Methods and compositions for treating non-small cell lung cancer

Abstract

To provide additional therapeutic approaches, in which since EGFR mutant NSCLC patients are initially responsive to EGFR targeted therapies, resistant disease inevitably emerges.SOLUTION: Aspects of the disclosure relate to a method for treating EGFR-mutant non-small-cell lung cancer (NSCLC) in a patient comprising the step of administering a CD70 targeting molecule to the patient. Further aspects of the disclosure relate to a method for treating an epithelial-to-mesenchymal transition (EMT)-positive NSCLC in a patient comprising the step of administering a CD70-targeting molecule to the patient.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 848,123, filed May 15, 2019, the entire disclosure of which is hereby incorporated by reference.

[0002] This invention was made with government support under grant number 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 Art

[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, NSCLC is relatively chemoresistant compared to small cell cancer. If possible, they are primarily treated by surgical resection with the goal of cure, but chemotherapy is increasingly being used both preoperatively (neoadjuvant chemotherapy) and postoperatively (adjuvant chemotherapy).

[0005] Patients with EGFR mutant NSCLC initially respond to EGFR targeted therapy. However, resistant disease inevitably emerges, and in nearly half of the resistant cases, the tumors lack secondary EGFR mutations such as T790M and are resistant to second and third generation EGFR tyrosine kinase inhibitors (TKIs). Further therapeutic approaches are needed in the art.

Summary of the Invention

[0006] Aspects of the present disclosure relate to methods for treating EGFR variant non-small cell lung cancer (NSCLC) in a patient, including the step of administering a CD70-targeted molecule to the patient. Further aspects of the present disclosure relate to methods for treating epithelial-mesenchymal transition (EMT)-positive NSCLC in a patient, including the step of administering a CD70-targeted molecule to the patient. Further aspects of the present disclosure relate to a composition comprising a CD70-targeted molecule and one or more additional therapeutic agents.

[0007] In some embodiments, the patient has been determined to have EGFR variant NSCLC. In some embodiments, the NSCLC includes lung adenocarcinoma. In some embodiments, the patient is a non-smoker. In some embodiments, the patient is human.

[0008] The term "EGFR variant cancer" refers to cancer in which the expression or activity of EGFR (epidermal growth factor receptor) has changed. The mutation may be in the coding region of EGFR and affects the expression level of endogenous EGFR or the activity level of the resulting protein. The mutation may also be in the non-coding portion of the gene, such as in the promoter region, 3' or 5' UTR, or intron region. 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 includes an activating mutation. In some embodiments, the activating mutation includes L858R. In some embodiments, the activating mutation includes a deletion in exon 19. In some embodiments, the EGFR mutation includes 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 includes 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 mutation comprises at least one of 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 at least one class II mutation selected from 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 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 one embodiment.

[0009] In some embodiments, the patient has not been tested for CD70 expression in patient-derived cancer cells. In some embodiments, the patient has been determined to have CD70-expressing cancer cells. In some embodiments, the patient has previously received treatment for NSCLC. In some embodiments, the patient has been determined to have acquired resistance to a previous treatment. In some embodiments, the previous treatment includes EGFR tyrosine kinase inhibitor (TKI) therapy, where this therapy includes one or more EGFR TKIs. In some embodiments, the previous treatment includes monotherapy EGFR TKI therapy. In some embodiments, the previous treatment includes 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, during ongoing EGFR TKI therapy, systemic disease progression has been determined. In some embodiments, the EGFR TKI therapy includes one or more of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. In some embodiments, the EGFR TKI therapy includes one or more of erlotinib, gefitinib, and osimertinib. In some embodiments, the EGFR TKI therapy includes at least two of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. In some embodiments, the EGFR TKI therapy includes at least three of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. In some embodiments, the EGFR TKI therapy includes at least four of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. It is particularly 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 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 the administration of adjuvant therapy and / or neoadjuvant therapy. In some embodiments, the additional therapy can be conjugated or linked to a CD70-targeted 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-targeted agent and an additional therapeutic agent).

[0011] In some embodiments, the patient has been determined to be an ALK variant. In some embodiments, the patient has been determined not to be an ALK variant.

[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 monomethyl auristatin E (MMAE), monomethyl auristatin 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 an anti-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 are from an anti-CD70 antibody or may be derived from an anti-CD70 antibody. In some embodiments, the CD70 targeting molecule comprises a single-chain variable fragment (scFV). The scFv may comprise hypervariable regions such as CDR1, CDR2, and CDR3 from the heavy chain variable region and / or CDR1, CDR2, and CDR3 from the light chain variable region derived from an anti-CD70 antibody. In some embodiments, the antibody comprises crizanlizumab or besilesomab. In some embodiments, the CD70 targeting molecule comprises a heavy chain variable region and / or a light chain variable region from crizanlizumab or besilesomab. In some embodiments, the CD70 targeting molecule comprises CDR1, CDR2, and CDR3 from the heavy chain variable region of crizanlizumab and / or CDR1, CDR2, and CDR3 from the light chain variable region of crizanlizumab. In some embodiments, the CD70 targeting molecule comprises CDR1, CDR2, and CDR3 from the heavy chain variable region of besilesomab and / or CDR1, CDR2, and CDR3 from the light chain variable region of besilesomab.

[0014] In some embodiments, additional therapies or agents in the methods and compositions of the disclosure include secondary antibodies conjugated to toxic molecules. For example, a combination therapy can include an agent comprising a conjugate that includes a secondary antibody that binds to a CD70 targeting antibody conjugated to a toxic molecule, in combination with a CD70 targeting antibody. Thus, such a combination of agents delivers the antibody conjugated 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 T cell comprising a CAR, 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 crizanlizumab or besilesizumab. In some embodiments, the BiTE, CAR, or TriNKET comprises a heavy chain comprising CDR1, CDR2, and CDR3 of crizanlizumab or besilesizumab, and / or a light chain comprising CDR1, CDR2, and CDR3 of crizanlizumab or besilesizumab. In some embodiments, the CD70-targeted molecule comprises crizanlizumab-MMAE, besilesizumab-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-targeted 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 embodiments of the present disclosure. Thus, in some embodiments, the CD70-targeted molecule includes CTX130. CTX130 is an allogeneic CRISPR / Cas9 gene-edited CAR-T cell therapy targeting CD70, created by CRISPR Therapeutics. In some embodiments, the CD70-targeted molecule includes ALLO-316. ALLO-316 is an anti-CD70 AlloCAR T cell therapy 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-targeted molecules described as useful for other indications may be used in embodiments of the methods and compositions of the present disclosure. In some embodiments, the CD70-targeted molecule includes a CD70 ligand such as CD27. In some embodiments, the CD70-targeted molecule includes truncated CD27. In some embodiments, the CD70-targeted molecule includes a CD27 CAR, which is a CD27 polypeptide fused to the transmembrane and intracellular signaling regions of a CAR molecule, such as 41BB and CD3-zeta. The CD27 polypeptide can be a full-length polypeptide or a fragment or truncated form thereof that interacts and binds to CD70. The CARs of the present disclosure can be expressed in T cells or NK cells.

[0017] In some embodiments, the CD70-targeted molecule comprises cells including BiTE, CAR, or TriNKET. In some embodiments, the cells include stem cells, progenitor cells, immune cells, or natural killer (NK) cells. In some embodiments, the cells include hematopoietic stem or progenitor cells, T cells, cells differentiated from mesenchymal stem cells (MSCs), or induced pluripotent stem cells (iPSCs). In some embodiments, the cells are isolated from or derived from peripheral blood mononuclear cells (PBMCs). In some embodiments, the T cells are cytotoxic T lymphocytes (CTLs), 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 includes a biopsy material. In some embodiments, the biological sample is obtained by methods such as fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, incisional biopsy, excisional biopsy, punch biopsy, shave biopsy, or skin biopsy. In certain embodiments, the sample is obtained from a biopsy material from lung tissue by any of the aforementioned biopsy methods. In other embodiments, the sample can be obtained from any of the tissues provided herein, including but not limited to non-cancerous or cancerous tissue, and non-cancerous or cancerous tissue 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 can be obtained from any other source, including but not limited to blood, sweat, hair follicles, buccal tissue, tears, menstrual secretions, feces, or saliva. In certain aspects of the method, any medical professional, such as a physician, nurse, or medical technician, may obtain the biological sample for testing. Additionally, the biological sample can be obtained without the assistance of a medical professional. The sample can include, but is not limited to, tissue, cells, or biological material derived from or of the cells of the subject. The biological sample can be a heterogeneous or homogeneous population of cells or tissue. The biological sample can be obtained using any method known in the art that can provide a sample suitable for the analysis methods described herein. The sample can be obtained by non-invasive methods, including but not limited to scraping of the skin or cervix, swabbing of the cheek, saliva collection, urine collection, fecal collection, menstrual secretions, tears, or semen collection. The sample can be obtained by methods known in the art. In certain embodiments, the sample is obtained by biopsy.

[0019] As used herein, the terms "or" and "and / or" are used to combine or describe multiple components in an exclusive manner. For example, "x, y, and / or z" can mean "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 its plain and ordinary sense in the field of cell biology to indicate that the value includes the standard deviation of error for the device or method used to determine that value.

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

[0022] It is specifically contemplated that any limitations discussed with respect to one aspect of the present invention may apply to any other aspect of the present invention. Further, 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 may also be implemented in the context of aspects discussed elsewhere in different examples or elsewhere in this application, such as in the context of the summary of the invention, the detailed description of the aspects, the claims, and the description of the legends of the figures.

[0023] [Invention 1001] A method for treating EGFR variant non-small cell lung cancer (NSCLC) in a patient, comprising administering a CD70-targeted molecule to the patient. [Invention 1002] A method for treating epithelial-mesenchymal transition (EMT)-positive NSCLC in a patient, comprising administering a CD70-targeted molecule to the patient. [Invention 1003] The method of Invention 1001 or 1002, wherein the patient has been determined to have EGFR variant NSCLC. [Invention 1004] The method according to any one of Inventions 1001 to 1003, wherein the NSCLC comprises lung adenocarcinoma. [Invention 1005] The method according to any one of Inventions 1001 to 1004, wherein the patient is a non-smoker. [Invention 1006] The method according to any one of Inventions 1001 to 1005, wherein the EGFR variant comprises an activating mutation. [Invention 1007] The method of Invention 1006, wherein the activating mutation comprises L858R or a deletion in exon 19. [Invention 1008] The method according to any one of Inventions 1001 to 1007, wherein the EGFR mutation comprises a class I, II or III EGFR mutation. [Invention 1009] The method according to any one of Inventions 1001 to 1008, wherein the patient has not been tested for CD70 expression in cancer cells. [The present invention 1010] Any of the methods of the present invention 1001 - 1008, wherein the patient has been determined to have CD70-expressing cancer cells. [The present invention 1011] Any of the methods of the present invention 1001 - 1010, wherein the patient has previously received treatment for NSCLC. [The present invention 1012] The method of the present invention 1011, wherein the patient has been determined to have acquired resistance to a previous treatment. [The present invention 1013] The method of the present invention 1011 or 1012, wherein the previous treatment includes EGFR tyrosine kinase inhibitor (TKI) therapy, and the therapy includes one or more EGFR TKIs. [The present invention 1014] Any of the methods of the present invention 1011 - 1013, wherein the previous treatment includes monotherapy EGFR TKI therapy. [The present invention 1015] Any of the methods of the present invention 1011 - 1013, wherein the previous treatment includes a combination of at least two EGFR TKIs. [The present invention 1016] Any of the methods of the present invention 1011 - 1015, wherein the patient has been determined to have systemic disease progression while receiving continuous EGFR TKI therapy. [The present invention 1017] Any of the methods of the present invention 1013 - 1016, wherein the EGFR TKI therapy includes one or more of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. [The present invention 1018] Any of the methods of the present invention 1001 - 1017, further comprising the implementation of an additional therapy. [The present invention 1019] The method of the present invention 1018, wherein the additional therapy includes chemotherapy, radiation, surgery, TKI therapy, or immunotherapy. [The present invention 1020] The method of the present invention 1018 or 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 one of the present inventions 1018 to 1020, wherein the additional therapy comprises one or more of carboplatin, pemetrexed, nab-paclitaxel, porfimer, cisplatin, docetaxel, gemcitabine, paclitaxel, and vinorelbine. [The present invention 1022] The method of any one of the present inventions 1018 to 1021, wherein the additional therapy comprises one or more of alectinib, lorlatinib, and ceritinib. [The present invention 1023] The method of any one of the present inventions 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 the present invention 1023, wherein the additional therapy comprises osimertinib. [The present invention 1025] The method of any one of the present inventions 1001 to 1022, further comprising administering adjuvant therapy and / or neoadjuvant therapy. [The present invention 1026] The method of any one of the present inventions 1001 to 1025, wherein the patient has been determined to be an ALK variant. [The present invention 1027] The method of any one of the present inventions 1001 to 1025, wherein the patient has been determined not to be an ALK variant. [The present invention 1028] The method of any one of the present inventions 1001 to 1027, wherein the CD70-targeted molecule comprises an anti-CD70 antibody or a CD70-binding fragment thereof. [The present invention 1029] The method of the present invention 1028, wherein the additional therapy comprises a secondary antibody conjugated to a toxic molecule. [The present invention 1030] The method of the present invention 1029, wherein the secondary antibody and the toxic molecule are linked through a cleavable linker. [The present invention 1031] The method according to any one of the present inventions 1028 to 1030, wherein the antibody is a humanized antibody or a chimeric antibody. [The present invention 1032] The method according to any one of the present inventions 1028 to 1031, wherein the antibody comprises trastuzumab or bortezomib. [The present invention 1033] The method according to any one of the present inventions 1028 to 1032, wherein the antibody is conjugated to a molecule. [The present invention 1034] The method of the present invention 1033, wherein the molecule is a toxic molecule. [The present invention 1035] The method of the present invention 1034, wherein the toxic molecule comprises monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), pyrrolobenzodiazepine (PBD), or duocarmycin. [The present invention 1036] The method according to the present invention 1034 or 1035, wherein the CD70 target-directed molecule comprises trastuzumab-MMAE, bortezomib-MMAE, or a combination thereof. [The present invention 1037] The method according to any one of the present inventions 1001 to 1036, wherein the CD70 target-directed molecule comprises a heavy chain variable region and / or a light chain variable region from a CD70 antibody. [The present invention 1038] The method according to any one of the present inventions 1001 to 1037, wherein the CD70 target-directed 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 present invention 1039] The method according to any one of the present inventions 1001 to 1038, wherein the CD70 target-directed molecule comprises a single-chain variable fragment (scFV). [The present invention 1040] A method according to any one of 1001 to 1039 of the present invention, wherein the CD70-targeted 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). [1041 of the present invention] A method according to 1040 of the present invention, wherein the CD70-targeted molecule comprises a cell comprising a BiTE, a CAR, or a TriNKET. [1042 of the present invention] A method according to 1041 of the present invention, wherein the cell comprises a stem cell, a progenitor cell, an immune cell, or a natural killer (NK) cell. [1043 of the present invention] A cell according to 1042 of the present invention, comprising a hematopoietic stem cell or progenitor cell, a T cell, a cell differentiated from a mesenchymal stem cell (MSC), or an induced pluripotent stem cell (iPSC). [1044 of the present invention] A cell according to 1042 or 1043 of the present invention, isolated from or derived from peripheral blood mononuclear cells (PBMC). [1045 of the present invention] The T cell is a cytotoxic T lymphocyte (CTL), CD8 + T cell, CD4 + A cell according to 1043 or 1044 of the present invention, comprising a T cell, an invariant NK T (iNKT) cell, a gamma-delta T cell, an NKT cell, or a regulatory T cell. [1046 of the present invention] A method according to any one of 1040 to 1045 of the present invention, wherein the CD70-targeted molecule comprises CTX130 or ALLO-316. [1047 of the present invention] A method according to any one of 1040 to 1045 of the present invention, wherein the CD70-targeted molecule comprises a CD27 CAR. [1048 of the present invention] A method according to any one of 1001 to 1039 of the present invention, wherein the CD70-targeted molecule comprises SGN-75, SGN-CD70A, AMG 172, and / or ARGX-110. [1049 of the present invention] Any method of the present invention from 1001 to 1048, wherein a patient-derived biological sample has been determined to be positive for one or more EMT markers. [The present invention 1050] The method of the present invention 1049, wherein the biological sample comprises tumor cells and / or tumor-related cells. [The present invention 1051] The method of the present invention 1049 or 1050, wherein the biological sample comprises a biopsy material. [The present invention 1052] Any method of the present invention from 1049 to 1051, wherein one or more EMT markers comprise a reduction of an epithelial marker and / or an increase of a mesenchymal marker. [The present invention 1053] The method of the present invention 1049 or 1052, wherein the EMT marker comprises 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 the present invention 1054 or 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 one of the present invention from 1054 to 1056, wherein the additional therapeutic agent comprises one or more of carboplatin, pemetrexed, nab-paclitaxel, porfimer sodium, cisplatin, docetaxel, gemcitabine, paclitaxel, and vinorelbine. [The present invention 1058] The composition of any one of the present invention from 1055 to 1057, wherein the additional therapeutic agent comprises one or more of alectinib, lorlatinib, and ceritinib. [The present invention 1059] A composition according to any one of aspects 1055 to 1058 of the present invention, wherein the additional therapeutic agent comprises one or more of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib. [Aspect 1060] A composition according to aspect 1059 of the present invention, wherein the additional therapeutic agent comprises osimertinib. [Aspect 1061] A composition according to any one of aspects 1055 to 1057 of the present invention, wherein the CD70-targeted molecule comprises an anti-CD70 antibody or a CD70-binding fragment thereof. [Aspect 1062] A composition according to aspect 1061 of the present invention, wherein the additional therapeutic agent comprises a secondary antibody conjugated to a toxic molecule. [Aspect 1063] A composition according to aspect 1062 of the present invention, wherein the secondary antibody and the toxic molecule are linked through a cleavable linker. [Aspect 1064] A composition according to any one of aspects 1061 to 1063 of the present invention, wherein the antibody is a humanized antibody or a chimeric antibody. [Aspect 1065] A composition according to any one of aspects 1061 to 1064 of the present invention, wherein the antibody comprises trastuzumab or bortezomib. [Aspect 1066] A composition according to any one of aspects 1061 to 1065 of the present invention, wherein the antibody is conjugated to a molecule. [Aspect 1067] A composition according to aspect 1066 of the present invention, wherein the molecule is a toxic molecule. [Aspect 1068] A composition according to aspect 1067 of the present invention, wherein the toxic molecule comprises monomethyl auristatin E (MMAE), duocarmycin, monomethyl auristatin F (MMAF), or pyrrolobenzodiazepine (PBD). [Aspect 1069] A composition according to aspect 1067 or 1068 of the present invention, wherein the CD70-targeted molecule comprises trastuzumab-MMAE, bortezomib-MMAE, or a combination thereof. [Aspect 1070] A composition according to any one of the present invention 1054-1069, wherein the CD70-targeted molecule comprises a heavy chain variable region and / or a light chain variable region from a CD70 antibody. [The present invention 1071] A composition according to any one of the present invention 1054-1070, wherein the CD70-targeted 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 present invention 1072] A composition according to any one of the present invention 1054-1071, wherein the CD70-targeted molecule comprises a single-chain variable fragment (scFV) that specifically binds to CD70. [The present invention 1073] A composition according to any one of the present invention 1054-1072, wherein the CD70-targeted 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 1074] A composition according to any one of the present invention 1054-1073, wherein the CD70-targeted 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. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration 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 a part of this specification and are included to further demonstrate certain aspects of the present invention. The present invention may be better understood by reference to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein.

[0025]

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Mode for Carrying Out the Invention

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

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

[0028] "Identity" or "identicalness" refers to sequence similarity between two peptides or between two nucleic acid molecules. Identity can be determined by comparing positions in each sequence that can be aligned for purposes of comparison. If a position in the sequences being compared is occupied by the same base or amino acid, then 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. "Unrelated" or "non-identical" sequences share 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] The terms "amino portion", "N-terminus", "amino terminus", etc., as used herein, are used to refer to the order of regions of a polypeptide. Further, if something is at the N-terminus of a region, it is only at the N-terminus of the region or domain, and not necessarily at the end (or terminus) of the entire polypeptide. Similarly, the terms "carboxy portion", "C-terminus", "carboxy terminus", etc., as used herein, are used to refer to the order of regions of a polypeptide, and if something is at the C-terminus of a region, it is only at the C-terminus of the region or domain, and not necessarily at the end (or terminus) of the entire polypeptide.

[0030] The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably and refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. A polynucleotide 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, ESTs 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. A polynucleotide can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. When present, the modifications to the nucleotide structure can be imparted before or after the assembly of the polynucleotide. The nucleotide sequence can be interrupted by non-nucleotide components. A polynucleotide can also be 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 aspect 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 the double-stranded form.

[0031] As used herein, a cell or cell culture is "substantially free" of a particular reagent or factor, such as serum, a signaling inhibitor, an animal component or feeder cell, a foreign genetic element, or a vector element, if it has less than 10% of that element, and "essentially free" of a particular reagent or factor if it has less than 1% of that element. However, a cell population in which the foreign genetic element or vector element present is less than 0.5% or less than 0.1% of the total cell population is even more desirable.

[0032] A cell or cell culture is "essentially free" of certain reagents or components, such as serum, signal transduction inhibitors, animal components, or feeder cells, if the levels of these reagents contained in the culture, matrix, or medium are each below the level detectable using conventional detection methods known to those skilled in the art, or if these reagents are not externally added to the culture, matrix, or medium. Serum-free media 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 appropriate regulatory sequences. The coding region can exist in any of cDNA form, genomic DNA form, or RNA form. When the nucleic acid molecule exists in DNA form, the nucleic acid molecule can be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of the coding region are determined by the start codon at the 5' (amino) terminus and the translation stop codon at the 3' (carboxy) terminus. Genes can include, but are not limited to, cDNA derived from prokaryotic mRNA or eukaryotic mRNA, genomic DNA sequences derived from prokaryotic DNA or eukaryotic DNA, and synthetic DNA sequences. Transcription termination sequences are typically located on the 3' side of the gene sequence.

[0034] The term "cell" is used herein in its broadest sense in the art, referring to a living entity that is a structural unit of a tissue of a multicellular organism, is surrounded by a membrane structure that isolates it from the outside, can self-replicate, and has genetic information and a mechanism for expressing it. The cells used herein can be naturally occurring cells or artificially modified cells (e.g., fused cells, genetically modified cells, etc.).

[0035] As used herein, the terms "treatment" and "treating" refer to obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in terms of completely or partially preventing a disease and / or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or its adverse effects resulting therefrom. "Treatment" as used herein encompasses any treatment of a disease in a mammal, e.g., a human, and includes (a) preventing a 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., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

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

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

[0038] "Antibody or its functional fragment means an immunoglobulin molecule that specifically binds to or immunologically reacts with a specific antigen or epitope, including both polyclonal antibodies 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 tetra-bodies). Antibodies may be derived from natural sources or may be produced partially or entirely by synthesis. Antibodies can be monoclonal or polyclonal. Antibodies can be members of any immunoglobulin class, including any human class: IgG, IgM, IgA, IgD, and IgE. The term "functional antibody fragment" includes antigen-binding fragments of an antibody, including, for example, Fab', F(ab') 2 , Fab, Fv, rlgG, and scFv fragments. The term "scFv" refers to a single-chain Fv antibody in which the variable domains of the heavy and light chains of a conventional two-chain antibody are linked to form a single chain. Antibody fragments may optionally be single-chain antibody fragments. Alternatively, the fragment may contain multiple chains linked together, for example, by disulfide bonds. The fragment may also optionally be a multimolecular complex. Functional antibody fragments retain the ability to bind to their cognate antigen with an affinity comparable to that of a complete antibody.

[0039] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. 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 comprise a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence is 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 pool of clones such as a hybridoma clone, a phage clone, or a recombinant DNA clone. The selected target-binding sequence can be further modified, e.g., to improve its 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 it should be understood that antibodies comprising the modified target-binding sequence are also monoclonal antibodies of the present disclosure. In contrast to polyclonal antibody preparations, which typically include several different antibodies raised against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is made against a single determinant on an antigen. Monoclonal antibody preparations are advantageous in that, in addition to their specificity, they are typically free of contamination with other immunoglobulins.

[0040] The terms "pharmaceutical composition" or "pharmacologically acceptable composition" refer, as appropriate, to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to animals such as humans. The preparation of pharmaceutical compositions that contain antibodies or additional active ingredients will be known to those of skill in the art in light of the present disclosure. Further, in the case of administration to animals (e.g., humans), it will be understood that the preparations must meet the standards of sterility, pyrogenicity, general safety, and purity as required by the FDA's Office of Biological Standards.

[0041] As used herein, "pharmaceutically acceptable carrier" includes, as is known to those of skill in the art, any and all aqueous solvents (e.g., water, alcohol / aqueous solutions, saline, parenteral vehicles such as sodium chloride, and dextrose in Ringer's solution), 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), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrating agents, lubricants, sweetening agents, flavoring agents, dyes, liquid and nutrient supplements, such like materials and combinations thereof. The pH and exact concentration of the various components in a pharmaceutical composition can be adjusted according to well-known parameters.

[0042] The term "unit dose" or "dosage" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of a therapeutic composition calculated to provide the desired response contemplated herein, with its administration, i.e., via the appropriate route and regimen. The amount administered depends on the desired effect, depending on both the number of treatments and the unit dose. The actual dosage of the composition of this aspect administered to a patient or subject depends on physical and physiological factors such as the subject's weight, age, health status and gender, the type of disease being treated, the degree of disease penetration, previous or concurrent therapeutic interventions, the subject's idiopathic diseases, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, the dosage can also include from about 1 μg / kg / body weight to about 1000 mg / kg / body weight per administration (such ranges include dosages intermediate therebetween) or more, and any specific dosage derivable therein. In non-limiting examples of ranges derivable from the numbers described herein, ranges such as 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. can be administered. In any event, the practitioner responsible for administration will determine the concentration of the active ingredient in the composition and the dosage appropriate for the individual subject.

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

[0044] As used herein, the term "binding affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (Kd). The 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) than the binding affinity of the antibody for an irrelevant amino acid sequence. As used herein, the term "binding strength" refers to the resistance to dissociation of a complex of two or more agents after dilution. The terms "immunoreactive" and "selectively binds" are used interchangeably herein with respect to antibodies and / or antigen-binding fragments.

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

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

[0047] "Subject" and "patient" refer to either a human or non-human, such as a primate, mammal, and vertebrate. In certain embodiments, the subject is human.

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

[0049] II. CD70 Targeting Agents A. Antibodies Aspects of the present disclosure relate to CD70 targeting agents. In some embodiments, the CD70 targeting agent comprises an anti-CD70 antibody or fragment thereof. The term "antibody" refers to any isotype of intact immunoglobulin that can compete with an intact antibody for specific binding to a target antigen, or fragment thereof, 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 the animal immune response, including IgG, IgD, IgE, IgA, IgM and related proteins, as well as polypeptides comprising antibody CDR domains that retain antigen-binding activity.

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

[0051] The term "epitope" includes any region or portion of a molecule that can elicit an immune response by binding to an immunoglobulin or a T cell receptor. An epitope determinant includes 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. Generally, an antibody specific for a particular target antigen selectively recognizes the epitope on the target antigen within a complex mixture.

[0052] The epitope region of a given polypeptide can be identified using a number of different epitope mapping techniques well known in the art, including X-ray crystallography, nuclear magnetic resonance spectroscopy, site-directed mutagenesis mapping, protein display arrays, see, for example, Epitope Mapping Protocols, (Johan Rockberg and Johan Nilvebrant, Ed., 2018) Humana Press, New York, N.Y. Such techniques are known in the art and are described, for example, in U.S. Patent 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, for example, Epitope Mapping Protocols, supra. Additionally, the antigenic regions of a protein can also be predicted and identified using standard antigenicity and hydrophobicity plots.

[0053] Intact antibodies generally consist of two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, such as antibodies that naturally occur in camels and contain only heavy chains. The antibodies disclosed herein may be derived from a single source only or may be "chimeric", i.e., different parts of the antibody may be derived from two different antibodies. For example, the variable region or CDR region may be derived from a rat or mouse source, while the constant region is derived from a different animal source, such as human. 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 having a variable region sequence sufficient to confer binding specificity. Full-length light chains have a molecular weight of approximately 25,000 daltons and include a variable region domain (abbreviated herein as VL) and a constant region domain (abbreviated herein as CL). There are two classifications of light chains, designated kappa (κ) and lambda (λ). The term "VL fragment" means a fragment of the light chain of a monoclonal antibody that includes all or part of the light chain variable region and includes the CDR. The VL fragment may further include the light chain constant region sequence. The variable region domain of the light chain is at the amino terminus of the polypeptide.

[0055] The term "heavy chain" includes full-length heavy chains and fragments thereof having a variable region sequence sufficient to confer binding specificity. The 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" means a fragment of the heavy chain of a monoclonal antibody that includes all or part of the heavy chain variable region, including the CDRs. The VH fragment can 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 domains are at the carboxy terminus, and CH3 is closest to the -COOH terminus. The isotype of the antibody can be IgM, IgD, IgG, IgA, or IgE, and is defined by the heavy chain, which has one of five 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] The antibodies can be of any isotype or class of immunoglobulins, chimeric antibodies, or hybrid antibodies having specificity for two or more antigens. They can also be fragments (e.g., F(ab')2, Fab', Fab, Fv, etc.) that include hybrid fragments. 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 immunoglobulins in genetically engineered or otherwise modified forms, such as the following.

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

[0058] The term "bivalent antibody" means an antibody containing 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 that have two paratopes with different binding sites for two or more different epitopes. In some embodiments, bispecific antibodies can be biparatopic, where the bispecific antibody 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 referred to as "nanobodies". Single-domain antibodies are sourced from cartilaginous fish and camelids and have been modified. Nanobodies can be linked together by a linker using techniques conventional to those skilled in the art; such methods for the selection and ligation of nanobodies are described in PCT Publication Nos. WO2015044386A1, WO2010037838A2, and Bever et al., Anal Chem. 86:7875-7882 (2014), which are hereby specifically incorporated by reference in their entireties.

[0060] Bispecific antibodies can be constructed as whole IgG, Fab'2, Fab'PEG, diabodies, or alternatively as scFv. Diabodies and scFv can be constructed using only the variable domains without the Fc region, which may reduce the influence of anti-idiotypic reactions. Bispecific antibodies can be produced by various methods including, but not limited to, fusion of hybridomas or ligation of Fab' fragments. See, for example, Songsivilai and Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992), which are specifically incorporated by reference in their entirety.

[0061] In certain aspects, antigen-binding domains can be multispecific or heterospecific by multimerizing VH and VL domain pairs that bind different antigens. For example, an 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. Accordingly, aspects can include, but are not limited to, bispecific, trispecific, tetra-specific, and other multispecific antibodies or antigen-binding fragments thereof that are directed to an epitope and other targets such as Fc receptors on effector cells.

[0062] In some embodiments, multispecific antibodies can be used and directly linked via short, flexible polypeptide chains using conventional methods known in the art. One such example is the diabody, a bivalent bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain and are too short to allow pairing between domains on the same chain, thereby forcing the domains to pair with complementary domains on a different chain to create two antigen-binding sites. Linker functionality is applicable to the triabody, tetrabody, and even higher-order antibody multimer formats (see, e.g., 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 may also be advantageous because they can be readily constructed and expressed in Escherichia coli (E. coli). Diabodies (and other polypeptides such as antibody fragments) with appropriate binding specificities can be readily selected using phage display from libraries (WO94 / 13804). If one arm of a diabody is held constant, for example, if the specificity for a protein is maintained, libraries can be created that vary the other arm and select antibodies with the appropriate specificity. Bispecific whole antibodies may be made by alternative methods of manipulation as described by Ridgeway et al. (Protein Eng., 9:616-621, 1996) and Krah et al. (N Biotechnol. 39:167-173, 2017), which are hereby incorporated by reference in their entireties.

[0064] Heteroconjugate antibodies are composed of two covalently joined monoclonal antibodies with different specificities. See, for example, U.S. Patent No. 6,010,902, which is hereby incorporated 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 herein referred to as a "paratope". A 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 is delimited by framework regions (FRs) and contains three adjacent hypervariable loops. Hypervariable loops are regions with the greatest variability in primary sequence among antibody molecules from any mammal. The term hypervariable loop may sometimes be used interchangeably with the term "complementary determining region (CDR)". The length of the hypervariable loops (or CDRs) varies among antibody molecules. All framework regions of antibody molecules from a given mammal have high primary sequence similarity / consensus. The consensus of the framework regions can be used by those 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 identifying names that distinguish their positions within the polypeptide and the domains in which they occur. The CDRs in the VL domain are identified as L1, L2, and L3, with L1 being at the most distal end 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 among all antibody molecules produced by a given organism. CDRs are regions of the polypeptide chain that are linearly arranged in the primary structure and separated from each other by the framework regions. The amino-terminal (N-terminal) end of the VL chain is named FR1. The region identified as FR2 is located between the L1 and L2 hypervariable loops. FR3 is located between the L2 and L3 hypervariable loops, and the FR4 region is closest to the CL domain. This structure and nomenclature are repeated for the VH chain, which contains three CDRs identified as H1, H2, and H3. Most of the amino acid residues in the variable domains, or Fv fragment (VH and VL), are part of the framework regions (approximately 85%).The three-dimensional or tertiary structure of the antibody molecule is such that the framework regions are more internal to the molecule and the CDRs are on the outer surface of the molecule, providing most of the structure.

[0066] To identify the exact amino acids that make up each of these regions, several methods have been developed and can be used by those skilled in the art. This is done using any of several multiple sequence alignment methods and algorithms that identify the conserved amino acid residues that make up the framework regions, and thus, although they may be of different lengths, the CDRs located between the framework regions can be identified. Three commonly used methods have been developed for the identification of the CDRs of antibodies: 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 (as described in 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 includes 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, although in some cases, residues within the framework regions contribute to antigen binding.

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

[0068] In certain situations, affinity matured antibodies are enhanced by one or more modifications in one or more of their CDRs that result in an improvement in the affinity of the antibody for the target antigen as compared to the parental antibody that does not possess those modifications. Certain affinity matured antibodies have nanomolar or picomolar affinities for 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 has been described by Rajpal et al., PNAS. 24: 8466-8471 (2005) and Thie et al., Methods Mol Biol. 525:309-22 (2009) in combination with computational methods demonstrated by Tiller et al., Front. Immunol. 8:986 (2017).

[0069] Chimeric immunoglobulins are products of fusion genes derived from different species; “humanized” chimeras generally have framework regions (FRs) from human immunoglobulins and one or more CDRs are from non-human sources.

[0070] In certain instances, 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 chain is derived from a different species or belongs to a different antibody class or subclass and is identical or homologous to the corresponding sequences in such antibodies and fragments of such antibodies so long as the desired biological activity is shown. U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984). For methods regarding chimeric antibodies, see, e.g., U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1985), which are hereby specifically incorporated by reference in their entireties. CDR grafting is described, e.g., 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 hereby incorporated by reference for all purposes.

[0071] In some embodiments, minimizing antibody polypeptide sequences from non-human species optimizes chimeric antibody function and reduces immunogenicity. Specific amino acid residues from non-antigen recognition regions of non-human antibodies 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, but the remainder of the antibody chain is identical or homologous to the corresponding sequences in an antibody from another species or belonging to another antibody class or subclass. For use in humans, the V region, composed of CDR1, CDR2, and partial CDR3 of both the light and heavy chain variable regions from non-human immunoglobulins, is grafted onto a human antibody framework region, replacing the native antigen receptor of the human antibody with the non-human CDRs. Optionally, the corresponding non-human residues are replaced with framework region residues of human immunoglobulins. Additionally, humanized antibodies may contain residues not found in the recipient or donor antibodies to further refine performance. Humanized antibodies may also include at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, for example, 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 intracellularly localized immunoglobulins that bind to intracellular antigens, as opposed to secreted antibodies that bind to antigens in the extracellular space.

[0073] Polyclonal antibody preparations usually contain different antibodies against different determinants (epitopes). To produce polyclonal antibodies, a host such as a rabbit or a goat is generally immunized with an antigen or antigen fragment, together with an adjuvant and, if necessary, conjugated to a carrier. Subsequently, the antibodies against the antigen are recovered from the host's serum. Polyclonal antibodies can be affinity purified against the antigen to make them monospecific.

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

[0075] 1. Functional antibody fragments and antigen-binding fragments a. Antigen-binding fragments Certain aspects relate to antibody fragments, such as antibody fragments that bind to and / or neutralize inflammatory mediators. The term functional antibody fragment includes antigen-binding fragments of antibodies that retain the ability to specifically bind to an antigen. These fragments are composed of various arrangements of variable heavy (VH) and / or light (VL) chains; in some embodiments, they include the constant heavy chain 1 (CH1) and light chain (CL). In some embodiments, they lack the Fc region composed of the heavy chain 2 (CH2) and 3 (CH3) domains. Antigen-binding fragments and modified forms thereof can include: (i) Fab fragment types composed of VL, VH, CL, and CH1 domains; (ii) Fd fragment types composed of VH and CH1 domains; (iii) Fv fragment types composed of VH and VL domains; (iv) single domain fragment types 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, R. A. (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, E. D., Advanced Immunochemistry, 2d ed., Wiley-Liss, Inc. New York, N.Y. (1990); Antibodies, 4:259-277 (2015). All citations in this paragraph are incorporated by reference.

[0076] The antigen-binding fragment also includes a fragment of an antibody that retains exactly, at least, or at most one, two, or three complementarity-determining regions (CDRs) from the variable region of the light chain. The fusion of a CDR-containing sequence to the Fc region (or its CH2 or CH3 region) is included within the scope of this definition, for example, including scFv directly or indirectly fused to the Fc region included herein.

[0077] The term Fab fragment means a monovalent antigen-binding fragment of an antibody that includes the VL, VH, CL, and CH1 domains. The term Fab' fragment means a monovalent antigen-binding fragment of a monoclonal antibody that is larger than the Fab fragment. For example, a Fab' fragment includes the VL, VH, CL, and CH1 domains and all or part of the hinge region. The term F(ab')2 fragment means a divalent antigen-binding fragment of a monoclonal antibody that includes two Fab' fragments linked by a disulfide bridge in the hinge region. An F(ab')2 fragment includes, for example, all or part of two VH and VL domains and may further include all or part of two CL and CH1 domains.

[0078] The term Fd fragment means a fragment of the heavy chain of a monoclonal antibody that includes all or part of the VH and includes a CDR. The Fd fragment can further include the CH1 region sequence.

[0079] The term "Fv fragment" means a monovalent antigen-binding fragment of a monoclonal antibody that includes all or part of VL and VH and lacks the CL and CH1 domains. VL and VH include, for example, CDRs. A single-chain antibody (sFv or scFv) is an Fv molecule that forms a single polypeptide chain in which the VL and VH regions are linked by a flexible linker 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. Patent Nos. 4,946,778 and 5,260,203, the disclosures of which are incorporated herein by reference. The term "(scFv)2" means a divalent or bispecific sFv polypeptide chain that is separated from the sFv by a hinge region and includes an oligomerization domain at the C-terminus (Pack et al. 1992). The oligomerization domain includes a self-associating a-helix, such as a leucine zipper, which can be further stabilized by additional disulfide bonds. The (scFv)2 fragment is also known as a "minibody" or "minibody".

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

[0081] b. Fragment crystallizable region, Fc The Fc region includes two heavy-chain fragments that include the CH2 and CH3 domains of the antibody. The two heavy-chain fragments are held together by two or more disulfide bonds and by hydrophobic interactions of the CH3 domains. The term "Fc polypeptide" as used herein includes the native and mutant forms of polypeptides derived from the Fc region of an antibody. Cleaved forms of such polypeptides that include a hinge region that promotes dimerization are included.

[0082] c. Polypeptides having antibody CDRs and a scaffold domain presenting the CDRs Antigen-binding peptide scaffolds, such as complementarity-determining regions (CDRs), are used to create protein-binding molecules according to embodiments. Generally, one of ordinary skill in the art can determine the type of protein scaffold onto which at least one CDR will be grafted. The scaffold should optimally meet several criteria, such as good phylogenetic conservation; known three-dimensional structure; small size; little or no post-translational modification; and ease of generation, expression, and purification. Skerra, J Mol Recognit, 13:167-87 (2000).

[0083] Protein scaffolds can be supplied from proteins having repetitive motifs such as fibronectin type III FN3 domains (known as "monobodies"), fibronectin type III domain 10, lipocalin, anticalin, the Z domain of protein A from Staphylococcus aureus, thioredoxin A or "ankyrin repeats", "armadillo repeats", "leucine-rich repeats" and "tetratricopeptide repeats", but are not limited thereto. 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, which are hereby specifically incorporated by reference in their entireties. Scaffolds derived from toxins from scorpions, insects, plants, mollusks, etc., and protein inhibitors of neuronal nitric oxide synthase (PINs) can also be used.

[0084] B. Chimeric Antigen Receptors In some embodiments, the CD70 targeting agent comprises and / or expresses a CD70-specific CAR molecule or cell, such as a T cell comprising the CD70-specific CAR molecule. A chimeric antigen receptor T cell, i.e., a CAR T cell, is a T cell that has been genetically modified to express a chimeric receptor specific for a tumor antigen, along with a signaling domain and a costimulatory molecule, and is derived from a patient, donor, or produced in vitro. This fusion of a single-chain variable fragment derived from an antibody and the intracellular signaling domain of a T cell confers upon the CAR T cell the ability to recognize tumor antigens in an MHC-unrestricted manner.

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

[0086] 1. Antigen-binding region The antigen-binding region can be a single-chain variable fragment (scFv) derived from a CD70 antibody. A "single-chain Fv" or "scFv" antibody fragment comprises the V 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 domain and the VL domain, which can facilitate the scFv forming a structure desirable for antigen binding.

[0087] The variable regions of the antigen-binding domains of the polypeptides of the present disclosure can be modified by mutating the amino acid residues within 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 complementarity-determining regions based on portions of the variable chains in immunoglobulins (antibodies) and T cell receptors, which are made by B cells and T cells, respectively, and these molecules bind to their specific antigens. Since most sequence variations associated with immunoglobulins and T cell receptors are found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations can be introduced by site-directed mutagenesis or mutagenesis via PCR, and the effects on antibody binding or other functional properties of interest can be evaluated in appropriate in vitro or in vivo assays. Preferably, conservative modifications are introduced, typically modifying one, two, three, four, or five or fewer residues within the CDR regions. The mutations can be amino acid substitutions, additions, or deletions.

[0088] For example, framework modifications can be added to the antibody by "reverting" one or more framework residues to their corresponding germline sequences to reduce immunogenicity.

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

[0090] 2. Extracellular Spacer The extracellular spacer can link the antigen-binding domain to the transmembrane domain. It must be sufficiently flexible to allow the antigen-binding domain to be oriented in different directions to facilitate antigen binding. In one embodiment, the spacer is the 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 mobile polypeptide connector region that provides structural mobility and spacing between adjacent polypeptide regions (also referred to herein as "hinge region" or "spacer"), and can consist of natural or synthetic polypeptides. A "hinge" derived from an immunoglobulin (e.g., IgG1) is generally defined as a stretch from Glu216 to Pro230 of human IgG1 (Burton (1985) Molec. Immunol., 22: 161-206). The 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 (S-S) bonds at the same positions. The hinge region may be naturally occurring or non-naturally occurring, and includes, but is not limited to, modified hinge regions such as those described in U.S. Patent No. 5,677,425. The hinge region can include a complete hinge region derived from an antibody of a different class or subclass than that of the CH1 domain. The term "hinge" can also include regions derived from CD8 and other receptors that provide a similar function of providing mobility and spacing to adjacent regions.

[0092] 3. Transmembrane domain The transmembrane domain is a hydrophobic alpha helix that spans the membrane. Different transmembrane domains can result in 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 co-stimulatory regions. In some embodiments, a linker is between the transmembrane domain and one or more co-stimulatory 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 receptor clusters and signals are transmitted to the cell through the cytoplasmic region. In some embodiments, the costimulatory domains described herein are part of the cytoplasmic region.

[0095] The cytoplasmic regions and / or costimulatory regions suitable for use with the polypeptides of the disclosure include any desired signaling domain that provides a distinct and detectable signal (e.g., an increase in the production of one or more cytokines by the cell; a change in the transcription of a target gene; a change in the activity of a protein; a change in cell behavior, such as cell death; cell proliferation; cell differentiation; cell survival; regulation of the cell signaling response, etc.) in response to activation by the binding of an antigen to the antigen-binding domain. In some embodiments, the cytoplasmic region includes at least one (e.g., 1, 2, 3, 4, 5, 6, etc.) ITAM motif as described herein. In some embodiments, the cytoplasmic region includes a DAP10 / CD28 type signaling chain.

[0096] The cytoplasmic regions suitable for use with the polypeptides of the disclosure include intracellular signaling polypeptides that contain immunoreceptor activation tyrosine motifs (ITAMs). The ITAM motif is YX 1 X 2 (L / I), where X 1 and X 2 are independently any amino acid. Optionally, the cytoplasmic region includes 1, 2, 3, 4, or 5 ITAM motifs. Optionally, 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 only 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 each of the 6 - 8 X 3 can be any amino acid.

[0097] A suitable cytoplasmic region can be an ITAM motif-containing portion derived from a polypeptide containing 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 need not 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 co-stimulatory regions, such as those included in the cytoplasmic region, include polypeptides from 4-1BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM, but are not limited thereto.

[0099] C. Bispecific T cell engager (BiTE) Bispecific T cell engagers are a new class of immunotherapy molecules for the treatment of cancer. These molecules, referred to as BiTEs, enhance the patient's immune response against tumors by retargeting T cells to tumor cells. BiTEs contain two single-chain variable fragments (scFvs) tandemly connected by a flexible linker. This structure and specificity allow BiTEs to physically link T cells to tumor cells, ultimately stimulating T cell activation, tumor cell death, and cytokine production. Embodiments include BiTEs that contain a CD70-specific targeting region, such as a CD70-specific scFV. BiTEs may further include specificity for additional cancer-related molecules, such as EGFR or AXL. Accordingly, 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 includes a tumor antigen associated with non-small cell lung cancer. Further embodiments relate to BiTEs that contain a CD70-specific targeting region and a TCR-specific targeting region. For example, the TCR-specific targeting region may target a TCR subunit on a T cell, such as CD3.

[0100] D. Tri-specific Natural Killer Cell Engager 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 bridge tumors and NK cells. In some embodiments, the NK cell activation region comprises NK activation molecules, such as cell surface molecules capable of activating cells. The NK cell activation region and / or the antigen binding region may each comprise an scFv specific for an NK activation protein and an scFv specific for a cancer antigen. In some embodiments, TriNKET comprises an scFv domain specific for CD16. In some embodiments, TriNKET comprises an scFV that specifically binds to CD70. TriNKET may further comprise an IL-15 or IL-2 molecule. TriNKET can serve to (a) direct NK cells towards tumors by promoting the formation of intracellular synapses; (b) bind to CD16 on NK cells to induce ADCC; and (c) drive the proliferation of NK cells in vivo through the expression of IL-15 or IL-2.

[0101] III. Cells Certain embodiments relate to cells comprising a polypeptide or nucleic acid of the present disclosure, such as a CD70 targeting agent. In some embodiments, the cell is an immune cell or a T cell. "T cell" includes all types of immune cells expressing CD3, including T helper cells, cytotoxic T cells, regulatory T 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, etc. Suitable mammalian cell lines include HeLa cells (e.g., American Type Culture Collection (ATCC) number CCL-2), CHO cells (e.g., ATCC numbers CRL9618, CCL61, CRL9096), human embryonic kidney (HEK) 293 cells (e.g., ATCC number CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC number CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC number CCL10), PC12 cells (ATCC number CRL1721), COS cells, COS-7 cells (ATCC number CRL1651), RATI cells, mouse L cells (ATCC number CCLI.3), HLHepG2 cells, Hut-78, Jurkat, HL-60, NK cell lines (e.g., NKL, NK92, and YTS), etc., but are not limited thereto.

[0103] In some cases, the cells are not immortalized cell lines but instead cells obtained from an individual (e.g., primary cells). 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 cells or progenitor cells obtained from an individual. In some embodiments, the cells used for treating a patient are autologous. In some embodiments, the cells used for treating a patient are allogeneic.

[0104] IV. Methods for Modifying Genomic DNA In certain embodiments, the genomic DNA is modified to include further mutations, insertions, or deletions, or is modified 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 incorporated into the genomic DNA of a cell. In some embodiments, the incorporation is targeted integration. In some embodiments, the targeted integration is achieved through the use of DNA cleavage agents / polynucleotide modifying enzymes such as site-specific recombinases and / or targeted endonucleases. The term "DNA cleavage agent" refers to an agent capable of cleaving the bonds between the nucleotide subunits of a nucleic acid (i.e., phosphodiester bonds). One particular target is the TRAC (T cell receptor alpha constant) locus. For example, cells are first electroporated with a ribonucleoprotein (RNP) complex consisting of Cas9 protein complexed with a single guide RNA (sgRNA) that targets the TRAC (T cell receptor alpha constant) locus. Fifteen minutes after electroporation, the cells are treated with AAV6 carrying an HDR template encoding the CAR.

[0105] Accordingly, in one aspect, the present disclosure includes targeted integration. One way to achieve this is by use of 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 targeted endonuclease. Site-specific recombinases are well known in the art and can generally be referred to as invertases, resolvases, or integrases. Non-limiting examples of site-specific recombinases can include lambda integrase, Cre recombinase, FLP recombinase, gamma-delta resolvase, Tn3 resolvase, ΦC31 integrase, Bxb1-integrase, and R4 integrase. Site-specific recombinases recognize specific recognition sequences (or recognition sites) or variants thereof, all of which are well known in the art. For example, Cre recombinase recognizes the LoxP site and FLP recombinase recognizes the FRT site.

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

[0107] The landing pad array 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 target-directed endonuclease. Generally, the recognition sequences in the landing pad array do not inherently exist in the genome of the cell to be modified. For example, if the cell to be modified is a CHO cell, the recognition sequences in the landing pad array do not exist in the endogenous CHO genome. The rate of target-directed integration can be improved by selecting recognition sequences for highly efficient polynucleotide modifying enzymes that do not inherently exist within the genome of the target cell. The selection of recognition sequences that do not inherently exist also reduces the likelihood of off-target integration. In other aspects, the use of recognition sequences that are native in the cell to be modified may be desired. For example, if multiple recognition sequences are used in the landing pad array, one or more may be exogenous and one or more may be native.

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

[0109] Multiple recognition arrays may be present in a single landing pad, thereby enabling the landing pad to be successively targeted by two or more polynucleotide modifying enzymes such that two or more distinct nucleic acids (especially those including receptor genes and / or inducible reporters) can be inserted. Alternatively, the presence of multiple recognition arrays in the landing pad enables 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 includes a first recognition array for a first polynucleotide modifying enzyme (e.g., a first pair of ZFNs), and a second recognition array for a second polynucleotide modifying enzyme (e.g., a second pair of ZFNs). Alternatively, or in addition, individual landing pads containing one or more recognition arrays may be integrated at multiple positions. Increased protein expression may be observed in cells transformed with multiple copies of the payload. As another approach, in cells modified with multiple copies, it is also possible to simultaneously express multiple gene products when multiple unique nucleic acid sequences constituting different expression cassettes are introduced into the same or different landing pads. Regardless of the number and type of nucleic acids, when the site-directed nuclease is a ZFN, exemplary ZFN pairs include hSIRT, hRSK4, and hAAVS1 and include recognition arrays.

[0110] Generally speaking, a landing pad used to facilitate site-directed integration may include at least one recognition array. For example, the landing pad may include 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 arrays. In embodiments including two or more recognition arrays, the recognition arrays may be unique to each other (i.e., recognized by different polynucleotide modifying enzymes), the same repeat sequence, or a combination of a repeat sequence and a unique sequence.

[0111] One of ordinary skill in the art can readily understand that exogenous nucleic acids used as landing pads can contain other sequences in addition to recognition sequences. For example, it may be convenient to include one or more sequences encoding selectable or screenable genes as described herein, such as antibiotic resistance genes, metabolic selection markers, or fluorescent proteins. 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) may also be present.

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

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

[0114] Another example of a target-directed endonuclease that can be used is an RNA-guided endonuclease that contains at least one nuclear localization signal that enables the endonuclease to enter the nucleus of a eukaryotic cell. The RNA-guided endonuclease also contains at least one nuclease domain and at least one domain that interacts with the guide RNA. The RNA-guided endonuclease is directed to a specific chromosomal sequence by the guide RNA such that the RNA-guided endonuclease cleaves the specific chromosomal sequence. Since the guide RNA provides specificity for target-directed cleavage, the endonuclease of the RNA-guided endonuclease is universal and can be used with different guide 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 a clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated (Cas) system.

[0115] The target-directed 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 site generally occurs only once in any given genome. Among meganucleases, the intron-encoded family named "LAGLIDADG" has become a valuable tool for genome and genome engineering research. 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 a targetable endonuclease that can be used is a transcription activator-like effector (TALE) nuclease. TALEs are transcription factors from the plant pathogen Xanthomonas, which can be easily engineered to bind to new DNA targets. A TALE or a truncated form thereof may be linked to the catalytic domain of an endonuclease, such as FokI, to create a targetable endonuclease called a TALE nuclease or TALEN. See, for example, Sanjana et al., 2012, Nature Protocols 7(1):171-192; Bogdanove A J, Voytas D F., 2011, Science, 333(6051):1843-6; Bradley P, Bogdanove A J, Stoddard B L., 2013, Curr Opin Struct Biol., 23(1):93-9.

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

[0118] In some embodiments, target-directed integration can be achieved by the use of integrase. For example, phiC31 integrase is a site-specific recombinase encoded within 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 integrated unidirectionally into the target genome through recombination at a site having sequence similarity to the native attP site (referred to as a 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, genomic integration of the polynucleotides of the present disclosure is achieved by the use of a transposase. For example, synthetic DNA transposons designed to introduce precisely defined DNA sequences into the chromosomes of vertebrates (e.g., the "Sleeping Beauty" transposon system) can be used. The Sleeping Beauty transposon system consists of a Sleeping Beauty (SB) transposase and a transposon designed to insert a specific sequence of DNA into the genome of a vertebrate. DNA transposons translocate from one DNA site to another in a simple cut-and-paste fashion. Translocation is an exact process in which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or a different DNA molecule or genome.

[0120] As is the case with all other Tc1 / mariner - type transposases, SB transposase inserts the transposon into the 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). The mammalian genome, including the human genome, has approximately 200 million TA sites. The TA insertion site is duplicated in the process of transposon integration. This duplication of the TA sequence is a prominent feature of transposition and is used to confirm the mechanism in some experiments. The transposase can be encoded within the transposon or the transposase 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 excise and reintegrate independently. All of the DNA transposons identified in the human genome and other mammalian genomes are non - autonomous because, even if they contain the transposase gene, the gene is non - functional and cannot produce a transposase that can mobilize the transposon.

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

[0122] Aspects of the present disclosure include ex vivo methods. For example, T lymphocytes, stem cells, or NK cells (or cells described herein) are obtained from an individual; the cells obtained from the individual are genetically modified to express a CD70 targeting molecule of the present disclosure. Optionally, the genetically modified cells are activated ex vivo. In other cases, the genetically modified cells are introduced into an individual (e.g., the individual from whom the cells were obtained); the genetically modified cells are activated in vivo.

[0123] In some aspects, the method relates to the administration of a cell or a CD70 targeting molecule for the treatment of cancer or to a person having cancer. In some aspects, the cancer is non-small cell lung cancer.

[0124] II. Pharmaceutical Compositions The present disclosure includes methods for treating a disease and for modulating an immune response in a subject in need thereof. The present disclosure includes cells that can be in the form of a pharmaceutical composition for use in inducing or modifying an immune response.

[0125] Administration of the compositions according to the present disclosure will typically be by any common route. This includes, but is not limited to, parenteral, topical, intradermal, subcutaneous, intramuscular, intraperitoneal, or intravenous injection.

[0126] Typically, the compositions of the present disclosure are administered in an amount that is therapeutically effective and immunomodulatory in a manner compatible with the dosage formulation. The amount administered depends on the subject being treated. The exact amount of the active ingredient that needs to be administered depends on the judgment of the practitioner.

[0127] The methods of application can vary widely. Any of the conventional methods for the 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 status of the subject.

[0128] In many cases, it may be desirable to have at most about or at least about 3, 4, 5, 6, 7, 8, 9, 10 or more administrations. The administrations can be in the range of intervals from 2 days to 12 weeks, more commonly from 1 to 2 weeks. Following the course of administration, assays of alloreactive immune responses and T cell activity can follow.

[0129] The terms "pharmaceutically acceptable" or "pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. The use thereof in immunogenic and therapeutic compositions is contemplated, except in cases where any conventional media or agent is incompatible with the active ingredient. The pharmaceutical compositions of the present disclosure are pharmaceutically acceptable compositions.

[0130] The compositions of the present disclosure can be formulated for parenteral administration, for example, formulated for injection by intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as an injectable, either as a liquid solution or a liquid suspension, and 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. Also, they should be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms, such as bacteria and fungi.

[0132] Sterile injectable solutions are prepared by incorporating the required amount of the active ingredient (i.e., the cells of the present disclosure) into a suitable solvent having the various other ingredients enumerated above, as required, and then filtering to sterilize. Generally, dispersions are prepared by incorporating the various sterile active ingredients into a sterile medium containing a base dispersion medium and the other ingredients required from those enumerated above.

[0133] The effective amount of the composition is determined based on the intended purpose. The terms "unit dose" or "dosage" refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired response considered herein, accompanied by its administration, i.e., the appropriate route and regimen. The amount administered depends on both the number of treatments and the unit dose, depending on the desired result and / or protection. Also, the exact amount of the composition depends on the judgment of the practitioner and is specific to each individual. Factors affecting the dosage include the physical and clinical condition of the subject, the route of administration, the intended purpose of the treatment (symptom relief or cure), and the potency, stability, and toxicity of the specific composition. Once formulated, the solution is administered therapeutically or prophylactically in an amount effective in a manner compatible with the dosage formulation. The formulation is readily administered in various dosage forms, such as an injectable solution of the type described above.

[0134] V. Further Therapeutic Methods The methods and compositions of the present disclosure may include one or more further therapeutic methods known in the art and / or described herein. In some embodiments, the further therapeutic method or agent includes a further cancer treatment. Examples of such treatments are described herein.

[0135] A. Immunotherapy In some embodiments, the further therapeutic method or agent includes cancer immunotherapy. Cancer immunotherapy (also called immuno - oncology and 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 utilize the fact that cancer cells often have on their surface molecules that can be detected by the immune system, known as tumor - associated antigens (TAAs); these are often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy instructs the immune system to attack tumor cells by targeting TAAs. Passive immunotherapy enhances existing anti - tumor responses and includes the use of monoclonal antibodies, lymphocytes, and cytokines. Immunotherapy is known in the art, and some are described below.

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

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

[0138] One way to induce dendritic cells to present tumor antigens is by vaccinating with autologous tumor lysates or short peptides (small portions of proteins corresponding to protein antigens on cancer cells). These peptides are often given in combination with adjuvants (substances with high immunogenicity) to enhance the immune and anti-tumor responses. Other adjuvants include proteins or other chemical substances such as granulocyte macrophage colony-stimulating factor (GM-CSF) that attract and / or activate dendritic cells.

[0139] Dendritic cells can also be activated in vivo by causing tumor cells to express GM-CSF. This can be achieved by genetically engineering tumor cells to produce GM-CSF or by infecting tumor cells with tumor-lytic viruses that express GM-CSF.

[0140] Another strategy is to remove dendritic cells from the patient's blood and activate them outside the body. Dendritic cells are activated in the presence of tumor antigens that can be a single tumor-specific peptide / protein or tumor cell lysate (solution of destroyed tumor cells). These cells (with selective adjuvants) are injected to induce 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, which can induce dendritic cells to mature and provide immunity against tumors. Dendritic cell receptors such as TLR3, TLR7, TLR8, or CD40 are used as antibody targets.

[0142] 3. CAR-T Cell Therapy Chimeric antigen receptors (CARs), also known as chimeric immunoreceptors, chimeric T cell receptors, or artificial T cell receptors, are engineered receptors that combine the specificity of immune cells targeting cancer cells with new specificities. Usually, these receptors transplant the specificity of monoclonal antibodies to T cells. The receptors are called chimeric because parts from different sources are fused. CAR-T cell therapy refers to a treatment that uses such transformed cells for cancer treatment.

[0143] The basic principle of CAR-T cell design includes recombinant receptors that combine antigen-binding function and T cell activation function. A 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. When T cells are engineered to become CAR-T cells, they act as "living drugs." CAR-T cells create a link between the extracellular ligand recognition domain and the intracellular signaling molecule, which activates the T cells. The extracellular ligand recognition domain is usually a single-chain variable fragment (scFv). An important aspect of the safety of CAR-T cell therapy is a way to ensure that only cancerous tumor cells, not normal cells, are targeted. The specificity of CAR-T cells is determined by the selection of the molecule to be targeted.

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

[0145] 4. Cytokine Therapy Cytokines are proteins produced by many types of cells present within tumors. They can regulate the immune response. Tumors often utilize cytokines to grow the tumor and reduce the immune response. Due to these immunomodulatory effects, it becomes possible to use them as drugs to elicit an immune response. Two commonly used cytokines are interferon and interleukin.

[0146] Interferons are produced by the immune system. They are usually involved in the antiviral response 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 numerous 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 immunity by transfusion of T cells (adoptive cell transfer). T cells are found in the blood and tissues and usually become activated when they encounter foreign pathogens. Specifically, T cells become activated when the surface receptors on the T cells encounter cells that present a portion of a foreign protein on the surface antigen. These can be either infected cells or antigen-presenting cells (APCs). T cells are found in normal tissues as well as in 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 the tumor, but the environment within the tumor is highly immunosuppressive and prevents immune-mediated tumor death.

[0149] Multiple methods have been developed to produce and obtain T cells targeting tumors. T cells specific to tumor antigens can be removed from tumor samples (TIL) or filtered from the blood. Subsequent activation and culturing are performed ex vivo, and as a result, they are reinjected. Activation can be carried out through gene therapy or by exposing T cells to tumor antigens.

[0150] 6. Checkpoint inhibitors and combination treatments In some embodiments, additional therapies or agents include immune checkpoint inhibitors. 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 tumors. 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 cells 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 the immune response. 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, a 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, a 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, a 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 of PDL1 or PDL2 or a PD-1 binding portion fused to a constant region (e.g., the 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 is also known as MK-3475, Merck3475, lambrolizumab, KEYTRUDA®, and SCH-900475, and is an anti-PD-1 antibody described in WO2009 / 114335. Pidilizumab is also known as CT-011, hBAT, or hBAT-1, and is an anti-PD-1 antibody described in WO2009 / 101611. AMP-224 is also known as B7-DCIg, and 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 combinations 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 with and / or binds to the same epitope on PD-1, PDL1, or PDL2 as the antibody described above. In another embodiment, the antibody has at least about 70, 75, 80, 85, 90, 95, 97, or 99% (or a range derivable therein) variable region amino acid sequence identity with the antibody described above.

[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 an inhibitory signal to T cells. CTLA4 is similar to the T cell co-stimulatory protein CD28, and both molecules bind to B7-1 and B7-2 on antigen-presenting cells. While CTLA-4 transmits an inhibitory signal to T cells, CD28 transmits a stimulatory signal. Intracellular CTLA-4 is also found in regulatory T cells and may be important for their function. When T cells are activated via the T cell receptor and CD28, the expression of CTLA-4, an inhibitory receptor for B7 molecules, increases. The inhibitors of the present disclosure can block one or more functions of CTLA-4, B7-1, and / or B7-2 activities. In some embodiments, the inhibitor blocks the CTLA-4 and B7-1 interaction. In some embodiments, the inhibitor blocks the 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] An anti-human - CTLA-4 antibody (or a VH domain and / or VL domain derived therefrom) suitable for use in the method of the present invention can be produced using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art 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. For binding to CTLA-4, antibodies that compete with any of these antibodies recognized in the art can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application Nos. WO2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0160] Further anti-CTLA-4 antibodies useful as checkpoint inhibitors in the methods and compositions of the present disclosure are 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 with the aforementioned antibody in binding to an epitope on PD-1, B7-1, or B7-2, and / or binds to an epitope on PD-1, B7-1, or B7-2 that is the same 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) variable region amino acid sequence identity with the aforementioned antibody.

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

[0163] C. Polysaccharide In some embodiments, the additional therapy or agent comprises a polysaccharide. Certain compounds found in mushrooms, mainly polysaccharides, can upregulate the immune system and may 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. Neoantigen In some embodiments, additional therapies or agents include the administration of neoantigens. Many tumors express mutations. These mutations potentially create antigens (neoantigens) that can be newly targeted 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 burdens. The levels of transcripts associated with the cytolytic activity of natural killer cells and T cells are positively correlated with the mutation burden in many human tumors.

[0165] E. Chemotherapy In some embodiments, additional therapies or agents include chemotherapy. Suitable classes of chemotherapeutic agents include: (a) alkylating agents such as nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil), ethyleneimines and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, chlorozotocin, streptozocin) and triazines (e.g., dacarbazine); (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 adrenocortical 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, for example, 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, for example, intravenous, subcutaneous, intratumoral or intraperitoneal injection. Cisplatin can be used alone or in combination with other agents, and in certain embodiments an effective dose is contemplated for clinical use including from about 15 mg / m2 to about 20 mg / m2 for 5 days every 3 weeks for a total of 3 cycles. In some embodiments, the amount of cisplatin delivered to cells and / or a subject in combination 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 were used alone.

[0167] Other suitable chemotherapeutic agents include antimicrotubule agents such as, for example, paclitaxel (“Taxol”) and doxorubicin hydrochloride (“doxorubicin”). The combination of an Egr-1 promoter / TNFα construct delivered via an adenovirus vector and doxorubicin has been confirmed to be effective in overcoming resistance to chemotherapy and / or TNF-α, suggesting that combination treatment with the construct and doxorubicin overcomes resistance to both doxorubicin and TNF-α.

[0168] Doxorubicin has poor absorption and is preferably administered intravenously. In certain embodiments, appropriate intravenous doses for adults include from about 60 mg / m2 to about 75 mg / m2 at about 21-day intervals or from about 25 mg / m2 to about 30 mg / m2 for 2 or 3 consecutive days repeated at about 3- to 4-week intervals or about 20 mg / m2 once a week. The lowest doses should be used in elderly patients in the presence of previous myelosuppression or neoplastic bone marrow infiltration caused by previous chemotherapy or if the drug is combined with other myelosuppressive agents.

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

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

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

[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 arrest or regression of cancer in the host when chemotherapy is administered with the construct. In other embodiments, the chemotherapeutic agent can be administered in any amount that is 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 that is 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 determination of an effective dosage. 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, additional therapies or agents or previous therapies include radiation such as ionizing radiation. As used herein, "ionizing radiation" means radiation that includes particles or photons that have sufficient energy or that can produce sufficient energy through nuclear interactions that produce ionization (acquisition or loss of electrons). Exemplary and preferred ionizing radiation is x-rays. Means for delivering x-rays to the target tissue or cell are well known in the art.

[0174] In some embodiments, the amount of ionizing radiation is greater than 20 Gy and is administered in a single 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). When more than one dose is administered, the doses may be spaced apart 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 the total dose of IR and is administered as a fractionated dose. For example, in some embodiments, the total dose is 50 Gy administered as 10 fractionated doses of 5 Gy each. In some embodiments, the total dose is 50 - 90 Gy administered as 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 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 of fractionated doses 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 (or any range derivable therein) fractional doses 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) fractional doses are administered per week.

[0176] G. Surgery Approximately 60% of people with cancer undergo some type of surgery, including prophylactic surgery, diagnostic or staging surgery, curative surgery, and palliative surgery. Curative surgery involves 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 the treatments, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies of the embodiments of the present invention. Tumor resection refers to the physical removal of at least a part of a tumor. In addition to tumor resection, surgical procedures include laser surgery, cryosurgery, electrocautery, and microsurgically controlled surgery (Mohs surgery).

[0177] Removing part or all of cancer cells, tissues, or tumors may create cavities in the body. The treatment may be performed by perfusing, directly injecting, or topically applying additional anti-cancer therapy to the area. Such treatment may be repeated, for example, daily, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, or every 7 days, or every week, every 2 weeks, every 3 weeks, every 4 weeks, and every 5 weeks, or every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months. These treatments may also be treatments 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, cell division arrest and differentiation agents, cell adhesion inhibitors, agents that enhance the sensitivity of hyperproliferative cells to apoptosis-inducing agents, or other biological agents. Increasing the number of gap junctions increases intercellular signaling, which increases the anti-hyperproliferative effect on nearby hyperproliferative cell populations. In other embodiments, a cell division arrest or differentiation agent can be used in combination with certain aspects of the embodiments of the present invention to improve the anti-hyperproliferative efficacy of the treatment. A cell adhesion inhibitor is contemplated to 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 the antibody c225, can be used in combination with certain aspects of the embodiments of the present invention to improve treatment efficacy.

[0179] VI. Administration of the Therapeutic Composition The methods of the present disclosure include the administration of combinations of therapeutic agents and / or the administration of therapeutic agents such as fecal matter and therapeutic regimens such as, for example, steroid therapy or anti-integrin therapy. The treatment can be administered by any suitable method known in the art. For example, the treatment can be administered continuously (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, by designating one treatment as "A" and another treatment as "B", various combinations of treatments can be utilized. TIFF2025084874000002.tif13128

[0181] The treatments of the present disclosure, such as fecal matter from a healthy subject, can be administered by the same route of administration or by different routes of administration. In some embodiments, the treatment is administered into the colon, intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by transplantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the microbial modulator is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by transplantation, by inhalation, intrathecally, intraventricularly, or intranasally.

[0182] The amount administered depends on the desired treatment effect, depending on both the number of treatments and the unit dose. The effective dose is understood to refer to the amount necessary to achieve a particular effect. In certain embodiments, it is contemplated that doses in the range of 10 mg / kg to 200 mg / kg may affect the protective ability of these agents. Thus, the dose may be 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 of doses derivable therein. Further, such doses can be administered once a day and / or multiple times over a plurality of days, weeks, or months.

[0183] In some embodiments, the therapeutically effective or sufficient amount of the therapeutic composition administered to a human will be in the range of about 0.01 to about 50 mg / kg patient body weight, whether by one 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 day 1 of a 21-day cycle. The dose can be administered as a single dose or multiple doses (e.g., two or three doses), such as by infusion. The progress of this treatment is readily monitored by conventional techniques.

[0184] In certain embodiments, the effective dosage of the pharmaceutical composition is capable of providing blood levels of from about 1 μM to 150 μM. In another embodiment, the effective dosage provides blood levels of from about 4 μM to 100 μM; or from about 1 μM to 100 μM; or from about 1 μM to 50 μM; or from about 1 μM to 40 μM; or from about 1 μM to 30 μM; or from about 1 μM to 20 μM; or from about 1 μM to 10 μM; or from about 10 μM to 150 μM; or from about 10 μM to 100 μM; or from about 10 μM to 50 μM; or from about 25 μM to 150 μM; or from about 25 μM to 100 μM; or from about 25 μM to 50 μM; or from about 50 μM to 150 μM; or from about 50 μM to 100 μM (or any range derivable therein). In other embodiments, the dosage is capable of providing 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, or 100 μM or any range derivable therein. In certain embodiments, the therapeutic agent administered to the subject is metabolized in vivo to the metabolized therapeutic agent, in which case the blood level refers to the amount of that agent. Alternatively, to the extent that the therapeutic agent is not metabolized by the subject, the blood levels discussed herein refer to the unmetabolized therapeutic agent.

[0185] The exact amount of the therapeutic composition also depends on the judgment of the practitioner and is specific to each individual. Factors that affect the dosage include the patient's physical and clinical condition, the route of administration, the intended treatment goal (symptom relief or cure), and the efficacy, stability, and toxicity of the specific therapeutic substance or other therapies the subject may be receiving.

[0186] Those skilled in the art will understand and recognize that dosage units of μg / kg or mg / kg body weight can be converted to equivalent concentration units such as μg / ml or mM (blood levels), such as from 4 μM to 100 μM. It is also understood that uptake depends on the species and organ / tissue. 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 dosages, efficacy, and results described herein.

[0187] VII. Kit Certain aspects of the present disclosure also include kits for performing the methods of the present disclosure, such as for the detection, diagnosis, or treatment of cancer. 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, surfactants, primers, probes, antibodies. In a preferred embodiment, these kits enable the practitioner to obtain samples of neoplastic cells from blood, tears, semen, saliva, urine, tissue, serum, feces, saliva, cerebrospinal fluid, and the supernatant from cell lysates. In another preferred embodiment, these kits include the equipment necessary to perform RNA extraction, RT-PCR, and gel electrophoresis. Instructions for use for performing the assay can also be included in the kit.

[0188] The kit may further include instructions for using the kit to evaluate an array and means for converting and / or analyzing array data for prognostic diagnosis. 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 biomarker. In another embodiment, the agent in the kit for measuring biomarker expression may include an array of polynucleotides complementary to the mRNA of the biomarker of the present invention. Also included may be possible means for converting expression data into expression values and generating a score for analyzing the expression values to predict survival or prognosis.

[0189] The kit may include a labeled container. 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 containing probes useful for prognostic or non-prognostic applications as described above. The label on the container indicates that the composition is used for a specific prognostic or non-prognostic application and may also indicate the method of use, either in vivo or in vitro, such as those described above. The kit may include one or more other containers containing materials desirable from a commercial and user perspective, including the above container, as well as buffers, diluents, filters, needles, syringes, and accompanying documents with instructions.

[0190] A further aspect of the kit relates to a kit comprising the therapeutic composition of the present disclosure. The kit is useful in the treatment methods of the present disclosure and may include instructions.

Examples

[0191] VIII. Examples The following examples are included to demonstrate preferred embodiments of the present invention. The techniques disclosed in the following examples show that the techniques discovered by the inventors function well in the practice of the present invention, and thus it should be understood by those skilled in the art that they constitute a preferred mode of practice thereof. However, in view of the present disclosure, it should be understood by those skilled in the art that many changes can be made in the specific embodiments disclosed without departing from the spirit and scope of the present invention, and still obtain similar or like results.

[0192] Example 1 - EGFR TKI resistance is associated with a mesenchymal phenotype and increased expression of CD70 Herein, the inventors demonstrate that CD70 is a therapeutic target for TKI-resistant EGFR mutant tumors, and that CD70-directed therapies, such as CD70-antibody drug conjugates, anti-CD70 CAR-T cells, or TriNKET, EGFR-CD70 BiTE, Axl-CD70 BiTE, or other approaches targeting CD70 (collectively referred to as CD70-directed therapies), may be effective, either alone or in combination with other treatments, against TKI-resistant EGFR mutant tumors. Furthermore, the EGFR mutation is a biomarker for selecting patients to be treated with CD70-targeting agents. Additionally, the inventors describe that CD70-targeting is a therapeutic strategy for mesenchymal NSCLC tumors, and that the mesenchymal state, 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-directed therapies.

[0193] As part of efforts to identify potential targets in TKI-resistant EGFR mutant NSCLC, the inventors 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. The inventors' finding that gene expression of CD70 was highly upregulated in NSCLC cells with acquired resistance to EGFR TKI was verified by flow cytometry, demonstrating that resistant cells expressed high levels of CD70 protein on the cell surface compared to parental (EGFR TKI-sensitive) cells. To evaluate whether CD70 was increased in NSCLC clinical specimens that had undergone EMT, the inventors evaluated RNAseq data from TCGA. Expression of CD70 correlated with a mesenchymal gene signature in NSCLC tumor specimens.

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

[0195] EGFR mutant NSCLC patients initially respond to EGFR tyrosine kinase inhibitors (TKIs), but drug-resistant disease inevitably emerges. The inventors derived a panel of NSCLC cell lines with acquired resistance to EGFR TKIs. The EGFR-TKI-resistant (ER) cells were negative for secondary EGFR mutations and were resistant to EGFR TKIs (Figure 1). Using RNAseq and gene expression analysis, the inventors 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 high expression of ZEB1 and ZEB2, which are major mediators of epithelial-mesenchymal transition (EMT) (Figures 2A - G). RNA expression analysis further revealed that EGFR TKI-resistant cells highly overexpressed CD70 (Figure 2H). Flow cytometry analysis revealed an increase in the surface CD70 protein level 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). Considering the finding that EGFR TKI resistance is associated with EMT and the finding that these cells overexpress CD70, the inventors next used the TCGA database to evaluate whether CD70 expression is associated with the mesenchymal phenotype in human lung adenocarcinoma. The inventors found that CD70 expression was significantly associated with the EMT gene expression signature in lung adenocarcinoma and in a broad panel of NSCLC cell lines (Figure 5). CD70 is normally expressed in T cells 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 affecting / 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 CD70 expression This example may include overlapping and / or rearranged data from Example 1.

[0197] EGFR mutant NSCLC patients initially respond to EGFR tyrosine kinase inhibitors (TKIs), but drug-resistant disease inevitably emerges. The inventors 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, the inventors 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 overexpression of ZEB1 and ZEB2, which are major 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 an increase in the surface CD70 protein level of EGFR TKI-resistant cells compared to EGFR TKI-sensitive parental cells (Figures 7B–F). To determine whether CD70 is elevated in an animal model of EGFR TKI resistance, the inventors utilized a doxycycline-inducible EGFR L858R GEMM model in which administration of doxycycline results in mutant EGFR expression and lung tumor development. 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, as determined by CT imaging, tumors began to regrow. Animals were treated with osimertinib to confirm the EGFR TKI-resistant phenotype. Tumors were collected and CD70 expression was analyzed by immunohistochemistry. CD70 expression was elevated in acquired EGFR-independent tumors (Figure 8). Next, the inventors evaluated CD70 expression in EGFR mutant EGFR TKI-untreated NSCLC clinical specimens and EGFR mutant NSCLC specimens collected after EGFR TKI resistance. CD70 expression was minimal in untreated tissues, but CD70 was highly expressed in EGFR TKI-resistant tumors (Figure 9).

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

[0199] The binding of CD27 to CD70 induces the activation of the signaling pathway downstream of CD70. To examine the potential effect of CD70 signaling on EGFR TKI-resistant cells, the inventors stimulated EGFR TKI-resistant cells with recombinant soluble CD27. CD27 treatment resulted in the activation of Akt and ERK, important signaling molecules known to be reactivated in EGFR TKI resistance (Figure 12). Next, the inventors used siRNA to knockdown CD70 expression and found that knockdown of CD70 impaired the proliferation of EGFR TKI-resistant cells by a clonogenic assay (Figure 13).

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

[0201] CD70 is normally expressed in T cells 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 affecting / attracting regulatory T cells and promoting apoptosis and depletion 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 in terms of preferred embodiments, it will be apparent to those skilled in the art that changes can be made in the methods described herein and in the steps or the order of steps of such methods without departing from the concept, spirit, and scope of the present invention. More specifically, it will be apparent that certain chemically and physiologically related agents can be used in place of the agents described herein, and that in so doing, the same or similar results can be obtained. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the scope of 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 procedures or other details that supplement what is described herein.

Claims

1. A method for treating EGFR mutant non-small cell lung cancer (NSCLC) in a patient, comprising administering to the patient a CD70 targeting molecule.

2. A method for treating epithelial-mesenchymal transition (EMT) positive NSCLC in a patient, comprising administering to the patient a CD70 targeting molecule.

3. 3. The method of claim 1 or 2, wherein the patient is determined to have EGFR mutant NSCLC.

4. The method of any one of claims 1 to 3, wherein the NSCLC comprises lung adenocarcinoma.

5. The method of any one of claims 1 to 4, wherein the patient is a non-smoker.

6. The method of any one of claims 1 to 5, wherein the EGFR mutant comprises an activating mutation.

7. The method of claim 6, wherein the activating mutation comprises L858R or a deletion in exon 19.

8. The method of any one of claims 1 to 7, wherein the EGFR mutation comprises a class I, II or III EGFR mutation.

9. The method of any one of claims 1 to 8, wherein the patient has not been tested for CD70 expression in the cancer cells.

10. The method of any one of claims 1 to 8, wherein the patient is determined to have CD70-expressing cancer cells.

11. The method of any one of claims 1 to 10, wherein the patient has previously undergone treatment for NSCLC.

12. 12. The method of claim 11, wherein the patient has been determined to have acquired resistance to a previous treatment.

13. 13. The method of claim 11 or 12, wherein the previous treatment comprises EGFR tyrosine kinase inhibitor (TKI) therapy, said therapy comprising one or more EGFR TKIs.

14. The method of any one of claims 11 to 13, wherein the previous treatment comprises single agent EGFR TKI therapy.

15. The method of any one of claims 11 to 13, wherein the previous treatment comprises a combination of at least two EGFR TKIs.

16. The method of any one of claims 11 to 15, wherein the patient has been determined to have systemic disease progression while receiving ongoing EGFR TKI therapy.

17. The method of any one of claims 13-16, wherein the EGFR TKI therapy comprises one or more of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib.

18. 18. The method of any one of claims 1 to 17, further comprising administration of a further therapy.

19. 20. The method of claim 18, wherein the additional therapy comprises chemotherapy, radiation, surgery, TKI therapy, or immunotherapy.

20. 20. The method of claim 18 or 19, wherein the additional therapy comprises one or more of durvalumab, atezolizumab, pembrolizumab, nivolumab, necitumumab, and bevacizumab.

21. 21. The method of any one of claims 18-20, wherein the additional therapy comprises one or more of carboplatin, pemetrexed, nab-paclitaxel, photofrin, cisplatin, docetaxel, gemcitabine, paclitaxel, and vinorelbine.

22. 22. The method of any one of claims 18-21, wherein the additional therapy comprises one or more of alectinib, lorlatinib, and ceritinib.

23. 23. The method of any one of claims 18-22, wherein the additional therapy comprises one or more of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib.

24. 24. The method of claim 23, wherein the additional therapy comprises osimertinib.

25. The method according to any one of claims 1 to 22, further comprising the administration of adjuvant and / or neoadjuvant therapy.

26. The method of any one of claims 1 to 25, wherein the patient has been determined to be ALK mutant.

27. The method of any one of claims 1 to 25, wherein the patient has been determined to not be ALK mutant.

28. The method of any one of claims 1 to 27, wherein the CD70 targeting molecule comprises an anti-CD70 antibody or a CD70-binding fragment thereof.

29. 30. The method of claim 28, wherein the additional therapy comprises a second antibody linked to a toxic molecule.

30. 30. The method of claim 29, wherein the second antibody and the toxic molecule are linked through a cleavable linker.

31. The method of any one of claims 28 to 30, wherein the antibody is a humanized antibody or a chimeric antibody.

32. The method of any one of claims 28-31, wherein the antibody comprises cusatuzumab or borsetuzumab.

33. The method of any one of claims 28 to 32, wherein the antibody is conjugated to the molecule.

34. 34. The method of claim 33, wherein the molecule is a toxic molecule.

35. 35. The method of claim 34, wherein the toxic molecule comprises monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), a pyrrolobenzodiazepine (PBD), or a duocarmycin.

36. The method of claim 34 or 35, wherein the CD70 targeting molecule comprises cusatuzumab-MMAE, borsetuzumab-MMAE, or a combination thereof.

37. The method of any one of claims 1 to 36, wherein the CD70 targeting molecule comprises a heavy chain variable region and / or a light chain variable region from a CD70 antibody.

38. The method of any one of claims 1 to 37, 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.

39. The method of any one of claims 1 to 38, wherein the CD70 targeting molecule comprises a single chain variable fragment (scFV).

40. The method of any one of claims 1-39, 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).

41. The method of claim 40, wherein the CD70 targeting molecule comprises a cell comprising a BiTE, a CAR, or a TriNKET.

42. 42. The method of claim 41, wherein the cells comprise stem cells, progenitor cells, immune cells, or natural killer (NK) cells.

43. 43. The cell of claim 42, comprising 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).

44. 44. The cell of claim 42 or 43, isolated or derived from a peripheral blood mononuclear cell (PBMC).

45. T cells are cytotoxic T lymphocytes (CTL), CD8 + T cells, CD4 + The cell of claim 43 or 44, comprising a T cell, an invariant NK T (iNKT) cell, a gamma-delta T cell, an NKT cell, or a regulatory T cell.

46. The method of any one of claims 40-45, wherein the CD70 targeting molecule comprises CTX130 or ALLO-316.

47. The method of any one of claims 40-45, wherein the CD70 targeting molecule comprises a CD27 CAR.

48. The method of any one of claims 1 to 39, wherein the CD70 targeting molecule comprises SGN-75, SGN-CD70A, AMG 172, and / or ARGX-110.

49. The method of any one of claims 1 to 48, wherein the biological sample from the patient has been determined to be positive for one or more EMT markers.

50. 50. The method of claim 49, wherein the biological sample comprises tumor cells and / or tumor-associated cells.

51. 51. The method of claim 49 or 50, wherein the biological sample comprises a biopsy.

52. The method of any one of claims 49-51, wherein the one or more EMT markers comprise a reduction in an epithelial marker and / or an increase in a mesenchymal marker.

53. The method of claim 49 or 52, wherein the EMT markers include one or more of CDH1, VIM, AXL, ZEB1, and ZEB2.

54. A composition comprising a CD70 targeting molecule and one or more additional therapeutic agents.

55. 55. The composition of claim 54, wherein the additional therapeutic agent comprises chemotherapy, radiation, surgery, TKI therapy, immunotherapy, or a combination thereof.

56. 56. The composition of claim 54 or 55, wherein the additional therapeutic agents comprise one or more of durvalumab, atezolizumab, pembrolizumab, nivolumab, necitumumab, and bevacizumab.

57. 57. The composition of any one of claims 54-56, wherein the additional therapeutic agents comprise one or more of carboplatin, pemetrexed, nab-paclitaxel, photofrin, cisplatin, docetaxel, gemcitabine, paclitaxel, and vinorelbine.

58. The composition of any one of claims 55-57, wherein the additional therapeutic agents comprise one or more of alectinib, lorlatinib, and ceritinib.

59. 59. The composition of any one of claims 55-58, wherein the additional therapeutic agent comprises one or more of gefitinib, erlotinib, afatinib, dacomitinib, osimertinib, and brigatinib.

60. 60. The composition of claim 59, wherein the additional therapeutic agent comprises osimertinib.

61. The composition of any one of claims 55-57, wherein the CD70 targeting molecule comprises an anti-CD70 antibody or a CD70-binding fragment thereof.

62. 62. The composition of claim 61, wherein the additional therapeutic agent comprises a second antibody linked to a toxic molecule.

63. The composition of claim 62, wherein the second antibody and the toxic molecule are linked through a cleavable linker.

64. The composition of any one of claims 61 to 63, wherein the antibody is a humanized antibody or a chimeric antibody.

65. The composition of any one of claims 61-64, wherein the antibody comprises cusatuzumab or borsetuzumab.

66. The composition of any one of claims 61 to 65, wherein the antibody is conjugated to a molecule.

67. 67. The composition of claim 66, wherein the molecule is a toxic molecule.

68. 68. The composition of claim 67, wherein the toxic molecule comprises monomethylauristatin E (MMAE), duocarmycin, monomethylauristatin F (MMAF), or pyrrolobenzodiazepine (PBD).

69. The composition of claim 67 or 68, wherein the CD70 targeting molecule comprises cusatuzumab-MMAE, borsetuzumab-MMAE, or a combination thereof.

70. The composition of any one of claims 54 to 69, wherein the CD70 targeting molecule comprises a heavy chain variable region and / or a light chain variable region from a CD70 antibody.

71. The composition of any one of claims 54 to 70, 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.

72. The composition of any one of claims 54-71, wherein the CD70 targeting molecule comprises a single chain variable fragment (scFV) that specifically binds to CD70.

73. The composition of any one of claims 54-72, 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).

74. The composition of any one of claims 54 to 73, wherein the CD70 targeting molecule comprises SGN-75, SGN-CD70A, AMG 172, and / or ARGX-110.

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