Methods and compositions for treating non-small cell lung cancer

Abstract

Aspects of the present disclosure relate to methods for treating EGFR-mutant non-small cell lung cancer (NSCLC) in a patient, comprising administering a CD70-targeting molecule to the patient. A further aspect of the present disclosure relates to methods for treating epithelial-mesenchymal transition (EMT)-positive NSCLC in a patient, comprising administering a CD70-targeting molecule to the patient. TIFF2022532402000003.tif82129

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 848,123, filed May 15, 2019, which is incorporated by reference in its entirety.

[0002] This invention was made with Government support under Grant No. CA190628 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] 1. Field of the Invention The present invention relates to the fields of molecular biology and medicine. [Background technology]

[0004] 2. Background Non-small cell lung cancer (NSCLC) is any type of epithelial lung cancer other than small cell lung cancer (SCLC). NSCLC accounts for approximately 85% of all lung cancers. As a class, NSCLCs are relatively insensitive to chemotherapy compared to small cell lung cancers. They are primarily treated by surgical resection with curative intent, when possible, although chemotherapy is increasingly being used both before (neoadjuvant chemotherapy) and after surgery (adjuvant chemotherapy).

[0005] Patients with EGFR mutant NSCLC initially respond to EGFR targeted therapy. However, resistant disease inevitably emerges, and in almost half of resistant cases, tumors lack secondary EGFR mutations such as T790M and are resistant to second and third generation EGFR tyrosine kinase inhibitors (TKIs). There is a need for further therapeutic approaches in the art. Summary of the Invention

[0006] An aspect of the present disclosure relates to a method for treating EGFR mutant non-small cell lung cancer (NSCLC) in a patient, comprising administering a CD70 targeting molecule to the patient.A further aspect of the present disclosure relates to a method for treating epithelial-mesenchymal transition (EMT) positive NSCLC in a patient, comprising administering a CD70 targeting molecule to the patient.A further aspect of the present disclosure relates to a composition comprising a CD70 targeting molecule and one or more additional therapeutic agents.

[0007] In some embodiments, the patient is determined to have EGFR mutant NSCLC. In some embodiments, the NSCLC comprises lung adenocarcinoma. In some embodiments, the patient is a non-smoker. In some embodiments, the patient is a human.

[0008] The term EGFR mutant cancer refers to cancers in which the expression or activity of EGFR (epidermal growth factor receptor) is altered. The mutations may be in the coding region of EGFR, affecting the expression level of endogenous EGFR or the resulting activity level of the protein. The mutations may also be in non-coding parts of the gene, such as in the promoter region, 3' or 5' UTR, or intronic regions. In some embodiments, the EGFR mutation is a gain-of-function mutation. In some embodiments, the EGFR mutation is a loss-of-function mutation. In some embodiments, the EGFR mutation comprises an activating mutation. In some embodiments, the activating mutation comprises L858R. In some embodiments, the activating mutation comprises a deletion in exon 19. In some embodiments, the EGFR mutations include one or more of the following mutations instead of or in addition to these other mutations: G719S (c.2155G>A), G719C (c.2155G>T), G719A (c.2156G>C), S720F (c.2159C>T), exon 19 deletion or partial deletion, D761Y (c.2281G>T), D770_N771 (insNPG), D770_N771 (insSVQ), D770_N771 (insG), V765A (c.2294T>C), T783A (c.2347A>G), S7681I (c.2303G>T), T790M (c.2369C>T), V769L (c.2305G>T), N771T (c.2312A>C), L858R (C.2573T>G), L861Q (c.2582T>A), L861R (c.2582T>G). In some embodiments, the EGFR mutation comprises a class I, II or III EGFR mutation.In some embodiments, the EGFR mutation comprises at least one of delE746-A750, delL747-P753insS, delL747-T751, delL747-A750insP, p.L747_S752del, K754insANKG, delT751_I759insN, delL747_A750insP, delE746_T751insV, delT751_I759insS, delE746_T751insI, delL747_A755insSKG, delE746_T751insVA, delL747_T751insP, delE746_S752insV, and delE746_A750insAP. In some embodiments, the EGFR mutation comprises at least one class I mutation selected from delE746-A750, delL747-P753insS, delL747-T751, delL747-A750insP, p.L747_S752del, K754insANKG, delT751_I759insN, delL747_A750insP, delE746_T751insV, delT751_I759insS, delE746_T751insI, delL747_A755insSKG, delE746_T751insVA, delL747_T751insP, delE746_S752insV and delE746_A750insAP.In some embodiments, the EGFR mutation comprises an in-frame deletion or partial deletion in exon 19. In some embodiments, the EGFR mutations are G719S (c.2155G>A), G719C (c.2155G>T), G719A (c.2156G>C), S720F (c.2159C>T), D761Y (c.2281G>T), V765A (c.2294T>C), T783A (c.2347A>G), S7681I (c.2303G>T), T790M (c.2369C>T), V769L (c.2305G>T), N771T (c.2312A>C), L858R (C.2573T>G), L861Q (c.2582T>A), and L861R Contains at least one of the following: (c.2582T>G).In some embodiments, the EGFR mutations are G719S (c.2155G>A), G719C (c.2155G>T), G719A (c.2156G>C), S720F (c.2159C>T), D761Y (c.2281G>T), V765A (c.2294T>C), T783A (c.2347A>G), S7681I (c.2303G>T), T790M (c.2369C>T), V769L (c.2305G>T), N771T (c.2312A>C), L858R (C.2573T>G), L861Q (c.2582T>A), and L861R (c.2582T>G). In some embodiments, the EGFR mutation comprises a single nucleotide substitution. In some embodiments, the EGFR mutation comprises at least one of D770_N771 (insNPG), D770_N771 (insSVQ), D770_N771 (insG). In some embodiments, the EGFR mutation comprises at least one of D770_N771 (insNPG), D770_N771 (insSVQ), D770_N771 (insG). In some embodiments, the EGFR mutation comprises at least one class III mutation selected from D770_N771 (insNPG), D770_N771 (insSVQ), D770_N771 (insG). In some embodiments, the EGFR mutation comprises an in-frame duplication or insertion in exon 20. It is contemplated that at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 (or any range derivable therein) of these mutations may be determined, known, or used in the embodiments described herein. In certain embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of these mutations may be excluded in an embodiment.

[0009] In some embodiments, the patient has not been tested for CD70 expression in cancer cells from the patient. In some embodiments, the patient has been determined to have CD70-expressing cancer cells. In some embodiments, the patient has previously been treated for NSCLC. In some embodiments, the patient has been determined to have acquired resistance to the previous treatment. In some embodiments, the previous treatment comprises EGFR tyrosine kinase inhibitor (TKI) therapy, where the therapy comprises one or more EGFR TKIs. In some embodiments, the previous treatment comprises single agent EGFR TKI therapy. In some embodiments, the previous treatment comprises a combination of at least two EGFR TKIs. In some embodiments, the patient has been determined to be resistant to at least two EGFR TKIs. In some embodiments, the patient has been determined to have systemic disease progression while undergoing ongoing EGFR TKI therapy. In some embodiments, the EGFR TKI therapy comprises one or more of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. In some embodiments, the EGFR TKI therapy comprises one or more of erlotinib, gefitinib, and osimertinib. In some embodiments, the EGFR TKI therapy comprises at least two of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. In some embodiments, the EGFR TKI therapy comprises at least three of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. In some embodiments, the EGFR TKI therapy comprises at least four of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. It is specifically contemplated that one or more of these may be excluded as EGFR TKI therapy.

[0010] In some embodiments, the method further comprises the administration of an additional therapy or the composition comprises an additional therapeutic agent.In some embodiments, the additional therapy or agent comprises chemotherapy, radiation, surgery, TKI therapy, or immunotherapy.In some embodiments, the additional therapy or agent comprises one or more of durvalumab, atezolizumab, pembrolizumab, nivolumab, necitumumab, and bevacizumab.In some embodiments, the additional therapy or agent comprises one or more of carboplatin, pemetrexed, nab-paclitaxel, photofrin, cisplatin, docetaxel, gemcitabine, paclitaxel, and vinorelbine.In some embodiments, the additional therapy or agent comprises one or more of alectinib, lorlatinib, and ceritinib. In some embodiments, the additional therapy or agent comprises one or more of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, brigatinib, and combinations thereof. In some embodiments, the additional therapy or agent comprises osimertinib. In some embodiments, the method further comprises administering adjuvant therapy and / or neoadjuvant therapy. In some embodiments, the additional therapy can be conjugated or linked to the CD70 targeting therapy. In some embodiments, the linkage is via a chemical linker. In some embodiments, the linkage is via a peptide bond (e.g., a fusion protein comprising a CD70 targeting agent and an additional therapeutic agent).

[0011] In some embodiments, the patient is determined to have an ALK mutant. In some embodiments, the patient is determined to not have an ALK mutant.

[0012] In some embodiments, the CD70 targeting molecule comprises an anti-CD70 antibody or a CD70-binding fragment thereof. In some embodiments, the antibody is a humanized antibody or a chimeric antibody. In some embodiments, the antibody is conjugated to a molecule. In some embodiments, the antibody is conjugated to a toxic molecule. In some embodiments, the toxic molecule comprises monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), pyrrolobenzodiazepine (PBD), duocarmycin, or a combination thereof.

[0013] In some embodiments, the CD70 targeting molecule comprises a heavy chain variable region and / or a light chain variable region from a CD70 antibody. In some embodiments, the CD70 targeting molecule comprises CDR1, CDR2 and CDR3 from the heavy chain variable region and / or CDR1, CDR2 and CDR3 from the light chain variable region. The heavy and light chain variable regions can be from or derived from a CD70 antibody. In some embodiments, the CD70 targeting molecule comprises a single chain variable fragment (scFv). The scFv can comprise a hypervariable region, such as CDR1, CDR2 and CDR3 from the heavy chain variable region and / or CDR1, CDR2 and CDR3 from the light chain variable region from an anti-CD70 antibody. In some embodiments, the antibody comprises cusatuzumab or borsetuzumab. In some embodiments, the CD70 targeting molecule comprises a heavy chain variable region and / or a light chain variable region from cusatuzumab or borsetuzumab. In some embodiments, the CD70 targeting molecule comprises CDR1, CDR2 and CDR3 from the heavy chain variable region of cusatuzumab and / or CDR1, CDR2 and CDR3 from the light chain variable region of cusatuzumab. In some embodiments, the CD70 targeting molecule comprises CDR1, CDR2 and CDR3 from the heavy chain variable region of borsetuzumab and / or CDR1, CDR2 and CDR3 from the light chain variable region of borsetuzumab.

[0014] In some embodiments, the additional therapy or agent in the disclosed methods and compositions comprises a secondary antibody linked to a toxic molecule. For example, the combination therapy may comprise a CD70 targeting antibody combined with an agent comprising a conjugate comprising a secondary antibody that binds to the CD70 targeting antibody conjugated to a toxic molecule. Thus, such a combination of agents delivers the antibody linked to the toxic molecule through the secondary antibody. In some embodiments, the secondary antibody and the toxic molecule are linked through a cleavable linker.

[0015] In some embodiments, the CD70-targeted molecule comprises a bispecific T cell engager (BiTE), a chimeric antigen receptor (CAR), a CAR-containing T cell, or a trispecific natural killer cell engager therapy (TriNKET). In some embodiments, the BiTE, CAR, or TriNKET is derived from the heavy chain variable region and / or the light chain variable region of ofatumumab or bortezomib. In some embodiments, the BiTE, CAR, or TriNKET comprises a heavy chain comprising CDR1, CDR2, and CDR3 of ofatumumab or bortezomib, and / or a light chain comprising CDR1, CDR2, and CDR3 of ofatumumab or bortezomib. In some embodiments, the CD70-targeted molecule comprises ofatumumab-MMAE, bortezomib-MMAE, or a combination thereof. In some embodiments, the CD70-targeted molecule comprises SGN-75, SGN-CD70A, AMG 172, and / or ARGX-110. SGN-75 is a CD70-blocking IgG1 antibody-drug conjugate (ADC) that releases a cell-killing agent upon internalization into CD70-expressing tumor cells. SGN-CD70A comprises a CD70-blocking antibody equipped with a cytotoxic agent. SGN-CD70A comprises a very potent cytotoxic pyrrolobenzodiazepine dimer stably linked to an antibody against CD70. AMG172 is an IgG1 ADC, and binding and internalization into CD70-expressing tumor cells induces cell cycle arrest, followed by cell apoptosis and ultimately tumor cell death. ARGX-110 comprises a CD70-blocking IgG1 monoclonal antibody (mAb), and its glycoengineered Fc domain mediates the targeted killing of CD70-expressing tumor cells via complement-dependent cytotoxicity (CDC), antibody-dependent cell phagocytosis (ADCP) properties, and enhanced antibody-dependent cell cytotoxicity (ADCC). In some embodiments, a patient-derived biological sample has been determined to be positive for one or more EMT markers. In some embodiments, the one or more EMT markers include a reduction of epithelial markers and / or an increase of mesenchymal markers.In some embodiments, the EMT markers include one, two, three, four, or all five of CDH1, VIM, AXL, ZEB1, and ZEB2. In some embodiments, the biological sample includes tumor cells and / or tumor-associated cells.

[0016] CD70 targeting molecules useful in the methods and compositions of the present disclosure are known in the art. For example, CD70 CARs have been developed and can be used in the embodiments of the present disclosure. Thus, in some embodiments, the CD70 targeting molecule comprises CTX130. CTX130 is an allogeneic CRISPR / Cas9 gene-edited CAR-T cell therapy that targets CD70, produced by CRISPR Therapeutics. In some embodiments, the CD70 targeting molecule comprises ALLO-316. ALLO-316 is an anti-CD70 AlloCAR T cell therapy being developed by Allogene. Further embodiments are described in Wang, QJ. et al., Clin Cancer Res. 2017 May 1;23(9):2267-2276, which is incorporated herein by reference. It is contemplated that CD70 targeting molecules described as useful for other indications can be used in the embodiments of the methods and compositions of the present disclosure. In some embodiments, the CD70 targeting molecule comprises a CD70 ligand, such as CD27. In some embodiments, the CD70 targeting molecule comprises a truncated CD27. In some embodiments, the CD70 targeting molecule comprises a CD27 CAR, which is a CD27 polypeptide fused to the transmembrane and intracellular signaling domains of a CAR molecule, such as 41BB and CD3-zeta. The CD27 polypeptide can be a full-length polypeptide, or a fragment or truncation thereof that interacts and binds to CD70. The CAR of the present disclosure can be expressed in T cells or NK cells.

[0017] In some embodiments, the CD70 targeting molecule comprises a cell comprising a BiTE, a CAR, or a TriNKET. In some embodiments, the cell comprises a stem cell, a progenitor cell, an immune cell, or a natural killer (NK) cell. In some embodiments, the cell comprises a hematopoietic stem or progenitor cell, a T cell, a cell differentiated from a mesenchymal stem cell (MSC), or an induced pluripotent stem cell (iPSC). In some embodiments, the cell is isolated or derived from a peripheral blood mononuclear cell (PBMC). In some embodiments, the T cell is a cytotoxic T lymphocyte (CTL), a CD8 + T cells, CD4 + T cells, invariant NK T (iNKT) cells, gamma-delta T cells, NKT cells, or regulatory T cells.

[0018] In some embodiments, the biological sample comprises a biopsy. In some embodiments, the biological sample is obtained by methods such as fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, incisional biopsy, excision biopsy, punch biopsy, shave biopsy or skin biopsy. In certain embodiments, the sample is obtained from a biopsy from lung tissue by any of the aforementioned biopsy methods. In other embodiments, the sample may be obtained from any of the tissues provided herein, including but not limited to non-cancerous or cancerous tissues and non-cancerous or cancerous tissues from serum, gallbladder, mucosa, skin, heart, lung, breast, pancreas, blood, liver, muscle, kidney, smooth muscle, bladder, colon, intestine, brain, prostate, esophagus, or thyroid tissue. Alternatively, the sample may be obtained from any other source, including but not limited to blood, sweat, hair follicle, cheek tissue, tears, menstrual secretions, feces, or saliva. In certain aspects of the method, any medical professional, such as a doctor, nurse, or medical technician, may obtain the biological sample for testing. Moreover, biological samples can be obtained without the assistance of a medical professional. Samples can include, but are not limited to, tissues, cells, or biological materials derived from cells or from the subject's cells. Biological samples can be heterogeneous or homogeneous populations of cells or tissues. Biological samples can be obtained using any method known in the art that can provide a sample suitable for the analytical methods described herein. Samples can be obtained by non-invasive methods, including, but not limited to, skin or cervical scraping, buccal swabbing, saliva collection, urine collection, fecal collection, menstrual secretions, tears, or semen collection. Samples can be obtained by methods known in the art. In certain embodiments, samples are obtained by biopsy.

[0019] As used herein, the terms "or" and "and / or" are utilized to describe multiple elements in combination or mutually exclusive. For example, "x, y, and / or z" can refer to "x" only, "y" only, "z" only, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." It is specifically contemplated that x, y, or z can be specifically excluded from an embodiment.

[0020] Throughout this application, the term "about" is used in accordance with its plain and ordinary meaning within the art of cell biology to indicate that a value includes the standard deviation of error for the device or method being utilized to determine the value.

[0021] The term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional unrecited elements or method steps. The phrase "consisting of" excludes any unspecified elements, steps, or ingredients. The phrase "consisting essentially of" limits the scope of the described subject matter to the specified materials or steps that do not materially affect its basic and novel characteristics. It is contemplated that embodiments described in the context of "comprising" can also be implemented in the context of the terms "consisting of" or "consisting essentially of."

[0022] It is specifically contemplated that any limitation discussed with respect to one embodiment of the present invention may be applied to any other embodiment of the present invention.Furthermore, any composition of the present invention may be used in any method of the present invention, and any method of the present invention may be used to make or utilize any composition of the present invention.Aspects of the embodiments described in the examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in different examples or elsewhere in this application, for example in the Summary of the Invention, Detailed Description of the Embodiments, Claims, and Figure Legend Descriptions.

[0023] [The present invention 1001] A method for treating EGFR mutant non-small cell lung cancer (NSCLC) in a patient, comprising administering to the patient a CD70 targeting molecule. [The present invention 1002] A method for treating epithelial-mesenchymal transition (EMT) positive NSCLC in a patient, comprising administering to the patient a CD70 targeting molecule. [The present invention 1003] The method of any one of claims 1001 to 1002, wherein the patient is determined to have EGFR mutant NSCLC. [The present invention 1004] The method of any one of claims 1001 to 1003, wherein the NSCLC comprises lung adenocarcinoma. [The present invention 1005] The method according to any one of 1001 to 1004, wherein the patient is a non-smoker. [The present invention 1006] The method of any one of claims 1001 to 1005, wherein the EGFR mutant comprises an activating mutation. [The present invention 1007] The method of claim 1006, wherein the activating mutation comprises L858R or a deletion in exon 19. [The present invention 1008] The method of any of claims 1001 to 1007, wherein the EGFR mutation comprises a class I, II or III EGFR mutation. [The present invention 1009] The method of any of claims 1001 to 1008, wherein the patient has not been tested for CD70 expression on the cancer cells. [The present invention 1010] The method of any of claims 1001 to 1008, wherein the patient is determined to have CD70-expressing cancer cells. [The present invention 1011] The method of any of claims 1001 to 1010, wherein the patient has previously undergone treatment for NSCLC. [The present invention 1012] The method of claim 1011, wherein the patient has been determined to have acquired resistance to a previous treatment. [The present invention 1013] The method of any one of claims 1011 to 1012, wherein the previous treatment comprises EGFR tyrosine kinase inhibitor (TKI) therapy, said therapy comprising one or more EGFR TKIs. [The present invention 1014] The method of any of claims 1011 to 1013, wherein the prior treatment comprises single agent EGFR TKI therapy. [The present invention 1015] The method of any of claims 1011 to 1013, wherein the previous treatment comprises a combination of at least two EGFR TKIs. [The present invention 1016] The method of any of claims 1011 to 1015, wherein the patient is determined to have systemic disease progression while receiving ongoing EGFR TKI therapy. [The present invention 1017] The method of any of claims 1013 to 1016, wherein the EGFR TKI therapy comprises one or more of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. [The present invention 1018] Any of the methods of 1001 to 1017, further comprising administration of an additional therapy. [The present invention 1019] The method of claim 1018, wherein the additional therapy comprises chemotherapy, radiation, surgery, TKI therapy, or immunotherapy. [The present invention 1020] The method of any one of claims 1018 to 1019, wherein the additional therapy comprises one or more of durvalumab, atezolizumab, pembrolizumab, nivolumab, necitumumab, and bevacizumab. [The present invention 1021] The method of any of claims 1018-1020, wherein the additional therapy comprises one or more of carboplatin, pemetrexed, nab-paclitaxel, photofrin, cisplatin, docetaxel, gemcitabine, paclitaxel, and vinorelbine. [The present invention 1022] The method of any of claims 1018-1021, wherein the additional therapy comprises one or more of alectinib, lorlatinib, and ceritinib. [The present invention 1023] The method of any of claims 1018 to 1022, wherein the additional therapy comprises one or more of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. [The present invention 1024] The method of claim 1023, wherein the further therapy comprises osimertinib. [The present invention 1025] The method of any of claims 1001 to 1022, further comprising administering adjuvant therapy and / or neoadjuvant therapy. [The present invention 1026] The method of any one of claims 1001 to 1025, wherein the patient is determined to be ALK mutant. [The present invention 1027] The method of any of claims 1001 to 1025, wherein the patient is determined not to be ALK mutant. [The present invention 1028] The method of any of claims 1001 to 1027, wherein the CD70 targeting molecule comprises an anti-CD70 antibody or a CD70-binding fragment thereof. [The present invention 1029] The method of claim 1028, wherein the further therapy comprises a second antibody linked to a toxic molecule. [The present invention 1030] The method of claim 1029, wherein the second antibody and the toxic molecule are linked via a cleavable linker. [The present invention 1031] The method of any of claims 1028 to 1030, wherein the antibody is a humanized antibody or a chimeric antibody. [The present invention 1032] The method of any of claims 1028 to 1031, wherein the antibody comprises cusatuzumab or borsetuzumab. [The present invention 1033] The method of any of claims 1028 to 1032, wherein the antibody is conjugated to the molecule. [The present invention 1034] The method of claim 1033, wherein the molecule is a toxic molecule. [The present invention 1035] The method of claim 1034, wherein the toxic molecule comprises monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), a pyrrolobenzodiazepine (PBD), or a duocarmycin. [The present invention 1036] The method of any one of claims 1034 to 1035, wherein the CD70 targeting molecule comprises cusatuzumab-MMAE, borsetuzumab-MMAE, or a combination thereof. [The present invention 1037] The method of any of claims 1001 to 1036, wherein the CD70 targeting molecule comprises a heavy chain variable region and / or a light chain variable region from a CD70 antibody. [The present invention 1038] The method of any of claims 1001 to 1037, wherein the CD70 targeting molecule comprises CDR1, CDR2 and CDR3 from a heavy chain variable region, and / or CDR1, CDR2 and CDR3 from a light chain variable region. [The present invention 1039] The method of any of claims 1001 to 1038, wherein the CD70 targeting molecule comprises a single chain variable fragment (scFV). [The present invention 1040] Any of the methods of claims 1001-1039, wherein the CD70 targeting molecule comprises a bispecific T cell engager (BiTE), a chimeric antigen receptor (CAR), a T cell comprising a CAR, or a trispecific natural killer cell engager therapy (TriNKET). [The present invention 1041] The method of claim 1040, wherein the CD70 targeting molecule comprises a cell comprising a BiTE, a CAR, or a TriNKET. [The present invention 1042] The method of claim 1041, wherein the cell comprises a stem cell, a progenitor cell, an immune cell, or a natural killer (NK) cell. [The present invention 1043] The cell of the present invention 1042, including a hematopoietic stem or progenitor cell, a T cell, a cell differentiated from a mesenchymal stem cell (MSC), or an induced pluripotent stem cell (iPSC). [The present invention 1044] The cell of the invention 1042 or 1043 isolated or derived from peripheral blood mononuclear cells (PBMC). [The present invention 1045] T cells are cytotoxic T lymphocytes (CTL), CD8 + T cells, CD4 + The cell of the invention 1043 or 1044, including a T cell, an invariant NK T (iNKT) cell, a gamma-delta T cell, an NKT cell, or a regulatory T cell. [The present invention 1046] The method of any of claims 1040 to 1045, wherein the CD70 targeting molecule comprises CTX130 or ALLO-316. [The present invention 1047] The method of any of claims 1040 to 1045, wherein the CD70 targeting molecule comprises a CD27 CAR. [The present invention 1048] The method of any of claims 1001 to 1039, wherein the CD70 targeting molecule comprises SGN-75, SGN-CD70A, AMG 172, and / or ARGX-110. [The present invention 1049] The method of any of claims 1001-1048, wherein the biological sample from the patient has been determined to be positive for one or more EMT markers. [The present invention 1050] The method of claim 1049, wherein the biological sample comprises tumor cells and / or tumor-associated cells. [The present invention 1051] The method of any one of claims 1049 to 1050, wherein the biological sample comprises a biopsy. [The present invention 1052] The method of any of claims 1049 to 1051, wherein the one or more EMT markers comprise a reduction in an epithelial marker and / or an increase in a mesenchymal marker. [The present invention 1053] The method of any one of claims 1049 to 1052, wherein the EMT markers include one or more of CDH1, VIM, AXL, ZEB1, and ZEB2. [The present invention 1054] A composition comprising a CD70 targeting molecule and one or more additional therapeutic agents. [The present invention 1055] The composition of the present invention 1054, wherein the additional therapeutic agent comprises chemotherapy, radiation, surgery, TKI therapy, immunotherapy, or a combination thereof. [The present invention 1056] The composition of any one of claims 1054 to 1055, wherein the additional therapeutic agent comprises one or more of durvalumab, atezolizumab, pembrolizumab, nivolumab, necitumumab, and bevacizumab. [The present invention 1057] The composition of any of claims 1054-1056, wherein the additional therapeutic agent comprises one or more of carboplatin, pemetrexed, nab-paclitaxel, photofrin, cisplatin, docetaxel, gemcitabine, paclitaxel, and vinorelbine. [The present invention 1058] The composition of any of claims 1055-1057, wherein the additional therapeutic agent comprises one or more of alectinib, lorlatinib, and ceritinib. [The present invention 1059] Any of the compositions of claims 1055-1058, wherein the additional therapeutic agent comprises one or more of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. [The present invention 1060] The composition of the present invention, wherein the further therapeutic agent comprises osimertinib. [The present invention 1061] The composition of any of claims 1055 to 1057, wherein the CD70 targeting molecule comprises an anti-CD70 antibody or a CD70-binding fragment thereof. [The present invention 1062] The composition of the present invention 1061, wherein the additional therapeutic agent comprises a second antibody linked to a toxic molecule. [The present invention 1063] The composition of the present invention 1062, wherein the second antibody and the toxic molecule are linked via a cleavable linker. [The present invention 1064] The composition of any of claims 1061 to 1063, wherein the antibody is a humanized antibody or a chimeric antibody. [The present invention 1065] The composition of any of claims 1061 to 1064, wherein the antibody comprises cusatuzumab or borsetuzumab. [The present invention 1066] The composition of any of claims 1061 to 1065, wherein the antibody is conjugated to a molecule. [The present invention 1067] The composition of the present invention 1066, wherein the molecule is a toxic molecule. [The present invention 1068] The composition of the present invention 1067, wherein the toxic molecule comprises monomethylauristatin E (MMAE), duocarmycin, monomethylauristatin F (MMAF), or pyrrolobenzodiazepine (PBD). [The present invention 1069] The composition of any one of claims 1067 to 1068, wherein the CD70 targeting molecule comprises cusatuzumab-MMAE, borsetuzumab-MMAE, or a combination thereof. [The present invention 1070] The composition of any of claims 1054 to 1069, wherein the CD70 targeting molecule comprises a heavy chain variable region and / or a light chain variable region from a CD70 antibody. [The present invention 1071] The composition of any of claims 1054 to 1070, wherein the CD70 targeting molecule comprises CDR1, CDR2 and CDR3 from a heavy chain variable region, and / or CDR1, CDR2 and CDR3 from a light chain variable region. [The present invention 1072] The composition of any of claims 1054 to 1071, wherein the CD70 targeting molecule comprises a single chain variable fragment (scFV) that specifically binds to CD70. [The present invention 1073] Any of the compositions of claims 1054 to 1072, wherein the CD70 targeting molecule comprises a bispecific T cell engager (BiTE), a chimeric antigen receptor (CAR), a T cell containing a CAR, or a trispecific natural killer cell engager therapy (TriNKET). [The present invention 1074] Any of the compositions of claims 1054 to 1073, wherein the CD70 targeting molecule comprises SGN-75, SGN-CD70A, AMG 172, and / or ARGX-110. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of example only, since various modifications and changes within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. [Brief description of the drawings]

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0025] [Figure 1] HCC827 and HCC4006 (both EGFR mutant) cells are sensitive to EGFR TKIs, including erlotinib and osimertinib, compared to EGFR TKI-resistant (ER) variants. [Diagram 2] EGFR TKI resistance is associated with transcriptomic changes, epithelial-mesenchymal transition (EMT) and upregulation of CD70. (A) We compared gene expression by RNA seq between parental HCC827 and HCC4006 cells and cells with acquired resistance to EGFR TKIs. (B) Gene Set enrichment analysis reveals increased EMT signatures in EGFR TKI-resistant (ER) cells compared to parental cells. ER cells expressed lower levels of epithelial marker CDH1 (C) and increased expression of mesenchymal markers VIM, AXL, ZEB1 and ZEB2 (D-G). (H) EGFR TKI-resistant cells significantly upregulated expression of CD70. [Diagram 3] CD70 protein levels are elevated on the surface of EGFR TKI-resistant NSCLC cells.Flow cytometry reveals increased protein levels of CD70 in EGFR TKI-resistant cells HCC827 ER1, ER3 and ER6 compared to parental HCC827 cells. [Figure 4] Induction of EMT is sufficient to induce EGFR TKI resistance. (A) Induction of ZEB1 expression in HCC827 (EGFR TKI-sensitive) cells resulted in a shift to a mesenchymal phenotype, as demonstrated by loss of E-cadherin and increased expression of N-cadherin, Axl, and vimentin. Expression of ZEB1 rendered HCC827 cells resistant to the EGFR TKIs erlotinib, osimertinib, and afatinib (B-D). [Diagram 5] In human NSCLC, EMT is associated with high expression of CD70. CD70 expression is significantly associated with the EMT gene expression signature in NSCLC cell lines (A) and clinical samples from TCGA (B). [Figure 6] EGFR TKI resistance is associated with epithelial-mesenchymal transition (EMT). (A) We compared gene expression by RNA seq between parental HCC827 and HCC4006 cells and cells with acquired resistance to EGFR TKIs. Gene Set enrichment analysis reveals an EMT signature in EGFR TKI-resistant (ER) cells compared to parental cells. ER cells expressed lower levels of epithelial marker CDH1 (C) and increased expression of mesenchymal markers VIM, AXL, ZEB1 and ZEB2 (B). (C and D) EGFR TKI-resistant cells, including erlotinib-resistant (ER) and osimertinib-resistant (OR) cells, displayed a proteomic signature consistent with EMT as determined by reverse-phase protein arrays. [Figure 7]CD70 is elevated in T790M-negative EGFR TKI-resistant cells. (A) RNAseq analysis revealed overexpression of CD70 in EGFR TKI-resistant cells compared to parental EGFR TKI-sensitive cells. (B-F) Cell surface expression of CD70 was assessed by flow cytometry. CD70 expression was elevated in EGFR TKI-resistant cells, including erlotinib-resistant (ER) and osimertinib-resistant (OR) lines, compared to parental EGFR TKI-sensitive cell lines (HCC4006, HCC827, H1975). CD70 expression was minimal in cells in which acquired resistance to EGFR TKIs was mediated by secondary EGFR mutations (T790M) or MET amplification. [Figure 8] CD70 is elevated in genetically engineered mouse models (GEMMs) of EGFR TKI resistance. To generate a mouse model of acquired EGFR-independent NSCLC, doxycycline (DOX) was used to induce EGFR mutant NSCLC tumors in a DOX-inducible L858R EGFR mouse model. DOX was removed from a subset of animals once tumors were visible on CT imaging. After a period of tumor regression, resistant tumors began to regrow. Animals were euthanized and tumors were collected for immunohistochemical staining of CD70. CD70 expression was elevated in tumors that had acquired EGFR independence. [Figure 9] CD70 is elevated in NSCLC clinical specimens following EGFR TKI resistance. Immunohistochemical staining for CD70 revealed minimal CD70 expression in EGFR mutant NSCLC tumors that had not received EGFR TKI treatment. However, CD70 expression was markedly elevated in EGFR mutant tumors following therapeutic resistance to EGFR TKI treatment. [Figure 10]Induction of EMT is sufficient to induce EGFR TKI resistance. (A) Induction of ZEB1 expression in HCC827 (EGFR TKI-sensitive) cells resulted in a shift to a mesenchymal phenotype, as demonstrated by loss of E-cadherin and increased expression of N-cadherin, Axl, and vimentin. (B) Expression of ZEB1 rendered HCC827 cells resistant to the EGFR TKIs erlotinib, osimertinib, and afatinib. (C) ZEB1 expression induces a significant increase in CD70 mRNA expression and cell surface expression of CD70. [Figure 11] EMT is associated with high expression of CD70 in human NSCLC clinical specimens and NSCLC cell lines. CD70 expression is significantly associated with the EMT gene expression signature and ZEB1 in TCGA NSCLC clinical specimens (A) and NSCLC cell lines (B). [Figure 12] Stimulation of CD70 activates signaling pathways in EGFR TKI-resistant cells. HCC4006 osimertinib-resistant (OR) cells were treated with soluble CD27, a binding partner of CD70, and the effects on downstream signaling pathways were assessed by Western blotting. CD70 activation led to phosphorylation of Akt and ERK. [Figure 13] Knockdown of CD70 impairs proliferation of EGFR TKI-resistant cells. siRNA-mediated knockdown of CD70 (A) led to decreased viability of H1975 OR5 cells (B). Similar results were obtained in HCC4006 OR cells (C). [Figure 14]CD70 antibody-drug conjugates (ADCs) target CD70+ EGFR TKI-resistant cells in vitro. The CD70 ADCs cusatuzumab-MMAE and borsetuzumab-MMAE demonstrated antitumor cell activity against H1975 osimertinib-resistant (OR) cells, including H1975 OR5 and H1975 OR16. (A) shows the CD70 positive cell rate in various cell lines, and line graphs show the relative cell viability of cell lines with the addition of (B) cusatuzumab-MMAE and (C) borsetuzumab-MMAE. [Figure 15] CD70 ADC enhances osimertinib (OSI) activity in EGFR TKI-resistant cells in vitro. Bar graph shows relative cell viability of OSI and ADC combinations. [Figure 16] CD70 ADCs have activity against EGFR TKI-resistant cells in vitro. The CD70 ADCs cusatuzumab-MMAE and borsetuzumab-MMAE have demonstrated anti-tumor cell activity against, for example, H1975 osimertinib-resistant (OR) cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Description of Exemplary Embodiments Although EGFR mutant NSCLC patients initially respond to EGFR tyrosine kinase inhibitors (TKIs), resistant disease inevitably emerges. The present disclosure provides a new therapeutic approach for NSCLC. Further embodiments are described below.

[0027] I. Definition The terms "protein," "polypeptide," and "peptide" are used interchangeably herein when referring to gene products.

[0028] "Homology" or "identity" refers to sequence similarity between two peptides or two nucleic acid molecules. Identity can be determined by comparing a position in each sequence that can be aligned for purposes of comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules share sequence identity at that position. The degree of identity between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 60% identity, less than 50% identity, less than 40% identity, less than 30% identity, or less than 25% identity with one of the sequences of the present disclosure.

[0029] As used herein, terms such as "amino moiety," "N-terminus," "amino terminus," and the like are used to refer to the order of a region of a polypeptide. Furthermore, if something is at the N-terminus of a region, it is not necessarily at the end (or end) of the entire polypeptide, but only at the N-terminus of the region or domain. Similarly, terms such as "carboxy moiety," "C-terminus," "carboxy terminus," and the like are used to refer to the order of a region of a polypeptide, and if something is at the C-terminus of a region, it is not necessarily at the end (or end) of the entire polypeptide, but only at the C-terminus of the region or domain.

[0030] The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute a double-stranded form.

[0031] As used herein, a cell or culture of cells is "substantially free" of a certain reagent or element, such as serum, signal transduction inhibitors, animal components or feeder cells, exogenous genetic elements, or vector elements, if it has less than 10% of that element, and is "essentially free" of a certain reagent or element if it has less than 1% of that element. However, more desirable are cell populations that contain less than 0.5% or less than 0.1% of the total cell population of exogenous genetic elements or vector elements.

[0032] A cell or culture of cells is "essentially free" of a certain reagent or component, such as serum, signal transduction inhibitors, animal components, or feeder cells, if the culture, matrix, or medium contains levels of these reagents below detectable levels using conventional detection methods known to those of skill in the art, respectively, or if these reagents are not added exogenously to the culture, matrix, or medium. Serum-free medium may be essentially free of serum.

[0033] A "gene," "polynucleotide," "coding region," "sequence," "segment," "fragment," or "transgene" that "encodes" a particular protein is a nucleic acid molecule that is transcribed in vitro or in vivo and, optionally, translated into a gene product (e.g., a polypeptide) when placed under the control of proper regulatory sequences. The coding region may be present in either cDNA, genomic DNA, or RNA form. If the nucleic acid molecule is present in DNA form, it may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene may include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence is typically located 3' to the gene sequence.

[0034] The term "cell" is used herein in its broadest sense in the art and refers to a living organism, which is a structural unit of tissue in a multicellular organism, surrounded by an insulating membrane structure, capable of self-replication, and which contains genetic information and mechanisms for expressing it. As used herein, a cell can be a naturally occurring cell or an artificially modified cell (e.g., a fused cell, a genetically modified cell, etc.).

[0035] As used herein, terms such as "treatment" and "treating" refer to obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in that it completely or partially prevents a disease or a symptom of the disease, and / or it may be therapeutic in that it partially or completely cures a disease and / or the adverse effects caused by the disease. As used herein, "treatment" includes any treatment of disease in a mammal, for example, a human, including (a) preventing the disease from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., preventing its onset; and (c) alleviating the disease, i.e., causing the regression of the disease.

[0036] In some embodiments, the methods are useful for reducing tumor size and / or cell number. In some embodiments, the methods of the present disclosure are useful for inhibiting the growth of a tumor, such as a solid tumor, in a subject.

[0037] The term "antibody" includes monoclonal, polyclonal, dimeric, multimeric, multispecific antibodies and antibody fragments, which may be human, murine, humanized, chimeric, or derived from another species. A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies which have been directed against a specific antigenic site.

[0038] "Antibody or functional fragment thereof means an immunoglobulin molecule that specifically binds to or immunologically reacts with a particular antigen or epitope, and includes both polyclonal and monoclonal antibodies. The term antibody includes genetically engineered or otherwise modified immunoglobulin forms, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, and tetrabodies). Antibodies may be derived from natural sources or may be partially or wholly synthetically produced. Antibodies may be monoclonal or polyclonal. Antibodies may be members of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. The term functional antibody fragment includes, for example, Fab', F(ab') 2 The term scFv refers to a single chain Fv antibody, in which the heavy and light chain variable domains of a conventional two-chain antibody are linked to form a single chain. An antibody fragment may optionally be a single chain antibody fragment. Alternatively, the fragment may contain multiple chains linked together, for example, by disulfide bonds. The fragment may also optionally be a multimolecular complex. A functional antibody fragment retains the ability to bind to its cognate antigen with an affinity comparable to that of the intact antibody.

[0039] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies that make up the population are identical except for mutations that may be present in minor amounts, e.g., naturally occurring mutations. Thus, the modifier "monoclonal" indicates the characteristic of the antibody that it is not a mixture of individual antibodies. In certain embodiments, such monoclonal antibodies typically include antibodies that include a polypeptide sequence that binds to a target, where the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that the selected target-binding sequence can be further modified, e.g., to improve affinity for the target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that antibodies that include modified target-binding sequences are also monoclonal antibodies of the present disclosure. In contrast to polyclonal antibody preparations, which typically include several different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.

[0040] The phrase "pharmaceutical composition" or "pharmacologically acceptable composition" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals, such as humans, as appropriate. Preparation of pharmaceutical compositions containing antibodies or additional active ingredients will be known to those skilled in the art in light of this disclosure. Furthermore, it will be understood that for administration to animals (e.g., humans), preparations must meet the standards of sterility, pyrogenicity, general safety, and purity as required by the FDA Office of Biological Standards.

[0041] As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., water, alcoholic / aqueous solutions, saline, parenteral vehicles such as sodium chloride, and Ringer's dextrose), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, and inert gases), isotonicity agents, absorption retarding agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, liquids, and nutritional supplements, such similar materials, and combinations thereof, as known to those skilled in the art. The pH and exact concentration of various components in the pharmaceutical composition can be adjusted according to well-known parameters.

[0042] The term "unit dose" or "dosage" refers to a physically separate unit suitable for use in a subject, each unit containing a predetermined amount of a therapeutic composition calculated to produce the desired response discussed herein with its administration, i.e., with an appropriate route and regimen. The amount administered depends on the desired effect, both according to the number of treatments and the unit dose. The actual dosage of the composition of this embodiment administered to a patient or subject can be determined by physical and physiological factors, such as the subject's weight, age, health, and sex, the type of disease being treated, the degree of disease penetration, previous or concurrent therapeutic interventions, the patient's idiopathic disease, the route of administration, and the potency, stability, and toxicity of the particular therapeutic agent. For example, the dosage can also include from about 1 μg / kg / body weight to about 1000 mg / kg / body weight per administration (including such ranges in between) or more, and any particular dosage derivable therein. Non-limiting examples of ranges derivable from the numbers set forth herein include ranges of from about 5 μg / kg / body weight to about 100 mg / kg / body weight, from about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose for the individual subject.

[0043] Of particular interest is the use of single chain variable fragments (scFv). scFvs are recombinant molecules in which the variable regions of an immunoglobulin light chain and an immunoglobulin heavy chain encoding an antigen binding domain have been engineered into a single polypeptide. Generally, V H and V LThe sequences are linked by a linker sequence. See, for example, Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, specifically incorporated herein by reference. Described herein are BCMA-specific scFv molecules that include variable regions of immunoglobulin light chains and immunoglobulin heavy chains that encode a BCMA-binding domain engineered into a single polypeptide. Similarly, described herein are CS1-specific scFv molecules that include variable regions of immunoglobulin light chains and immunoglobulin heavy chains that encode a CS1-binding domain engineered into a single polypeptide.

[0044] As used herein, the term "binding affinity" refers to the equilibrium constant of reversible binding of two agents, expressed as a dissociation constant (Kd). Binding affinity can be at least 1-fold higher, at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 20-fold higher, at least 30-fold higher, at least 40-fold higher, at least 50-fold higher, at least 60-fold higher, at least 70-fold higher, at least 80-fold higher, at least 90-fold higher, at least 100-fold higher, or at least 1000-fold higher, or more (or any range derivable therein) higher than the binding affinity of an antibody to an unrelated amino acid sequence. As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation after dilution. The terms "immunoreactive" and "selectively bind" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

[0045] The term "bond" refers to a direct association between two molecules, for example, through covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bonding interactions, including interactions such as salt bridges and water bridges.

[0046] "Therapeutically effective amount" or "effective amount" refers to the amount of an agent, or the combined amount of two agents, that when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment of the disease. A "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0047] "Subject" and "patient" refer to either humans or non-humans, such as primates, mammals, and vertebrates. In certain embodiments, the subject is a human.

[0048] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method utilized to determine the value, or the variation that exists among study subjects.

[0049] II. CD70 targeting agent A. Antibodies An aspect of the present disclosure relates to a CD70 targeting agent. In some embodiments, the CD70 targeting agent comprises an anti-CD70 antibody or a fragment thereof. The term "antibody" refers to any isotype of intact immunoglobulin, or a fragment thereof, that can compete with intact antibody for specific binding to target antigen, including chimeric antibodies, humanized antibodies, fully human antibodies, and bispecific antibodies. As used herein, the terms "antibody" or "immunoglobulin" are used interchangeably and refer to any of several classes of structurally related proteins that function as part of an animal's immune response, including IgG, IgD, IgE, IgA, IgM and related proteins, as well as polypeptides that contain antibody CDR domains that retain antigen-binding activity.

[0050] The term "antigen" refers to a molecule or portion of a molecule capable of being bound by a selective binding agent, such as an antibody. An antigen can possess one or more epitopes capable of interacting with different antibodies.

[0051] The term "epitope" includes any region or portion of a molecule capable of eliciting an immune response by binding to an immunoglobulin or T-cell receptor. Epitopic determinants include chemically active surface groups such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three-dimensional structural characteristics and / or specific charge characteristics. In general, an antibody specific for a particular target antigen selectively recognizes an epitope on the target antigen within a complex mixture.

[0052] Epitope regions of a given polypeptide can be identified using many different epitope mapping techniques that are well known in the art, including x-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, and protein display arrays, see, e.g., Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, NY. Such techniques are known in the art and are described, for example, in U.S. Pat. No. 4,708,871; Geysen et al. Proc. Natl. Acad. Sci. USA 81:3998-4002 (1984); Geysen et al. Proc. Natl. Acad. Sci. USA 82:178-182 (1985); Geysen et al. Molec. Immunol. 23:709-715 (1986). See, e.g., Epitope Mapping Protocols, supra. Additionally, antigenic regions of proteins can be predicted and identified using standard antigenicity and hydrophobicity plots.

[0053] An intact antibody is generally composed of two full-length heavy chains and two full-length light chains, but may contain fewer chains in some cases, such as antibodies naturally occurring in camelids, which may contain only heavy chains. The antibodies disclosed herein may be derived from only a single source, or may be "chimeric", i.e., different portions of the antibody may be derived from two different antibodies. For example, the variable or CDR regions may be derived from a rat or mouse source, while the constant regions are derived from different animal sources, such as humans. Antibodies or binding fragments may be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes its derivatives, variants, fragments, and muteins, examples of which are described below (Sela-Culang et al. Front Immunol. 2013; 4: 302; 2013).

[0054] The term "light chain" includes full-length light chains and fragments thereof that have sufficient variable region sequence to confer binding specificity. Full-length light chains have a molecular weight of around 25,000 daltons and contain a variable region domain (abbreviated herein as VL) and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, identified as kappa (κ) and lambda (λ). The term "VL fragment" refers to a fragment of the light chain of a monoclonal antibody that contains all or a portion of the light chain variable region, including the CDRs. The VL fragment may further contain a light chain constant region sequence. The light chain variable region domain is at the amino terminus of the polypeptide.

[0055] The term "heavy chain" includes full-length heavy chains and fragments thereof that have sufficient variable region sequence to confer binding specificity. A full-length heavy chain has a molecular weight of around 50,000 daltons and includes a variable region domain (abbreviated herein as VH), and three constant region domains (abbreviated herein as CH1, CH2, and CH3). The term "VH fragment" refers to a fragment of a heavy chain of a monoclonal antibody that includes all or a portion of the heavy chain variable region, including the CDRs. A VH fragment may further include a heavy chain constant region sequence. The number of heavy chain constant region domains will depend on the isotype. The VH domain is at the amino terminus of the polypeptide, the CH domain is at the carboxy terminus, and CH3 is closest to the -COOH terminus. The isotype of an antibody can be IgM, IgD, IgG, IgA, or IgE, and is defined by the presence of five heavy chain classifications: mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε) chains, respectively. IgG has several subtypes, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes include IgM1 and IgM2. IgA subtypes include IgA1 and IgA2.

[0056] Antibodies can be whole immunoglobulins of any isotype or classification, chimeric antibodies, or hybrid antibodies having specificity for two or more antigens. They can also be fragments, including hybrid fragments (e.g., F(ab')2, Fab', Fab, Fv, etc.). Immunoglobulins also include natural, synthetic, or genetically engineered proteins that act like antibodies by binding to a specific antigen to form a complex. The term antibody includes genetically engineered or otherwise modified forms of immunoglobulins, such as:

[0057] The term "monomer" refers to an antibody that contains only one Ig unit. A monomer is the basic functional unit of an antibody. The term "dimer" refers to an antibody that contains two Ig units bound to each other via the constant domains (Fc regions or fragment crystallizable regions) of the antibody heavy chains. The complex may be stabilized by a joining (J) chain protein. The term "multimer" refers to an antibody that contains more than two Ig units bound to each other via the constant domains (Fc regions) of the antibody heavy chains. The complex may be stabilized by a joining (J) chain protein.

[0058] The term "bivalent antibody" refers to an antibody that comprises two antigen-binding sites. The two binding sites may have the same antigen specificity or they may be bispecific, meaning that the two antigen-binding sites have different antigen specificities.

[0059] Bispecific antibodies are a class of antibodies with two paratopes that have different binding sites for two or more different epitopes. In some embodiments, bispecific antibodies can be biparatopic, where the bispecific antibodies can specifically recognize different epitopes from the same antigen. In some embodiments, bispecific antibodies can be constructed from a pair of different single domain antibodies, called "nanobodies." Single domain antibodies are sourced and engineered from cartilaginous fish and camelids. Nanobodies can be linked together by linkers using techniques common to those skilled in the art; such methods for the selection and linking of nanobodies are described in PCT Publication Nos. WO2015044386A1, WO2010037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), each of which is specifically incorporated herein by reference in its entirety.

[0060] Bispecific antibodies can be constructed as whole IgG, Fab'2, Fab'PEG, diabodies, or alternatively as scFvs. Diabodies and scFvs can be constructed without Fc regions using only variable domains, potentially reducing the effects of anti-idiotypic reactions. Bispecific antibodies can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), each of which is specifically incorporated by reference in its entirety.

[0061] In certain aspects, the antigen-binding domain can be multispecific or heterospecific by multimerizing with VH and VL domain pairs that bind different antigens. For example, the antibody can bind to or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, or (c) at least one other component. Thus, aspects can include, but are not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies or antigen-binding fragments thereof that are directed to epitopes and other targets, such as Fc receptors on effector cells.

[0062] In some embodiments, multispecific antibodies can be used and directly linked via a flexible short polypeptide chain using conventional methods known in the art. One such example is a diabody, a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain that is too short to allow pairing between the domains on the same chain, thereby pairing the domains with complementary domains on another chain to create two antigen binding sites. Linker functionality is applicable to triabodies, tetrabodies, and higher antibody multimer embodiments (see, for example, Hollinger et al., Proc Natl. Acad. Sci. USA 90:6444-6448 (1993); Polijak et al., Structure 2:1121-1123 (1994); Todorovska et al., J. Immunol. Methods 248:47-66 (2001)).

[0063] In contrast to bispecific whole antibodies, bispecific diabodies can also be advantageous because they can be easily constructed and expressed in E. coli. Diabodies (and other polypeptides, such as antibody fragments) of appropriate binding specificity can be easily selected using phage display (WO94 / 13804) from libraries. If one arm of the diabody is held constant, e.g., specificity for a protein is maintained, a library can be created in which the other arm is varied and antibodies of appropriate specificity are selected. Bispecific whole antibodies can also be produced by alternative engineering methods, as described in Ridgeway et al. (Protein Eng., 9:616-621, 1996) and Krah et al. (N Biotechnol. 39:167-173, 2017), each of which is incorporated herein by reference in its entirety.

[0064] Heteroconjugate antibodies are composed of two covalently linked monoclonal antibodies with different specificities, see, e.g., U.S. Patent No. 6,010,902, incorporated herein by reference in its entirety.

[0065] The portion of the Fv fragment of an antibody molecule that binds with high specificity to an epitope of an antigen is referred to herein as the "paratope". The paratope consists of amino acid residues that contact the epitope of the antigen and promote antigen recognition. Each of the two Fv fragments of an antibody is composed of two variable domains, VH and VL, in a dimerized configuration. The primary structure of each of the variable domains includes three hypervariable loops bounded and flanked by framework regions (FR). The hypervariable loops are the regions of greatest primary sequence variability among antibody molecules from any mammal. The term hypervariable loops is sometimes used interchangeably with the term "complementarity determining region (CDR)". The length of the hypervariable loops (or CDRs) varies from one antibody molecule to another. The framework regions of all antibody molecules from a given mammal have a high degree of primary sequence similarity / consensus. The consensus of the framework regions can be used by one skilled in the art to identify both the framework regions and the hypervariable loops (or CDRs) interspersed between the framework regions. The hypervariable loops are given identifiers that distinguish their position in the polypeptide and the domain in which they occur. The CDRs in the VL domain are identified as L1, L2, and L3, with L1 being the most distal and L3 being closest to the CL domain. The CDRs may also be named CDR-1, CDR-2, and CDR-3. L3 (CDR-3) is generally the most variable region of all antibody molecules produced by a given organism. The CDRs are regions of the polypeptide chain that are linearly arranged in the primary structure and separated from each other by framework regions. The amino-terminal (N-terminal) end of the VL chain is named FR1. The region identified as FR2 is between the L1 and L2 hypervariable loops. FR3 is between the L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. This structure and nomenclature is repeated for the VH chain, which contains three CDRs identified as H1, H2, and H3. The majority of amino acid residues in the variable domains, or Fv fragments (VH and VL), are part of framework regions (approximately 85%).The three-dimensional or tertiary structure of an antibody molecule is such that the framework regions are more interior to the molecule and provide the majority of the structure, with the CDRs on the exterior of the molecule.

[0066] Several methods have been developed and can be used by those skilled in the art to identify the exact amino acids that make up each of these regions. This can be done using any of several sequence alignment methods and algorithms that identify the conserved amino acid residues that make up the framework regions, and therefore identify the CDRs that are located between the framework regions, which may vary in length. Three commonly used methods have been developed for the identification of antibody CDRs: Kabat (as described in T. T. Wu and E. A. Kabat, "AN ANALYSIS OF THE SEQUENCES OF THE VARIABLE REGIONS OF BENCE JONES PROTEINS AND MYELOMA LIGHT CHAINS AND THEIR IMPLICATIONS FOR ANTIBODY COMPLEMENTARITY", J Exp Med, vol. 132, no. 2, pp. 211-250, Aug. 1970); Chothia (as described in C. Chothia et al., "Conformations of immunoglobulin hypervariable regions", Nature, vol. 342, no. 6252, pp. 877-883, Dec. 1989); and IMGT (M.-P. Lefranc et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Developmental & Comparative Immunology, vol. 27, no. 1, pp. 55-77, Jan. 2003). Each of these methods involves a unique numbering system for the identification of the amino acid residues that make up the variable regions. In most antibody molecules, the amino acid residues that actually contact the epitope of the antigen are present in the CDRs, but in some cases, residues in the framework regions contribute to antigen binding.

[0067] One skilled in the art can use any of several methods to determine the paratope of an antibody. These methods include; 1) computational prediction of the tertiary structure of the antibody / epitope binding interaction based on the chemical nature of the amino acid sequence of the antibody variable region and the composition of the epitope; 2) hydrogen-deuterium exchange and mass spectrometry; 3) polypeptide fragmentation and peptide mapping approaches, in which multiple overlapping peptide fragments are generated from the full length of the polypeptide and the binding affinity of these peptides to the epitope is evaluated; 4) antibody phage display library analysis, in which mammalian antibody Fab fragment-encoding genes are expressed by bacteriophage so that they are incorporated into the coat of the phage. This population of Fab-expressing phages is then allowed to interact with the antigen, which may be immobilized or expressed by a different exogenous expression system. The non-binding Fab fragments are washed away, thereby leaving only the specific binding Fab fragments attached to the antigen. The binding Fab fragments can be easily isolated and the genes encoding them can be determined. This approach can also be used for Fv fragments or even smaller regions of Fab fragments containing specific VH and VL domains, if desired.

[0068] In certain aspects, affinity matured antibody is enhanced with one or more modifications in one or more of its CDRs, which results in improved affinity of the antibody to the target antigen, compared to the parent antibody that does not carry these modifications.Some affinity matured antibodies have nanomolar or picomolar affinity to the target antigen.Affinity matured antibodies are produced by procedures known in the art, for example, Marks et al., Bio / Technology 10:779 (1992) describes affinity maturation by VH and VL domain shuffling, and random mutagenesis of CDR and / or framework residues utilized in phage display is described by Rajpal et al., PNAS. 24: 8466-8471 (2005) and Thie et al., Methods Mol Biol. 525:309-22 (2009) in combination with the computational method demonstrated in Tiller et al., Front. Immunol. 8:986 (2017).

[0069] Chimeric immunoglobulins are the product of fusion genes derived from different species; a "humanized" chimera generally has framework regions (FR) from a human immunoglobulin and one or more CDRs are from a non-human source.

[0070] In certain aspects, portions of the heavy and / or light chains are identical or homologous to corresponding sequences from another particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity. U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984). For methods relating to chimeric antibodies, see, for example, U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985), each of which is specifically incorporated herein by reference in its entirety. CDR grafting is described, for example, in U.S. Patent Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, all of which are incorporated by reference herein for all purposes.

[0071] In some embodiments, the antibody polypeptide sequence from the non-human species is minimized to optimize chimeric antibody function and reduce immunogenicity. Certain amino acid residues from non-antigen recognition regions of the non-human antibody are modified to be homologous to the corresponding residues in human antibodies or isotypes. One example is a "CDR-grafted" antibody, where the antibody contains one or more CDRs from a particular species or belonging to a particular antibody class or subclass, while the remainder of the antibody chain is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass. For use in humans, the V-regions, consisting of CDR1, CDR2, and partial CDR3 of both the light and heavy chain variable regions from a non-human immunoglobulin, are grafted with human antibody framework regions, replacing the natural antigen receptor of the human antibody with the non-human CDRs. In some cases, the corresponding non-human residues replace the framework region residues of the human immunoglobulin. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or donor antibody to further refine performance. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Presta, Curr. Op. Struct. Biol. 2:593 (1992); Vaswani and Hamilton, Ann. Allergy, Asthma and Immunol. 1:105 (1998); Harris, Biochem. Soc. Transactions 23; 1035 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428 (1994); Verhoeyen et al., Science 239:1534-36 (1988).

[0072] Intrabodies are immunoglobulins located within cells that bind to intracellular antigens, as opposed to secreted antibodies, which bind to antigens in the extracellular space.

[0073] A polyclonal antibody preparation usually contains different antibodies against different determinants (epitopes). To produce polyclonal antibodies, a host, such as a rabbit or goat, is immunized with an antigen or an antigen fragment, generally with an adjuvant, optionally bound to a carrier. Antibodies against the antigen are then recovered from the host's serum. Polyclonal antibodies can be affinity purified against the antigen to render them monospecific.

[0074] A monoclonal antibody or "mAb" refers to an antibody obtained from a homogeneous population of antibodies from a single parent cell, i.e., the population is identical except for natural mutations that may be present in minor amounts. Each monoclonal antibody is directed against a single antigenic determinant.

[0075] 1. Functional antibody and antigen-binding fragments a. Antigen-binding fragment Certain aspects relate to antibody fragments, such as antibody fragments that bind and / or neutralize inflammatory mediators. The term functional antibody fragment includes antigen-binding fragments of antibodies that retain the ability to specifically bind to antigens. These fragments are composed of various arrangements of variable regions heavy chain (VH) and / or light chain (VL); in some embodiments, they contain constant regions heavy chain 1 (CH1) and light chain (CL). In some embodiments, they lack the Fc region, which is composed of heavy chain 2 (CH2) and 3 (CH3) domains. Embodiments of antigen-binding fragments and modifications thereof may include: (i) Fab fragment type composed of VL, VH, CL and CH1 domains; (ii) Fd fragment type composed of VH and CH1 domains; (iii) Fv fragment type composed of VH and VL domains; (iv) single domain fragment type dAb composed of a single VH or VL domain (Ward, 1989; McCafferty et al., 1990; Holt et al., 2003); (v) isolated complementarity determining region (CDR) regions. Such terms are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, NY (1989); Molec. Biology and Biotechnology: A Comprehensive Desk Reference (Myers, RA (ed.), New York: VCH Publisher, Inc.); Huston et al., Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymol., 178:497-515 (1989) and Day, ED, Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, NY (1990); Antibodies, 4:259-277 (2015). All citations in this paragraph are incorporated by reference.

[0076] Antigen-binding fragments also include fragments of antibodies that retain at least or exactly one, two, or three complementarity determining regions (CDRs) from the light chain variable region. Fusions of CDR-containing sequences to an Fc region (or its CH2 or CH3 regions) are included within this definition, including, for example, scFvs fused directly or indirectly to an Fc region as included herein.

[0077] The term Fab fragment refers to a monovalent antigen-binding fragment of an antibody that contains the VL, VH, CL and CH1 domains. The term Fab' fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody that is larger than the Fab fragment. For example, a Fab' fragment contains the VL, VH, CL and CH1 domains and all or part of the hinge region. The term F(ab')2 fragment refers to a bivalent antigen-binding fragment of a monoclonal antibody that contains two Fab' fragments linked by a disulfide bridge at the hinge region. The F(ab')2 fragment, for example, contains all or part of two VH and VL domains and can further contain all or part of two CL and CH1 domains.

[0078] The term Fd fragment refers to a fragment of a heavy chain of a monoclonal antibody that contains all or a portion of the VH, including the CDRs. The Fd fragment may further contain a CH1 region sequence.

[0079] The term Fv fragment refers to a monovalent antigen-binding fragment of a monoclonal antibody, comprising all or part of VL and VH, but lacking CL and CH1 domains. VL and VH, for example, comprise CDRs. Single-chain antibodies (sFv or scFv) are Fv molecules in which the VL and VH regions are linked by a flexible linker to form a single polypeptide chain to form an antigen-binding fragment. Single-chain antibodies are discussed in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference. The term (scFv)2 refers to a bivalent or bispecific sFv polypeptide chain comprising an oligomerization domain at the C-terminus, separated from the sFv by a hinge region (Pack et al. 1992). The oligomerization domain contains self-associating a-helices, e.g., leucine zippers, which can be further stabilized by additional disulfide bonds. (scFv)2 fragments are also known as "mini-antibodies" or "mini-bodies".

[0080] A single domain antibody is an antigen-binding fragment that contains only a VH or VL domain. In some cases, two or more VH regions are covalently linked with a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0081] b. Fragment crystallizable region, Fc The Fc region comprises two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic interaction of the CH3 domain. The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. It includes truncated forms of such polypeptides, including the hinge region that promotes dimerization.

[0082] c. A polypeptide having an antibody CDR and a scaffold domain that displays the CDR Antigen-binding peptide scaffolds, such as complementarity determining regions (CDRs), are used to create protein-binding molecules according to the embodiment.Generally, those skilled in the art can determine the type of protein scaffold to graft at least one CDR.It is known that optimally, scaffolds must meet several criteria, such as good phylogenetic conservation; known three-dimensional structure; small size; little or no post-transcriptional modification; easy to produce, express and purify.Skerra, J Mol Recognit, 13:167-87 (2000).

[0083] Protein scaffolds can be provided by, but are not limited to, fibronectin type III FN3 domain (known as "monobody"), fibronectin type III domain 10, lipocalins, anticalins, the Z domain of Staphylococcus aureus protein A, thioredoxin A or proteins with repeat motifs such as "ankyrin repeats", "armadillo repeats", "leucine-rich repeats" and "tetratricopeptide repeats". Such proteins are described in U.S. Patent Application Publication Nos. 2010 / 0285564, 2006 / 0058510, 2006 / 0088908, 2005 / 0106660, and PCT Publication No. WO2006 / 056464, each of which is specifically incorporated herein by reference in its entirety. Scaffolds derived from toxins from scorpions, insects, plants, mollusks, etc., and protein inhibitors of neuronal NO synthase (PIN) may also be used.

[0084] B. Chimeric Antigen Receptors In some embodiments, the CD70 targeting agent comprises a CD70-specific CAR molecule or cell, such as a T cell that comprises and / or expresses a CD70-specific CAR molecule. Chimeric antigen receptor T cells, or CAR T cells, are T cells that are either patient-derived, donor-derived, or in vitro-produced, that are genetically modified to express a chimeric receptor specific for tumor antigens, together with signaling domains and costimulatory molecules. This fusion of a single-chain variable fragment derived from an antibody with the intracellular signaling domain of a T cell provides the CAR T cell with the ability to recognize tumor antigens in an MHC-inhibited manner.

[0085] CAR molecules typically comprise one or more antibody binding regions, an extracellular spacer, a transmembrane domain, and a cytoplasmic region, which are further described below.

[0086] 1. Antigen binding region The antigen-binding region may be a single chain variable fragment (scFv) derived from a CD70 antibody. "Single chain Fv" or "scFv" antibody fragments are fragments of the V of an antibody. H and V L domains, where these domains are present in a single polypeptide chain. In some embodiments, the antigen-binding domain further comprises a peptide linker between the VH and VL domains, which can facilitate the formation of the desired structure for antigen binding of the scFv.

[0087] The variable regions of the antigen-binding domain of the polypeptide of the present disclosure can be modified by mutating amino acid residues in the VH and / or VL CDR 1, CDR 2 and / or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term "CDR" refers to a complementarity determining region based on a portion of the variable chain in immunoglobulins (antibodies) and T cell receptors, which are produced by B cells and T cells, respectively, and these molecules bind to their specific antigens. Most sequence variations associated with immunoglobulins and T cell receptors are found in the CDRs, so these regions are sometimes referred to as hypervariable regions. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis, and the effect on antibody binding, or other functional properties of interest, can be evaluated in suitable in vitro or in vivo assays. Preferably, conservative modifications are introduced, typically modifying no more than one, two, three, four or five residues in the CDR regions. Mutations can be amino acid substitutions, additions or deletions.

[0088] For example, framework modifications can be made to the antibody to reduce immunogenicity by "backmutating" one or more framework residues to the corresponding germline sequence.

[0089] It is also contemplated that an antigen-binding domain may be multispecific or multivalent by multimerizing the antigen-binding domain with VH and VL domain pairs that bind either the same antigen (multivalent) or different antigens (multispecific).

[0090] 2. Extracellular spacer The extracellular spacer may connect the antigen-binding domain to the transmembrane domain. It must be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen binding. In one embodiment, the spacer is a hinge region from IgG. Alternatives include the CH2CH3 region of immunoglobulins and a portion of CD3.

[0091] As used herein, the term "hinge" refers to a flexible polypeptide connector region (also referred to herein as "hinge region" or "spacer") that provides structural flexibility and spacing between adjacent polypeptide regions, and can be composed of natural or synthetic polypeptides. A "hinge" derived from an immunoglobulin (e.g., IgG1) is generally defined as stretching from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec. Immunol., 22: 161-206). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide (SS) bonds in the same positions. Hinge regions can be naturally occurring or non-naturally occurring, including, but not limited to, modified hinge regions as described in U.S. Pat. No. 5,677,425. The hinge region may include a complete hinge region derived from an antibody of a different class or subclass than that of the CH1 domain. The term "hinge" may also include regions derived from CD8 and other receptors which provide similar functions of providing flexibility and spacing between adjacent regions.

[0092] 3. Transmembrane domain Transmembrane domains are hydrophobic alpha helices that span the membrane. Different transmembrane domains may confer different receptor stabilities.

[0093] The transmembrane domain is inserted between the extracellular spacer and the cytoplasmic region. In some embodiments, the transmembrane domain is inserted between the extracellular spacer and one or more costimulatory regions. In some embodiments, a linker is between the transmembrane domain and one or more costimulatory regions. In some embodiments, the transmembrane domain is derived from CD28, CD8, CD4, CD3 zeta, CD134, or CD7.

[0094] 4. Cytoplasmic region After antigen recognition, the receptors cluster and a signal is transmitted to the cell through the cytoplasmic region. In some embodiments, the costimulatory domain described herein is part of the cytoplasmic region.

[0095] The cytoplasmic region and / or costimulatory region suitable for use in the polypeptide of the present disclosure include any desired signaling domain that provides a clear and detectable signal in response to activation by binding of an antigen to the antigen-binding domain (e.g., increased production of one or more cytokines by the cell; altered transcription of a target gene; altered activity of a protein; altered cell behavior, e.g., cell death; cell proliferation; cell differentiation; cell survival; modulation of cell signaling responses, etc.). In some embodiments, the cytoplasmic region includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motif as described herein. In some embodiments, the cytoplasmic region includes a DAP10 / CD28 type signaling chain.

[0096] Cytoplasmic domains suitable for use in the polypeptides of the present disclosure include intracellular signaling polypeptides that contain an immunoreceptor tyrosine-based activation motif (ITAM). An ITAM motif is YX 1 X 2 (L / I), where X 1 and X 2 are independently any amino acid. In some cases, the cytoplasmic region contains one, two, three, four, or five ITAM motifs. In some cases, the ITAM motif is repeated twice in the endodomain, where the ITAM motifs in the first and second instances are separated from each other by 6-8 amino acids, e.g., (YX 1 X 2 (L / I))(X3) n( YX 1 X 2 (L / I)), where n is an integer from 6 to 8, and 6 to 8 X 3 Each of can be any amino acid.

[0097] A suitable cytoplasmic region can be an ITAM motif-containing portion derived from a polypeptide that contains an ITAM motif. For example, a suitable cytoplasmic region can be an ITAM motif-containing domain derived from any ITAM motif-containing protein. Thus, a suitable endodomain does not need to include the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to, DAP12, DAP10, FCER1G (Fc epsilon receptor I gamma chain); CD3D (CD3 delta); CD3E (CD3 epsilon); CD3G (CD3 gamma); CD3 zeta; and CD79A (antigen receptor complex-associated protein alpha chain).

[0098] Non-limiting examples of suitable costimulatory regions, such as those contained in the cytoplasmic domain, include, but are not limited to, polypeptides from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0099] C. Bispecific T Cell Engagers (BiTEs) Bispecific T cell engagers are a new class of immunotherapeutic molecules aimed at the treatment of cancer. These molecules, termed BiTEs, enhance a patient's immune response against tumors by retargeting T cells to tumor cells. BiTEs contain two single-chain variable fragments (scFvs) connected in tandem by a flexible linker. Due to their structure and specificity, BiTEs physically link T cells to tumor cells, ultimately stimulating T cell activation, tumor killing and cytokine production. Embodiments include BiTEs that contain a CD70-specific targeting region, such as a CD70-specific scFV. BiTEs may further contain specificity for additional cancer-associated molecules, such as EGFR or AXL. Thus, embodiments of the present disclosure relate to BiTEs that contain a CD70-specific scFV and an EGFR-specific scFv. Further embodiments of the present disclosure relate to BiTEs that contain a CD70-specific scFv and an AXL scFV. Further embodiments of the present disclosure relate to BiTEs that contain a CD70-specific scFv and a tumor antigen-specific scFv. In some embodiments, the tumor antigen comprises a tumor antigen associated with non-small cell lung cancer.Another embodiment relates to a BiTE comprising a CD70-specific targeting region and a TCR-specific targeting region.For example, the TCR-specific targeting region can target a TCR subunit on T cells, such as CD3.

[0100] D. Trispecific Natural Killer Cell Engagement Therapy (TriNKET) TriNKET comprises an NK cell activation region and an antigen binding region, where the antigen binding region binds to CD70. TriNKET is designed to crosslink tumors and NK cells. In some embodiments, the NK cell activation region comprises an NK activation molecule, such as a cell surface molecule that can activate cells. The NK cell activation region and / or the antigen binding region can comprise an scFv specific for an NK activation protein and an scFv specific for a cancer antigen, respectively. In some embodiments, TriNKET comprises an scFv domain specific for CD16. In some embodiments, TriNKET comprises an scFV that specifically binds to CD70. TriNKET can further comprise an IL-15 or IL-2 molecule. TriNKET can be useful for (a) directing NK cells to tumors by promoting the formation of intracellular synapses; (b) binding to CD16 on NK cells to trigger ADCC; and (c) promoting the proliferation of NK cells in vivo through the expression of IL-15 or IL-2.

[0101] III. Cells Certain embodiments relate to cells that contain the polypeptides or nucleic acids of the present disclosure, such as CD70 targeting agents. In some embodiments, the cells are immune cells or T cells. "T cells" include all types of immune cells that express CD3, including T helper cells, cytotoxic T cells, T regulatory cells (Tregs), gamma-delta T cells, natural killer (NK) cells, and neutrophils. T cells can refer to CD4+ or CD8+ T cells.

[0102] Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), and the like.

[0103] In some cases, the cells are not immortalized cell lines, but are instead cells (e.g., primary cells) obtained from an individual. For example, in some cases, the cells are immune cells obtained from an individual. As an example, the cells are T lymphocytes obtained from an individual. As another example, the cells are cytotoxic cells obtained from an individual. As another example, the cells are stem or progenitor cells obtained from an individual. In some embodiments, the cells used to treat a patient are autologous. In some embodiments, the cells used to treat a patient are non-autologous.

[0104] IV. Methods for Modifying Genomic DNA In certain embodiments, the genomic DNA is modified to include additional mutations, insertions, or deletions, or to incorporate certain molecular constructs of the present disclosure such that the constructs are expressed from the genomic DNA. In some embodiments, the nucleic acid encoding the polypeptide of the present disclosure is integrated into the genomic DNA of the cell. In some embodiments, the integration is targeted integration. In some embodiments, the targeted integration is achieved through the use of a DNA digesting agent / polynucleotide modifying enzyme, such as a site-specific recombinase and / or a targeting endonuclease. The term "DNA digesting agent" refers to an agent that can cleave the bond (i.e., phosphodiester bond) between the nucleotide subunits of a nucleic acid. One particular target is the TRAC (T cell receptor alpha constant) locus. For example, the cells are first electroporated with a ribonucleoprotein (RNP) complex consisting of a Cas9 protein complexed with a single guide RNA (sgRNA) that targets the TRAC (T cell receptor alpha constant) locus. 15 minutes after electroporation, the cells are treated with an AAV6 carrying a HDR template that encodes a CAR.

[0105] Therefore, in one aspect, the present disclosure includes targeted integration. One way to achieve this is by using an exogenous nucleic acid sequence (i.e., a landing pad) that includes at least one recognition sequence for at least one polynucleotide-modifying enzyme, such as a site-specific recombinase and / or a targeting endonuclease. Site-specific recombinases are well known in the art and may be generally referred to as invertases, resolvases, or integrases. Non-limiting examples of site-specific recombinases may include lambda integrase, Cre recombinase, FLP recombinase, gamma-delta resolvase, Tn3 resolvase, ΦC31 integrase, Bxb1-integrase, and R4 integrase. Site-specific recombinases recognize a specific recognition sequence (or recognition site) or a variant thereof, all of which are well known in the art. For example, Cre recombinase recognizes LoxP sites, and FLP recombinase recognizes FRT sites.

[0106] Contemplated targeting endonucleases include zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs), CRIPSR / Cas-like endonucleases, I-Tevl nucleases or related monomeric hybrids, or artificial target DNA double-strand break inducers.Exemplary targeting endonucleases are described further below.For example, typically, zinc finger nucleases include a DNA binding domain (i.e., zinc finger) and a cleavage domain (i.e., nuclease), both of which are described below.The definition of polynucleotide modifying enzyme also includes any other useful fusion protein known to those skilled in the art, which may include a DNA binding domain and a nuclease.

[0107] A landing pad sequence is a nucleotide sequence that contains at least one recognition sequence that is selectively bound and modified by a specific polynucleotide modifying enzyme, such as a site-specific recombinase and / or a targeting endonuclease. In general, the recognition sequence in the landing pad sequence is not endogenously present in the genome of the cell to be modified. For example, if the cell to be modified is a CHO cell, the recognition sequence in the landing pad sequence is not endogenously present in the endogenous CHO genome. The rate of targeted integration can be improved by selecting a recognition sequence of a highly efficient nucleotide modifying enzyme that is not endogenously present in the genome of the target cell. The selection of a recognition sequence that is not endogenously present also reduces the possibility of off-target integration. In other aspects, it may be desirable to use a recognition sequence that is native to the cell to be modified. For example, when multiple recognition sequences are used in a landing pad sequence, one or more can be exogenous and one or more can be natural.

[0108] One of skill in the art can readily determine the sequences bound and cleaved by a site-specific recombinase and / or a targeting endonuclease.

[0109] Multiple recognition sequences may be present in a single landing pad, allowing the landing pad to be targeted sequentially by two or more polynucleotide modifying enzymes so that two or more unique nucleic acids (including, inter alia, a receptor gene and / or an inducible reporter) can be inserted. Alternatively, the presence of multiple recognition sequences in a landing pad allows multiple copies of the same nucleic acid to be inserted into the landing pad. When two nucleic acids are targeted to a single landing pad, the landing pad contains a first recognition sequence for a first polynucleotide modifying enzyme (e.g., a first ZFN pair) and a second recognition sequence for a second polynucleotide modifying enzyme (e.g., a second ZFN pair). Alternatively, or in addition, individual landing pads containing one or more recognition sequences may be integrated at multiple locations. Increased protein expression may be observed in cells transformed with multiple copies of a payload. Alternatively, it is also possible to simultaneously express multiple gene products in cells transformed with multiple copies when multiple unique nucleic acid sequences constituting different expression cassettes are loaded into the same or different landing pads. Regardless of the number and type of nucleic acids, when the targeting endonuclease is a ZFN, an exemplary ZFN pair includes hSIRT, hRSK4, and hAAVS1, along with their recognition sequences.

[0110] Generally speaking, the landing pad used to facilitate targeted integration can comprise at least one recognition sequence.For example, the landing pad can comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more recognition sequences.In the embodiment that comprises two or more recognition sequences, the recognition sequences can be unique to each other (i.e., recognized by different polynucleotide modifying enzymes), or can be the same repeat sequence, or a combination of repeat sequence and unique sequence.

[0111] Those skilled in the art will easily understand that the exogenous nucleic acid used as a landing pad can also contain other sequences in addition to the recognition sequence.For example, it may be advantageous to include one or more sequences that code for selectable or screenable genes as described herein, such as antibiotic resistance genes, metabolic selection markers, or fluorescent proteins.There may also be the use of other supplementary sequences, such as transcriptional regulatory elements and control elements (i.e., promoters, partial promoters, promoter traps, start codons, enhancers, introns, insulators and other expression elements).

[0112] In addition to selecting an appropriate recognition sequence, selecting a targeting endonuclease with high cleavage efficiency also improves the rate of targeted integration of the landing pad. The cleavage efficiency of a targeting endonuclease can be determined using methods well known in the art, including, for example, using an assay such as the CEL-1 assay or direct sequencing of insertions / deletions (Indels) in PCR amplicons.

[0113] The type of targeting endonuclease used in the methods and cells disclosed herein can vary or vary.Targeting endonuclease can be a naturally occurring protein or an engineered protein.One example of a targeting endonuclease is zinc finger nuclease, which will be discussed in more detail below.

[0114] Another example of a targeting endonuclease that can be used is an RNA-guided endonuclease that contains at least one nuclear localization signal, which allows the endonuclease to enter the nucleus of eukaryotic cells. The RNA-guided endonuclease also contains at least one nuclease domain and at least one domain that interacts with the guided RNA. The RNA-guided endonuclease is directed to a specific chromosomal sequence by the guided RNA, so that the RNA-guided endonuclease cleaves the specific chromosomal sequence. Since the guided RNA provides specificity for targeting cleavage, the endonuclease of the RNA-guided endonuclease is universal and can be used with different guided RNAs to cleave different target chromosomal sequences. Exemplary RNA-guided endonuclease proteins are discussed in more detail below. For example, the RNA-guided endonuclease can be a CRISPR / Cas protein or a CRISPR / Cas-like fusion protein, an RNA-guided endonuclease derived from clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system.

[0115] The targeting endonuclease can also be a meganuclease. Meganucleases are endodeoxyribonucleases characterized by large recognition sites, i.e., the recognition sites generally range from about 12 base pairs to about 40 base pairs. As a result of this requirement, the recognition sites generally occur only once in any given genome. Among the meganucleases, the endonucleases family, named "LAGLIDADG", have become valuable tools for the study of genomes and genome engineering. Meganucleases can be targeted to specific chromosomal sequences by modifying their recognition sequences using techniques well known to those skilled in the art. See, for example, Epinat et al., 2003, Nuc. Acid Res., 31(11):2952-62 and Stoddard, 2005, Quarterly Review of Biophysics, pp. 1-47.

[0116] Another example of targeting endonucleases that can be used is transcription activator-like effector (TALE) nuclease. TALE is a transcription factor from plant pathogen Xanthomonas, which can be easily engineered to bind to new DNA targets. TALE or its truncated form can be linked to the catalytic domain of endonucleases such as FokI to create targeting endonucleases called TALE nucleases or TALENs. See, for example, Sanjana et al., 2012, Nature Protocols 7(1):171-192; Bogdanove AJ, Voytas D F., 2011, Science, 333(6051):1843-6; Bradley P, Bogdanove AJ, Stoddard B L., 2013, Curr Opin Struct Biol., 23(1):93-9.

[0117] Another exemplary targeting endonuclease is a site-specific nuclease. In particular, the site-specific nuclease can be a "low-frequency cutter" endonuclease whose recognition sequence is rarely found in genome. Preferably, the recognition sequence of the site-specific nuclease is found only once in genome. Alternatively, the targeting nuclease can be an artificial targeting DNA double strand break inducer.

[0118] In some embodiments, targeted integration can be achieved by the use of integrase. For example, phiC31 integrase is a sequence-specific recombinase encoded in the genome of bacteriophage phiC31. phiC31 integrase mediates recombination between two 34-base pair sequences called attachment sites (att), one found in the phage and the other in the bacterial host. This serine integrase has been shown to function efficiently in many different cell types, including mammalian cells. In the presence of phiC31 integrase, an attB-containing donor plasmid can be directionally integrated into the target genome through recombination at a site with sequence similarity to the natural attP site (called the pseudo attP site). phiC31 integrase can integrate plasmids of any size as a single copy and does not require cofactors. The integrated transgene is stably expressed and heritable.

[0119] In one embodiment, genome integration of the polynucleotide of the present disclosure is achieved by using transposase. For example, synthetic DNA transposons designed to introduce precisely defined DNA sequences into vertebrate chromosomes (e.g., "Sleeping Beauty" transposon system) can be used. The Sleeping Beauty transposon system is composed of Sleeping Beauty (SB) transposase and a transposon designed to insert a specific sequence of DNA into the genome of a vertebrate. DNA transposons transpose from one DNA site to another in a simple cut-and-paste manner. Transposition is the precise process by which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or different DNA molecule or genome.

[0120] Like all other Tc1 / mariner type transposases, SB transposase inserts the transposon into a TA dinucleotide base pair in the recipient DNA sequence. The insertion site can be elsewhere in the same DNA molecule or in another DNA molecule (or chromosome). There are approximately 200 million TA sites in mammalian genomes, including humans. The TA insertion site is duplicated in the process of transposon integration. This duplication of TA sequences is a hallmark of transposition and is used to confirm the mechanism in some experiments. The transposase can be encoded within the transposon, or it can be supplied by another source, in which case the transposon becomes a non-autonomous element. Non-autonomous transposons are the most useful as genetic tools because, after insertion, they cannot continue to excise and reinsert independently. All of the DNA transposons identified in the human genome and in the genomes of other mammals are non-autonomous. Because, even though they contain a transposase gene, the gene is non-functional and unable to produce a transposase capable of mobilizing the transposon.

[0121] I. Treatment Method Aspects of the present disclosure relate to methods for treating cancer, such as non-small cell lung cancer. In further embodiments, the CD70 targeting molecules described herein can be used to stimulate an immune response. The immune response stimulation can be in vitro, in vivo, or ex vivo. In some embodiments, the CD70 targeting molecules described herein are for preventing recurrence. The method generally involves administering a CD70 targeting molecule to a patient. In some embodiments, the CD70 targeting molecule is a genetically modified mammalian cell carrying an expression vector or RNA (e.g., in vitro transcribed RNA) that includes a nucleotide sequence encoding a polypeptide that targets CD70. The cell can be an immune cell (e.g., T lymphocyte or NK cell), stem cell, progenitor cell, etc. In some embodiments, the cell is a cell described herein or its progeny.

[0122] The embodiment of the present disclosure includes ex vivo method.For example, T lymphocyte, stem cell, or NK cell (or the cell described herein) is obtained from an individual; The cell obtained from the individual is genetically modified to express the CD70 targeting molecule of the present disclosure.In some cases, the genetically modified cell is activated ex vivo.In other cases, the genetically modified cell is introduced into an individual (e.g., the individual from whom the cell is obtained); The genetically modified cell is activated in vivo.

[0123] In some embodiments, the methods relate to the administration of cells or CD70 targeting molecules for the treatment of cancer or to a person with cancer. In some embodiments, the cancer is non-small cell lung cancer.

[0124] II. Pharmaceutical Compositions The disclosure includes methods for treating disease and modulating immune responses in subjects in need thereof. The disclosure includes cells, which may be in the form of pharmaceutical compositions that can be used to induce or modify an immune response.

[0125] Administration of compositions according to the present disclosure will generally be by any common route, including, but not limited to, parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

[0126] Typically, compositions of the present disclosure are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunomodulatory. The amount administered will depend on the subject being treated. Precise amounts of active ingredient required to be administered will depend on the judgment of the practitioner.

[0127] The method of application can vary widely. Any of the conventional methods for administration of pharmaceutical compositions containing cellular components are applicable. The dosage of the pharmaceutical composition will depend on the route of administration and will vary according to the size and health of the subject.

[0128] In many cases, it will be desirable to have multiple administrations, at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more. Administration can range from 2 days to 12 weeks apart, more usually 1 to 2 weeks apart. The course of administration can be followed by assays of alloreactive immune responses and T cell activity.

[0129] The phrase "pharmacologically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other untoward reactions when administered to animals or humans. As used herein, "pharmacologically acceptable carriers" include any and all solvents, dispersion media, coatings, antibacterial and antifungal substances, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmacoactive substances is well known in the art. Except in the case where any conventional media or agent is incompatible with the active ingredient, its use in immunogenic and therapeutic compositions is contemplated. The pharmaceutical compositions of the present disclosure are pharmacologic acceptable compositions.

[0130] The compositions of the present disclosure can be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, or even intraperitoneal routes.Typically, such compositions can be prepared as injectables, either as liquid solutions or liquid suspensions, and the preparations can also be emulsified.

[0131] Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol. It should also be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0132] Sterile injectable solutions are prepared by incorporating the required amount of active ingredient (i.e., cells of the present disclosure) in a suitable solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above.

[0133] The effective amount of the composition is determined based on the intended purpose. The term "unit dose" or "dosage" refers to a physically separate unit suitable for use in a subject, each unit containing a predetermined amount of the composition calculated to bring about the desired response discussed herein with its administration, i.e., with a suitable route and regimen. The amount administered depends on the desired outcome and / or protection, both according to the number of treatments and the unit dose. The exact amount of the composition also depends on the judgment of the practitioner and is specific to each individual. Factors that affect the dosage include the subject's physical and clinical condition, the route of administration, the intended purpose of the treatment (symptom relief or cure), and the efficacy, stability and toxicity of the particular composition. Once the solution is formulated, it is administered in a manner compatible with the dosage formulation and in such an amount that is therapeutically or prophylactically effective. The formulation is easily administered in a variety of dosage forms, for example, in the type of injectable solutions described above.

[0134] V. Further Treatments The disclosed method and composition may comprise one or more additional therapeutics known in the art and / or described herein.In some embodiments, the additional therapeutics or agents comprise additional cancer treatments.Examples of such treatments are described herein.

[0135] A. Immunotherapy In some embodiments, the additional therapy or agent comprises cancer immunotherapy. Cancer immunotherapy (sometimes called immuno-oncology, abbreviated as IO) is the use of the immune system to treat cancer. Immunotherapy can be classified as active, passive or hybrid (active and passive). These approaches take advantage of the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAA); they are often proteins or other macromolecules (e.g. carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting TAA. Passive immunotherapy enhances existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes and cytokines. Immunotherapies are known in the art, and some are described below.

[0136] 1. Inhibition of costimulatory molecules In some embodiments, the immunotherapy comprises inhibitors of costimulatory molecules. In some embodiments, the inhibitors comprise inhibitors of B7-1 (CD80), B7-2 (CD86), CD28, ICOS, OX40 (TNFRSF4), 4-1BB (CD137; TNFRSF9), CD40L (CD40LG), GITR (TNFRSF18), and combinations thereof. Inhibitors include inhibitory antibodies, polypeptides, compounds, and nucleic acids.

[0137] 2. Dendritic cell therapy Dendritic cell therapy induces an anti-tumor response by having dendritic cells present tumor antigens to lymphocytes, thereby activating the lymphocytes and stimulating them to kill other cells that present the antigens. Dendritic cells are antigen-presenting cells (APCs) in the mammalian immune system. In cancer treatment, dendritic cells help target cancer antigens. One example of a dendritic cell-based cellular cancer therapy is sipuleucel-T.

[0138] One way to induce dendritic cells to present tumor antigens is by vaccination with autologous tumor lysates or short peptides (small portions of proteins that correspond to protein antigens on cancer cells). These peptides are often given in combination with adjuvants (highly immunogenic substances) to boost immune and antitumor responses. Other adjuvants include proteins or other chemicals that attract and / or activate dendritic cells, such as granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0139] Dendritic cells can also be activated in vivo by expressing GM-CSF in tumor cells, which can be accomplished by genetically engineering the tumor cells to produce GM-CSF or by infecting the tumor cells with an oncolytic virus that expresses GM-CSF.

[0140] Another strategy is to remove dendritic cells from the patient's blood and activate them ex vivo. The dendritic cells are activated in the presence of tumor antigens, which can be single tumor-specific peptides / proteins or tumor cell lysates (a solution of destroyed tumor cells). These cells (with optional adjuvants) are injected to elicit an immune response.

[0141] Dendritic cell therapy involves the use of antibodies that bind to receptors on the surface of dendritic cells. Antigens can be added to the antibodies, inducing dendritic cells to mature and provide immunity against tumors. Dendritic cell receptors such as TLR3, TLR7, TLR8 or CD40 have been used as antibody targets.

[0142] 3. CAR-T cell therapy Chimeric antigen receptors (CARs, also known as chimeric immune receptors, chimeric T cell receptors or artificial T cell receptors) are engineered receptors that combine immune cells with new specificities to target cancer cells. Typically, these receptors transfer the specificity of a monoclonal antibody onto a T cell. The receptors are called chimeric because parts from different sources are fused together. CAR-T cell therapy refers to the treatment using such transformed cells for cancer treatment.

[0143] The basic principle of CAR-T cell design involves a recombinant receptor that combines antigen-binding and T-cell activation functions. The general premise of CAR-T cells is to artificially create T cells that are targeted to markers found on cancer cells. Scientists can remove T cells from a person, genetically modify them, and return them to the patient to attack cancer cells. Once a T cell is engineered to become a CAR-T cell, it acts as a "living drug". CAR-T cells create a link between an extracellular ligand recognition domain and an intracellular signaling molecule, which activates the T cell. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). A key aspect of the safety of CAR-T cell therapy is how to ensure that only cancerous tumor cells are targeted, and not normal cells. The specificity of CAR-T cells is determined by the choice of the molecule that is targeted.

[0144] Exemplary CAR-T therapies include tisagenlecleucel (Kymriah) and axicabtageneciloreucel (Yescarta). In some embodiments, the CAR-T therapy targets CD19.

[0145] 4. Cytokine therapy Cytokines are proteins produced by many types of cells present in tumors. They can modulate the immune response. Tumors often use cytokines to promote tumor growth and reduce immune responses. These immunomodulatory effects allow them to be used as drugs to elicit an immune response. Two commonly used cytokines are interferons and interleukins.

[0146] Interferons are produced by the immune system. They are usually involved in antiviral responses, but are also used in cancer. They are classified into three groups: type I (IFNα and IFNβ), type II (IFNγ) and type III (IFNλ).

[0147] Interleukins have a number of immune system effects. IL-2 is an exemplary interleukin cytokine therapy.

[0148] 5. Adoptive T cell therapy Adoptive T cell therapy is a form of passive immunization by transfusion of T cells (adoptive cell transfer). T cells are found in blood and tissues and are usually activated when they find a foreign pathogen. Specifically, T cells become activated when their surface receptors encounter cells that present a portion of a foreign protein on their surface antigen. These can be either infected cells or antigen-presenting cells (APCs). T cells are found in normal and tumor tissues, in which case they are known as tumor-infiltrating lymphocytes (TILs). They are activated by the presence of APCs, such as dendritic cells, that present tumor antigens. These cells can attack tumors, but the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.

[0149] Several methods have been developed to produce and obtain tumor-targeted T cells. T cells specific for tumor antigens can be extracted from tumor samples (TILs) or filtered from the blood. Subsequent activation and culture is performed ex vivo, followed by reinfusion. Activation can be achieved through gene therapy or by exposing the T cells to tumor antigens.

[0150] 6. Checkpoint Inhibitors and Combination Treatments In some embodiments, the additional therapy or agent comprises an immune checkpoint inhibitor. Certain embodiments are further described below.

[0151] a. PD-1, PDL1, and PDL2 inhibitors PD-1 can act in the tumor microenvironment where T cells encounter infection or tumor. Activated T cells upregulate PD-1 and continue to express PD-1 in peripheral tissues. Cytokines such as IFN-gamma induce the expression of PDL1 in epithelial and tumor cells. PDL2 is expressed in macrophages and dendritic cells. The main role of PD-1 is to limit the activity of effector T cells in the periphery and prevent excessive damage to tissues during immune responses. The inhibitors of the present disclosure can block one or more functions of PD-1 and / or PDL1 activity.

[0152] Alternative names for "PD-1" include CD279 and SLEB2. Alternative names for "PDL1" include B7-H1, B7-4, CD274, and B7-H. Alternative names for "PDL2" include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PDL1, and PDL2 are human PD-1, PDL1, and PDL2.

[0153] In some embodiments, the PD-1 inhibitor is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In certain aspects, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 inhibitor is a molecule that inhibits the binding of PDL1 to its ligand binding partner. In certain aspects, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 inhibitor is a molecule that inhibits the binding of PDL2 to its ligand binding partner. In certain aspects, the PDL2 binding partner is PD-1. The inhibitor may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 inhibitors for use in the methods and compositions provided herein are known in the art, as described in U.S. Patent Application Publication Nos. US2014 / 0294898, US2014 / 022021, and US2011 / 0008369, all of which are incorporated herein by reference.

[0154] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and pidilizumab. In some embodiments, the PD-1 inhibitor is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular portion or a PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence). In some embodiments, the PDL1 inhibitor comprises AMP-224. Nivolumab is also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, and is an anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab, also known as MK-3475, Merck3475, Lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009 / 114335. Pidilizumab, also known as CT-011, hBAT, or hBAT-1, is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342. Additional PD-1 inhibitors include MEDI0680, also known as AMP-514, and REGN2810.

[0155] In some embodiments, the immune checkpoint inhibitor is a PDL1 inhibitor, such as durvalumab, also known as MEDI4736, atezolizumab, also known as MPDL3280A, avelumab, also known as MSB00010118C, MDX-1105, BMS-936559, or a combination thereof. In certain aspects, the immune checkpoint inhibitor is a PDL2 inhibitor, such as rHIgM12B7.

[0156] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of nivolumab, pembrolizumab, or pidilizumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of nivolumab, pembrolizumab, or pidilizumab, and the CDR1, CDR2, and CDR3 domains of the VL region of nivolumab, pembrolizumab, or pidilizumab. In another embodiment, the antibody competes for binding to the same epitope on PD-1, PDL1, or PDL2 as the aforementioned antibody, and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the aforementioned antibody. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or a range derivable therein) of variable region amino acid sequence identity with the aforementioned antibody.

[0157] b. CTLA-4, B7-1, and B7-2 Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when it binds to B7-1 (CD80) or B7-2 (CD86) on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is similar to CD28, a T cell costimulatory protein, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. CTLA-4 transmits inhibitory signals to T cells, whereas CD28 transmits stimulatory signals. Intracellular CTLA-4 is also found in regulatory T cells and may be important for their function. Activation of T cells through T cell receptor and CD28 increases the expression of CTLA-4, which is an inhibitory receptor for B7 molecules. The inhibitor of the present disclosure can block one or more functions of CTLA-4, B7-1, and / or B7-2 activity. In some embodiments, the inhibitor blocks CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks CTLA-4 and B7-2 interaction.

[0158] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0159] Anti-human-CTLA-4 antibodies (or VH domains and / or VL domains derived therefrom) suitable for use in the methods of the present invention can be produced using methods well known in the art. Alternatively, art-recognized anti-CTLA-4 antibodies can be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, tremelimumab; formerly also known as ticilimumab), U.S. Patent No. 6,207,156; Hurwitz et al., 1998 can be used in the methods disclosed herein. The disclosure of each of the aforementioned publications is incorporated herein by reference. Antibodies that compete with any of these art-recognized antibodies for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, WO2000 / 037504, and US Pat. No. 8,017,114, all of which are incorporated herein by reference.

[0160] An additional anti-CTLA-4 antibody useful as a checkpoint inhibitor in the methods and compositions of the disclosure is ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and variants thereof (see, e.g., WO01 / 14424).

[0161] In some embodiments, the inhibitor comprises the heavy and light chain CDRs or VRs of tremelimumab or ipilimumab. Thus, in one embodiment, the inhibitor comprises the CDR1, CDR2, and CDR3 domains of the VH region of tremelimumab or ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of tremelimumab or ipilimumab. In another embodiment, the antibody competes for binding to the same epitope on PD-1, B7-1, or B7-2 as the aforementioned antibodies and / or binds to the same epitope on PD-1, B7-1, or B7-2 as the aforementioned antibodies. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or a range derivable therein) of variable region amino acid sequence identity with the aforementioned antibodies.

[0162] B. Oncolytic Viruses In some embodiments, the additional therapy or agent comprises oncolytic virus.Oncolytic virus is a virus that selectively infects and kills cancer cells.When infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy remaining tumors.Oncolytic virus is not only believed to cause direct destruction of tumor cells, but also to stimulate host anti-tumor immune response for long-term immunotherapy.

[0163] C. Polysaccharides In some embodiments, the additional treatment or drug comprises polysaccharides.Certain compounds found in mushrooms, mainly polysaccharides, can upregulate immune system and have anti-cancer properties.For example, beta-glucans such as lentinan have been shown in laboratory studies to stimulate macrophages, NK cells, T cells and immune system cytokines, and are being investigated in clinical trials as immunological adjuvants.

[0164] D. Neoantigens In some embodiments, the additional therapy or agent comprises the administration of neoantigens. Many tumors express mutations. These mutations potentially create new targetable antigens (neoantigens) for use in T cell immunotherapy. The presence of CD8+ T cells in cancer lesions, identified using RNA sequencing data, is higher in tumors with high mutation load. The levels of transcripts associated with natural killer cell and T cell cytolytic activity are positively correlated with mutation load in many human tumors.

[0165] E. Chemotherapy In some embodiments, the additional therapy or agent comprises chemotherapy. Suitable classes of chemotherapeutic agents include: (a) alkylating agents, such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethylenimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozotocin, streptozocin) and triazines (e.g., dicarbazine); (b) antimetabolites, such as folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g., 5-fluorouracil, floxuridine, cytarabine, azauridine) and purine analogs and related substances (e.g., 6-mercaptopurine, 6-thioguanine, pentostatin); (c) Natural Products, such as Vinca Alkaloids (e.g., Vinblastine, Vincristine), Epipodophyllotoxins (e.g., Etoposide, Teniposide), Antibiotics (e.g., Dactinomycin, Daunorubicin, Doxorubicin, Bleomycin, Plicamycin, and Mitoxantrone), Enzymes (e.g., L-Asparaginase), and Biological Response Modifiers (e.g., Interferon-α), and (d) Miscellaneous Agents, such as platinum coordination complexes (e.g., Cisplatin, Carboplatin), Substituted Ureas (e.g., Hydroxyurea), Methylhydrazine Derivatives (e.g., Procarbazine), and Adrenal Cortical Suppressants (e.g., Taxol and Mitotane). In some embodiments, Cisplatin is a particularly suitable chemotherapeutic agent.

[0166] Cisplatin has been widely used to treat cancers such as metastatic testicular or ovarian cancer, advanced bladder cancer, head and neck cancer, cervical cancer, lung cancer or other tumors. Cisplatin is not absorbed orally and must therefore be delivered via other routes, such as intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, and in certain embodiments, effective doses used in clinical applications include about 15 mg / m2 to about 20 mg / m2 for 5 days every 3 weeks for a total of 3 courses. In some embodiments, the amount of cisplatin delivered to cells and / or subjects in conjunction with a construct comprising an Egr-1 promoter operably linked to a polynucleotide encoding a therapeutic polypeptide is less than the amount that would be delivered if cisplatin was used alone.

[0167] Other suitable chemotherapeutic agents include anti-microtubule agents, such as paclitaxel ("taxol") and doxorubicin hydrochloride ("doxorubicin"). The combination of an Egr-1 promoter / TNFα construct delivered via an adenoviral vector and doxorubicin has been found to be effective in overcoming resistance to chemotherapy and / or TNF-α, suggesting that combined treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-α.

[0168] Doxorubicin is poorly absorbed and is preferably administered intravenously.In certain embodiments, suitable intravenous doses for adults include about 60 mg / m2 to about 75 mg / m2 at intervals of about 21 days, or about 25 mg / m2 to about 30 mg / m2 on each of two or three consecutive days repeated at intervals of about 3 weeks to about 4 weeks, or about 20 mg / m2 once a week.In cases where there is previous myelosuppression or neoplastic bone marrow infiltration caused by previous chemotherapy, or when the drug is combined with other myelopoiesis suppressing drugs, the lowest dose should be used in elderly patients.

[0169] Nitrogen mustard is another suitable chemotherapeutic agent useful in the methods of the present disclosure. Nitrogen mustards may include, but are not limited to, mechlorethamine (HN2), cyclophosphamide and / or ifosfamide, melphalan (L-sarcolysin), and chlorambucil. Cyclophosphamide (CYTOXAN® available from Mead Johnson, NEOSTAR® available from Adria) is another suitable chemotherapeutic agent. Suitable oral doses for adults include, for example, about 1 mg / kg / day to about 5 mg / kg / day, and intravenous doses include, for example, about 40 mg / kg to about 50 mg / kg in divided doses for about 2 to about 5 days initially, or about 10 mg / kg to about 15 mg / kg every about 7 to about 10 days, or about 3 mg / kg to about 5 mg / kg twice weekly, or about 1.5 mg / kg / day to about 3 mg / kg / day. Due to adverse gastrointestinal effects, the intravenous route is preferred. Drugs may also be administered intramuscularly, by infiltration, or into body cavities.

[0170] Further suitable chemotherapeutic agents include pyrimidine analogs, such as cytarabine (cytosine arabinoside), 5-fluorouracil (fluorouracil; 5-FU) and floxuridine (fluorodeoxyuridine; FudR). 5-FU may be administered to a subject at a dosage of about 7.5 to about 1000 mg / m2. Furthermore, 5-FU dosing schedules may be for various periods, for example, up to 6 weeks, or as determined by one of skill in the art to which this disclosure pertains.

[0171] Another suitable chemotherapeutic agent, gemcitabine diphosphate (GEMZAR®, Eli Lilly & Co., "gemcitabine"), is recommended for the treatment of advanced and metastatic pancreatic cancer and therefore would be useful in the present disclosure for these cancers as well.

[0172] The amount of chemotherapeutic agent delivered to the patient can be variable. In one suitable embodiment, the chemotherapeutic agent can be administered in an amount effective to cause the arrest or regression of cancer in the host when the chemotherapy is administered with the construct. In other embodiments, the chemotherapeutic agent can be administered anywhere from 2 to 10,000 times less than the chemotherapeutic effective amount of the chemotherapeutic agent. For example, the chemotherapeutic agent can be administered in an amount about 20 times less, about 500 times less, or even about 5000 times less than the chemotherapeutic effective amount of the chemotherapeutic agent. The chemotherapeutic agents of the present disclosure can be tested in vivo for the desired therapeutic activity in combination with the construct and for determining effective dosages. For example, such compounds can be tested in suitable animal model systems, including but not limited to rats, mice, chickens, cows, monkeys, rabbits, etc., prior to testing in humans. As described in the examples, in vitro testing can also be used to determine suitable combinations and dosages.

[0173] F. Radiation Therapy In some embodiments, the additional therapy or agent or previous therapy comprises radiation, such as ionizing radiation.As used herein, "ionizing radiation" refers to radiation that comprises particles or photons that have sufficient energy or can produce sufficient energy through nuclear interaction to produce ionization (gain or loss of electrons).An exemplary and preferred ionizing radiation is x-rays.Means for delivering x-rays to target tissue or cells are well known in the art.

[0174] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in one dose. In some embodiments, the amount of ionizing radiation is 18 Gy and is administered in three doses. In some embodiments, the amount of ionizing radiation is at least, at most, or exactly 2, 4, 6, 8, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 18, 19, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 40 Gy (or any range derivable therein). In some embodiments, the ionizing radiation is administered in at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses (or any range derivable therein). If more than one dose is administered, the doses may be separated by about 1, 4, 8, 12, or 24 hours, or 1, 2, 3, 4, 5, 6, 7, or 8 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or 16 weeks, or any range derivable therein.

[0175] In some embodiments, the amount of IR may be presented as a total dose of IR, which is administered in fractionated doses. For example, in some embodiments, the total dose is 50 Gy administered in 10 fractionated doses of 5 Gy each. In some embodiments, the total dose is 50-90 Gy administered in 20-60 fractionated doses of 2-3 Gy each. In some embodiments, the total dose of IR is at least, at most, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, or 150 (or any range derivable therein). In some embodiments, the total dose is administered in fractions of at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 20, 25, 30, 35, 40, 45, or 50 Gy (or any range derivable therein).In some embodiments, at least, at most, or exactly 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 fractional doses are administered (or any range derivable therein). In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 fractional doses (or any range derivable therein) are administered per day. In some embodiments, at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 (or any range derivable therein) fractionated doses are administered per week.

[0176] G. Surgery Approximately 60% of people with cancer undergo some type of surgery, including preventive, diagnostic, or staging surgery, curative surgery, and palliative surgery. Curative surgery includes resection, in which all or part of the cancerous tissue is physically removed, excised, and / or destroyed, and may be used in conjunction with other therapies, such as treatments of the present invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor resection refers to the physical removal of at least a portion of the tumor. In addition to tumor resection, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microscopically controlled surgery (Mohs surgery).

[0177] Removal of part or all of the cancer cells, tissues, or tumors may result in the formation of a cavity in the body. Treatment may be performed by perfusion, direct injection, or local application of additional anticancer therapy to the area. Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments may also be at various dosages.

[0178] H. Other Agents It is contemplated that other agents may be used in combination with certain aspects of the embodiments of the present invention to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cytostatic and differentiation agents, cell adhesion inhibitors, agents that enhance the sensitivity of hyperproliferative cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions increases the anti-hyperproliferative effect on nearby hyperproliferative cell populations. In other embodiments, cytostatic or differentiation agents may be used in combination with certain aspects of the embodiments of the present invention to improve the anti-hyperproliferative efficacy of the treatment. It is contemplated that cell adhesion inhibitors improve the efficacy of the embodiments of the present invention. Examples of cell adhesion inhibitors are focal adhesion kinase (FAK) inhibitors and lovastatin. It is further contemplated that other agents that enhance the sensitivity of hyperproliferative cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of the embodiments of the present invention to improve the efficacy of the treatment.

[0179] VI. Administration of Therapeutic Compositions The disclosed methods include administration of a combination of therapeutic agents and / or administration of therapeutic agents such as fecal matter and a therapeutic regimen such as, for example, steroid therapy or anti-integrin therapy. The treatments can be administered in any suitable manner known in the art. For example, the treatments can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the treatments are in separate compositions. In some embodiments, the treatments are in the same composition.

[0180] For example, various combinations of therapies may be utilized, with one therapy designated as "A" and another designated as "B." TIFF0007682104000001.tif13128

[0181] Therapeutic agents of the present disclosure, such as fecal material from healthy subjects, may be administered by the same or different routes of administration. In some embodiments, therapeutic agents are administered intracolonically, intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, microbial regulators are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally.

[0182] The amount to be administered depends on the desired therapeutic effect, depending on both the number of treatments and the unit dose. Effective dose is understood to refer to the amount required to achieve a specific effect. In the practice of certain embodiments, it is contemplated that a dose within the range of 10 mg / kg to 200 mg / kg can affect the protective capacity of these agents. Thus, doses are contemplated to include doses of about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such doses can be administered multiple times during the day and / or on multiple days, weeks, or months.

[0183] In some embodiments, a therapeutically effective or sufficient amount of a therapeutic composition administered to a human will be within the range of about 0.01 to about 50 mg / kg of patient body weight, whether by single or multiple administrations. In some embodiments, the therapeutic agent used is, for example, about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg, about 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg administered daily. In some embodiments, the therapeutic agent is administered at 15 mg / kg. However, other dosing regimens may be useful. In one embodiment, the therapeutic agent described herein is administered to a subject at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on the first day of a 21-day cycle.The dose can be administered as a single dose or as multiple doses (e.g., two or three doses), such as by infusion.The progress of this therapy can be easily monitored by conventional techniques.

[0184] In certain embodiments, an effective dose of the pharmaceutical composition is one that can provide a blood level of about 1 μM to 150 μM. In other embodiments, an effective dose provides a blood level of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to 50 μM; or about 25 μM to 150 μM; or about 25 μM to 100 μM; or about 25 μM to 50 μM; or about 50 μM to 150 μM; or about 50 μM to 100 μM (or any range derivable therein). In other embodiments, the dose can provide the following drug blood levels resulting from the therapeutic agent administered to the subject: about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 μM or any range derivable therein. In certain embodiments, the therapeutic agent administered to the subject is metabolized in the body to a metabolized therapeutic agent, in which case blood level can refer to the amount of the agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, blood level discussed herein can refer to the unmetabolized therapeutic agent.

[0185] The precise amount of a therapeutic composition also depends on the judgment of the practitioner and is peculiar to each individual. Factors affecting dosage include the physical and clinical condition of the patient, the route of administration, the intended goal of treatment (either symptomatic relief or cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other treatments the subject may be undergoing.

[0186] Those skilled in the art will understand and appreciate that dosage units of μg / kg or mg / kg body weight can be converted and expressed in equivalent concentration units of μg / ml or mM (blood levels), such as 4 μM to 100 μM. It is also understood that uptake is species and organ / tissue dependent. Applicable conversion factors and physiological assumptions made regarding uptake and concentration measurements are well known, and those skilled in the art will be able to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacy and results described herein.

[0187] VII. Kits Certain aspects of the present disclosure also include kits for carrying out the methods of the present disclosure, such as cancer detection, diagnosis, or treatment. Such kits can be prepared from readily available materials and reagents. For example, such kits can include any one or more of the following materials: enzymes, reaction tubes, buffers, detergents, primers, probes, antibodies. In preferred embodiments, these kits allow practitioners to obtain samples of neoplastic cells in blood, tears, semen, saliva, urine, tissue, serum, stool, saliva, cerebrospinal fluid, and supernatants from cell lysates. In another preferred embodiment, these kits include the equipment required to carry out RNA extraction, RT-PCR, and gel electrophoresis. Instructions for carrying out the assay can also be included in the kit.

[0188] The kit may further include instructions for using the kit to evaluate the sequence, and means for converting and / or analyzing the sequence data to perform a prognosis. The agents in the kit for measuring biomarker expression may include a plurality of PCR probes and / or primers for qRT-PCR, and / or a plurality of antibodies or fragments thereof for evaluating the expression of the biomarkers. In another embodiment, the agents in the kit for measuring biomarker expression may include an array of polynucleotides complementary to the mRNA of the biomarkers of the invention. Possible means for converting expression data into expression values ​​and analyzing the expression values ​​to generate a score predictive of viability or prognosis may also be included.

[0189] The kit may include a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The container may be formed of various materials, such as glass or plastic. The container may hold a composition that includes a probe useful for prognostic or non-prognostic applications, as described above. The label on the container may indicate that the composition is used for a particular prognostic or non-prognostic application, and may indicate either in vivo or in vitro use, such as those described above. The kit may include the container described above, as well as one or more other containers that contain materials that are desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0190] Further kit aspects relate to kits that include the therapeutic compositions of the present disclosure. The kits are useful in the treatment methods of the present disclosure and can include instructions for use. EXAMPLES

[0191] VIII. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples demonstrate techniques that the inventors have discovered work well in the practice of the invention, and thus are believed to constitute preferred modes for its practice. However, in light of this disclosure, it should be understood by those skilled in the art that many changes can be made in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the invention.

[0192] Example 1 - EGFR TKI resistance is associated with a mesenchymal phenotype and increased expression of CD70 Herein, we demonstrate that CD70 is a therapeutic target for TKI-resistant EGFR mutant tumors, and that CD70-antibody drug conjugates, anti-CD70 CAR-T cells, or approaches targeting CD70 such as TriNKET, EGFR-CD70 BiTE, Axl-CD70 BiTE, or other approaches targeting CD70 (collectively referred to as CD70-directed therapy) can be effective against TKI-resistant EGFR mutant tumors, either alone or in combination with other treatments. Furthermore, EGFR mutation is a biomarker for selecting patients to be treated with CD70-targeted drugs. Furthermore, we describe that CD70 targeting is a therapeutic strategy for mesenchymal NSCLC tumors, and that mesenchymal status determined by gene expression or protein markers (collectively referred to as epithelial-mesenchymal transition (EMT) biomarkers) is a biomarker for selecting patients for treatment with CD70-targeted therapy.

[0193] As part of an effort to identify potential targets in TKI-resistant EGFR mutant NSCLC, we derived a panel of NSCLC cell lines with acquired resistance to the EGFR TKIs erlotinib, gefitinib, and osimertinib. Transcriptomics and proteomics profiling revealed that the resistant cells had undergone epithelial-mesenchymal transition (EMT). Gene expression analysis revealed that CD70 was significantly overexpressed in EGFR TKI-resistant cells compared to parental (EGFR TKI-sensitive) cells. Our finding that CD70 gene expression was highly upregulated in NSCLC cells with acquired resistance to EGFR TKIs was validated by flow cytometry, demonstrating that resistant cells express higher levels of CD70 protein on the cell surface compared to parental (EGFR TKI-sensitive) cells. To assess whether CD70 is increased in NSCLC clinical specimens that have undergone EMT, we evaluated RNAseq data from TCGA. CD70 expression correlated with a mesenchymal gene signature in NSCLC tumor samples.

[0194] CD70 is known to be expressed on T cells and B cells, as well as on some malignant cells, including leukemia cells and renal cell carcinoma. CD70 expression is believed to contribute to the immunosuppressive environment by influencing / attracting regulatory T cells and promoting apoptosis and exhaustion of T cells. Furthermore, CD70-expressing tumor cells can be directly targeted using anti-CD70 antibody-drug conjugates or CAR T cells. Taken together, the data presented herein demonstrate that CD70 is overexpressed in NSCLC cells with acquired resistance to EGFR TKIs, and suggest that CD70 targeting may be an effective therapeutic strategy in this setting.

[0195] EGFR mutant NSCLC patients initially respond to EGFR tyrosine kinase inhibitors (TKIs), but resistant disease inevitably emerges. We derived a panel of NSCLC cell lines with acquired resistance to EGFR TKIs. EGFR-TKI-resistant (ER) cells were negative for secondary EGFR mutations and resistant to EGFR TKIs (Figure 1). Using RNAseq and gene expression analysis, we confirmed that EGFR TKI-resistant cells exhibited a mesenchymal gene expression signature, including loss of CDH1 expression and increased expression of VIM and AXL, as well as ZEB1 and ZEB2, key mediators of epithelial-mesenchymal transition (EMT) (Figure 2A-G). RNA expression analysis further revealed that EGFR TKI-resistant cells highly overexpressed CD70 (Figure 2H). Flow cytometry analysis revealed increased CD70 protein levels on the surface of EGFR TKI-resistant cells compared to EGFR TKI-sensitive parental cells (Figure 3). Furthermore, induction of EMT by forced expression of ZEB1 in HCC827 parental (EGFR TKI-sensitive) cells was sufficient to render the cells resistant to EGFR inhibition by erlotinib, osimertinib or afatinib (Figure 4). Given the findings that EGFR TKI resistance is associated with EMT, and that these cells overexpress CD70, we next used the TCGA database to evaluate whether expression of CD70 is associated with a mesenchymal phenotype in human lung adenocarcinoma. We found that CD70 expression was significantly associated with an EMT gene expression signature in lung adenocarcinoma and in a broad panel of NSCLC cell lines (Figure 5). CD70 is typically expressed in T and B cells, but can also be expressed in some malignant cells. Expression of CD70 by tumor cells is thought to contribute to an immunosuppressive environment by influencing / attracting regulatory T cells and promoting apoptosis and exhaustion of T cells. These findings that CD70 expression is enhanced in EGFR TKI-resistant cells suggest that CD70 targeting may be clinically useful in the setting of EGFR TKI-resistant NSCLC.

[0196] Example 2: EGFR TKI resistance is associated with a mesenchymal phenotype and increased expression of CD70 This embodiment may include duplicate data and / or rearranged data of the first embodiment.

[0197] EGFR mutant NSCLC patients initially respond to EGFR tyrosine kinase inhibitors (TKIs), but resistant disease inevitably emerges. We derived a panel of NSCLC cell lines with acquired resistance to EGFR TKIs. EGFR-TKI-resistant (ER) cells were negative for secondary EGFR mutations and resistant to EGFR TKIs (Figure 1). Using RNAseq and gene expression analysis, we confirmed that EGFR TKI-resistant cells exhibited a mesenchymal gene expression signature, including loss of CDH1 expression and increased expression of VIM and AXL, as well as ZEB1 and ZEB2, key mediators of epithelial-mesenchymal transition (EMT) (Figure 6). RNA expression analysis further revealed that EGFR TKI-resistant cells highly overexpressed CD70 (Figure 7A). Flow cytometry analysis revealed increased CD70 protein levels on the surface of EGFR TKI-resistant cells compared to EGFR TKI-sensitive parental cells (Figure 7B-F). To determine whether CD70 is elevated in animal models of EGFR TKI resistance, we utilized a doxycycline-inducible EGFR L858R GEMM model in which administration of doxycycline results in mutant EGFR expression and the development of lung tumors. Once tumors were visualized by CT imaging, doxycycline was removed from a subset of animals to mimic EGFR inhibition. After a period of tumor regression, tumors began to regrow, as determined by CT imaging. Animals were treated with osimertinib to confirm the EGFR TKI resistance phenotype. Tumors were collected and CD70 expression was analyzed by immunohistochemistry. CD70 expression was elevated in acquired EGFR-independent tumors (Figure 8). Next, we evaluated CD70 expression in EGFR mutant EGFR TKI-naive NSCLC clinical specimens and EGFR mutant NSCLC specimens collected after EGFR TKI resistance. While CD70 expression was minimal in untreated tissues, CD70 was highly expressed in EGFR TKI-resistant tumors (Figure 9).

[0198] Next, we investigated the effect of EMT on CD70 expression in EGFR mutant NSCLC cell lines. We induced EMT through forced expression of ZEB1 in HCC827 parental (EGFR TKI-sensitive) cells. ZEB1 expression was sufficient to induce mesenchymal phenotype and render cells resistant to EGFR inhibition by erlotinib, osimertinib or afatinib (Figure 10A and B). Expression of ZEB1 induced a significant increase in CD70 mRNA levels and cell surface expression of CD70. We next used the TCGA database to evaluate whether CD70 expression is associated with mesenchymal phenotype in NSCLC cell lines and human lung adenocarcinoma. We found that CD70 expression was significantly associated with EMT gene expression signature and ZEB1 expression in lung adenocarcinoma and in a broad panel of NSCLC cell lines (Figure 11).

[0199] Binding of CD27 to CD70 induces activation of signaling pathways downstream of CD70. To investigate the potential impact of CD70 signaling on EGFR TKI-resistant cells, we stimulated EGFR TKI-resistant cells with recombinant soluble CD27. CD27 treatment resulted in activation of key signaling molecules Akt and ERK, which are known to be reactivated in EGFR TKI resistance (Figure 12). Next, we knocked down CD70 expression using siRNA and found that knockdown of CD70 impaired the proliferation of EGFR TKI-resistant cells by clonogenic assay (Figure 13).

[0200] To determine whether CD70 antibody drug conjugates (ADCs) are an effective approach to target EGFR TKI-resistant cells, we treated H1975 cells (CD70 low and EGFR TKI sensitive) as well as H1975 OR5 and H1975 OR16 (both EGFR TKI resistant and CD70 high) with increasing concentrations of the CD70 ADCs cusatuzumab-MMAE or borsetuzumab-MMAE. As expected, H1975 OR5 and OR16 cells were more sensitive to the CD70 ADCs than the H1975 parental cells (Figures 14 and 16). We further observed an additive anti-tumor cell effect when osimertinib was combined with anti-CD70 ADCs (Figure 15).

[0201] CD70 is usually expressed on T and B cells, but can also be expressed on some malignant cells. Expression of CD70 by tumor cells is thought to contribute to an immunosuppressive environment by influencing / attracting regulatory T cells and promoting apoptosis and exhaustion of T cells. These findings that CD70 expression is enhanced in EGFR TKI-resistant cells suggest that CD70 targeting may be clinically useful in the setting of EGFR TKI-resistant NSCLC.

[0202] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present invention. Although the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to the methods described herein and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain agents that are chemically and physiologically related can be substituted for the agents described herein while still achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present invention as defined by the appended claims. Publications described in this application are specifically incorporated herein by reference to the extent that they provide exemplary procedural or other details supplementary to those set forth herein.

Claims

1. 1. A pharmaceutical composition comprising a CD70 targeting molecule, The pharmaceutical composition comprises: (a) for treating EGFR mutant non-small cell lung cancer (NSCLC) in patients who are resistant to EGFR TKI treatment, or (b) for the treatment of epithelial-mesenchymal transition (EMT)-positive NSCLC in patients resistant to EGFR TKI treatment A pharmaceutical composition comprising: the CD70 targeting molecule comprises an anti-CD70 antibody or a CD70-binding fragment thereof; The anti-CD70 antibody or CD70-binding fragment thereof is conjugated to a toxic molecule. The pharmaceutical composition.

2. (a) the patient has been determined to have EGFR mutant NSCLC; and / or (b) the NSCLC includes lung adenocarcinoma; and / or (c) the patient is a non-smoker; and / or (d) the EGFR mutant contains an activating mutation; and / or (e) the EGFR mutation comprises a class I, II, or III EGFR mutation; and / or (f) the patient has not been tested for CD70 expression on the cancer cells or the patient has been determined to have CD70-expressing cancer cells; 2. The pharmaceutical composition of claim 1.

3. The pharmaceutical composition of claim 2(d), wherein the activating mutation comprises L858R or a deletion in exon 19.

4. (a) the patient has previously undergone treatment for NSCLC; and / or (b) the previous treatment included EGFR tyrosine kinase inhibitor (TKI) therapy, the therapy including administration of one or more EGFR TKIs; and / or (c) the prior treatment included single-agent EGFR TKI therapy or the prior treatment included administration of a combination of at least two EGFR TKIs; and / or (d) the patient is determined to have systemic disease progression while receiving ongoing EGFR TKI therapy; A pharmaceutical composition according to any one of claims 1 to 3.

5. The pharmaceutical composition of claim 4(a), wherein the patient has been determined to have acquired resistance to a previous treatment.

6. The pharmaceutical composition of any of claims 4(b) to 4(d), wherein the EGFR TKI therapy comprises administration of one or more of osimertinib, gefitinib, erlotinib, afatinib, dacomitinib, and brigatinib.

7. The pharmaceutical composition of any one of claims 1 to 6, for use in combination with a further therapy.

8. (a) the further therapy comprises chemotherapy, radiation, surgery, TKI therapy, or immunotherapy; and / or (b) the additional therapy includes administration of one or more of durvalumab, atezolizumab, pembrolizumab, nivolumab, necitumumab, and bevacizumab; and / or (c) the additional therapy comprises administration of one or more of carboplatin, pemetrexed, nab-paclitaxel, photofrin, cisplatin, docetaxel, gemcitabine, paclitaxel, and vinorelbine; and / or (d) the additional therapy comprises administration of one or more of alectinib, lorlatinib, and ceritinib; and / or (e) the additional therapy comprises administration of one or more of osimertinib, gefitinib, erlotinib, afatinib, dacomitinib, and brigatinib; 8. The pharmaceutical composition of claim 7.

9. 9. The pharmaceutical composition of claim 8, wherein the additional therapy comprises administration of osimertinib.

10. A pharmaceutical composition according to any one of claims 1 to 7 and claims 8(a) to 8(d) for use in combination with adjuvant and / or neoadjuvant therapy.

11. The pharmaceutical composition of any one of claims 1 to 10, wherein the patient has been determined to have an ALK mutant or the patient has been determined not to have an ALK mutant.

12. 12. The pharmaceutical composition of claim 11, wherein the additional therapy comprises administration of a second antibody linked to a toxic molecule.

13. 13. The pharmaceutical composition of claim 12, wherein the second antibody and the toxic molecule are linked through a cleavable linker.

14. (i) the antibody is a humanized or chimeric antibody, and / or (ii) the antibody comprises cusatuzumab or borsetuzumab; A pharmaceutical composition according to any one of claims 1 to 13.

15. (I) the toxic molecule comprises monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), a pyrrolobenzodiazepine (PBD), or a duocarmycin; and / or (II) the CD70 targeting molecule comprises cusatuzumab-MMAE, borsetuzumab-MMAE, or a combination thereof; 15. The pharmaceutical composition of any one of claims 1 to 14.

16. (a) the CD70 targeting molecule comprises a heavy chain variable region and / or a light chain variable region from a CD70 antibody; and / or (b) the CD70 targeting molecule comprises CDR1, CDR2 and CDR3 from a heavy chain variable region, and / or CDR1, CDR2 and CDR3 from a light chain variable region; and / or (c) the CD70 targeting molecule comprises a single chain variable fragment (scFV); 16. The pharmaceutical composition of any one of claims 1 to 15.

17. The pharmaceutical composition of any one of claims 1 to 16, wherein a biological sample from the patient has been determined to be positive for one or more EMT markers.

18. (a) the biological sample contains tumor cells and / or tumor-associated cells; and / or (b) the biological sample includes a biopsy; and / or (c) one or more EMT markers include a decrease in epithelial markers and / or an increase in mesenchymal markers; 18. The pharmaceutical composition of claim 17.

19. The pharmaceutical composition of claim 17 or 18, wherein the EMT markers include one or more of CDH1, VIM, AXL, ZEB1, and ZEB2.

20. 20. The pharmaceutical composition of any one of claims 1 to 19, comprising one or more additional therapeutic agents.

21. (a) the additional therapeutic agent comprises chemotherapy, radiation, surgery, immunotherapy, or a combination thereof; and / or (b) the additional therapeutic agents include one or more of durvalumab, atezolizumab, pembrolizumab, nivolumab, necitumumab, and bevacizumab; and / or (c) the additional therapeutic agents comprise one or more of carboplatin, pemetrexed, nab-paclitaxel, photofrin, cisplatin, docetaxel, gemcitabine, paclitaxel, and vinorelbine; 21. The pharmaceutical composition of claim 20.

22. (a) the additional therapeutic agents comprise one or more of alectinib, lorlatinib, and ceritinib; and / or (b) the additional therapeutic agent comprises one or more of osimertinib, gefitinib, erlotinib, afatinib, dacomitinib, and brigatinib; 22. The pharmaceutical composition of claim 21.

23. 23. The pharmaceutical composition of claim 21 or 22, wherein the additional therapeutic agent comprises osimertinib.

24. (a) the additional therapeutic agent comprises a second antibody linked to a toxic molecule; and / or (b) the antibody is a humanized or chimeric antibody, and / or (c) the antibody comprises cusatuzumab or borsetuzumab; and / or (d) the antibody is conjugated to a molecule; 22. The pharmaceutical composition of claim 21.

25. The pharmaceutical composition of claim 24(a), wherein the second antibody and the toxic molecule are linked through a cleavable linker.

26. 26. The pharmaceutical composition of claim 24 or 25, wherein the molecule is a toxic molecule.

27. 27. The pharmaceutical composition of claim 26, wherein the toxic molecule comprises monomethylauristatin E (MMAE), duocarmycin, monomethylauristatin F (MMAF), or pyrrolobenzodiazepine (PBD) and / or the CD70 targeting molecule comprises cusatuzumab-MMAE, borsetuzumab-MMAE, or a combination thereof.

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