, mitotane, omacetaxine, pegaspargase, procarbazine, romidepsin, and vorinostat.

[0066] By radiation or radiotherapy is meant a cancer treatment that uses radiation to kill or control the growth of cancer cells. Radiotherapy uses high doses of radiation from sources like X-rays, gamma rays, neutrons, or protons to damage and destroy cancer cells. Radiation can be delivered externally with a machine outside the body, or internally with radioactive material placed in the body.

[0067] Immunotherapy is a treatment that uses substances to activate or suppress the immune system to help fight disease. Immunotherapies can be used to treat a variety of conditions, including cancer, infections, and other diseases. Immunotherapy can target and destroy proteins or receptors on cancer cells to prevent them from evading the immune system. Examples of immunotherapies include but are not limited to immune checkpoint inhibitors, T-cell transfer therapy, interleukins (11-2, 11-7, 11-12), cytokines (Interferons, G-CSF, imiquimod), chemokines (CCL3, CC126, CXCL7), immunomodulatory imide drugs (thalidomide and its analogues), and antibodies.

[0068] Resection surgery or surgery is a surgical procedure that removes tissue, part, or all of an organ. It can be performed for a variety of reasons, including to remove diseased or cancerous tissue, or to treat or cure a disease process.

[0069] In particular embodiments, the additional therapy includes pembrolizumab, nivolumab, durvalumab, atezolizumab, carboplatin, pemetrexed, nab-paclitaxel, photofrin, cisplatin, docetaxel, gemcitabine, paclitaxel, vinorelbine, alectinib, lorlatinib, ceritinib, gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, brigatinib, cusatuzumab, vorsetuzumab, auristatin E (MMAE), duocarmycin, monomethyl auristatin F (MMAF) or pyrrolobenzodiazepine (PBD) or a combination thereof.

[0070] In some embodiments, the method further includes administering an adjuvant, a neoadjuvant, or a combination thereof to the subject. As used herein, the term “adjuvant” can denote a species that enhances an immune or anti-tumor response that is administeredconcurrently with or after a therapy, while the term “neoadjuvant” can denote a species that is administered prior to therapy. Typically, an adjuvant exhibits minimal effects when administered on its own. Non-limiting examples of adjuvants include aluminum salts such as aluminum hydroxide, Freund’s adjuvants, Freund’s complete adjuvants, lipopolysaccharides, saponins, cholera toxin, unmethylated CpG dinucleotide-containing DNA, monophosphoryl lipid A (MPL), QS21, CpG 1018, matrix-MTM, MF59, AS03, AS04, and matrix M.

[0071] The term “cancer” refers to a group diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to other sites (secondary sites, metastases) which differentiate cancer (malignant tumor) from benign tumor. Virtually all the organs can be affected, leading to more than 100 types of cancer that can affect humans. Cancers can result from many causes including genetic predisposition, viral infection, exposure to ionizing radiation, exposure environmental pollutant, tobacco and or alcohol use, obesity, poor diet, lack of physical activity or any combination thereof.

[0072] Examples of cancers include but are not limited to Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS-Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma / Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma / Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas / Carcinoids, Childhood: Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma / Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer;Esophageal Cancer, Childhood; Ewing's Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary); Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin's Lymphoma, Adult; Hodgkin's Lymphoma, Childhood; Hodgkin's Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi's Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS-Related; Lymphoma, Central Nervous System (Primary); Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin's, Adult; Lymphoma, Hodgkin's; Childhood; Lymphoma, Hodgkin's During Pregnancy; Lymphoma, Non-Hodgkin’s, Adult; Lymphoma, Non-Hodgkin's, Childhood; Lymphoma, Non-Hodgkin's During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom’s; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma / Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin's Lymphoma, Adult; Non-Hodgkin's Lymphoma, Childhood; Non-Hodgkin's Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer; Osteosarcoma / Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian GermCell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood', Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm / Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin’s Lymphoma; Pregnancy and Non-Hodgkin’s Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma; Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi’s; Sarcoma Osteosarcoma / Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom’s Macro globulinemia; and Wilms’ Tumor.

[0073] In some embodiments, the cancer is selected from bile duct cancer, bladder cancer, brain cancer, breast cancer, carcinoma, cervical cancer, colorectal cancer, endometrial cancer, epitheloid carcinoma of the bone, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, hepatocellular cancer, large cell lung carcinoma, leukemia, lung cancer, medulloblastoma, melanoma, ovarian cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, renal cell cancer, or thyroid cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the NSCLC is lung adenocarcinoma. In some embodiments, the subject is a non-smoker.

[0074] In some embodiments, the cancer includes a RAS, RET, or ALK activating mutation. In some embodiments, the cancer includes a RAS or RET activating mutation. In someembodiments, the cancer has not been tested for CD70 expression. In some embodiments, the cancer expresses CD70. In some embodiments, the cancer is not an EGFR mutant cancer.

[0075] As used herein the term “EGFR” refers to epidermal growth factor receptor. EGFR is a protein found in some cells that control cell division and survival. Mutations in the EGFR gene can cause cancer cells to divide more rapidly. EGFR mutant cancer is a type of cancer that occurs when the gene for the EGFR protein is mutated, causing cells to grow abnormally, and spread.

[0076] In some embodiments, the RAS is KRAS. In some embodiments, the KRAS includes a mutation selected from G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61R, Q61Y, or a combination thereof; the KRAS is a KRAS fusion selected from an ALK fusion, a BRAF fusion, an FGFR2 fusion, an NRF1 fusion, an NTRK1 fusion, an NTRK3 fusion, a RAFI fusion, a ROS1 fusion, or a UBEL2L3 fusion; the RET is a RET fusion selected from a CCDC6 fusion, a CUX1 fusion, a KIAA1468 fusion, a KIF5B fusion, an NCOA4 fusion, or a TRIM33 fusion; the ALK is an ALK fusion selected from an EMIL4 fusion, a KIF5B fusion, a KLC1 fusion, a PTPN3 fusion, or a TGF fusion; or a combination thereof.

[0077] Leveraging the discovery that CD70 upregulation is an early event during RAS, RET, and ALK inhibitor resistance development, it is contemplated herein that blocking CD70 signaling (for example with a CD70-targeted antibody or mutated form of CD27) during RAS, RET, and ALK inhibitor-treatment can prevent drug resistance and associated phenotypic changes such as epithelial to mesenchymal transition (EMT). Accordingly, in another aspect, the present disclosure provides a method for treating cancer in a subject that includes administering to the subject a CD70-targeted therapy; and a RAS inhibitor, a RET inhibitor, or an ALK inhibitor, thereby treating the cancer in the subject.

[0078] The term “treatment” is used interchangeably herein with the term “therapeutic method” or “therapy” and refers to 1) therapeutic treatments or measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic conditions or disorder, and / or 2) prophylactic / preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (i.e. , those needing preventive measures).

[0079] In some embodiments, the CD70-targeted therapy is administered concurrently with the RAS inhibitor, the RET inhibitor, or the ALK inhibitor. In some embodiments, the CD70- targeted therapy blocks CD70 signaling. In some embodiments, the CD70-targeted therapy includes a CD70 antagonist or a CD27 antagonist. For example, in some embodiments, the CD70-targeted therapy comprises an antibody or a fragment thereof.

[0080] In some embodiments, the cancer is not resistant to the RAS inhibitor, the RET inhibitor, or the ALK inhibitor; CD70 is not upregulated in the cancer; the cancer has not undergone an epithelial to mesenchymal transition (EMT); or a combination thereof. In such cases, the CD70-targeted therapy can prevent or slow the development of one or more of these processes.

[0081] In some embodiments, the RAS inhibitor is selected from adagrasib, sotorasib, MRTX1257, JNJ-74699157, LY3499446, KRAS (G12C) inhibitor 6, ARS-853, BPI-421286, LY3537982, JDQ443, JAB-21000, RMC-6291, or GDC-6036. In some embodiments, the RET inhibitor is selected from BLU6864, cabozantinib, dovitinib, foretinib, lenvatinib, ponatinib, pralsetinib, selpercatinib, sorafenib, sunitinib, or vandetanib. In some embodiments, the ALK inhibitor is selected from alectinib, alkotinib, belizatinib, brigatinib, ceritinib, conteltinib, crizotinib, ensartinib, entrectinib, foritinib, lorlatinib, repotrectinib, or zotizalkib.

[0082] In some embodiments, the RAS is KRAS, hi some embodiments, the KRAS includes a mutation selected from G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61R, Q61Y, or a combination thereof; the KRAS is a KRAS fusion selected from an ALK fusion, a BRAF fusion, an FGFR2 fusion, an NRF1 fusion, an NTRK1 fusion, an NTRK3 fusion, a RAFI fusion, a ROS1 fusion, or a UBEL2L3 fusion; the RET is a RET fusion selected from a CCDC6 fusion, a CUX1 fusion, a KIAA1468 fusion, a KIF5B fusion, an NCOA4 fusion, or a TRIM33 fusion; the ALK is an ALK fusion selected from an EMIL4 fusion, a KIF5B fusion, a KLC1 fusion, a PTPN3 fusion, or a TGF fusion; or a combination thereof. In some embodiments, the cancer is not an EGFR mutant cancer.

[0083] In a further aspect, the present disclosure provides a method of identifying a subject with cancer as a candidate for treatment with a CD70-targeted therapy that includes a) determining whether the cancer is resistant to a RAS inhibitor, a RET inhibitor, or an ALK inhibitor; and b) classifying the subject as a likely responder to the CD70-targeted therapy if the cancer is resistant the RAS inhibitor, the RET inhibitor, or the ALK inhibitor, thereby identifying subject as suitable for treatment with the CD70-targeted therapy; or c) classifying the subject as an unlikely responder to the CD70-targeted therapy if the cancer is not resistant to the RAS inhibitor, the RET inhibitor, or the ALK inhibitor, thereby identifying subject as an unlikely responder and unsuitable for treatment with the CD70-targeted therapy, thereby identifying a subject with cancer as a candidate for a CD70-targeted treatment.

[0084] In some embodiments, determining includes in vivo tumor imaging, a solid tumor biopsy, a liquid tumor biopsy, a cancer biomarker panel, circulating tumor cell analysis, circulating tumor DNA analysis, circulating tumor RNA analysis, mRNA sequencing, DNAsequencing, protein expression measurements, in vitro inhibitor analysis, or a combination thereof.

[0085] In some embodiments, the cancer is not an EGFR mutant cancer.

[0086] The following examples are provided to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.EXAMPLES EXAMPLE 1CD70 and Endothelial to Mesenchymal Transition in KRAS Inhibitor Resistant Cancer

[0087] This example covers whether tumor cells with a driver oncogene upregulates CD70 at the time of therapeutic resistance to KRAS targeted agents. H358 (KRAS mutant) parental cells (P) were treated with the KRAS G12C inhibitor ARS-1620 until resistant cells (R) emerged. Transcriptomic data from these cells were then used to identify expression-level changes in H358 cells with acquired resistance to the KRAS inhibitor ARS-1620.

[0088] Transcriptomic analyses of the parental cells, resistant cells, and cells 24 and 48 hours after ARS-1620 treatment are summarized in FIG. 1A-1C. FIG. 1A is a plot of EMT scores from RNAseq analysis. This plot shows that the ARS-1620 resistant cells had upregulated epithelial to mesenchymal (EMT) gene expression signatures, and thus had undergone EMT. FIG. IB is a plot of CD70 expression levels from RNAseq, showing that KRAS inhibitor resistant H358 cells had upregulated CD70 RNA expression.

[0089] A panel of H358 cells with acquired resistance to the KRAS inhibitor adagrasib was developed by continuously culturing cells in adagrasib until resistant clones emerged. CD70 cell surface expression in these cells was evaluated by flow cytometry. It was found that CD70 was highly expressed on the adagrasib resistant cells (FIG. 1C). These data indicate that CD70 is a target on cells with acquired resistance to KRAS inhibitors.EXAMPLE 2CD70 and ZEB1 Upregulation in KRAS Inhibitor Resistant Cancer Cells

[0090] This example addresses the possibility that upregulation of CD70 is an early event in the evolution of resistance to KRAS inhibitors. For these analyses, KRAS mutant NSCLC cells (H1373 and Calul) and KRAS mutant pancreatic cancer cells (PACA2) were treated with theKRAS inhibitor adagrasib for 14 days, after which time the majority of cells had been killed and only drug-tolerant persister cells (DTPCs) remained.

[0091] FIG. 2A-2B are series of plots of ZEB 1 mRNA and CD70 mRNA expression levels, respectively, in untreated and KRAS inhibitor-treated cells from the three cancer cell lines. It was found that KRAS inhibitor derived DTPCs upregulated ZEB1, indicating that these cells had initiated EMT (FIG. 2A). Moreover, the KRAS inhibitor resistant DTPCs upregulated CD70 (FIG. 2B).EXAMPLE 3CD70-Targeted Treatments of KRAS Inhibitor Resistant Cancers

[0092] This example is directed to CD70 targeting of KRAS inhibitor resistant DTPCs. H1373 (KRAS mutant) cells were first treated with the KRAS inhibitor adagrasib for 14 days to generate KRAS inhibitor resistant DTPCs. CD70 was found to be upregulated on the DTPCs by flow cytometry (FIG. 3A-3B) relative to unstained (US) and stained parental / untreated H1373 cells.

[0093] Next, the activities of CD70-targeting CAR T cells were measured on untreated H1373 cells (FIG. 3C) and KRAS inhibitor resistant Hl 373 DTPCs (FIG. 3D). While the CAR T cells had limited activity on untreated H1373 parental cells (FIG. 3C), the CAR T cells displayed potent anti-tumor cell activity against CD70 positive Hl 373 KRAS inhibitor DTPCs (FIG. 3D).

[0094] Similar analyses were then performed on Calul cells (FIG. 4A-4D). As with the H1373 cells, CD70 was upregulated on Calul KRAS inhibitor DTPCs (FIG. 4A-4B) as compared to unstained US) and stained parental / untreated Calul cells. It was further shown that CD70-targeting CAR T cells had limited activity on untreated Calul cells (FIG. 4C), but effectively targeted Calu-1 adagrasib-treated DTPCs (FIG. 4D).

[0095] Next, KRAS mutant non-small cell lung cancer (NSCLC) H358 cells were treated with the CD70-targeted antibody-drug conjugate (ADC) vorsetuzumab-MMAE. Prior to these treatments, the cells were either left untreated or were converted into KRAS inhibitor (adagrasib) resistant variants. Relative cell viability was evaluated with Cell Titer Gio following 24 hours of treatment with 0 or 20 pg / ml CD70-targeted ADCs, and summarized in FIG. 5. While the ADCs did not impact the viability of parental H358 (CD70 negative) cells, the ADCs impaired the viability of cells with acquired resistance to KRAS inhibitors.

[0096] In addition, CD70 targeting using the CD70 antibody-drug conjugate (ADC) vorsetuzumab-MMAE induced a significant reduction in the viability of KRAS inhibitor resistant H358 Ada-Res cells as compared to parental H358 NSCLC cells (FIG. 5).EXAMPLE 4CD70 and ZEB1 Upregulation in RET Inhibitor Resistant Cancer Cells

[0097] This example assesses whether CD70 is similarly upregulated on tumor cells bearing RET-fusion mutations following treatment with a RET inhibitor. (RET fusion positive) Lc2 cells were treated with the RET inhibitor selpercatinib for 14 days, after which time mRNA levels were evaluated by real time PCR. The results of these analyses are shown in FIG. 6A- 6B. Consistent with mRNA expression in KRAS inhibitor resistant DTPCs, the EMT regulator ZEB1 was upregulated in selpercatinib-treated cells (FIG. 6A). CD70 was also significantly upregulated in the selpercatinib-treated cells (FIG. 6B). Moreover, CD70 was upregulated on the cell surface of RET fusion positive cells with acquired resistance to selpercatinib (FIG 7A), and CD70 targeting CAR cells effectively killed RET fusion positive cells with acquired resistance to selpercatinib (FIG 7B).Example 5CD70 Upregulation in ALK Inhibitor Drug Tolerant Persister Cells and Resistant Cancer Cells

[0098] This example assesses whether CD70 is similarly upregulated on tumor cells bearing ALK fusion mutations following treatment with an ALK inhibitor. ALK fusion positive H3122 cells were treated with the ALK inhibitor crizotinib for 14 days, after which time CD70 was significantly upregulated (FIG 8). Analysis of RNAseq data from ALK fusion positive cells (H2228) with acquired resistance to the ALK inhibitor alectinib indicated that ALK inhibitor resistant cells acquire an EMT gene expression signature with increased expression of EMT markers vimentin and AXL (FIG. 9A-9C). These ALK inhibitor resistant cells also upregulate expression of CD70 (FIG 9D).Example 6CD70 Upregulation on ALK Inhibitor Resistant NSCLC Cells

[0099] NSCLC cells with acquired resistance to ALK inhibitors including alectinib, brigatinib, crizotinib, and lorlatinib were generated by continuously culturing DFCI032 cells in ALK inhibitors until resistant cells emerged. Alectanib resistant (AR), brigatinib resistant (BR), crizotinib resistant (CR), and lorlatinib resistant (LR) were confirmed to be resistant to ALK inhibitors in vitro by Cell Titer Gio assay (Figure 10A-10D). Quantitative real time PCRanalysis revealed that ALK inhibitor resistant NSCLC cells significantly upregulated expression of CD70 as compared to parental cells (Figure 10E).

[0100] Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What Is Claimed Is:

1. A method for treating a RAS, RET, or ALK mutant cancer in a subject comprising administering a CD70-targeted therapy to the subject, thereby treating the RAS, RET, or ALK-mutant cancer in the subject.

2. The method of claim 1, wherein the subject was previously administered a RAS inhibitor, RET inhibitor, or ALK inhibitor or a combination thereof for the cancer.

3. The method of claim 2, wherein the cancer progressed during the treatment with the RAS inhibitor, the RET inhibitor, or the ALK inhibitor or a combination thereof.

4. The method of claim 2 or claim 3, wherein the cancer is resistant to the RAS inhibitor, the RET inhibitor, or the ALK inhibitor or a combination thereof.

5. The method of any one of claims 1-4, wherein CD70 is upregulated in the cancer.

6. The method of any one of claims 1-5, wherein the cancer has undergone an epithelial to mesenchymal transition.

7. The method of claim 6, wherein the cancer expresses an EMT marker selected from CDH1, BIM, AXL, ZEB1, or ZEB2.

8. The method of any one of claims 1-7, wherein the CD70-targeted therapy comprises a CD70 antagonist, an antibody-drug conjugate, a chimeric antigen receptor (CAR) T cell, a CAR natural killer (NK) cell, a tyrosine kinase inhibitor therapy (TKI) or a combination thereof.

9. The method of any one of claim 1, further comprising administering a RAS inhibitor, a RET inhibitor, an ALK inhibitor, or a combination thereof to the subject.

10. The method of claim 9, wherein i) the RAS inhibitor is selected from adagrasib, sotorasib, MRTX1257, JNJ-74699157, LY3499446, KRAS (G12C) inhibitor 6, ARS-853, BPI- 421286, LY3537982, IDQ443, IAB-21000, RMC-6291, or GDC-6036; ii) the RET inhibitor is selected from BLU6864, cabozantinib, dovitinib, foretinib, lenvatinib, ponatinib, pralsetinib, selpercatinib, sorafenib, sunitinib, or vandetanib;iii) the ALK inhibitor is selected from alectinib, alkotinib, belizatinib, brigatinib, ceritinib, conteltinib, crizotinib, ensartinib, entrectinib, foritinib, lorlatinib, repotrectinib, or zotizalkib; or iv) a combination thereof.

11. The method of claim 9 or claim 10, wherein the CD70-targeted therapy is administered concurrently with and / or subsequently to the RAS inhibitor, the RET inhibitor, the ALK inhibitor, or the combination thereof.

12. The method of any one of claims 1-11, wherein the RAS is KRAS.

13. The method of claim 12, wherein i) the KRAS comprises a mutation selected from G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61R, Q61Y, or a combination thereof; ii) the KRAS is a KRAS fusion selected from an ALK fusion, a BRAF fusion, an FGFR2 fusion, an NRF1 fusion, an NTRK1 fusion, an NTRK3 fusion, a RAFI fusion, a ROSl fusion, or a UBEL2L3 fusion; iii) the RET is a RET fusion selected from a CCDC6 fusion, a CUX1 fusion, a KIAA1468 fusion, a KIF5B fusion, an NCOA4 fusion, or a TRIM33 fusion; iv) the ALK is an ALK fusion selected from an EMIL4 fusion, a KIF5B fusion, a KLC1 fusion, a PTPN3 fusion, or a TGF fusion; or v) a combination thereof.

14. The method of any one of claims 1-13, further comprising administering an additional therapy selected from chemotherapy, radiation, surgery, tyrosine kinase inhibitor therapy, immunotherapy, or a combination thereof.

15. The method of claim 14, wherein the additional therapy comprises pembrolizumab, nivolumab, durvalumab, atezolizumab, carboplatin, pemetrexed, nab-paclitaxel, photofrin, cisplatin, docetaxel, gemcitabine, paclitaxel, vinorelbine, alectinib, lorlatinib, ceritinib, gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, brigatinib, cusatuzumab, vorsetuzumab, auristatin E (MMAE), duocarmycin, monomethyl auristatin F (MMAF) or pyrrolobenzodiazepine (PBD) or a combination thereof.

16. The method of any one of claims 1-15, further comprising administering an adjuvant, a neoadjuvant, or a combination thereof to the subject.

17. The method of any one of claims 1-16, wherein the cancer is selected from bile duct cancer, bladder cancer, brain cancer, breast cancer, carcinoma, cervical cancer, colorectal cancer, endometrial cancer, epitheloid carcinoma of the bone, esophageal cancer, gallbladder cancer, gastric cancer, glioblastoma, hepatocellular cancer, large cell lung carcinoma, leukemia, lung cancer, medulloblastoma, melanoma, ovarian cancer, non-small cell lung cancer, pancreatic cancer, prostate cancer, renal cell cancer, or thyroid cancer.

18. The method of claim 17, wherein the cancer is non-small cell lung cancer (NSCLC).

19. The method of claim 18, wherein the NSCLC comprises lung adenocarcinoma.

20. The method of any one of claims 1-19, wherein the subject is a non-smoker.

21. The method of any one of claims 1-20, wherein the cancer comprises a RAS, RET, or ALK activating mutation.

22. The method of any one of claims 1-21, wherein the cancer has not been tested for CD70 expression.

23. The method of any one of claims 1-21, wherein the cancer expresses CD70.

24. A method for treating cancer in a subject comprising: administering to the subject a CD70-targeted therapy; and a RAS inhibitor, a RET inhibitor, or an ALK inhibitor, thereby treating the cancer in the subject.

25. The method of claim 24, wherein the CD70-targeted therapy is administered concurrently with the RAS inhibitor, the RET inhibitor, or the ALK inhibitor.

26. The method of claim 24 or claim 25, wherein the CD70-targeted therapy blocks CD70 signaling.

27. The method of any one of claims 24-26, wherein the CD70-targeted therapy comprises a CD70 antagonist or a CD27 antagonist.

28. The method of any one of claims 24-27, wherein:i) the cancer is not resistant to the RAS inhibitor, the RET inhibitor, or the ALK inhibitor; ii) CD70 is not upregulated in the cancer; iii) the cancer has not undergone an epithelial to mesenchymal transition (EMT); or iv) a combination thereof.

29. The method of any one of claims 24-28, wherein i) the RAS inhibitor is selected from adagrasib, sotorasib, MRTX1257, JNJ-74699157, LY3499446, KRAS (G12C) inhibitor 6, ARS-853, BPI- 421286, LY3537982, JDQ443, JAB-21000, RMC-6291, or GDC-6036; ii) the RET inhibitor is selected from BLU6864, cabozantinib, dovitinib, foretinib, lenvatinib, ponatinib, pralsetinib, selpercatinib, sorafenib, sunitinib, or vandetanib; iii) the ALK inhibitor is selected from alectinib, alkotinib, belizatinib, brigatinib, ceritinib, conteltinib, crizotinib, ensartinib, entrectinib, foritinib, lorlatinib, repotrectinib, or zotizalkib; or iv) a combination thereof.

30. The method of any one of claims 24-29, wherein the RAS is KRAS.

31. The method of claim 30, wherein i) the KRAS comprises a mutation selected from G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61R, Q61Y, or a combination thereof; ii) the KRAS is a KRAS fusion selected from an ALK fusion, a BRAF fusion, an FGFR2 fusion, an NRF1 fusion, an NTRK1 fusion, an NTRK3 fusion, a RAFI fusion, a ROSl fusion, or a UBEL2L3 fusion; iii) the RET is a RET fusion selected from a CCDC6 fusion, a CUX1 fusion, a KIAA1468 fusion, a KIF5B fusion, an NCOA4 fusion, or a TRIM33 fusion; iv) the ALK is an ALK fusion selected from an EMIL4 fusion, a KIF5B fusion, a KLC1 fusion, a PTPN3 fusion, or a TGF fusion; or v) a combination thereof.

32. A method of identifying a subject with cancer as a candidate for treatment with a CD70- targeted therapy comprising: a) determining whether the cancer is resistant to a RAS inhibitor, a RET inhibitor, or an ALK inhibitor; and b) classifying the subject as a likely responder to the CD70-targeted therapy if the cancer is resistant the RAS inhibitor, the RET inhibitor, or the ALK inhibitor, thereby identifying subject as suitable for treatment with the CD70-targeted therapy; or c) classifying the subject as an unlikely responder to the CD70-targeted therapy if the cancer is not resistant to the RAS inhibitor, the RET inhibitor, or the ALK inhibitor, thereby identifying subject as an unlikely responder and unsuitable for treatment with the CD70-targeted therapy, thereby identifying a subject with cancer as a candidate for a CD70-targeted treatment.

33. The method of claim 32, wherein determining comprises in vivo tumor imaging, a solid tumor biopsy, a liquid tumor biopsy, a cancer biomarker panel, circulating tumor cell analysis, circulating tumor DNA analysis, circulating tumor RNA analysis, mRNA sequencing, DNA sequencing, protein expression measurements, in vitro inhibitor analysis, or a combination thereof.

34. The method of any one of claims 1-33, with the proviso that the cancer is not an EGFR mutant cancer.

Images