Use of anti-CD137 antibody drug conjugates (ADCs) in allogeneic cell therapy

Abstract

The present invention provides a method for using allogeneic cells in treatment, the method being performed by combining allogeneic cell therapy with an anti-CD137 antibody drug conjugate (ADC). A method for treating or preventing host-versus-graft (HvG) rejection in a human subject receiving allogeneic cell therapy is disclosed, the method being performed by administering an anti-CD137 ADC to the human subject.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 702,292, filed on July 23, 2018. The contents of the foregoing application are hereby incorporated by reference. Field of the Invention

[0003] The present invention provides methods of treating or preventing rejection of allogeneic cell therapy (e.g., chimeric antigen receptor (CAR) cell therapy) in human subjects by administering an antibody drug conjugate (ADC) that binds to an antigen expressed by a host immune system cell that is responsible for mediating rejection of the cell therapy. Background of the Invention

[0005] Allogeneic cell therapy includes transplanting cells to patients, wherein the transplanted cells are derived from donors other than patients. Common types of allogeneic donors for allogeneic cell therapy include HLA-matched brothers and sisters (siblings) donors, matching unrelated donors, partially matched family member donors, related cord blood donors and unrelated cord blood donors. An ultimate goal of cell therapy is to identify allogeneic cell therapy (Brandenberger, et al. (2011). BioProcess International.9 (supplement 1): 30-37) that can form a "off the shelf" product basis, which will expand the purposes of allogeneic cell therapy.

[0006] One therapeutic area with great potential for allogeneic cell therapy is chimeric antigen receptor (CAR) therapy. CAR therapy is an immunotherapy that uses the body's own immune system to destroy cells expressing specific antigens associated with a disease (such as cancer). For example, in cancer, CAR therapy recruits and enhances the ability of the patient's immune system to attack tumors. CAR therapy is based on immune cells such as T cells that express CAR, and CAR is generally a transmembrane fusion protein that combines an extracellular antigen binding domain (such as scFv) with a cytoplasmic active signal transduction and a "co-stimulatory" domain (transduction signal from a surface receptor to a cell). Therefore, when an immune cell (such as a T cell) expresses CAR, the immune cell can recognize and kill cells expressing an antigen (e.g., a tumor-associated antigen) targeted by the antigen binding domain of CAR (Geyer and Brentjens (2016) Cytotherapy 18 (11): 1393-1409).

[0007] To date, approved CAR therapies rely on obtaining autologous cells from the patient and converting the patient's own cells into cells expressing CAR, which are then used for treatment. However, one challenge with CAR therapy is the time and cost associated with obtaining autologous cells, as autologous cells generally require an individualized regimen. Using allogeneic cells will reduce the time and cost associated with CAR therapy.

[0008] Despite its promise, current therapeutic use of allogeneic cells can have complications that make this therapy challenging. In an immune-competent host, transplanted allogeneic cells are rapidly rejected, a process known as host-versus-graft rejection (HvG). HvG can significantly reduce the efficacy of the transferred cells, as well as produce adverse events in the recipient, limiting the use of allogeneic cells. There remains a need for methods that can overcome HvG rejection in order to further the potential of allogeneic cell therapy. SUMMARY OF THE INVENTION

[0010] The present invention provides a method for preventing or treating the rejection of allogeneic cells transplanted in human subjects. In particular, the method disclosed herein can be used to treat or prevent host-versus-graft (HvG) rejection in patients receiving allogeneic cell therapy. The present invention is characterized in that anti-CD137 antibody drug conjugates (ADC) are administered to deplete immune cell populations such as activated T cells expressing CD137 in patients to promote the method for administering allogeneic cell therapy. This selective consumption of CD137+ cells (e.g., activated T cells) of the patient's immune system prevents the rejection of cell therapy or significantly reduces the risk of cell therapy rejection.

[0011] In a first aspect, the invention features a method for treating or preventing rejection of allogeneic cells transplanted into a human subject, the method being performed by the following steps such that allogeneic cell rejection in the human subject is treated or prevented: (a) administering a first amount of allogeneic cells to the human subject, wherein the first amount is sufficient to elicit a priming response to the allogeneic cells in the human subject; (b) administering an anti-CD137 antibody drug conjugate (ADC) to the human subject such that endogenous CD137+ activated T cells are depleted, wherein the anti-CD137 ADC comprises an anti-CD137 antibody or antigen-binding fragment thereof conjugated to a cytotoxin via a linker; and (c) administering a therapeutically effective amount of allogeneic cells expressing a CAR to the human subject, wherein the allogeneic cells are of the same type as the allogeneic cells administered above, and wherein the CAR comprises an extracellular domain that binds to a tumor antigen, a transmembrane domain, and a cytoplasmic signaling domain. In one embodiment, the method includes administering a first amount of allogeneic cells to a human subject between about 48 hours to about 7 days before step (b). In another embodiment, the method includes administering a therapeutically effective amount of allogeneic cells expressing CAR to a human subject between about 24 hours to about 14 days after step (b).

[0012] On the other hand, the present invention features a method for treating or preventing rejection of allogeneic cells transplanted into a human subject, the method being performed by the following steps so that rejection of transplanted allogeneic cells in a human subject is treated or prevented: (a) administering an anti-CD137 ADC to a human subject, wherein the anti-CD137 ADC comprises an anti-CD137 antibody or an antigen-binding fragment thereof conjugated to a cytotoxin via a linker, and wherein the human subject is characterized by having activated T cells for allogeneic cells previously administered to the human subject; and (b) administering a therapeutically effective amount of allogeneic cells expressing CAR to a human subject, wherein the allogeneic cells are of the same type as the allogeneic cells described in (a), and wherein the CAR comprises an extracellular domain, a transmembrane domain, and a cytoplasmic signaling domain that binds to a tumor antigen. In one embodiment, the method includes administering an effective amount of allogeneic cells expressing CAR to a human subject between about 24 hours to about 14 days after step (a).

[0013] In certain embodiments, the allogeneic cells are allogeneic T cells or allogeneic NK cells. In other embodiments, the effective amount of allogeneic cells expressing CAR is about 2×10 6 cells / kg to about 3.0×10 8In another embodiment, allogeneic cell rejection is characterized by cytokine release syndrome (CRS). In other embodiments, human subjects have cancer or autoimmune diseases. In another embodiment, anti-CD137 antibodies or their antigen-binding fragments include heavy chain variable regions, which include CDR1, CDR2 and CDR3 with amino acid sequences listed in SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:21, and anti-CD137 antibodies or their antigen-binding fragments include light chain variable regions, which include CDR1, CDR2 and CDR3 with amino acid sequences listed in SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25, respectively. In other embodiments, CD137 antibodies or their antigen-binding fragments are chimeric or humanized.

[0014] In yet another embodiment, the anti-CD137 antibody or antigen-binding fragment thereof is of the IgG1 isotype or the IgG4 isotype.In other embodiments, the cytotoxin is an anti-mitotic agent or an RNA polymerase inhibitor.

[0015] In other embodiments, the cytotoxin is maytansine, calicheamicin, pyrrolobenzodiazepine, indolebenzodiazepine, or auristatin. In one embodiment, the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE). In one embodiment, the cytotoxin is maytansine. In one embodiment, the cytotoxin is a pyrrolobenzodiazepine (PBD). For example, in some embodiments, the PBD can be selected from tesirine or talirine. In some embodiments, the cytotoxin can be calicheamicin. For example, in some embodiments, the calicheamicin can be ozogamicin.

[0016] In another embodiment, the RNA polymerase inhibitor is an amanitin. In yet another embodiment, the RNA polymerase inhibitor is amanitin. In another embodiment, the amanitin is selected from the group consisting of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, amanullinic acid, and proamanullin. In yet another embodiment, the anti-CD137 ADC is represented by the formula Ab-ZL-Am, wherein Ab is an antibody or antigen-binding fragment thereof that binds to CD137, L is a linker, Z is a chemical moiety, and Am is an amanitin toxin. In certain embodiments, the linker-amanitin toxin conjugate Am-LZ is represented by formula (III)

[0017]

[0018] Where R1 is H, OH, OR A OR C ;

[0019] R2 is H, OH, OR B OR C ;

[0020] R A and R B When present, taken together with the oxygen atom to which they are attached, form an optionally substituted 5-membered heterocycloalkyl group;

[0021] R3 is H, R C or R D ;

[0022] R4, R5, R6 and R7 are each independently H, OH, OR C , OR D , R C or R D ;

[0023] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0024] R9 is H, OH, OR C OR D ;

[0025] Q is -S-, -S(O)- or -SO2-;

[0026] R C Yes - LZ;

[0027] R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0028] L is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl; or includes a dipeptide; or includes -((CH2) m O) n (CH2) m -, wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; or a combination thereof; and

[0029] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the anti-CD137 antibody or antigen-binding fragment thereof.

[0030] In certain embodiments, the linker-amanitin conjugate Am-LZ is represented by formula (III)

[0031]

[0032] Where R1 is H, OH, OR A OR C ;

[0033] R2 is H, OH, OR B OR C ;

[0034] R A and R B When present, taken together with the oxygen atom to which they are attached, form an optionally substituted 5-membered heterocycloalkyl group;

[0035] R3 is H, R C or R D ;

[0036] R4, R5, R6 and R7 are each independently H, OH, ORC , OR D , R C or R D ;

[0037] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0038] R9 is H, OH, OR C OR D ;

[0039] Q is -S-, -S(O)- or -SO2-;

[0040] R C Yes - LZ;

[0041] R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0042] L is a connector; and

[0043] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the anti-CD137 antibody or antigen-binding fragment thereof.

[0044] In one embodiment of Formula (III), L is a peptide comprising a linker.

[0045] In some embodiments, the linker includes one or more of the following: a dipeptide, a p-aminobenzyl (PAB) group, an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted C3-C6 cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, a solubility enhancing group, -(C=O)-, -(CH2CH2O) p - group, wherein p is an integer from 1 to 6, ((CH2) m O) n (CH2) m-, wherein n and each m are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; or a combination thereof.

[0046] In some embodiments, the linker comprises ((CH2) m O) n (CH2) m -group and a heteroaryl group, wherein the heteroaryl group is triazole. In some embodiments, ((CH2) m O) n (CH2) m - group and triazole together include

[0047] wherein n is 1 to 10 and the wavy line indicates the point of attachment to an additional linker component, chemical moiety Z, or Amanita toxin.

[0048] In some embodiments, Am contains exactly one R C Substituent.

[0049] In other embodiments, the antimitotic agent is a maytansine, a calicheamicin, a pyrrolobenzodiazepine, an indolebenzodiazepine, or an auristatin. In another embodiment, the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE).

[0050] In certain embodiments, the linker of the ADC is N-β-maleimidopropionyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB). In one embodiment, the linker of the ADC is N-β-maleimidopropionyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB). In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0051]

[0052] wherein S is a sulfur atom and represents a reactive substituent (eg, a -SH group from a cysteine ​​residue) present in the antibody or antigen-binding fragment thereof that binds CD5.

[0053] In some embodiments, LZ is

[0054]

[0055] In one embodiment, ADC is represented by any of the following structures:

[0056]

[0057] In one embodiment, ADC is represented by one of the following structures:

[0058]

[0059]

[0060] In other embodiments, ADC has a serum half-life of 3 days or less.In other embodiments, the extracellular domain includes scFv antibody or single-chain T cell receptor (scTCR).In another embodiment, the extracellular domain includes non-immunoglobulin scaffold protein.In other embodiments, the extracellular domain of CAR is combined with the tumor antigen selected from the group consisting of: CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D or CD123. In other embodiments, the cytoplasmic signaling domain of CAR includes a CD28 cytoplasmic signaling domain, a CD3 δ cytoplasmic signaling domain, an OX40 cytoplasmic signaling domain and / or a CD137 cytoplasmic signaling domain. In yet another embodiment, the cytoplasmic signaling domain of CAR includes a CD3 δ cytoplasmic signaling domain.In certain embodiments, the cancer is leukemia, advanced adult cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, cancer), metastatic colorectal cancer, ovarian cancer, triple-negative breast cancer, hematopoietic / lymphoid cancer, liver metastasis from colorectal cancer, small cell lung cancer, non-small cell lung cancer, B-cell lymphoma, relapsed or refractory B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, relapsed or refractory diffuse large cell lymphoma, anaplastic large cell lymphoma, primary mediastinal B-cell lymphoma, relapsed mediastinal large B-cell lymphoma, refractory mediastinal large B-cell lymphoma, large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, colorectal epithelial cancer (colorectal carcinoma), gastric cancer, pancreatic cancer, triple-negative invasive breast cancer, renal cell carcinoma, squamous cell lung carcinoma, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, prolymphocytic leukemia Baxyleukemia, chronic lymphocytic leukemia, acute myeloid leukemia, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, multiple myeloma of bone, malignant glioma of brain, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasms, blastic plasmacytoid dendritic cell neoplasm, liver metastasis, solid tumors, advanced solid tumors, mesothelin-positive tumors, hematological malignancies, or other advanced malignancies. Sequence Listing <110> Migenda Therapeutics <120> Use of anti-CD137 antibody drug conjugates (ADCs) in allogeneic cell therapy <130> M103034 2030WO <150> 62 / 702,292 <151> 2018-07-23 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 255 <212> PRT <213> Homo sapiens <400> 1 Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu 1 5 10 15 Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro 20 25 30 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys 35 40 45 Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile 50 55 60 Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser 65 70 75 80 Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly 85 90 95 Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu 100 105 110 Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln 115 120 125 Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys 130 135 140 Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro 145 150 155 160 Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255 <210> 2 <211> 46 <212> PRT <213> Homo sapiens <400> 2 Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 1 5 10 15 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 20 25 30 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 35 40 45 <210> 3 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 3 Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 1 5 10 15 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 20 25 30 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Pro Arg 35 40 45 Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 50 55 60 Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 65 70 75 80 Leu Phe Pro Gly Pro Ser Lys Pro 85 <210> 4 <211> 28 <212> PRT <213> Homo sapiens <400> 4 Leu Asp Pro Lys Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr 1 5 10 15 Gly Val Ile Leu Thr Ala Leu Phe Leu Arg Val Lys 20 25 <210> 5 <211> 21 <212> PRT <213> Homo sapiens <400> 5 Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu 1 5 10 15 Thr Ala Leu Phe Leu 20 <210> 6 <211> 27 <212> PRT <213> Homo sapiens <400> 6 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 7 <211> 66 <212> PRT <213> Homo sapiens <400> 7 Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 35 40 45 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 50 55 60 Trp Val 65 <210> 8 <211> 112 <212> PRT <213> Homo sapiens <400> 8 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 9 <211> 42 <212> PRT <213> Homo sapiens <400> 9 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 10 <211> 41 <212> PRT <213> Homo sapiens <400> 10 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 11 <211> 453 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 11 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Val Tyr 65 70 75 80 Met Gln Leu Asn Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asn Gly Val Glu Gly Tyr Pro His Tyr Tyr Ala Met Glu Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 12 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Description of synthetic polypeptide: artificial sequence <400> 12 Asp Ile Gln Met Thr Gln Thr Thr Ser Ala Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Gly Cys Arg Ala Ser Gln Asp Leu Ser Asn His 20 25 30 Leu Tyr Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Arg Asn Leu Glu Gln 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 13 Ser Gly Tyr Thr Phe Thr Ser Tyr Trp 1 5 <210> 14 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 14 Asn Ile Tyr Pro Ser Asp Ser Tyr Thr 1 5 <210> 15 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 15 Thr Arg Asn Gly Val Glu Gly Tyr Pro His Tyr Tyr Ala Met Glu 1 5 10 15 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 16 Ser Gln Asp Leu Ser Asn His 1 5 <210> 17 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 17 Tyr Tyr Thr Ser 1 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 18 Cys Gln Gln Gly Tyr Thr Leu Pro Tyr 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 19 Ser Tyr Trp Ile Asn 1 5 <210> 20 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 20 Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> twenty one <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> twenty one Asn Gly Val Glu Gly Tyr Pro His Tyr Tyr Ala Met Glu Tyr 1 5 10 <210> twenty two <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> twenty two Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Asn Thr Val Tyr 65 70 75 80 Met Gln Leu Asn Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asn Gly Val Glu Gly Tyr Pro His Tyr Tyr Ala Met Glu Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> twenty three <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> twenty three Arg Ala Ser Gln Asp Leu Ser Asn His Leu Tyr 1 5 10 <210> twenty four <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> twenty four Tyr Thr Ser Arg Leu His Ser 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 25 Gln Gln Gly Tyr Thr Leu Pro Tyr Thr 1 5 <210> 26 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 26 Asp Ile Gln Met Thr Gln Thr Thr Ser Ala Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Gly Cys Arg Ala Ser Gln Asp Leu Ser Asn His 20 25 30 Leu Tyr Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Arg Asn Leu Glu Gln 65 70 75 80 Glu Asp Val Ala Thr Tyr Phe Cys Gln Gln Gly Tyr Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 27 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 27 Gly Gly Gly Gly Ser 1 5 <210> 28 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 28 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1A and Figure 1B The graphic depicts the results of an in vitro T cell killing assay that included comparing an anti-CD137-amanitin ADC (i.e., "CD137-amanitin") and an anti-T cell specific-amanitin ADC (i.e., "anti-T cell-amanitin") to a negative control (i.e., "hIgG-amanitin"). The results show the number of viable activated cells ( Figure 1A ) or the number of viable non-activated cells ( Figure 1B ) (y-axis) versus ADC concentration (x-axis).

[0063] Figure 2Graph depicts the results of an in vivo assay comparing the percentage of mouse survival (y-axis) of anti-CD137-amanitin ADC to control PBS, anti-CD137 antibody (naked), and isotype-amanitin in the NSG mouse xeno-GvHD model as a function of days post-transplantation (x-axis).

[0064] Figure 3 Graph depicts the results of an in vivo assay comparing the number of human T cells detected in peripheral blood (y-axis) as a function of post-transplantation days (x-axis) for anti-CD137-amanitin ADC versus control PBS, anti-CD137 antibody (naked), and isotype-amanitin in the NSG mouse xeno-GvHD model.

[0065] Figure 4A and Figure 4B Graph depicts the method used to determine engraftment rate in the humanized NSG-SGM3 mouse model ( Figure 4A ) and T cell frequency ( Figure 4B ), where engraftment rates and T cell frequencies were measured on days 5, 9, 14, 22, and 27 post-transplant in a NSG mouse xeno-GvHD model, and anti-CD137-amanitin ADCs were compared to control PBS, anti-CD137-BBK antibody (naked), isotype-amanitin, and anti-T cell-amanitin ADCs.

[0066] Details

[0067] Disclosed herein is a method for treating or preventing the rejection of transplanted allogeneic cells in human subjects, i.e., a method for treating or preventing host-versus-graft (HvG) diseases. The method disclosed herein includes administering an anti-CD137 antibody drug conjugate (ADC) (which is combined with endogenous CD137+ immune cells such as activated T cells) and allogeneic cell therapy. By administering anti-CD137 ADC to human patients receiving allogeneic cell transplantation, endogenous CD137+ T cells are consumed, thereby reducing the risk of endogenous cells reacting to allogeneic cell therapy. The method disclosed herein can be used in combination with allogeneic chimeric antigen receptor (CAR) therapy, because the combined use of anti-CD137 ADC and allogeneic cells expressing CAR provides acceptance of allogeneic cells by recipient patients, and reduces the risk of host-versus-graft (HvG) reactions that may invalidate cells expressing CAR.

[0068] I. Definitions

[0069] As used herein, the term "about" refers to a value within 5% above or below the stated value.

[0070] As used herein, the term "allogeneic", when used in the context of transplantation, is used to define cells (or tissues or organs) that are transplanted from a donor to a recipient, where the donor and recipient are different individuals of the same species.

[0071] As used herein, the term "autologous" refers to cells or transplants where the donor and recipient are the same subject.

[0072] As used herein, the term "xenogeneic" refers to cells where the donor and recipient species are different.

[0073] As used herein, the term "immune cell" is intended to include, but is not limited to, cells of hematopoietic origin and that play a role in an immune response. Immune cells include, but are not limited to, T cells and natural killer (NK) cells. Natural killer cells are well known in the art. In one embodiment, natural killer cells include cell lines, such as NK-92 cells. Additional examples of NK cell lines include NKG cells, YT cells, NK-YS cells, HANK-1 cells, YTS cells, and NKL cells. Immune cells can be allogeneic or autologous.

[0074] "Engineered cell" means any cell of any organism modified, transformed or manipulated by adding a gene, DNA or RNA sequence or protein or polypeptide or modifying a gene, DNA or RNA sequence or protein or polypeptide. The separated cells, host cells and genetically engineered cells of the present disclosure include immune cells, such as NK cells and T cells, comprising a DNA or RNA sequence encoding CAR and expressing CAR on the cell surface. The separated host cells and engineered cells can be used, for example, to enhance NK cell activity or T lymphocyte activity, treat cancer, and treat autoimmune diseases. In embodiments, engineered cells include immune cells, such as T cells or natural killer (NK) cells.

[0075] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or immunoreacts with a specific antigen. Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired antigen-binding activity.

[0076] Typically, antibodies comprise heavy and light chains containing antigen-binding regions. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

[0077] As used herein, the term "antigen binding fragment" refers to one or more portions of an antibody that retain the ability to specifically bind to a target antigen. The antigen binding function of an antibody can be performed by a fragment of a full-length antibody. An antibody fragment can be, for example, a Fab, F(ab')2, scFv, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb comprising the VH and VL domains; (vi) a dAb fragment consisting of the VH domain (see, e.g., Ward et al., Nature 341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) isolated complementarity determining regions (CDRs); and (ix) a combination of two or more (e.g., two, three, four, five or six) isolated CDRs, which may optionally be linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected by a linker using recombinant methods, which enables them to become a single protein chain, wherein the VL and VH regions are paired to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of these fragments can be screened in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or in some cases, by chemical peptide synthesis procedures known in the art.

[0078] As used herein, an "intact" or "full length" antibody refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds.

[0079] As used herein, the term "anti-CD137 antibody" or "antibody that binds to CD137" or "anti-CD137 ADC" or "ADC that binds to CD137" refers to an antibody or ADC that specifically binds to human CD137 when CD137 is present on the cell surface of a cell such as a T cell. The amino acid sequence of the human CD137 extracellular domain is described in SEQ ID NO: 1 below.

[0080]

[0081] As used herein, the term "specifically binds" refers to the ability of an antibody (or ADC) to recognize and bind to a specific protein structure (epitope) rather than to proteins in general. If the antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of molecules containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody. By way of example, if the antibody can be competed away from its target by the corresponding unlabeled antibody when labeled, then the antibody "specifically binds" to the target. In one embodiment, if the antibody has at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or smaller (smaller means less than 10 -12 Number, such as 10 -13 ) D , then the antibody specifically binds to the target, such as CD137. In one embodiment, as used herein, the term "specific binding to CD137" or "specific binding to CD137" refers to binding to CD137 and having a specific affinity of 1.0×10 -7 M or less dissociation constant (K D ) as determined by surface plasmon resonance. In one embodiment, K D According to standard biolayer interferometry (BLI) determination. However, it should be understood that the antibody may be able to specifically bind to two or more sequence-related antigens. For example, in one embodiment, the antibody can specifically bind to both human orthologs and non-human (e.g., mouse or non-human primate) orthologs of CD137.

[0082] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies are obtained from a single clone, including any eukaryotic clone, prokaryotic clone, or phage clone, by any available or known means in the art. Monoclonal antibodies useful in the present disclosure can be prepared using a variety of techniques known in the art, including the use of hybridoma technology, recombinant technology, and phage display technology, or a combination thereof.

[0083] As used herein, the term "chimeric" antibody refers to an antibody having a variable sequence derived from a non-human immunoglobulin (such as a rat or mouse antibody) and a human immunoglobulin constant region (usually selected from a human immunoglobulin template). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719): 1202-7; Oi et al., 1986, BioTechniques 4: 214-221; Gillies et al., 1985, J. Immunol. Methods 125: 191-202; U.S. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397.

[0084] As used herein, the term "half-life" refers to the time required for the plasma concentration of an antibody drug in the body to decrease by half or 50%. This 50% decrease in serum concentration reflects the amount of drug circulating.

[0085] As used herein, the term "host versus graft disease" or "HvG" refers to the rejection of a transplant whereby allogeneic cells, tissues, or organs are rejected by the recipient's immune system.

[0086] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins that contain minimal sequence derived from a non-human immunoglobulin. Typically, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370 to Queen et al.; EP 239400; PCT Publication No. WO 91 / 09967; U.S. Patent No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Patent No. 5,565,332.

[0087] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide comprising at least one extracellular domain, a transmembrane domain and at least one intracellular signaling domain that can specifically bind to an antigen. Typically, CAR is a genetically engineered receptor that redirects the cytotoxicity of immune effector cells toward cells presenting a given antigen. CAR is a molecule that combines the specificity of antibodies based on the desired antigen (eg, tumor antigen) with the intracellular domain of the activated T cell receptor to produce a chimeric protein that exhibits specific cellular immune activity. In a specific embodiment, CAR comprises an extracellular domain (also referred to as a binding domain or an antigen-specific binding domain), a transmembrane domain and an intracellular (cytoplasmic) signaling domain. The engagement of the antigen binding domain of CAR with the target antigen on the surface of the target cell results in the aggregation of CAR, and the activation stimulus is delivered to the cell comprising CAR. The main feature of CAR is that it uses the cell-specific targeting ability of monoclonal antibodies, soluble ligands or cell-specific co-receptors to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or molecular production (capable of mediating cell death of cells expressing target antigens in a manner independent of major histocompatibility (MHC)). In some embodiments, CAR comprises an extracellular binding domain that specifically binds to a tumor antigen; a transmembrane domain and one or more intracellular signaling domains. In various embodiments, CAR comprises an extracellular binding domain, a transmembrane domain and one or more intracellular signaling domains that specifically bind to human CD137.

[0088] As used herein, the term "CAR therapy" refers to the administration of engineered immune cells expressing CAR to human subjects to treat a given disease, such as cancer or autoimmune diseases. CAR therapy refers to the specific treatment of patients with engineered immune cells, and is not intended to include therapies commonly used in conjunction with CAR cell therapy, such as lymphodepleting chemotherapy. It is worth noting that, unless otherwise stated, in the case of the term "cell" used throughout the text, the term also includes cell colonies. For example, because CAR therapy requires the administration of engineered cell colonies.

[0089] As used herein, the term "combination" or "combination therapy" refers to the use of two (or more) therapies in a single human patient. These terms are not intended to refer to the combined components. For example, a combination therapy comprising the administration of an anti-CD137 ADC and an allogeneic CAR therapy is described herein.

[0090] As used herein, the term "sensitization response" refers to the immune response caused in a subject when an antigen is presented to the immune system of a human subject for the first time. The first contact of a T cell or a B cell with a specific antigen is called sensitization, and causes alloantigen (allo-antigen) T cell activation. The sensitization dose can be a single dose or multiple doses required for the elicitation response. "Sensitizing amount" is the amount of an antigen (e.g., allogeneic cells) used to induce a sensitization response. The sensitization amount is generally not a therapeutically effective amount, but an amount for activating a subject's T cells. In some embodiments, the sensitization amount of allogeneic cells is less than the therapeutically effective amount of allogeneic cells. In one embodiment, a sensitization response is induced after allogeneic cells are administered to a human subject, so that CD137+ T cells are activated.

[0091] In the context of the effect of an anti-CD137 antibody or ADC on cells expressing CD137, the term "depletion" refers to a decrease or elimination in the number of cells expressing CD137.

[0092] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to achieve a desired result or have an effect on an autoimmune disease or cancer.

[0093] As used herein, the terms "subject" and "patient" refer to an organism, such as a human, who is receiving treatment for a particular disease or condition as described herein.

[0094] As used herein, "to treat" or "treatment" refers to any improvement in disease outcomes, such as prolonged survival, less morbidity, and / or mitigation of side effects as a byproduct of alternative treatment modalities; as is readily understood in the art, complete elimination of the disease is preferred, but not a requirement for therapeutic behavior. Beneficial or desired clinical outcomes include, but are not limited to, promoting the acceptance of allogeneic immune cells expressing CAR. Insofar as the methods of the present invention are intended to prevent disorders, it should be understood that the term "prevention" does not require that the disease state be completely prevented. Rather, as used herein, the term prevention refers to the ability of a technician to identify a population susceptible to the disorder so that administration of the compounds of the present invention can be performed before the onset of the disease. The term does not mean that the disease state is completely avoided.

[0095] As used herein, the term "vector" includes nucleic acid vectors, such as plasmids, DNA vectors, plasmids, RNA vectors, viruses or other suitable replicons. The expression vectors described herein may contain polynucleotide sequences and, for example, other sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that may be used to express CAR include plasmids containing regulatory sequences (such as promoters and enhancer regions) for guiding gene transcription. Other useful vectors for antibody or CAR expression include polynucleotide sequences that enhance the translation rate of these genes or improve the stability of mRNA produced by gene transcription or nuclear export. These sequence elements may include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to guide the effective transcription of genes carried on the expression vector. The expression vectors described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin and nourseothricin.

[0096] As used herein, the term "antibody drug conjugate" or "ADC" refers to an antibody connected to a cytotoxin. ADC is formed by chemical bonding of a reactive functional group of a molecule (such as an antibody or its antigen-binding fragment) to an appropriate reactive functional group of another molecule (such as a cytotoxin described herein). The conjugate can include a joint between two molecules bound to each other (e.g., between an antibody and a cytotoxin). It is worth noting that the term "conjugate" (when referring to a compound) is also referred to interchangeably herein as "drug conjugate" or "antibody drug conjugate" or "ADC". Examples of joints that can be used to form conjugates include peptide-containing joints, such as joints containing naturally occurring or non-naturally occurring amino acids such as D-amino acids. Joints can be prepared using a variety of strategies described herein and known in the art. Depending on the reactive components therein, the linker can be cleaved by, for example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0097] As used herein, the term "coupling reaction" refers to a chemical reaction in which two or more substituents suitable for reacting with each other react to form a chemical moiety that links together (e.g., covalently) the molecular fragments bound to each substituent. Coupling reactions include those reactions in which a reactive substituent bound to a fragment of a cytotoxin (such as a cytotoxin known in the art or described herein) reacts with a suitable reactive substituent bound to a fragment of an antibody or its antigen-binding fragment (such as an antibody, an antigen-binding fragment thereof, or a specific anti-CD137 antibody known in the art or described herein in conjunction with CD137). Examples of suitable reactive substituents include nucleophiles / electrophiles pairs (e.g., especially thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs), dienes / dienophiles pairs (e.g., especially azide / alkyne pairs), etc. Coupling reactions include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine condensation, amidation, esterification, disulfide formation, cycloadditions (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, in particular), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reactive modes known in the art or described herein. As used herein, the term "microtubule-binding agent" refers to a compound that acts by disrupting the microtubule network, which is essential for mitotic and interphase cell function in cells. Examples of microtubule-binding agents include, but are not limited to, maytansine, maytansine alkaloids and derivatives thereof, such as those described herein or known in the art, vinca alkaloids, such as vinblastine, vinblastine sulfate, vincristine, vincristine sulfate, vindesine, and vinorelbine, taxanes, such as docetaxel and paclitaxel, macrolides, such as discodermolide, colchicine, and epothilones and derivatives thereof, such as epothilone B or a derivative thereof.

[0098] As used herein, the term "amanitin" refers to a member of the amanita toxin family of peptides produced by the Amanita phalloides mushroom or a derivative thereof, such as a variant or derivative thereof that is capable of inhibiting RNA polymerase II activity. Amanita toxins can be isolated from a variety of mushroom species (e.g., Amanita phalloides, Galerina marginata, Lepiota brunneo-incarnata), or can be prepared semi-synthetically or synthetically. A member of this family, α-amanitin, is described in Wieland, Int. J. Pept. Protein Res. 1983, 22 (3): 257-276. Derivatives of amanita toxins can be obtained by chemically modifying naturally occurring compounds ("semi-synthesizing"), or can be obtained from completely synthetic sources. Various synthetic pathways for amanita ani toxin derivatives are disclosed, for example, in U.S. Pat. No. 9,676,702 and Perrin et al., J. Am. Chem. Soc. 2018, 140, pp. 6513-6517, each of which is incorporated herein by reference in its entirety for synthetic methods for preparing and deriving amanita ani toxins.

[0099] Amanita toxins useful in combination with the compositions and methods described herein include compounds such as, but not limited to, compounds of formula (II), for example, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin non-toxic cyclic peptide, monohydroxyamanitin carboxylic acid, and proamanitin non-toxic cyclic peptide. As described herein, amanitin toxins can be conjugated to antibodies or antigen-binding fragments thereof (thereby forming ADCs) via, for example, a linker portion (L). Exemplary methods of amanitin toxin conjugation and linkers useful for such methods are described below. The structures of exemplary amanitin toxin-linker conjugates are represented by formula (III), formula (IIIA), and formula (IIIB). Also described herein are exemplary amanitin toxins containing linkers that can be used for conjugation to antibodies or antigen-binding fragments according to the compositions and methods.

[0100] As used herein, the term "acyl" refers to -C(=O)R, wherein R is hydrogen ("aldehyde"), C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C7 carbocyclic group, C6-C 20 Aryl, 5-10 membered heteroaryl or 5-10 membered heterocyclyl. Non-limiting examples include formyl, acetyl, propionyl, benzoyl and acryloyl.

[0101] As used herein, the term "C1-C 12"Alkyl" refers to a straight or branched chain saturated hydrocarbon having from 1 to 12 carbon atoms. Representative C1-C 12 Alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; and branched C1-C 12 Alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl and 2-methylbutyl. 12 Alkyl groups can be unsubstituted or substituted.

[0102] As used herein, the term "alkenyl" refers to a group comprising a hydrocarbon having at least one site of unsaturation (ie, a carbon-carbon sp 2 double bond) of a normal, secondary or tertiary carbon atom 12 Examples include, but are not limited to, ethylene or vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, etc. The alkenyl group may be unsubstituted or substituted.

[0103] As used herein, "alkynyl" refers to a C2-C6 group containing a normal, secondary, or tertiary carbon atom with at least one site of unsaturation (i.e., a carbon-carbon sp triple bond). 12 Examples include, but are not limited to, acetylenic and propargyl. Alkynyl groups can be unsubstituted or substituted.

[0104] As used herein, "aryl" refers to a C6-C 20 Carbocyclic aromatic groups. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. Aryl groups may be unsubstituted or substituted.

[0105] As used herein, "arylalkyl" refers to a group in which the carbon atom (typically a terminal carbon atom or sp 3 The alkyl radical of aryl group is substituted with 2- phenylethyl-1-yl, 2-phenylethane-1-yl, 2-naphthylethene-1-yl, naphthylbenzyl, 2-naphthylphenylethane-1-yl, etc. The arylalkyl group comprises 6 to 20 carbon atoms, and the alkyl moiety (comprising alkyl (alkanyl), alkenyl or alkynyl group) of arylalkyl group is 1 to 6 carbon atoms, and the aryl moiety is 5 to 14 carbon atoms. The alkaryl group can be unsubstituted or substituted.

[0106] As used herein, "cycloalkyl" refers to a saturated carbocyclic group, which may be monocyclic or bicyclic. Cycloalkyl groups include rings having 3 to 7 carbon atoms as monocyclic or 7 to 12 carbon atoms as bicyclic. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be unsubstituted or substituted.

[0107] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic group, which may be monocyclic or bicyclic. The cycloalkenyl group includes a ring having 3 to 6 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. Examples of monocyclic cycloalkenyl groups include 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl and 1-cyclohex-3-enyl. The cycloalkenyl group may be unsubstituted or substituted.

[0108] As used herein, "heteroaralkyl" refers to an alkyl radical in which the carbon atom (typically a terminal carbon atom or sp 3 Acyclic alkyl groups in which one hydrogen atom bonded to a heteroaryl group (carbon atom) is replaced by a heteroaryl group. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, etc. The heteroarylalkyl group contains 6 to 20 carbon atoms, for example, the alkyl portion of the heteroarylalkyl group (including alkyl, alkenyl or alkynyl groups) is 1 to 6 carbon atoms, and the heteroaryl portion is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S. The heteroaryl portion of the heteroarylalkyl group can be a monocyclic ring with 3 to 7 ring members (2 to 6 carbon atoms) or a bicyclic ring with 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example: bicyclic [4,5], [5,5], [5,6] or [6,6] systems.

[0109] As used herein, "heteroaryl" and "heterocycloalkyl" refer to an aromatic ring system or a non-aromatic ring system, respectively, in which one or more ring atoms are heteroatoms, such as nitrogen, oxygen and sulfur. The heteroaryl or heterocycloalkyl group contains 2 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S. The heteroaryl or heterocycloalkyl group can be a monocyclic ring with 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S) or a bicyclic ring with 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example: a bicyclic [4,5], [5,5], [5,6] or [6,6] system. Heteroaryl and heterocycloalkyl can be unsubstituted or substituted.

[0110] Heteroaryl groups and heterocycloalkyl groups are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), especially Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.

[0111] For example, examples of heteroaryl groups include, but are not limited to, pyridyl, thiazolyl, tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolinyl, quinolyl, isoquinolyl, benzimidazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl (4H-quinolizinyl), 2H-dole-2-yl, 2H-dole-3-yl, 2H-dole-4-yl, 2H-dole-5-yl, 2H-dole-6-yl, 2H-dole-7-yl, 2H-dole-8-yl, 2H-dole-9-yl, 2H-dole-10-yl, 2H-dole-21-yl, 2H-dole-12-yl, 2H-dole-13-yl, 2H-dole-14-yl, 2H-dole-15-yl

[0063] The invention also includes phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, benzotriazolyl, benzisoxazolyl and isatinoyl.

[0112] For instance, examples of heterocycloalkyl include, but are not limited to, dihydropyridinyl, tetrahydropyridinyl (piperidyl), tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidinonyl, tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, piperazinyl, quinuclidinyl, and morpholinyl.

[0113] For example, but not limited to, carbon-bonded heteroaryl and heterocycloalkyl are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole, or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline, or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more typically, the carbon-bonded heterocycle includes 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl.

[0114] For example, but not limited to, nitrogen-bonded heteroaryl and heterocycloalkyl are bonded at position 1 of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, dihydroindoline, 1H-indazole, position 2 of isoindole or isoindoline, position 4 of morpholine, and position 9 of carbazole or β-carboline. Still more typically, nitrogen-bonded heterocycles include 1-aziridinyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0115] As used herein and applied to any of the above alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, etc., "substituted" means that one or more hydrogen atoms are each independently replaced by a substituent. Unless otherwise limited by the definition of the individual substituent, the aforementioned chemical moieties, such as "alkyl", "alkylene", "heteroalkyl", "heteroalkylene", "alkenyl", "alkenylene", "heteroalkenyl", "heteroalkenylene", "alkynyl", "alkynylene", "heteroalkynyl", "heteroalkynylene", "cycloalkyl", "cycloalkylene", "heterocycloalkyl", "heterocycloalkylene", "aryl", "arylene", "heteroaryl" and "heteroarylene" groups may be optionally substituted. Typical substituents include, but are not limited to, -X, -R, -OH, -OR, -SH, -SR, NH2, -NHR, -N(R)2, -N +(R)3, -CX3, -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO2, -N3, -NC(=O)H, -NC(=O)R, -C(=O)H, -C(=O)R, -C(=O)NH2, -C(=O)N (R)2, -SO3-, -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NH2, -S(=O)2N(R)2, -S(=O)R, -OP(=O)(OH)2, -OP(=O)(OR)2, -P( =O)(OR)2, -PO3, -PO3H2, -C(=O)X, -C(=S)R, -CO2H, -CO2R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NH2, -C(=O)N(R)2, -C(=S)NH2, -C(=S)N(R)2, -C(=NH)NH2, and -C(=NR)N(R)2; wherein for each occurrence each X is independently selected from F, Cl, Br, and I; and for each occurrence each R is independently selected from Cl-C 12 Alkyl, C6-C 20 Aryl, C3-C 14 Heterocycloalkyl or heteroaryl, protecting groups and prodrug moieties. In all cases where a group is described as "optionally substituted", the group may, independently for each occurrence, be substituted with one or more of the above substituents.

[0116] It is understood that certain group naming conventions may include monovalent groups or divalent groups, depending on the context. For example, where a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a divalent group. For example, a substituent of an alkyl group identified as requiring two points of attachment includes a divalent group, such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc. Other group naming conventions explicitly indicate that the group is a divalent group, such as "alkylene", "alkenylene", "arylene", "heterocycloalkylene", etc.

[0117] In all cases where a substituent is described as a diradical (ie, having two points of attachment to the rest of the molecule), it is understood that the substituent may be attached in any orientation configuration unless otherwise specified.

[0118] "Isomeric" means compounds that have the same molecular formula but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers." Stereoisomers that are not mirror images of one another are termed "diastereoisomers," and stereoisomers that are non-superimposable mirror images of one another are termed "enantiomers" or sometimes "optical isomers."

[0119] A carbon atom bonded to four non-identical substituents is called a "chiral center." A "chiral isomer" means a compound having at least one chiral center. Compounds having more than one chiral center may exist as individual diastereomers or as a mixture of diastereomers (called a "diastereomeric mixture"). When one chiral center is present, the stereoisomers may be characterized by the absolute configuration (R or S) of that chiral center. The absolute configuration refers to the spatial arrangement of the substituents attached to the chiral center in question. The substituents attached to the chiral center in question are ordered according to the sequence rules of Cahn, Ingold, and Prelog. (Cahn et al., Angew. Chem. Inter. ed. 1966, 5, 385; corrigendum 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116.) A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is called a "racemic mixture".

[0120] The compounds disclosed in this specification and claims may contain one or more asymmetric centers, and different diastereomers and / or enantiomers may exist for each compound. Unless otherwise stated, the description of any compound in this specification and claims is intended to include all enantiomers, diastereomers and mixtures thereof. In addition, unless otherwise stated, the description of any compound in this specification and claims is intended to include both a single enantiomer and any racemic or other mixtures of enantiomers. When the structure of a compound is described as a specific enantiomer, it should be understood that the invention of the present application is not limited to the specific enantiomer. Accordingly, enantiomers, optical isomers and diastereomers of each structural formula of the present disclosure are considered herein. In this specification, for convenience, the structural formula of a compound represents a certain isomer in some cases, but the present disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, etc., and it should be understood that not all isomers can have the same activity level. Compounds may exist in different tautomeric forms. Unless otherwise stated, compounds according to the present disclosure are intended to include all tautomeric forms. When the structure of a compound is described as a specific tautomer, it should be understood that the invention of the present application is not limited to this specific tautomer.

[0121] Compounds of any formula described herein include the compounds themselves, and if applicable, include their salts and their solvates. For example, salts can be formed between anions and positively charged groups (e.g., amino) on the compounds of the present disclosure. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, mesylate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, toluenesulfonate, salicylate, lactate, naphthylidenesulfonate and acetate (e.g., trifluoroacetate). The term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Similarly, salts can also be formed between negatively charged groups (e.g., carboxylates) on cations and compounds of the present disclosure. Suitable cations include sodium, potassium, magnesium, calcium and ammonium cations, such as tetramethylammonium ions. Examples of some suitable substituted ammonium ions are those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and amino acids such as lysine and arginine. Compounds of the present disclosure also include those salts containing quaternary nitrogen atoms.

[0122] Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid and phosphorous acid. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0123] Additionally, the compounds of the present disclosure, for example, salts of the compounds, may exist in hydrated or non-hydrated (anhydrous) forms, or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc. "Solvate" means a solvent addition form containing a stoichiometric or non-stoichiometric amount of solvent. Some compounds have a tendency to capture a fixed molar ratio of solvent molecules in a crystalline solid state, thereby forming a solvate. If the solvent is water, the solvate formed is a hydrate; and if the solvent is an alcohol, the solvate formed is an alcoholate. Hydrates are formed by combining one or more water molecules with a substance molecule, wherein the water maintains its molecular state as H2O. Hydrates refer to, for example, monohydrates, dihydrates, trihydrates, etc.

[0124] In addition, the compounds represented by the formula disclosed herein or salts thereof may exist in crystal polymorphism. It should be noted that any crystal form, mixture of crystal forms or anhydrides or hydrates thereof are included within the scope of the present disclosure.

[0125] The following sections provide descriptions of ADCs that can be administered to human patients to treat or prevent rejection of allogeneic cell therapy, as well as methods of administering such treatments to patients.

[0126] II. Allogeneic Cell Therapy Approach

[0127] In an immunocompetent host, transplanted allogeneic cells are often rapidly rejected. Such rejection is often referred to as host-versus-graft (HvG) rejection. One challenge of allogeneic cell therapy is identifying means by which human recipients can accept allogeneic cells. Such acceptance of allogeneic cells can affect the efficacy of treatment and the outcome of adverse side effects to patients.

[0128] Described herein are methods of treating or preventing allogeneic cellular rejection in human subjects by administering an anti-CD137 antibody drug conjugate (ADC) that targets endogenous CD137+ cells (eg, activated T cells).

[0129] In certain embodiments, prior to administering the anti-CD137 ADC, a sensitization response is first induced in a human subject. The "sensitization" of the patient helps to generate activated anti-alloantigen T cells, which are generated after the administration of allogeneic cells. It is expected that the infusion of allogeneic cells will stimulate the patient's immune system to react against the allogeneic cells. A sensitizing amount of allogeneic cells is administered to a human subject in order to induce a "sensitized" immune response.

[0130] In the sensitization step, allogeneic cells are administered to the patient, for example, intravenously, in order to elicit an immune response, thereby generating endogenously activated CD137+ T cells. Thus, the sensitizing dose of allogeneic cells is preferably an amount effective to elicit an immune response, including activation of endogenous CD137+ T cells. In an exemplary embodiment, the effective sensitizing dose is an aliquot of allogeneic cells for intravenous infusion, which is at least about 1×10 5 cells / kg, 0.5×10 6 cells / kg, about 1×10 6 cells / kg, about 1×10 7 cells / kg, about 1×10 8 Allogeneic cells / kg, about 1×10 9 Allogeneic cells / kg, about 1×10 10 Allogeneic cells / kg, about 0.5×10 6 cells / kg to about 1×10 10 cells / kg, about 0.5×10 6 cells / kg to about 1×10 8 cells / kg, about 1×10 7 cells / kg to about 1×10 10 cells / kg, about 1×10 8 cells / kg to about 1×10 10 cells / kg or about 1×10 9 cells / kg to about 1×10 10 In other exemplary embodiments, the sensitizing dose comprises about 1×10 5 Allogeneic cells / kg to 1×10 12 Allogeneic cells / kg, for example, 1×10 5 cells / kg to 1×10 6 cells / kg, 1×10 5 cells / kg to 1×10 7 cells / kg, 1×10 5 cells / kg to 1×10 8 cells / kg, 1×10 5 cells / kg to 1×10 9 cells / kg, 1×10 5 cells / kg to 1×10 10 cells / kg, 1×10 5 cells / kg to 1×10 11 cells / kg or 1×10 5 cells / kg to 1×1012 In other exemplary embodiments, the sensitizing dose of allogeneic cells comprises about 1×10 6 cells / kg to 1×10 12 cells / kg, 1×10 7 cells / kg to 1×10 12 cells / kg, 1×10 8 cells / kg to 1×10 12 cells / kg, 1×10 9 cells / kg to 1×10 12 cells / kg, 1×10 10 cells / kg to 1×10 12 cells / kg or 1×10 11 cells / kg to 1×10 12 Cells / kg. Doses intermediate to the above ranges are also contemplated for use in the methods of the invention. Doses of allogeneic cells capable of eliciting an immune response outside of these ranges are also within the scope of the invention.

[0131] In one embodiment, the allogeneic cells include, but are not limited to, allogeneic T cells or allogeneic NK cells.In an exemplary embodiment, the allogeneic cells express a chimeric antigen receptor, as described herein.

[0132] After administering a sensitized amount of allogeneic cells, an anti-CD137 antibody drug conjugate (ADC) is administered to a human subject. CD137 is expressed on activated T cells. Therefore, anti-CD137 ADC can be used to consume the number of activated T cells in a human subject before administering allogeneic cell therapy. Exemplary anti-CD137 antibodies, cytotoxins, and connectors suitable for use in the methods described herein are described in detail below.

[0133] After CD137 ADC is administered, a therapeutically effective amount of allogeneic cells is administered to a human subject. Before administering allogeneic cells (e.g., allogeneic cells expressing CAR), activated T cell populations are selectively consumed to improve the overall efficacy of allogeneic cell therapy by treating or preventing allogeneic cell rejection. The therapeutically effective amount of allogeneic cells can be an amount that effectively achieves therapeutic benefit in a patient. For example, if allogeneic cells are used in the treatment of a subject with cancer, a therapeutically effective amount can be an amount that effectively treats cancer, reduces the size or volume of cancer, reduces one or more symptoms associated with cancer, induces cancer remission, and / or prolongs the duration of survival or disease-free survival of a subject with cancer. In one embodiment, the therapeutically effective amount of allogeneic cells is greater than the number of cells administered to the subject during the sensitization step. In another embodiment, the therapeutically effective amount of allogeneic cells is approximately equal to the number of cells administered to the subject during the sensitization step. In another embodiment, the therapeutically effective amount of allogeneic cells is less than the number of cells administered to the subject during the sensitization step.

[0134] In an exemplary embodiment, the therapeutically effective amount of allogeneic cells comprises at least 1×10 5 cells / kg, for example, at least 1×10 6 cells / kg, at least 1×10 7 cells / kg, at least 1×10 8 cells / kg, at least 1×10 9 cells / kg, at least 1×10 10 cells / kg, at least 1×10 11 cells / kg, at least 1×10 12 cells / kg, at least 1×10 13 cells / kg, at least 1×10 14 cells / kg, at least 1×10 15 cells / kg, at least 1×10 16 cells / kg, at least 1×10 17 In other exemplary embodiments, the therapeutically effective amount of allogeneic cells comprises up to 1×10 5 cells / kg, e.g., up to 1×10 6 cells / kg, up to 1×10 7 cells / kg, up to 1×10 8 cells / kg, up to 1×10 9 cells / kg, up to 1×10 10 cells / kg, up to 1×10 11 cells / kg, up to 1×1012 cells / kg up to 1×10 13 cells / kg, up to 1×10 14 cells / kg, up to 1×10 15 cells / kg, up to 1×10 16 cells / kg or up to 1×10 17 In other exemplary embodiments, the therapeutically effective amount of allogeneic cells comprises 1×10 5 cells / kg to 1×10 17 cells / kg, for example, 1×10 6 cells / kg to 1×10 17 cells / kg, 1×10 7 cells / kg to 1×10 17 cells / kg, 1×10 8 cells / kg to 1×10 17 cells / kg, 1×10 9 cells / kg to 1×10 17 cells / kg, 1×10 10 cells / kg to 1×10 17 cells / kg, 1×10 11 cells / kg to 1×10 17 cells / kg, 1×10 12 cells / kg to 1×10 17 cells / kg, 1×10 13 cells / kg to 1×10 17 cells / kg, 1×10 14 cells / kg to 1×10 17 cells / kg, 1×10 15 cells / kg to 1×10 17 cells / kg or 1×10 16 cells / kg to 1×10 17 In other exemplary embodiments, the therapeutically effective amount of allogeneic cells comprises 1×10 5 cells / kg to 1×10 17 cells / kg, for example, 1×10 5 cells / kg to 1×10 16 cells / kg, 1×10 5 cells / kg to 1×10 15 cells / kg, 1×10 5 cells / kg to 1×10 14 cells / kg, 1×10 5 cells / kg to 1×1013 cells / kg, 1×10 5 cells / kg to 1×10 12 cells / kg, 1×10 5 cells / kg to 1×10 11 cells / kg, 1×10 5 cells / kg to 1×10 10 cells / kg, 1×10 5 cells / kg to 1×10 9 cells / kg, 1×10 5 cells / kg to 1×10 8 cells / kg, 1×10 5 cells / kg to 1×10 7 cells / kg or 1×10 5 cells / kg to 1×10 6 In other exemplary embodiments, the therapeutically effective amount of allogeneic cells comprises 1×10 8 cells / kg to 1×10 10 In other exemplary embodiments, the therapeutically effective amount of allogeneic cells comprises 1×10 10 cells / kg to 1×10 12 In other exemplary embodiments, the therapeutically effective amount of allogeneic cells comprises 1×10 12 cells / kg to 1×10 14 cells / kg.

[0135] The allogeneic cells used in the initial sensitization step of therapy can be the same (or similar) type as the allogeneic cells to be applied to human subjects as part of allogeneic cell therapy.For example, the allogeneic cells used in the sensitization step can be derived from the same donor as the cells used in the allogeneic cell therapy.In another embodiment, the allogeneic cells used in the sensitization step can express the same or similar MHC molecule complement as the cells used in the allogeneic cell therapy.The MHC molecule complement expressed by sensitized cells and / or therapeutic cells can be naturally occurring, or can be modified, for example, using genetic engineering.In one embodiment, in allogeneic CAR cell therapy, allogeneic cells of the same type as the allogeneic cells used in CAR cell therapy are administered to human subjects to induce sensitization responses.In an alternative, sensitization responses can be triggered with allogeneic cells expressing CAR, which are the same type as the cells expressing CAR used to treat patients.

[0136] After administering CAR cell therapy, the risk of rejection of allogeneic cells expressing CAR may be high. Anti-CD137 ADC can be used to selectively target activated T cells in patients who will receive allogeneic CAR cell therapy. In certain embodiments, the methods disclosed herein can be used to inhibit or prevent rejection of allogeneic cells expressing CAR in human patients.

[0137] Anti-CD137 ADCs can be administered to human patients in need before, at the same time as, or after administering one or more sensitizing doses of allogeneic cell therapy. In one embodiment, anti-CD137 ADCs are administered to human patients in need before administering a sensitizing dose of allogeneic cells. For example, in some embodiments, anti-CD137 ADCs can be administered up to about 10 days before administering a sensitizing dose of allogeneic cells, for example, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, about 24 hours, about 12 hours, about 6 hours, about 3 hours, about 2 hours, or about 1 hour before administering a sensitizing dose of allogeneic cells. In some embodiments, the anti-CD137 ADC is administered to a human patient in need thereof about 1 hour to about 48 hours; about 1 hour to about 24 hours; about 1 hour to about 18 hours; about 1 hour to about 12 hours; about 1 hour to about 10 hours; about 1 hour to about 6 hours; about 1 hour to about 3 hours; about 1 hour to about 2 hours; about 1 day to 2 days; about 1 day to 3 days; about 1 day to 4 days; about 1 day to 7 days; about 1 day to 10 days; about 3 days to 5 days; about 3 days to 7 days; about 3 days to 10 days; about 12 hours to about 48 hours; about 12 hours to about 36 hours, etc., prior to administration of a sensitizing dose of allogeneic cells.

[0138] In another embodiment, the anti-CD137 ADC is administered to a human patient in need thereof simultaneously with administration of a sensitizing dose of allogeneic cells.

[0139] In other embodiments, the anti-CD137 ADC is administered to a human patient in need thereof after administration of a sensitizing dose of allogeneic cells. For example, in some embodiments, the anti-CD137 ADC can be administered to a human patient in need thereof after administration of a sensitizing dose of allogeneic cells about 1 hour, 4 hours, 6 hours, 10 hours, 12 hours, 24 hours (1 day), 36 hours, 48 ​​hours (2 days), 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 21 days or more. In other embodiments, the anti-CD137 ADC can be administered to a human patient in need thereof from about 1 hour to about 10 days after administration of a sensitizing dose of allogeneic cells, for example, from about 1-6 hours; about 1-12 hours; about 12 hours to about 7 days; about 12 hours to about 6 days; about 12 hours to about 5 days; about 12 hours to about 4 days; about 12 hours to about 3 days; about 12 hours to about 2 days; about 12 hours to about 24 hours; about 1 day to about 10 days; about 1 day to about 9 days; about 1 day to about 8 days; about 1 day to about 7 days; about 1 day to about 6 days; about 1 day to about 5 days; about 1 day to about 4 days; about 1 day to about 3 days; about 1 day to about 2 days, about 3 days to about 10 days; about 5 days to about 7 days, etc. after administration of a sensitizing dose of allogeneic cells.

[0140] In one embodiment, a single dose of an anti-CD137 ADC is administered to a human patient prior to, subsequent to, or concurrently with administration of a sensitizing dose of allogeneic cells, wherein such a single dose is sufficient to selectively deplete a population of T cells activated in response to administration of the allogeneic cells.

[0141] Anti-CD137 ADC can be administered to human patients with corresponding needs before, at the same time or after administering a therapeutically effective amount of allogeneic cells (e.g., allogeneic cells expressing CAR). In one embodiment, anti-CD137 ADC is administered to human patients with corresponding needs before administering allogeneic cells expressing CAR. In one embodiment, allogeneic cell therapy is administered to patients after anti-CD137 ADC has been removed or substantially removed from the patient's blood. In one embodiment, anti-CD137 ADC is administered to human patients with corresponding needs before administering allogeneic cell therapy (e.g., CAR cell therapy) (e.g., about 14 days ago, about 13 days ago, about 12 days ago, about 11 days ago, about 10 days ago, about 9 days ago, about 8 days ago, about 7 days ago, about 6 days ago, about 5 days ago, about 4 days ago, about 2 days ago, or about 1 day ago). In one embodiment, the anti-CD137 ADC is administered to a human patient in need thereof about 1 day to about 14 days; about 2 days to about 13 days; about 3 days to about 12 days; about 3 days to about 11 days; about 4 days to about 10 days; about 5 days to about 9 days; about 6 days to about 8 days; about 1 day to about 13 days; about 1 day to about 12 days; about 1 day to about 11 days; about 1 day to about 10 days; about 1 day to about 9 days; about 1 day to about 8 days; about 1 day to about 7 days; about 1 day to about 6 days; about 1 day to about 5 days; about 1 day to about 4 days; about 1 day to about 3 days; or about 1 day to about 2 days prior to administration of the allogeneic cell therapy.

[0142] In one embodiment, the anti-CD137 ADC is administered to a human patient before allogeneic cells expressing CAR, wherein the anti-CD137 ADC is administered to a human subject about 12 hours to about 14 days before the administration of allogeneic cells expressing CAR. In one embodiment, the anti-CD137 ADC is administered to a human patient before allogeneic cells expressing CAR, wherein the anti-CD137 ADC is administered to a human subject about 24 hours to about 12 days before the administration of allogeneic cells expressing CAR. In one embodiment, the anti-CD137 ADC is administered to a human patient before allogeneic cells expressing CAR, wherein the anti-CD137 ADC is administered to a human subject about 1 day to about 10 days before the administration of allogeneic cells expressing CAR. In one embodiment, the anti-CD137 ADC is administered to a human patient before allogeneic cells expressing CAR, wherein the anti-CD137 ADC is administered to a human subject about 2 days to about 8 days before the administration of allogeneic cells expressing CAR.

[0143] In one embodiment, the anti-CD137 ADC is administered to a human patient in need thereof simultaneously with administration of allogeneic cells expressing CAR.

[0144] After anti-CD137 ADC is applied, human patients can be tested to confirm that before allogeneic CAR therapy is started, the CD137+ activated T cells of the patient have been substantially consumed. The level of CD137+ activated T cells in the biological sample (e.g., blood) of the human patient after applying CD137 ADC can be determined, and compared with the level of CD137+ activated T cells from the biological sample (same source, e.g., blood) before applying anti-CD137 ADC (but after allogeneic cell sensitization). The reduction of endogenous CD137+ activated T cell number indicates that anti-CD137 ADC is effective in reducing the risk of rejection of allogeneic (using the same or substantially similar allogeneic cells) CAR therapy. In one embodiment, the level of endogenous CD137+ activated T cells in a biological sample (of the same type, e.g., blood) from a human patient is reduced by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, 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%, relative to the level of CD137+ activated T cells in a biological sample from a human patient immediately before the administration of an anti-CD137 ADC. In one embodiment, the level of endogenous CD137+ activated T cells is determined one day or less before the administration of an anti-CD137 ADC.

[0145] Alternatively, the total level of T cells in a biological sample from a human patient can be tested after administration of an anti-CD137 ADC (after allogeneic cell sensitization), wherein the total number of T cells in a human patient after administration of an anti-CD137 ADC relative to the reduction in the level before administration indicates the efficacy of the anti-CD137 ADC for preventing rejection of allogeneic cell therapy. In one embodiment, the level of endogenous T cells in a biological sample from a human patient (of the same type, such as blood) is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, relative to the level of T cells in a biological sample from a human patient before the anti-CD137 ADC is administered. In one embodiment, the level of endogenous T cells in a biological sample from a human patient (of the same type, such as blood) is reduced by about 5% to 25%, about 5% to 20%, about 5% to 15%, or about 5% to 10%, relative to the level of T cells in a biological sample from a human patient before the anti-CD137 ADC is administered. In one embodiment, the level of endogenous T cells is determined one day or less prior to administration of the anti-CD137 ADC.

[0146] The level of T cells, including CD137+ activated T cells, can be determined according to standard methods known in the art, including but not limited to in vitro antigen stimulation assays, in which the number of alloantigenic T cells activated after exposure to allogeneic cells is compared to the number of alloantigenic T cells activated after exposure to allogeneic cells in the presence of an anti-CD137 ADC.

[0147] The methods disclosed herein can be used to deliver allogeneic cells expressing CAR to a subject. Importantly, the anti-CD137 ADC modulation methods described herein can be used to expand the types of immune cells that can be used in CAR therapy by providing a method by which allogeneic cell tolerance can be provided. In one embodiment, allogeneic cells expressing CAR include but are not limited to allogeneic T cells or allogeneic NK cells.

[0148] In one embodiment, the anti-CD137 antibody drug conjugate is used to deplete CD137-expressing T cells, e.g., activated CD137-expressing T cells, by administering the anti-CD137 antibody drug conjugate before, simultaneously with, or after administration of a sensitizing dose of allogeneic cells. In particular, the anti-CD137 ADC can be used to deplete host reactive T cells (activated T cells after sensitization).

[0149] The methods disclosed herein are particularly useful for treating cancer or an autoimmune disease in a human subject having one of these disorders.

[0150] In one embodiment, the methods disclosed herein are used to treat cancer. Examples of cancer types that can be treated using the methods disclosed herein include, but are not limited to, adult advanced cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple-negative breast cancer, hematopoietic / lymphoid cancer, colon cancer liver metastasis, small cell lung cancer, non-small cell lung cancer, B cell lymphoma, relapsed or refractory B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, relapsed or refractory diffuse large cell lymphoma, B-cell lymphoma, anaplastic large cell lymphoma, primary mediastinal B-cell lymphoma, relapsed mediastinal large B-cell lymphoma, refractory mediastinal large B-cell lymphoma, large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, colorectal epithelial carcinoma, gastric cancer, pancreatic cancer, triple-negative invasive breast cancer, renal cell carcinoma , squamous cell carcinoma of the lung, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, multiple myeloma of the bone, malignant glioma of the brain, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasms, blastic plasmacytoid dendritic cell tumor, liver metastasis, solid tumors, advanced solid tumors, mesothelin-positive tumors, hematological malignancies, and other advanced malignancies.

[0151] In one embodiment, the method disclosed herein is used to treat autoimmune diseases. More specifically, anti-CD137 ADC is administered in combination with allogeneic cell therapy (e.g., allogeneic cells expressing CAR) to human subjects with autoimmune diseases. Examples of autoimmune diseases that can be treated using the combined methods disclosed herein include, but are not limited to, multiple sclerosis, Crohn's disease, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, lupus, and psoriasis.

[0152] In certain embodiments, an anti-CD137 ADC is administered to a human patient in combination with a CAR-T cell therapy. In one embodiment, an anti-CD137 ADC is administered to a human patient prior to administration of a CAR-T therapy.

[0153] Examples of CAR-T constructs that can be expressed in allogeneic cells and used with the methods disclosed herein include, but are not limited to, CD19 CAR-T (e.g., CART-19-01, 02, 03 (Fujian Medical University); daopeicart (Hebei Senlang Biotechnology Inc.); IM19CART / 001, YMCART201702 (Beijing Immunochina Medical Science & Technology Co.); CART-CD19-02, 03 (Wuhan Sian Medical Technology Co.); Universal CD19-CART / SHBYCL001, 002 (Shanghai Bioray Laboratory Inc.); UnicarTherapy201701 (Shanghai Unicar-Therapy BiomedicineTechnology Co.); Genechem / NCT02672501 (Shanghai GeneChem Co.); SenL_19 (Hebei Senlang Biotechnology Inc.); PCAR-019 (PersonGen BioTherapeutics (Suzhou); ICAR19 (Immune Cell, Inc.); WM-CART-02 (Sinobioway Cell Therapy Co.); HenanCH080,109,152 (Henan Cancer Hospital / The Pregene (ShenZhen) Biotechnology Co.); IM19-CD28 andIM19-41BB CAR-T cells (Beijing Immunochina Medical Science & Technology Company); CTL019 / IT1601-CART19 (Beijing Sanwater Biological Technology Co.);CTL019 / CCTL019C2201 (Novartis Pharmaceuticals); CD19:4-1BB:CD28:CD3 / FirstShenzhen01 (Shenzhen Second People’s Hospital / The Beijing Pregene Science and Technology Company); MB-CART19.1 (Shanghai Children’s Medical Center / Miltenyi Biotec GmbH); PZ01 CAR-T cells (Pinze Lifetechnology Co.); YMCART201701 (Beijing Immunochina Medical Science & Technology Co.); 2016YJZ12 (Peking University / Marino Biotechnology Co.); EGFRt / 19-28z / 4-1BBL CAR T cells (Memorial Sloan Kettering Cancer Center / Juno Therapeutics, Inc.); Doing-002 (Beijing Doing Biomedical Co.); PCAR-019 (PersonGen BioTherapeutics (Suzhou) Co.); C-CAR011 (Peking Union Medical College Hospital / Cellular Biomedicine Group Ltd.); iPD1CD19 eCAR T cells (Peking University / Marino Biotechnology Co.); 2013-1018 / NCT02529813 (M.D. Anderson Cancer Center / Ziopharm / Intrexon Corp.); HenanCH CAR2-1 (Henan Cancer Hospital / The Pregene (ShenZhen) Biotechnology Co.); JCAR015 (Juno Therapeutics, Inc.); JCAR017 / 017001,004,006 (Juno Therapeutics, Inc.); JCAR017 (Celgene); TBI-1501 (Takara Bio Inc.); JMU-CD19CAR (Jichi Medical University); KTE-C19 (Kite, A Gilead Company); TriCAR-T-CD19 (Timmune Biotech Inc.); PF-05175157 (Fred Hutchinson Cancer Research Center)); CD22 / CD30 / CD7 / BCMA / CD123 (for example, 2016040 / NCT03121625 (Hebei Senlang Biotechnology Inc.)); CD22 (for example, Ruijin-CAR-01 (Ruijin Hospital / Shanghai Unicar-Therapy Bio-medicine Technology Co.); AUTO-PA1, DB1 (Autolus Limited)), CD20 (e.g., Doing-006 (BeijingDoing Biomedical Co.)) or CD20 / CD22 / CD30 (e.g., SZ5601 (The First AffiliatedHospital of Soochow University Shanghai / Unicar-Therapy Bio-medicineTechnology Co.)). .

[0154] Chimeric Antigen Receptor (CAR)

[0155] The present invention includes the use of allogeneic cell therapy (e.g., allogeneic CAR cell therapy) combined with anti-CD137 immunosuppressive ADC to treat or prevent the rejection of allogeneic cells required for therapy. The present invention is generally not limited to specific CAR constructs, such as specific antigen binding regions or intracellular signaling domains, because the present invention is at least partially based on such a discovery that anti-CD137 ADC can promote the acceptance of cells expressing CAR by eliminating endogenous CD137+ immune cells, such as endogenous T cells, thereby being used as a regulator of CAR therapy. Specific CARs, such as CD19-specific CARs, are contemplated herein, and are included in the methods disclosed herein, but are not intended to be limited.

[0156] CAR constructs are known in the art and typically include (a) an extracellular region including an antigen binding domain, (b) a transmembrane domain, and (c) a cytoplasmic signaling domain. Exemplary CAR configurations are known in the art, and any suitable configuration can be used in the methods described herein. For example, CAR can be a first generation CAR, a second generation CAR, or a third generation CAR, for example, as described in: Guedan et al. Molecular Therapy-Methods & Clinical Development. 12: 145-156 (2019) or Sadelain et al. Cancer discovery 3.4: 388-398 (2013), the entire contents of which are incorporated herein by reference. In short, a "first generation" CAR may include (a) an extracellular antigen binding domain, (b) a transmembrane domain, (c) one or more intracellular signaling domains, and optionally (d) a hinge region connecting an antigen binding domain to a transmembrane domain. "Second generation" CAR may include elements (a), (b), (c) and optionally (d), and also include a costimulatory domain, such as a costimulatory domain of CD28 or 4-1BB. "Third generation" CAR may include elements (a), (b), (c) and optionally (d), and also include more than one costimulatory domain, such as a costimulatory domain of CD28 and 4-1BB, or a costimulatory domain of CD28 and OX40. Each of the above elements is described in detail below. It should be understood that in some embodiments, the CAR molecules described by the following exemplary non-limiting arrangements are from left to right, N-terminal to C-terminal of CAR. CAR as described in the present disclosure may include or also include any other combination of elements as described herein. Other exemplary chimeric antigen receptor constructs are disclosed in: U.S. Patent No. 9,328,156; U.S. Patent No. 9,783,591; U.S. Patent No. 9,714,278; U.S. Patent No. 9,765,156; U.S. Patent No. 10,117,896; U.S. Patent No. 9,573,988; U.S. Patent No. 10,308,717; U.S. Patent No. 10,221,245; U.S. Patent No. 10,040,865; U.S. Patent No. 2018 / 0256712A1; U.S. Patent Publication No. 2018 / 0271907A1; U.S. Patent Publication No. 2016 / 0046724A1; U.S. Patent Publication No. 2018 / 0044424A1; U.S. Patent Publication No. 2018 / 0258149A1; U.S. Patent Publication No. 2019 / 0151363A1 and U.S. Patent Publication No. 2018 / 0273601A1; the contents of each of the foregoing patents and patent publications are incorporated herein by reference in their entirety.

[0157] The CAR used in the methods disclosed herein may include an extracellular antigen binding domain. The extracellular antigen binding domain may be any molecule that binds to an antigen, including but not limited to human antibodies, humanized antibodies, or any functional fragments thereof. In certain embodiments, the antigen binding domain is a scFv. In other embodiments, the extracellular antigen binding domain is a non-immunoglobulin scaffold protein. In other embodiments, the extracellular binding domain of CAR includes a single-chain T cell receptor (scTCR). As described in U.S. Patents Nos. 5,359,046, 5,686,281, and 6,103,521, the extracellular domain may also be obtained from any of a variety of extracellular domains or secreted proteins associated with ligand binding and / or signal transduction.

[0158] The selection of the molecular target (antigen) of the extracellular binding domain depends on the type and number of the ligands defining the target cell surface. For example, antigen binding domains can be selected to identify the ligands acting as cell surface markers on target cells related to a specific disease state. Therefore, on the one hand, CAR-mediated immune cells (such as T cells) can be directed to the antigen of interest by engineering the extracellular antigen binding domains specifically binding to the desired antigen into the CAR. For example, antigen binding domains can be selected to identify the ligands acting as cell surface markers on target cells related to a specific disease state (such as cancer or autoimmune diseases). Therefore, examples of cell surface markers that can serve as ligands of the antigen binding domains in CAR include those associated with cancer cells and other forms of diseased cells (such as autoimmune disease cells and pathogen-infected cells). In some embodiments, by engineering the desired antigen binding domains specifically bound to the antigen on tumor cells, CAR is engineered to target tumor antigens of interest. In the context of the present invention, "tumor antigens" refer to antigens common to specific hyperproliferative disorders such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or CD123. In one embodiment, the CAR comprises a scFv that binds to CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or CD123.

[0159] On the other hand, the extracellular binding domain of CAR binds to: AFP (e.g., ETCH17AFPCAR01 (AeonTherapeutics (Shanghai) Co. / Eureka Therapeutics Inc.)), GPC3 (e.g., GeneChem GPC-3CART (Shanghai GeneChem Co.); 302GPC3-CART (Shanghai GeneChem Co.); CAR-T for liver cancer (Shanghai GeneChem Co.); CAR-GPC3 T cells (Carsgen Therapeutics)), MUC1 (e.g., PG-021-001,002 (PersonGen BioTherapeutics (Suzhou) Co.)), mesothelin (e.g., H2017-01-P01 (Ningbo Cancer Hospital); TAI-meso-CART (Shanghai GeneChem Co.); K16-4 / NCT02930993 (China Meitan General Hospital / Marino Biotechnology Co.)), CD38 (e.g., Anti-CD38 A2 CAR-T / SOR-CART-MM-001 (Sorrento Therapeutics, Inc.)), herinCAR-PD1 (e.g., herinCAR-PD1 / NBWYKY2016-06-001, 002, 003 (Ningbo Cancer Hospital); SIMC-20160101, 02, 03 (Shanghai International Medical Center)), BCMA (e.g., P-BCMA-101 autologous memory T stem cell (Tscm) CAR-T cells / P-BCMA-101-001 (Poseida Therapeutics, Inc.); HenanCH284 (Henan Cancer Hospital / The Pregene (ShenZhen) Biotechnology Company); LCAR-B38MCAR-T cells (Nanjing Legend Biotech Co.); 9762 / NCT03338972 (Fred Hutchinson Cancer Research Center / Juno Therapeutics, Inc.); Descartes-08 (Cartesian Therapeutics); KITE-585 (Kite, A Gilead Company); bb21217 (bluebirdbio); bb21217 (Celgene); JCARH125 (Juno Therapeutics, Inc.)), CD30 (e.g., ICAR30 T cells (Immune) Cell, Inc.)), EGFR (for example, EGFR:4-1BB:CD28:CD3 modified T cells / FirstShenzhen02 (Shenzhen Sceond People's Hospital / The Beijing Pregene Science and Technology Company); EGFR-IL12-CART (Shenzhen Second People's Hospital / ThePregene (ShenZhen) Biotechnology Co.); SBNK-2016-015-01 (Beijing Sanbo BrainHospital / Marino Biotechnology Co.)), MG7 (e.g., MG7-CART (Xijing Hospital / Shanghai GeneChem Co.)), BCMA / TACI (e.g., AUTO2-MM1 (Autolus Limited)), CEA (e.g., 383-74 / NCT02416466 (Roger Williams Medical Center / Sirtex Medical)), mesothelin / PSCA / CEA / HER2 / MUC1 / EGFRvIII (e.g., NCT03267173 (First Affiliated Hospital of Harbin Medical University / Shanghai Unicar-Therapy Bio-medicine Technology Co.)), CD20 (e.g., EY201605-19 (Beijing Biohealthcare Biotechnology Co.)), CD33 (e.g., 2016-0341 / NCT03126864 (MDAnderson Cancer Center / Intrexon Corp. / Ziopharm)), EGFR / BCMA (e.g., EGFRt / BCMA-41BBz CAR T cells (Memorial Sloan Kettering Cancer Center / Juno Therapeutics, Inc.)), ROR2 (e.g., autologous CCT301-38 or CCT301-59 T cells (Shanghai Sinobioway Sunterra Biotech)), NKR-2 (e.g., CYAD-N2T-002, 003, 004 (Celyad)), PSCA (e.g., BP-012 (Bellicum Pharmaceuticals)), CD28 (e.g., autologous CSR T cells (Beijing Sanbo Brain Hospital / Marino Biotechnology Co.)), TAA (e.g., AMG 119 (Amgen)), NKG2D (e.g., CM-CS1 (Celyad)) or CD123 (e.g., UCART123 (Cellectis SA)). The foregoing also provides examples of CARs that bind to the antigen (e.g., AMG 119 (Amgen)). These CAR constructs can be used in conjunction with anti-CD137 antibodies in the methods disclosed herein. .

[0160] The CAR construct may also include a transmembrane domain that connects the extracellular antigen binding domain to the cytoplasmic signaling domain (either literally or generally close, such as with a spacer). In some embodiments, the extracellular antigen binding domain of CAR (e.g., scFv, Fab or other antigen binding moieties) can be connected to the transmembrane domain using a hinge or other linker. A spacer, linker or hinge may be introduced between the extracellular antigen binding domain and the transmembrane domain to provide flexibility that allows the antigen binding domain to be oriented in different directions, thereby promoting antigen recognition and binding. As discussed below, the cytoplasmic side of the transmembrane domain may be attached to an intracellular signaling domain, such as an intracellular signaling domain of CD28 or CD3δ (CD3-δ), and may additionally include one or more costimulatory domains.

[0161] Therefore, in certain embodiments, CAR may also include a hinge region between the extracellular antigen binding domain and the transmembrane domain. For example, the hinge region may be derived from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28 or CD8α. In a specific embodiment, the hinge region is derived from the hinge region of IgG4. In another embodiment, the hinge region is a CD8 hinge domain (see SwissProt / GenBank accession number P01732).

[0162] In one embodiment, the CAR comprises an extracellular antigen binding domain and a transmembrane domain connected via a CD8 hinge: AKPTTTPAPR PPTPAPTIAS QPLSLRPEAC RPAAGGAVHT RGLDFA (SEQ ID NO: 2).

[0163] In one embodiment, the CAR comprises an extracellular antigen binding domain and a transmembrane domain connected via a hybrid CD8-CD28 hinge: AKPTTTPAPR PPTPAPTIAS QPLSLRPEAC RPAAGGAVHT RGLDFAPRKIEVMYPPPYLD NEKSNGTIIH VKGKHLCPSP LFPGPSKP (SEQ ID NO: 3).

[0164] The transmembrane domain can be derived from the sequence of the protein that contributes the extracellular antigen binding domain, the sequence of the protein that contributes the effector function signaling domain, the sequence of the protein that contributes the proliferation signaling part, or the sequence of a completely different protein. In some embodiments, the transmembrane domain is naturally associated with one of the other domains of CAR. For example, the transmembrane domain and the cytoplasmic domain can be derived from the transmembrane region and the cytoplasmic region of the same protein. In one embodiment, the transmembrane domain and the cytoplasmic domain of CAR include a continuous portion of the CD28 sequence. Any transmembrane domain can be used in the CAR constructs described herein, provided that the domain is capable of anchoring the CAR comprising the antigen binding domain to the cell membrane.

[0165] Transmembrane domains are derived from natural sources or from synthetic sources. When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. The exemplary transmembrane domains that can be used in the methods provided herein can be derived from (for example, at least comprising the following transmembrane domains): α, β or δ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, LFA-1T cell co-receptors, CD2 T cell co-receptors / adhesion molecules, CD8α and fragments thereof. Any method known in the art (e.g., hydrophobicity analysis, structural analysis, etc.) or by using a public database such as the UniProt database to identify the transmembrane domain of the protein can be used.

[0166] In some embodiments, the transmembrane domain can be synthetic.In an exemplary embodiment, the transmembrane domain can mainly include hydrophobic residues, such as leucine and valine.In one embodiment, the triplets of phenylalanine, tryptophan and valine can be located at each end of the synthetic transmembrane domain.Optionally, a short oligopeptide or polypeptide linker (preferably, a length of 2 to 10 amino acids) can form a connection between the transmembrane domain and the cytoplasmic signaling domain of CAR.Glycine-serine doublets provide particularly suitable linkers.

[0167] In some embodiments, the transmembrane domain in the CAR used herein is a CD8 transmembrane domain or a portion thereof. The sequence of CD8 for this purpose is taught in PCT Publication No. WO2014 / 055771A1.

[0168] In some embodiments, the transmembrane domain in CAR is a CD8 transmembrane domain or a functional portion thereof. For example, CAR can include a CD3 transmembrane domain or a functional portion thereof having an amino acid sequence LDPKLCYLLD GILFIYGVIL TALFLRVK (SEQ ID NO: 4), such as LCYLLDGILF IYGVILTALFL (SEQ ID NO: 5).

[0169] In some embodiments, the transmembrane domain of the CAR of the present invention is a CD28 transmembrane domain. Exemplary sequences of CD28 and exemplary transmembrane domain sequences are provided below. In some embodiments, the CD28 transmembrane domain comprises the following exemplary transmembrane domain sequence or a fragment or variant thereof, and the CAR comprising the sequence can be anchored to the cell membrane. Therefore, in some embodiments, the transmembrane domain of CAR is a CD28 transmembrane domain comprising the following amino acid sequence: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 6). In one embodiment, the transmembrane domain of CAR is a CD28 transmembrane domain comprising the following amino acid sequence: IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWV (SEQ ID NO: 7) or a functional fragment thereof, such as SEQ ID NO: 6.

[0170] In addition to the extracellular antigen-binding domain and the transmembrane domain, CARs also contain an intracellular (or cytoplasmic) signaling domain.

[0171] It is known that the signal generated by the endogenous TCR alone is insufficient to fully activate T cells, and that secondary or co-stimulatory signals may also be required. Therefore, T cell activation can be mediated by two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0172] As used herein, the term "intracellular signaling domain" or "cytoplasmic signaling domain" refers to the intracellular portion of CAR. The intracellular signaling domain can generate signals that promote the immune effector function of immune allogeneic cells (e.g., CAR-T cells or NK cells expressing CAR) containing CAR. Examples of immune effector functions, such as in CART cells or NK cells expressing CAR, include cytolytic activity and auxiliary activity, including the secretion of cytokines. In certain embodiments, the intracellular signaling domain transduces effector function signals and instructs cells to perform specific functions. Although the entire intracellular signaling domain can be used, in many cases, it is not necessary to use the entire chain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used to replace a complete chain, provided that the truncated portion transduces an effector function signal. Therefore, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce an effector function signal.

[0173] In one embodiment, the intracellular signaling domain of the CAR comprises a CD3 delta signaling region as depicted in SEQ ID NO: 8, or a signaling portion thereof.

[0174]

[0175] The cytoplasmic signaling domain may also include, but is not limited to, those derived from CD3δ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, CD278 ("ICOS"), Fc.ε.RI, CD66d, DAP10, and DAP12.

[0176] CAR can also include an "intracellular co-stimulatory domain", which is a polypeptide chain derived from an intracellular signaling domain of a co-stimulatory protein or more co-stimulatory proteins (such as CD28 and 4-1BB), which enhances cytokine production. Exemplary co-stimulatory signaling regions include 4-1BB, CD21, CD28, CD27, CD127, ICOS, IL-15Rα and OX40.

[0177] In certain embodiments, the cytoplasmic costimulatory domain of CAR comprises the 4-1BB signaling domain itself or in combination with any other desired cytoplasmic domain that can be used in the context of CAR. 4-1BB is a member of the TNFR superfamily, having an amino acid sequence provided with GenBank accession number AAA62478.2 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.); and "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.).

[0178] In one embodiment, the intracellular co-stimulatory domain of the CAR is a 4-1BB (CD137) co-stimulatory signaling region or a signaling portion thereof:

[0179]

[0180] In one embodiment, the costimulatory signaling domain of the CAR is a CD28 costimulatory signaling region sequence. For example, the costimulatory signaling domain can include the following CD28 costimulatory signaling region or a signaling portion thereof:

[0181]

[0182] In an exemplary embodiment, the cytoplasmic domain of CAR can include a CD3 δ signaling domain in combination with any other desired cytoplasmic domain that can be used in the context of the CAR of the present invention. In certain embodiments, the cytoplasmic domain of CAR can include a CD3 δ domain and a costimulatory signaling region, including but not limited to the costimulatory signaling region of 4-1BB, CD28 and / or CD27.

[0183] The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR of the present invention can be connected to each other in a random or specific order. Optionally, a short oligopeptide or polypeptide linker or spacer preferably having a length between 5 and 20 amino acids can be inserted between the cytoplasmic domains. GGGGS (SEQ ID NO: 27) or (GGGGS) × 3 (SEQ ID NO: 28) provides a particularly suitable linker.

[0184] In one embodiment, the CAR used herein comprises an extracellular domain containing a single-chain variable domain of an anti-CD19 monoclonal antibody, a transmembrane domain containing a hinge and a transmembrane domain of CD8α, and a cytoplasmic domain containing a signaling domain of CD3δ and a signaling domain of 4-1BB. An exemplary CAR comprises an anti-CD19 extracellular domain, which is included in Nicholson IC et al., Mol Immunol 34: 1157-1165 (1997) and an anti-CD19 monoclonal antibody plus a 21 amino acid signal peptide of CD8α (translated from 63 nucleotides at positions 26-88 of GenBank accession number NM_001768). The CD8α hinge and transmembrane domain consist of 69 amino acids translated from 207 nucleotides at positions 815-1021 of GenBank accession number NM_001768. The CD3 delta signaling domain of a preferred embodiment comprises 112 amino acids translated from 339 nucleotides at positions 1022-1360 of GenBank Accession No. NM_000734.

[0185] Between the extracellular domain (including antigen binding domain) and the transmembrane domain (as described above) of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a spacer or hinge domain can be incorporated. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide whose function is to connect the transmembrane domain in the polypeptide chain to the extracellular domain and / or the cytoplasmic domain. As used herein, the hinge domain generally means any oligopeptide or polypeptide whose function is to provide flexibility for CAR or its domain and / or to prevent the steric hindrance of CAR or its domain. In some embodiments, the spacer or hinge domain may include up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 5 to 20 amino acids. Typically, a spacer or hinge is introduced between the extracellular antigen binding domain and the transmembrane domain to provide flexibility allowing the antigen binding domain to be oriented in different directions, thereby promoting antigen recognition and binding. It should also be understood that one or more spacer domains may be included in other regions of CAR, because aspects of the present disclosure are not limited to this aspect.

[0186] It should be understood that CAR may include a region having a sequence provided herein (e.g., an antigen binding domain, a transmembrane domain, a cytoplasmic domain, a signaling domain, a safety domain and / or a joint or any combination thereof), or a variant thereof or a fragment of any of them (e.g., a variant and / or a fragment that maintains the required function of CAR activity) may be included in the CAR protein described herein. In some embodiments, the variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes relative to the sequence shown. In some embodiments, the variant has a sequence that is at least 80%, at least 85%, at least 90%, 90%-95%, at least 95%, or at least 99% identical to the sequence shown. In some embodiments, the fragment is 1-5, 5-10, 10-20, 20-30, 30-40, or 40-50 amino acids shorter than the sequence provided herein. In some embodiments, the fragment is shorter at the N-terminus, C-terminus, or two terminal regions of the provided sequence. In some embodiments, fragments comprise 80%-85%, 85%-90%, 90%-95%, or 95%-99% of the number of amino acids in the sequences provided herein.

[0187] In other embodiments, the present invention includes nucleic acid sequences encoding the amino acid sequences disclosed herein.

[0188] In some embodiments, the above exemplary, non-limiting arrangement is from left to right, N-terminal to C-terminal of CAR.CAR can include or also include any other combination of elements as described herein.

[0189] After identifying each part of the CAR construct, immune cells expressing CAR are produced, and thus immune cells express CAR. The method includes introducing immune cells with nucleic acid molecules (e.g., RNA molecules, e.g., mRNA) described herein or vectors comprising nucleic acid molecules encoding CAR (e.g., CAR described herein), e.g., transducing immune cells. The present invention also provides a method for producing a cell colony (e.g., RNA engineered cells transiently expressing exogenous RNA). The method includes introducing RNA (e.g., RNA transcribed in vitro or synthetic RNA; mRNA sequences encoding CAR polypeptides as described herein) as described herein into cells. In embodiments, RNA transiently expresses CAR polypeptides. In one embodiment, the cell is a cell as described herein, such as an immune effector cell (e.g., T cell or NK cell, or cell colony).

[0190] Anti-CD137 Antibody Drug Conjugate (ADC)

[0191] As described herein, anti-CD137 ADC can be used in combination with allogeneic cell therapy to treat cancer or autoimmune diseases in human patients. More specifically, anti-CD137 ADC can be used to consume CD137+ cells (e.g., CD137+ T cells) in human subjects who also receive allogeneic cell therapy. Anti-CD137 ADC targets endogenous T cells and kills these cells so that the patient's immune system will not attack the allogeneic cell therapy (e.g., allogeneic cells expressing CAR) administered to the subject. Therefore, anti-CD137 ADC is used in combination with CAR therapy to prevent cell therapy rejection in recipient patients or reduce the risk of cell therapy rejection in recipient patients. One advantage of using anti-CD137 ADC as a regulatory scheme is that activated T cells expressing CD137 can be specifically targeted for consumption.

[0192] Anti-CD137 antibody

[0193] The present invention is based in part on the discovery that antibodies and antigen-binding fragments thereof that are capable of binding to CD137 (also known as CDw137, TNFRSF9, 4-1BB and ILA) can be used as therapeutic agents to prevent or reduce the risk of rejection of cell therapy in patients in need of cell therapy.

[0194] T cells have been shown to express CD137, which is a transmembrane TNF receptor superfamily of co-stimulatory molecules and is expressed on a variety of hematopoietic cells and promotes T cell activation, as well as regulates the proliferation and survival of T cells (see, e.g., Cannons et al., J. Immunol. 167: 1313-1324, 2001, the disclosure of which is incorporated herein by reference when it relates to the expression of CD137 by T cells). The amino acid sequence of human CD137 is provided below:

[0195]

[0196] Antibodies and antigen-binding fragments thereof specific for human CD137 can be identified using techniques known in the art and described herein, such as by immunization, computational modeling techniques, in vitro selection methods, such as the phage display platforms and cell-based display platforms described below.

[0197] In one embodiment, the anti-CD137 antibody that can be used for the methods and compositions described herein (including ADC) is a mouse anti-CD137 antibody BBK2 (Thermo Fisher; MS621PABX) or an anti-CD137 antibody comprising an antigen binding region corresponding to the BBK2 antibody. The BBK2 antibody (also referred to as the BBK-2 antibody or the anti-4-1BB antibody) is a mouse monoclonal antibody (IgG1, κ) that binds to the extracellular domain of the human 4-1BB recombinant protein (4-1BB is also referred to as CD137). In certain embodiments, the methods and compositions of the present disclosure include an anti-CD137 antibody comprising a binding region (e.g., CDR) of a BBK2 antibody. In another embodiment, the methods and compositions of the present disclosure include an antibody that competitively inhibits the binding of the BBK2 antibody to its epitope on CD137. In certain embodiments, the anti-CD137 antibody is a humanized BBK2 or a chimeric BBK2.

[0198] In one embodiment, the methods and compositions described herein include chimeric anti-CD137 (ch-BBK2) antibodies comprising variable heavy and light chain regions of BBK2. In certain embodiments, the chimeric BBK2 antibody is an IgG1 antibody comprising a human constant region. The heavy chain amino acid sequence of ch-BBK2 is described in SEQ ID NO: 11, and the light chain amino acid sequence of ch-BBK2 is described in SEQ ID NO: 12. The CDR regions (CDR1, CDR2, and CDR3) of each heavy chain sequence and light chain sequence are described in bold below. The variable regions are italic.

[0199]

[0200] The aforementioned CDR regions (and BBK2 antibodies) are described in Lee et al. (2002) European J of Immunogenetics 29(5):449-452. Thus, in one embodiment, the VH CDR amino acid sequences of the anti-CD137 antibody BBK2 (including ch-BBK2) are as follows: SGYTFTSYW (VH CDR1; SEQ ID NO: 13); NIYPSDSYT (VH CDR2; SEQ ID NO: 14) and TRNGVEGYPHYYAME (VH CDR3; SEQ ID NO: 15). The VLCDR amino acid sequences of the anti-CD137 antibody BBK2 (including ch-BBK2) are as follows: SQDLSNH (VL CDR1; SEQ ID NO: 16); YYTS (VL CDR2; SEQ ID NO: 17) and CQQGYTLPY (VL CDR3; SEQ ID NO: 18).

[0201] Alternatively, the CDR regions of BBK2 may be defined according to Kabat numbering. The CDRs defined by Kabat numbering for each heavy and light chain sequence are described below (described in bold below). The variable regions of BBK2 are in italics.

[0202]

[0203]

[0204] Thus, in one embodiment, the VH CDR amino acid sequences of the anti-CD137 antibody BBK2 (including ch-BBK2) are as follows: SYWIN (VH CDR1; SEQ ID NO: 19); NIYPSDSYTNYNQKFKD (VH CDR2; SEQ ID NO: 20) and NGVEGYPHYYAMEY (VH CDR3; SEQ ID NO: 21), and the VL CDR amino acid sequences of the anti-CD137 antibody BBK2 (including ch-BBK2) are as follows: RASQDLSNHLY (VL CDR1; SEQ ID NO: 23); YTSRLHS (VL CDR2; SEQ ID NO: 24) and QQGYTLPYT (VL CDR3; SEQ ID NO: 25).

[0205] The heavy chain variable region of BBK2 is listed in SEQ ID NO: 22 as QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSNTVYMQLNSPTSEDSAVYYCTRNGVEGYPHYYAMEYWGQGTSVTVSS. The light chain variable region of BBK2 is listed in SEQ ID NO: 26 as DIQMTQTTSALSASLGDRVTIGCRASQDLSNHLYWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDVATYFCQQGYTLPYTFGGGTKLEIK. Anti-CD137 antibodies (including anti-CD137 ADCs) may comprise the heavy and light chain variable region amino acid sequences set forth in SEQ ID No: 22 and SEQ ID No: 26, respectively.

[0206] In one embodiment, an anti-CD137 antibody, e.g., a chimeric (ch-BBK2) antibody or a humanized BBK2 antibody, comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 19, a CDR2 comprising the amino acid sequence of SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 21; and a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 25.

[0207] In one embodiment, an anti-CD137 antibody, e.g., a chimeric (ch-BBK2) antibody or a humanized BBK2 antibody, comprises a heavy chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and a light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 16, a CDR2 comprising the amino acid sequence of SEQ ID NO: 17, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 18.

[0208] Thus, BBK2, humanized BBK2, or chimeric BBK2 antibodies can be used in the anti-CD137 ADCs and methods described herein. Each of these antibodies can be conjugated to any of the cytotoxins described below using methods known in the art and described herein.

[0209] Various other anti-CD137 antibodies are known in the art and can be used in the methods described herein. For example, in certain embodiments, the anti-CD137 antibody is selected from ADG106 (as described in, for example, WO2019105468, US20190055314, WO2019037711, WO2019036855); AGEN2373 (as described in, for example, WO2018191502, US20180344870); ATOR-1017 (as described in, for example, WO2018091740, US20180118841), PE0166 (as described in, for example, Song et al. AACR 2019, Abstract 2397 / 21), urelumab (also known as BMS-663513; as described in, e.g., WO2004010947, WO2005035584, US20090068192, US7659384, US8475790, US8137667, US20100183621, US8716452, US20120141494, US9382328, US20140193422, WO2016029073, US20160368998, WO2017181034, US20190062445, Chin et al. Nature communications. 9.1 (2018): 4679.; Segal et al. Clinical Cancer Research.23.8(2017):1929-1936.); and utomilumab (also known as PF-05082566, MOR-7480.; as described in, e.g., WO2012032433, US20120237498, US20140178368, WO2012145183, WO2015119923, WO2015179236, US20160152722, US20190031765, WO2017130076, Chin et al. Nature communications.9.1(2018):4679.; Segal et al. Clinical Cancer Research.24.8(2018):1816-1823; Fisher et al. Cancer Immunology, Immunotherapy. 61.10 (2012): 1721-1733), each of which is incorporated by reference.

[0210] Other anti-CD137 antibodies have been described in, for example, WO2019020774, WO2017077085, US20180327504, US20190099488, US2019006045, US20190015508, WO2019014328, US20190071510, WO2018127787, US20180258177, US10174122, WO2016110584, WO2018017761, WO2018098370, US20130149301, WO2019027754, WO2018156740, US20160244528, WO 2016134358, US10233251, US20170226215, US20160083474, WO2017049452, US2 0180282422, WO2015188047, WO2010132389, US20120076722, US20110177104, W O2011031063, US20080305113, US20080008716, US7829088, US20090041763, WO 2006126835, US20030096976, US20030223989, US20060182744, WO1996029348, et al. Circulation J. 81.12 (2017): 1945-1952; Makkouk, et al. Annals of Oncology 28.2 (2016): 415-420; Martinez-Forero et al. J. of Immunology. 190.12 (2013): 6694-6706; Dubrot et al. Cancer immunology, immunotherapy. 59.8 (2010): 1223-1233, each of which is incorporated by reference.

[0211] In one embodiment, anti-CD137 antibodies include the heavy chain of anti-CD137 antibodies described herein and the light chain variable region of anti-CD137 antibodies described herein. In one embodiment, anti-CD137 antibodies include the heavy chain containing CDR1, CDR2 and CDR3 of anti-CD137 antibodies described herein and the light chain variable region containing CDR1, CDR2 and CDR3 of anti-CD137 antibodies described herein.

[0212] In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to an anti-CD137 antibody herein, e.g., at least 95%, 96%, 97%, 98%, 99% or 100% identical to an anti-CD137 antibody herein. In certain embodiments, the antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain of an anti-CD137 antibody herein or a variant thereof, the variant (i) differing from an anti-CD137 antibody by 1, 2, 3, 4 or 5 amino acid substitutions, additions or deletions; (ii) differing from an anti-CD137 antibody by at most 5, 4, 3, 2 or 1 amino acid substitutions, additions or deletions; (iii) differing from an anti-CD137 antibody by 1-5, 1-3, 1-2 , 2-5 or 3-5 amino acid substitutions, additions or deletions and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an anti-CD137 antibody, wherein in any of (i)-(iv), the amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified heavy chain variable region may have enhanced biological activity relative to the heavy chain variable region of an anti-CD137 antibody while maintaining the CD137 binding specificity of the antibody.

[0213] Other anti-CD137 antibodies that can be used in combination with the cytotoxins described herein can be identified using techniques known in the art (e.g., hybridoma production). Hybridomas can be prepared using a murine system. Protocols for immunization and subsequent isolation of splenocytes for fusion are known in the art. Fusion partners and procedures for hybridoma production are also known. Human anti-CD137 antibodies can also be produced in or XenoMouse TMIn the preparation of anti-CD137 antibodies, the CD137 antigen is separated and / or purified. The CD137 antigen can be a CD137 fragment from the extracellular domain of CD137. Immunization of animals can be carried out by any method known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, for example, Harlow and Lane, supra, and U.S. Patent No. 5,994,619. The CD137 antigen can be administered with an adjuvant to stimulate an immune response. Adjuvants known in the art include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulatory complex). After immunizing animals with CD137 antigens, immortalized cell lines producing antibodies are prepared by cells isolated from immunized animals. After immunization, the animal is sacrificed and lymph node and / or spleen B cells are immortalized by methods known in the art (e.g., oncogene transfer, oncogenic viral transduction, exposure to carcinogenic or mutagenic compounds, fusion with immortalized cells such as myeloma cells, and inactivation of tumor suppressor genes). See, e.g., Harlow and Lane, supra. Hybridomas can be selected, cloned, and further screened for desired properties, including robust growth, high antibody production, and desired antibody properties.

[0214] Anti-CD137 antibodies can be produced by an isolated nucleic acid molecule comprising a nucleotide sequence encoding the amino acid sequence of the CD137 binding molecules provided by the present disclosure. The amino acid sequence encoded by the nucleotide sequence can be any part of the antibody, such as CDR, comprising one, two or three CDR sequences, the variable region of the heavy chain, the variable region of the light chain, or can be a full-length heavy chain or a full-length light chain. The nucleic acid of the present disclosure can be, for example, DNA or RNA, and may or may not include intron sequences. Typically, the nucleic acid is a cDNA molecule.

[0215] In some embodiments, anti-CD137 antibodies (or fragments thereof) have a determined serum half-life. For example, in human patients, anti-CD137 antibodies (or fragments thereof) can have a serum half-life of about 1-24 hours. For example, in human patients, ADCs containing such anti-CD137 antibodies can also have a serum half-life of about 1-24 hours. Pharmacokinetic analysis by measuring serum levels can be performed by assays known in the art.

[0216] Antibodies for use in conjunction with the ADCs described herein include variants of those described above, such as antibody fragments comprising or lacking an Fc domain, antibody fragments comprising one or more or all of the CDRs of an antibody described herein or their equivalent regions.

[0217] Methods for identifying antibodies

[0218] Methods for high-throughput screening of antibody libraries and antibody fragment libraries capable of binding to CD137 can be used to identify agents and affinity mature agents, which can be used, for example, to treat or prevent allogeneic cell therapy rejection. Such methods include in vitro display techniques known in the art, such as, in particular, phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display. The use of phage display to separate antibodies or antigen-binding fragments that bind to biologically relevant molecules has been reviewed in, for example, Felici et al., Biotechnol. Annual Rev. 1: 149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26: 27-45, 1997; and Hoogenboom et al., Immunotechnology 4: 1-20, 1998, the disclosure of each of which is incorporated herein by reference when it relates to in vitro display techniques. Randomized combinatorial peptide libraries have been constructed to select polypeptides that bind to cell surface antigens, such as Kay, Perspect. Drug Discovery Des. 2: 251-268, 1995 and Kay et al., Mol. Divers. 1: 139-140, 1996, the disclosure of each of which is incorporated herein by reference when it relates to the discovery of antigen binding molecules. Proteins, such as multimeric proteins, have been successfully displayed on phages as functional molecules (see, e.g., EP 0 349 578, EP 4 527 839 and EP 0 589 877 and Chiswell and McCafferty, Trends Biotechnol. 10: 80-84 1992, the disclosure of each of which is incorporated herein by reference when it relates to the use of in vitro display technology for the discovery of antigen binding molecules). In addition, functional antibody fragments such as Fab and scFv fragments have been expressed in in vitro display formats (see, e.g., McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, the disclosures of each of which are incorporated herein by reference as they relate to in vitro display platforms for discovering antigen-binding molecules). Human anti-CD137 antibodies can also be expressed, e.g., in or XenoMouse TM These techniques can be used, among other things, to identify and improve the affinity of antibodies or antibody fragments that bind CD137, and which in turn can be used to deplete hematopoietic cells in a patient.

[0219] In addition to in vitro display techniques, computer modeling techniques can be used to design and identify anti-CD137 antibodies or antibody fragments in a computer (in silico), for example, using the program described in US 2013 / 0288373, the disclosure of which is incorporated herein when it relates to molecular modeling methods for identifying anti-CD137 antibodies. For example, using computer modeling techniques, one skilled in the art can screen an antibody library or antibody fragment library in a computer for molecules that can bind to a specific epitope on CD137, such as an extracellular epitope of CD137.

[0220] Additional techniques can be used to identify antibodies or antibody fragments that bind to CD137 on the surface of cells (e.g., T cells) and are internalized by cells, for example, through receptor-mediated endocytosis. For example, the in vitro display techniques described above can be modified to screen for antibodies or antibody fragments that bind to CD137 on the surface of hematopoietic stem cells and are subsequently internalized. Phage display represents such a technique that can be used in conjunction with this screening paradigm. In order to identify anti-CD137 antibodies or antibody fragments that bind to CD137 and are subsequently internalized by hematopoietic stem cells, those skilled in the art can use the phage display techniques described in Williams et al., Leukemia 19: 1432-1438, 2005, the disclosure of which is incorporated herein by reference in its entirety. For example, using mutagenesis methods known in the art, recombinant phage libraries can be generated, the recombinant phage libraries encoding antibodies, antibody fragments, such as scFv fragments, Fab fragments, double bodies, triple bodies and antibodies containing randomized amino acid boxes (e.g., in one or more or all of the CDRs or their equivalent regions or antibodies or antibody fragments). 10 Fn3 domains or ligands. The framework regions, hinges, Fc domains and other regions of antibodies or antibody fragments can be designed so that they are non-immunogenic in humans by, for example, having human germline antibody sequences or sequences that exhibit only minor variations relative to human germline antibodies.

[0221] Using phage display technology described herein or known in the art, for example, by first incubating the phage library with a blocking agent (such as, for example, milk protein, bovine serum albumin and / or IgG) to remove the phage encoding the antibody or antibody fragment that exhibits non-specific protein binding and the phage encoding the antibody or its fragment that binds the Fc domain, and then incubating the phage library with a hematopoietic stem cell population of CD137+, the phage library containing the randomized antibody or antibody fragment covalently bound to the phage particle can be incubated with the CD137 antigen. The phage library can be incubated with hematopoietic stem cells for a period sufficient to allow CD137-specific antibodies or antibody fragments to bind to cell surface CD137 and subsequently be internalized by the hematopoietic stem cells (e.g., from 30 minutes to 6 hours at 4°C, such as 1 hour at 4°C). Phages containing antibodies or antibody fragments that do not show enough affinity to CD137 to allow binding to hematopoietic stem cells and internalization thereof can then be removed by, for example, washing cells with a cold (4°C) 0.1M glycine buffer of pH 2.8. Phages that bind to antibodies or antibody fragments that have been internalized by hematopoietic stem cells can be identified by, for example, lysing cells and recovering internalized phages from cell culture medium. Phages can then be amplified in bacterial cells, for example, by incubating bacterial cells with recovered phages in 2xYT culture medium using methods known in the art. Then, phages recovered from the culture medium can, for example, be characterized by determining the nucleic acid sequence of the gene encoding antibodies or antibody fragments inserted into the phage genome. Encoded antibodies or antibody fragments can then be prepared from scratch by chemical synthesis (for example, chemically synthesized antibody fragments, such as scFv fragments) or by recombinant expression (for example, recombinantly expressed full-length antibodies).

[0222] The internalization ability of the prepared antibodies or antibody fragments can be evaluated, for example, using radionuclide internalization assays known in the art. For example, an anti-CD137 antibody or antibody fragment identified using an in vitro display technique described herein or known in the art can be functionalized by incorporating a radioisotope such as 18 F. 75 Br, 77 Br, 122 I. 123 I. 124 I. 125 I. 129 I. 131 I. 211 At 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu,177 Lu, 77 As, 72 As, 86 Y. 90 Y. 89 Zr, 212 Bi, 213 Bior 225 Ac. For example, a radioactive halogen, such as Ac, can be introduced into the cell using beads containing an electrophilic halogen reagent, such as polystyrene beads (e.g., Iodination Beads, Thermo Fisher Scientific, Inc., Cambridge, MA). 18 F. 75 Br, 77 Br, 122 I. 123 I. 124 I. 125 I. 129 I. 131 I. 211 At is incorporated into the antibody or antibody fragment. Radiolabeled antibodies, fragments thereof or ADCs can be incubated with hematopoietic stem cells for a time sufficient to allow internalization (e.g., from 30 minutes to 6 hours at 4°C, such as 1 hour at 4°C). The cells can then be washed to remove uninternalized antibodies or fragments thereof (e.g., using a cold (4°C) 0.1M glycine buffer at pH 2.8). By detecting the emission radiation (e.g., γ-radiation) of the resulting hematopoietic stem cells compared to the emission radiation (e.g., γ-radiation) of the recovered washing buffer, the internalized antibody or antibody fragment can be identified. The aforementioned internalization assay can also be used to characterize ADCs.

[0223] Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, there is provided an isolated nucleic acid encoding an anti-CD137 antibody described herein. Such nucleic acid can encode an amino acid sequence comprising VL of an antibody and / or an amino acid sequence comprising VH (for example, a light chain and / or a heavy chain of an antibody). In another embodiment, there is provided one or more vectors (for example, an expression vector) comprising such nucleic acid. In another embodiment, there is provided a host cell comprising such nucleic acid. In one such embodiment, the host cell comprises (for example, having been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (for example, a Y0, NS0, Sp20 cell). In one embodiment, a method for preparing an anti-CLL-1 antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0224] In order to recombinantly produce anti-CD137 antibodies, nucleic acids encoding antibodies such as those described above are separated and inserted into one or more vectors to further clone and / or express in a host cell. Such nucleic acids can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can be specifically bound to genes encoding antibody heavy and light chains).

[0225] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. Regarding the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents No. 5,648,237, No. 5,789,199 and No. 5,840,523. (See also: Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in Escherichia coli (E. coli.). After expression, antibodies can be separated from the soluble fraction of bacterial cell mass (paste), and can be further purified.

[0226] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7), human embryonic kidney cell line (293 or 293 cells, as described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK, Buffalo rat liver cells (BRL 3A); human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells (as described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)), MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)) and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., Y0, NS0, Sp20 cell).

[0227] Cytotoxins

[0228] Various cytotoxins can be conjugated to anti-CD137 antibodies via a linker for use in the combination therapy described herein. In particular, anti-CD137 ADCs include antibodies (or antigen-binding fragments thereof) conjugated to a cytotoxic moiety (or cytotoxin) (i.e., covalently attached via a linker). As used herein, the terms "cytotoxin," "cytotoxic moiety," and "drug" are used interchangeably. In various embodiments, the cytotoxic moiety exhibits reduced cytotoxicity or no cytotoxicity when incorporated into the conjugate, but recovers cytotoxicity after cleavage from the linker. In various embodiments, the cytotoxic moiety maintains cytotoxicity without cleavage from the linker. In some embodiments, a cytotoxic molecule is conjugated to a cell-internalizing antibody or its antigen-binding fragment as disclosed herein, so that after the cell takes up the antibody or its fragment, the cytotoxin can approach its intracellular target and, for example, mediate T cell death.

[0229] Thus, the ADC of the present invention may have the general formula I, wherein the antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently linked) to a cytotoxic moiety ("drug", D) via a chemical moiety (Z) and a linker (L),

[0230] Ab-(ZLD) n (I).

[0231] Accordingly, the antibody or its antigen-binding fragment can be conjugated with a plurality of drug moieties as indicated by an integer n, the integer n representing the average number of cytotoxins per antibody, and the integer n range can be, for example, from about 1 to about 20. Any number of cytotoxins can be conjugated to the antibody, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In some embodiments, n is 1 to 4. In some embodiments, n is 1 to 3. In some embodiments, n is about 2. In some embodiments, n is about 1. The average number of drug moieties per antibody in the ADC prepared by the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of ADC in terms of n can also be determined. In some cases, separation, purification, and characterization of homogeneous ADCs with a certain value of n from ADCs with other drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis.

[0232] For some anti-CD137 ADCs, n can be limited by the number of attachment sites on the antibody. For example, in the case where the attachment is a cysteine ​​thiol, the antibody may have only one or several cysteine ​​thiol groups, or may have only one or several sufficiently reactive thiol groups through which the linker can be attached. Typically, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be connected to the drug moiety; the cysteine ​​thiol residues in the antibody are mainly present in the form of disulfide bridges. In certain embodiments, the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or full reducing conditions to produce reactive cysteine ​​thiol groups. In certain embodiments, higher drug loading, such as n>5, can cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody drug conjugates.

[0233] In certain embodiments, during the conjugation reaction, less than the theoretical maximum number of drug moieties is conjugated to the antibody. The antibody may contain, for example, lysine residues that do not react with drug-linker intermediates or linker reagents, as described below. Only the most reactive lysine groups can react with amine-reactive linker reagents. In certain embodiments, the antibody undergoes denaturation conditions to expose reactive nucleophilic groups, such as lysine or cysteine.

[0234] The loading capacity (drug / antibody ratio) of the ADC can be controlled in different ways, for example by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limited reducing conditions for modification of cysteine ​​thiol groups, (iv) engineering the amino acid sequence of the antibody by recombinant technology so that the number and position of cysteine ​​residues are modified to control the number and / or position of linker-drug attachments.

[0235] Cytotoxins suitable for use with the compositions and methods described herein include, among others known in the art, DNA intercalators (e.g., anthracyclines), agents capable of disrupting the mitotic spindle (e.g., vinca alkaloids, maytansine, maytansine alkaloids, and derivatives thereof), RNA polymerase inhibitors (e.g., amanita toxins such as α-amanitin and its derivatives), and agents capable of disrupting protein biosynthesis (e.g., agents exhibiting rRNA N-glycosidase activity such as saporin and ricin A chain).

[0236] In some embodiments, the cytotoxin is a microtubule-binding agent (e.g., maytansine or a maytansine alkaloid), amanita anitoxin, Pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolebenzodiazepines, and indolebenzodiazepine dimers, or variants thereof, or another cytotoxic compound described herein or known in the art.

[0237] In some embodiments, the cytotoxin of the antibody drug conjugate is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is an amanita toxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody drug conjugate as disclosed herein is an amanita toxin or a derivative thereof, such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin non-toxic cyclic peptide, monohydroxyamanitin carboxylic acid, proamanitin non-toxic cyclic peptide or a derivative thereof.

[0238] Additional details regarding cytotoxins that may be used in anti-CD137 ADCs useful in the methods of the invention are described below.

[0239] Amanita phalloides

[0240] In some embodiments, the RNA polymerase inhibitor is an amanita toxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody drug conjugate as disclosed herein is an amanita toxin or a derivative thereof, such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin non-toxic cyclic peptide, monohydroxyamanitin carboxylic acid, proamanitin non-toxic cyclic peptide or a derivative thereof. The structures of a variety of naturally occurring amanitin toxins are represented by formula II and the accompanying Table 1, and are disclosed in, for example, Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459.

[0241]

[0242] Table 1. Structures of Amanita phalloides toxins.

[0243]

[0244] In one embodiment, the cytotoxin is amanitin or a derivative thereof. In one embodiment, the cytotoxin is alpha-amanitin or a derivative thereof.

[0245] Many positions on the Amanita ani toxin or derivatives thereof can serve as positions for covalent bonding to the linking moiety L and thus to the antibody or antigen-binding fragment thereof. In some embodiments, the cytotoxin in the ADC of Formula I is an Amanita ani toxin or derivative thereof according to Formula (II). In one embodiment, the ADC is represented by the formula Ab-ZL-Am, wherein Ab is an antibody or antigen-binding fragment thereof that binds CD137, L is a linker, Z is a chemical moiety, and Am is an Amanita ani toxin. In certain embodiments, the linker-Amanita ani toxin conjugate Am-LZ is represented by Formula (III)

[0246]

[0247] in:

[0248] R1 is H, OH, OR A OR C ;

[0249] R2 is H, OH, OR B OR C ;

[0250] R A and R B When present, together with the oxygen atoms to which they are attached, they combine to form a 5-membered heterocycloalkyl group;

[0251] R3 is H, R C or R D ;

[0252] Each of R4, R5, R6 and R7 is independently H, OH, OR C , OR D , R C or R D ;

[0253] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0254] R9 is H, OH, OR C OR D ;

[0255] Q is -S-, -S(O)- or -SO2-;

[0256] R Cis -LZ' or -LZ-Ab, wherein L is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl; or includes a dipeptide; or includes -((CH2) m O) n (CH2) m -, wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; or a combination thereof; Z' is a reactive moiety, and Z is a chemical moiety resulting from a coupling reaction between Z' and a functional group on Ab; and

[0257] R D is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or a combination thereof, wherein each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally substituted by 1 to 5 Substituents are selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0258] Formula (III) comprises an amanita ani toxin and a linker, and in some embodiments, comprises a linker, a chemical moiety, and an antibody.

[0259] In some embodiments, the cytotoxin is Amanita toxin, and the linker-Amanita toxin conjugate or antibody-linker-Amanita toxin conjugate is represented by formula (IIIA):

[0260]

[0261] in:

[0262] R1 is H, OH, OR A OR C ;

[0263] R2 is H, OH, OR B OR C ;

[0264] R A and R B When present, together with the oxygen atoms to which they are attached, they combine to form a 5-membered heterocycloalkyl group;

[0265] R3 is H, R C or R D ;

[0266] Each of R4, R5, R6 and R7 is independently H, OH, OR C , OR D , R C or R D ;

[0267] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0268] R9 is H, OH, OR C OR D ;

[0269] Q is -S-, -S(O)- or -SO2-;

[0270] R C is -LZ' or -LZ-Ab, wherein L is a linker, an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl; or includes a dipeptide; or includes -(C=O)-, -((CH2) m O) n (CH2) m -, wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; or a combination thereof; Z' is a reactive moiety, and Z is a chemical moiety resulting from a coupling reaction between Z' and a functional group on Ab; and

[0271] R Dis C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or a combination thereof, wherein each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally substituted by 1 The invention further comprises a cycloalkyl radical, a cycloalkyl radical, a heterocycloalkyl radical, a ...

[0272] In some embodiments, the conjugate comprises one R C Substituent.

[0273] In some embodiments, R A and R B Together with the oxygen atom to which they are attached, they combine to form a 5-membered heterocycloalkyl group of the formula:

[0274]

[0275] wherein Y is -(C=O)-, -(C=S)-, -(C=NR E) -or-(CR E R E’ )-;and

[0276] Where R E and R E’ are independently H, C1-C6 alkylene-R C 、C1-C6 heteroalkylene-R C 、C2-C6 alkenylene-R C 、C2-C6 heteroalkenylene-R C 、C2-C6 alkynylene-R C 、C2-C6 heteroalkynylene-R C , cycloalkylene-R C , heterocycloalkylene-R C , arylene-R C or heteroarylene-R C , or a combination thereof; wherein each C1-C6 alkylene-R C 、C1-C6 heteroalkylene-R C 、C2-C6 alkenylene-R C 、C2-C6 heteroalkenylene-R C 、C2-C6 alkynylene-RC 、C2-C6 heteroalkynylene-R C , cycloalkylene-R C , heterocycloalkylene-R C , arylene-R C or heteroarylene-R C Optionally substituted with 1 to 5 substituents, the substituents being independently selected for each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxy, mercapto and nitro.

[0277] Formula (IIIA) comprises an amanita ani toxin and a linker, and in some embodiments, comprises a linker, a chemical moiety, and an antibody.

[0278] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIA, wherein:

[0279] R1 is H, OH, OR A OR C ;

[0280] R2 is H, OH, OR B OR C ;

[0281] R A and R B When present, they combine with the oxygen atoms to which they are bound to form:

[0282]

[0283] Where R3 is H or R C .

[0284] In some embodiments, the cytotoxin is amanita ani toxin or a derivative thereof, and the conjugate is represented by Formula IIIA, wherein:

[0285] R1 is H, OH, OR A OR C ;

[0286] R2 is H, OH, OR B OR C ;

[0287] R A and R B When present, they combine with the oxygen atoms to which they are bound to form:

[0288]

[0289] in

[0290] R3 is H or R C ;

[0291] R4 and R5 are each independently H, OH, OR C , R C OR D ;

[0292] R6 and R7 are each H;

[0293] R8 is OH, NH2, OR C or NHR C ;

[0294] R9 is H or OH; and

[0295] Where R C and R D As defined above.

[0296] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIA, wherein:

[0297] R1 is H, OH or OR A ;

[0298] R2 is H, OH or OR B ;

[0299] R A and R B When present, they combine with the oxygen atoms to which they are bound to form:

[0300]

[0301] in

[0302] R3, R4, R6 and R7 are each H;

[0303] R5 is OR C ;

[0304] R8 is OH or NH2;

[0305] R9 is H or OH;

[0306] Q is -S-, -S(O)- or -SO2-; and

[0307] Where R C and R DAs defined above. Such Amanita ani toxin conjugates are described, for example, in U.S. Patent Application Publication No. 2016 / 0002298, the disclosure of which is incorporated herein by reference in its entirety.

[0308] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIA, wherein:

[0309] R1 and R2 are each independently H or OH;

[0310] R3 is R C ;

[0311] R4, R6 and R7 are each H;

[0312] R5 is H, OH or OC1-C6 alkyl;

[0313] R8 is OH or NH2;

[0314] R9 is H or OH;

[0315] Q is -S-, -S(O)- or -SO2-; and

[0316] Where R C and R D As defined above. Such Amanita anitoxin-linker conjugates are described, for example, in US Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated herein by reference in its entirety.

[0317] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIA, wherein:

[0318] R1 and R2 are each independently H or OH;

[0319] R3, R6 and R7 are each H;

[0320] R4 and R5 are each independently H, OH, OR C or R C ;

[0321] R8 is OH or NH2;

[0322] R9 is H or OH;

[0323] Q is -S-, -S(O)- or -SO2-; and

[0324] Where R C and R DAs defined above. Such amanita anitoxin-linker conjugates are described, for example, in US Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0325] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIA, wherein:

[0326] R1 and R2 are each independently H or OH;

[0327] R3, R6 and R7 are each H;

[0328] R4 and R5 are each independently H or OH;

[0329] R8 is OH, NH2, OR C or NHR C ;

[0330] R9 is H or OH;

[0331] Q is -S-, -S(O)- or -SO2-; and

[0332] Where R C and R D As defined above. Such Amanita ani toxin conjugates are described, for example, in US Pat. Nos. 9,233,173 and 9,399,681, the disclosures of each of which are incorporated herein by reference in their entirety.

[0333] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein:

[0334]

[0335] in:

[0336] R1 is H, OH, OR A OR C ;

[0337] R2 is H, OH, OR B OR C ;

[0338] R A and R B When present, together with the oxygen atoms to which they are attached, they combine to form a 5-membered heterocycloalkyl group;

[0339] R3 is H, R C or R D ;

[0340] Each of R4, R5, R6 and R7 is independently H, OH, OR C , OR D , R C or R D ;

[0341] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0342] R9 is H, OH, OR C OR D ;

[0343] Q is -S-, -S(O)- or -SO2-;

[0344] R C is -LZ' or -LZ-Ab, wherein L is a linker, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, a peptide, a dipeptide, -(C=O)-, -((CH2) m O) n (CH2) m -, wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; or a combination thereof; Z' is a reactive moiety, and Z is a chemical moiety resulting from a coupling reaction between Z' and a functional group on Ab; and

[0345] R D is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or a combination thereof, wherein each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally substituted by 1 The invention further comprises a cycloalkyl radical, a cycloalkyl radical, a heterocycloalkyl radical, a alkylaryl radical, a alkylheteroaryl radical, a cycloalkyl ...

[0346] Formula (IIIB) comprises an amanita ani toxin and a linker, and in some embodiments, comprises a linker, a chemical moiety, and an antibody.

[0347] In some embodiments, R A and R B Together with the oxygen atom to which they are attached, they form a 5-membered heterocycloalkyl group of the following formula:

[0348]

[0349] wherein Y is -(C=O)-, -(C=S)-, -(C=NR E) -or-(CR E R E’ )-;and

[0350] Where R E and R E’ are independently H, C1-C6 alkylene-R C 、C1-C6 heteroalkylene-R C 、C2-C6 alkenylene-R C 、C2-C6 heteroalkenylene-R C 、C2-C6 alkynylene-R C 、C2-C6 heteroalkynylene-R C , cycloalkylene-R C , heterocycloalkylene-R C , arylene-R C or heteroarylene-R C , or a combination thereof; wherein each C1-C6 alkylene-R C 、C1-C6 heteroalkylene-R C 、C2-C6 alkenylene-R C 、C2-C6 heteroalkenylene-R C 、C2-C6 alkynylene-R C 、C2-C6 heteroalkynylene-R C , cycloalkylene-R C , heterocycloalkylene-R C , arylene-R C or heteroarylene-R C Optionally substituted with 1 to 5 substituents, the substituents being independently selected for each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxy, mercapto and nitro.

[0351] In some embodiments, an antibody or antigen-binding fragment thereof as described herein is conjugated to an amanita ani toxin-linker conjugate represented by Formula IIIB or a derivative thereof, wherein

[0352] R1 is H, OH, OR A OR C ;

[0353] R2 is H, OH, OR B OR C ;

[0354] R A and R B When present, together with the oxygen atom to which they are attached, they combine to form a 5-membered heterocycloalkyl group of the formula:

[0355]

[0356] Where R3 is H or R C .

[0357] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein:

[0358] R1 is H, OH, OR A OR C ;

[0359] R2 is H, OH, OR B OR C ;

[0360] R A and R B When present, together with the oxygen atom to which they are attached, they combine to form a 5-membered heterocycloalkyl group of the formula:

[0361]

[0362] in

[0363] R3 is H or R C ;

[0364] R4 and R5 are each independently H, OH, OR C , R C OR D ;

[0365] R6 and R7 are each H;

[0366] R8 is OH, NH2, OR C or NHR C ;

[0367] R9 is H or OH; and

[0368] Where R C and R D As defined above.

[0369] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein:

[0370] R1 is H, OH or OR A ;

[0371] R2 is H, OH or OR B ;

[0372] R A and R B When present, together with the oxygen atom to which they are attached, they combine to form a 5-membered heterocycloalkyl group of the formula:

[0373]

[0374] in

[0375] R3, R4, R6 and R7 are each H;

[0376] R5 is OR C ;

[0377] R8 is OH or NH2;

[0378] R9 is H or OH;

[0379] Q is -S-, -S(O)- or -SO2-; and

[0380] Where R C and R D As defined above. Such Amanita anitoxin-linker conjugates are described, for example, in U.S. Patent Application Publication No. 2016 / 0002298, the disclosure of which is incorporated herein by reference in its entirety.

[0381] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein:

[0382] R1 and R2 are each independently H or OH;

[0383] R3 is R C ;

[0384] R4, R6 and R7 are each H;

[0385] R5 is H, OH or OC1-C6 alkyl;

[0386] R8 is OH or NH2;

[0387] R9 is H or OH;

[0388] Q is -S-, -S(O)- or -SO2-; and

[0389] Where R C and R D As defined above. Such Amanita ani toxin conjugates are described, for example, in US Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated herein by reference in its entirety.

[0390] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein:

[0391] R1 and R2 are each independently H or OH;

[0392] R3, R6 and R7 are each H;

[0393] R4 and R5 are each independently H, OH, OR C or R C ;

[0394] R8 is OH or NH2;

[0395] R9 is H or OH;

[0396] Q is -S-, -S(O)- or -SO2-; and

[0397] Where R C and R D As defined above. Such amanita anitoxin-linker conjugates are described, for example, in US Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0398] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein:

[0399] R1 and R2 are each independently H or OH;

[0400] R3, R6 and R7 are each H;

[0401] R4 and R5 are each independently H or OH;

[0402] R8 is OH, NH2, OR C or NHR C ;

[0403] R9 is H or OH;

[0404] Q is -S-, -S(O)- or -SO2-; and

[0405] Where R C and R D As defined above. Such amanita anitoxin-linker conjugates are described, for example, in U.S. Patent Nos. 9,233,173 and 9,399,681, the disclosures of each of which are incorporated herein by reference in their entirety.

[0406] Auristatin

[0407] The anti-CD137 antibodies and antigen-binding fragments thereof described herein can be conjugated to a cytotoxin that is auristatin (U.S. Pat. Nos. 5,635,483 and 5,780,588). Auristatin is an antimitotic agent that interferes with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584), and has anticancer activity (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965). (U.S. Pat. Nos. 5,635,483 and 5,780,588). The auristatin drug moiety can be attached to the antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172).

[0408] Exemplary auristatin embodiments include the N-terminally linked monomethyl auristatin drug moieties DE and DF (MMAE and MMAF, respectively), which are disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Vol. 45, Abstract No. 623, filed March 28, 2004, the disclosure of which is expressly incorporated by reference in its entirety.

[0409] An exemplary auristatin embodiment is MMAE:

[0410]

[0411] Therein the wavy line indicates the point of covalent attachment of the linker of the antibody-drug or drug-linker conjugate (-LZ-Ab or -LZ', as described herein).

[0412] Another exemplary auristatin embodiment is MMAF,

[0413]

[0414] Wherein the wavy line indicates the point of covalent attachment of the linker of the antibody-linker conjugate (-LZ-Ab or -LZ', as described herein), as disclosed in US 2005 / 0238649.

[0415] Auristatins can be prepared according to the following methods: U.S. Patent No. 5,635,483; U.S. Patent No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit, GR, et al. Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 15:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784.

[0416] Maytansine alkaloids

[0417] Antibodies and Fabs thereof described herein can be conjugated with cytotoxins that are microtubule binding agents. In some embodiments, the microtubule binding agent is an analog of maytansine, maytansine alkaloids or maytansine alkaloids. Maytansine alkaloids are mitotic inhibitors that bind microtubules and play a role by inhibiting tubulin polymerization. For the first time, maytansine (U.S. Patent No. 3,896,111) was isolated from the East African shrub Maytenus serrata. Subsequently, it was found that some microorganisms also produce maytansine alkaloids, such as maytansinol and C-3 maytansine alcohol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed in, for example, U.S. Pat. Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,3 No. 09,428, No. 4,313,946, No. 4,315,929, No. 4,317,821, No. 4,322,348, No. 4,331,598, No. 4,361,650, No. 4,364,866, No. 4,424,219, No. 4,450,254, No. 4,362,663 and No. 4,371,533. Maytansine alkaloid drug moieties are attractive drug moieties in antibody drug conjugates because they are: (i) relatively easy to prepare by fermentation or chemical modification, derivatization of fermentation products, (ii) easily derivatized with functional groups suitable for conjugation to antibodies via non-disulfide linkers, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.

[0418] Examples of suitable maytansine alkaloids include esters of maytansinol, synthetic maytansinol, and maytansinol analogs and derivatives. Included herein are any cytotoxins that inhibit microtubule formation and are highly toxic to mammalian cells, such as maytansine alkaloids, maytansinol, and maytansinol analogs and derivatives.

[0419] The example of suitable maytansinol ester includes those with modified aromatic ring and those with modified maytansinol ester at other positions. Such suitable maytansine alkaloids are disclosed in the following: U.S. Patent Nos. 4,137,230, 4,151,042, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,36 No. 1,650, 4,362,663, 4,364,866, 4,424,219, 4,450,254, 4,322,348, 4,362,663, 4,371,533, 5,208,020, 5,416,064, 5,475,092, 5,585,499, 5,846,545, 6,333,410, 7,276,497, and 7,473,796, the disclosures of each of which are incorporated herein by reference as they relate to maytansine alkaloids and derivatives thereof.

[0420] In some embodiments, the antibody drug conjugates (ADCs) of the present disclosure utilize a thiol-containing maytansine alkaloid (DM1) as a cytotoxic agent, which maytansine alkaloid is formally known as N 2 ′-Deacetyl-N 2 '-(3-mercapto-1-oxopropyl)-maytansine. DM1 is represented by the following structural formula IV:

[0421]

[0422] In another embodiment, the conjugates of the invention utilize thiol-containing maytansine alkaloids N 2 ′-Deacetyl-N 2 '(4-methyl-4-mercapto-1-oxopentyl)-maytansine (e.g., DM4) as a cytotoxic agent. DM4 is represented by the following structural formula V:

[0423]

[0424] Another maytansine alkaloid containing a side chain containing a sterically hindered thiol bond is N 2 ′-Deacetyl-N- 2 '(4-mercapto-1-oxopentyl)-maytansine (referred to as DM3) is represented by the following structural formula VI:

[0425]

[0426] Each of the maytansine alkaloids taught in U.S. Pat. Nos. 5,208,020 and 7,276,497 may also be used in the conjugates of the present disclosure. In this regard, the entire disclosures of 5,208,020 and 7,276,697 are incorporated herein by reference.

[0427] Many positions on the maytansine alkaloid can be used as positions for covalent bonding to the linking moiety, and thus covalent bonding to the antibody or its antigen-binding fragment (-LZ-Ab or -LZ ', as described herein). For example, the C-3 position with a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group, and the C-20 position with a hydroxyl group are all expected to be useful. In some embodiments, the C-3 position is used as a position for covalent bonding to the linking moiety, and in some specific embodiments, the C-3 position of maytansine is used as a position for covalent bonding to the linking moiety. There are many known linker groups in the art for preparing antibody-maytansine alkaloid conjugates, including, for example, those disclosed in U.S. Pat. Nos. 5,208,020, 6,441,163, and EP Patent No. 0425235B1; Chari et al., Cancer Research 52:127-131 (1992); and US2005 / 0169933A, the disclosure of which is expressly incorporated by reference herein. Additional linker groups are described and exemplified herein.

[0428] The present invention also includes multiple isomers and mixtures of maytansine alkaloids and conjugates. Certain compounds of the present invention and conjugates can exist in the form of multiple stereoisomers, enantiomers and diastereomers. Some descriptions for producing such antibody-maytansine alkaloid conjugates are provided below: U.S. Patent No. 5,208,020, No. 5,416,064, No. 6,333,410, No. 6,441,163, No. 6,716,821 and No. 7,368,565, each of which is incorporated herein in its entirety.

[0429] Anthracyclines

[0430] In other embodiments, the antibodies and antigen-binding fragments thereof described herein can be conjugated to cytotoxins that are anthracycline molecules. Anthracyclines are antibiotic compounds that exhibit cytotoxicity. Studies have shown that anthracyclines can operate to kill cells by many different mechanisms, including: 1) embedding drug molecules into the DNA of cells, thereby inhibiting nucleic acid synthesis that depends on DNA; 2) generating free radicals from drugs, which then react with cell macromolecules to cause damage to cells, or 3) interactions between drug molecules and cell membranes [see, e.g., C. Peterson et al. in Anthracycline Antibiotics In Cancer Therapy, "Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia"; NRBachur, "Free Radical Damage" supra, see pages 97-102]. Due to the cytotoxic potential of anthracyclines, they have been used to treat many cancers, such as leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma. [See, e.g., PH-Wiernik in Anthracycline: Current Status And New Developments, page 11]. Commonly used anthracyclines include doxorubicin, epirubicin, idarubicin, and daunomycin.

[0431] Representative examples of anthracyclines include, but are not limited to, daunorubicin (Cerubidine; Bedford Laboratories), doxorubicin (Adriamycin; Bedford Laboratories; also known as doxorubicin hydrochloride, hydroxydaunorubicin and Rubex), epirubicin (Ellence; Pfizer) and idarubicin (Idamycin; Pfizer Inc.). The anthracycline analog doxorubicin (ADRIAMYCINO) is believed to interact with DNA by intercalation and inhibit the process of topoisomerase II, which unwinds DNA for transcription. After the topoisomerase II complex breaks the DNA strand for replication, doxorubicin stabilizes the topoisomerase II complex, preventing the DNA double helix from being resealed, thereby stopping the replication process. Doxorubicin and daunorubicin (DAUNOMYCIN) are the prototypical cytotoxic natural product anthracycline chemotherapeutic agents (Sessa et al., (2007) Cardiovasc. Toxicol. 7:75-79).

[0432] A non-limiting example of a suitable anthracycline for use herein is PNU-159682 ("PNU"). PNU exhibits greater than 3000-fold cytotoxicity relative to the parent nemorubicin (Quintieri et al., Clinical Cancer Research 2005, 11, 1608-1617). PNU is represented by the following structural formula:

[0433]

[0434] More than one position on an anthracycline such as PNU can serve as a location for covalent bonding to a linking moiety L and thus to an anti-CD137 antibody or antigen-binding fragment thereof as described herein. For example, a linker can be introduced by modification of the hydroxymethylketone side chain.

[0435] In some embodiments, the cytotoxin is a PNU derivative represented by the following structural formula:

[0436]

[0437] Therein the wavy line indicates the point of covalent attachment of a linker of an ADC as described herein.

[0438] In some embodiments, the cytotoxin is a PNU derivative represented by the following structural formula:

[0439]

[0440] Therein the wavy line indicates the point of covalent attachment of a linker of an ADC as described herein.

[0441] Pyrrolobenzodiazepines (PBDs)

[0442] In other embodiments, anti-CD117 antibodies or their antigen-binding fragments described herein can be conjugated to cytotoxins for pyrrolobenzodiazepines (PBD) or to cytotoxins comprising PBD. PBD can be produced by certain actinomycetes, and has been shown to be a sequence-selective DNA alkylation compound. PBD cytotoxins include, but are not limited to, anthramycin (anthramycin), dimer PBD, and those disclosed in, for example, Hartley, JA (2011) The development of pyrrolobenzodiazepines as antitumor agents. Expert Opin Inv Drug, 20 (6), 733-744 and Antonow D, Thurston DE (2011) Synthesis of DNA-interactive pyrrolo [2, 1-c] [1, 4] benzodiazepines (PBDs). Chem Rev 111: 2815-2864.

[0443] In some embodiments, the cytotoxin may be a pyrrolobenzodiazepine dimer represented by the following structural formula:

[0444]

[0445] Wherein the wavy line indicates the covalent attachment point of the linker of the ADC as described herein. ADCs based on this PBD are disclosed in, for example, Sutherland et al., Blood 2013 122: 1455-1463, which is incorporated herein by reference in its entirety.

[0446] In some embodiments, the cytotoxin may be a PBD dimer represented by the following structural formula:

[0447]

[0448] wherein n is 3 or 5, and wherein the wavy line indicates the point of covalent attachment of a linker of an ADC as described herein.

[0449] In some embodiments, the cytotoxin may be a PBD dimer represented by the following structural formula:

[0450]

[0451] Therein the wavy line indicates the point of covalent attachment of a linker of an ADC as described herein.

[0452] In a specific embodiment, the cytotoxin may be a PBD dimer, which, when used with a linker and a reactive moiety Z', as described herein, each may be represented by the following structure:

[0453]

[0454] This particular cytotoxin-linker conjugate is called tesirine (SG3249) and has been described, for example, in Howard et al., ACS Med. Chem. Lett. 2016, 7(11), 983-987, the disclosure of which is incorporated herein by reference in its entirety.

[0455] In certain embodiments, the cytotoxin may be a PBD dimer, which, when taken together with a linker and a reactive moiety Z', as described herein, each may be represented by the following structure:

[0456]

[0457] This particular cytotoxic linker conjugate is called talirine and has been described, for example, in Mantaj et al., Angewandte Chemie International Edition English 2017, 56, 462-488, the disclosure of which is incorporated herein by reference in its entirety.

[0458] Gary car Mycin

[0459] In other embodiments, the antibodies and antigen-binding fragments thereof described herein can be conjugated to cytotoxins that are enediyne antitumor antibiotics (e.g., calicheamicin, ozogamicin). Antibiotics of the calicheamicin family are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. Examples of calicheamicins suitable for use in the present invention are disclosed in, for example, U.S. Patent No. 4,671,958, U.S. Patent No. 4,970,198, U.S. Patent No. 5,053,394, U.S. Patent No. 5,037,651, and U.S. Patent No. 5,079,233, which are incorporated herein in their entirety.

[0460] An exemplary calicheamicin is designated γ1, which is referred to herein as γ, and has the structural formula:

[0461]

[0462] In some embodiments, calicheamicin is a gamma calicheamicin derivative or an N-acetyl gamma calicheamicin derivative. Structural analogs of calicheamicin that can be used include, but are not limited to, those disclosed in, for example, Hinman et al., Cancer Research 53:3336-3342 (1993); Lode et al., Cancer Research 58:2925-2928 (1998); and the aforementioned U.S. patents. Calicheamicin includes a methyl trisulfide moiety that can react with an appropriate thiol to form a disulfide while introducing a functional group that can be used to attach a calicheamicin derivative to an anti-CD137 antibody or antigen-binding fragment thereof as described herein via a linker. For the preparation of calicheamicin family conjugates, see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all to American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, those disclosed in, for example, Hinman et al., Cancer Research 53:3336-3342 (1993); Lode et al., Cancer Research 58:2925-2928 (1998); and the aforementioned U.S. patents of American Cyanamid.

[0463] In one embodiment, the cytotoxin of the ADC as disclosed herein is a calicheamicin disulfide derivative represented by the formula:

[0464]

[0465] The wavy lines indicate the attachment points of the connectors.

[0466] Other cytotoxins

[0467] In other embodiments, the antibodies and antigen-binding fragments thereof described herein can be conjugated to cytotoxins other than or in addition to those disclosed herein above. Other cytotoxins suitable for use with the compositions and methods described herein include, but are not limited to, 5-ethynyluracil, abiraterone, acylfulvene, adecypenol, adozelesin, aldesleukin, hexamethylmelamine, aminostine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antarelix, anti-dorsalizing morphogenetic protein-1, antiandrogens, prostate cancer, antiestrogens, antineoplastons, antisense oligonucleotides, glycine aphidicolin glycinate), apoptosis gene regulators, apoptosis regulators, apurinic nucleic acids, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, diazotyrosine, baccatin III derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorins, benzoylstaurosporine, beta-lactam derivatives, beta-alethine, beta-betamycin B B), betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, bisaziridinylspermine, binasafide, bistrateneA, bizelesin, breflate, bleomycin A2, bleomycin B2, bropirimine, budotitane, buthionine sulfoxide, calcipotriol, calphostin C, camptothecin derivatives (e.g., 10-hydroxy-camptothecin), capecitabine, carboxamide-amino-triazole, carboxyamidotriazole, carzelesin, casein kinase inhibitors, castanespermine, cecropin B, cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene and its analogs, clotrimazole, collismycin A, collismycin B B), combretastatin A4, combretastatin analogs, conagenin, crambescidin 816, crisnatol, scutellariae cyclotide 8, scutellariae cyclotide A derivatives, curacin A, cyclopentanthraquinone, cycloplatam, cypemycin, cytarabineocfosfate, cytolytic factor, cytostatin, dacximab, decitabine, dehydrogenated ecteinascidin B, 2'deoxycoformycin (DCF), deslorelin, dexifosfamide, dexrazoxane, dexverapamil exverapamil), diaziquone, tunicain B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin, diphenylspiromustine, spondylolipidol, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocannycin SASA), ebselen, ecomustine, edilfosine, edrecolomab, eflornithine, elemene, ethiflu, epothilones, epithilones, epristeride, estramustine and its analogs, etoposide, etoposide 4'-phosphate (also known as etopofos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezellastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, homoharringtonine (HHT), hypericin, ibandronic acid, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridones, imiquimod, immunostimulatory peptides, iodobenzylguanidine, iodoxorubicin, irinotecan, iroplact, irsogladine, isobengazole, jasplakinolide, kahalalide F, lamellarin-N triacetate triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol, lonidamine, losoxantrone, loxoribine, lurtotecan, lutetiumtexaphyrin, lysofylline, masorofol, maspin, matrix metalloproteinase inhibitors, menogaril, rnerbarone, meterelin, methioninease, metoclopramide, MIF inhibitors, ifepristone, miltefosine, mirimostim, mithracin, mitoguazone, dibromodulanol, mitomycin and its analogs, mitonafide, mitoxantrone, mofarotene, molgramostim, myloroxideB), myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagrestip, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, nilutamide, nisamycin, nitrullyn, octreotide, okicenone, onapristone, ondansetron, oracin, ormaplatin, oxaliplatin, oxaunomycin, paclitaxel and its analogs, palauamine, hexadecene palmitoylrhizoxin, pamidronate, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, sodium pentosan polysulfate, pentostatin, pentrozole, perflubron, perphosphamide, phenazinomycin, picibanil, pirarubicin, piritrexim, podophyllotoxin, porfiromycin, purine nucleoside phosphorylase inhibitors, raltitrexed, rhizotomycin, rogletimide, roxitocin, rubiginone B1 B1), ruboxyl, safingol, saintopin, sarcophytol A, sargramostim, sobuzoxane, sonermin, sparlfosic acid, spicamycin DD), spiromustine, stipiamide, sulfinosine, tallimustine, tegafur, temozolomide, teniposide, thaliblastine, thiocoraline, tirapazamine, topotecan, topsentin, triciribine, trimetrexate, veramine, vinorelbine, vinxaltine, vorozole, zeniplatin, and zilascorb.

[0468] Connectors

[0469] As used herein, the term "linker" means a divalent chemical part comprising a chain of covalent bonds or atoms, which covalently attaches an anti-CD137 antibody or fragment thereof (Ab) to a drug portion (D) to form an antibody drug conjugate (ADC) of Formula I. Suitable linkers have two reactive ends, one for conjugation with an antibody, and the other for conjugation with a cytotoxin. The antibody conjugation reactive end (reactive moiety, Z') of the linker is typically a site that can be conjugated to an antibody through a cysteine ​​thiol or lysine amine group on the antibody, and is therefore typically a thiol reactive group such as a double bond (as in maleimide) or a leaving group such as chlorine, bromine, iodine or R-sulfonyl group, or an amine reactive group such as a carboxyl group; and the cytotoxin conjugation reactive end of the linker is typically a site that can be conjugated with a cytotoxin. Non-limiting examples of linker-cytotoxin conjugation include, for example, respectively via a carboxyl or basic amine group on a linker and a basic amine or carboxyl group on a cytotoxin to form an amide bond, or via the alkylation of an OH group on a cytotoxin (e.g., via a leaving group on a linker) to form an ether, etc. In some embodiments, the cytotoxin linker conjugation is by forming an amide bond with a basic amine or carboxyl group on a cytotoxin, and thus the reactive substituent on the linker is a carboxyl or basic amine group, respectively. When the term "linker" is used to describe a linker in a conjugated form, due to the formation of a bond between a linker and / or a cytotoxin and between a linker and / or an antibody or its antigen-binding fragment, one or two reactive ends will be non-existent (such as reactive moiety Z', which has been converted into a chemical moiety Z) or incomplete (such as only a carbonyl group of a carboxylic acid). Such a conjugation reaction is further described below.

[0470] A variety of joints can be used to conjugate the described antibodies or antibody fragments to cytotoxic molecules. In some embodiments, the joint is cleavable under intracellular conditions, so that the cleavage of the joint in the intracellular environment releases the drug unit from the antibody. In yet other embodiments, the joint unit is non-cleavable, and the drug is released by, for example, antibody degradation. The joints that can be used for the ADC of the present invention are preferably stable outside the cell, prevent ADC molecules from aggregating, and keep ADC easily soluble in aqueous media and in a monomeric state. Before transport or delivery to the cell, preferably ADC is stable and remains intact, that is, the antibody remains connected to the drug portion. The joint is stable outside the target cell and can be cleaved at a certain effective rate within the cell. An effective joint will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug portion; (iii) remain stable and intact until the conjugate has been delivered or transported to its targeted site, that is, not cleaved; and (iv) maintain the cytotoxicity, cell killing or cell inhibition of the cytotoxic portion. The stability of the ADC can be measured by standard analytical techniques such as mass spectrometry, HPLC and separation / analysis techniques LC / MS. Covalent attachment of the antibody and drug moieties requires that the linker has two reactive functional groups, i.e., divalent in the sense of reactivity. Bivalent linker reagents useful for attaching two or more functional or biologically active moieties (such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups) are known, and methods for their production of conjugates have been described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, pp. 234-242).

[0471] Suitable cleavable linkers include linkers that can be cleaved by, for example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference when it relates to linkers suitable for covalent conjugation). Suitable cleavable linkers may include, for example, chemical moieties such as hydrazines, disulfides, thioethers, or dipeptides.

[0472] Linkers that can be hydrolyzed under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, and the like. (See, for example, U.S. Pat. Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661, the disclosures of each of which are incorporated herein by reference in their entirety as they relate to linkers suitable for covalent conjugation). Such linkers are relatively stable under neutral pH conditions (such as under neutral pH conditions in blood), but are unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes).

[0473] Linkers that can be cleaved under reducing conditions include, for example, disulfides. A variety of disulfide linkers are known in the art, including, for example, disulfide linkers formed by: SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate) and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio) toluene), SPDB and SMPT (see, for example, Thorpe et al., 1987, Cancer Res. 47: 5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford U. Press, 1987). See also U.S. Pat. No. 4,880,935, the disclosure of each of which is incorporated herein by reference in its entirety as it relates to linkers suitable for covalent conjugation).

[0474] The linker susceptible to enzymatic hydrolysis can be, for example, a peptide-containing linker that is cleaved by an intracellular peptidase or protease (including but not limited to a lysosomal or endosomal protease). An advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is usually weakened when conjugated, and the serum stability of the conjugate is usually high. In some embodiments, the length of the peptidyl linker is at least two amino acids or at least three amino acids. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides or pentapeptides. Examples of suitable peptides include peptides containing the following amino acids: such as valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine and glycine. The amino acid residues constituting the amino acid linker component include naturally occurring amino acid residues, as well as a small amount of amino acids and non-naturally occurring amino acid analogs, such as citrulline. Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). In some embodiments, the linker comprises a dipeptide such as Val-Cit, Ala-Val or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg or Trp-Cit. Linkers containing dipeptides such as Val-Cit or Phe-Lys are disclosed in, for example, U.S. Pat. No. 6,214,345, the disclosure of which is incorporated herein by reference in its entirety when it relates to linkers suitable for covalent conjugation. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit.

[0475] Suitable linkers for conjugating the antibodies or antibody fragments described herein to cytotoxic molecules include those capable of releasing cytotoxins by a 1,6-elimination process. Chemical moieties capable of such an elimination process include p-aminobenzyl (PAB) groups, 6-maleimidohexanoic acid, pH-sensitive carbonates, and other reagents as described in Jain et al. Pharm. Res. 32: 3526-3540, 2015, the disclosure of which is incorporated herein by reference in its entirety when it relates to linkers suitable for covalent conjugation.

[0476] In some embodiments, the linker includes a "self-immolative" group, such as the aforementioned PAB or PABC (p-aminobenzyloxycarbonyl), which is disclosed in, for example, Carl et al., J. Med. Chem. (1981) 24: 479-480; Chakravarty et al. (1983) J. Med. Chem. 26: 638-644; US Other such chemical moieties capable of undergoing this process ("self-immolative linkers") include methylene carbamates and heteroaryl groups such as aminothiazoles, aminoimidazoles, aminopyrimidines, and the like. Linkers containing such heterocyclic self-immolative groups are disclosed in, for example, U.S. Patent Publication Nos. 20160303254 and 20150079114 and U.S. Patent No. 7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237; US 2005 / 0256030; de Groot et al. (2001) J. Org. Chem. 66:8815-8830; and US 7223837. In some embodiments, a dipeptide is used in combination with a self-immolative linker.

[0477] The linker suitable for use herein may also include one or more groups selected from the following: C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene and combinations thereof, each of which may be optionally substituted. Non-limiting examples of such groups include (CH2) p 、(CH2CH2O) p and -(C=O)(CH2) p- Units, wherein for each occurrence p is an independently selected integer from 1 to 6.

[0478] In some embodiments, each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocycloalkyl, aryl or heteroaryl group can be optionally substituted with 1 to 5 substituents independently selected for each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxyl, thiol and nitro.

[0479] In some embodiments, each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocycloalkyl, aryl or heteroaryl group can be optionally interrupted by one or more heteroatoms selected from O, S and N.

[0480] In some embodiments, each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocycloalkyl, aryl or heteroaryl group may be optionally interrupted by one or more heteroatoms selected from O, S and N and may be optionally substituted with 1 to 5 substituents, the substituents being independently selected for each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxyl, thiol and nitro.

[0481] Suitable linkers may contain groups with solubility enhancing properties. For example, (CH2CH2O) p Unit (polyethylene glycol, PEG) linker can enhance solubility, as can alkyl chains substituted with amino, sulfonic acid, phosphonic acid or phosphoric acid residues. Linkers containing such moieties are disclosed, for example, in U.S. Pat. Nos. 8,236,319 and 9,504,756, the disclosure of each of which is incorporated herein by reference in its entirety when it relates to linkers suitable for covalent conjugation. Other groups that enhance solubility include, for example, acyl and carbamoylsulfonamide groups, which have the following structure:

[0482]

[0483] where a is 0 or 1; and

[0484] R 10 Selected from the group consisting of: hydrogen, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C1-C 24 (Hetero)aryl groups, C1-C 24 Alkyl (hetero) aryl groups and C1-C 24 (Hetero)arylalkyl group, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero) aryl groups and C3-C 24 (Hetero)arylalkyl groups, each of which may be optionally substituted and / or optionally interrupted by one or more heteroatoms selected from O, S and NR 11 R 12 , where R 11 and R 12 are independently selected from the group consisting of: hydrogen and a C1-C4 alkyl group; or R 10 is a cytotoxin, wherein the cytotoxin is optionally linked to N via a spacer moiety. Linkers comprising such groups are described, for example, in U.S. Pat. No. 9,636,421 and U.S. Patent Application Publication No. 2017 / 0298145, the disclosures of which are incorporated herein by reference in their entireties as they relate to linkers suitable for covalent conjugation to cytotoxins and antibodies or antigen-binding fragments thereof.

[0485] In some embodiments, the linker may include one or more of the following: hydrazine, disulfide, thioether, dipeptide, p-aminobenzyl (PAB) group, heterocyclic self-immolative group, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, a solubility enhancing group, acyl, -(C=O)- or -(CH2CH2O) p -group, wherein p is an integer from 1 to 6. Those skilled in the art will recognize that one or more of the groups listed may exist in the form of a divalent (diradical) species, such as C1-C6 alkylene, etc.

[0486] In some embodiments, the linker includes a p-aminobenzyl group (PAB). In one embodiment, the p-aminobenzyl group is disposed between the cytotoxic drug and the protease cleavage site in the linker. In one embodiment, the p-aminobenzyl group is part of a p-aminobenzyloxycarbonyl unit. In one embodiment, the p-aminobenzyl group is part of a p-aminobenzylamido unit.

[0487] In some embodiments, the linker comprises a dipeptide selected from the group consisting of: Phe-Lys, Val-Lys, Phe-Ala, Phe-Cit, Val-Ala, Val-Cit, and Val-Arg. In some embodiments, the linker comprises one or more of: PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0488] In some embodiments, the linker comprises PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0489] In some embodiments, the linker comprises a combination of one or more of the following: a peptide, an oligosaccharide, -(CH2) p -、-(CH2CH2O) p -, PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB or Ala-PAB.

[0490] In some embodiments, the linker comprises -(C=O)(CH2) p -unit, wherein p is an integer from 1 to 6.

[0491] In some embodiments, the linker comprises -(CH2) n -unit, wherein n is an integer from 2 to 6. In some embodiments, the linker comprises -((CH2) n , wherein n is 6. In some embodiments, LZ is

[0492]

[0493] wherein S is a sulfur atom and represents a reactive substituent (eg, a -SH group from a cysteine ​​residue) present in the antibody or antigen-binding fragment thereof that binds CD137.

[0494] In some embodiments, the linker comprises ((CH2) m O) n (CH2) m -group and heteroaryl group, wherein n and m are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein the heteroaryl group is triazole. In some embodiments, ((CH2) m O) n (CH2) m - group and triazole together include

[0495] Wherein n is 1 to 10, and the wavy line indicates the point of attachment to an additional linker component, chemical moiety Z, or amanita phalloidin. Other linkers that can be used in the methods and compositions described herein are described in US2019 / 0144504, which is incorporated herein by reference.

[0496] In one specific embodiment, the linker comprises PAB-Ala-Val-propionyl represented by the structure

[0497]

[0498] wherein the wavy lines indicate the points of attachment of the cytotoxin and the reactive moiety Z'. In another specific embodiment, the linker comprises PAB-Cit-Val-Propionyl represented by the structure

[0499]

[0500] Wherein the wavy line indicates the attachment point of the cytotoxin and the reactive moiety Z'. Such a PAB-dipeptide-propionyl linker is disclosed, for example, in Patent Application Publication No. WO2017 / 149077, which is incorporated herein by reference in its entirety. In addition, the cytotoxins disclosed in WO2017 / 149077 are incorporated herein by reference.

[0501] Those skilled in the art will recognize that any one or more of the chemical groups, moieties, and features disclosed herein can be combined in a variety of ways to form linkers useful for conjugation of antibodies and cytotoxins as disclosed herein. Other linkers that can be used in conjunction with the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0502] In certain embodiments, the intermediate as the joint precursor reacts with the drug moiety under appropriate conditions. In certain embodiments, a reactive group is used on the drug and / or intermediate or joint. The reaction product between the drug and the intermediate or the derivatized drug reacts with the antibody or Fab under appropriate conditions subsequently. Alternatively, the joint or intermediate can react with the antibody or the derivatized antibody at first, and then react with the drug or the derivatized drug. Such conjugation reaction will now be described more fully.

[0503] Many different reactions can be used to covalently attach a joint or drug-joint conjugate to an antibody or its Fab. Suitable attachment points on the antibody molecule include the amine group of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the thiol group of cysteine, and various parts of aromatic amino acids. For example, a carbodiimide reaction can be used to connect the carboxyl (or amino) group on the compound to the amino (or carboxyl) group on the antibody portion for non-specific covalent attachment. In addition, bifunctional agents such as dialdehydes or imidoesters can also be used to connect the amino group on the compound to the amino group on the antibody portion. Schiff base reaction can also be used for the attachment of drugs to binding agents. This method includes periodic acid oxidation of drugs containing ethylene glycol or hydroxyl groups to form aldehydes, which then react with the binding agent. Attachment occurs via the formation of Schiff bases with the amino groups of the binding agent. Isothiocyanates can also be used as coupling agents for covalently attaching drugs to binding agents. Other techniques are known to the technician and are within the scope of the present disclosure.

[0504] Linkers useful for conjugation to the antibodies or antigen-binding fragments described herein include, but are not limited to, linkers comprising a chemical moiety Z formed by a coupling reaction as depicted below in Table 2. The curved lines represent the points of attachment to the antibody or antigen-binding fragment and the cytotoxic molecule, respectively.

[0505] Table 2. Exemplary chemical moieties Z formed by coupling reactions in the formation of antibody drug conjugates

[0506]

[0507]

[0508]

[0509]

[0510] Those skilled in the art will recognize that the reactive substituent Z' attached to the linker and the reactive substituent on the antibody or antigen-binding fragment thereof participate in a covalent coupling reaction to produce the chemical moiety Z, and will recognize the reactive substituent Z'. Thus, an antibody drug conjugate useful in conjunction with the methods described herein can be formed by reacting the antibody or antigen-binding fragment thereof with a linker or cytotoxin-linker conjugate as described herein, the linker or cytotoxin-linker conjugate comprising the reactive substituent Z', which is suitable for reacting with the reactive substituent on the antibody or antigen-binding fragment thereof to form the chemical moiety Z.

[0511] As depicted in Table 2, examples of suitable reactive substituents on the linker and the antibody or antigen-binding fragment thereof include nucleophile / electrophile pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, etc.), diene / dienophile pairs (e.g., especially, azide / alkyne pairs or diene / α,β-unsaturated carbonyl pairs), etc. Coupling reactions between reactive substituents to form the chemical moiety Z include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine or hydroxylamine condensation, hydrazine formation, amidation, esterification, disulfide formation, cycloaddition (e.g., especially, [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reaction forms known in the art or described herein. Preferably, the linker comprises an electrophilic functional group to react with a nucleophilic functional group on the antibody or antigen-binding fragment thereof.

[0512] Reactive substituents that may be present in an antibody or antigen-binding fragment thereof as disclosed herein include, but are not limited to, nucleophilic groups such as (i) an N-terminal amine group, (ii) a side chain amine group, e.g., lysine, (iii) a side chain thiol group, e.g., cysteine, and (iv) a sugar hydroxyl or amino group, wherein the antibody is glycosylated. Reactive substituents that may be present in an antibody or antigen-binding fragment thereof as disclosed herein include, but are not limited to, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine ​​residues, as well as propargyl, azido, halogenated aryl (e.g., fluoroaryl), halogenated heteroaryl (e.g., fluoroheteroaryl), halogenated alkyl, and halogenated heteroalkyl moieties of non-naturally occurring amino acids. In some embodiments, reactive substituents present in an antibody or antigen-binding fragment thereof as disclosed herein include amine or thiol moieties, are amine or thiol moieties. Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive to be conjugated with linker reagents by treating with a reducing agent such as DTT (dithiothreitol). Therefore, in theory, each cysteine ​​bridge will form two reactive thiol nucleophiles. Other nucleophilic groups can be introduced into antibodies by the reaction of lysine and 2-iminothiolane (2-iminothiolane) (Traut reagent), resulting in amine conversion into thiol. Reactive thiol groups can be introduced into antibodies (or their fragments) by introducing one, two, three, four or more cysteine ​​residues (for example, preparing mutant antibodies comprising one or more non-natural cysteine ​​amino acid residues). U.S. Patent No. 7,521,541 teaches antibody engineering by introducing reactive cysteine ​​amino acids.

[0513] In some embodiments, the reactive moiety Z' attached to the linker is a nucleophilic group that reacts with an electrophilic group present on the antibody. Useful electrophilic groups on the antibody include, but are not limited to, aldehyde and keto carbonyl groups. The heteroatom of the nucleophilic group can react with the electrophilic group on the antibody and form a covalent bond with the antibody. Useful nucleophilic groups include, but are not limited to, hydrazides, oximes, amino groups, hydroxyls, hydrazines, thiosemicarbazones, hydrazide carboxylates, and aryl hydrazides.

[0514] In some embodiments, Z is a reaction product between a reactive nucleophilic substituent (such as an amine and thiol moiety) present in an antibody or antigen-binding fragment thereof and a reactive electrophilic substituent Z'. For example, Z' can be, among others, a Michael acceptor (e.g., maleimide), an activated ester, an electron-deficient carbonyl compound, or an aldehyde. Several representative and non-limiting examples of reactive substituents Z' and resulting chemical moieties Z are provided in Table 3.

[0515] Table 3. Complementary reactive substituents and chemical moieties

[0516]

[0517] For example, suitable linkers for synthesizing ADCs include, but are not limited to, reactive substituents Z', such as maleimide or haloalkyl groups. These can be attached to linkers by reagents such as, inter alia, succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, which are described, for example, in Liu et al., 18: 690-697, 1979, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation.

[0518] In some embodiments, the reactive substituent Z' attached to the linker L is a maleimide, an azide, or an alkyne. An example of a maleimide-containing linker is a non-cleavable maleimidocaproyl-based linker, which is particularly useful for conjugation of microtubule disrupting agents such as auristatins. Such a linker is described by Doronina et al. Bioconjugate Chem. 17: 14-24, 2006, the disclosure of which is incorporated herein by reference when it relates to linkers for chemical conjugation.

[0519] In some embodiments, the reactive substituent Z′ is -(C=O)- or -NH(C=O)-, such that the linker can be attached to the antibody or its antigen-binding fragment via an amide or urea moiety, respectively, resulting from the reaction of the -(C=O)- or -NH(C=O)- group with an amino group of the antibody or its antigen-binding fragment.

[0520] In some embodiments, the reactive substituent is an N-maleimido group, a halo-substituted N-alkylamido group, a sulfonyloxy N-alkylamido group, a carbonate group, a sulfonyl halide group, a thiol group or a derivative thereof, an alkynyl group containing an internal carbon-carbon triple bond, a (hetero)cycloalkynyl group, a bicyclo[6.1.0]non-4-yn-9-yl group, an alkenyl group containing an internal carbon-carbon double bond, a cycloalkenyl group, a tetrazinyl group, an azido group, a phosphine group, a nitrile oxide group, a nitrone group, a nitrile imine group, a diazo group, a ketone group, an (O-alkyl)hydroxyamino group, a hydrazine group, a halo-substituted N-maleimido group, a 1,1-bis(sulfonylmethyl)methylcarbonyl group or an elimination derivative thereof, a carbonyl halide group, or an allenamide group, each of which can be optionally substituted. In some embodiments, the reactive substituents include a cycloalkene group, a cycloalkynyl group, or an optionally substituted (hetero)cycloalkynyl group.

[0521] Non-limiting examples of Amanita toxin-linker conjugates comprising a reactive substituent Z' suitable for reacting with a reactive residue on an antibody or antigen-binding fragment thereof include, but are not limited to, 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-Amanita toxin, 7'C-(4-(6-(maleimido)hexanoylamino)piperidin-1-yl)-Amanita toxin, 7'C-(4-(6-(6-(maleimido)hexanoylamino)hexanoyl)piperazin-1-yl)-Amanita toxin, 7'C-(4-(6-(6-(maleimido)hexanoylamino)hexanoyl)piperazin-1-yl)-Amanita toxin, 7'C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperazin-1-yl)-Amanita toxin, 7'C-(4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)piperazin-1-yl)- 'C-(4-(2-(6-(maleimido)hexanoylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(6-(maleimido)hexanoylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin, yl)-amanitin toxin, 7'C-(4-(2-(2-bromoacetylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(3-(pyridin-2-yldisulfanyl)propionylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(4-(maleimido)butyrylamino)ethyl)piperidin-1-yl)-amanitin toxin 1-yl)-amanitin toxin, 7'C-(4-(2-(maleimido)acetyl)piperazin-1-yl)-amanitin toxin, 7'C-(4-(3-(maleimido)propionyl)piperazin-1-yl)-amanitin toxin, 7'C-(4-(4-(maleimido)butyryl)piperazin-1-yl)-amanitin toxin, 7'C-(4 -(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(3-((6-(maleimido)hexanoylamino)methyl)pyrrolidin-1-yl)-amanitin toxin, 7'C-(3-((6-(6-(maleimido)hexanoylamino)hexanoylamino)methyl)pyrrolidin-1-yl)-amanitin toxin, 7'C-(3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrolidin-1-yl)-amanitin toxin, 7'C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)methyl)pyrrolidin-1-yl)-amanitin toxin,7'C-(4-(2-(6-(2-(aminooxy)acetylamino)hexanoylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(4-(2-(aminooxy)acetylamino)butyrylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(4-(2-(aminooxy)acetylamino)butyryl)piperazin-1-yl)-amanitin toxin, 7'C-(4-(6-(2-(aminooxy)acetylamino)hexanoyl)piperazin-1-yl)-amanitin toxin, 7'C-((4-(6-(maleimido)hexanoylamino)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(maleimido)hexanoylamino)piperidin-1-yl)methyl)-amanitin toxin methyl)-amanitin toxin, (S)-7'C-((3-((6-(maleimido)hexanoylamino)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(6-(maleimido)hexanoylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(maleimido)hexanoylamino)hexanoylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, (R)-7'C-((3-((6-(maleimido)hexanoylamino)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, (S)-7'C-((3-((6-(maleimido)hexanoylamino)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(6-(maleimido)hexanoylamino)hexanoylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(6-(maleimido)hexanoylamino)ethyl)piperazin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(6-(6-(maleimido)hexanoylamino)ethyl)piperazin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(6-(6-(maleimido)hexanoylamino)hexanoylamino)ethyl)piperazin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl)-Amanita toxin ) methyl)-amanitin toxin, 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)ethyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((3-((6-(6-(maleimido)hexanoylamino)hexanoylamino)-S-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((6-(6-(maleimido)hexanoylamino)hexanoylamino)-R-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin,7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-R-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(3-carboxypropionamido)ethyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(6-(6-(maleimido)hexanoylamino)hexanoyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin )hexanoyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(maleimido)acetyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(3-(maleimido)propionyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(4-(maleimido)butyryl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(2-(maleimido)acetylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(4-(maleimido)butyrylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C- ((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((6-(maleimido)hexanoylamino)methyl)azetidin-1-yl)methyl)-amanitin toxin, 7'C-((3-(2-(6-(maleimido)hexanoylamino)ethyl)azetidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((2-(6-(maleimido)hexanoylamino)ethyl)azetidin-1-yl)methyl)-amanitin toxin toxin, 7'C-(((4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)azetidin-1-yl)methyl)-amanitin toxin, 7'C-(((2-(6-(maleimido)-N-methylhexanoylamino)ethyl)(methyl)amino)methyl)-amanitin toxin, 7'C-(((4-(6-(maleimido)-N-methylhexanoylamino)butyl(methyl)amino)methyl)-amanitin toxin, 7'C-((2-(2-(6-(maleimido)hexanoylamino)ethyl)aziridine-1-yl)methyl)-amanitin toxin,7'C-((2-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)ethyl)azetidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(6-(6-(2-(aminooxy)acetylamino)hexanoylamino)hexanoyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-oyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(2-(aminooxy)acetylamino)acetyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(3-(2-(aminooxy)acetylamino)acetyl)piperazin-1-yl)methyl)-amanitin toxin oxy)acetylamino)propionyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(4-(2-(aminooxy)acetylamino)butyryl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(2-(aminooxy)acetylamino)hexanoylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(2-(2-(aminooxy)acetylamino)acetylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(4-(2-(aminooxy)acetylamino)butyrylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(20-(aminooxy)acetylamino)butyrylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin methyl)-amanitin toxin, 7'C-(((2-(6-(2-(aminooxy)acetylamino)-N-methylhexanoylamino)ethyl)(methyl)amino)methyl)-amanitin toxin, 7'C-(((4-(6-(2-(aminooxy)acetylamino)-N-methylhexanoylamino)butyl)(methyl)amino)methyl)-amanitin toxin, 7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)methyl)pyrrolidin-1-yl)-S-methyl)-amanitin toxin, 7'C-((3-((6-(4-(( 7'C-((4-(2-(2-bromoacetylamino)ethyl)piperazin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(2-bromoacetylamino)ethyl)piperidin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(2-bromoacetylamino)ethyl)piperidin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(2-bromoacetylamino)ethyl)piperidin-1-yl)methyl)-Amanita toxin, 6'O-(6-(6-(maleimido)hexanoylamino)hexyl)-Amanita toxin, 6'O-(5-(4-((maleimido)methyl)cyclohexanecarboxamido)pentyl)-Amanita toxin,6'O-(2-((6-(maleimido)hexyl)oxy)-2-oxoethyl)-amanitin toxin, 6'O-((6-(maleimido)hexyl)carbamoyl)-amanitin toxin, 6'O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexyl)carbamoyl)-amanitin toxin, 6'O-(6-(2-bromoacetamido)hexyl)-amanitin toxin, 7'C-(4-(6-(azido)hexanoylamino)piperidin-1-yl)-amanitin toxin, 7'C-(4-(hex-5-ynoylamino)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(maleimido)hexanoylamino)piperidin-1-yl)-amanitin toxin The toxins of the present invention are 6'O-(6-((6-(11,12-didehydro-5,6-dihydro-dibenzo[b,f]azocin-5-yl)-6-oxohexanoylamino)hexyl)-amanitin toxin, 6'O-(6-(hex-5-ynylamino)hexyl)-amanitin toxin, 6'O-(6-(2-(aminooxy)acetylamido)hexyl)-amanitin toxin, 6'O-((6-aminooxy)hexyl)-amanitin toxin and 6'O-(6-(2-iodoacetylamido)hexyl)-amanitin toxin. ,

[0522] In some embodiments, the chemical moiety Z is selected from Table 2 or Table 3. In some embodiments, the chemical moiety Z is:

[0523]

[0524] wherein S is a sulfur atom and represents a reactive substituent present in the antibody or antigen-binding fragment thereof (such as an anti-CD-137 antibody).

[0525] In some embodiments, an Amanita toxin as disclosed herein is conjugated to a linker-reactive moiety-LZ′ having the formula:

[0526]

[0527] Wherein the wavy line indicates the point of attachment to the substituent on the cytotoxin (eg, amanita toxin).The linker-reactive substituent group LZ' may alternatively be referred to as N-β-maleimidopropionyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB).

[0528] In some embodiments, an Amanita toxin as disclosed herein is conjugated to a linker-reactive moiety-LZ′ having the formula:

[0529]

[0530] Wherein the wavy line indicates the point of attachment to the substituent on the cytotoxin (eg, amanita toxin).The linker reactive substituent group LZ' may alternatively be referred to as N-β-maleimidopropyl-Val-Cit-p-aminobenzyl (BMP-Val-Cit-PAB).

[0531] In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0532]

[0533] Wherein S is a sulfur atom, representing a reactive substituent present in an antibody or antigen-binding fragment thereof (such as an anti-CD-5 antibody). The wavy line at the end of the linker indicates the point of attachment to the Amanita phalloidin toxin.

[0534] In some embodiments, the linker L and chemical moiety Z after conjugation to the antibody (collectively referred to as LZ-Ab) have the structure:

[0535]

[0536] Wherein S is a sulfur atom, representing a reactive substituent present in an antibody or antigen-binding fragment thereof, such as an anti-CD-137 antibody. The wavy line at the end of the linker indicates the point of attachment to the Amanita phalloidin toxin.

[0537] The foregoing linker moieties and amanita toxin-linker conjugates, as well as other linker moieties and amanita toxin-linker conjugates that can be used in conjunction with the compositions and methods described herein, are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220 and Patent Application Publication No. WO2017 / 149077, the disclosures of each of which are incorporated herein by reference in their entirety.

[0538] The foregoing linker moieties and amanita toxin-linker conjugates, as well as other linker moieties and amanita toxin-linker conjugates that can be used in conjunction with the compositions and methods described herein, are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220 and Patent Application Publication No. WO2017 / 149077, the disclosures of each of which are incorporated herein by reference in their entirety.

[0539] In one embodiment, a CD137 antibody or antigen-binding fragment described herein can be conjugated to an Amanita ani toxin to form a conjugate represented by the formula Ab-ZL-Am, wherein Ab is a CD137 antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an Amanita ani toxin, each as described herein.

[0540] In some embodiments, Am-LZ-Ab is:

[0541]

[0542] In some embodiments, Am-LZ-Ab is:

[0543] In some embodiments, Am-LZ-Ab is:

[0544]

[0545] In some embodiments, Am-LZ-Ab is:

[0546]

[0547] In some embodiments, Am-LZ-Ab is:

[0548]

[0549] Preparation of Antibody Drug Conjugates

[0550] In the ADC of Formula I as disclosed herein, the anti-CD137 antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxic drug moieties (D) via a linker L and a chemical moiety Z as disclosed herein, for example, each antibody is conjugated to about 1 to about 20 drug moieties. The ADC of the present disclosure can be prepared by several routes using organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) reacting a reactive substituent of an antibody or antigen-binding fragment thereof with a divalent linker reagent to form Ab-ZL as described above, followed by reaction with a drug moiety D; or (2) reacting a reactive substituent of a drug moiety with a divalent linker reagent to form DLZ', followed by reaction with a reactive substituent of an antibody or antigen-binding fragment thereof as described above. Additional methods for preparing ADCs are described herein.

[0551] On the other hand, the anti-CD137 antibody or its antigen-binding fragment has one or more lysine residues, which can be chemically modified to introduce one or more sulfhydryl groups. The ADC is then formed by conjugation of the sulfur atom of the sulfhydryl group as described above. Reagents that can be used to modify lysine include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut reagent).

[0552] In another aspect, the anti-CD137 antibody or antigen-binding fragment thereof may have one or more carbohydrate groups that may be chemically modified to have one or more sulfhydryl groups. The ADC is then formed by conjugation via the sulfur atom of the sulfhydryl group as described above.

[0553] In yet another aspect, the anti-CD137 antibody may have one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) group (see, e.g., Laguzza et al., J. Med. Chem. 1989, 32(3), 548-55). The ADC is then formed by conjugation with the corresponding aldehyde as described above. Other protocols for modifying proteins to attach or associate cytotoxins are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), incorporated herein by reference.

[0554] Methods for conjugating linker-drug moieties to cell-targeting proteins such as antibodies, immunoglobulins, or fragments thereof are found in, e.g., U.S. Pat. No. 5,208,020; U.S. Pat. No. 6,441,163; WO2005037992; WO2005081711; and WO2006 / 034488, all of which are expressly incorporated herein by reference in their entirety.

[0555] Route of administration and dosage

[0556] Alternatively, a fusion protein comprising the antibody and the cytotoxic agent may be prepared, for example, by recombinant techniques or peptide synthesis. The length of DNA may comprise the corresponding regions encoding the two parts of the conjugate, adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0557] The ADC described herein can be administered to a patient (e.g., a human patient suffering from an immune disease or cancer) in a variety of dosage forms. For example, the ADC described herein can be administered to a patient suffering from an immune disease or cancer in the form of an aqueous solution (such as an aqueous solution containing one or more pharmaceutically acceptable excipients). Suitable pharmaceutically acceptable excipients for use with the compositions and methods described herein include viscosity modifiers. The aqueous solution can be sterilized using techniques known in the art.

[0558] Pharmaceutical formulations containing anti-CD137 ADCs as described herein are prepared by mixing such ADCs with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 1 0 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0559] The amount of the ADC administered should be sufficient to consume cells, such as activated T cells, which reject autologous cell therapy, such as CAR cell therapy. The determination of the therapeutic effective dose is within the ability of practitioners in the art, however, as an example, in the embodiments of the method for treating immune diseases or cancer using systemic administration of ADC described herein, effective human doses will be within the range of 0.1mg / kg-150mg / kg (e.g., 5mg / kg, 10mg / kg, 25mg / kg, 50mg / kg, 75mg / kg, 100mg / kg, 150mg / kg, etc.). The route of administration can affect the recommended dose. Depending on the mode of administration adopted, it is envisioned that repeated systemic doses are in order to maintain effective levels, for example, in order to reduce the risk of CAR-T cell rejection.

[0560] The anti-CD137 ADC described herein can be administered by a variety of routes, such as oral administration, transdermal administration, subcutaneous administration, intranasal administration, intravenous administration, intramuscular administration, intraocular administration or parenteral administration. In any given case, the most suitable route of administration will depend on the specific ADC, the patient, the drug formulation method, the method of administration (e.g., administration time and route of administration), the patient's age, weight, sex, severity of the disease being treated, the patient's diet and the patient's excretion rate.

[0561] The effective dose of the anti-CD137 ADC described herein can range from about 0.001 mg / kg body weight to about 100 mg / kg body weight per single (e.g., bolus) administration, multiple administrations, or continuous administration, or can be in the range of reaching the optimal serum concentration of anti-CD137 ADC (e.g., 0.0001 μg / mL-5000 μg / mL serum concentration). The dose of anti-CD137 ADC can be administered once or more (e.g., 2-10 times) per day, week, or month to human subjects who have received CAR therapy, are receiving CAR therapy at the same time, or will receive CAR therapy at a time point after the delivery of anti-CD137 ADC. Anti-CD137 ADC can be administered to human patients at one or more doses. In one embodiment, anti-CD137 ADC can be administered before CAR therapy in an amount sufficient to reduce the amount of host reactive T cells, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. Example

[0562] The following examples are provided to provide a person of ordinary skill in the art with a description of how the compositions and methods described herein may be used, prepared and evaluated, and are intended to be mere illustrations of the invention and are not intended to limit the scope of what the inventors regard as their invention. The data in Examples 1-3 were previously presented in International Patent Application No. WO2018 / 134787 (published on July 26, 2018), the entire contents of which are incorporated herein by reference.

[0563] Example 1. In vitro analysis of anti-CD137-amanitin antibody drug conjugate (ADC) using in vitro T cell killing assay

[0564] Cryopreserved negatively selected primary human T cells were thawed and stimulated with anti-CD3 / anti-CD28 beads (Invitrogen) at a bead:cell ratio of 0.5:1. At the start of the assay, 2e4 T cells were seeded in each well of a 384-well plate, and ADC was added to the wells at different concentrations between 0.003nm and 30nm and then placed in an incubator at 37°C and 5% CO2. After 4 days in culture, cells were analyzed by flow cytometry. Cells were stained with the viability marker Live / Dead Yellow (Invitrogen) and run on a volume flow cytometer. Surviving activated cells ( Figure 1A ) and viable non-activated cells ( Figure 1B) was determined by FSC and SSC. Nonspecific human IgG conjugated to amanitin (hIgG-amanitin) was used as a negative control. Therefore, the control was compared with two different ADCs: 1) chimeric anti-CD137 antibody BBK2 conjugated to amanitin (CD137-amanitin); and 2) ADCs comprising antibodies specific for T cell antigens conjugated to amanitin (anti-T cell-amanitin). The anti-T cell-amanitin ADC was used as a positive control because it is expected to bind and kill both activated and non-activated T cells.

[0565] like Figure 1A and Figure 1B As shown in , the anti-CD137-amanitin ADC (i.e., "CD137-amanitin") specifically kills activated T cells and does not significantly kill unactivated (resting, steady-state) T cells. In addition, the positive control (i.e., "anti-T cell-amanitin" ADC), as an ADC that specifically targets both activated T cells and unactivated T cells, kills both activated T cells and unactivated T cells.

[0566] Example 2. Analysis of GvHD Prevention Using a Xenogeneic-GvHD Mouse Model

[0567] The ability of anti-CD137-amanitin ADC to prevent the development or occurrence of GvHD was evaluated in vivo using a xeno-GvHD mouse model. Female 6-8 week old NSG mice were irradiated (200 cGy) and transplanted with 6 × 10 6 Individual human peripheral blood mononuclear cells (PBMCs) were used to generate a GvHD mouse model. One day later, the animals were administered (at 3 mg / kg) anti-CD137-amanitin ADC (i.e., "CD137-amanitin") or various control agents (i.e., buffer alone ("PBS"), anti-CD137 antibody ("naked CD137"), or an amanitin-based ADC that is not specific for CD137 ("isotype-amanitin")). The anti-CD137 antibody in the ADC used in this example and the anti-CD137 antibody as a naked antibody is chimeric BBK2.

[0568] After dosing, animals were closely followed daily to determine signs of GvHD and / or diminished physical condition, including but not limited to hunched posture, ruffled fur, weight loss, and / or limited activity. Animals with severe physical condition issues or those showing weight loss >20% were sacrificed, and their tissues were analyzed for human cells. The peripheral blood and spleen of mice were stained with an antibody cocktail comprising hCD45 antibodies, mCD45 antibodies, hCD3 antibodies, and hCD137 antibodies, the red blood cells were lysed, and analyzed by flow cytometry. The number of human T cells in the blood was defined as hCD45+CD3+ and normalized for the volume of input blood. Figure 2 The percentage survival as a function of days after treatment is provided in Figure 3 The number of human T cells in peripheral blood changes with the number of days after transplantation are provided in .

[0569] like Figure 2 As shown, animals treated with a single dose of anti-CD137-amanitin ADC ("CD137-amanitin") showed essentially complete prevention of GvHD, even 80 days after transplantation, while animals treated with controls (i.e., PBS, anti-CD137 antibody (naked) and isotype-amanitin) all died within 11 to 13 days after transplantation. These results also indicate that anti-CD137-amanitin ADC was well tolerated in all animals and that a single dose of anti-CD137-amanitin ADC was sufficient to completely prevent GvHD (as opposed to requiring multiple doses). Figure 3 As shown, no human T cells were detected in the peripheral blood of mice for a period of at least 70 days after transplantation in anti-CD137-amanitin-treated mice, while animals treated with controls were characterized by detectable human T cells in the peripheral blood of mice several days after transplantation, indicating the development of GvHD in animals treated with controls.

[0570] Example 3. Determination of engraftment rate and steady-state T cell depletion in the hNSG-SGM3 mouse model

[0571] Baseline human hematopoietic cell (total and T cell) engraftment rates in the peripheral blood of female humanized NSG-SGM3 mice were assessed by flow cytometry using hCD45, mCD45, hCD3, and hCD137 antibodies. Mice were randomized and then treated with CD137-amanitin ADC (chimeric BBK2-amanitin ADC), isotype-amanitin ADC, or anti-T cell amanitin ADC, each at a dose of 3 mg / kg. Engraftment and T cell depletion in the periphery of mice were measured on days 5, 9, 14, 22, and 27 after treatment ( Figure 4A and Figure 4BPeripheral blood was stained to assess chimerism and changes in T cell numbers. Equal amounts of blood were run on a volume flow cytometer and absolute counts were determined based on the number of events. Engraftment and reduction in T cell numbers were normalized to baseline values.

[0572] Figure 4A and Figure 4B The results in show that anti-CD137-amanitin ADC is well tolerated in this mouse model. In addition, the implantation rate and T cell frequency (after normalization to baseline) remain near the baseline level, and there is a moderate to short-term decline in the implantation rate and T cell frequency of mice treated with anti-CD137-amanitin ADC. These data show that T cell consumption is generally lacking in the humanized NSG mice of steady state, which indicates that immune function can be maintained in mice treated with anti-CD137-amanitin ADC because most T cells are not consumed. On the contrary, mice treated with ADC ("anti-T cell-amanitin") that specifically targets alternative T cell markers show complete elimination of implantation and T cell number.

[0573] The heavy chain and light chain amino acid sequences of the chimeric BBK2 described in the above examples are set forth in SEQ ID NO: 11 and SEQ ID NO: 12, respectively.

[0574] Example 4: Administration of allogeneic CAR-T cells in a mouse model

[0575] The following studies were performed to evaluate the levels of CAR-T cells present in allogeneic recipients under different conditions.

[0576] This study used a murine allogeneic CAR-T model. A suitable model is described in Ghosh et al., Nature Medicine (2017), 23(2): 242-249.

[0577] On day 0, mice in the first treatment group were administered 1×10 7 cells / kg to 1×10 9 The mice were treated with a sensitizing dose of allogeneic T cells at a dose of 10 cells / kg of allogeneic T cells. On day 3, the mice were administered anti-CD137-α-amanitin ADC at a dose of 3 mg / kg. On day 10, after the ADC was substantially cleared from the blood of the mice, the mice were administered allogeneic CAR-T cells. The CAR-T cells were from the same donor as the allogeneic T cells administered on day 0.

[0578] Mice in the second treatment group were treated using the same regimen as the first treatment group, but non-conjugated anti-CD137 antibody was administered on day 3 instead of the anti-CD137 ADC.

[0579] Mice in the third treatment group were treated using the same regimen as the first treatment group, but an isotype control antibody conjugated to α-amanitin was administered on day 3 instead of the anti-CD137 ADC.

[0580] Mice in the fourth treatment group were treated using the same regimen as the first treatment group, but a priming dose of autologous T cells was administered on day 0 instead of allogeneic T cells.

[0581] Mice in the fifth treatment group were administered allogeneic CAR-T cells on day 10 without pre-treatment.

[0582] Mice in the sixth treatment group were administered autologous CAR-T cells on day 10 without pre-treatment.

[0583] The number of CAR-T cells present in the spleen and peripheral blood of mice from each treatment group was determined on days 14, 17, and 30. The number of CD137+ activated T cells in the spleen and peripheral blood of mice from each treatment group was determined on day 9. Throughout the study, mice were monitored for symptoms of GVHD and the presence of CAR-T cells.

[0584] Example 5: Administration of anti-CD137 antibody drug conjugates to human patients to prevent rejection of allogeneic cell therapy

[0585] Human patients are selected to receive allogeneic cell therapy, such as allogeneic CAR cell therapy. In order to inhibit or prevent rejection of allogeneic cells, anti-CD137 antibody drug conjugates (ADC) are administered according to the methods disclosed herein. The doctor performs the following treatment steps.

[0586] First, an initial amount of allogeneic cells is intravenously administered to the patient in an amount sufficient to elicit a sensitizing immune response to the allogeneic cells. In the sensitization step, the allogeneic cells are administered to the patient in order to elicit an immune response, thereby generating endogenously activated CD137+ T cells.

[0587] Subsequently, an anti-CD137 ADC is administered to the patient, the anti-CD137 ADC comprising an anti-CD137 antibody conjugated to a cytotoxin via a linker. The anti-CD137 ADC is administered in an amount effective to deplete endogenous CD137+ activated T cells. After administration of the anti-CD137 ADC, the level of CD137+ activated T cells in the patient is assessed to confirm depletion.

[0588] Next, a therapeutically effective amount of allogeneic cells expressing CAR is administered to the patient. The allogeneic cells are derived from the same donor as the cells administered to the patient during the sensitization step. Relative to patients receiving allogeneic cell therapy without sensitization and administration of anti-CD137 ADC, the recipient patient's acceptance of allogeneic cells is promoted, and the risk of host-versus-graft (HvG) reactions is reduced. Table 4: Sequence overview

[0589]

[0590]

[0591] The present invention also provides the following items:

[0592] 1. A method for treating or preventing rejection of allogeneic cells transplanted into a human subject, the method comprising:

[0593] (a) administering a first amount of allogeneic cells to the human subject, wherein the first amount is sufficient to elicit a sensitization response to the allogeneic cells in the human subject;

[0594] (b) administering an anti-CD137 antibody drug conjugate (ADC) to the human subject such that endogenous CD137+ activated T cells are depleted, wherein the anti-CD137 ADC comprises an anti-CD137 antibody or an antigen-binding fragment thereof conjugated to a cytotoxin via a linker; and

[0595] (c) administering a therapeutically effective amount of an allogeneic cell expressing a CAR to the human subject, wherein the allogeneic cell is of the same type as the allogeneic cell administered in (a), and wherein the CAR comprises an extracellular domain that binds to a tumor antigen, a transmembrane domain, and a cytoplasmic signaling domain;

[0596] Such that allogeneic cell rejection in said human subject is treated or prevented.

[0597] 2. The method according to item 1, comprising administering the first amount of allogeneic cells to the human subject between about 48 hours and about 7 days before step (b).

[0598] 3. The method according to item 1 comprises administering the therapeutically effective amount of allogeneic cells expressing CAR to the human subject between about 24 hours to about 14 days after step (b).

[0599] 4. A method of treating or preventing rejection of allogeneic cells transplanted into a human subject, the method comprising:

[0600] (a) administering an anti-CD137 ADC to the human subject, wherein the anti-CD137 ADC comprises an anti-CD137 antibody or antigen-binding fragment thereof conjugated to a cytotoxin via a linker, and wherein the human subject is characterized by having activated T cells against allogeneic cells previously administered to the human subject; and

[0601] (b) administering a therapeutically effective amount of an allogeneic cell expressing a CAR to the human subject, wherein the allogeneic cell is of the same type as the allogeneic cell described in (a), and wherein the CAR comprises an extracellular domain that binds to a tumor antigen, a transmembrane domain, and a cytoplasmic signaling domain,

[0602] Such that rejection of the transplanted allogeneic cells in the human subject is treated or prevented.

[0603] 5. The method according to item 4 comprises administering the effective amount of allogeneic cells expressing CAR to the human subject between about 24 hours and about 14 days after step (a).

[0604] 6. The method according to any one of items 1-5, wherein the allogeneic cells are allogeneic T cells or allogeneic NK cells.

[0605] 7. The method according to any one of items 1-6, wherein the effective amount of the allogeneic cells expressing CAR is about 2×10 6 cells / kg to about 3.0×10 8 cells / kg.

[0606] 8. A method according to any one of items 1-7, wherein the allogeneic cell rejection is characterized by cytokine release syndrome (CRS).

[0607] 9. The method according to any one of items 1 to 8, wherein the human subject has cancer or an autoimmune disease.

[0608] 10. The method according to any one of items 1 to 9, wherein the anti-CD137 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences listed in SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, and the anti-CD137 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences listed in SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, respectively.

[0609] 11. The method according to item 10, wherein the anti-CD137 antibody or antigen-binding fragment thereof is chimeric or humanized.

[0610] 12. The method according to any one of items 1-11, wherein the anti-CD137 antibody or antigen-binding fragment thereof is of IgG1 isotype or IgG4 isotype.

[0611] 13. The method according to items 1-12, wherein the cytotoxin is an antimitotic agent or an RNA polymerase inhibitor.

[0612] 14. The method according to item 13, wherein the RNA polymerase inhibitor is Amanita ani toxin.

[0613] 15. The method according to item 13, wherein the RNA polymerase inhibitor is amanitin.

[0614] 16. The method according to item 15, wherein the amanitin is selected from the group consisting of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin, monohydroxyamanitin carboxylic acid and proamanitin.

[0615] 17. The method according to item 14, wherein the anti-CD137 antibody or antigen-binding fragment thereof conjugated to Amanita phalloides toxin is represented by the formula Ab-ZL-Am, wherein Ab is the antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an Amanita phalloides toxin represented by formula (III)

[0616]

[0617] in

[0618] R1 is H, OH, OR A OR C ;

[0619] R2 is H, OH, ORB OR C ;

[0620] R A and R B When present, taken together with the oxygen atom to which they are attached, form an optionally substituted 5-membered heterocycloalkyl group;

[0621] R3 is H, R C or R D ;

[0622] R4, R5, R6 and R7 are each independently H, OH, OR C , OR D , R C or R D ;

[0623] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0624] R9 is H, OH, OR C OR D ;

[0625] Q is -S-, -S(O)- or -SO2-;

[0626] R C Yes - LZ;

[0627] R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0628] L is a connector; and

[0629] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the anti-CD137 antibody or antigen-binding fragment thereof, wherein Am comprises exactly one R c Substituent.

[0630] 18. The method according to item 17, wherein the linker is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, a dipeptide, -(C=O)-, -((CH2) m O) n (CH2) m -, wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; or a combination thereof.

[0631] 19. The method according to item 13, wherein the antimitotic agent is maytansine, calicheamicin, pyrrolobenzodiazepines, indolebenzodiazepines or auristatin.

[0632] 20. The method according to item 19, wherein the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE).

[0633] 21. The method according to any one of items 1-20, wherein the linker of the ADC is N-β-maleimidopropionyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB).

[0634] 22. The method according to any one of items 1-5, wherein the ADC is represented by any one of the following structures:

[0635]

[0636] 23. The method according to any one of items 1-5, wherein the ADC is represented by:

[0637]

[0638]

[0639] 24. A method according to any one of items 1-23, wherein the ADC has a serum half-life of 3 days or less.

[0640] 25. A method according to any one of items 1-24, wherein the extracellular domain comprises a scFv antibody or a single-chain T cell receptor (scTCR).

[0641] 26. A method according to any one of items 1-24, wherein the extracellular domain comprises a non-immunoglobulin scaffold protein.

[0642] 27. A method according to any one of items 1-26, wherein the extracellular domain of the CAR binds to a tumor antigen selected from the group consisting of: CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28.

[0643] 28. A method according to any one of items 1-27, wherein the cytoplasmic signaling domain of the CAR includes a CD28 cytoplasmic signaling domain, a CD3δ cytoplasmic signaling domain, an OX40 cytoplasmic signaling domain and / or a CD137 cytoplasmic signaling domain.

[0644] 29. A method according to any one of items 1-28, wherein the cytoplasmic signaling domain of the CAR includes a CD3δ cytoplasmic signaling domain.

[0645] 30. The method according to item 9, wherein the cancer is leukemia, adult advanced cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple-negative breast cancer, hematopoietic / lymphoid cancer, colon cancer liver metastasis, small cell lung cancer, non-small cell lung cancer, B cell lymphoma, relapsed or refractory B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, relapsed or refractory diffuse large cell Lymphoma, anaplastic large cell lymphoma, primary mediastinal B-cell lymphoma, relapsed mediastinal large B-cell lymphoma, refractory mediastinal large B-cell lymphoma, large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, colorectal epithelial carcinoma, gastric cancer, pancreatic cancer, triple-negative invasive breast cancer, renal cell carcinoma, Squamous cell lung carcinoma, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, multiple myeloma of bone, malignant glioma of brain, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasms, blastic plasmacytoid dendritic cell neoplasm, liver metastasis, solid tumors, advanced solid tumors, mesothelin-positive tumors, hematological malignancies or other advanced malignancies.

[0646] Other Implementations

[0647] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0648] Although the present invention has been described in conjunction with specific embodiments thereof, it will be appreciated that the present invention is capable of further modification and that this application is intended to cover any changes, uses or modifications of the present invention which generally follow the principles of the present invention and include such deviations from the present invention as are known or within customary practice in the field to which the present invention pertains and which may be applied to the essential features set forth hereinabove and which fall within the scope of the claims of this application.

[0649] Other embodiments are within the scope of the following claims.

Claims

1. Use of an anti-CD137 antibody drug conjugate (ADC) in the preparation of a composition for treating or preventing rejection of allogeneic cells transplanted into a human subject, wherein the anti-CD137 ADC comprises an anti-CD137 antibody or an antigen-binding fragment thereof conjugated to amanitin via a linker, wherein when the composition is used, (a) a first amount of allogeneic cells is administered to the human subject, wherein the first amount is sufficient to elicit a sensitization response to the allogeneic cells in the human subject; (b) the anti-CD137 ADC is administered to the human subject such that endogenous CD137+ activated T cells are depleted; and (c) administering to the human subject a therapeutically effective amount of an allogeneic cell expressing a chimeric antigen receptor (CAR), wherein the allogeneic cell is of the same type as the allogeneic cell administered in (a), and wherein the CAR comprises an extracellular domain that binds to a tumor antigen, a transmembrane domain, and a cytoplasmic signaling domain; such that allogeneic cell rejection in said human subject is treated or prevented, wherein the anti-CD137 antibody or antigen-binding fragment thereof conjugated to amanitin is represented by the formula Ab-ZL-Am, wherein Ab is the antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is amanitin; Wherein the ADC is represented by any of the following structures: The heavy chain amino acid sequence of the anti-CD137 antibody is listed in SEQ ID NO:11, and the light chain amino acid sequence of the anti-CD137 antibody is listed in SEQ ID NO:

12.

2. The use according to claim 1, wherein the first amount of allogeneic cells is administered to the human subject between 48 hours and 7 days prior to step (b).

3. The use according to claim 1, wherein the therapeutically effective amount of allogeneic cells expressing CAR is administered to the human subject between 24 hours and 14 days after step (b).

4. Use of an anti-CD137 antibody drug conjugate (ADC) in the preparation of a composition for treating or preventing rejection of allogeneic cells transplanted into a human subject, wherein the anti-CD137 ADC comprises an anti-CD137 antibody or an antigen-binding fragment thereof conjugated to amanitin via a linker, wherein when the composition is used, (a) the anti-CD137 ADC is administered to the human subject, and wherein the human subject is characterized by having activated T cells against allogeneic cells previously administered to the human subject; and (b) a therapeutically effective amount of an allogeneic cell expressing a chimeric antigen receptor (CAR) is administered to the human subject, wherein the allogeneic cell is of the same type as the allogeneic cell described in (a), and wherein the CAR comprises an extracellular domain that binds to a tumor antigen, a transmembrane domain, and a cytoplasmic signaling domain, such that rejection of the transplanted allogeneic cells in the human subject is treated or prevented, wherein the anti-CD137 antibody or antigen-binding fragment thereof conjugated to amanitin is represented by the formula Ab-ZL-Am, wherein Ab is the antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is amanitin; Wherein the ADC is represented by any of the following structures: Wherein the anti-CD137 antibody is a chimeric BBK2 antibody, the heavy chain amino acid sequence of the chimeric BBK2 antibody is listed in SEQ ID NO:11, and the light chain amino acid sequence of the chimeric BBK2 antibody is listed in SEQ ID NO:

12.

5. The use according to claim 4, wherein the effective amount of allogeneic cells expressing CAR is administered to the human subject between 24 hours and 14 days after step (a).

6. The use according to claim 1 or claim 4, wherein the allogeneic cells are allogeneic T cells or allogeneic NK cells.

7. The use according to claim 1 or claim 4, wherein the effective amount of the allogeneic cells expressing CAR is 2×10 6 cells / kg to 3.0×10 8 cells / kg.

8. The use of claim 1 or claim 4, wherein the allogeneic cell rejection is characterized by cytokine release syndrome (CRS).

9. The use of claim 1 or claim 4, wherein the human subject has cancer or an autoimmune disease.

10. The use according to claim 1 or claim 4, wherein the ADC has a serum half-life of 3 days or less.

11. The use according to claim 1 or claim 4, wherein the extracellular domain comprises a single-chain Fv (scFv) antibody or a single-chain T cell receptor (scTCR).

12. The use of claim 1 or claim 4, wherein the extracellular domain comprises a non-immunoglobulin scaffold protein.

13. The use according to claim 1 or claim 4, wherein the extracellular domain of the CAR binds to a tumor antigen selected from the group consisting of: CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR, MG7, TACI, CEA, PSCA, HER2, CD33, ROR2, NKR-2, CD28, TAA, NKG2D or CD123.

14. The use according to claim 13, wherein the EGFR is EGFRvIII.

15. The use according to claim 1 or claim 4, wherein the cytoplasmic signaling domain of the CAR comprises a CD28 cytoplasmic signaling domain, a CD3ζ cytoplasmic signaling domain, an OX40 cytoplasmic signaling domain and / or a CD137 cytoplasmic signaling domain.

16. The use according to claim 1 or claim 4, wherein the cytoplasmic signaling domain of the CAR comprises a CD3ζ cytoplasmic signaling domain.

17. The method of claim 9, wherein the cancer is leukemia, pancreatic cancer, colorectal cancer, ovarian cancer, triple-negative breast cancer, hematopoietic / lymphoid cancer, liver metastasis of colon cancer, small cell lung cancer, non-small cell lung cancer, B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, anaplastic large cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, gastric cancer, renal cell carcinoma, squamous cell carcinoma of the lung, hepatocellular carcinoma, urothelial carcinoma, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, malignant glioma of the brain, myelodysplastic syndrome, glioblastoma multiforme, blastic plasmacytoid dendritic cell tumor, mesothelin-positive tumor.

18. The use according to claim 9, wherein the cancer is unresectable pancreatic cancer, metastatic colorectal cancer, relapsed or refractory B-cell lymphoma, relapsed or refractory diffuse large cell lymphoma, primary mediastinal B-cell lymphoma, relapsed or refractory non-Hodgkin's lymphoma, triple-negative invasive breast cancer, B-cell leukemia, prolymphocytic leukemia, acute myeloid leukemia or relapsed or refractory multiple myeloma.

19. The use according to claim 9, wherein the cancer is large B-cell lymphoma, refractory aggressive non-Hodgkin's lymphoma, colorectal epithelial carcinoma, B-cell acute lymphoblastic leukemia or multiple myeloma of bone.

20. The use according to claim 9, wherein the cancer is relapsed mediastinal large B-cell lymphoma, refractory mediastinal large B-cell lymphoma or B-cell non-Hodgkin's lymphoma.

21. The use according to claim 9, wherein the cancer is B-cell acute lymphoblastic leukemia, B-cell prolymphocytic leukemia or EGFR-positive colorectal cancer.

22. The use according to claim 9, wherein the cancer is acute leukemia.

23. The use according to claim 9, wherein the cancer is acute lymphoblastic leukemia, acute lymphocytic leukemia or chronic lymphocytic leukemia.

24. The use according to claim 9, wherein the cancer is adult acute lymphoblastic leukemia or childhood acute lymphoblastic leukemia.

25. The use according to claim 9, wherein the cancer is refractory childhood acute lymphoblastic leukemia.

26. The use according to claim 9, wherein the cancer is an adult advanced cancer.

27. The use according to claim 9, wherein the cancer is a solid tumor.

28. The use according to claim 9, wherein the cancer is an advanced solid tumor.

29. The use according to claim 9, wherein the cancer is a hematological malignancy.

30. The use according to claim 1 or claim 4, wherein the sensitizing dose of the allogeneic cells is at least 1×10 5 cells / kg to 1×10 10 cells / kg, and the therapeutically effective amount of the allogeneic cells comprises 1×10 12 cells / kg to 1×10 14 cells / kg.

Images