Administration Regimen of CD19-Targeted Cancer Immunotherapy

Abstract

Some embodiments of the methods and compositions disclosed herein relate to immune cells engineered to express a cytotoxic chimeric receptor, and various dosing regimens for administering such cells. In some embodiments, the immune cells express a chimeric receptor that targets CD19 on tumor cells. In some embodiments, the cancer is a blood cancer, such as a B cell malignancy. In some embodiments, immune cells engineered to express a CD19-directed CAR are administered (before, after, or simultaneously) in combination with an antibody treatment, such as an anti-CD20-directed antibody.

Description

Technical Field

[0001] Some embodiments disclosed herein relate to methods and compositions comprising genetically engineered cells for cancer immunotherapy. In some embodiments, the disclosure relates to cells engineered to express a chimeric antigen receptor directed to a specific tumor marker, such as CD19, and the administration of such cells according to certain dosing regimens for successful cancer immunotherapy.

Background Art

[0002] Additional knowledge has been obtained regarding the characteristics of cancer cells that can be used to specifically distinguish those cells from various cancers and healthy cells, and thus treatments that utilize the prominent properties of cancer cells are under development. Immunotherapy using engineered immune cells is one approach to treating cancer. Incorporation by reference of materials in the sequence listing file

[0003] This application incorporates by reference the sequence listing contained in the following XML text file, which was filed simultaneously: File name: NKT.088WO_ST26.xml, created on June 29, 2023, and having a size of 60180 bytes.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Immunotherapy represents a new technological advance in the treatment of disease, where immune cells are engineered to express certain targeting and / or effector molecules that specifically identify and respond to diseased or damaged cells. This represents an expected advance, at least in part, due to the ability to specifically target diseased or damaged cells, as opposed to conventional approaches such as chemotherapy, where all cells are affected and the desired outcome is the survival of sufficient healthy cells to enable the patient's survival. One immunotherapy approach is the recombinant expression of a chimeric antigen receptor, also referred to as a CAR or chimeric receptor, in immune cells, which achieves target recognition and destruction of abnormal cells in a subject.

Means for Solving the Problem

[0005] In some embodiments, a population of genetically engineered natural killer (NK) cells for cancer immunotherapy is provided herein, comprising a plurality of NK cells expanded by culture, wherein the plurality of NK cells are engineered to express a chimeric antigen receptor comprising a CD19-targeting extracellular domain, a transmembrane domain, and a cytotoxic signaling complex. In some embodiments, the CD19-targeting extracellular domain binds to CD19. In some embodiments, the CD19-targeting extracellular domain binds to human CD19.

[0006] In some embodiments, a dosing regimen for cancer immunotherapy comprising at least a first dosing cycle, wherein the first dosing cycle comprises a first dose of genetically engineered natural killer (NK) cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, the first dose being administered to a subject in need of cancer immunotherapy at a first time point, the second dose being administered to the subject between 5 and 10 days after the first time point, the third dose being administered to the subject between 5 and 10 days after the second dose; each of the first, second, and third doses comprising about 1.5×10 9 NK cells, at least a portion of the genetically engineered NK cells being engineered to express a chimeric antigen receptor (CAR) directed against the CD19 tumor marker, and the first dosing cycle being initiated after the subject has undergone a lymphodepletion process to reduce the number of natural immune cells. In some embodiments, the first dosing cycle is followed by one or more additional dosing cycles.

[0007] In some embodiments, a dosing regimen for cancer immunotherapy, comprising at least a first dosing cycle, wherein the first dosing cycle comprises a first dose of genetically engineered natural killer (NK) cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, the first dose being administered to a subject in need of cancer immunotherapy at a first time point, the second dose being administered to the subject between 5 and 10 days after the first time point, the third dose being administered to the subject between 5 and 10 days after the second dose; each of the first, second, and third doses comprising about 1.5×10 9 NK cells, at least a portion of the genetically engineered NK cells being engineered to express a chimeric antigen receptor (CAR) directed against the CD19 tumor marker, the first dosing cycle being initiated after the subject has undergone a lymphodepletion process to reduce the number of natural immune cells, and the first dosing cycle may be followed by one or more additional dosing cycles, providing a dosing regimen. In some embodiments, a subject showing at least a partial response receives at least one additional dosing cycle. The dosing cycle may continue according to the embodiment as long as the subject shows an anti-tumor response and tolerates the engineered NK cells. In some embodiments, the subject does not receive an additional dosing cycle if the subject does not respond (e.g., no tumor response) and / or the treatment is not tolerated. However, as discussed herein, in some embodiments, the dosing regimens of the present disclosure have limited or no adverse effects or toxicity. In some embodiments, the decision to receive / administer an additional dosing cycle is made at an evaluation 30 days after the start of the dosing cycle (the first dosing cycle or a subsequent cycle). In some embodiments, fewer than 5 additional cycles are given to the subject.

[0008] In some embodiments, the dosing regimen provided herein further comprises the administration of an additional therapeutic agent targeting the CD20 tumor marker. In some such embodiments, at least about 1.0×10 9 NK cells are administered at each dose in such combination therapy. In some such embodiments, about 1.0×109 Individual NK cells are administered at each dose in such combination therapies. In some embodiments, however, at least about 1.5×10 9 individual NK cells are used in combination therapy with an agent targeting CD20. In some embodiments, however, about 1.5×10 9 individual NK cells are used in combination therapy with an agent targeting CD20. According to embodiments, the further therapeutic agent is an antibody or a biologic follow-on. In some embodiments, the further therapeutic agent is administered in an amount between about 150 mg / m 2 and about 500 mg / m 2 . In some embodiments, the further therapeutic agent is administered in an amount between about 200 mg / m 2 and about 400 mg / m 2 . In some embodiments, the further therapeutic agent is administered in an amount between about 350 mg / m 2 and about 425 mg / m 2 . In some embodiments, the further therapeutic agent is administered in an amount of about 375 mg / m 2 . In some embodiments, the further therapeutic agent is administered at least 1 time and at least 2 days prior to administration of the first dose of the dosing cycle. In some embodiments, the further therapeutic agent is administered as a single dose, and the further treatment is administered 3 days prior to administration of the first dose of the dosing cycle. In some embodiments, the further therapeutic agent is a biologic follow-on selected from rituximab-abb, rituximab-arrx, and / or rituximab-pvvr. In some embodiments, the further therapeutic agent is an anti-CD20 monoclonal antibody. In some embodiments, the anti-CD20 monoclonal antibody is selected from rituximab, ofatumumab, obinutuzumab, ibritumomab, ibritumomab or combinations thereof. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the anti-CD20 antibody is obinutuzumab.

[0009] As used herein, a dosing regimen for cancer immunotherapy comprising at least a first dosing cycle, wherein the first dosing cycle comprises a first dose of genetically engineered natural killer (NK) cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, the first dose being administered to a subject in need of cancer immunotherapy at a first time point, the second dose being administered to the subject between 5 and 10 days after the first time point, and the third dose being administered to the subject between 5 and 10 days after the second dose; each of the first, second, and third doses comprising about 1.5×10 9 NK cells, at least a portion of the engineered NK cells being engineered to express a chimeric antigen receptor (CAR) directed against the CD19 tumor marker, and the first dosing cycle being initiated after the subject has undergone a lymphodepletion process comprising at least two doses of cyclophosphamide and fludarabine, and an anti-CD20 antibody being administered during the lymphodepletion process, also provides a dosing regimen. In some embodiments, the first dosing cycle is followed by one or more additional dosing cycles.

[0010] As used herein, a dosing regimen for cancer immunotherapy comprising at least a first dosing cycle, wherein the first dosing cycle comprises a first dose of genetically engineered natural killer (NK) cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, the first dose being administered to a subject in need of cancer immunotherapy at a first time point, the second dose being administered to the subject between 5 and 10 days after the first time point, and the third dose being administered to the subject between 5 and 10 days after the second dose; each of the first, second, and third doses comprising about 1.5×10 9 NK cells, at least a portion of the engineered NK cells being engineered to express a chimeric antigen receptor (CAR) directed against the CD19 tumor marker, and the first dosing cycle being initiated after the subject has undergone a lymphodepletion process comprising at least two doses of cyclophosphamide and fludarabine, and an anti-CD20 antibody being administered during the lymphodepletion process, and the first dosing cycle may be followed by one or more additional dosing cycles, also provides a dosing regimen.

[0011] In some embodiments, the NK cells are CAR-expressing NK cells. In some embodiments, each of the first, second, and third doses comprises about 1.5×10 9 CAR-expressing NK cells.

[0012] In some embodiments, among the subjects treated according to the dosing regimen, the overall response rate (ORR) is at least about 50%, at least about 60%, at least about 70%, or at least about 80%. In some embodiments, among the subjects treated according to the dosing regimen, the ORR is at least about 50%. In some embodiments, among the subjects treated according to the dosing regimen, the ORR is at least about 60%. In some embodiments, among the subjects treated according to the dosing regimen, the ORR is at least about 70%. In some embodiments, among the subjects treated according to the dosing regimen, the ORR is at least about 80%.

[0013] In some embodiments, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the subjects treated according to the dosing regimen exhibit a complete response (CR). In some embodiments, at least about 50% of the subjects treated according to the dosing regimen exhibit a CR. In some embodiments, at least about 60% of the subjects treated according to the dosing regimen exhibit a CR. In some embodiments, at least about 70% of the subjects treated according to the dosing regimen exhibit a CR. In some embodiments, at least about 80% of the subjects treated according to the dosing regimen exhibit a CR.

[0014] In some embodiments, if the subject shows a clinical response after the first dosing cycle, the dosing regimen includes additional dosing cycles. In some embodiments, if the subject shows a complete response (CR) after the first dosing cycle, the dosing regimen includes additional dosing cycles. In some embodiments, if the subject shows a clinical response after a dosing cycle and subsequently shows disease progression, the dosing regimen includes additional dosing cycles. In some embodiments, the dosing regimen includes between 1 and 5 dosing cycles. In some embodiments, the dosing regimen consists of between 1 and 5 dosing cycles. In some embodiments, the dosing regimen consists of between 1 and 5 dosing cycles. In some embodiments, the dosing regimen consists of 1 dosing cycle. In some embodiments, the dosing regimen consists of 2 dosing cycles. In some embodiments, the dosing regimen consists of 3 dosing cycles. In some embodiments, the dosing regimen consists of 4 dosing cycles. In some embodiments, the dosing regimen consists of 5 dosing cycles. In some embodiments, the subject undergoes a lymphocyte depletion process prior to each dosing cycle.

[0015] In some embodiments, cancer cells do not express CD58 or express a mutant form of CD58. In some embodiments, cancer cells do not express CD58. In some embodiments, cancer cells express a mutant form of CD58. In some embodiments, prior to administration of the first dosing cycle to the subject, it is determined that the cancer cells do not express CD58 or express a mutant form of CD58. In some embodiments, prior to administration of the first dosing cycle to the subject, the subject is selected for treatment with a dosing regimen based on cancer cells showing a CD58 deficiency or mutation. In some embodiments, the CD58 mutation includes a deficiency of a functional mutation.

[0016] In some embodiments, one dose of each dosing cycle is administered to the subject exogenously. In some embodiments, each dose of each dosing cycle is administered to the subject exogenously.

[0017] Disclosed herein is a method for treating cancer, comprising administering to a subject having cancer genetically engineered natural killer (NK) cells that express a chimeric antigen receptor (CAR) directed against an antigen associated with cancer cells or expressed by cancer cells, wherein the cancer cells do not express CD58 or express a mutant form of CD58, and wherein the subject is refractory and / or relapsed to genetically engineered T cells that express a CAR directed against the antigen.

[0018] Disclosed herein is also a method for treating cancer, comprising administering to a subject having cancer that does not express CD58 or expresses a mutant form of CD58 genetically engineered natural killer (NK) cells that express a chimeric antigen receptor (CAR) directed against CD19.

[0019] Disclosed herein is also a method for treating cancer, comprising: (a) identifying a subject having cancer that does not express CD58 or expresses a mutant form of CD58; (b) selecting the identified subject for treatment with genetically engineered natural killer (NK) cells that express a chimeric antigen receptor (CAR) directed against CD19; and (c) administering the genetically engineered NK cells to the selected subject.

[0020] In some embodiments, prior to identifying the subject, the method further comprises determining whether the cancer expresses CD58 or a mutant form of CD58. In some embodiments, the subject has been previously treated with genetically engineered T cells that express a CAR directed against CD19 due to cancer. In some embodiments, the subject is refractory and / or relapsing with genetically engineered T cells. In some embodiments, administering comprises administering to a selected subject at least a first dosing cycle, the first dosing cycle comprising a first dose of genetically engineered NK cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, the first dose being administered to the subject at a first time point, the second dose being administered to the subject between 5 and 10 days after the first time point, and the third dose being administered to the subject between 5 and 10 days after the second dose; each of the first, second, and third doses comprising at least about 1.5×10 9 NK cells.

[0021] Disclosed herein is a method of treating cancer, comprising: (a) selecting a subject for treatment of cancer if the cancer cells do not express CD58 or express a mutant form of CD58; (b) administering to the selected subject at least a first dosing cycle, the first dosing cycle comprising a first dose of genetically engineered natural killer (NK) cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein the first dose is administered to the subject in need of cancer immunotherapy at a first time point, the second dose is administered to the subject between 5 and 10 days after the first time point, and the third dose is administered to the subject between 5 and 10 days after the second dose; each of the first, second, and third doses comprising at least about 1.5×10 9 NK cells, and at least a portion of the genetically engineered NK cells are engineered to express a chimeric antigen receptor (CAR) directed against the CD19 tumor marker. In some embodiments, the first dosing cycle is initiated after the subject has undergone a lymphodepletion process to reduce the number of natural immune cells. In some embodiments, the mutant form of CD58 comprises a loss of function.

[0022] In some embodiments, provided herein is a method of treating cancer, comprising administering to a subject having cancer a lymphodepletion regimen comprising at least two doses of cyclophosphamide and at least two doses of fludarabine, and administering to the subject at least first, second, and third doses of genetically engineered NK cells, wherein the first dose of genetically engineered NK cells is administered to the subject after the final dose of fludarabine, the second dose of genetically engineered NK cells is administered to the subject between 6 and 8 days after the first dose, the third dose of genetically engineered NK cells is administered to the subject between 6 and 8 days after the second dose, and each of the first, second, and third doses comprises about 1.5×10 9 NK cells, and wherein the genetically engineered NK cells are allogeneic to the subject and are engineered to express a chimeric antigen receptor (CAR) that binds to CD19.

[0023] Also provided herein is a method of treating cancer, comprising administering to a subject having cancer a lymphodepletion regimen comprising at least two doses of cyclophosphamide and at least two doses of fludarabine, administering to the subject an agent that binds to CD20, and administering to the subject at least first, second, and third doses of genetically engineered NK cells, wherein the first dose of genetically engineered NK cells is administered to the subject after the final dose of fludarabine, the second dose of genetically engineered NK cells is administered to the subject between 6 and 8 days after the first dose, the third dose of genetically engineered NK cells is administered to the subject between 6 and 8 days after the second dose, and each of the first, second, and third doses comprises at least 1.0×10 9 NK cells, and wherein the genetically engineered NK cells are allogeneic to the subject and are engineered to express a chimeric antigen receptor (CAR) that binds to CD19. In some embodiments, about 1.5×10 9 NK cells are administered at each of the three doses during the cycle.

[0024] Use of a population of engineered NK cells expressing a chimeric antigen receptor targeting CD19 for treating cancer by administration of at least first, second, and third doses of the engineered NK cells, wherein the first dose of engineered NK cells is administered to a subject after a final dose lymphodepletion process comprising at least two doses of cyclophosphamide and at least two doses of fludarabine, the second dose is administered to the subject between 6 and 8 days after the first dose, the third dose is administered to the subject between 6 and 8 days after the second dose, and each of the first, second, and third doses comprises about 1.5×10 9 Also provided is use comprising the engineered NK cells.

[0025] Use of a population of engineered NK cells expressing a chimeric antigen receptor targeting CD19 for treating cancer by administration of at least first, second, and third doses of the engineered NK cells, wherein the first dose of engineered NK cells is administered to a subject after a final dose lymphodepletion process comprising at least two doses of cyclophosphamide and at least two doses of fludarabine, the first dose of engineered NK cells is administered to the subject after administration of an agent that binds to CD20, the second dose is administered to the subject between 6 and 8 days after the first dose, the third dose is administered to the subject between 6 and 8 days after the second dose, and each of the first, second, and third doses comprises at least 1.0×10 9 Also provided is use comprising the engineered NK cells. In some embodiments, each of the first, second, and third doses comprises about 1.5×10 9 engineered NK cells.

[0026] In some embodiments, the NK cells are CAR-expressing NK cells. In some embodiments, each of the first, second, and third doses comprises about 1.5×10 9 CAR-expressing NK cells.

[0027] Also provided herein is the use of genetically engineered natural killer (NK) cells that express a chimeric antigen receptor (CAR) directed against an antigen associated with cancer cells or expressed by cancer cells for treating a subject having cancer, wherein the cancer cells do not express CD58 or express a mutant form of CD58, and the subject relapses and / or is refractory to genetically engineered T cells that express a CAR directed against the antigen. In some embodiments, the antigen is CD19.

[0028] Also provided herein is the use of genetically engineered natural killer (NK) cells that express a chimeric antigen receptor (CAR) directed against CD19 for treating a subject having cancer, wherein the cancer does not express CD58 or expresses a mutant form of CD58.

[0029] Also provided herein is the use of genetically engineered natural killer (NK) cells that express a chimeric antigen receptor (CAR) directed against CD19 for treating a subject having cancer, wherein the subject is selected for treatment based on having cancer that does not express CD58 or expresses a mutant form of CD58.

[0030] In some embodiments, the dosing cycle (e.g., 3 doses of genetically engineered cells) has a duration between about 14 days and about 35 days. In some embodiments, the dosing cycle is about 21 days. In some embodiments, the dosing cycle is about 28 days.

[0031] In some embodiments, the lymphodepletion process comprises at least two doses of cyclophosphamide and at least two doses of fludarabine. In some embodiments, the lymphodepletion process comprises three doses of cyclophosphamide and three doses of fludarabine, wherein the first dose of cyclophosphamide and fludarabine are administered 5 days prior to the start of the administration cycle, the second dose of cyclophosphamide and fludarabine are administered 4 days prior to the start of the administration cycle, and the third dose of cyclophosphamide and fludarabine are administered 3 days prior to the start of the administration cycle. In some embodiments, a break of about 2 days is provided between the third dose of cyclophosphamide and fludarabine and the start of the administration cycle.

[0032] In some embodiments, cyclophosphamide is administered in an amount between about 100 mg / m 2 and about 600 mg / m 2 and fludarabine is administered in an amount between about 10 mg / m 2 and about 60 mg / m 2 In some embodiments, cyclophosphamide is administered in an amount between about 200 mg / m 2 and about 600 mg / m 2 and fludarabine is administered in an amount between about 20 mg / m 2 and about 40 mg / m 2 In some embodiments, cyclophosphamide is administered in an amount of about 300 mg / m 2 In some embodiments, cyclophosphamide is administered in an amount of about 500 mg / m 2 In some embodiments, fludarabine is administered in an amount of about 30 mg / m 2 In some embodiments, cyclophosphamide is administered in an amount of about 300 mg / m 2 and fludarabine is administered in an amount of about 30 mg / m 2 In some embodiments, cyclophosphamide is administered in an amount of about 500 mg / m 2 and fludarabine is administered in an amount of about 30 mg / m 2

[0033] ​In some embodiments, the first and second doses of genetically engineered NK cells are administered to a subject before the subject's natural immune cell population recovers from a lymphodepletion process.

[0034] In some embodiments, the provided dosing regimens, methods, and uses are for the treatment of cancer or a tumor. In some embodiments, the provided dosing regimens, methods, and uses are for the treatment of cancer. In some embodiments, the provided dosing regimens, methods, and uses are for the treatment of blood cancers. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the cancer is a B cell cancer. In some embodiments, the cancer is large cell type B cell lymphoma (LBCL). In some embodiments, the cancer is aggressive LBCL. In some embodiments, the cancer is non-Hodgkin lymphoma (NHL). In some embodiments, the cancer is diffuse large cell type B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), or B cell acute lymphoblastic leukemia (B-ALL). In some embodiments, the cancer is diffuse large cell type B cell lymphoma (DLBCL). In some embodiments, the cancer is follicular lymphoma (FL). In some embodiments, the FL is high grade FL (e.g., FL grade 3b). In some embodiments, the cancer is indolent lymphoma (IL). In some embodiments, the FL is grade 1, 2, or 3a FL. In some embodiments, the cancer is marginal zone lymphoma (MZL). In some embodiments, the cancer is mantle cell lymphoma (MCL). In some embodiments, the cancer is B cell acute lymphoblastic leukemia (B-ALL). In some embodiments, the cancer is Waldenström macroglobulinemia (WM). In some embodiments, the cancer is chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). In some embodiments, the cancer is CLL. In some embodiments, the cancer is SLL. In some embodiments, the cancer is primary mediastinal large cell type B cell lymphoma (PMBCL).

[0035] In some embodiments, the cancer is relapsed / refractory (R / R). In some embodiments, the cancer is R / R NHL. In some embodiments, the cancer is R / R B-ALL. In some embodiments, the cancer is R / R LBCL. In some embodiments, the cancer is R / R aggressive LBCL. In some embodiments, the cancer is R / R MCL. In some embodiments, the cancer is R / R IL. In some embodiments, the cancer is R / R WM. In some embodiments, the cancer is R / R CLL. In some embodiments, the cancer is R / R SLL. In some embodiments, the subject has less than or equal to 5% peripheral blood blasts. In some embodiments, the subject has less than 5% peripheral blood blasts. In some embodiments, at the time of administration of lymphodepleting therapy, the subject has less than or equal to 5% peripheral blood blasts. In some embodiments, at the time of administration of the first dose administration cycle, the subject has less than or equal to 5% peripheral blood blasts. In some embodiments, at the time of administration of lymphodepleting therapy, the subject has less than 5% peripheral blood blasts. In some embodiments, at the time of administration of the first dose administration cycle, the subject has less than 5% peripheral blood blasts. In some embodiments, the subject has no evidence of extramedullary disease. In some embodiments, the subject has no other evidence of extramedullary disease. In some embodiments, the subject has no evidence of extramedullary disease, including lymphoblastic lymphoma. In some embodiments, the subject has no other evidence of extramedullary disease, including lymphoblastic lymphoma.

[0036] In some embodiments, the subject to be treated received prior treatment of at least 1 line but not more than 7 lines. In some embodiments, the subject received prior treatment of at least 1 line but not more than 4 lines. In some embodiments, the subject received prior treatment of at least 1 line. In some embodiments, the subject received prior treatment of at least 2 lines. In some embodiments, the subject received prior treatment of at least 3 lines. In some embodiments, the subject received prior treatment of at least 4 lines. In some embodiments, the subject received prior treatment of 1 line. In some embodiments, the subject received prior treatment of 2 lines. In some embodiments, the subject received prior treatment of 3 lines. In some embodiments, the subject received prior treatment of 4 lines.

[0037] In some embodiments, the subject to be treated received prior treatment lines. In some embodiments, the prior treatment lines include treatment with 1 prior line. In some embodiments, the prior treatment lines include treatment with 2 prior lines. In some embodiments, the prior treatment lines include treatment with 3 prior lines. In some embodiments, the prior treatment lines include treatment with 4 prior lines.

[0038] In some embodiments, the prior treatment lines include anti-CD20 monoclonal antibody and cytotoxic chemotherapy. In some embodiments, the cytotoxic treatment is anthracycline. The prior treatment lines include an inhibitor of Bruton's tyrosine kinase (BTKi). In some embodiments, the BTKi is ibrutinib. In some embodiments, the prior treatment lines include an inhibitor of Bcl-2. In some embodiments, the Bcl-2 inhibitor is venetoclax.

[0039] In some embodiments, the line of prior treatment includes chimeric antigen receptor (CAR) T cells. In some embodiments, the subject has been exposed to CAR T cells. In some embodiments, the CAR T cells are autologous CAR T cells. In some embodiments, the subject has been exposed to autologous CAR T cells. In some embodiments, the CAR T cells target CD19. In some embodiments, the line of prior treatment includes autologous anti-CD19 CAR T cells. In some embodiments, the line of prior treatment does not include CAR T cells. In some embodiments, the subject is CAR T naive. In some embodiments, the subject is autologous CAR T naive.

[0040] In some embodiments, the first, second, and third doses of genetically engineered NK cells are administered to the subject within about 21 days of the first time point (e.g., the first dose). In some embodiments, the first, second, and third doses of genetically engineered NK cells are administered to the subject within about 14 days after the first time point (e.g., the first dose).

[0041] In some embodiments, the CAR comprises (a) an antigen-binding portion that targets CD19; (b) a transmembrane domain; and (c) an intracellular signaling domain comprising an OX40 domain and a CD3 zeta domain. In some embodiments, the antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), and VH comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively; and VL comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NOs: 48, 49, and 16, respectively. In some embodiments, the antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), and VH comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 23, and 24, respectively; and VL comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively. In some embodiments, the antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL), and VH comprises the amino acid sequence set forth in SEQ ID NO: 21 and / or VL comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the antigen-binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 6.

[0042] In some embodiments, the genetically engineered NK cells express a chimeric receptor encoded by a polynucleotide having at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity with SEQ ID NO: 3. In some embodiments, the genetically engineered NK cells express a chimeric receptor having at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity with SEQ ID NO: 4. In some embodiments, the genetically engineered NK cells express a chimeric receptor comprising the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the genetically engineered NK cells express a chimeric receptor having at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity with SEQ ID NO: 43. In some embodiments, the genetically engineered NK cells express a chimeric receptor comprising the amino acid sequence set forth in SEQ ID NO: 43. In some embodiments, the genetically engineered NK cells are also engineered to express membrane-bound interleukin 15 (mbIL15). In some embodiments, mbIL15 has at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity with SEQ ID NO: 40. In some embodiments, mbIL15 comprises the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, mbIL15 has at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity with SEQ ID NO: 44. In some embodiments, mbIL15 comprises the amino acid sequence set forth in SEQ ID NO: 44.

[0043] In some embodiments, the dosing regimens, methods, and uses do not result in cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) / neurotoxicity, and / or graft-versus-host disease. In some embodiments, the dosing regimens, methods, and uses do not result in cytokine release syndrome. In some embodiments, the dosing regimens, methods, and uses do not result in immune effector cell-associated neurotoxicity syndrome (ICANS) / neurotoxicity. In some embodiments, the dosing regimens, methods, and uses do not result in graft-versus-host disease.

[0044] In some embodiments, the genetically engineered NK cells are allogeneic with respect to the subject.

[0045] In some embodiments, the subject is human. In some embodiments, the subject is at least 18 years old.

[0046] In some embodiments, the subject has a 158V / 158V CD16 genotype. In some embodiments, the subject has a 158F / 158F CD16 genotype.

[0047] In some embodiments, the subject has a Performance Status (PS) of the Eastern Cooperative Oncology Group (ECOG) that is lower than or equal to 1. In some embodiments, the subject has an ECOG PS that is lower than or equal to 2. In some embodiments, the subject has an ECOG status of 0. In some embodiments, the subject has an ECOG status of 1. In some embodiments, the subject has an ECOG status of 2.

[0048] In some embodiments, if the subject shows a clinical response after the first dosing cycle, the subject is administered additional dosing cycles. In some embodiments, if the subject shows a complete response (CR) after the first dosing cycle, the subject is administered additional dosing cycles. In some embodiments, if the subject shows a clinical response after a dosing cycle and subsequently shows disease progression, the subject is administered additional dosing cycles. In some embodiments, the subject is administered between 1 and 5 dosing cycles. In some embodiments, the subject is administered 1 dosing cycle. In some embodiments, the subject is administered 2 dosing cycles. In some embodiments, the subject is administered 3 dosing cycles. In some embodiments, the subject is administered 4 dosing cycles. In some embodiments, the subject is administered 5 dosing cycles. In some embodiments, the subject undergoes a lymphodepletion process prior to each dosing cycle.

[0049] In some embodiments, cancer cells do not express CD58 or express a mutant form of CD58. In some embodiments, cancer cells do not express CD58. In some embodiments, cancer cells express a mutant form of CD58. In some embodiments, prior to administration of the first dosing cycle to a subject, it is determined that cancer cells do not express CD58 or express a mutant form of CD58. In some embodiments, prior to administration of the first dosing cycle to a subject, the subject is selected for treatment with a dosing regimen based on cancer cells that exhibit a CD58 deficiency or mutation. In some embodiments, the CD58 mutation comprises a loss of functional mutation. In some embodiments, the CD58 mutation is a loss of functional mutation.

[0050] In some embodiments, a single dose of genetically engineered NK cells is administered to a subject exogenously. In some embodiments, each dose of genetically engineered NK cells is administered to a subject exogenously. BRIEF DESCRIPTION OF THE DRAWINGS

[0051]

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[0052] Some embodiments of the methods and compositions provided herein relate to engineered immune cells and their combinations for use in immunotherapy. In some embodiments, the engineered cells are engineered in multiple ways, for example, expressing a chimeric antigen receptor (CAR) that targets a tumor antigen.

[0053] The term "anticancer effect" refers to a biological effect that can be achieved by various means, including, but not limited to, reduction of tumor volume, reduction of cancer cell number, reduction of metastasis number, increase of average life span, reduction of cancer cell proliferation, reduction of cancer cell survival, and / or improvement of various physiological symptoms associated with the cancer state.

[0054] Cell type Some embodiments of the methods and compositions provided herein relate to cells such as immune cells. For example, immune cells such as NK cells or T cells can be engineered to express a tumor-targeting CAR.

[0055] In contrast to traditional anticancer treatments such as surgical approaches, radiation therapy, chemotherapy, or combinations of these methods, targeting therapy is a cancer treatment that uses specific drugs that target specific genes or proteins found in cancer cells or cells that support cancer growth (such as vascular cells) to reduce or stop cancer cell growth. More recently, genetic engineering has enabled the development of approaches that utilize certain aspects of the immune system to fight cancer. In some embodiments, the patient's own immune cells, donor immune cells, or cells derived from pluripotent cells are modified to specifically eradicate the patient's cancer type. As described in more detail below, various types of immune cells, such as T cells, natural killer (NK) cells, or combinations thereof, can be used.

[0056] To promote cancer immunotherapy, the present specification also provides polynucleotides, polypeptides, and vectors encoding chimeric antigen receptors (CARs) comprising a target-binding moiety (e.g., the extracellular binding moiety of a ligand expressed by cancer cells) operably linked to a cytotoxic signaling complex. For example, some embodiments include polynucleotides, polypeptides, or vectors encoding extracellular domains directed against, for example, tumor markers, such as CD19, which promote targeting of immune cells to CD19-expressing cancers and exerting a cytotoxic effect on cancer cells. Engineered immune cells (e.g., NK cells and / or T cells) expressing such CARs are also provided. Compositions (e.g., pharmaceutical compositions) comprising engineered immune cells (e.g., NK cells) expressing such CARs are also provided. Methods of treating cancer and other uses of such cells for cancer immunotherapy are also provided herein.

[0057] Cells engineered for immunotherapy In some embodiments, cells of the immune system are engineered to have an enhanced cytotoxic effect against target cells such as tumor cells. For example, cells of the immune system can be engineered to include a tumor-directed chimeric receptor and / or a tumor-directed CAR as described herein. In some embodiments, white blood cells or leukocytes are used because their natural function is to defend the body against abnormal cell growth and infectious diseases. There are various types of white blood cells that contribute to specific roles in the human immune system, and thus there are preferred starting points for engineering the cells disclosed herein. White blood cells include granulocytes and agranulocytes (presence or absence of granules in the cytoplasm, respectively). Granulocytes include basophils, eosinophils, neutrophils, and mast cells. Agranulocytes include lymphocytes and monocytes. Cells such as those listed above or according to or otherwise described herein can be engineered to express a chimeric antigen receptor, for example, by providing the cell with a nucleic acid encoding the CAR. In some embodiments, the cells may be engineered to co-express a membrane-bound interleukin-15 (mbIL15) domain. Thus, in some embodiments, the cells are engineered to express a CAR and a membrane-bound interleukin-15 (mbIL15) domain. As discussed in more detail below, in some embodiments, the therapeutic cells are further genetically modified to enhance cytotoxicity and / or cell persistence. In some embodiments, the genetic modification enhances the ability of the cells to resist signals released from the tumor microenvironment that can cause a decrease in the effectiveness or a shortening of the lifespan of the therapeutic cells.

[0058] Monocytes for immunotherapy Monocytes are a subtype of white blood cells. Monocytes can differentiate into macrophages and myeloid dendritic cells. Monocytes are associated with the adaptive immune system and contribute to the main functions of phagocytosis, antigen presentation, and cytokine production. Phagocytosis is a process of taking in cellular material or entire cells and subsequently digesting and destroying the ingested cellular material. In some embodiments, monocytes are used in combination with one or more further engineered cells, as disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to monocytes engineered to express a chimeric antigen receptor (CAR) that targets a ligand on tumor cells, such as CD19, and optionally, a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, monocytes are engineered to express a CAR that targets CD19. In some embodiments, monocytes are engineered to express a CAR that targets CD19 and a membrane-bound interleukin 15 (mbIL15) domain.

[0059] Lymphocytes for Immunotherapy Lymphocytes, another major subtype of leukemia, include T cells (cell-mediated, cytotoxic adaptive immunity), natural killer cells (cell-mediated, cytotoxic innate immunity), and B cells (humoral, antibody-driven adaptive immunity). B cells are engineered according to some embodiments disclosed herein, but some embodiments also relate to engineered T cells or engineered NK cells (in some embodiments, a mixture of T cells and NK cells, either from the same donor or different donors, is used). Some embodiments of the methods and compositions disclosed herein relate to lymphocytes engineered to express a chimeric antigen receptor (CAR) that targets a ligand on tumor cells, such as CD19, and optionally, a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, lymphocytes are engineered to express a CAR that targets CD19. In some embodiments, lymphocytes are engineered to express a CAR that targets CD19 and a membrane-bound interleukin 15 (mbIL15) domain.

[0060] T Cells for Immunotherapy T cells are distinguished from other lymphocyte subtypes (e.g., B cells or NK cells) based on the presence of T cell receptors on their cell surface. T cells can be divided into various different subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, mucosal-associated invariant T cells, and gamma-delta T cells. In some embodiments, a particular subtype of T cell is manipulated. In some embodiments, a mixed pool of T cell subtypes is manipulated. In some embodiments, there is no specific selection of the type of T cell to be manipulated to express the cytotoxic receptor complex disclosed herein. In some embodiments, specific techniques, such as the use of cytokine stimulation, are used to enhance the proliferation / recruitment of T cells based on a particular marker profile. For example, in some embodiments, the activation of certain human T cells, e.g., CD4+ T cells, CD8+ T cells, is achieved by the use of CD3 and / or CD28 as stimulatory molecules. In some embodiments, provided are methods of treating or preventing cancer or infectious diseases, including administering a therapeutically effective amount of T cells that express a cytotoxic receptor complex and / or a homing moiety as described herein. In some embodiments, the manipulated T cells are autologous cells, but in some embodiments, the T cells are allogeneic cells. Some embodiments of the methods and compositions disclosed herein are manipulated to express a chimeric antigen receptor (CAR) that targets a ligand on tumor cells, e.g., CD19, and optionally, a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, the T cells are manipulated to express a CAR that targets CD19. In some embodiments, the T cells are manipulated to express a CAR that targets CD19 and a membrane-bound interleukin 15 (mbIL15) domain.

[0061] NK Cells for Immunotherapy In some embodiments, provided is a method of treating or preventing cancer or an infectious disease, comprising administering a therapeutically effective amount of natural killer (NK) cells that express a cytotoxic receptor complex and / or a homing moiety, as described herein. In some embodiments, the engineered NK cells are autologous cells, while in some embodiments, the NK cells are allogeneic cells. In some embodiments, the engineered NK cells are autologous cells. In some embodiments, the engineered NK cells are allogeneic cells. In some embodiments, NK cells are preferred because of their relatively high natural cytotoxic ability. In some embodiments, it is unexpectedly beneficial that the engineered cells disclosed herein further upregulate the cytotoxic activity of NK cells and can provide even more effective activity against target cells (e.g., tumors or other diseased cells). Some embodiments of the methods and compositions disclosed herein relate to NK cells engineered to express a chimeric antigen receptor (CAR) that targets a ligand on tumor cells, such as CD19, and optionally, a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, immortalized NK cells are used and are engineered as disclosed herein. In some embodiments, primary NK cells are used and are engineered as disclosed herein. In some embodiments, the NK cells are derived from the cell line NK-92. NK-92 cells are derived from NK cells but lack the major inhibitory receptors presented by normal NK cells while retaining most of the activating receptors. Some embodiments of the NK-92 cells described herein relate to NK92 cells engineered to silence a particular additional inhibitory receptor, such as SMAD3, that enables upregulation of interferon-γ (IFNγ), granzyme B, and / or perforin production. Further information regarding the NK-92 cell line is disclosed in WO1998 / 49268 and US Patent Application Publication No. 2002 / 0068044, which are hereby incorporated by reference in their entirety. In some embodiments, NK-92 cells are used in combination with one or more other cell types disclosed herein.For example, in one embodiment, NK-92 cells are used in combination with NK cells as disclosed herein. In further embodiments, NK-92 cells are used in combination with T cells as disclosed herein. In some embodiments, NK cells are engineered to express a CAR targeting CD19. In some embodiments, NK cells are engineered to express a CAR targeting CD19 and the membrane-bound interleukin 15 (mbIL15) domain.

[0062] Hematopoietic stem cells for cancer immunotherapy In some embodiments, hematopoietic stem cells (HSCs) are used in the methods of immunotherapy disclosed herein. In some embodiments, the cells are engineered to express a homing moiety and / or a cytotoxic receptor complex. In some embodiments, HSCs are used to take advantage of their ability to engraft for long-term blood cell production, which can provide a persistent source of targeted anti-cancer effector cells, for example, effective in cancer remission. In some embodiments, this persistent production, for example, is assisted by the tumor microenvironment to abrogate anergy or depletion of other cell types. In some embodiments, allogeneic HSCs are used, while in some embodiments, autologous HSCs are used. In some embodiments, HSCs are used in combination with one or more of the further engineered cell types disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to hematopoietic stem cells engineered to express a CAR targeting a ligand on tumor cells, such as CD19, and optionally, the membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, HSCs are engineered to express a CAR targeting CD19. In some embodiments, HSCs are engineered to express a CAR targeting CD19 and the membrane-bound interleukin 15 (mbIL15) domain.

[0063] Induced pluripotent stem cells In some embodiments, NK, T, or other immune cells derived from pluripotent stem cells (iPSCs) are used in the methods of immunotherapy disclosed herein. In some embodiments, induced pluripotent stem cells (iPSCs) are used in the methods of immunotherapy disclosed herein. In some embodiments, iPSCs are used, and, without limitation, the differentiating iPSCs are utilized for their ability to differentiate and drive into non-pluripotent cells, including CD34 cells, hematopoietic endothelial cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell precursors, NK cell precursors, T cells, NKT cells, NK cells, and B cells, or less differentiated cells containing the same genetic modifications at the same selected location, which contain one or several genetic modifications at the selected location. In some embodiments, iPSCs are used to generate iPSC-derived NK or T cells. In some embodiments, the cells are engineered to express a homing moiety and / or a cytotoxic receptor complex. In some embodiments, iPSCs are used in combination with one or more of the further engineered cell types disclosed herein. Some embodiments of the methods and compositions disclosed herein relate to induced pluripotent stem cell-derived NK, T, or other immune cells engineered to express a chimeric antigen receptor (CAR) that targets a ligand on tumor cells, such as CD19, and optionally, a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, iPSCs are engineered to express a CAR that targets a ligand on tumor cells, such as CD19, and optionally, a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, iPSCs are engineered to express a CAR that targets CD19. In some embodiments, iPSCs are engineered to express a CAR that targets CD19 and a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, the engineered iPSCs are differentiated into NK, T, or other immune cells, such as for use in the compositions or methods provided herein.

[0064] Chimeric antigen receptor Some embodiments of the compositions and methods described herein relate to chimeric receptors comprising an extracellular domain that includes a tumor-binding domain (also referred to as an antigen-binding protein or antigen-binding domain) as described herein. Depending on the embodiment, the tumor-binding domain targets, for example, CD19. In some embodiments, the tumor-binding domain binds to CD19. In some embodiments, the tumor-binding domain binds to human CD19. CAR constructs according to some embodiments are schematically illustrated in FIGS. 1A-1B. FIG. 1A shows a non-limiting CAR construct (e.g., amino acid or nucleic acid) that includes a CD19-binding domain, a CD8 alpha hinge and transmembrane domain, an OX40 co-stimulatory domain, and a CD3 zeta signaling domain. FIG. 1B shows a non-limiting nucleic acid construct described in some embodiments, where membrane-bound interleukin 15 (mbIL15) is bicistronically encoded by a nucleic acid sequence encoding the CAR. In some embodiments, mbIL15 is bicistronically encoded by a bicistronic element between the nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding mbIL15. It should be understood that translation of the nucleic acid of FIG. 1B results in the CAR construct shown in FIG. 1A, together with mbIL15 expressed separately on the cell. In some embodiments, mbIL15 is provided on a separate nucleic acid. In some embodiments, the CARs disclosed herein have the general structure of an extracellular antigen-binding protein (targeting a cancer antigen such as CD19), one or both of a hinge and transmembrane domain, optionally a co-stimulatory domain, and a signaling domain.

[0065] Antigen-binding protein In some embodiments, an antigen-binding protein is provided. As used herein, the term "antigen-binding protein" refers to its ordinary meaning and also refers to an antigen-binding fragment that binds to an antigen and, optionally, a protein that includes a scaffold or framework portion that allows the antigen-binding fragment to adopt a structure that facilitates the binding of the antigen-binding protein to the antigen. In some embodiments, the antigen is a cancer antigen or a fragment thereof. In some embodiments, the antigen-binding fragment includes at least one CDR derived from an antibody that binds to the antigen. In some embodiments, the antigen-binding fragment includes all three CDRs derived from the heavy chain of an antibody that binds to the antigen or all three CDRs derived from the light chain of an antibody that binds to the antigen. In still some embodiments, the antigen-binding fragment includes all six CDRs derived from an antibody that binds to the antigen (three from the heavy chain and three from the light chain). In some embodiments, the antigen-binding fragment includes one, two, three, four, five, or six CDRs derived from an antibody that binds to the antigen, and in some embodiments, the CDRs can be any combination of heavy chain and / or light chain CDRs. In some embodiments, the antigen-binding fragment is an antibody fragment.

[0066] Non-limiting examples of antigen-binding proteins include antibodies, antibody fragments (e.g., antigen-binding fragments of antibodies), antibody derivatives, and antibody mimetics. Further specific examples include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., VH domains of camelid heavy-chain antibodies; VHH fragments), Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments. These molecules can be of any mammalian origin, e.g., human, mouse, rat, rabbit, or pig, dog, or camel. Antibody fragments can compete with intact (e.g., native) antibodies for binding to the target antigen and the fragments can be produced by modification (e.g., enzymatic or chemical cleavage) of intact antibodies or can be synthesized de novo using recombinant DNA technology or peptide synthesis. Antigen-binding proteins can include, for example, alternative protein scaffolds or artificial scaffolds having linked CDRs or CDR derivatives. Such scaffolds include, but are not limited to, antibody-derived scaffolds that stabilize the overall three-dimensional structure of the antigen-binding protein and that include, for example, mutations introduced to stabilize synthetic scaffolds including biocompatible polymers. Further, peptide antibody mimetics (“PAMs”), as well as scaffolds based on antibody mimetics that utilize fibronectin components as scaffolds, can be used.

[0067] In some embodiments, the antigen-binding protein comprises one or more antibody fragments incorporated into a single polypeptide chain or multiple polypeptide chains. For example, the antigen-binding protein can include, but is not limited to, diabodies; intrabodies; domain antibodies (a single VL or VH domain or two or more VH domains connected by a peptide linker); maxibodies (two scFvs fused to the Fc region); tribodies; tetrabodies; minibodies (scFvs fused to the CH3 domain); peptibodies (one or more peptides attached to the Fc region); linear antibodies (a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with a complementary light chain polypeptide); small modular immunopharmaceuticals; and immunoglobulin fusion proteins (e.g., IgG-scFv, IgG-Fab, 2scFv-IgG, 4scFv-IgG, VH-IgG, IgG-VH, and Fab-scFv-Fc).

[0068] In some embodiments, the antigen-binding protein has the structure of an immunoglobulin. As used herein, the term "immunoglobulin" has its ordinary meaning and also refers to a trimeric molecule having each trimer comprising two identical pairs of polypeptide chains, one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids that is mainly responsible for antigen recognition. The carboxyl-terminal portion of each chain defines a constant region that is mainly responsible for effector functions.

[0069] Within the light and heavy chains, the variable (V) and constant regions I are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region that is about 10 amino acids longer. The variable regions of each light / heavy chain pair form an antibody binding site such that the intact immunoglobulin has two binding sites.

[0070] Immunoglobulin chains exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also known as complementarity-determining regions or CDRs. From the N-terminus to the C-terminus, both light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, and FR4.

[0071] Human light chains are classified as kappa and lambda light chains. The antibody "light chain" refers to the smaller of the two types of polypeptide chains present in the antibody molecule in their native structure. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes. A light chain can contain a polypeptide that includes a single immunoglobulin light chain variable region (VL) and a single immunoglobulin light chain constant domain (CL) from the amino terminus to the carboxyl terminus.

[0072] Heavy chains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), which define the isotypes of antibodies as IgM, IgD, IgG, IgA, and IgE, respectively. The antibody "heavy chain" refers to the larger of the two types of polypeptide chains present in the antibody molecule in their native structure and usually determines the class to which the antibody belongs. A heavy chain can contain a polypeptide that includes a single immunoglobulin heavy chain variable region (VH), an immunoglobulin heavy chain constant domain 1 (CH1), an immunoglobulin hinge region, an immunoglobulin heavy chain constant domain 2 (CH2), an immunoglobulin heavy chain constant domain (CH3), and, optionally, an immunoglobulin heavy chain constant domain 4 (CH4) from the amino terminus to the carboxyl terminus.

[0073] The IgG class is further divided into subclasses, namely, IgG1, IgG2, IgG3, and IgG4. The IgA class is further divided into subclasses, namely, IgA1 and IgA2. IgM has subclasses including, but not limited to, IgM1 and IgM2. The heavy chains of IgG, IgA, and IgD antibodies have three domains (CH1, CH2, and CH3), while the heavy chains of IgM and IgE antibodies have four domains (CH1, CH2, CH3, and CH4). The immunoglobulin heavy chain constant domains can be from any immunoglobulin isotype, including subtypes. Antibody chains are linked together via inter-polypeptide disulfide bonds between the CL and CH1 domains (e.g., between the light and heavy chains) and between the hinge regions of the antibody heavy chains.

[0074] In some embodiments, the antigen-binding protein is an antibody. As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be monoclonal, or polyclonal, multi-chain or single-chain, or intact immunoglobulins, and can be of natural or recombinant origin. Antibodies can be trimers of immunoglobulin molecules. Antibodies can be "humanized," "chimeric," or non-human. Antibodies can include intact immunoglobulins of any isotype, for example, chimeric, humanized, human, and bispecific antibodies. Intact antibodies typically include at least two full-length heavy chains and two full-length light chains. Antibody sequences can be derived from a single species alone or can be "chimeric," i.e., different portions of the antibody can be from two different primary sources as further described below. Unless otherwise indicated, the term "antibody" includes antibodies that contain two substantially full-length heavy chains and two substantially full-length light chains if the antibody retains the same or similar binding and / or function as an antibody that contains two full-length light chains and heavy chains. For example, antibodies having 1, 2, 3, 4, or 5 amino acid residue substitutions, insertions, or deletions at the N-terminus and / or C-terminus of the heavy chain and / or light chain are included in the definition so long as the antibody retains the same or similar binding and / or function as an antibody that contains two full-length heavy chains and two full-length light chains. Examples of antibodies include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, and synthetic antibodies. In some embodiments, monoclonal and polyclonal antibodies are provided. As used herein, the term "polyclonal antibody" refers to its ordinary meaning and typically also refers to a population of antibodies with widely different compositions and binding specificities. As used herein, the term "monoclonal antibody ("mAb")" refers to its ordinary meaning and also refers to one or more populations of antibodies having the same sequence. Monoclonal antibodies bind to an antigen at a specific epitope on the antigen.

[0075] In some embodiments, the antigen-binding protein is an antibody fragment or antigen-binding fragment. The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CHI domains, linear antibodies, single-domain antibodies such as sdAb (either vL or vH), camelid vHH domains, bispecific antibodies formed from antibody fragments such as a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region, and isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetra-bodies, v-NAR, and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies). Antibody fragments can include Fab, Fab’, F(ab’)2, and / or Fv fragments containing at least one CDR of an immunoglobulin sufficient to confer specific antigen binding to a cancer antigen (e.g., CD19). Antibody fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.

[0076] In some embodiments, a Fab fragment is provided. A Fab fragment is a monovalent fragment having VL, VH, CL, and CH1 domains; an F(ab’)2 fragment is a divalent fragment having two Fab fragments linked by a disulfide bond in the hinge region; an Fd fragment has VH and CH1 domains; an Fv fragment has the VL and VH domains of a single arm of an antibody; and a dAb fragment has a VH domain, a VL domain, or an antigen-binding fragment of a VH or VL domain. In some embodiments, these antibody fragments can be incorporated into single-domain antibodies, single-chain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs. In some embodiments, an antibody comprises at least one CDR as described herein.

[0077] In some embodiments, single-chain variable fragments are also provided herein. As used herein, the term “single-chain variable fragment” (“scFv”) refers to its ordinary meaning, in which the VL and VH regions are connected via a linker (e.g., a synthetic sequence of amino acid residues), and the linker forms a continuous protein chain long enough for the protein chain to fold back on itself to form a monovalent antigen-binding site, and also refers to a fusion protein. For clarity, unless otherwise indicated as such, “single-chain variable fragment” is not an antibody or antibody fragment as defined herein. A diabody is a divalent antibody comprising two polypeptide chains, each polypeptide chain comprising a VH and a VL domain connected by a linker configured to reduce or prevent pairing between the two domains on the same chain, thus allowing each domain to pair with a complementary domain on a different polypeptide chain. In some embodiments, when the two polypeptide chains of a diabody are identical, then the diabody resulting from their pairing has two identical antigen-binding sites. Polypeptide chains having different sequences can be used to create a diabody having two different antigen-binding sites. Similarly, a triabody and a tetrabody are antibodies comprising three or four polypeptide chains, respectively, and forming three and four antigen-binding sites, respectively, which can be the same or different.

[0078] In some embodiments, the antigen-binding protein comprises one or more CDRs. As used herein, the term "CDR" refers to its ordinary meaning and also refers to the complementarity-determining regions (also referred to as "minimal recognition units" or "hypervariable regions") within an antibody variable sequence. CDRs enable the antigen-binding protein to specifically bind to a particular antigen of interest. There are three heavy-chain variable region CDRs (CDR-H1, CDR-H2, and CDR-H3) and three light-chain variable region CDRs (CDR-L1, CDR-L2, and CDR-L3). Each CDR of the two chains is typically aligned in a framework region and forms a structure that specifically binds to a particular epitope or domain on the target protein. From the N-terminus to the C-terminus, both the naturally occurring light-chain and heavy-chain variable regions typically constitute these elements in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, FW4. In the heavy-chain variable region, the order is typically: from the N-terminus to the C-terminus, FW-H1, CDR-H1, FW-H2, CDR-H2, FW-H3, CDR-H3, and FW-H4. In the light-chain variable region, the order is typically: from the N-terminus to the C-terminus, FW-L1, CDR-L1, FW-L2, CDR-L2, FW-L3, CDR-L3, and FW-L4. A numbering system was devised to assign numbers to the amino acids occupying positions within each of these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, MD), or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883. The complementarity-determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using this system.Other numbering systems for the amino acids of the immunoglobulin chains include IMGT® (the international ImMunoGeneTics information system; Lefranc et al, Dev. Comp. Immunol. 29:185-203; 2005) and Aho (Honegger and Pluckthun, J. Mol. Biol. 309(3):657-670; 2001). The binding domains disclosed herein may utilize the CDRs defined by any of these systems. In any given embodiment containing more than one CDR, the CDRs may be defined according to any of Kabat, Chothia, extended, IMGT, Paratome, AbM, and / or structure definition, or any combination of the foregoing. Any CDR may be interpreted by one of ordinary skill in the art under any of these numbering systems as being appropriate, either separately from or within the context of the variable domain. One or more CDRs are incorporated into the molecule by covalent or non-covalent bonds to make it an antigen-binding protein.

[0079] In some embodiments, the various components of the antigen-binding portion are separated by a linker, such as a G4S linker. In some embodiments, the G4S linker comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 26. In some embodiments, the G4S linker is encoded by a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 25. In some embodiments, a (G n S) x linker is used (wherein "n" represents the number of glycine residues following serine and "X" represents the G in the linker n(representing the number of S repeats). Such linkers may be used elsewhere in the CAR and, for example, provide the ability to separate various component parts of the receptor complex along the polynucleotide that may enhance the expression, stability, and / or functionality of the receptor complex.)

[0080] In some embodiments, the antigen-binding portion comprises a binding portion that targets CD19. In some embodiments, the antigen-binding portion binds to CD19. In some embodiments, the antigen-binding portion binds to human CD19. In some embodiments, the anti-CD19 binding portion comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions (CDRs) 1, 2, and 3 (HCDR1, HCDR2, and HCDR3), and a light chain variable region (VL) comprising light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, and LCDR3).

[0081] In some embodiments, HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 22. In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 23. In some embodiments, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 24. In some embodiments, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively. In some embodiments, VH comprises HCDR1, HCDR2, and HCDR3, each of which comprises the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively. In some embodiments, VH comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 21. In some embodiments, VH comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, HCDR1 is encoded by a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 18. In some embodiments, HCDR2 is encoded by a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 19. In some embodiments, HCDR3 is encoded by a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 20.In some embodiments, VH is encoded by a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 17.

[0082] In some embodiments, LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 14. In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15. In some embodiments, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16. In some embodiments, LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively. In some embodiments, VL comprises LCDR1, LCDR2, and LCDR3, each of which comprises the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively. In some embodiments, VL comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13. In some embodiments, VL comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, LCDR1 is encoded by a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 10. In some embodiments, LCDR2 is encoded by a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 11. In some embodiments, LCDR3 is encoded by a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 12.In some embodiments, VL is encoded by a nucleic acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 9.

[0083] In some embodiments, VL comprises CDR-L1, CDR-L2, and CDR-L3, each comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively; VH comprises CDR-H1, CDR-H2, and CDR-H3, each comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively.

[0084] In some embodiments, LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 48. In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 49. In some embodiments, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16. In some embodiments, LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences set forth in SEQ ID NOs: 48, 49, and 16, respectively. In some embodiments, VL comprises LCDR1, LCDR2, and LCDR3, each of which comprises the amino acid sequences set forth in SEQ ID NOs: 48, 49, and 16, respectively. In some embodiments, VL comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13. In some embodiments, VL comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, VL comprises CDR-L1, CDR-L2, and CDR-L3 of the VL sequence set forth in SEQ ID NO: 13. In some embodiments, HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 45. In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45. In some embodiments, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 46. In some embodiments, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 46.In some embodiments, HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 47. In some embodiments, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively. In some embodiments, VH comprises HCDR1, HCDR2, and HCDR3, each of which comprises the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively. In some embodiments, VH comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 21. In some embodiments, VH comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, VH comprises CDR-H1, CDR-H2, and CDR-H3 of the VH sequence set forth in SEQ ID NO: 21.

[0085] In some embodiments, VL comprises CDR-L1, CDR-L2, and CDR-L3, each of which comprises the amino acid sequences set forth in SEQ ID NOs: 48, 49, and 16, respectively; and VH comprises CDR-H1, CDR-H2, and CDR-H3, each of which comprises the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively.

[0086] In some embodiments, LCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 14. In some embodiments, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, LCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 15. In some embodiments, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, LCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 16. In some embodiments, LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, LCDR1, LCDR2, and LCDR3 each comprise the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively. In some embodiments, VL comprises LCDR1, LCDR2, and LCDR3, each of which comprises the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively. In some embodiments, VL comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13. In some embodiments, VL comprises the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, VL comprises CDR-L1, CDR-L2, and CDR-L3 of the VL sequence set forth in SEQ ID NO: 13. In some embodiments, HCDR1 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 50. In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 50. In some embodiments, HCDR2 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 23. In some embodiments, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 23.In some embodiments, HCDR3 comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 24. In some embodiments, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, HCDR1, HCDR2, and HCDR3 each comprise the amino acid sequences set forth in SEQ ID NOs: 50, 23, and 24, respectively. In some embodiments, VH comprises HCDR1, HCDR2, and HCDR3, each of which comprises the amino acid sequences set forth in SEQ ID NOs: 50, 23, and 24, respectively. In some embodiments, VH comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 21. In some embodiments, VH comprises the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, VH comprises CDR-H1, CDR-H2, and CDR-H3 of the VH sequence set forth in SEQ ID NO: 21.

[0087] In some embodiments, VL comprises CDR-L1, CDR-L2, and CDR-L3, each of which comprises the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively; and VH comprises CDR-H1, CDR-H2, and CDR-H3, each of which comprises the amino acid sequences set forth in SEQ ID NOs: 50, 23, and 24, respectively.

[0088] In some embodiments, the anti-CD19 binding portion comprises a VH comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 21. In some embodiments, the anti-CD19 binding portion comprises a VL comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13. In some embodiments, the anti-CD19 binding portion comprises a VH comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 21 and a VL comprising a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 13. In some embodiments, the anti-CD19 binding portion comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the anti-CD19 binding portion comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the anti-CD19 binding portion comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 21 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0089] Embodiments of the anti-CD19 binding portion include various arrangements of the VH and VL disclosed herein. In some embodiments, the anti-CD19 binding portion is a single-chain variable fragment (scFv). In some embodiments, the VH and VL are separated by a linker. In some embodiments, the linker comprises the sequence of SEQ ID NO: 26. In some embodiments, the linker is encoded by the nucleic acid of SEQ ID NO: 25. In some embodiments, the anti-CD19 binding portion comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 21, a linker comprising the nucleic acid sequence set forth in SEQ ID NO: 26, and a VL comprising the amino acid sequence set forth in SEQ ID NO: 13.

[0090] In some embodiments of the anti-CD19 binding portion, the VH is the N-terminus of the VL. In some embodiments of the anti-CD19 binding portion, the VL is the N-terminus of the VH. In some embodiments, the anti-CD19 binding portion comprises a sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 6. In some embodiments, the anti-CD19 binding portion comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the anti-CD19 binding portion is encoded by a nucleic acid having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 5.

[0091] In some embodiments, the antigen-binding proteins provided herein comprise one or more CDRs as part of a larger polypeptide chain. In some embodiments, the antigen-binding protein covalently attaches one or more CDRs to another polypeptide chain. In some embodiments, the antigen-binding protein incorporates one or more CDRs by non-covalent binding. In some embodiments, the antigen-binding protein may comprise at least one CDR described herein incorporated into a biocompatible framework structure. In some embodiments, the biocompatible framework structure is a structurally stable structural support, or framework, or scaffold that can present one or more sequences of amino acids that bind to an antigen (e.g., CDR, variable region, etc.) in a localized surface region, or a polypeptide or portion thereof that is sufficient to form a scaffold. Such a structure can be a naturally occurring polypeptide or polypeptide “fold” (structural motif), or can have one or more modifications such as additions, deletions and / or substitutions of amino acids compared to a naturally occurring polypeptide or fold. Depending on the embodiment, the scaffold can be derived from polypeptides of various different species (or more than one species), such as human, non-human primate or other mammalian, other vertebrate, invertebrate, plant, bacterial or viral polypeptides.

[0092] Depending on the embodiment, the biocompatible framework structure is based on a protein scaffold or backbone other than the immunoglobulin domain. In some such embodiments, those framework structures are based on fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain, and / or tendamistat domain.

[0093] Transmembrane domain Some embodiments of the compositions and methods described herein relate to tumor-targeting CARs that include a transmembrane domain. Some embodiments include a transmembrane domain derived from NKG2D or another transmembrane protein. In some embodiments in which a transmembrane domain is used, the portion of the transmembrane protein used retains at least a portion of its normal transmembrane domain.

[0094] In some embodiments, however, the transmembrane domain comprises at least a portion of the transmembrane glycoprotein CD8, which is normally expressed in both T cells and NK cells. In some embodiments, the transmembrane domain comprises CD8α. In some embodiments, the transmembrane domain comprises a hinge and a transmembrane region. In some embodiments, the transmembrane domain comprises a CD8α hinge and a CD8α transmembrane region. In some embodiments, the transmembrane domain comprises a "hinge" (e.g., a CD8α hinge). In some embodiments, the "hinge" of CD8α is encoded by a nucleic acid sequence comprising the sequence set forth in SEQ ID NO: 27. In some embodiments, the "hinge" of CD8α is encoded by the nucleic acid sequence of SEQ ID NO: 27. In some embodiments, the CD8α hinge is truncated or modified and encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to CD8α having the sequence of SEQ ID NO: 27. In some embodiments, the "hinge" of CD8α comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, CD8α can be truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO: 28.

[0095] In some embodiments, the transmembrane domain comprises the CD8α transmembrane region. In some embodiments, the CD8α transmembrane region is encoded by the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, the CD8α transmembrane region has the nucleic acid sequence of SEQ ID NO: 29. In some embodiments, the CD8α transmembrane region is truncated or modified and is encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to CD8α having the sequence of SEQ ID NO: 29. In some embodiments, the CD8α transmembrane region comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the CD8α transmembrane region is truncated or modified and has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to CD8α having the sequence of SEQ ID NO: 30. In some embodiments, the transmembrane region comprises a short portion of the intracellular region of the CD8α sequence (e.g., the C-terminal LTC (leucine-threonine-cysteine) motif of SEQ ID NO: 30).

[0096] In some embodiments, the transmembrane domain comprises the CD8α hinge and the CD8α transmembrane region. In some embodiments, the transmembrane domain comprises the amino acid sequences of SEQ ID NOs: 28 and 30. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 51 or 52. In some embodiments, the transmembrane region comprises a short portion of the intracellular region of the CD8α sequence (e.g., the C-terminal LTC (leucine-threonine-cysteine) motif of SEQ ID NO: 30). In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 52. In some embodiments, the motif is absent. In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 51.

[0097] Signal transduction domain Some embodiments of the compositions and methods described herein relate to tumor antigen-directed CARs that include a signaling domain. For example, immune cells engineered according to some embodiments disclosed herein can include at least one subunit (or a fragment thereof) of the CD3 T cell receptor complex. In some embodiments, the signaling domain includes the CD3 zeta subunit. In some embodiments, CD3 zeta is encoded by the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, CD3 zeta can be truncated or modified to be encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to CD3 zeta having the sequence of SEQ ID NO: 33. In some embodiments, the CD3 zeta domain includes the amino acid sequence of SEQ ID NO: 34. In some embodiments, the CD3 zeta domain is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the CD3 zeta domain having the sequence of SEQ ID NO: 34.

[0098] In some embodiments, unexpectedly enhanced signaling is achieved by the use of multiple signaling domains whose activities act synergistically. For example, in some embodiments, the signaling domain further comprises a co-stimulatory intracellular signaling domain. For example, in some embodiments, the signaling domain further comprises an OX40 domain. In some embodiments, the OX40 domain is an intracellular signaling domain. In some embodiments, the OX40 intracellular signaling domain is encoded by the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the OX40 intracellular signaling domain can be truncated or modified to be encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO: 31. In some embodiments, the OX40 intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the OX40 intracellular signaling domain is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the OX40 intracellular signaling domain having the sequence of SEQ ID NO: 32. In some embodiments, OX40 is used as the sole intracellular signaling domain in the construct, but in some embodiments, OX40 can be used with one or more other signaling domains. For example, a combination of OX40 and CD3 zeta is used in some embodiments. Thus, in some embodiments, the CAR comprises an anti-CD19 antibody portion, a CD8a transmembrane domain, a CD3 zeta signaling domain, and an OX40 domain. In some embodiments, the CAR comprises the amino acid sequence set forth in SEQ ID NO: 43.

[0099] In some embodiments, alternative co-stimulatory intracellular signaling domains are incorporated into the signaling domain. By way of further example, one or more of the CD28, OX40, or 4-1BB intracellular signaling domains are used in combination with CD3 zeta. By way of further example, combinations of CD28, OX40, 4-1BB, and / or CD3 zeta are used in some embodiments.

[0100] Stimulatory molecule Some embodiments of the compositions and methods described herein relate to vectors encoding tumor antigen-directed CARs and stimulatory molecules. In addition to CARs that include various transmembrane domains and signaling domains (and combinations of transmembrane / signaling domains), in some embodiments, stimulatory molecules can be provided for expression by immune cells. These can be, for example, certain molecules that further enhance the activity of immune cells. Cytokines can be used in some embodiments. For example, by way of non-limiting example, certain interleukins such as IL-2 and / or IL-15 are used. In some embodiments, immune cells for treatment are engineered to express such molecules in secreted form. In further embodiments, such stimulatory molecules are engineered to be membrane-bound and act as autocrine stimulatory molecules (or even as paracrine stimulators to neighboring cells).

[0101] In some embodiments, the NK cells disclosed herein are engineered to express interleukin 15 (IL15, IL-15). In some embodiments, IL15 is expressed from a separate cassette on a construct comprising any one of the CARs disclosed herein. In some embodiments, IL15 is expressed in the same cassette as any one of the CARs disclosed herein and may be separated by a cleavage site, for example, a proteolytic cleavage site or a T2A, P2A, E2A, or F2A self-cleaving peptide cleavage site. In some embodiments, IL-15 is expressed in the same cassette as any one of the CARs disclosed herein. In some embodiments, IL15 is expressed in the same cassette as any one of the CARs disclosed herein, and IL15 and the CAR are separated by a cleavage site. In some embodiments, the cleavage site is a proteolytic cleavage site. In some embodiments, the cleavage site is a T2A, P2A, E2A, or F2A site. In some embodiments, the cleavage site is T2A. In some embodiments, IL15 is membrane-bound IL15 (mbIL15). In some embodiments, mbIL15 comprises a native IL15 sequence, such as a human native IL15 sequence, and at least one transmembrane domain. In some embodiments, the native IL15 sequence is encoded by a sequence having at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO: 37. In some embodiments, the native IL15 sequence comprises a peptide sequence having at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO: 38. In some embodiments, the native IL15 sequence comprises the amino acid sequence set forth in SEQ ID NO: 38.

[0102] In some embodiments, IL15 is membrane-bound by its coupling to at least one transmembrane domain. In some embodiments, the at least one transmembrane domain includes a CD8 transmembrane domain. In some embodiments, mbIL15 can include additional components such as a leader sequence and / or a hinge sequence. In some embodiments, the leader sequence is a CD8 leader sequence. In some embodiments, the hinge sequence is a CD8 hinge sequence. In some embodiments, mbIL15 includes an amino acid sequence having at least 85%, at least 90%, at least 95% sequence identity with SEQ ID NO: 44. In some embodiments, mbIL15 includes the amino acid sequence set forth in SEQ ID NO: 44.

[0103] In some embodiments, the tumor antigen-directed CAR and / or the tumor ligand-directed chimeric receptor are encoded by a polynucleotide encoding one or more cytosolic protease cleavage sites. Such sites can be recognized and cleaved by cytosolic proteases, resulting in the separation (and separate expression) of the various component parts of the receptor encoded by the polynucleotide. In some embodiments, the tumor antigen-directed CAR and / or the tumor ligand-directed chimeric receptor are encoded by a polynucleotide encoding one or more self-cleaving peptides, such as a T2A cleavage site, a P2A cleavage site, an E2A cleavage site, and / or an F2A cleavage site. As a result, depending on the embodiment, the various components of the engineered cytotoxic receptor complex can be delivered to NK cells or T cells by a single vector or by multiple vectors. Thus, as schematically shown in the figure, the construct can be encoded by a single polynucleotide, but also includes a cleavage site (in some embodiments, as in the case of IL-15) such that elements downstream of the construct are expressed by the cell as a separate protein. In some embodiments, the T2A cleavage site is used. In some embodiments, the T2A cleavage site has the nucleic acid sequence of SEQ ID NO: 35. In some embodiments, the T2A cleavage site is encoded by the nucleic acid sequence of SEQ ID NO: 35. In some embodiments, the T2A cleavage site can be truncated or modified to be encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO: 35. In some embodiments, the T2A cleavage site comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the T2A cleavage site is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the T2A cleavage site having the sequence of SEQ ID NO: 36.

[0104] In some embodiments, NK cells are engineered to express membrane-bound interleukin 15 (mbIL15). In such embodiments, mbIL15 expression on the NK enhances the cytotoxic effect of the engineered NK cells by enhancing the proliferation and / or lifespan of the NK cells. In some embodiments, mbIL15 is encoded by the same polynucleotide as the CAR. In some embodiments, IL15 is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 37 and a sequence encoding a transmembrane domain. In some embodiments, IL15 comprises the amino acid sequence of SEQ ID NO: 38 coupled to the amino acid sequence of the transmembrane domain. In some embodiments, IL15 comprises the amino acid sequence of SEQ ID NO: 38 functionally coupled to the amino acid sequence of the transmembrane domain. In some embodiments, mbIL15 is encoded by a polynucleotide comprising the sequence of SEQ ID NO: 39. In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 44 (e.g., SEQ ID NO: 40). In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, mbIL15 can be truncated or modified to be encoded by a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO: 39. In some embodiments, mbIL15 comprises the amino acid sequence of SEQ ID NO: 44 (e.g., SEQ ID NO: 40). In some embodiments, mbIL15 is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO: 44. In some embodiments, mbIL15 is truncated or modified to have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO: 44 (e.g., SEQ ID NO: 40). The membrane-bound IL15 sequences are also described in PCT applications WO2018 / 183385 and WO2020 / 056045, each of which is hereby incorporated by reference in its entirety.

[0105] Chimeric antigen receptor construct Some embodiments of the compositions and methods described herein relate to chimeric receptors, such as CARs that target (e.g., bind) CD19. Expression of these CARs in immune cells such as genetically modified non-alloreactive T cells and / or NK cells enables targeting and destruction of specific target cells such as cancer cells. Non-limiting examples of such cytotoxic receptor complexes are discussed in more detail below.

[0106] In some embodiments, a polynucleotide (and encoded amino acids) encoding a tumor-binding / hinge transmembrane domain / signal transduction complex is provided. Various component parts can be used according to the embodiments disclosed herein. In some embodiments, the polynucleotide further encodes a further construct or molecule, such as a stimulatory molecule such as IL15. Thus, in some embodiments, the polynucleotide encodes a CAR and IL15 (e.g., membrane-bound IL15). In some embodiments, the polynucleotide thus includes, for example, a sequence encoding a T2A cleavage site. In some embodiments, the sequence encoding the T2A cleavage site is between the sequences encoding the CAR and IL15 (e.g., mbIL15) such that the CAR and IL15 are expressed bicistronically.

[0107] In some embodiments, this CAR complex is encoded by a nucleic acid molecule comprising a sequence obtained from a combination of the sequences disclosed herein, or comprises an amino acid sequence obtained from a combination of the sequences disclosed herein. In some embodiments, the encoding nucleic acid sequence, or amino acid sequence, comprises a sequence according to one or more of the SEQ ID NOs described herein, such as those included herein as examples of components. In some embodiments, the encoding nucleic acid sequence, or amino acid sequence, comprises a sequence that shares at least about 90%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity, homology, and / or functional equivalence with a sequence resulting from a combination of one or more of the SEQ ID NOs described herein. It should be understood that certain sequence variabilities, extensions, and / or truncations of the disclosed sequences can occur when the sequences are combined, for example, as a result of ease or effectiveness in cloning (e.g., for creating restriction sites). In some embodiments, the chimeric receptor comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or an identity within a range defined by any two of the foregoing percentages, with a sequence of one or more of the SEQ ID NOs provided herein, or a portion thereof (e.g., a portion excluding the mbIL15 sequence and / or the self-cleaving peptide sequence).

[0108] In some embodiments, a polynucleotide encoding a CD19 binder / CD8α hinge / CD8α transmembrane domain / OX40 / CD3 zeta CAR is provided (see FIG. 1A). The polynucleotide comprises, or consists of, a CD19 binding domain, a CD8 alpha hinge, a CD8α transmembrane domain, an OX40 domain, and a CD3 zeta domain, as described herein. In some embodiments, the CAR comprises, in order from the N-terminus to the C-terminus, a CD19 binding domain, a CD8 alpha hinge, a CD8α transmembrane region, an OX40 co-stimulatory intracellular signaling domain, and a CD3 zeta domain.

[0109] In some embodiments, this receptor complex is encoded by a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, the CD19 CAR is encoded by a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 3. In some embodiments, the CD19 CAR comprises the amino acid sequence of SEQ ID NO: 43 (e.g., SEQ ID NO: 4). In some embodiments, the CD19 CAR comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, the CD19 CAR comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, the CD19 CAR comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 43 (e.g., SEQ ID NO: 4). In some embodiments, the CAR may vary from SEQ ID NO: 3, 4, or 43, but the CAR retains NK cell activation and / or cytotoxic function, or in some embodiments, has enhanced NK cell activation and / or cytotoxic function. Further, in some embodiments, this CD19 CAR construct may be co-expressed with, for example, mbIL15 encoded by SEQ ID NO: 39, on immune cells. In some embodiments, the CD19 CAR is co-expressed with mbIL15 encoded by a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 39. In some embodiments, the co-expressed mbIL15 comprises the amino acid sequence of SEQ ID NO: 44 (e.g., SEQ ID NO: 40). In some embodiments, the co-expressed mbIL15 comprises the amino acid sequence of SEQ ID NO: 44. In some embodiments, the co-expressed mbIL15 comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the co-expressed mbIL15 has an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 44 (e.g., SEQ ID NO: 40).mbIL15 may be introduced into cells by another vector encoding the CAR. However, in some embodiments, mbIL15 is bicistronically encoded on the same nucleic acid sequence as the CD19 CAR. See, for example, FIG. 1B. In some embodiments, the CD19 CAR and mbIL15 are encoded by a nucleic acid of any of SEQ ID NO: 1, 41, or 53. In some embodiments, the CD19 CAR and mbIL15 are encoded by the nucleic acid of SEQ ID NO: 1. In some embodiments, the CD19 CAR and mbIL15 are encoded by the nucleic acid of SEQ ID NO: 41. In some embodiments, the CD19 CAR and mbIL15 are encoded by the nucleic acid of SEQ ID NO: 53. In some embodiments, the CD19 CAR and mbIL15 are encoded by a nucleic acid having at least , at least 75%, at least 80%, at least 8%, at least 90%, at least 95% sequence identity with any of the sequences of SEQ ID NO: 1, SEQ ID NO: 41, or SEQ ID NO: 53. In some embodiments, the CD19 CAR and mbIL15 are encoded by a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 1. In some embodiments, the CD19 CAR and mbIL15 are encoded by a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 41. In some embodiments, the CD19 CAR and mbIL15 are encoded by a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 53. In some embodiments, the co-encoded CD19 CAR and mbIL15 have the amino acid sequence of SEQ ID NO: 2 or 42 (although they are ultimately expressed separately). In some embodiments, the co-encoded CD19 CAR and mbIL15 comprise the amino acid sequence set forth in SEQ ID NO: 2. In some embodiments, the co-encoded CD19 CAR and mbIL15 comprise the amino acid sequence set forth in SEQ ID NO: 42.In some embodiments, the co-encoded CD19 CAR and mbIL15 comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 2 or 42 (although they are ultimately expressed separately). In some embodiments, the co-encoded CD19 CAR and mbIL15 comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 2. In some embodiments, the co-encoded CD19 CAR and mbIL15 comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity with the sequence of SEQ ID NO: 42. In some embodiments, the co-encoded CD19 CAR and mbIL15 comprise the amino acid sequence of SEQ ID NO: 42.

[0110] Further information about chimeric receptors for use in the presently disclosed methods and compositions can be found in U.S. Patent Nos. 11,253,547, 11,141,436, or 11,153,575, the entire contents of each of which are hereby incorporated by reference in their entirety.

[0111] Methods of Treatment, Use, Administration, and Dosage Some embodiments relate to methods of treating, ameliorating, inhibiting, or preventing cancer with cells or immune cells comprising a chimeric antigen receptor, as disclosed herein. In some embodiments, the method comprises treating or preventing cancer. In some embodiments, the method comprises administering a therapeutically effective amount of immune cells that express a tumor-directed chimeric antigen receptor, as described herein. Examples of types of cancer that can be so treated are described herein.

[0112] This specification discloses a method of treating cancer in a subject. In some embodiments, the method comprises administering to the subject any one of the CD19 binding domains disclosed herein, any one of the CD19-directed CARs disclosed herein, any one of the CAR-expressing cells disclosed herein, or any combination thereof.

[0113] Also disclosed herein is the use of any one of the CD19 binding domains disclosed herein, any one of the CD19-directed CARs disclosed herein, any one of the cells disclosed herein, or any combination thereof, for the treatment of cancer.

[0114] Also disclosed herein is the use of any one of the CD19 binding domains disclosed herein, any one of the CD19-directed CARs disclosed herein, any one of the cells disclosed herein, or any combination thereof, in the manufacture of a medicament for the treatment of cancer.

[0115] In certain embodiments, treatment of a subject with the genetically engineered cells described herein achieves one, two, three, four, or more of the following effects, including, for example: (i) a decrease or improvement in the severity of a disease or a symptom associated therewith; (ii) a decrease in the duration of a symptom associated with the disease; (iii) protection against the progression of a disease or a symptom associated therewith; (iv) regression of a disease or a symptom associated therewith; (v) protection against the occurrence or onset of a symptom associated with the disease; (vi) protection against recurrence of a symptom associated with the disease; (vii) a decrease in hospitalization of the subject; (viii) a decrease in the length of hospitalization; (ix) an increase in survival of a subject having the disease; (x) a decrease in the number of symptoms associated with the disease; and (xi) enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect of another treatment. Each of these comparisons includes, for example, cell-based immunotherapy of a disease using cells that do not express the constructs disclosed herein, relative to different treatments of the disease. Advantageously, the engineered NK and / or T cells disclosed herein further enhance one or more of the above. In particular, the methods of treating a subject by the dosing regimens provided herein and described in the examples (e.g., NK cells engineered to express a CD19 CAR) have been surprisingly found to provide unexpected efficacy and safety, including an efficacy and safety profile that allows for administration ex vivo. Furthermore, the inventors have surprisingly found that the potency of engineered NK cells and compositions containing the same as described herein is not impaired when CD58 expression is absent in tumor cells, whereas the potency of engineered T cells is impaired. CD58 is a costimulatory receptor that activates T cells and NK cells through its interaction with CD2 (Zhang et al., Front Immunol. (2021) 12: 705260).CD58 deficiency or mutation is associated with poor response to CD19 CAR-T therapy and decreased survival in clinical and preclinical models of blood cancer. Thus, the data provided herein demonstrate the unexpected finding that CD19 CAR NK cells are not defective in their efficacy in the same manner as CD19 CAR T cells against tumor cells lacking CD58 expression (Majzner et al., Blood (2020) 136 (Supplement 1): 53-54). Thus, without wishing to be bound by theory, the engineered NK cells and related compositions, dosing regimens, methods, and uses described herein may provide superior anti-tumor activity compared to CD19 CAR T cells in patients having cancer that exhibits CD58 deficiency or mutation (e.g., lack of a functional mutation).

[0116] Administration can be obtained by a variety of routes including, but not limited to, intravenous, intraarterial, subcutaneous, intramuscular, intrahepatic, intraperitoneal, and / or local delivery to affected tissue. Cells engineered to express the chimeric receptor complexes described herein (specifically, NK cells and / or T cells) can be formulated for parenteral administration by injection, e.g., by bolus injection or infusion. More particularly discussed, the cell therapies provided herein can be delivered to a subject as a monotherapy or as a co-therapy with one or more additional anti-cancer agents or pre-treatments.

[0117] The dosage of immune cells such as NK cells and / or T cells can be readily determined for a given subject based on the subject's body weight, disease type and condition, and desired aggressiveness of treatment, but depending on the embodiment, the range is from about 10 5 cells per kg to about 10 12 cells per kg (e.g., 10 5 to 10 7 , 10 7 to 10 10 10 10 to 10 12 and overlapping ranges thereof). In one embodiment, a dose escalation regimen is used. In some embodiments, the range of immune cells such as NK and / or T cells is, for example, about 1×108 cells / kg to about 1×10 10 cells / kg are administered.

[0118] In some embodiments, 1×10 8 NK cells are administered three times over a 28-day cycle (2×10 6 cells / kg for subjects under 50 kg). In some embodiments, 3×10 8 NK cells are administered three times over a 28-day cycle (6×10 6 cells / kg for subjects under 50 kg). In some embodiments, 1×10 9 NK cells are administered three times over a 28-day cycle (2×10 7 cells / kg for subjects under 50 kg). In some embodiments, 1.5×10 9 NK cells are administered three times over a 28-day cycle (3×10 7 cells / kg for subjects under 50 kg).

[0119] In some embodiments, 1×10 8 CAR NK cells are administered three times over a 28-day cycle (2×10 6 cells / kg for subjects under 50 kg). In some embodiments, 3×10 8 CAR NK cells are administered three times over a 28-day cycle (6×10 6 cells / kg for subjects under 50 kg). In some embodiments, 1×10 9 CAR NK cells are administered three times over a 28-day cycle (2×10 7 cells / kg for subjects under 50 kg). In some embodiments, 1.5×10 9 CAR NK cells are administered three times over a 28-day cycle (3×10 7 cells / kg for subjects under 50 kg).

[0120] Optionally, lower (or higher) doses may be used. For example, in some embodiments, 1.5×10 8Individual NK cells are administered twice over a 28-day cycle (3×10 6 cells / kg for subjects under 50 kg). In some embodiments, 4.5×10 8 cells of NK cells are administered twice over a 28-day cycle. In some embodiments, 1.5×10 9 cells of NK cells are administered twice over a 28-day cycle.

[0121] In some embodiments, 0.3×10 9 cells of NK cells are administered three times over a 28-day cycle. In some embodiments, 0.5×10 9 cells of NK cells are administered three times over a 28-day cycle. In some embodiments, 1.0×10 9 cells of NK cells are administered three times over a 28-day cycle. In some embodiments, 1.5×10 9 cells of NK cells are administered three times over a 28-day cycle (3×10 7 cells / kg for subjects under 50 kg). In some embodiments, 3×10 9 cells of NK cells are administered three times over a 28-day cycle. In some embodiments, 1.5×10 10 cells of NK cells are administered three times over a 28-day cycle. In some embodiments, at least 4.5×10 9 cells of NK cells are administered over a cycle.

[0122] In some embodiments, 0.3×10 9 cells of CAR NK cells are administered three times over a 28-day cycle. In some embodiments, 0.5×10 9 cells of CAR NK cells are administered three times over a 28-day cycle. In some embodiments, 1.0×10 9 cells of CAR NK cells are administered three times over a 28-day cycle. In some embodiments, 1.5×10 9 cells of CAR NK cells are administered three times over a 28-day cycle (3×10 7 cells / kg for subjects under 50 kg). In some embodiments, 3×109 Individual CAR NK cells are administered three times over a 28-day cycle. In some embodiments, 1.5×10 10 Individual CAR NK cells are administered three times over a 28-day cycle. In some embodiments, at least 4.5×10 9 CAR NK cells are administered over the cycle.

[0123] In some embodiments, the dose of NK cells in the administration cycle is administered exogenously. In some embodiments, two doses of NK cells in the administration cycle are administered exogenously. In some embodiments, each dose of NK cells in the administration cycle is administered exogenously.

[0124] In some embodiments, the administration of engineered NK cells is preceded by one or more pretreatment steps. In some embodiments, the administration of engineered NK cells is preceded by lymphodepletion. In some embodiments, each administration cycle is preceded by lymphodepletion. In some embodiments, a combination of chemotherapeutic agents is used for lymphodepletion. In some embodiments, a single chemotherapeutic agent is used for lymphodepletion. In some embodiments, a combination of chemotherapeutic agents is used, and agents having different mechanisms of action may be used. In some embodiments, different classes of agents may be used. In some embodiments, antimetabolites are used. In some embodiments, the antimetabolites inhibit and / or prevent cell replication.

[0125] In some embodiments, cyclophosphamide, an alkylating agent that reduces tumor growth, is used for lymphodepletion. In some embodiments, lymphodepletion includes cyclophosphamide. In some embodiments, a dose of cyclophosphamide between about 200 mg / m 2 and 600 mg / m 2 is administered, about 200 mg / m 2 , about 225 mg / m 2 , about 250 mg / m 2 , about 275 mg / m 2 , about 300 mg / m 2, approximately 325 mg / m 2 , approximately 350 mg / m 2 , approximately 400 mg / m 2 , approximately 450 mg / m 2 , approximately 475 mg / m 2 , approximately 500 mg / m 2 , approximately 525 mg / m 2 , approximately 550 mg / m 2 , approximately 600 mg / m 2 , or approximately 700 mg / m 2 , or includes any dosage in between the recited ones. In some embodiments, a dosage of cyclophosphamide of approximately 300 mg / m 2 is administered. In some embodiments, a dosage of cyclophosphamide of approximately 500 mg / m 2 is administered. In some embodiments, the dosage of cyclophosphamide is administered daily for several days (e.g., before CAR-NK or CAR-T administration). In some embodiments, the dosage of cyclophosphamide is administered daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days (e.g., before CAR-NK or CAR-T administration). In some embodiments, cyclophosphamide is administered daily for 3 days. In some embodiments, if necessary, the dosage can be administered, for example, in two divided doses per day. In some embodiments, cyclophosphamide is initiated 5 days before the first administration of CD-19 CAR-expressing immune cells and administered daily for 3 days. In some embodiments, cyclophosphamide is initiated 5 days before the first administration of CD-19 CAR-expressing immune cells and administered daily for 3 days at a dosage of approximately 300 mg / m 2 . In some embodiments, cyclophosphamide is initiated 5 days before the first administration of CD-19 CAR-expressing immune cells and administered daily for 3 days at a dosage of approximately 500 mg / m 2 . In some embodiments, cyclophosphamide is administered in combination with another agent.

[0126] In some embodiments, the additional agent is also an antimetabolite. In some embodiments, the additional agent inhibits one or more of DNA polymerase alpha, ribonucleotide reductase and / or DNA primase, and thus inhibits DNA synthesis. In some embodiments, the additional agent is fludarabine. In some embodiments, a dose of fludarabine between about 5.0 mg / m 2 ~ about 200 mg / m 2 is administered, including a dose of about 5.0 mg / m 2 , about 10.0 mg / m 2 , about 15.0 mg / m 2 , about 20.0 mg / m 2 , about 25.0 mg / m 2 , about 30.0 mg / m 2 , about 35.0 mg / m 2 , about 40.0 mg / m 2 , about 45.0 mg / m 2 , about 50.0 mg / m 2 , about 60.0 mg / m 2 , about 70.0 mg / m 2 , about 80.0 mg / m 2 , about 90.0 mg / m 2 , about 100.0 mg / m 2 , about 125.0 mg / m 2 , about 150.0 mg / m 2 , about 175.0 mg / m 2 , about 200.0 mg / m 2 or any dose in between the recited. In some embodiments, a dose of about 30.0 mg / m 2 of fludarabine is administered. In some embodiments, the dose of fludarabine is administered daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, the dose of fludarabine is administered daily for about 3 days. In some embodiments, a dose of about 30.0 mg / m 2 of fludarabine is administered daily for about 3 days. In some embodiments, if necessary, the dose can be administered, for example, twice a day.

[0127] In some embodiments, about 300 mg / m2 Cyclophosphamide of 2 and fludarabine of about 30 mg / m 2 are each administered daily for about 3 days. In some embodiments, prior to each dosing cycle, cyclophosphamide of about 300 mg / m 2 and fludarabine of about 30 mg / m 2 are each administered daily for about 3 days. In some embodiments, cyclophosphamide of about 500 mg / m 2 and fludarabine of about 30 mg / m 2 are each administered daily for about 3 days. In some embodiments, prior to each dosing cycle, cyclophosphamide of about 500 mg / m 2 and fludarabine of about 30 mg / m

[0128] are each administered daily for about 3 days. 2 In some embodiments, the engineered cells expressing the CD19-directed CAR described in the embodiments disclosed herein are administered in combination with additional agents. For example, in some embodiments, another anti-cancer agent is administered (in addition to those used in the lymphodepletion process). In some embodiments, an antibody (e.g., a monoclonal antibody or a biosimilar) is used in combination with the engineered immune cells. In some embodiments, the antibody targets CD20. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the biosimilar rituximab-abb, rituximab arrx, and / or rituximab pvvr are used. In some embodiments, ocrelizumab, ofatumumab, obinutuzumab, ibritumomab, ibritumomab or combinations thereof are used. In some embodiments, the dose of the anti-CD20 antibody ranges between about 150 mg / m 2 and about 500 mg / m 2 and is about 150 mg / m 2 about 200 mg / m 2 about 250 mg / m 2 about 300 mg / m 2 about 350 mg / m 2 about 375 mg / m 2, about 425 mg / m 2 , about 450 mg / m 2 , or about 500 mg / m 2 (or any dose in between those recited). In some embodiments, the dose of the anti-CD20 antibody is about 375 mg / m 2 . In some embodiments, the anti-CD20 antibody is rituximab and the dose is about 375 mg / m 2 . In some embodiments, the dose of the anti-CD20 antibody is about 500 mg / m 2 . In some embodiments, the anti-CD20 antibody is rituximab and the dose is about 500 mg / m 2 . In some embodiments, the anti-CD20 antibody is administered 1, 2, 3 times, or more. In some embodiments, the anti-CD20 antibody is administered 1, 2, 3, 4 days or more before the administration of the cells at the first time point of the administration cycle. In some embodiments, a single dose of 375 mg / m 2 rituximab is administered during the administration cycle. In some embodiments, a single dose of 375 mg / m 2 rituximab is administered during the first administration cycle, and a single dose of 500 mg / m 2 rituximab is administered during each of the subsequent administration cycles. In some embodiments, a single dose of rituximab is administered before the administration of the engineered immune cells (e.g., about 3 days before administration). In some embodiments, a single dose of rituximab is administered about 3 days before the administration of the engineered immune cells. In some embodiments, a single dose of rituximab is administered on about -3 days. In some embodiments, the anti-CD20 antibody is administered 3 days before the administration of the cells.

[0129] In certain embodiments, the dosage of the genetically engineered cells or their compositions described herein is administered to a subject daily, once every two days, every two to three days, every three days, once a week, twice a week, three times a week, or once every two weeks. In other embodiments, two, three, or four dosages of the genetically engineered cells or their compositions described herein are administered to a subject daily, every two to three days, every three days, once a week, or once every two weeks. In some embodiments, the dosage of the genetically engineered cells or their compositions described herein is administered for two days, three days, five days, seven days, fourteen days, twenty-one days, or twenty-eight days. In certain embodiments, the dosage of the genetically engineered cells or their compositions described herein is administered for one month, one and a half months, two months, two and a half months, three months, four months, five months, six months, or longer. In some embodiments, a dosing period is set and a specific number of dosages are administered within that period. For example, in some embodiments, the dosing cycle is 28 days in length with dosages of engineered immune cells administered on days 0, 7, and 14.

[0130] In some embodiments, the subject is lymphodepleted at least once prior to administration of the genetically engineered cells disclosed herein. In some embodiments, lymphodepletion is performed prior to administration of one or more additional doses of engineered cells. In some embodiments, a dosing cycle including lymphodepletion prior to at least two doses of engineered cells disclosed herein is used with two doses separated by a time interval. In some embodiments, the time interval is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or more days (including the interval between the last administration and the time marking the interval, e.g., 84 hours or 3.5 days). In some embodiments, the dosing cycle itself is approximately 14, 21, 28, 35, 42, or more days. In some embodiments, three doses are administered approximately one week apart from each other. In some embodiments, two doses are administered approximately one week apart from each other. In some embodiments, the subject receives a first dose on day 0 of the cycle, a second dose on day 7 of the cycle, and a third dose on day 14 of the cycle. In some such embodiments, a 28-day cycle is used with a primary outcome measure evaluated on day 28 (see, e.g., Figure 2). In some embodiments, lymphodepletion is performed prior to the start of each dosing cycle if subsequent dosing cycles are required (e.g., if the subject requires further treatment). For example, in some embodiments, the subject undergoes lymphodepletion, receives multiple doses of engineered cells according to a cycle, is evaluated at the end of the cycle time, and if deemed necessary, undergoes a second lymphodepletion prior to a second dosing cycle. In some embodiments, fludarabine / cyclophosphamide is used to achieve lymphodepletion. In some embodiments, cyclophosphamide (500 mg / m 2 ) and fludarabine (30 mg / m 2) is administered daily for 5 days. Depending on the embodiment, different concentrations may be used. In such embodiments where multiple dosing cycles are used, the first and second dosing cycles need not be the same (e.g., the first cycle may have 2 doses, while the second uses 3 doses). Depending on the subject, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dosing cycles are carried out.

[0131] Advantageously, in some embodiments, the treatments and dosing regimens provided herein provide an effective anti-cancer treatment without certain CAR-T toxicities, such as cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) or neurotoxicity, or graft-versus-host disease. In some embodiments, complete remission is achieved. In some embodiments, complete response (CR) is achieved. In some embodiments, partial response (PR) is achieved. In some embodiments, stable disease (SD) or limited disease progression is achieved.

[0132] Clinical outcomes can be evaluated by any method known in the art, including differentiation according to the Lugano classification according to the lymphoma response to immunomodulatory therapy criteria (LYRIC) for subjects with non-Hodgkin lymphoma (NHL); the 2018 International Workshop (iwCLL) guidelines for chronic lymphocytic leukemia for subjects with chronic lymphocytic leukemia (CLL) or small lymphocytic leukemia (SLL); Version 1.2020 National Comprehensive Cancer Network (NCCN) for subjects with B-cell lymphoblastic leukemia (B-ALL); or the 6th International Workshop on Waldenström macroglobulinemia (WM) for subjects with Waldenström macroglobulinemia (WM). See Cheson et al., Blood (2016) 128(21):2489-96; Cheson et al., J Clin Oncol (2014) 32(27):3059-68; Hallek et al., Blood (2018) 131(25):2745-60; NCCN Guidelines for Acute Lymphoblastic Leukemia 1.2020; and Owen et al., Br J Haematol (2013) 160(2):171-6.

[0133] In some embodiments, when compared to each of the nucleic acid or amino acid sequences of SEQ ID NO: 1-44 (or a combination of two or more of SEQ ID NO: 1-44), nucleic acid and amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (and ranges thereof) sequence identity and / or homology are also provided herein, and when compared to each of SEQ ID NO: 1-44 (or a combination of two or more of SEQ ID NO: 1-44), although not limited to, (i) enhanced proliferation, (ii) enhanced activation, (iii) enhanced cytotoxic activity against cells presenting ligands to which NK cells bearing receptors encoded by the nucleic acid and amino acid sequences bind, (iv) enhanced homing to tumor or infection sites, (v) reduced off-target cytotoxic effects, (vi) enhanced secretion of immunostimulatory cytokines and chemokines (including, but not limited to, IFNγ, TNFα, IL-22, CCL3, CCL4, and CCL5), (vii) enhanced ability to stimulate additional innate and adaptive immune responses, and (viii) one or more functions including combinations thereof are also shown.

[0134] Furthermore, in some embodiments, amino acid sequences are provided that correspond to any of the nucleic acids disclosed herein but exhibit degeneracy of the nucleic acid code. Furthermore, these sequences (either nucleic acid or amino acid), which are different from those explicitly disclosed herein but have functional similarity or equivalence, are also considered to be within the scope of the present disclosure. The foregoing includes variants, truncations, substitutions, or other types of modifications.

[0135] In some embodiments, the polynucleotide encoding the disclosed cytotoxic receptor complex is mRNA. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is operably linked to at least one control element for expression of the cytotoxic receptor complex.

[0136] According to some embodiments, there is further provided a vector comprising a polynucleotide encoding any polynucleotide provided herein, wherein the polynucleotide may be operably linked to at least one control element for the expression of a cytotoxic receptor complex. In some embodiments, the vector is a retrovirus.

[0137] Also provided herein are engineered immune cells (such as NK cells and / or T cells) comprising a polynucleotide, vector, or cytotoxic receptor complex disclosed herein. Also provided herein are engineered NK cells comprising a polynucleotide, vector, or cytotoxic receptor complex disclosed herein. Also provided herein are compositions comprising a mixture of engineered immune cells (such as NK cells and / or engineered T cells) wherein each population comprises a polynucleotide, vector, or cytotoxic receptor complex disclosed herein. Also provided herein are compositions comprising engineered NK cells comprising a polynucleotide, vector, or cytotoxic receptor complex disclosed herein.

[0138] subject Some embodiments of the compositions and methods described herein relate to administering to a subject having cancer an immune cell comprising a chimeric antigen receptor.

[0139] In some embodiments, the subject has large B-cell lymphoma (LBCL). In some embodiments, the subject has aggressive LBCL. In some embodiments, the subject has non-Hodgkin lymphoma (NHL). In some embodiments, the subject has diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), or B-cell acute lymphoblastic leukemia (B-ALL). In some embodiments, the subject has diffuse large B-cell lymphoma (DLBCL). In some embodiments, the subject has follicular lymphoma (FL). In some embodiments, the subject has high-grade FL (e.g., FL grade 3b). In some embodiments, the subject has indolent lymphoma (IL). In some embodiments, the subject has grade 1, 2, or 3a FL. In some embodiments, the subject has marginal zone lymphoma (MZL). In some embodiments, the subject has mantle cell lymphoma (MCL). In some embodiments, the subject has B-cell acute lymphoblastic leukemia (B-ALL). In some embodiments, the subject has Waldenström macroglobulinemia (WM). In some embodiments, the subject has chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). In some embodiments, the subject has CLL. In some embodiments, the subject has SLL. In some embodiments, the subject has primary mediastinal large B-cell lymphoma (PMBCL). In some embodiments, the cancer is relapsed / refractory (r / r) cancer.

[0140] In some embodiments, the subject has a bone marrow-localized disease (e.g., ≤5% peripheral blood blasts without other evidence of extramedullary disease, including lymphoblastic lymphoma). In some embodiments, the subject has ≤5% peripheral blood blasts. In some embodiments, the subject has <5% peripheral blood blasts. In some embodiments, the subject has r / r B-ALL. In some embodiments, the subject has r / r B-ALL with ≤5% peripheral blood blasts. In some embodiments, the subject has r / r B-ALL with <5% peripheral blood blasts. In some embodiments, the subject has no evidence of extramedullary disease. In some embodiments, the subject has no other evidence of extramedullary disease. In some embodiments, the subject has no evidence of extramedullary disease including lymphoblastic lymphoma. In some embodiments, the subject has no other evidence of extramedullary disease including lymphoblastic lymphoma.

[0141] In some embodiments, the subject has a measurable disease defined by any method known in the art for diagnosis and staging, including the WHO 2016 classification for r / r B-cell NHL or B-ALL (Quintanilla-Martinez, Hematological Oncology (2017) 35:37-4); the Lugano classification for NHL (Cheson et al., J. Clin. Oncol (2014) 32(27):3059-68); the iwCLL for CLL and SLL (Hallek et al., Blood (2018) 131(25):2745-60); and the Second International Workshop on Waldenström macroglobulinemia for WM (Owen et al., Semin Oncol (2003) 30(2):110-15).

[0142] In some embodiments, the subject was treated with prior line therapy. In some embodiments, the subject is refractory / relapsed (R / R) to prior line therapy. In some embodiments, prior line therapy includes one prior line of therapy. In some embodiments, the subject has MCL, the prior line of therapy is one prior line of therapy, and the one prior line of therapy is not CAR T cells. In some embodiments, the subject has WM and the prior line of therapy is one prior line of therapy. In some embodiments, prior line therapy includes two prior lines of therapy. In some embodiments, prior line therapy includes three prior lines of therapy. In some embodiments, prior line therapy includes four prior lines of therapy. In some embodiments, the subject did not respond or relapsed within 12 months of completion of prior line therapy. In some embodiments, the subject did not respond to prior line therapy. In some embodiments, the subject relapsed within 12 months of completion of prior line therapy.

[0143] In some embodiments, prior line therapy includes a Bruton's tyrosine kinase inhibitor (BTKi). In some embodiments, the subject was previously treated with a BTKi. In some embodiments, the subject is R / R to a BTKi. In some embodiments, the BTKi includes ibrutinib. In some embodiments, the BTKi is ibrutinib. In some embodiments, the subject was previously treated with ibrutinib. In some embodiments, the subject is R / R to ibrutinib.

[0144] In some embodiments, prior line therapy includes a tyrosine kinase inhibitor. In some embodiments, the subject has Philadelphia chromosome (Ph+) B-ALL and prior line therapy includes a tyrosine kinase inhibitor.

[0145] In some embodiments, the prior line of treatment comprises a Bcl-2 inhibitor. In some embodiments, the subject has been previously treated with a Bcl-2 inhibitor. In some embodiments, the subject is R / R to a Bcl-2 inhibitor. In some embodiments, the Bcl-2 inhibitor comprises venetoclax. In some embodiments, the Bcl-2 inhibitor is venetoclax. In some embodiments, the subject has been previously treated with venetoclax. In some embodiments, the subject is R / R to venetoclax.

[0146] In some embodiments, the prior line of treatment comprises a BTKi and a Bcl-2 inhibitor. In some embodiments, the subject has been previously treated with a BTKi and a Bcl-2 inhibitor. In some embodiments, the subject is R / R to a BTKi and a Bcl-2 inhibitor. In some embodiments, the BTKi is ibrutinib. In some embodiments, the Bcl-2 inhibitor is venetoclax.

[0147] In some embodiments, the prior line of treatment comprises CD20-targeted therapy and cytotoxic chemotherapy (e.g., anthracycline). In some embodiments, the CD20-targeted therapy is an anti-CD20 antibody. In some embodiments, the anti-CD20 antibody is an anti-CD20 monoclonal antibody. In some embodiments, the anti-CD20 antibody comprises rituximab. In some embodiments, the anti-CD20 antibody is rituximab. In some embodiments, the cytotoxic chemotherapy comprises anthracycline. In some embodiments, the cytotoxic chemotherapy is anthracycline. In some embodiments, the subject has been previously treated with an anti-CD20 monoclonal antibody and cytotoxic therapy (e.g., anthracycline). In some embodiments, the subject is R / R to an anti-CD20 monoclonal antibody and cytotoxic therapy (e.g., anthracycline). In some embodiments, when the prior line of treatment comprises CD20-targeted therapy, the cancer cells are CD20+ (e.g., when locally evaluated).

[0148] In some embodiments, the prior line of treatment includes CD19-directed therapy. In some embodiments, the subject has been previously treated with CD19-directed therapy. In some embodiments, the prior line of treatment includes chimeric antigen receptor (CAR) T cells. In some embodiments, the subject has been previously treated with CAR T cells (CAR T exposure). In some embodiments, the subject has been previously treated with anti-CD19 CAR T cells. In some embodiments, the CAR T cells are autologous CAR T cells. In some embodiments, the subject has been previously treated with autologous CAR T cells. In some embodiments, the subject has been previously treated with autologous anti-CD19 CAR T cells. In some embodiments, when the prior line of treatment includes CD19-directed therapy, the cancer cells are CD19+ (e.g., when locally evaluated).

[0149] In some embodiments, the prior line of treatment does not include CD19-directed therapy. In some embodiments, the subject has not been previously treated with CD19-directed therapy. In some embodiments, the prior line of treatment does not include chimeric antigen receptor (CAR) T cells. In some embodiments, the subject has not been previously treated with CAR T cells (CAR T naive). In some embodiments, the subject has not been previously treated with autologous CAR T cells. In some embodiments, the subject has not been previously treated with anti-CD19 CAR T cells. In some embodiments, the subject has not been previously treated with autologous anti-CD19 CAR T cells.

[0150] In some embodiments, the subject is human. In some embodiments, the subject is an adult. In some embodiments, the subject is at least 18 years old.

[0151] In some embodiments, the subject has a Performance Status of 0, 1, or 2 of the Eastern Cooperative Oncology Group (ECOG). In some embodiments, the subject has a Performance Status of 0 or 1 of the Eastern Cooperative Oncology Group (ECOG). In some embodiments, the subject has an ECOG of 0. In some embodiments, the subject has an ECOG of 1. In some embodiments, the subject has an ECOG of 2.

[0152] In some embodiments, the subject has sufficient organ function. In some embodiments, sufficient organ function includes a platelet count ≥ 30,000 cells / μL. In some embodiments, sufficient organ function includes a serum creatinine value ≤ 1.5 × the upper limit of normal (ULN). In some embodiments, in subjects with hereditary benign hyperbilirubinemia, sufficient organ function includes a total bilirubin value ≤ 1.5 × ULN or ≤ 3.0 × ULN. In some embodiments, sufficient organ function includes an aspartate aminotransferase (AST) / serum glutamate oxaloacetate transaminase (SGOT) value ≤ 3 × ULN and an alanine aminotransferase (ALT) / serum glutamate pyruvate transaminase (SGPT) value ≤ 3 × ULN. In some embodiments, sufficient organ function includes a baseline international normalized ratio (INR) ≤ 2 or an activated partial thromboplastin time (aPTT) ≤ 2 times the ULN. In some embodiments, sufficient organ function includes subjects who do not require oxygen therapy.

[0153] In some embodiments, the subject does not have Burkitt lymphoma. In some embodiments, the subject does not have primary central nervous system (CNS) lymphoma. In some embodiments, the subject does not have Richter transformation to Hodgkin lymphoma.

[0154] Cancer type Some embodiments of the compositions and methods described herein relate to administering immune cells comprising a tumor-directed chimeric antigen receptor and / or a tumor-directed chimeric receptor to a subject having cancer.

[0155] B-cell lineage-derived cancers are a global healthcare burden. More than 500,000 new cases of non-Hodgkin lymphoma (median age 69 years) and 50,000 new cases of acute lymphoblastic leukemia (ALL) (median age 16 years) are expected worldwide each year (seer.cancer.gov, Smith Br J Cancer. 2015;112(9):1575-84, Solomon, paper presented at: 11th International Conference on Hematology & Hematological Oncology; November 08-09, 2017). Despite advances in treatment, many patients diagnosed with these heterogeneous groups of cancers still succumb to these diseases. Approximately 30% - 50% of newly diagnosed patients with advanced large cell lymphoma are not cured by first-line treatment (Gisselbrecht J Clin Oncol. 2010 Sep 20;28(27):4184-9, Kenkre Curr Oncol Rep. 2008:10:393-403, Sehn Blood 2006;109(5):1857-1861, Sinha Expert Opin Investig Drugs 2011 May 20(5):669-80). Similarly, more than half of adults with ALL ultimately relapse (Malard 2020).

[0156] In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is leukemia or lymphoma. In some embodiments, the lymphoma is double-hit / double-expressor lymphoma. In some embodiments, the lymphoma is triple-hit / triple-expressor lymphoma. In some embodiments, the cancer includes Richter transformation.

[0157] The various embodiments provided herein include the treatment or prevention of various malignancies, such as non-Hodgkin lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), mantle cell lymphoma, marginal zone lymphoma, Burkitt lymphoma, lymphoblastic lymphoma, hairy cell leukemia, primary central nervous system lymphoma, and primary intraocular lymphoma. In some embodiments, the cancer is non-Hodgkin lymphoma. In some embodiments, the cancer is B-cell lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma. In some embodiments, the cancer is follicular lymphoma. In some embodiments, the cancer is chronic lymphocytic leukemia. In some embodiments, the cancer is chronic myelogenous leukemia. In some embodiments, the cancer is mantle cell lymphoma. In some embodiments, the cancer is marginal zone lymphoma. Further types of cancer include, but are not limited to, Hodgkin lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), adrenocortical carcinoma, Kaposi sarcoma, lymphoma, gastrointestinal cancer, appendiceal cancer, central nervous system cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (including, but not limited to, astrocytoma, spinal cord tumor, brainstem glioma, craniopharyngioma, ependymoma, ependymoblastoma, medulloblastoma, and medulloepithelioma), breast cancer, bronchial tumor, cervical cancer, colon cancer, chronic myeloproliferative disorder, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, renal cell cancer, leukemia, oral cancer, hypopharyngeal cancer, liver cancer, lung cancer (including, but not limited to, non-small cell lung cancer (NSCLC) and small cell lung cancer), pancreatic cancer, intestinal cancer, lymphoma, melanoma, eye cancer, ovarian cancer, pancreatic cancer, prostate cancer, pituitary cancer, uterine cancer, and vaginal cancer.

[0158] In some embodiments, the cancer cells do not express CD58 or express a mutant form of CD58. In some embodiments, the cancer cells do not express CD58. In some embodiments, the cancer cells express a mutant form of CD58. In some embodiments, the mutant form of CD58 includes a loss of function mutation. In some embodiments, the CD58 mutation is a loss of function mutation.

[0159] In some embodiments, the cancer cells are B cell-derived NHL, such as aggressive large cell type B cell lymphoma (LBCL). In some embodiments, LBCL is diffuse large cell type B cell lymphoma (DLBCL) unless otherwise specified; high-grade B cell lymphoma; DLBCL derived from follicular lymphoma (FL) (FL grade 3b); DLBCL derived from Richter transformation from chronic lymphocytic leukemia (CLL) to DLBCL; primary mediastinal LBCL; and DLBCL derived from Waldenström macroglobulinemia (WM). In some embodiments, the cancer is NHL. In some embodiments, the cancer is LBCL. In some embodiments, the cancer is aggressive LBCL. In some embodiments, the cancer is DLBCL. In some embodiments, the cancer is FL grade 3b.

[0160] In some embodiments, the cancer is indolent lymphoma (IL). In some embodiments, IL is low-grade FL (FL grades 1, 2, and 3a), MCL, or MZL. In some embodiments, IL is low-grade FL (FL grades 1, 2, and 3a). In some embodiments, IL is MCL. In some embodiments, IL is MZL. In some embodiments, the cancer is low-grade FL (FL grades 1, 2, and 3a). In some embodiments, the cancer is FL grade 1. In some embodiments, the cancer is FL grade 2. In some embodiments, the cancer is FL grade 3a. In some embodiments, the cancer is MCL. In some embodiments, the cancer is MZL.

[0161] In some embodiments, the cancer is CLL or SLL. In some embodiments, the cancer is CLL. In some embodiments, the cancer is SLL. In some embodiments, the cancer is B-ALL.

[0162] In some embodiments, the cancer is relapsed / refractory (R / R). In some embodiments, the cancer is R / R NHL. In some embodiments, the cancer is R / R LBCL. In some embodiments, the cancer is R / R CLL. In some embodiments, the cancer is R / R SLL. In some embodiments, the cancer is R / R B-ALL.

[0163] In some embodiments, the cancer cells express CD19. In some embodiments, the cancer cells express CD19 at the time of administration of a dose of genetically engineered NK cells (e.g., the first dose). The expression of CD19 can be determined by any method known in the art, including flow cytometry.

[0164] In some embodiments, the cancer cells express CD20. In some embodiments, the cancer cells express CD20 at the time of administration of a dose of genetically engineered NK cells (e.g., the first dose). The expression of CD20 can be determined by any method known in the art, including flow cytometry.

[0165] In some embodiments, the cancer has been previously treated with CAR T cells (CAR T exposure). In some embodiments, the cancer is relapsed / refractory to CAR T cells. In some embodiments, the CAR T cells are anti-CD19 CAR T cells. In some embodiments, the CAR T cells are autologous CAR T cells. In some embodiments, the CAR T cells are autologous anti-CD19 CAR T cells. Thus, in some embodiments, the cancer has been previously treated with autologous anti-CD19 CAR T cells. In some embodiments, the cancer is R / R to autologous anti-CD19 CAR T cells. In some embodiments, the cancer has not been previously treated with CAR T cells (CAR T naive). In some embodiments, the cancer has not been previously treated with anti-CD19 CAR T cells, optionally autologous anti-CD19 CAR T cells. Thus, in some embodiments, the cancer is not R / R to anti-CD19 CAR T cells.

[0166] In some embodiments, the cancer is aggressive LBCL that has not been previously treated with CAR T cells (CAR T naive), optionally autologous CAR T cells. In some embodiments, the cancer is aggressive LBCL that has not been previously treated with anti-CD19 CAR T cells (CAR T naive), optionally autologous anti-CD19 CAR T cells. In some embodiments, the cancer is R / R LBCL that has not been previously treated with CAR T cells (CAR T naive), optionally autologous CAR T cells. In some embodiments, the cancer is R / R LBCL that has not been previously treated with anti-CD19 CAR T cells (CAR T naive), optionally autologous anti-CD19 CAR T cells. In some embodiments, the cancer is R / R NHL that has not been previously treated with CAR T cells (CAR T naive), optionally autologous CAR T cells. In some embodiments, the cancer is R / R NHL that has not been previously treated with anti-CD19 CAR T cells (CAR T naive), optionally autologous anti-CD19 CAR T cells. In some embodiments, the cancer is MCL that has not been previously treated with CAR T cells (CAR T naive), optionally autologous CAR T cells. In some embodiments, the cancer is MCL that has not been previously treated with anti-CD19 CAR T cells (CAR T naive), optionally autologous anti-CD19 CAR T cells. In some embodiments, the cancer is IL that has not been previously treated with CAR T cells (CAR T naive), optionally autologous CAR T cells. In some embodiments, the cancer is IL that has not been previously treated with anti-CD19 CAR T cells (CAR T naive), optionally autologous anti-CD19 CAR T cells. In some embodiments, the cancer is B-ALL that has not been previously treated with CAR T cells (CAR T naive), optionally autologous CAR T cells. In some embodiments, the cancer is B-ALL that has not been previously treated with anti-CD19 CAR T cells (CAR T naive), optionally autologous anti-CD19 CAR T cells.

[0167] In some embodiments, the cancer is aggressive LBCL that has been previously treated, as appropriate, with autologous CAR T cells (CAR T exposure). In some embodiments, the cancer is aggressive LBCL that has been previously treated, as appropriate, with autologous anti-CD19 CAR T cells (CAR T exposure). In some embodiments, the cancer is R / R LBCL that has been previously treated, as appropriate, with autologous CAR T cells (CAR T exposure). In some embodiments, the cancer is R / R LBCL that has been previously treated, as appropriate, with autologous anti-CD19 CAR T cells (CAR T exposure). In some embodiments, the cancer is R / R NHL that has been previously treated, as appropriate, with autologous CAR T cells (CAR T exposure). In some embodiments, the cancer is R / R NHL that has been previously treated, as appropriate, with autologous anti-CD19 CAR T cells (CAR T exposure). In some embodiments, the cancer is MCL that has been previously treated, as appropriate, with autologous CAR T cells (CAR T exposure). In some embodiments, the cancer is MCL that has been previously treated, as appropriate, with autologous anti-CD19 CAR T cells (CAR T exposure). In some embodiments, the cancer is IL that has been previously treated, as appropriate, with autologous CAR T cells (CAR T exposure). In some embodiments, the cancer is IL that has been previously treated, as appropriate, with autologous anti-CD19 CAR T cells (CAR T exposure). In some embodiments, the cancer is B-ALL that has been previously treated, as appropriate, with autologous CAR T cells (CAR T exposure). In some embodiments, the cancer is B-ALL that has been previously treated, as appropriate, with autologous anti-CD19 CAR T cells (CAR T exposure).

[0168] In some embodiments, the cancer is LBCL (e.g., DLBCL) that has been previously treated with an anti-CD20 monoclonal antibody and cytotoxic chemotherapy (e.g., anthracycline). In some embodiments, the cancer is LBCL (e.g., DLBCL) that has been previously treated with an anti-CD20 monoclonal antibody and anthracycline. In some embodiments, the cancer is IL that has been previously treated with an anti-CD20 monoclonal antibody and cytotoxic chemotherapy (e.g., anthracycline). In some embodiments, the cancer is IL that has been previously treated with an anti-CD20 monoclonal antibody and anthracycline.

[0169] In some embodiments, the cancer is MCL, CLL, SLL, or WM that has been previously treated with an inhibitor of Bruton's tyrosine kinase (BTKi) (e.g., ibrutinib). In some embodiments, the cancer is MCL that has been previously treated with a BTKi (e.g., ibrutinib). In some embodiments, the cancer is MCL that has been previously treated with a BTKi (e.g., ibrutinib) and anti-CD19 CAR T cells. In some embodiments, the cancer is CLL that has been previously treated with a BTKi (e.g., ibrutinib). In some embodiments, the cancer is SLL that has been previously treated with a BTKi (e.g., ibrutinib). In some embodiments, the cancer is WM that has been previously treated with a BTKi (e.g., ibrutinib).

[0170] In some embodiments, the cancer is CLL or SLL that has been previously treated with a Bcl-2 inhibitor (e.g., venetoclax). In some embodiments, the cancer is CLL that has been previously treated with a Bcl-2 inhibitor (e.g., venetoclax). In some embodiments, the cancer is SLL that has been previously treated with a Bcl-2 inhibitor (e.g., venetoclax). In some embodiments, the cancer is CLL or SLL that has been previously treated with a Bcl-2 inhibitor (e.g., venetoclax) and a BTKi (e.g., ibrutinib). In some embodiments, the cancer is CLL that has been previously treated with a Bcl-2 inhibitor (e.g., venetoclax) and a BTKi (e.g., ibrutinib). In some embodiments, the cancer is SLL that has been previously treated with a Bcl-2 inhibitor (e.g., venetoclax) and a BTKi (e.g., ibrutinib).

[0171] Cancer target Some embodiments of the compositions and methods described herein relate to immune cells comprising chimeric receptors that target (e.g., bind to) cancer antigens such as CD19, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and / or ULBP6. In some embodiments, the chimeric receptor targets (e.g., binds to) CD19, such as human CD19. Further non-limiting examples of target antigens are: CD70, CD5; CD123; CD22; CD30; CD171; CS1 (also called CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); TNF receptor family member B cell maturation (BCMA); CD38; DLL3; G protein-coupled receptor class C group 5, member D (GPRC5D); epidermal growth factor receptor (EGFR) CD138; prostate-specific membrane antigen (PSMA); Fms-like tyrosine kinase 3 (FLT3); KREMEN2 (kringle-containing transmembrane protein 2), ALPPL2, Claudin4, Claudin6, C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRviii); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer)); Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; a glycosylated CD43 epitope expressed in acute leukemia or lymphoma but not in hematopoietic progenitor cells, a glycosylated CD43 epitope expressed in non-hematopoietic cancers, carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin 11 receptor alpha (IL-IIRa); prostate stem cell antigen (PSCA); serine protease 21 (trypsin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen;CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha (Fra or FR1); folate receptor beta (FRb); receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostate-specific acid phosphatase (PAP); elongation factor 2 variant (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-1 receptor), carbonic anhydrase IX (CAIX); proteasome (Prosome, Macropain) subunit, beta type, 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (AbI) (bcr-ab1); tyrosine kinase; ephrin A receptor type 2 (EphA2); sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDClalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OacGD2); tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycosphingolipid (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1);Cancer / Testis Antigen 2 (LAGE-Ia); Melanoma-Associated Antigen 1 (MAGE-A1); ETS Variant Gene 6 Located on Chromosome 12p (ETV6-AML); Spermatid Protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); Angiopoietin-Binding Cell Surface Receptor 2 (Tie2); Melanoma Cancer Testis Antigen-1 (MAD-CT-1); Melanoma Cancer Testis Antigen-2 (MAD-CT-2); Fos-Related Antigen 1; Tumor Protein p53 (p53); p53 Variant; Prostein; 56urviving; Telomerase; Prostate Cancer Tumor Antigen-1 (PCT A-I or Galectin8), Melanoma Antigen Recognized by T Cells 1 (MelanA or MARTI); Rat Sarcoma (Ras) Variant; Human Telomerase; Reverse Transcriptase (hTERT); Sarcoma Translocation Breakpoint; Melanoma Inhibitor of Apoptosis (ML-IAP); ERG (Membrane-Transporter Protease, Serine 2 (TMPRSS2) ETS Fusion Gene); N-Acetylglucosaminyltransferase V (NA17); Paired Box Protein Pax-3 (PAX3); Androgen Receptor; Cyclin B1; v-myc Avian Myelocytomatosis Virus Oncogene Neuroblastoma-Derived Homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-Related Protein 2 (TRP-2); Cytochrome P450 IB1 (CYPIB 1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized by T Cells 3 (SART3); Paired Box Protein Pax-5 (PAX5); Proacrosin-Binding Protein sp32 (OY-TES1); Lymphocyte-Specific Protein Tyrosine Kinase (LCK); Kinase Anchor Protein 4 (AKAP-4); Synovial Sarcoma, X Breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); Kidney Ubiquitin 1 (RU1); Kidney Ubiquitin 2 (RU2); Legumain; Human Papillomavirus E6 (HPV E6);Human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 variant (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILR2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLLI), MPL, biotin, c-MYC epitope tag, CD34, LAMP1, TROP2, GFR alpha 4, CDH17, CDH6, NYBR1, CDH19, CD200R, Slea (CA19.9; Sialyl Lewis antigen);Including fucosyl-GMI, PTK7, gpNMB, CDH1-CD324, DLL3, CD276 / B7H3, Ill LRa, IL13Ra2, CD179b-IGLll, TCR gamma-delta, NKG2D, CD32(FCGR2A), Tn Ag, Timl- / HVCR1, CSF2RA(GM-CSFR-alpha), TGF beta R2, Lews Ag, TCR-beta1 chain, TCR-beta2 chain, TCR-gamma chain, TCR-delta chain, FITC, luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin hormone receptor (CGHR or GR), CCR4, GD3, SLAMF6, SLAMF4, HIV1 envelope glycoprotein, HTLV1-Tax, CMV pp65, EBV-EBNA3c, KSHV K8.1, KSHV-gH, influenza A hemagglutinin (HA), GAD, PDL1, guanylyl cyclase C (GCC), autoantibody against desmoglein 3 (Dsg3), autoantibody against desmoglein 1 (Dsg1), HLA, HLA-A, HLA-A2, HLA-B, HLA-C, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, HLA-DR, HLA-G, IgE, CD99, Ras G12V, tissue factor 1 (TF1), AFP, GPRC5D, Claudinl 8.2(CLD18A2 or CLDN18A.2)), P-glycoprotein, STEAP1, Livl, Nectin-4, Cripto, gpA33, BST1 / CD157, low-conductance chloride channel, and antigens recognized by TNT antibodies.; Non-limiting embodiments

[0172] The embodiments provided herein are as follows: 1. A cancer immunotherapy dosing regimen comprising at least a first dosing cycle, wherein: The first dosing cycle includes a first dose of genetically engineered natural killer (NK) cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, The first dose is administered to a subject in need of cancer immunotherapy at a first time point, The second dose is administered to the subject between 5 and 10 days after the first time point, the third dose is administered to the subject between 5 and 10 days after the second dose; each of the first, second, and third doses contains at least about 1.5×10 9 NK cells, at least a portion of the genetically engineered NK cells are engineered to express a chimeric antigen receptor (CAR) directed against the CD19 tumor marker, the first dosing cycle is initiated after the subject has undergone a lymphodepletion process to reduce the number of natural immune cells, dosing regimen. 2. The dosing regimen according to embodiment 1, wherein the first dosing cycle follows one or more additional dosing cycles. 3. The dosing regimen according to embodiment 1 or embodiment 2, wherein if the subject exhibits a clinical response that may be a complete response (CR) after the first dosing cycle, the dosing regimen includes additional dosing cycles. 4. The dosing regimen according to any one of embodiments 1 to 3, wherein if the subject exhibits a clinical response after a dosing cycle and subsequently shows disease progression, the dosing regimen includes additional dosing cycles. 5. The dosing regimen according to any one of embodiments 1 to 4, wherein the dosing regimen includes between 1 dosing cycle and 5 dosing cycles. 6. The dosing regimen according to any one of embodiments 1 to 5, wherein the subject undergoes a lymphodepletion process prior to each dosing cycle. 7. The dosing regimen according to any one of embodiments 1 to 6, wherein each dosing cycle is between about 14 days and about 35 days. 8. The dosing regimen according to any one of embodiments 1 to 7, wherein each dosing cycle is about 21 days. 9. The dosing regimen according to any one of embodiments 1 to 7, wherein each dosing cycle is about 28 days. 10. The dosing regimen according to any one of embodiments 1 to 9, wherein the lymphodepletion process includes at least 2 doses of cyclophosphamide and at least 2 doses of fludarabine. 11. The lymphocyte depletion process comprises 3 doses of cyclophosphamide and 3 doses of fludarabine, wherein the first dose of cyclophosphamide and fludarabine is administered 5 days before the start of the first administration cycle, the second dose of cyclophosphamide and fludarabine is administered 4 days before the start of the first administration cycle, and the third dose of cyclophosphamide and fludarabine is administered 3 days before the start of the first administration cycle, the dosing regimen according to embodiment 10. 12. The dosing regimen according to embodiment 10 or 11, wherein about 2 days are given for interruption between the third dose of cyclophosphamide and fludarabine and the start of the administration cycle. 13. Cyclophosphamide is administered in an amount between about 100 mg / m 2 and about 600 mg / m 2 , and fludarabine is administered in an amount between about 10 mg / m 2 and about 60 mg / m 2 , the dosing regimen according to any one of embodiments 10 to 12. 14. Cyclophosphamide is administered in an amount between about 200 mg / m 2 and about 600 mg / m 2 , and fludarabine is administered in an amount between about 20 mg / m 2 and about 40 mg / m 2 , the dosing regimen according to any one of embodiments 10 to 13. 15. Cyclophosphamide is administered in an amount of about 500 mg / m 2 , and fludarabine is administered in an amount of about 30 mg / m 2 , the dosing regimen according to any one of embodiments 10 to 14. 16. The dosing regimen according to any one of embodiments 1 to 15, further comprising administration of a therapeutic agent targeting CD20. 17. The dosing regimen according to any one of embodiments 1 to 15, wherein the subject is administered a therapeutic agent targeting CD20. 18. The dosing regimen according to embodiment 16 or 17, wherein the therapeutic agent is an anti-CD20 monoclonal antibody. 19. The dosing regimen according to embodiment 18, wherein the anti-CD20 antibody is rituximab. 20. The therapeutic agent is administered in an amount between about 150 mg / m 2 and about 500 mg / m 2 for the dosing regimen according to any one of embodiments 16-19. 21. The therapeutic agent is administered in an amount of about 375 mg / m 2 for the dosing regimen according to any one of embodiments 16-20. 22. The therapeutic agent is administered to the subject at least once, and at least one administration is at least 2 days prior to the administration of the first dose of the dosing cycle for the dosing regimen according to any one of embodiments 16-21. 23. The therapeutic agent is administered to the subject once 3 days prior to the administration of the first dose of the dosing cycle for the dosing regimen according to any one of embodiments 16-22. 24. The genetically engineered NK cells of the first dose are administered to the subject before the subject's natural immune cell population has recovered from the lymphodepletion process, and the genetically engineered NK cells of the first and second doses may be administered to the subject before the subject's natural immune cell population has recovered from the lymphodepletion process for the dosing regimen according to any one of embodiments 1-23. 25. The genetically engineered NK cells of the first dose are administered to the subject about 2-5 days after the completion of the lymphodepletion process for the dosing regimen according to any one of embodiments 1-24. 26. The cancer is a blood cancer for the dosing regimen according to any one of embodiments 1-25. 27. The cancer is leukemia or lymphoma for the dosing regimen according to any one of embodiments 1-26. 28. The cancer is a B cell cancer for the dosing regimen according to any one of embodiments 1-27. 29. The cancer is non-Hodgkin lymphoma (NHL) for the dosing regimen according to any one of embodiments 1-28. 30. The cancer is large cell type B cell lymphoma (LBCL), which may be aggressive LBCL, for the dosing regimen according to any one of embodiments 1-29. 31. The dosing regimen according to any one of Embodiments 1 to 30, wherein the cancer is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia (MW), or B-cell acute lymphoblastic leukemia (B-ALL). 32. The dosing regimen according to any one of Embodiments 1 to 28, wherein the cancer is chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). 33. The dosing regimen according to any one of Embodiments 1 to 32, wherein the cancer is relapsed / refractory (R / R) cancer. 34. The dosing regimen according to any one of Embodiments 1 to 33, wherein the subject has peripheral blood blasts that are less than or equal to 5%. 35. The dosing regimen according to any one of Embodiments 1 to 34, wherein the subject has received at least 1 line of prior treatment but not more than 7 lines of prior treatment, and the subject may have received at least 1 line of prior treatment but not more than 4 lines of prior treatment. 36. The dosing regimen according to any one of Embodiments 1 to 35, wherein the subject has received at least 1 line of prior treatment. 37. The dosing regimen according to any one of Embodiments 1 to 36, wherein the subject has received at least 2 lines of prior treatment. 38. The dosing regimen according to any one of Embodiments 35 to 37, wherein the lines of prior treatment include an anti-CD20 monoclonal antibody and cytotoxic chemotherapy, and the cytotoxic treatment may be an anthracycline. 39. The dosing regimen according to any one of Embodiments 35 to 38, wherein the lines of prior treatment include chimeric antigen receptor-expressing T (CAR-T) cells, and the lines of prior treatment may include autologous anti-CD19 CAR T cells. 40. The dosing regimen according to any one of Embodiments 35 to 38, wherein the lines of prior treatment do not include CAR T cells and do not include autologous anti-CD19 CAR T cells. 41. The dosing regimen according to any one of embodiments 35-40, wherein the prior treatment line includes an inhibitor of Bruton's tyrosine kinase (BTKi), and the BTKi may be ibrutinib. 42. The dosing regimen according to any one of embodiments 35-41, wherein the prior treatment line includes an inhibitor of Bcl-2, and the Bcl-2 inhibitor may be venetoclax. 43. The dosing regimen according to any one of embodiments 1-42, wherein the second and third doses of genetically engineered NK cells are administered to the subject within about 21 days of the first time point. 44. The dosing regimen according to any one of embodiments 1-43, wherein the second and third doses of genetically engineered NK cells are administered to the subject within about 14 days after the first time point. 45. The CAR is: (a) an antigen-binding portion targeting CD19; (b) a transmembrane domain; and (c) an intracellular signaling domain comprising an OX40 domain and a CD3 zeta domain The dosing regimen according to any one of embodiments 1-44. 46. The antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL): VH comprises CDR-1, CDR-2, and CDR-3 each comprising the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47; VL comprises CDR-1, CDR-2, and CDR-3 each comprising the amino acid sequences set forth in SEQ ID NOs: 48, 49, and 16; VH comprises CDR-1, CDR-2, and CDR-3 each comprising the amino acid sequences set forth in SEQ ID NOs: 50, 23, and 24; VL comprises CDR-1, CDR-2, and CDR-3 each comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16; VH comprises the amino acid sequence set forth in SEQ ID NO: 21 and / or VL comprises the amino acid sequence set forth in SEQ ID NO: 13; and / or The antigen-binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 6 The dosing regimen according to embodiment 45. 47. The dosing regimen according to any one of embodiments 1 to 46, wherein the genetically engineered NK cells are also engineered to express membrane-bound interleukin 15 (mbIL15). 48. The dosing regimen according to embodiment 47, wherein the mbIL15 has at least about 95% sequence identity with SEQ ID NO: 44. 49. The dosing regimen according to any one of embodiments 1 to 48, wherein the dosing regimen does not result in cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) / neurotoxicity, and / or graft-versus-host disease. 50. The dosing regimen according to any one of embodiments 1 to 49, wherein the engineered NK cells are allogeneic with respect to the subject. 51. The dosing regimen according to any one of embodiments 1 to 50, wherein the subject has a 158V / 158V CD16 genotype. 52. The dosing regimen according to any one of embodiments 1 to 51, wherein the subject has a 158F / 158F CD16 genotype. 53. A dosing regimen for cancer immunotherapy comprising at least a first dosing cycle, wherein: The first dosing cycle comprises a first dose of genetically engineered natural killer (NK) cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells. The first dose is administered to a subject in need of cancer immunotherapy at a first time point. The second dose is administered to the subject between 5 and 10 days after the first time point. The third dose is administered to the subject between 5 and 10 days after the second dose. Each of the first, second, and third doses comprises at least 1.0×10 9 NK cells. At least a portion of the engineered NK cells are engineered to express a chimeric antigen receptor (CAR) directed against the CD19 tumor marker. The first dosing cycle is initiated after the subject undergoes a lymphodepletion process comprising at least two doses of cyclophosphamide and fludarabine. An anti-CD20 antibody is administered during a lymphocyte depletion process, Dosage regimen. 54. The dosage regimen according to embodiment 53, wherein a first dosing cycle follows one or more additional dosing cycles. 55. If the subject exhibits a clinical response that may be a complete response (CR) after the first dosing cycle, the dosage regimen is the dosage regimen according to embodiment 53 or embodiment 54, which includes additional dosing cycles. 56. If the subject exhibits a clinical response after a dosing cycle and subsequently shows disease progression, the dosage regimen is the dosage regimen according to any one of embodiments 53 to 55, which includes additional dosing cycles. 57. The dosage regimen according to any one of embodiments 53 to 56, wherein the dosage regimen includes between one dosing cycle and five dosing cycles. 58. The dosage regimen according to any one of embodiments 53 to 57, wherein the subject undergoes a lymphocyte depletion process prior to each dosing cycle. 59. Each of the three doses of NK cells contains about 1.5×10 9 NK cells, and the dosage regimen is the dosage regimen according to any one of embodiments 1 to 58. 60. The lymphocyte depletion process includes three doses of cyclophosphamide and three doses of fludarabine, The first dose of cyclophosphamide and fludarabine is administered 5 days before the start of the dosing cycle, The second dose of cyclophosphamide and fludarabine is administered 4 days before the start of the dosing cycle, The third dose of cyclophosphamide and fludarabine is administered 3 days before the start of the dosing cycle, The anti-CD20 antibody is administered on the same day as the third dose of cyclophosphamide and fludarabine, The dosage regimen according to any one of embodiments 53 to 59. 61. Cyclophosphamide is administered in an amount between about 300 mg / m 2 and about 600 mg / m 2 and fludarabine is administered in an amount between about 20 mg / m 2 and about 40 mg / m 2administered in an amount between, and the anti-CD20 antibody is about 350 mg / m 2 and about 425 mg / m 2 administered in an amount between, and each of the three doses of NK cells contains at least 1.5×10 9 NK cells, and the dosing regimen according to any one of embodiments 53 to 60. 62. The dosing regimen according to any one of embodiments 53 to 61, wherein the anti-CD20 antibody comprises rituximab or obinutuzumab. 63. The dosing regimen according to any one of embodiments 1 to 62, wherein the cancer cells do not express CD58 or express a mutant form of CD58. 64. The dosing regimen according to any one of embodiments 1 to 63, wherein it is determined that the cancer cells do not express CD58 or express a mutant form of CD58 prior to administration of the first dosing cycle to the subject. 65. The dosing regimen according to any one of embodiments 1 to 64, wherein the subject is selected for treatment with a dosing regimen based on cancer cells that exhibit a CD58 deficiency or mutation prior to administration of the first dosing cycle to the subject. 66. The dosing regimen according to any one of embodiments 1 to 65, wherein each dosing cycle of a dose is administered to the subject ex vivo, and each dosing cycle of each dose may be administered to the subject ex vivo. 67. The dosing regimen according to any one of embodiments 1 to 66, wherein the overall response rate (ORR) among the subjects treated according to the dosing regimen is at least about 50%, at least about 60%, at least about 70%, or at least about 80%. 68. The dosing regimen according to any one of embodiments 1 to 67, wherein at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the subjects treated according to the dosing regimen exhibit a complete response (CR). 69. A method of treating cancer, comprising administering to a subject having cancer genetically engineered natural killer (NK) cells that express a chimeric antigen receptor (CAR) directed against an antigen associated with or expressed by the cancer cells: the cancer does not express CD58 or expresses a mutant form of CD58; a method for a subject to relapse and / or be refractory in genetically engineered T cells expressing a chimeric antigen receptor (CAR) directed against an antigen. 70. The method according to embodiment 69, wherein the antigen is CD19. 71. A method for treating cancer, comprising administering genetically engineered natural killer (NK) cells expressing a chimeric antigen receptor (CAR) directed against CD19 to a subject having cancer, wherein the cancer does not express CD58 or expresses a mutant form of CD58. 72. Determining whether the cancer does not express CD58 or expresses a mutant form of CD58; identifying a subject as having cancer that does not express CD58 or expresses a mutant form of CD58; and / or selecting the identified subject for treatment with the genetically engineered NK cells The method according to any one of embodiments 69 to 71, further comprising. 73. The method according to embodiment 71 or embodiment 72, wherein the subject has been previously treated with genetically engineered T cells expressing a CAR directed against CD19 for cancer, and the subject may be refractory and / or relapsed in the genetically engineered T cells. 74. The genetically engineered NK cells are administered in a dosing regimen comprising at least a first dosing cycle, the first dosing cycle comprising a first dose of genetically engineered NK cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, the first dose is administered to the subject at a first time point, the second dose is administered to the subject between 5 and 10 days after the first time point, the third dose is administered to the subject between 5 and 10 days after the second dose; each of the first, second, and third doses comprises at least about 1.5×10 9 cells of NK, and optionally at least about 1.5×10 9 cells of CAR-expressing NK. The method according to any one of Embodiments 69 to 73. 75. A method for treating cancer, comprising: (a) selecting a subject for treatment of cancer when the cancer cells do not express CD58 or express a mutant form of CD58; (b) administering to the selected subject at least a first administration cycle, the first administration cycle comprising administering a first dose of genetically engineered natural killer (NK) cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, comprising the first dose being administered to a subject in need of cancer immunotherapy at a first time point, the second dose being administered to the subject between 5 and 10 days after the first time point, the third dose being administered to the subject between 5 and 10 days after the second dose; each of the first, second, and third doses comprising at least about 1.5×10 9 NK cells, at least a portion of the genetically engineered NK cells being engineered to express a chimeric antigen receptor (CAR) directed against the CD19 tumor marker, a method. 76. The method according to Embodiment 74 or Embodiment 75, wherein the first administration cycle is initiated after the subject has undergone a lymphocyte depletion process so as to reduce the number of natural immune cells. 77. A method for treating cancer, comprising: administering to a subject having cancer a lymphocyte depletion regimen comprising at least two doses of cyclophosphamide and at least two doses of fludarabine; administering to the subject an administration cycle comprising at least a first, second, and third dose of genetically engineered NK cells, comprising the first dose of genetically engineered NK cells being administered to the subject after the final dose of fludarabine, the second dose of genetically engineered NK cells being administered to the subject between 6 and 8 days after the first dose of genetically engineered NK cells, The genetically engineered NK cells of the third dose are administered to the subject between 6 and 8 days after the genetically engineered NK cells of the second dose, Each of the first, second, and third doses of genetically engineered NK cells contains approximately 1.5×10 9 NK cells, The genetically engineered NK cells are allogeneic to the subject and are engineered to express a chimeric antigen receptor (CAR) that binds to CD19, Method. 78. A method for treating cancer, administering to a subject having cancer a lymphodepletion regimen comprising at least two doses of cyclophosphamide and at least two doses of fludarabine, administering to the subject an agent that binds to CD20, administering to the subject an administration cycle comprising at least first, second, and third doses of genetically engineered NK cells comprising, The genetically engineered NK cells of the first dose are administered to the subject after the final dose of fludarabine, The genetically engineered NK cells of the second dose are administered to the subject between 6 and 8 days after the genetically engineered NK cells of the first dose, The genetically engineered NK cells of the third dose are administered to the subject between 6 and 8 days after the genetically engineered NK cells of the second dose, Each of the first, second, and third doses of genetically engineered NK cells contains at least 1.0×10 9 NK cells, The genetically engineered NK cells are allogeneic to the subject and are engineered to express a chimeric antigen receptor (CAR) that binds to CD19, Method. 79. A method according to any one of embodiments 74, 76, and 78, wherein each of the first, second, and third doses contains at least 1.5×10 9 cells, and optionally at least 1.5×10 9 CAR-expressing NK cells. 80. The lymphocyte depletion regimen includes 3 doses of cyclophosphamide and 3 doses of fludarabine, wherein the first dose of cyclophosphamide and fludarabine are administered 5 days before the first dose of genetically engineered NK cells, the second dose of cyclophosphamide and fludarabine are administered 4 days before the genetically engineered NK cells, and the third dose of cyclophosphamide and fludarabine are administered 3 days before the start of the administration cycle, according to any one of embodiments 76 to 79. 81. Cyclophosphamide is administered in an amount between about 100 mg / m 2 and about 600 mg / m 2 and fludarabine is administered in an amount between about 20 mg / m 2 and about 40 mg / m 2 according to any one of embodiments 77 to 80. 82. Cyclophosphamide is administered in an amount of about 500 mg / m 2 and fludarabine is administered in an amount of about 30 mg / m 2 according to any one of embodiments 77 to 81. 83. The agent that binds to CD20 is an anti-CD20 monoclonal antibody, and the anti-CD20 monoclonal antibody is administered 3 days before the first dose of genetically engineered NK cells is administered to the subject, and the anti-CD20 monoclonal antibody is administered in an amount between about 350 mg / m 2 and about 425 mg / m 2 according to any one of embodiments 77 to 82. 84. The anti-CD20 monoclonal antibody includes rituximab or obinutuzumab, according to the method of embodiment 83. 85. The cancer is a blood cancer, according to any one of embodiments 69 to 84. 86. The cancer is leukemia or lymphoma, according to any one of embodiments 69 to 85. 87. The cancer is a B-cell cancer, according to any one of embodiments 69 to 86. 88. The cancer is non-Hodgkin lymphoma (NHL), according to any one of embodiments 69 to 87. 89. The method according to any one of embodiments 69 to 88, wherein the cancer is large cell type B cell lymphoma (LBCL), which may also be aggressive LBCL. 90. The method according to any one of embodiments 69 to 89, wherein the cancer is diffuse large cell type B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia (MW), or B cell acute lymphoblastic leukemia (B-ALL). 91. The method according to any one of embodiments 69 to 87, wherein the cancer is chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). 92. The method according to any one of embodiments 69 to 91, wherein the cancer is relapsed / refractory (R / R) cancer. 93. The method according to any one of embodiments 69 to 92, wherein the subject has peripheral blood blasts that are less than or equal to 5%. 94. The CAR is: (a) an antigen-binding portion that targets CD19; (b) a transmembrane domain; and (c) an intracellular signaling domain comprising an OX40 domain and a CD3 zeta domain The method according to any one of embodiments 69 to 93. 95. The antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL): VH comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively; VL comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 48, 49, and 16, respectively; VH comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 50, 23, and 24, respectively; VL comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively; VH comprises the amino acid sequence set forth in SEQ ID NO: 21, and / or VL comprises the amino acid sequence set forth in SEQ ID NO: 13; and / or The antigen-binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 6 The method according to embodiment 94 96. The method according to any one of embodiments 69 - 95, wherein the genetically engineered NK cells are also engineered to express membrane-bound interleukin-15 (mbIL15). 97. The method according to embodiment 96, wherein mbIL15 has at least about 95% sequence identity with SEQ ID NO: 44 98. The method according to any one of embodiments 74 - 97, wherein administration in the dosing cycle does not result in cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) / neurotoxicity, and / or graft-versus-host disease 99. The method according to any one of embodiments 75 - 98, wherein the cancer cells do not express CD58 or express a mutant form of CD58 100. (a) Prior to administration of the first dose of genetically engineered NK cells to a subject, it is determined that the cancer cells do not express CD58 or express a mutant form of CD58; and / or (b) The method further comprising selecting a subject for treatment based on cancer cells showing a deficiency or mutation in CD58, according to any one of embodiments 77 - 99 101. The method according to any one of embodiments 74 - 100, wherein one dose of the dosing cycle is administered to the subject ex vivo, and each dose of each dosing cycle may be administered to the subject ex vivo 102. Use of a population of engineered NK cells expressing a chimeric antigen receptor targeting CD19 for treating cancer by administration of a dosing cycle comprising at least the first, second, and third doses of said genetically engineered NK cells, wherein The first dose of genetically engineered NK cells is administered to the subject after administration of a final dose of lymphodepletion regimen comprising at least 2 doses of cyclophosphamide and at least 2 doses of fludarabine The second dose of genetically engineered NK cells is administered to the subject between 6 - 8 days after the first dose of genetically engineered NK cells The genetically engineered NK cells of the third dose are administered to the subject between 6 and 8 days after the genetically engineered NK cells of the second dose, Each of the first, second, and third doses of genetically engineered NK cells contains approximately 1.5 × 10 9 genetically engineered NK cells, for use. 103. Use of a population of engineered NK cells expressing a chimeric antigen receptor targeting CD19 for treating cancer by administration of an administration cycle comprising at least the first, second, and third doses of said genetically engineered NK cells, The genetically engineered NK cells of the first dose are administered to the subject after administration of a final dose of lymphodepletion regimen comprising at least 2 doses of cyclophosphamide and at least 2 doses of fludarabine, The genetically engineered NK cells of the first dose are administered to the subject after administration of an agent that binds to CD20, The genetically engineered NK cells of the second dose are administered to the subject between 6 and 8 days after the first dose, The genetically engineered NK cells of the third dose are administered to the subject between 6 and 8 days after the second dose, Each of the first, second, and third doses of genetically engineered NK cells contains approximately 1.5 × 10 9 genetically engineered NK cells, for use. 104. Each of the first, second, and third doses of genetically engineered NK cells contains at least 1.5 × 10 9 genetically engineered NK cells, and optionally at least 1.5 × 10 9 CAR-expressing NK cells, for the use of Embodiment 102 or Embodiment 103. 105. The use according to any one of embodiments 102 to 104, wherein the lymphocyte depletion regimen comprises 3 doses of cyclophosphamide and 3 doses of fludarabine, and the first dose of cyclophosphamide and fludarabine are administered 5 days before the first dose of genetically engineered NK cells, the second dose of cyclophosphamide and fludarabine are administered 4 days before the first dose of genetically engineered NK cells, and the third dose of cyclophosphamide and fludarabine are administered 3 days before the first dose of genetically engineered NK cells. 106. The use according to any one of embodiments 102 to 105, wherein the agent that binds to CD20 is administered 3 days before the first dose of genetically engineered NK cells. 107. Cyclophosphamide is administered in an amount between about 100 mg / m 2 and about 600 mg / m 2 and fludarabine is administered in an amount between about 10 mg / m 2 and about 60 mg / m 2 The use according to any one of embodiments 102 to 106. 108. Cyclophosphamide is administered in an amount between about 300 mg / m 2 and about 600 mg / m 2 and fludarabine is administered in an amount between about 20 mg / m 2 and about 40 mg / m 2 The use according to any one of embodiments 102 to 107. 109. Cyclophosphamide is administered in an amount of about 500 mg / m 2 and fludarabine is administered in an amount of about 30 mg / m 2 The use according to any one of embodiments 102 to 108. 110. The agent that binds to CD20 is an anti-CD20 monoclonal antibody selected from rituximab, obinutuzumab, and combinations thereof, and the anti-CD20 monoclonal antibody is administered in an amount between about 350 mg / m 2 and about 425 mg / m 2 The use according to any one of embodiments 102 to 119. 111. The anti-CD20 monoclonal antibody comprises rituximab, and rituximab is about 375 mg / m2 The use according to embodiment 110, administered in the amount of. 112. The use according to any one of embodiments 102 to 111, wherein the cancer is a blood cancer. 113. The use according to any one of embodiments 102 to 112, wherein the cancer is a B cell cancer. 114. The use according to any one of embodiments 120 to 113, wherein the cancer is leukemia or lymphoma. 115. The use according to any one of embodiments 102 to 114, wherein the cancer is non-Hodgkin lymphoma (NHL). 116. The use according to any one of embodiments 102 to 115, wherein the cancer is large cell type B cell lymphoma (LBCL), which may be aggressive LBCL. 117. The use according to any one of embodiments 102 to 116, wherein the cancer is diffuse large cell type B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia (MW), or B cell acute lymphoblastic leukemia (B-ALL). 118. The use according to any one of embodiments 102 to 114, wherein the cancer is chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). 119. The use according to any one of embodiments 102 to 118, wherein the cancer is relapsed / refractory (R / R) cancer. 120. The use according to any one of embodiments 102 to 119, wherein the subject has peripheral blood blasts less than or equal to 5%. 121. The CAR is: (a) an antigen-binding portion targeting CD19; (b) a transmembrane domain; and (c) an intracellular signaling domain comprising an OX40 domain and a CD3 zeta domain The use according to any one of embodiments 102 to 120, comprising. 122. The antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL): VH comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47; VL comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 48, 49, and 16; VH comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 50, 23, and 24; VL comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16; VH comprises the amino acid sequence set forth in SEQ ID NO: 21 and / or VL comprises the amino acid sequence set forth in SEQ ID NO: 13; and / or The antigen-binding domain is an scFv comprising the amino acid sequence of SEQ ID NO: 6. Use according to embodiment 121. 123. Use according to any one of embodiments 102 to 122, wherein the genetically engineered NK cells are also engineered to express membrane-bound interleukin 15 (mbIL15). 124. Use according to embodiment 123, wherein mbIL15 has at least about 95% sequence identity with SEQ ID NO: 44. 125. Use according to any one of embodiments 102 to 124, wherein the administration cycle does not result in cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) / neurotoxicity, and / or graft-versus-host disease. 126. Use of a population of engineered NK cells expressing a chimeric antigen receptor targeting CD19 for treating cancer in a subject by intravenous administration of an administration cycle comprising at least three consecutive doses of genetically engineered NK cells, wherein the first dose of genetically engineered NK cells is administered to the subject at a first time point and comprises at least 1.5×10 9 engineered NK cells, the second dose of genetically engineered NK cells is administered to the subject between 6 and 8 days after the first dose of genetically engineered NK cells and comprises at least 1.5×10 9 engineered NK cells, The genetically engineered NK cells of the third dose are administered to the subject between 6 and 8 days after the genetically engineered NK cells of the second dose and contain at least 1.5×10 9 engineered NK cells, wherein the engineered NK cells express a CD19 CAR having at least 95% sequence identity with SEQ ID NO: 43. Use. 127. The use according to embodiment 126, further comprising administering at least one agent targeting CD20 before the genetically engineered NK cells of the first dose. 128. The use according to embodiment 127, wherein the agent targeting CD20 comprises rituximab. 129. Use of genetically engineered natural killer (NK) cells expressing a chimeric antigen receptor (CAR) directed against an antigen associated with or expressed by cancer cells of a cancer for treating a subject having the cancer, wherein: the cancer does not express CD58 or expresses a mutant form of CD58; the subject is recurrent and / or refractory to genetically engineered T cells expressing a CAR directed against the antigen. Use. 130. Use of genetically engineered natural killer (NK) cells expressing a chimeric antigen receptor (CAR) directed against CD19 for treating a subject having cancer, wherein the cancer does not express CD58 or expresses a mutant form of CD58. Use. 131. The genetically engineered NK cells are for administration in a dosing regimen comprising at least a first dosing cycle, the first dosing cycle comprising a first dose of genetically engineered NK cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, the first dose being administered to the subject at a first time point, the second dose being administered to the subject between 5 and 10 days after the first time point, the third dose being administered to the subject between 5 and 10 days after the second dose; each of the first, second, and third doses being at least about 1.5×10 9Individual NK cells, optionally at least about 1.5×10 9 CAR-expressing NK cells, and The use according to embodiment 129 or embodiment 130.

Examples

[0173] The following is a non-limiting description of the experimental methods and materials used in the examples disclosed below.

[0174] [Example 1] The first dosing regimen for NK cell immunotherapy As discussed in more detail herein, certain cancer types express elevated selection markers. In some embodiments, the cytotoxic receptor construct is generated by the sequences disclosed herein to specifically target a given cancer. For example, many cancers express elevated levels of CD19, including non-Hodgkin lymphoma. Thus, in some embodiments, as discussed in detail above, a CD19-targeting CAR construct is provided. In some embodiments, the polynucleotides encoding these constructs are engineered to express mbIL15 bicistronically (e.g., SEQ ID NO: 1 or SEQ ID NO: 53). Dosing regimens were tested and the efficacy of cells expressing such constructs was evaluated. The dosing regimen used NK cells (derived from healthy donors) engineered to express CD19 CAR. In some embodiments, the engineered NK cells express a cytotoxic receptor encoded by SEQ ID NO: 3 (including degenerate or codon-optimized versions of SEQ ID NO: 3). In some embodiments, the engineered NK cells express a cytotoxic receptor comprising the amino acid sequence of SEQ ID NO: 43 (e.g., SEQ ID NO: 4) and, optionally, mbIL15 comprising the amino acid sequence of SEQ ID NO: 44 (e.g., SEQ ID NO: 40).

[0175] Design a dosing regimen, administer three times in a 28-day dosing cycle, and evaluate engineered NK cells for treating non-Hodgkin lymphoma. All subjects had relapsed / refractory CD19+ B-cell malignancies, including large B-cell lymphoma (LBCL, including diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma grade 3b (FL3b)), mantle cell lymphoma (MCL), follicular lymphoma (FL), and marginal zone lymphoma (MZL); had received two or more prior lines of treatment; had an ECOG status of 0 or 1; and were naive to CAR T-cell therapy. The dosing cycle was preceded by a conditioning phase during which subjects received lymphocyte depletion (cyclophosphamide (300 mg / m 2 ) and fludarabine (30 mg / m 2 ) on days -5, -4, and -3). On day 0, subjects received the first dose of 1×10 9 CD19 CAR-NK cells. Dose 2 was administered on day 7 and dose 3 on day 14. On day 28, outcome measures were evaluated.

[0176] Primary endpoints included the following: (1) the incidence, nature, and severity of treatment-related adverse events, as evaluated by adverse events defined as any unwanted unintended signs, including clinically significant abnormal laboratory findings, symptoms, or diseases, measured 30 days after the final dose of NK cells; and (2) the proportion of subjects experiencing dose-limiting toxicity (DLT) of NK cells, defined as an adverse event attributable to treatment occurring during cycle 1 and meeting protocol-specified criteria measured 28 days from the first dose of NK cells.

[0177] Secondary outcome measures included the following evaluations: (1) without limitation, maximum concentration (C max ), time to reach maximum concentration (T max ), area under the concentration-time curve (AUC), half-life (t 1 / 2Pharmacokinetic parameters in the context of the immune system, including the duration of CD19 CAR-NK cells in other target tissues such as peripheral blood and bone marrow; (2) Humoral and cellular immunogenicity against CD19 CAR-NK cells; (3) Changes in serum cytokine levels such as interferon gamma (IFN-γ) and other host responses to CD19 CAR-NK cells in the periphery; (4) Best overall response rate in the dose setting and safety lead-in cohort; and / or (5) Other anti-tumor measurements that may include duration of response (DOR), time to first response, time to best response, transplant bridge utilization rate, event-free survival (EFS), progression-free survival (PFS), and overall survival (OS) using standard disease-specific response evaluation criteria.

[0178] After evaluation of the primary evaluation items, CD19 CAR NK was determined to be sufficiently tolerant of the 1×10 9 dose (3 doses over 28 days). No DLT was identified. Myelosuppression (consistent with lymphopenia) was the most common high-level toxicity. One subject (out of 6 subjects) experienced a grade 1 infusion reaction with transient fever. CAR T-like cytotoxicity was not detected.

[0179] Figure 3 shows data summarizing the pre-response rates in six NHL patients treated according to this dosing regimen. Overall, more than 80% of NHL patients demonstrated a complete response (CR) or partial response (PR). Considering all NHL subtypes, three out of six subjects showed a complete response, and 100% of subjects had marginal zone lymphoma (MZL) and mantle cell lymphoma (MCL) that showed a complete response. Figures 4A - 4B show data from a 53-year-old male subject with extensive DLBCL that recurred after treatment with R-EPOCH (rituximab, etoposide phosphate, prednisone, vincristine sulfate (Oncovin®), cyclophosphamide, and doxorubicin hydrochloride (hydroxydaunorubicin)) and R-ICE (rituximab, ifosfamide, carboplatin, and etoposide phosphate). These data demonstrate engineered immune cells (e.g., NK cells) expressing a CD19-directed CAR that provide effective treatment of NHL, including various subtypes of NHL.

[0180] Four additional subjects were treated according to the same dosing regimen, except that each of the three doses contained 1.5×10 9 individual CD19 CAR-NK cells. The 1.5×10 9 cell dose was well tolerated, and no immune effector cell-associated neurotoxicity syndrome (ICANS) / neurotoxicity, GvHD, or DLT was observed. No treatment-related adverse events resulting in treatment discontinuation were observed. One subject showed a grade ≥3 infection. Myelosuppression consistent with lymphopenia was the most frequent grade ≥3 toxicity with the 1×10 9 cell dose and was manageable. A minority of patients experienced transient and manageable infusion-related events (fever within 8 hours resolving within 24 hours), but no association between symptoms and response was observed. Grade 3 CRS was not observed. Cytokine elevations were generally modest across all subjects, and no observations were made between serum cytokine elevations and clinical response (Figure 4C; for each cytokine, left is CR and right is non-CR).

[0181] Table E1 shows 1×109 individuals and 1.5×10 9 Data summarizing responses from subjects treated with cell doses of 1×10

[0182]

Table 1

[0183] The overall response rate (ORR) was 8 / 10 (80%) at cell dose levels of 1×10 9 individuals and 1.5×10 9 individuals, and 7 of these subjects achieved CR. Four of the 7 subjects achieved CR after a single dosing cycle, and 3 subjects (including 1 subject with FL treated at a cell dose level of 1×10 9 individuals) were deepened from an initial PR to CR after additional cycles. Six subjects received a consolidation cycle after achieving CR. The median interval between treatment cycles was 8 days, and lymphodepletion was provided at the start of each 28-day cycle. 40% of eligible patients received CD19 CAR-NK cells in the outpatient setting after the first cycle.

[0184] Follow-up analysis identified multiple patients showing response durability beyond 6 months, and 1 subject with follicular lymphoma (FL) treated at a dose of 1×10 9 individuals demonstrated a PR that was subsequently deepened to CR. Peak cell concentrations trended higher in patients achieving CR. Retreatments were planned for 2 subjects showing an initial clinical response and subsequent disease progression. Nearly all subjects receiving CD19 CAR-NK cells showed a decrease in tumor size. A summary of the clinical responses over time is shown by a swimmer plot in Figure 4D.

[0185] In summary, the data was consistent with the finding that continuous rounds of dosing cycles with CD19 CAR-NK cells were feasible and effective in achieving a clinical response.

[0186] [Example 2] Second dosing regimen for NK cell immunotherapy As discussed in more detail herein, certain cancer types express elevated selection markers. In some embodiments, the cytotoxic receptor constructs are generated by the sequences disclosed herein to specifically target a given cancer. For example, many cancers express elevated levels of CD19, including non-Hodgkin lymphoma (including various subtypes of NHL). Thus, in some embodiments, as discussed in detail above, a CD19-targeting CAR construct is provided. In some embodiments, the polynucleotides encoding these constructs are engineered to express mbIL15 bicistronically (e.g., SEQ ID NO: 1 or SEQ ID NO: 53). Dosing regimens were tested and the efficacy of cells expressing such constructs was evaluated. In some embodiments, the dosing regimen used NK cells (derived from healthy donors) engineered to express a CD19 CAR. In some embodiments, the engineered NK cells express a cytotoxic receptor encoded by SEQ ID NO: 3 (including degenerate or codon-optimized versions of SEQ ID NO: 3). In some embodiments, the engineered NK cells express a cytotoxic receptor (and optionally mbIL15 comprising the amino acid sequence of SEQ ID NO: 44 (e.g., SEQ ID NO: 40)) comprising the amino acid sequence of SEQ ID NO: 43 (e.g., SEQ ID NO: 4).

[0187] A dosing regimen was designed to evaluate engineered NK cells that were administered three times in 28-day dosing cycles to treat non-Hodgkin lymphoma (NHL), including LBCL (e.g., aggressive LBCL). Subjects with LBCL included those naive and experienced to CD19 CAR T cell therapy. The dosing cycle involved lymphodepletion of the subject during which cyclophosphamide (500 mg / m2 ) and fludarabine (30 mg / m 2 ) was preceded by a conditioning phase (using). On day 0, each subject received a first dose of 1.5 × 10 9 CD19 CAR-NK cells. Dose 2 was administered on day 7 and dose 3 on day 14 (both dose 2 and dose 3 were 1.5 × 10 9 CD19 CAR-NK cells). Approximately on day 28, outcome measures were evaluated.

[0188] The primary evaluation items include the following: (1) the incidence rate, nature, and severity of treatment-related adverse events, which are evaluated by adverse events defined as any unfavorable unintended signs, including clinically significant abnormal test findings, symptoms, or diseases measured 30 days after the final dose of NK cells; and (2) the proportion of subjects experiencing dose-limiting toxicity (DLT) of NK cells, which is defined as an adverse event resulting from the treatment occurring during cycle 1 and meets the protocol-specific criteria measured 28 days after the first dose of NK cells.

[0189] The secondary outcome measures include the following evaluations: (1) although not limited, maximum concentration (C max ), time to reach the maximum concentration (T max ), area under the concentration-time curve (AUC), half-life (t 1 / 2 ), and the duration of CD19 CAR-NK cells in other target tissues such as peripheral blood and bone marrow, pharmacokinetic parameters in the context of the immune system; (2) humoral immunogenicity and cellular immunogenicity against CD19 CAR-NK cells; (3) changes in serum cytokine levels such as interferon gamma (IFN-γ) and other host responses to CD19 CAR-NK cells in the periphery; (4) the best overall response rate in the dose setting and safety lead-in cohort; and / or (5) other anti-tumor measurements that may include duration of response (DOR), time to first response, time to best response, transplant bridge utilization rate, event-free survival (EFS), progression-free survival (PFS), and overall survival (OS) using standard disease-specific response evaluation criteria.

[0190] Administration of three doses of engineered NK cells expressing a CD19-targeting CAR and also expressing mbIL15 is thought to be preferably well tolerated and exhibit limited adverse events. Administration of three doses of engineered NK cells expressing a CD19-targeting CAR and also expressing mbIL15 is also thought to result in limited DLT. An increase in the concentration of cyclophosphamide is thought to enhance the anti-cancer effect of the engineered NK cells. The NK cells are thought to exhibit an extended half-life as well as an extended duration. The NK cells are thought to induce a limited host immune response and a clinically significant objective response rate (e.g., reduction in tumor burden).

[0191] [Example 3] Basics of the combination administration regimen Combination therapies using therapeutic agents with mechanisms of action that are not mutually exclusive (or mutually exclusive) may enable synergistic activity and enhancement of cancer immunotherapy. As discussed in more detail herein, certain cancer types express elevated selection markers. In some embodiments, the cytotoxic receptor construct is generated by the sequences disclosed herein to specifically target a given cancer. For example, many cancers express elevated levels of CD19, including non-Hodgkin lymphoma (including various subtypes of NHL). In some embodiments, an anti-CD20 antibody (or biologic follow-on) is utilized in combination with the engineered NK cells provided herein to enhance the antitumor effect. In some embodiments, the anti-CD20 antibody is rituximab, obinutuzumab, or a combination thereof. Anti-CD20 antibodies (e.g., monoclonal antibody-like rituximab or obinutuzumab) can function to affect the cytotoxic effect in various ways. For example, direct binding of the CD20 monoclonal antibody to tumor cells can initiate cross-linking of multiple CD20 molecules and result in cell death through induction of non-classical apoptosis. Similarly, antibody binding can result in activation of the complement system, leading to complement-dependent cytotoxicity. Furthermore, immune effector cells (e.g., NK cells) can recognize opsonized tumor cells via FcγRs expressed on the immune effector cells, thereby initiating antibody-dependent cell-mediated cytotoxicity (ADCC). Antibody-initiated complement activation also results in deposition of complement cleavage fragments and can enhance tumor killing through complement receptors, known as complement-enhanced ADCC. Additionally, FcγRs can contribute as a cross-linking platform to enhance antigen signaling in tumor cells.

[0192] To investigate the possible enhanced efficacy of the CD19-CD20 targeting combination therapy, the efficacy of the various compositions disclosed herein was tested in cell lines derived from B cell malignancies expressing CD19 and CD20. These include, as non-limiting examples, Raji lymphoblastoid cells (Burkitt lymphoma), DOHH-2 (follicular centroblastic / centrocytic lymphoma), and EHEB (B-CLL chronic lymphocytic leukemia). Cytotoxicity and ADCC were measured in 4-hour and extended assays when CD19 CAR NK cells were cultured with tumor cells in the presence or absence of anti-CD20 monoclonal antibodies, non-limiting examples of which are rituximab and obinutuzumab.

[0193] Figure 5A shows a plot of the cytotoxicity of various concentrations of rituximab against Raji tumor cells in vitro. Raji cells were cultured with the designated concentrations of rituximab over a 7-day culture period. Figure 5B shows similar data for obinutuzumab. These data indicate that both of these anti-CD20 antibodies are active against Raji tumor cells (as a non-limiting example of a B cell tumor), despite the type I mechanism of action of rituximab and the type II mechanism of action of obinutuzumab. Both antibodies demonstrated control of tumor growth over the assay, with only the lowest concentration (0.1 μg / mL) of rituximab allowing a slight increase in the number of tumor cells.

[0194] To investigate possible enhancements using combination therapies (e.g., synergy between agents), co-cultures were performed where Raji cells were co-cultured with anti-CD20 antibody and NK cells engineered to express the CD19 targeting CAR provided herein for 4 hours. Figure 6A shows summary data of the resulting cytotoxicity at effector:target (E:T) ratios of 1:1 (left), 1:2 (center), or 1:4 (right). In each dot plot, the leftmost group is the CD19 CAR NK cells and antibody isotype control, the center is the CD19 CAR NK cells and rituximab (1 μg / ml), and the right is the CD19 CAR NK cells and obinutuzumab (1 μg / ml). As shown in Figure 6A, combination therapy with CD19 CAR NK cells and either of two non-limiting examples of CD20 antibodies appears to increase the cytotoxic effect of the CD19 NK CAR against CD19 + / CD20 + target cells, such as the non-limiting example of Raji cells used herein. As the E:T ratio decreased, the contribution of the anti-CD20 antibody became more discernible. The isotype control group at an E:T of 1:4 was still able to kill ~50% of the target cells, but the addition of rituximab increased cytotoxicity to nearly the same level (~80%) as seen in the 1:2 E:T group. The addition of obinutuzumab surprisingly resulted in even further increased cytotoxicity of the CD19 NK CAR (~90%; nearly the same as the isotype control at 1:1 E:T. N = 3, representative donors are shown, E:1 = 1:4). * p < 0.05, ** p < 0.01, *** p < 0.001, independent t-test). These data indicate that the combined approach targeting CD19 and CD20 resulted in an unexpected enhancement of cytotoxicity.

[0195] Based on this initial data, longer-term experiments were conducted. Figure 6B shows the cytotoxicity profile of rituximab (10 μg / mL) in combination with CD19 CAR NK cells in a 4-day killing assay using Raji cells at an E:T ratio of 1:4. As expected, the isotype antibody control and CD19 CAR NK cells (due to the low E:T ratio) promoted Raji cell growth. Rituximab alone initially appeared to prevent Raji cell growth and increased slightly over a series of assays. The combination of CD19 CAR NK and rituximab showed the strongest control of tumor growth. Similar results were seen using obinutuzumab and CD19 CAR NK (see Figure 6C).

[0196] Interestingly, further enhancement of the combination was observed in 10-day co-culture assays when either low concentrations of rituximab or obinutuzumab were used in combination with CD19 CAR NK cells. These data are shown in Figures 7A and 7B, respectively. The antibodies were used at 0.01 μg / mL in these assays and the E:T ratio was 1:4. In contrast to the data in Figures 6B - 6C, the combination of antibody and NK cells resulted in a similar control of tumor growth compared to antibody alone, and these data showed a clear distinction between these groups. The combination of anti-CD20 antibody and CD19 CAR NK cells, respectively, clearly improved tumor control, demonstrating the potential for a synergistic effect between the two agents and providing a particular surprise with regard to the reduction in antibody concentration.

[0197] Further experiments were conducted to evaluate the efficacy of combination therapy when CD19 CAR NK cells were re-challenged with additional Raji cells. On day 3 after the start of co-culture at an E:T ratio of 1:1, 10×10 3Additional Raji cells were added to the CD19 CAR NK cells and cultured for an additional 6 days (9 days total) with either an isotype control antibody, rituximab, or obinutuzumab (each at 10 μg / ml). As shown in Figure 7C, both rituximab alone and rituximab in combination with CD19 CAR NK cells showed effective control of tumor cell proliferation, and the combination therapy provided slightly greater control than CD20 antibody alone. Similarly, in Figure 7D, both obinutuzumab alone and obinutuzumab in combination with CD19 CAR NK cells showed effective control of tumor cell proliferation.

[0198] In addition to initial experiments into the potential mechanisms of action, assays were performed using both normal rituximab and mutant rituximab. The mutant antibody has an asparagine residue within the constant region of human IgG1 that contains a putative glycosylation site replaced by a glutamine residue. This results in a non-glycosylated antibody that exhibits a defective ADCC function. Figure 8A shows the normalized cytotoxic effect of CD19 CAR NK when used in combination with normal rituximab (left) or mutant rituximab (right). This assay used Raji cells at an E:T ratio of 1:4 and rituximab at a concentration of 1 μg / mL. As seen in the figure, the cytotoxicity of CD19 CAR NK cells was unchanged, indicating that at least in this cell type, NK cells kill without relying on ADCC as a mechanism of action. In contrast, Figure 8B shows corresponding data (at various E:T ratios) using the EHEB tumor cell line. Some tumor cells are resistant to certain treatments based on their mechanism of action, for example, due to resistance to complement-dependent cytotoxicity. This means that knowledge of their mechanism can be useful for a given treatment in terms of performing in vitro screening prior to administering the treatment. Figure 8B shows data regarding ADCC disruption and cytotoxicity against EHEB cells. As shown, at each E:T ratio, the use of the mutant form of rituximab significantly decreased the degree of cytotoxicity shown by CD19 CAR NK cells. These data indicated that screening for various tumor sensitivities prior to the initiation of combination therapy can be useful in predicting the success of the treatment.

[0199] Further investigations were conducted regarding the mechanism of action useful in combination therapy. CD19 CAR NK cells were co-cultured at a 1:1 ratio with target cells (either Raji (lymphoblastoid), DOHH-2 (follicular lymphoma) or EHEB (B-lymphoblastoid) cells) in the presence of 1 μg / mL of anti-CD20 antibody (rituximab, the variant rituximab of Figure 8, or obinutuzumab), and LAMP-1 was stained after 4 hours. LAMP-1 (also known as CD107a) is a marker of NK cell degranulation. The increase in LAMP-1-expressing cells in the antibody groups was normalized to the isotype antibody groups. Figure 9A shows LAMP-1 expression from three donors after co-culture with one of various tumor cell types. These data indicate that obinutuzumab appears to enhance NK cell degranulation regardless of tumor type. NK cells were activated (in the absence of tumor cells) using a combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin. NK cell LAMP-1 expression was measured. As shown in Figure 9B, stimulation of NK cells by this combination of agents induced a substantial increase in LAMP-1 expression, with nearly 90% of NK cells expressing elevated levels of LAMP-1. Tumor cells, like PMA / ionomycin, appear to induce NK cell activation and degranulation, which is enhanced by the addition of anti-CD20 antibodies, such as obinutuzumab, in non-limiting examples, supporting the rationale and increased cytotoxicity of the combination therapy provided herein.

[0200] The non-limiting example CD20 targeting antibodies used in these experiments are known to be able to mediate antibody-dependent cellular cytotoxicity (ADCC), which is an important effector mechanism of NK cells and is promoted by the Fc receptor, CD16a. A common polymorphism is found in CD16 that affects the affinity of the Fc and can affect the ADCC response. Specifically, the polymorphism at position 158 of phenylalanine (158F) or valine (158V) results in CD16 with decreased or increased affinity for the Fc, respectively. To determine the potential impact of CD16 polymorphism on combination therapy, leukemia cells from two CLL subjects were incubated with CD19 CAR NK cells derived from donors expressing the 158V high-affinity CD16 variant (Figure 10A) or the 158F low-affinity CD16 variant (Figure 10B) in the presence of 1 μg / mL of anti-CD20 antibody (rituximab or mutant rituximab in Figure 8) or the corresponding irrelevant isotype control. The co-culture was for 4 hours at the indicated E:T ratio. The data shown are the average % cytotoxicity from two CLL subjects. The data are presented as the normalized frequency of remaining target cells, and the isotype control without target cells + effector cells = 100%. * p < 0.05, *** p < 0.001, independent t-test. As seen in Figures 10A and 10B, the addition of rituximab to CD19 CAR NK cells enhanced the cytotoxic activity of CD19 CAR NK cells compared to the control or when mutant rituximab was used. This increase occurred regardless of whether the CD16 variant was the high or low affinity variant. NK cells with the high-affinity CD16 variant showed greater overall cytotoxicity, but even NK cells with the low-affinity variant showed a significant increase in combination with rituximab across the isotype controls at E:T ratios of 1:1 and 1:2. Collectively, these data support the use of anti-CD20 antibodies in combination with CD19 CAR NK cells to provide an enhanced treatment for B cell malignancies.

[0201] [Example 4] Combination administration regimen for NK cell immunotherapy As discussed in more detail herein, certain cancer types express elevated selection markers. In some embodiments, cytotoxic receptor constructs are generated by the sequences disclosed herein to specifically target a given cancer. For example, many cancers express elevated levels of CD19, including non-Hodgkin lymphoma (including various subtypes of NHL). Thus, as discussed in detail above, in some embodiments, a CD19-targeting CAR construct is provided. In some embodiments, the polynucleotides encoding these constructs are engineered to express mbIL15 bicistronically (e.g., SEQ ID NO: 1 or SEQ ID NO: 53). Administration regimens were tested and the efficacy of cells expressing such constructs was evaluated. In some embodiments, the administration regimen uses NK cells engineered to express a CD19 CAR. This study uses haplo-matched NK cells as discussed herein, but in some embodiments, the NK cells are commercially available allogeneic engineered NK cells (from unrelated donors) and may be compared to a matched dose of engineered NK cells from a haplo-matched related donor. In some embodiments, the engineered NK cells express a cytotoxic receptor encoded by SEQ ID NO: 3 (including degenerate or codon-optimized versions of SEQ ID NO: 3). In some embodiments, the engineered NK cells express a cytotoxic receptor comprising the amino acid sequence of SEQ ID NO: 43 (e.g., SEQ ID NO: 4) (and optionally mbIL15 comprising the amino acid sequence of SEQ ID NO: 44 (e.g., SEQ ID NO: 40)). Further, tumor cells may express another marker at elevated levels that can be targeted by another agent (e.g., not CD19, targeted by the CARs described above). For example, some tumor types that show elevated CD19 also show elevated levels of CD20. Thus, in some embodiments, a CD19-targeting CAR NK cell population is administered in combination with a CD20-targeting agent, which can be an antibody (e.g., a monoclonal antibody such as rituximab or obinutuzumab) or a CD20-targeting CAR NK (and / or T) cell.

[0202] Design a dosing regimen that is administered three times over a 28-day dosing cycle and evaluate engineered NK cells that treat non-Hodgkin lymphoma, including LBCL (e.g., aggressive LBCL). The dosing regimen may be administered exogenously. Subjects include those who are naive and experienced to CD19 CAR T cell therapy. The dosing cycle is preceded by a conditioning phase during which the subject receives lymphodepletion (cyclophosphamide (500 mg / m 2 ) and fludarabine (30 mg / m 2 )). Three days prior to the first administration of CD19-targeting CAR NK cells (-3 days), the subject receives a single dose of anti-CD20 monoclonal antibody (e.g., rituximab at a dose of 375 mg / m 2 ). On day 0, each subject receives a first dose of 1.5×10 9 CD19 CAR-NK cells. Dose 2 is administered on day 7 and dose 3 is administered on day 14 (both dose 2 and dose 3 are 1.5×10 9 CD19 CAR-NK cells). Approximately on day 28, outcome measures were evaluated.

[0203] Primary evaluation items include the following: (1) Evaluate the incidence, nature, and severity of treatment-related adverse events. Adverse events are any unwanted unintended signs, including clinically significant abnormal test findings, symptoms, or diseases. This was measured 30 days after the final dose of NK cells. And (2) The proportion of subjects who experience DLT of NK cells due to treatment-induced adverse events occurring during cycle 1 and meeting the protocol-specified criteria for dose-limiting toxicity (DLT). Measured 28 days from the first dose of NK cells.

[0204] Secondary outcome measures include the following evaluations: (1) Although not limited, maximum concentration (C max ), time to reach maximum concentration (T max ), area under the concentration-time curve (AUC), half-life (t 1 / 2Pharmacokinetic parameters in the context of the immune system, including the duration of CD19 CAR-NK cells in other target tissues such as peripheral blood and bone marrow; (2) humoral and cellular immunogenicity against CD19 CAR-NK cells; (3) changes in serum cytokine levels such as interferon gamma (IFN-γ) and other host responses to CD19 CAR-NK cells peripherally; (4) best overall response rate in dose setting and safety lead-in cohorts; and / or (5) other anti-tumor measurements that may include duration of response (DOR), time to first response, time to best response, transplant bridge utilization rate, event-free survival (EFS), progression-free survival (PFS), and overall survival (OS) using standard disease-specific response evaluation criteria.

[0205] Administration of three doses of engineered NK cells expressing a CD19 targeting CAR and also expressing mbIL15 is thought to be preferably tolerable and exhibit limited adverse events. Administration of three doses of engineered NK cells expressing a CD19 targeting CAR and also expressing mbIL15 is also thought to result in limited DLT. An increase in the concentration of cyclophosphamide is thought to enhance the anti-cancer effect of the engineered NK cells. An increase in the concentration of cyclophosphamide is thought to enhance the anti-cancer effect of the engineered NK cells. The use of anti-CD20 antibodies is also thought to enhance the overall treatment outcome based on a different but synergistic mechanism of action compared to CAR NK cells. NK cells are thought to exhibit an extended half-life as well as an extended duration. NK cells are thought to induce a limited host immune response and a clinically significant objective response rate (e.g., reduction in tumor burden).

[0206] This is an in-flight example.

[0207] [Example 5] Treatment of CD58 knockout cells with CD19 CAR NK cells The efficacy of natural killer (NK) cells and T cells expressing non-limiting embodiments of CD19-directed CARs was evaluated in vivo and in vitro against target tumor cells knocked out for CD19, CD58, or both CD19 and CD58.

[0208] Natural killer (NK) cells were isolated from peripheral blood mononuclear cells (PBMCs) from healthy human donors by immunoaffinity. The isolated NK cells were transduced with a viral vector encoding a CD19-directed CAR and membrane-bound interleukin 15 (mbIL15; e.g., SEQ ID NO: 44), separated by T2A (e.g., SEQ ID NO: 36). The CD19-directed CAR contained an anti-CD19 scFv (e.g., SEQ ID NO: 6), a CD8 alpha hinge (e.g., SEQ ID NO: 28), and a transmembrane region (e.g., SEQ ID NO: 30), and an intracellular signaling domain containing an OX40 co-stimulatory region (e.g., SEQ ID NO: 32) and a CD3 zeta (e.g., SEQ ID NO: 34). Separately, T cells were isolated from PBMCs from healthy human donors by immunoaffinity and then transduced with a viral vector encoding a CD19-directed CAR. The CD19-directed CAR contained an FMC63 anti-CD19 scFv, a CD8 alpha transmembrane domain, and an intracellular signaling domain containing a 4-1BB co-stimulatory region and a CD3 zeta.

[0209] Nalm6 / Luc2.eGFP target tumor cells were stably knocked out for CD19 (CD19KO), CD58 (CD58KO), or both CD19 and CD58 (CD19KOCD58KO) by gene editing with Cas9 and commercially available CD19- and CD58-targeting gRNA sequences. Unedited Nalm6 / Luc2.eGFP cells expressing CD19 and CD58 (WT) served as controls. A. Characterization of CD19- and CD58-Knockout Tumor Cells

[0210] Flow cytometry analysis revealed that the cell surface expression of CD19 and CD58 was absent in cells stably knocked out of CD19 and CD58, respectively, including after extended cell culture (representative images are shown in Fig. 11A). CD2 binding was evaluated by flow cytometry in CD19KO, CD58KO, and CD19 / CD58KO cells. Briefly, target cells were first incubated with IgG (which enables CD2-CD58 binding) or a CD58 blocking antibody (which blocks CD2-CD58 binding), and then with biotinylated CD2-Fc fusion protein. CD2 binding was detected by staining with a PE anti-biotin antibody. CD2 binding was observed to be absent in CD58KO and CD19KO CD58KO cells, and in CD19KO and WT cells incubated in the presence of the CD58 blocking antibody (representative images are shown in Fig. 11B).

[0211] The cell surface expression of NKG2D ligands was evaluated by flow cytometry in target cells. Staining with anti-MICA, MICB, ULBP-1, ULBP-3, and ULBP-2 / 5 / 6 antibodies revealed that the expression of these ligands was equivalent among WT, CD19KO, CD58KO, and CD19KO CD58KO cells (data not shown). The expression of ULBP-4 was observed to be increased in CD19KO and CD58KO cells compared to CD19KO CD58KO cells and WT cells (representative images are shown in Fig. 11C).

[0212] Chromosomal analysis and cell identity assays of Nalm6 / Luc2.eGFP WT, CD19KO, CD58KO, and CD19KO CD58KO cells showed that WT cells were genetically similar to CD19KO, CD58KO, and CD19KO CD58KO cells.

[0213] Collectively, these data indicate that CD19 and CD58 can be stably knocked out in target tumor cells, either separately or in combination, without significant effects on all phenotypic and genotypic aspects. In vitro cytotoxicity of CD19 CAR NK and CD19 CAR T cells against BC CD19- and CD58-knockout tumor cells

[0214] CD19 CAR-expressing NK and T cell populations were incubated with target tumor cells at various effector:target (E:T) ratios, and cytotoxicity against the target cells was evaluated by BrightGlo® and Incucyte® assays at various time points.

[0215] CD19 knockout effectively eliminated the cytotoxicity of CD19 CAR T cells against target tumor cells in the 24-hour and 96-hour BrightGlo® assays (Figure 12A; the 96-hour time point is not shown). CD58 knockout was also observed to significantly reduce the cytotoxicity of CD19 CAR T cells at the 24-hour and 96-hour time points across almost all E:T ratios tested (Figure 12A; the 96-hour time point is not shown). These data are consistent with the findings of others that in vitro and in vivo CD19 CAR T cell efficacy is lost due to CD58 deficiency, and that CD58 deficiency or mutation is associated with poor outcomes in patients with large B cell lymphoma (LBCL) (Majzner et al., Blood (2020) 136(Suppl. 1):53-54).

[0216] In contrast, CD19 knockout was observed to significantly reduce the cytotoxicity of CD19 CAR NK cells against target tumor cells in the 24-hour BrightGlo® assay, and CD58KO was not observed to affect the cytotoxicity of CD19 CAR NK cells (Figure 12B).

[0217] The Incucyte® assay was performed to monitor target cell proliferation over time by co-culturing 20,000 target cells with CD19 CAR T cells or CD19 CAR NK cells at various E:T ratios for 72 hours. After 72 hours, an additional 10,000 target cells were added to the co-culture to re-challenge the CD19 CAR T cells or CD19 CAR NK cells. Target cell proliferation was monitored for an additional 72 hours after re-challenge. As with the BrightGlo® assay, CD19 CAR T cells demonstrated a substantial decrease in efficacy against CD19KO target cells at higher E:T ratios (e.g., 1:16), as well as a decrease in efficacy against CD58KO target cells, whereas only a decrease in efficacy against CD19KO cells was observed for CD19 CAR NK cells (Figures 13A - F). Further analysis confirmed that the sensitivity of target cells to CD19 CAR NK cells was not significantly affected by CD58 knockout at the time of re-challenge or 72 hours after re-challenge, whereas the sensitivity of target cells to CD19 CAR T cells was significantly decreased by CD58 knockout at both time points (data not shown). Supernatants from co-cultures of CD19 CAR T cells and target cells were harvested at the time of re-challenge, and T cell production of interferon gamma was evaluated by ELISA. Across various E:T ratios, CD19 CAR T cells produced significantly less interferon gamma in the presence of CD58KO target cells and only a small amount of interferon gamma in the presence of CD19 KO cells (Figure 14). C. In vivo cytotoxicity of CD19 CAR NK and CD19 CAR T cells against CD19- and CD58-knockout tumor cells

[0218] The efficacy of the CD19 CAR-expressing NK and T cell populations described in this example was measured against a mouse Nalm6 tumor model. On day - 1, NOD scid gamma (NSG) mice were injected with 2 × 10 5 WT or CD19KO luciferase-labeled Nalm6 cells or 1 × 10 6 CD58KO luciferase-labeled Nalm6 cells. Mice were then, on day 0, given a single dose of 1 × 107 individual CD19 CAR NK cells or 1.2×10 6 individual CD19 CAR T cells (or vehicle as a control) were injected. Tumor volume was monitored by bioluminescence imaging (BLI) for 28 days.

[0219] Substantial CD19KO tumor cell growth was observed in mice treated with CD19 CAR NK or CD19 CAR T cells (Figure 15A). CD19 CAR T cells showed a decrease in tumor control against CD58KO tumor cells compared to WT tumor cells (Figure 15B), while tumor control by CD19 CAR NK cells did not decrease against CD58KO tumor cells (Figure 15C).

[0220] In further in vivo experiments, NSG mice were injected with 2×10 5 individual WT or CD19KO luciferase-labeled Nalm6 cells or 1×10 6 individual CD58KO luciferase-labeled Nalm6 cells. Mice were subsequently injected with a single dose of 1×10 7 individual CD19 CAR NK cells or vehicle on days 0, 7, and 14 respectively. Tumor volume was monitored by BLI for 61 days. Consistent with other findings described herein, a decrease in tumor control by CD19 CAR NK cells against CD19KO tumor cells compared to WT tumor cells was observed (Figure 16). In particular, in this long-term experiment, it was observed that CD19 CAR NK cells showed significantly greater tumor control against CD58KO tumor cells compared to WT tumor cells (Figure 16).

[0221] Without wishing to be bound by theory, these data are consistent with the finding that the activation and anti-tumor activity of CD19 CAR NK cells are not lost due to CD58 deficiency or mutation in tumor cells.

[0222] Various combinations or sub - combinations of the specific features and aspects of the embodiments disclosed above may be made and are considered to be included within one or more of the present inventions. Further, the disclosure herein of any specific feature, aspect, method, property, characteristic, quality, attribute, element, etc. associated with an embodiment may be used in all other embodiments described herein. Thus, it is understood that the various features and aspects of the disclosed embodiments may be combined with or substituted for one another to form various modes of the disclosed invention. Accordingly, it is intended that the scope of the invention disclosed herein is not limited by the specific disclosed embodiments above. Further, the invention is subject to various modifications and alternative forms, and these specific examples are shown in the drawings and described in detail herein. However, the invention is not limited to the specific forms or methods disclosed, but on the contrary, the invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the described various embodiments and the appended claims. Any method disclosed herein need not be performed in the order recited. The methods disclosed herein include certain actions performed by a physician, but they may also include, explicitly or implicitly, the instructions of any third party for those actions. Further, the features or aspects of the present disclosure are described with respect to Markush groups, and those skilled in the art will recognize that the present disclosure is also described with respect to any individual member or subgroup of members of the Markush group.

[0223] The scope disclosed in this specification includes any and all overlaps, subranges, and combinations thereof. Words such as "up to", "at least", "greater than", "less than", "between", etc. include the recited numbers. Terms such as "about" or "approximately" preceding a number include the recited number. For example, "about 90%" includes "90%". In some embodiments, at least 95% sequence identity or homology includes 96%, 97%, 98%, 99%, and 100% sequence identity or homology to a reference sequence. Further, when a sequence is disclosed as "comprising" a nucleotide or amino acid sequence, such reference includes, unless otherwise specified, sequences that "comprise", "consist of", "consist essentially of" the recited sequence. Any headings or subheadings used herein are for organizational purposes and are not used to limit the scope of the embodiments disclosed herein.

[0224] For example, articles such as "a", "a", "the", etc. may mean one or more than one, unless indicated to the contrary or not apparent from the context. In the specification and claims, the phrase "and / or" as used herein is understood to mean "either or both" of the elements being combined. Multiple elements listed by "and / or" are to be construed in the same manner, i.e., "one or more" of the elements are combined. Other elements may be present in addition to those specifically identified by the "and / or" clause. In the specification and claims, when used herein, "or" is understood to have the same meaning as "and / or" as defined above. For example, when used in a listing of elements, "or" or "and / or" is construed as inclusive, i.e., a listing of elements and, optionally, at least one but more than one of any additional unlisted elements. Unless the terms clearly indicate to the contrary, for example, "only one of" or "exactly one of" refers to the inclusion of exactly one element of a number or listing of elements. Thus, claims that include "or" among one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, used in, or otherwise relevant to a given product or process, unless indicated to the contrary. Embodiments are provided where exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. Embodiments are provided where more than one or all of the group members are present in, used in, or otherwise relevant to a given product or process. Any one or more of the claims may be amended to expressly exclude any embodiment, aspect, feature, element, or characteristic, or any combination thereof. Any one or more of the claims may be amended to exclude any agent, composition, amount, dosage, route of administration, cell type, target, cell marker, antigen, targeting moiety, or any combination thereof.

[0225] All papers, including patent documents, scientific papers, and databases, referenced in this specification are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual paper were incorporated by reference individually. If the definitions set forth in this specification conflict with or are inconsistent with the definitions set forth in patents, applications, published applications, and other papers incorporated by reference herein, the definitions set forth in this specification shall control over the definitions incorporated by reference herein.

[0226]

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

[0227] In some embodiments, amino acid sequences corresponding to any of the nucleic acids disclosed herein (and / or included in the accompanying sequence listing) are provided, explaining the degeneracy of the nucleic acid code. Further, sequences (either nucleic acid or amino acid) that have functional similarity or equivalence, although different from those explicitly disclosed herein (and / or included in the accompanying sequence listing), are also contemplated within the scope of the present disclosure. The foregoing includes variants, truncations, substitutions, codon optimization, or other types of modifications.

[0228] According to some embodiments described herein, any array may be used, and truncations or variants of any array disclosed herein (and / or included in the accompanying sequence listing) may be used in any combination.

[0229] An electronic format sequence listing may be submitted as an attachment. Some of the sequences provided in the sequence listing may be designated as Artificial Sequences by being non-natural occurring fragments or portions of other sequences, including naturally occurring sequences. Some of the sequences provided in the sequence listing may be designated as Artificial Sequences by being combinations of sequences from different origins, such as humanized antibody sequences. Supplementary A is attached and incorporated herein by reference and provides sequence information for the nucleic acid and amino acid sequences provided herein.

Claims

**Claim 1** A dosing regimen for cancer immunotherapy comprising at least a first dosing cycle, wherein: The first dosing cycle comprises a first dose of genetically engineered natural killer (NK) cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells; The first dose is administered to a subject in need of cancer immunotherapy at a first time point; The second dose is administered to the subject between 5 and 10 days after the first time point; The third dose is administered to the subject between 5 and 10 days after the second dose; Each of the first, second, and third dosages contains at least about 1.5×10 ９ NK cells, At least a portion of the genetically engineered NK cells are engineered to express a chimeric antigen receptor (CAR) directed against the CD19 tumor marker; The first dosing cycle is initiated after the subject has undergone a lymphocyte depletion process so as to reduce the number of natural immune cells; The dosing regimen. **Claim 2** The dosing regimen according to claim 1, wherein the first dosing cycle is followed by one or more additional dosing cycles. **Claim 3** The dosing regimen according to claim 1, wherein if the subject exhibits a clinical response that may be a complete response (CR) after the first dosing cycle, the dosing regimen includes additional dosing cycles. **Claim 4** The dosing regimen according to claim 1, wherein if the subject exhibits a clinical response after a dosing cycle and subsequently exhibits disease progression, the dosing regimen includes additional dosing cycles. **Claim 5** The dosing regimen according to claim 1, wherein the dosing regimen includes between 1 and 5 dosing cycles. **Claim 6** The dosing regimen according to claim 1, wherein the subject undergoes a lymphocyte depletion process prior to each dosing cycle. **Claim 7** The dosing regimen according to claim 1, wherein each dosing cycle is between about 14 days and about 35 days. **Claim 8** The dosing regimen according to claim 1, wherein each dosing cycle is about 21 days. **Claim 9** The dosing regimen according to claim 1, wherein each dosing cycle is about 28 days. **Claim 10** The dosing regimen according to claim 1, wherein the lymphocyte depletion process includes at least two doses of cyclophosphamide and at least two doses of fludarabine. **Claim 11** The lymphocyte depletion process comprises three doses of cyclophosphamide and three doses of fludarabine, with the first dose of cyclophosphamide and fludarabine being administered 5 days before the start of the first administration cycle, the second dose of cyclophosphamide and fludarabine being administered 4 days before the start of the first administration cycle, and the third dose of cyclophosphamide and fludarabine being administered 3 days before the start of the first administration cycle, the dosing regimen according to claim 10.

12. The dosing regimen according to claim 10, wherein about 2 days are given for interruption between the third dose of cyclophosphamide and fludarabine and the start of the administration cycle.

13. Cyclophosphamide is administered in an amount between about 100 mg / m ２ and about 600 mg / m ２ and fludarabine is administered in an amount between about 10 mg / m ２ and about 60 mg / m ２ The dosing regimen according to claim 10, wherein the dosing regimen is administered.

14. Cyclophosphamide is administered in an amount between about 200 mg / m ２ and about 600 mg / m ２ and fludarabine is administered in an amount between about 20 mg / m ２ and about 40 mg / m ２ The dosing regimen according to claim 10, wherein the dosing regimen is administered.

15. Cyclophosphamide is administered in an amount of about 500 mg / m ２ and fludarabine is administered in an amount of about 30 mg / m ２ The dosing regimen according to claim 10, wherein the dosing is performed.

16. The dosing regimen according to any one of claims 1 to 15, further comprising administration of a therapeutic agent targeting CD20.

17. The dosing regimen according to any one of claims 1 to 15, wherein the subject is administered a therapeutic agent targeting CD20.

18. The dosing regimen according to claim 16, wherein the therapeutic agent is an anti-CD20 monoclonal antibody.

19. The dosing regimen according to claim 18, wherein the anti-CD20 antibody is rituximab.

20. The therapeutic agent is administered in an amount between about 150 mg / m ２ and about 500 mg / m ２ The dosing regimen according to claim 16, wherein the dosing regimen is administered.

21. The therapeutic agent is administered in an amount of about 375 mg / m ２ The dosing regimen according to claim 16, wherein the dosing is carried out in an amount of.

22. The dosing regimen according to claim 16, wherein the therapeutic agent is administered to the subject at least once, and at least one time is at least 2 days before the administration of the first dose administration cycle.

23. The dosing regimen according to claim 16, wherein the therapeutic agent is administered to the subject once 3 days before the administration of the first dose administration cycle.

24. The first dose of genetically engineered NK cells is administered to the subject before the subject's natural immune cell population recovers from the lymphocyte depletion process, and the first and second doses of genetically engineered NK cells may be administered to the subject before the subject's natural immune cell population recovers from the lymphocyte depletion process, the dosing regimen according to claim 1.

25. The dosing regimen according to claim 1, wherein the first dose of genetically engineered NK cells is administered to the subject about 2 to 5 days after the completion of the lymphocyte depletion process.

26. The dosing regimen according to any one of claims 1 to 25, wherein the cancer is a blood cancer.

27. The dosing regimen according to any one of claims 1 to 26, wherein the cancer is leukemia or lymphoma.

28. The dosing regimen according to any one of claims 1 to 27, wherein the cancer is B-cell cancer. **Claim 29** The dosing regimen according to any one of claims 1 to 28, wherein the cancer is non-Hodgkin lymphoma (NHL). **Claim 30** The dosing regimen according to any one of claims 1 to 29, wherein the cancer is large cell type B-cell lymphoma (LBCL), which may be aggressive LBCL. **Claim 31** The dosing regimen according to any one of claims 1 to 30, wherein the cancer is diffuse large cell type B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia (MW), or B-cell acute lymphoblastic leukemia (B-ALL). **Claim 32** The dosing regimen according to any one of claims 1 to 28, wherein the cancer is chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). **Claim 33** The dosing regimen according to any one of claims 1 to 32, wherein the cancer is relapsed / refractory (R / R) cancer. **Claim 34** The dosing regimen according to any one of claims 1 to 33, wherein the subject has peripheral blood blasts that are less than or equal to 5%. **Claim 35** The dosing regimen according to any one of claims 1 to 34, wherein the subject has received at least one line of prior treatment but not more than 7 lines of prior treatment, and the subject may have received at least one line of prior treatment but not more than 4 lines of prior treatment. **Claim 36** The dosing regimen according to any one of claims 1 to 35, wherein the subject has received at least one line of prior treatment. **Claim 37** The dosing regimen according to any one of claims 1 to 36, wherein the subject has received at least two lines of prior treatment. **Claim 38** The dosing regimen according to any one of claims 35 to 37, wherein the lines of prior treatment include anti-CD20 monoclonal antibody and cytotoxic chemotherapy, and the cytotoxic treatment may be anthracycline. **Claim 39** The dosing regimen according to any one of claims 35 to 38, wherein the lines of prior treatment include chimeric antigen receptor-expressing T (CAR-T) cells, and the lines of prior treatment may include autologous anti-CD19 CAR T cells. **Claim 40** The dosing regimen according to any one of claims 35 to 38, wherein the lines of prior treatment do not include CAR T cells and the lines of prior treatment do not include autologous anti-CD19 CAR T cells. **Claim 41** The dosing regimen according to any one of claims 35 to 40, wherein the prior treatment line includes an inhibitor of Bruton's tyrosine kinase (BTKi), and the BTKi may be ibrutinib.

42. The dosing regimen according to any one of claims 35 to 41, wherein the prior treatment line includes an inhibitor of Bcl-2, and the Bcl-2 inhibitor may be venetoclax.

43. The dosing regimen according to any one of claims 1 to 42, wherein the genetically engineered NK cells of the second and third doses are administered to the subject within about 21 days of the first time point.

44. The dosing regimen according to any one of claims 1 to 43, wherein the genetically engineered NK cells of the second and third doses are administered to the subject within about 14 days after the first time point.

45. The CAR is: (a) an antigen-binding portion targeting CD19; (b) a transmembrane domain; and (c) an intracellular signaling domain comprising an OX40 domain and a CD3 zeta domain The dosing regimen according to any one of claims 1 to 44, comprising.

46. The antigen-binding portion comprises a heavy-chain variable region (VH) and a light-chain variable region (VL): VH comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47, respectively; VL comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NOs: 48, 49, and 16, respectively; VH comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NOs: 50, 23, and 24, respectively; VL comprises CDR-1, CDR-2, and CDR-3 comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16, respectively; VH comprises the amino acid sequence set forth in SEQ ID NO: 21, and / or VL comprises the amino acid sequence set forth in SEQ ID NO: 13; and / or The antigen-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO:

6. The dosing regimen according to claim 45.

47. The dosing regimen according to any one of claims 1 to 46, wherein the genetically engineered NK cells are also engineered to express membrane-bound interleukin 15 (mbIL15).

48. The dosing regimen according to claim 47, wherein mbIL15 has at least about 95% sequence identity with SEQ ID NO:

44.

49. The dosing regimen according to any one of claims 1 to 48, wherein the dosing regimen does not result in cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) / neurotoxicity, and / or graft-versus-host disease.

50. The dosing regimen according to any one of claims 1 to 49, wherein the engineered NK cells are allogeneic with respect to the subject.

51. The dosing regimen according to any one of claims 1 to 50, wherein the subject has a 158V / 158V CD16 genotype.

52. The dosing regimen according to any one of claims 1 to 51, wherein the subject has a 158F / 158F CD16 genotype.

53. A dosing regimen for cancer immunotherapy comprising at least a first dosing cycle, wherein: The first dosing cycle comprises a first dose of genetically engineered natural killer (NK) cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells; The first dose is administered to a subject in need of cancer immunotherapy at a first time point; The second dose is administered to the subject between 5 and 10 days after the first time point; The third dose is administered to the subject between 5 and 10 days after the second dose; Each of the first, second, and third dosages contains at least 1.0×10 ９ NK cells, and At least a portion of the engineered NK cells are engineered to express a chimeric antigen receptor (CAR) directed against the CD19 tumor marker; The first dosing cycle is initiated after the subject has undergone a lymphocyte depletion process comprising at least two doses of cyclophosphamide and fludarabine; An anti-CD20 antibody is administered during the lymphocyte depletion process; The dosing regimen.

54. The dosing regimen according to claim 53, wherein the first dosing cycle is followed by one or more additional dosing cycles.

55. The dosing regimen according to claim 53 or claim 54, wherein if the subject exhibits a clinical response that may be a complete response (CR) after the first dosing cycle, the dosing regimen comprises additional dosing cycles.

56. The dosing regimen according to any one of claims 53 to 55, wherein if the subject exhibits a clinical response after a dosing cycle and subsequently exhibits disease progression, the dosing regimen comprises additional dosing cycles.

57. The dosing regimen according to any one of claims 53 to 56, wherein the dosing regimen comprises between one dosing cycle and five dosing cycles.

58. The dosing regimen according to any one of claims 53 to 57, wherein the subject undergoes a lymphodepletion process prior to each dosing cycle.

59. Each of the NK cells in an amount of 3 contains about 1.5×10 ９ NK cells, and appropriately about 1.5×10 ９ CAR-expressing NK cells, and the administration regimen according to any one of claims 1 to 58.

60. The lymphodepletion process comprises 3 doses of cyclophosphamide and 3 doses of fludarabine, The first dose of cyclophosphamide and fludarabine is administered 5 days before the start of the dosing cycle, The second dose of cyclophosphamide and fludarabine is administered 4 days before the start of the dosing cycle, The third dose of cyclophosphamide and fludarabine is administered 3 days before the start of the dosing cycle, The anti-CD20 antibody is administered on the same day as the third dose of cyclophosphamide and fludarabine, The dosing regimen according to any one of claims 53 to 59.

61. Cyclophosphamide is administered in an amount between about 300 mg / m ２ and about 600 mg / m ２ , fludarabine is administered in an amount between about 20 mg / m ２ and about 40 mg / m ２ , the anti-CD20 antibody is administered in an amount between about 350 mg / m ２ and about 425 mg / m ２ , and each of the three doses of NK cells contains at least 1.5×10 ９ NK cells. The administration regimen according to any one of claims 53 to 60.

62. The dosing regimen according to any one of claims 53 to 61, wherein the anti-CD20 antibody comprises rituximab or obinutuzumab.

63. The dosing regimen according to any one of claims 1 to 62, wherein the cancer cells do not express CD58 or express a mutant form of CD58.

64. The dosing regimen according to any one of claims 1 to 63, wherein it is determined that the cancer cells do not express CD58 or express a mutant form of CD58 prior to administration of the first dosing cycle to the subject.

65. The dosing regimen according to any one of claims 1 to 64, wherein the subject is selected for treatment with a dosing regimen based on cancer cells showing a CD58 deficiency or mutation prior to administration of the first dosing cycle to the subject.

66. The dosing regimen according to any one of claims 1 to 65, wherein each dosing cycle of one dose is administered to the subject in an outpatient setting, and each dosing cycle of each dose may be administered to the subject in an outpatient setting.

67. The dosing regimen according to any one of claims 1 to 66, wherein the overall response rate (ORR) among the subjects treated according to the dosing regimen is at least about 50%, at least about 60%, at least about 70%, or at least about 80%.

68. The dosing regimen according to any one of claims 1 to 67, wherein at least about 50%, at least about 60%, at least about 70%, or at least about 80% of the subjects treated according to the dosing regimen show a complete response (CR).

69. A method of treating cancer, comprising administering to a subject having cancer genetically engineered natural killer (NK) cells that express a chimeric antigen receptor (CAR) directed against an antigen associated with cancer cells or expressed by cancer cells: wherein the cancer does not express CD58 or expresses a mutant form of CD58; wherein the subject relapses and / or is refractory to genetically engineered T cells that express a CAR directed against the antigen.

70. The method according to claim 69, wherein the antigen is CD19.

71. A method of treating cancer, comprising administering to a subject having cancer genetically engineered natural killer (NK) cells that express a chimeric antigen receptor (CAR) directed against CD19, wherein the cancer does not express CD58 or expresses a mutant form of CD58.

72. Determining whether the cancer does not express CD58 or expresses a mutant form of CD58; Identifying a subject as having cancer that does not express CD58 or expresses a mutant form of CD58; and / or Selecting the identified subject for treatment with the genetically engineered NK cells The method according to any one of claims 69 to 71, further comprising.

73. The method according to claim 71 or claim 72, wherein the subject has been previously treated with genetically engineered T cells that express a CAR directed against CD19 due to cancer, and the subject may be refractory and / or relapse with the genetically engineered T cells.

74. The genetically engineered NK cells are administered in a dosing regimen comprising at least a first dosing cycle, the first dosing cycle comprising a first dose of genetically engineered NK cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, the first dose being administered to the subject at a first time point, the second dose being administered to the subject between 5 and 10 days after the first time point, the third dose being administered to the subject between 5 and 10 days after the second dose; Each of the first, second, and third dosages comprises at least about 1.5×10 ９ NK cells, and optionally at least about 1.5×10 ９ CAR-expressing NK cells The method according to any one of claims 69 to 73.

75. A method of treating cancer, comprising: (a) selecting a subject for treatment of cancer if the cancer cells do not express CD58 or express a mutant form of CD58; (b) administering to a subject selected for at least a first dosing cycle, the first dosing cycle comprising a first dose of genetically engineered natural killer (NK) cells, a second dose of genetically engineered NK cells, and a third dose of genetically engineered NK cells, including, the first dose being administered to a subject in need of cancer immunotherapy at a first time point, the second dose being administered to the subject between 5 and 10 days after the first time point, the third dose being administered to the subject between 5 and 10 days after the second dose; Each of the first, second, and third dosages contains at least about 1.5 x 10 ９ NK cells, at least a portion of the genetically engineered NK cells being engineered to express a chimeric antigen receptor (CAR) directed against the CD19 tumor marker, method.

76. the first dosing cycle being initiated after the subject has undergone a lymphocyte depletion process so as to reduce the number of natural immune cells, The method according to claim 74 or claim 75, wherein the method is initiated after the subject has undergone a lymphocyte depletion process.

77. A method for treating cancer, comprising: administering to a subject having cancer a lymphocyte depletion regimen comprising at least two doses of cyclophosphamide and at least two doses of fludarabine; administering to the subject a dosing cycle comprising at least first, second, and third doses of genetically engineered NK cells, including, the first dose of genetically engineered NK cells being administered to the subject after the final dose of fludarabine, the second dose of genetically engineered NK cells being administered to the subject between 6 and 8 days after the first dose of genetically engineered NK cells, the third dose of genetically engineered NK cells being administered to the subject between 6 and 8 days after the second dose of genetically engineered NK cells, Each of the first, second, and third doses of genetically engineered NK cells contains approximately 1.5 × 10 ９ NK cells, and the genetically engineered NK cells being allogeneic to the subject and engineered to express a chimeric antigen receptor (CAR) that binds to CD19, method.

78. A method for treating cancer, administering to a subject having cancer a lymphocyte depletion regimen comprising at least two doses of cyclophosphamide and at least two doses of fludarabine, administering to the subject an agent that binds to CD20, administering to the subject a dosing cycle comprising at least first, second, and third doses of genetically engineered NK cells including, the first dose of genetically engineered NK cells being administered to the subject after the final dose of fludarabine, the second dose of genetically engineered NK cells being administered to the subject between 6 and 8 days after the first dose of genetically engineered NK cells, The genetically engineered NK cells of the third dose are administered to the subject between 6 and 8 days after the genetically engineered NK cells of the second dose, Each of the first, second, and third doses of genetically engineered NK cells contains at least 1.0×10 ９ NK cells, and wherein the genetically engineered NK cells are allogeneic to the subject and are engineered to express a chimeric antigen receptor (CAR) that binds to CD19, method.

79. Each of the first, second, and third dosages is at least 1.5×10 ９ cells, and appropriately at least 1.5×10 ９ CAR-expressing NK cells, the method according to any one of claims 74, 76, and 78.

80. The lymphodepletion regimen comprises 3 doses of cyclophosphamide and 3 doses of fludarabine, wherein the first dose of cyclophosphamide and fludarabine is administered 5 days before the first dose of genetically engineered NK cells, the second dose of cyclophosphamide and fludarabine is administered 4 days before the genetically engineered NK cells, and the third dose of cyclophosphamide and fludarabine is administered 3 days before the start of the administration cycle. The method according to any one of claims 76 to 79.

81. Cyclophosphamide is administered in an amount between about 100 mg / m ２ and about 600 mg / m ２ and fludarabine is administered in an amount between about 20 mg / m ２ and about 40 mg / m ２ The method according to any one of claims 77 to 80, wherein the administration is carried out.

82. Cyclophosphamide is administered in an amount of about 500 mg / m ２ and fludarabine is administered in an amount of about 30 mg / m ２ The method according to any one of claims 77 to 81, wherein the method is carried out.

83. The agent that binds to CD20 is an anti-CD20 monoclonal antibody, the anti-CD20 monoclonal antibody is administered 3 days before the first dose of genetically engineered NK cells is administered to the subject, and the anti-CD20 monoclonal antibody is administered in an amount between about 350 mg / m ２ and about 425 mg / m ２ The method according to any one of claims 77 to 82, which is administered in an amount of.

84. The method according to claim 83, wherein the anti-CD20 monoclonal antibody comprises rituximab or obinutuzumab.

85. The method according to any one of claims 69 to 84, wherein the cancer is a blood cancer.

86. The method according to any one of claims 69 to 85, wherein the cancer is leukemia or lymphoma.

87. The method according to any one of claims 69 to 86, wherein the cancer is a B cell cancer.

88. The method according to any one of claims 69 to 87, wherein the cancer is non-Hodgkin lymphoma (NHL).

89. The method according to any one of claims 69 to 88, wherein the cancer is large cell type B cell lymphoma (LBCL), which may be aggressive LBCL.

90. The method according to any one of claims 69 to 89, wherein the cancer is diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia (MW), or B cell acute lymphoblastic leukemia (B-ALL).

91. The method according to any one of claims 69 to 87, wherein the cancer is chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL).

92. The method according to any one of claims 69 to 91, wherein the cancer is a recurrent / refractory (R / R) cancer.

93. The method according to any one of claims 69 to 92, wherein the subject has peripheral blood blasts that are less than or equal to 5%.

94. The CAR is: (a) an antigen-binding portion that targets CD19; (b) a transmembrane domain; and (c) an intracellular signaling domain comprising an OX40 domain and a CD3 zeta domain The method according to any one of claims 69 to 93, comprising. **Claim 95** The antigen-binding portion comprises a heavy chain variable region (VH) and a light chain variable region (VL): VH comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 45, 46, and 47; VL comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 48, 49, and 16; VH comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 50, 23, and 24; VL comprises CDR-1, CDR-2, and CDR-3, each comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16; VH comprises the amino acid sequence set forth in SEQ ID NO: 21, and / or VL comprises the amino acid sequence set forth in SEQ ID NO: 13; and / or The antigen-binding domain is a scFv comprising the amino acid sequence of SEQ ID NO: 6 The method according to claim 94. **Claim 96** The method according to any one of claims 69 to 95, wherein the genetically engineered NK cells are also engineered to express membrane-bound interleukin 15 (mbIL15). **Claim 97** The method according to claim 96, wherein mbIL15 has at least about 95% sequence identity with SEQ ID NO:

44. **Claim 98** The method according to any one of claims 74 to 97, wherein administration of the dosing cycle does not result in cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) / neurotoxicity, and / or graft-versus-host disease. **Claim 99** The method according to any one of claims 75 to 98, wherein the cancer cells do not express CD58 or express a mutant form of CD58. **Claim 100** (a) prior to administration of the first dose of genetically engineered NK cells to a subject, it is determined that the cancer cells do not express CD58 or express a mutant form of CD58; and / or (b) The method further comprises selecting a subject for treatment based on cancer cells that exhibit a CD58 deficiency or mutation. The method according to any one of claims 77 to 99. **Claim 101** The method according to any one of claims 74 to 100, wherein the administration cycle of the 1 dosage is administered to the subject in an outpatient setting, and each administration cycle of each dosage may be administered to the subject in an outpatient setting.

102. Use of a population of engineered NK cells expressing a chimeric antigen receptor targeting CD19 for treating cancer by administration of an administration cycle comprising at least first, second, and third dosages of said engineered NK cells, wherein the first dosage of engineered NK cells is administered to the subject after administration of a final dosage of lymphodepleting regimen comprising at least 2 dosages of cyclophosphamide and at least 2 dosages of fludarabine, the second dosage of engineered NK cells is administered to the subject between 6 and 8 days after the first dosage of engineered NK cells, the third dosage of engineered NK cells is administered to the subject between 6 and 8 days after the second dosage of engineered NK cells, Each of the first, second, and third doses of genetically engineered NK cells comprises about 1.5 × 10 ９ genetically engineered NK cells for use.

103. Use of a population of engineered NK cells expressing a chimeric antigen receptor targeting CD19 for treating cancer by administration of an administration cycle comprising at least first, second, and third dosages of said engineered NK cells, wherein the first dosage of engineered NK cells is administered to the subject after administering a final dosage of lymphodepleting regimen comprising at least 2 dosages of cyclophosphamide and at least 2 dosages of fludarabine to the subject, the first dosage of engineered NK cells is administered to the subject after administration of an agent that binds to CD20, the second dosage of engineered NK cells is administered to the subject between 6 and 8 days after the first dosage, the third dosage of engineered NK cells is administered to the subject between 6 and 8 days after the second dosage, Use, wherein each of the first, second, and third doses of genetically engineered cells comprises at least about 1.5×10 ９ genetically engineered NK cells.

104. Use of a population of engineered NK cells expressing a chimeric antigen receptor targeting CD19 for treating cancer in a subject by intravenous administration of an administration cycle comprising at least three consecutive dosages of engineered NK cells, Genetically engineered NK cells of the first dose are administered to the subject at the first time point and contain at least 1.5×10 ９ operated NK cells, The genetically engineered NK cells of the second dose are administered to the subject between 6 and 8 days after the genetically engineered NK cells of the first dose and contain at least 1.5 × 10 ９ engineered NK cells, The genetically engineered NK cells of the third dose are administered to the subject between 6 and 8 days after the genetically engineered NK cells of the second dose and contain at least 1.5 × 10 ９ engineered NK cells, wherein the engineered NK cells express a CD19 CAR having at least 95% sequence identity with SEQ ID NO:

43.

105. Use of genetically engineered natural killer (NK) cells expressing a chimeric antigen receptor (CAR) directed against an antigen associated with or expressed by cancer cells for treating a subject having cancer, wherein: the cancer does not express CD58 or expresses a mutant form of CD58; the subject is refractory and / or relapsing to genetically engineered T cells expressing a CAR directed against the antigen, use. **Claim 106** Use of genetically engineered natural killer (NK) cells expressing a chimeric antigen receptor (CAR) directed against CD19 for treating a subject having cancer, wherein the cancer does not express CD58 or expresses a mutant form of CD58.

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