Use of Anti-CD137 Antibody Drug Conjugates (ADCs) in Allogeneic Cell Therapy

Abstract

The present invention provides methods of using allogeneic cells in therapy by combining allogeneic cell therapy with anti-CD137 antibody drug conjugates (ADCs). Methods of treating or preventing host versus graft (HvG) rejection in a human subject receiving allogeneic cell therapy by administering an anti-CD137 ADC to the human subject are disclosed.

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 62 / 702,292, filed July 23, 2018, the contents of which are incorporated herein by reference.

[0002] [Technical field] The present invention provides methods for treating or preventing rejection of allogeneic cell therapy (chimeric antigen receptor (CAR) cell therapy) in a human subject by administering an antibody-drug conjugate (ADC) that binds to an antigen expressed by cells of the host's immune system that are responsible for mediating rejection of the cell therapy. [Background technology]

[0003] Allogeneic cell therapy involves transplanting cells into a patient, the cells being derived from a donor other than the patient. Common types of allogeneic donors used in allogeneic cell therapy include HLA-matched siblings, matched but unrelated donors, partially matched family member donors, related cord blood donors, and unrelated cord blood donors. The ultimate goal in cell therapy is to identify allogeneic cell therapies that can form the basis of "off-the-shelf" products (Brandenberger, et al. (2011). BioProcess International. 9 (suppl. I): 30-37), which will expand the use of allogeneic cell therapy.

[0004] One therapeutic area with great potential for allogeneic cell therapy is chimeric antigen receptor (CAR) therapy. CAR therapy is an immunological treatment that harnesses the body's own immune system to destroy cells expressing specific antigens associated with a certain disease, such as cancer. For example, in cancer, CAR therapy harnesses and strengthens the patient's immune system to attack the cancer. CAR therapy is based on immune cells, such as T-cells, that express CARs, which are typically transmembrane fusion proteins that combine an extracellular antigen-binding domain (such as an scFv) with a cytoplasmic active signaling and "co-stimulatory" domain that signals the cell from a surface receptor. Thus, when an immune cell, such as a T-cell, expresses a CAR, the immune cell can recognize and kill cells that express the antigen (e.g., a tumor-associated antigen) targeted by the antigen-binding domain of the CAR (Geyer and Brentjens (2016) Cytotherapy 18(11): 1393-1409).

[0005] To date, approved CAR therapy relies on obtaining autologous cells from the patient and transforming the patient's own cells into CAR-expressing cells, which are then used for treatment.However, CAR therapy has the challenge of obtaining autologous cells, as they often require separate protocols, and the time and cost associated with obtaining autologous cells.If allogeneic cells can be used, this will reduce the time and cost associated with CAR therapy.

[0006] Despite their promise, the therapeutic use of allogeneic cells currently suffers from complications that make this treatment difficult. In immunocompetent hosts, transplanted allogeneic cells are rapidly rejected, a process called host versus graft rejection (HvG). HvG can substantially reduce the efficacy of transferred cells and can cause adverse events in the recipient, limiting the use of allogeneic cells. Methods that can overcome HvG rejection remain necessary to further enhance the potential of allogeneic cell therapy. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides methods for preventing or treating rejection of transplanted allogeneic cells in a human subject. In particular, the methods disclosed herein may be used to treat or prevent host versus graft (HvG) in patients undergoing allogeneic cell therapy. The present invention features a method of administering an anti-CD137 antibody-drug conjugate (ADC) to reduce the population of immune cells, such as activated T cells, expressing CD137 in the patient to facilitate administration of the allogeneic cell therapy. Selectively reducing CD137+ cells (e.g., activated T cells) of the patient's immune system prevents or significantly reduces the risk of rejection of the cell therapy.

[0008] In a first aspect, the invention features a method for treating or preventing rejection of allogeneic cells transplanted into a human subject, the method comprising: (a) administering to the human subject a first amount of allogeneic cells, wherein the first amount is sufficient to induce a priming response to the allogeneic cells in the human subject; (b) administering to the human subject an anti-CD137 antibody drug conjugate (ADC), wherein the anti-CD137 ADC comprises an anti-CD137 antibody, or an antigen-binding fragment thereof, conjugated to a cytotoxin via a linker, to treat or prevent rejection of the allogeneic cells in the human subject; (c) administering to the human subject an anti-CD137 antibody drug conjugate (ADC) to deplete endogenous CD137+ activated T cells, wherein the anti-CD137 ADC comprises an anti-CD137 antibody, or an antigen-binding fragment thereof, conjugated to a cytotoxin via a linker; administering to the human subject a therapeutically effective amount of allogeneic cells expressing a CAR, 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 an intracellular signaling domain. In one embodiment, the method comprises administering to the human subject the first amount of allogeneic cells about 48 hours to about 7 days prior to step (b). In another embodiment, the method comprises administering to the human subject a therapeutically effective amount of allogeneic cells expressing a CAR about 24 hours to about 14 days after step (b).

[0009] In another aspect, the invention features a method for treating or preventing rejection of allogeneic cells transplanted into a human subject, the method comprising: (a) administering to the human subject an anti-CD137 antibody drug conjugate (ADC), 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 has activated T cells against allogeneic cells previously administered to the human subject; and (b) administering to the human subject a therapeutically effective amount of allogeneic cells expressing a CAR, wherein the allogeneic cells are of the same type as the allogeneic cells described in (a), and wherein the CAR comprises an extracellular domain that binds to a tumor antigen, a transmembrane domain, and an intracellular signaling domain, so as to treat or prevent rejection of the allogeneic cells in the human subject. In one embodiment, the method includes administering to the human subject a therapeutically effective amount of allogeneic cells expressing the CAR about 24 hours to about 14 days after step (a).

[0010] In certain embodiments, the allogeneic cells are allogeneic T cells or allogeneic NK cells. In other embodiments, the therapeutically effective amount of allogeneic cells expressing the CAR is about 2×10 6 ~Approx. 3.0×10 8 cells / kg. In yet another embodiment, the rejection of the allogeneic cells is characterized by cytokine release syndrome (CRS). In another embodiment, the human subject has cancer or an autoimmune disease. In another embodiment, 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 set forth in SEQ ID NOs: 19, 20, and 21, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively. In another embodiment, the anti-CD137 antibody or antigen-binding fragment thereof is chimerized or humanized.

[0011] In yet other embodiments, the anti-CD137 antibody or antigen-binding fragment thereof is an IgG1 or IgG4 isotype, hi other embodiments, the cytotoxin is an antimitotic agent or an RNA polymerase inhibitor.

[0012] In other embodiments, the cytotoxin is a maytansine, a calicheamicin, a pyrrolobenzodiazepine, an indolinobenzodiazepine, or an auristatin. In some embodiments, the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE). In some embodiments, the cytotoxin is a maytansine. In some embodiments, the cytotoxin is a pyrrolobenzodiazepine (PBD). For example, in some embodiments, the PBD may be selected from tesirine or taliline. In some embodiments, the cytotoxin may be a calicheamicin. For example, in some embodiments, the calicheamicin may be ozogamicin.

[0013] In another embodiment, the RNA polymerase inhibitor is an amatoxin. In yet another embodiment, the RNA polymerase inhibitor is amanitin. In a further embodiment, the amanitin is selected from the group consisting of α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, and proamanuline. In yet another embodiment, the anti-CD137 ADC is represented by the formula Ab-ZL-Am, where 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 amatoxin. In certain embodiments, the linker-amatoxin conjugate Am-LZ is represented by the formula (III): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R5, R 6、 and R7 are each independently H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , N.H.R. C , or NR C R D is; R9 is H, OH, OR C OR D is; Q is -S-, -S(O)-, or -SO2-; R C is -LZ; R D 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, or an optionally substituted heteroaryl; L 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; or comprises a dipeptide; or -((CH2) m O) n (CH2) m where m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; or combinations thereof; and Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an anti-CD137 antibody, or antigen-binding fragment thereof.

[0014] In a particular embodiment, the linker-amatoxin conjugate Am-LZ is represented by formula (III): [ka] where R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R5, R 6、 and R7 are each independently H, OH, OR C , OR D , R C , or RD is; R8 is OH, NH2, OR C , OR D , N.H.R. C , or NR C R D is; R9 is H, OH, OR C OR D is; Q is -S-, -S(O)-, or -SO2-; R C is -LZ; R D 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, or an optionally substituted heteroaryl; L is a linker; and Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an anti-CD137 antibody, or antigen-binding fragment thereof.

[0015] In certain embodiments of Formula (III), L is a linker that comprises a peptide.

[0016] In some embodiments, the linker is one or more of 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)-, -(CH2CHO) 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 combinations thereof.

[0017] In some embodiments, the linker is ((CH2) m O) n (CH2) m - group and a heteroaryl group, where the heteroaryl group is triazole. In some embodiments, ((CH2) m O) n (CH2) m The - group and the triazole together include [ka] where n is 1 to 10, and the wavy line indicates the position of attachment to a further linker moiety, chemical substructure Z, or amatoxin.

[0018] In some embodiments, Am is exactly one R C Contains substituents.

[0019] In another embodiment, the antimitotic agent is a maytansine, a calicheamicin, a pyrrolobenzodiazepine, an indolinobenzodiazepine, or an auristatin, hi another embodiment, the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE).

[0020] In certain embodiments, the linker of the ADC is N-beta-maleimidopropionyl-Val-Ala-para-aminobenzyl (BMP-Val-Ala-PAB). In certain embodiments, the linker of the ADC is N-beta-maleimidopropionyl-Val-Ala-para-aminobenzyl (BMP-Val-Ala-PAB). In some embodiments, the linker L and the chemical moiety Z, taken together as LZ, are [ka] Here, S is a sulfur atom representing a reactive substituent present in an antibody or antigen-binding fragment thereof that binds to CD5 (eg, resulting from the -SH group of a cysteine ​​residue).

[0021] In some embodiments, the LZ is [ka] It is.

[0022] In one embodiment, the ADC is represented by any one of the following structures: [ka] .

[0023] In one embodiment, the ADC is represented by any one of the following structures: [ka] or [ka] 。

[0024] In another embodiment, the ADC has a serum half-life of 3 days or less. In another embodiment, the extracellular domain is an scFv antibody or a single chain T cell receptor (scTCR). In yet another embodiment, the extracellular domain comprises a non-immunoglobulin scaffold protein. In another embodiment, 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, TAA, NKG2D, or CD123. In other embodiments, the intracellular signaling domain of the CAR comprises a CD28 intracellular signaling domain, a CD3 zeta intracellular signaling domain, an OX40 intracellular signaling domain, and / or a CD137 intracellular signaling domain. In yet another embodiment, the intracellular signaling domain of the CAR comprises a CD3 zeta intracellular signaling domain. In certain embodiments, the cancer is leukemia, advanced adult cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple-negative breast cancer, hematopoietic / lymphatic 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, recurrent mediastinal, refractory mediastinal large B-cell lymphoma, large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, recurrent or refractory non-Hodgkin's lymphoma, refractory progressive non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, refractory non-Hodgkin's lymphoma, colorectal carcinoma, gastric carcinoma, pancreatic carcinoma, triple-negative invasive breast cancerbreast carcinoma), renal cell carcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphocytic leukemia, B-cell acute lymphoblastic leukemia leukemia), adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia leukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, recurrent 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, neoplasm, blastic plasmacytoid dendritic cell neoplasm, liver metastasis, solid tumor, advanced solid tumor, mesothelin positive tumor tumor, hematologic malignancy, or other advanced malignancy. [Brief description of the drawings]

[0025] [Figure 1]Figures 1A and 1B graphically show the results of an in vitro T cell cytotoxicity assay including anti-CD137-amanitin ADCs (i.e., "CD137-amanitin") and anti-T cell specific-amanitin ADCs (i.e., "anti-T cell-amanitin") compared to a negative control (i.e., "hIgG-amanitin"). The results show the number of viable activated cells (Figure 1A) or viable non-activated cells (Figure 1B) (y-axis) for each ADC versus ADC concentration (x-axis). [Diagram 2] FIG. 2 graphically depicts the results of an in vivo assay comparing mouse survival (y-axis) versus days post-implantation (x-axis) for anti-CD137-amanitin ADC compared to control PBS, anti-CD137 antibody (naked), and isotype-amanitin in a xeno-GvHD model in NSG mice. [Diagram 3] FIG. 3 graphically depicts the results of an in vivo assay comparing the number of human T cells detected in peripheral blood (y-axis) versus days post-transplant (x-axis) for anti-CD137-amanitin ADC compared to control PBS, anti-CD137 antibody (naked), and isotype-amanitin in a xeno-GvHD model in NSG mice. [Figure 4] Figures 4A and 4B graphically depict the results of in vivo assays measuring engraftment rate (Figure 4A) and T cell frequency (Figure 4B) in the humanized NSG-SGM3 mouse model, where engraftment and T cell frequency were measured at days 5, 9, 14, 22, and 27 post-implantation for the anti-CD137-amanitin ADC compared to control PBS, anti-CD137-BBK antibody (naked), and isotype-amanitin and anti-T cell-amanitin ADC. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] [Detailed Description] Disclosed herein are methods of treating or preventing rejection of transplanted allogeneic cells in a human subject (i.e., treating or preventing host versus graft (HvG) disease). The methods disclosed herein include administering both an anti-CD137 antibody drug conjugate (ADC), which binds to endogenous CD137+ immune cells (e.g., activated T cells), and an allogeneic cell therapy. Administering an anti-CD137 ADC to a human undergoing an allogeneic cell transplant depletes endogenous CD137+ T cells, reducing the risk of a response by endogenous cells to the allogeneic cell therapy. The methods disclosed herein may be used in combination with allogeneic chimeric antigen receptor (CAR) therapy, as the combination of anti-CD137 ADCs and allogeneic cells expressing CARs provides for acceptance of the allogeneic cells in the recipient patient and reduces the risk of host-versus-graft (HvG) reactions that can render CAR-expressing cells ineffective.

[0027] I. Definition As used herein, the term "about" refers to a value 5% above or below the stated value.

[0028] 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 the recipient are different individuals of the same species.

[0029] As used herein, the term "autologous" refers to cells or grafts where the donor and recipient are the same subject.

[0030] As used herein, the term "xenogeneic" refers to cells where the donor and recipient species are different.

[0031] As used herein, the term "immune cells" is intended to include, but is not limited to, cells that are of hematopoietic origin and play a role in the 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 certain embodiments, natural killer cells include cell lines such as NK-92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells. Immune cells can be allogeneic or autologous.

[0032] "Engineered cell" refers to any cell of any organism that has been modified, transformed, or treated by the addition or modification of a gene, DNA or RNA sequence, or a protein or polypeptide. The isolated cells, host cells, and engineered cells of the present disclosure include isolated immune cells, such as NK cells and T cells, that contain a DNA or RNA sequence encoding a CAR and express the CAR on the cell surface. The isolated host cells and engineered cells may be used, for example, to enhance NK cell activity or T lymphocyte activity, treat cancer, and treat autoimmune diseases. In one embodiment, the engineered cells include immune cells, such as T-cells or natural killer (NK) cells.

[0033] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen. Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0034] Generally, an antibody comprises a heavy chain and a light chain, which contain an antigen-binding region. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability (called complementarity determining regions (CDRs)), interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of 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 the binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0035] 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. The 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 within 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) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an 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) an isolated complementarity determining region (CDR); and (ix) a combination of two or more (e.g., 2, 3, 4, 5, or 6) isolated CDRs, optionally linked by a synthetic linker. Furthermore, although the two domains of an Fv fragment (VL and VH) are encoded by separate genes, they can be joined using recombinant methods with a linker to result in a single protein chain (wherein the VL and VH regions pair to form a monovalent molecule) (known as a 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 of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies.Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis procedures known in the art.

[0036] 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.

[0037] As used herein, the term "anti-CD137 antibody" or "antibody that binds to CD137" or "anti-CD137 ADC" or "ADC that binds CD137" refers to an antibody or ADC that specifically binds to human CD137 when CD137 is found on the cell surface of a cell, such as a T cell. The amino acid sequence of the extracellular domain of human CD137 is set forth below as SEQ ID NO: 1. [ka]

[0038] The term "specifically binds" as used herein refers to the ability of an antibody (or ADC) to recognize and bind to a specific protein structure (epitope), rather than proteins in general. If an antibody is specific for epitope "A", then in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody. For example, an antibody (if labeled) "specifically binds" to a target if it can be competed away from its target by a corresponding unlabeled antibody. In one embodiment, an antibody binds to a target (e.g., CD137) at least about 10 times the amount of the target that the antibody binds to. -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 less (less than 10 -12 means a number less than, e.g. 10 -13 ) for the target D In one embodiment, the term "specific binding to CD137" or "specifically binds to CD137" as used herein refers to a specific antibody that binds to CD137 and has a specific binding activity of 1.0×10 or more as measured by surface plasmon resonance. -7 The dissociation constant (K D In one embodiment, the antibody has the following structure: D is measured according to standard Bio-Layer Interferometry (BLI). However, it is understood that the antibody may specifically bind to more than one antigen that is related in sequence. For example, in one embodiment, an antibody may specifically bind to both human and non-human (e.g., mouse or non-human primate) orthologs of CD137.

[0039] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies may be obtained from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art. Monoclonal antibodies useful with the present disclosure can be prepared using a wide variety of techniques known in the art, including using hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0040] The term "chimeric" antibody, as used herein, refers to an antibody having variable sequences derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region, typically 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; US Pat. Nos. 5,807,715; 4,816,567; and 4,816,397.

[0041] As used herein, the term "half-life" refers to the time it takes for the plasma concentration of an antibody drug in the body to decrease by half or by 50%. This 50% decrease in serum concentration reflects the amount of drug in circulation.

[0042] As used herein, the term "host-versus-graft disease" or "HvG" refers to transplant rejection in which allogeneic cells, tissues, or organs are rejected by the recipient's immune system.

[0043] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins that contain minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody will contain substantially all of at least one, and typically two, variable domains, in which 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 from human immunoglobulin sequences. The humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. For example, Riechmann et al., 1988, Nature 332:323-7; US Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 to Queen et al.; EP239400; PCT Publication WO 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 US Pat. No. 5,565,332.

[0044] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide that includes at least one extracellular domain capable of specifically binding to an antigen, a transmembrane domain, and at least one intracellular signaling domain. In general, CARs are engineered receptors that direct the cytotoxicity of immune effector cells to cells that present a given antigen. CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., a tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits specific cellular immune activity. In certain embodiments, a CAR includes an extracellular domain (also called a binding domain or an antigen-specific binding domain), a transmembrane domain, and an intracellular (cytoplasmic) signaling domain. When the antigen-binding domain of the CAR binds to a target antigen on the surface of a target cell, the CAR clusters and delivers an activating stimulus to the CAR-containing cell. A key feature of CARs is their ability to switch the specificity of immune effector cells, thereby triggering the production of molecules that can mediate proliferation, cytokine production, phagocytosis, or major histocompatibility (MHC)-independent cell death of cells expressing a target antigen, utilizing the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors. In some embodiments, a CAR comprises an extracellular binding domain that specifically binds a tumor antigen; a transmembrane domain; and one or more intracellular signaling domains. In various embodiments, a CAR comprises an extracellular binding domain that specifically binds human CD137; a transmembrane domain; and one or more intracellular signaling domains.

[0045] As used herein, the term "CAR therapy" refers to the administration of immune cells engineered to express a CAR to a human subject to treat a given disease (e.g., cancer or autoimmune disease). CAR therapy refers to the use of engineered immune cells to specifically treat the patient, and is not intended to include therapies commonly used in conjunction with CAR cell therapy (e.g., lymphodepleting chemotherapy). In particular, when the term "cell" is used throughout, a population of cells is also included in this term unless otherwise specified. For example, CAR therapy requires the administration of a population of engineered cells.

[0046] 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 a combined drug composition. For example, described herein is a combination therapy that includes administering an anti-CD137 ADC and an allogeneic CAR therapy.

[0047] The term "priming response" as used herein refers to an immune response elicited in a human subject when the subject's immune system is first presented with an antigen. The initial contact of T or B cells with a specific antigen is called priming, resulting in activated allo-antigen T cells. A priming dose can be a single dose or multiple doses required to elicit a response. A "priming amount" is the amount of antigen (e.g., allogeneic cells) used to elicit a priming response. A priming amount is generally not a therapeutically effective amount, but rather an amount used to activate a subject's T cells. In some embodiments, the priming amount of allogeneic cells is less than a therapeutically effective amount of allogeneic cells. In certain embodiments, a priming response is elicited upon administration of allogeneic cells to a human subject, resulting in activation of CD137+ T cells.

[0048] The term "deplete," in the context of the effect of an anti-CD137 antibody or ADC on CD137-expressing cells, refers to reducing in number or eliminating CD137-expressing cells.

[0049] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to achieve a desired result or to have an effect against an autoimmune disease or cancer.

[0050] As used herein, the terms "subject" and "patient" refer to an organism, such as a human, that undergoes treatment for a particular disease or condition as described herein.

[0051] As used herein, "treat" or "treatment" refers to any improvement in any outcome of a disease (e.g., increased survival, less morbidity, and / or reduced side effects that are a by-product of alternative treatment modalities); as will be readily appreciated in the art, complete eradication of a disease is preferred, but is not a prerequisite for therapeutic intervention. Beneficial or desired clinical outcomes include, but are not limited to, promoting the reception of immune cells expressing allogeneic CARs. To the extent that the method of the present invention is directed to preventing a disorder, it is understood that the term "prevent" does not require that the pathology be completely prevented. Rather, as used herein, the term prevent refers to the ability of one of skill in the art to identify a population susceptible to a disorder, such that a compound of the present invention may be administered before the disease develops. The term does not imply a complete avoidance of the pathology.

[0052] 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 include polynucleotide sequences and additional sequence elements used, for example, to express proteins and / or to integrate these polynucleotide sequences into the genome of a mammalian cell. Particular vectors that can be used to express a CAR include plasmids that include regulatory sequences (e.g., promoter and enhancer regions, etc.) that direct gene transcription. Other useful vectors for expressing antibodies or CARs include polynucleotide sequences that increase the translation efficiency of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements may include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to ensure efficient transcription of genes present on the expression vector. The expression vectors described herein may also include a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.

[0053] As used herein, the term "antibody drug conjugate" or "ADC" refers to an antibody linked to a cytotoxin. An ADC is formed by chemical conjugation of a reactive functional group of one molecule (e.g., an antibody or an antigen-binding fragment thereof) with a suitable reactive functional group of another molecule (e.g., a cytotoxin as described herein). A conjugate may include a linker between the two molecules bound to each other (e.g., between the antibody and the cytotoxin). In particular, the term "conjugate" (when referring to a compound) is also referred to herein interchangeably as "drug conjugate," "antibody drug conjugate," or "ADC." Examples of linkers that can be used to form conjugates include peptide-containing linkers (e.g., linkers containing naturally occurring or non-naturally occurring amino acids, e.g., D-amino acids). Linkers may be prepared using a variety of strategies, as described herein and known in the art. Depending on the reactive moieties therein, the linker may be cleaved, for example, by 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).

[0054] 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 joins (e.g., covalently) the molecular fragments attached to the respective substituents. Coupling reactions include reactions in which a reactive substituent attached to a fragment that is a cytotoxin (e.g., a cytotoxin known in the art or described herein) reacts with a suitable reactive substituent attached to a fragment that is an antibody, or an antigen-binding fragment thereof (e.g., an antibody, antigen-binding fragment thereof, or a specific anti-CD137 antibody that binds to CD137, as known in the art or described herein). Examples of suitable reactive substituents include nucleophilic / electrophilic pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, among others), diene / dienophile pairs (e.g., azide / alkyne pairs, among others), and the like. Coupling reactions include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine condensation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, among others), aromatic nucleophilic substitution, aromatic electrophilic substitution, and other reaction 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 cellular functions in cells. Examples of microtubule binding agents include, but are not limited to, maytasine, maytansinoids, 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, cochicine, and epothilones, and derivatives thereof (such as epothilone B or derivatives thereof).

[0055] As used herein, the term "amatoxin" refers to a member of the amatoxin family of peptides produced by Amanita phalloides mushrooms, or a derivative thereof (e.g., a variant or derivative thereof capable of inhibiting RNA polymerase II activity). Amatoxins may be isolated from various mushroom species (e.g., Amanita phalloides, Galerina marginata, Lepiota brunneo-incarnata) or may be prepared semisynthetically or synthetically. A member of this family (α-amanitin) is described in Wieland, Int. J. Pept. Protein Res. 1983, 22(3):257-276. Amatoxin derivatives may be obtained by chemically modifying the naturally occurring compound ("semisynthetic") or may be obtained from fully synthetic sources. Synthetic routes to various amatoxin derivatives are described, for example, in U.S. Pat. No. 9,676,702 and Perrin et al., J. Am. Chem. Soc. 2018, 140, p. 6513-6517, each of which is incorporated by reference in its entirety herein with respect to synthetic methods for preparing and derivatizing amatoxins.

[0056] Amatoxins useful in conjunction with the compositions and methods described herein include, but are not limited to, compounds of formula (II) (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, or proamanutin). As described herein, amatoxins may be conjugated to an antibody, or an antigen-binding fragment thereof (thereby forming an ADC), for example, via a linker moiety (L). Exemplary methods of conjugating amatoxins and linkers useful in such processes are described below. Exemplary amatoxin-linker conjugate structures are represented by formulae (III), (IIIA), and (IIIB). Exemplary linker-containing amatoxins useful for conjugation to an antibody, or an antigen-binding fragment thereof, according to the compositions and methods are also described herein.

[0057] The term "acyl" as used herein refers to -C(=O)R, where R is hydrogen (an "aldehyde"), C1-C2 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C7 carbocyclyl, C6-C 20 aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl. Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, and acryloyl.

[0058] As used herein, the term "C 12 "Alkyl" refers to a straight or branched chain saturated hydrocarbon having 1 to 12 carbon atoms. Typical C1-C 12 Alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while branched C-C 12 Alkyl includes, but is not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl.12 The alkyl group may be unsubstituted or substituted.

[0059] As used herein, the term "alkenyl" refers to an alkyl group having at least one site of unsaturation, i.e., carbon-carbon, sp 2 C2-C containing normal, secondary, or tertiary carbon atoms with double bonds 12 It refers to a hydrocarbon. Examples include, but are not limited to, ethylene or vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, etc. Alkenyl groups can be unsubstituted or substituted.

[0060] As used herein, "alkynyl" refers to a C-C alkyl group containing normal, secondary, or tertiary carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon, sp triple bond. 12 Refers to a hydrocarbon. Examples include, but are not limited to, acetylene and propargyl. Alkynyl groups can be unsubstituted or substituted.

[0061] As used herein, "aryl" refers to any group having a C6-C 20 It refers to a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and anthracenyl. Aryl groups can be unsubstituted or substituted.

[0062] As used herein, "arylalkyl" refers to an alkyl group having a carbon atom (typically a terminal or sp 3A non-cyclic alkyl radical in which one of the hydrogen atoms bonded to the aryl group (carbon atom) is replaced with an aryl radical. Exemplary arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethene-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like. The arylalkyl group contains 6 to 20 carbon atoms, e.g., the alkyl portion of the arylalkyl group (e.g., alkanyl, alkenyl, or alkynyl groups, etc.) is 1 to 6 carbon atoms, and the aryl portion is 5 to 14 carbon atoms. The alkaryl group can be unsubstituted or substituted.

[0063] As used herein, "cycloalkyl" refers to a saturated carbocyclic radical, which may be monocyclic or bicyclic. Cycloalkyl groups include rings having 3 to 7 carbon atoms as a monocycle, or rings having 7 to 12 carbon atoms as a bicycle. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be unsubstituted or substituted.

[0064] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic radical, which may be monocyclic or bicyclic. Cycloalkenyl groups include rings having 3 to 6 carbon atoms as a monocycle, or rings having 7 to 12 carbon atoms as a bicycle. 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. Cycloalkenyl groups may be unsubstituted or substituted.

[0065] As used herein, a "heteroaralkyl" refers to an aryl group having a carbon atom (typically a terminal or sp 3A heteroaryl alkyl group refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to the heteroaryl group (carbon atom) is replaced with a heteroaryl radical. Exemplary heteroaryl alkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroaryl alkyl group contains 6-20 carbon atoms, e.g., the alkyl portion of the heteroaryl alkyl group (including, e.g., alkanyl, alkenyl, or alkynyl groups) is 1-6 carbon atoms, and the heteroaryl portion is 5-14 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S. The heteroaryl portion of the heteroaryl alkyl group can be a monocyclic ring (2-6 carbon atoms) having 3-7 ring members, or a bicyclic ring (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S) having 7-10 ring members, e.g., bicyclic [4,5], [5,5], [5,6], or [6,6] systems.

[0066] As used herein, "heteroaryl" and "heterocycloalkyl" refer to an aromatic or non-aromatic ring system, respectively, in which one or more ring atoms are heteroatoms, such as nitrogen, oxygen, and sulfur. The heteroaryl or heterocycloalkyl radicals contain 2-20 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S. The heteroaryl or heterocycloalkyl can be monocyclic (2-6 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S) having 3-7 ring members, or bicyclic (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S) having 7-10 ring members, such as: bicyclic [4,5], [5,5], [5,6], or [6,6] systems. The heteroaryl and heterocycloalkyl can be unsubstituted or substituted.

[0067] Heteroaryl 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.

[0068] Examples of heteroaryl groups include, but are not limited to, pyridyl, thiazolyl, tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H- Examples include quinolidinyl, 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.

[0069] Examples of heterocycloalkyl include, but are not limited to, dihydropyridyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinuclidinyl, and morpholinyl.

[0070] By way of example and not limitation, carbon-linked heteroaryls and heterocycloalkyls are bonded at the 2-, 3-, 4-, 5-, or 6-position of a pyridine, the 3-, 4-, 5-, or 6-position of a pyridazine, the 2-, 4-, 5-, or 6-position of a pyrimidine, the 2-, 3-, 5-, or 6-position of a pyrazine, the 2-, 3-, 4-, or 5-position of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2-, 4-, or 5-position of an oxazole, imidazole, or thiazole, the 3-, 4-, or 5-position of an isoxazole, pyrazole, or isothiazole, the 2-, or 3-position of an aziridine, the 2-, 3-, or 4-position of an azetidine, the 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of a quinoline, or the 1-, 3-, 4-, 5-, 6-, 7-, or 8-position of an isoquinoline. More typically, carbon-linked heterocyclyl 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.

[0071] By way of example and not limitation, nitrogen-linked heteroaryls and heterocycloalkyls are bonded at the 1-position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole, 2-position of isoindole or isoindoline, 4-position of morpholine, and 9-position of carbazole or beta-carboline. More typically, nitrogen-linked heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0072] As used herein and as 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 with a substituent. Unless otherwise constrained by the definition of individual substituents, the above 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. Exemplary 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)2NH 2, -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)NH 2, -C(=O)N(R)2, -C(=S)NH 2, -C(=S)N(R)2, -C(=NH)NH 2, and -C(=NR)N(R)2; where each X is independently selected at each occurrence from F, Cl, Br, and I; and each R is C1-C 12 Alkyl, C6-C 20 Aryl, C3-C 14 is independently selected at each occurrence from heterocycloalkyl or heteroaryl, a protecting group, and a prodrug moiety. Wherever a group is described as "optionally substituted," that group may be substituted, independently at each occurrence, with one or more of the above substituents.

[0073] It should be understood that a given radical naming convention may include either mono-radicals or di-radicals, depending on the context. For example, if a substituent requires two points of attachment to the remainder of the molecule, the substituent is understood to be a di-radical. For example, a substituent identified as an alkyl, which requires two points of attachment, includes di-radicals such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc. Other radical naming conventions clearly indicate that the radical is a di-radical, such as "alkylene," "alkenylene," "arylene," "heterocycloalkylene," etc.

[0074] Wherever a substituent is shown as a di-radical (i.e., having two points of attachment to the remainder of the molecule), it is understood that said substituent can be attached in any directional orientation unless otherwise indicated.

[0075] "Isomerism" means compounds that have identical molecular formulae but differ in the sequence of bonds of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are called "stereoisomers". Stereoisomers that are not mirror images of one another are called "diastereomers", and stereoisomers that are non-superimposable mirror images of each other are called "enantiomers" or sometimes "optical isomers".

[0076] A carbon atom attached to four non-identical substituents is called a "chiral center." "Chiral isomer" means a compound having at least one chiral center. Compounds having one or more chiral centers may exist as individual diastereomers or as a mixture of diastereomers called a "diastereomeric mixture." When one chiral center is present, a stereoisomer can be characterized by the absolute configuration (R or S) of its chiral center. Absolute configuration refers to the spatial arrangement of the substituents attached to said chiral center. Substituents attached to the chiral center under consideration are ranked according to the sequencing rules of Cahn, Ingold and Prelog. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 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".

[0077] The compounds disclosed in this specification and claims may contain one or more asymmetric centers, and different diastereomers and / or enantiomers of each compound may exist. The description of any compound in this specification and claims is meant to include all enantiomers, diastereomers and mixtures thereof, unless otherwise stated. Furthermore, the description of any compound in this specification and claims is meant to include each individual enantiomer, as well as any mixture of said enantiomers, racemic or otherwise, unless otherwise stated. When a compound structure is depicted as a specific enantiomer, it is to be understood that the invention of this application is not limited to that specific enantiomer. Thus, the enantiomers, optical isomers, and diastereomers of each structural formula of this disclosure are contemplated herein. In this specification, the structural formula of the compound may, for convenience, in some cases, represent a specific isomer, but the present disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., with the understanding that not all isomers have the same level of activity. These compounds may exist in different tautomeric forms. Compounds according to the present disclosure are meant to include all tautomeric forms, unless otherwise stated. When the structure of a compound is depicted as a specific tautomer, it should be understood that the invention of the present application is not limited to that specific tautomer.

[0078] The compounds of any formula described herein include the compounds themselves, as well as their salts and solvates, if applicable. For example, salts may 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, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate and acetate (e.g., trifluoroacetate). The term "pharmaceutically acceptable anion" refers to a suitable anion for forming a pharmaceutically acceptable salt. Similarly, salts may be formed between cations and negatively charged groups (e.g., carboxylate) on the compounds of the present disclosure. Suitable cations include ammonium cations such as sodium, potassium, magnesium, calcium, and tetramethylammonium ions. Some suitable examples of substituted ammonium ions include those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, amino acids such as lysine and arginine. Compounds of the present disclosure also include salts containing quaternary nitrogen atoms.

[0079] Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous. 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, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, gluceptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalene carboxylic 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, phenylsulfonic 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.

[0080] Further, the compounds of the present disclosure (e.g., salts of the compounds) may exist in hydrous or non-hydrous (anhydrous) form, or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, and the like. Non-limiting examples of solvates include ethanol solvates, acetone solvates, and the like. "Solvate" refers to a solvent addition form that contains either a stoichiometric or non-stoichiometric amount of solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid phase, thereby forming a solvate. When the solvent is water, the solvate formed is a hydrate; and when the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of one molecule of a substance with one or more molecules of water, in which the water retains its molecular state as H2O. Hydrates refer to, for example, monohydrates, dihydrates, trihydrates, and the like.

[0081] Furthermore, for the compounds represented by the formulas disclosed herein or salts thereof, crystalline polymorphism may exist. It is noted that any crystalline form, mixture of crystalline forms, or anhydrates or hydrates thereof are included within the scope of the present disclosure.

[0082] The following sections provide a description of ADCs that can be administered to human patients to treat or prevent rejection of allogeneic cell therapy, as well as methods of prescribing such therapy to said patients.

[0083] II. Methods of Allogeneic Cell Therapy In immunocompetent hosts, transplanted allogeneic cells are often quickly rejected. Such rejection is commonly referred to as host-versus-graft (HvG) rejection. One challenge of allogeneic cell therapy is identifying a means by which the human recipient will accept the allogeneic cells. Such acceptance of the allogeneic cells may affect the efficacy of the therapy and may also result in adverse side effects for the patient.

[0084] Described herein are methods for treating or preventing allogeneic cell rejection in a human subject by administering an anti-CD137 antibody drug conjugate (ADC) that targets endogenous CD137+ cells (e.g., activated T cells).

[0085] In certain embodiments, a priming response is first induced in a human subject prior to administration of anti-CD137 ADC. "Priming" the patient promotes the development of activated anti-alloantigen T cells produced following administration of allogeneic cells. Injection of allogeneic cells is desired to stimulate a response of the patient's immune system against the allogeneic cells. To induce a "priming" immune response, a priming amount of allogeneic cells is administered to the human subject.

[0086] In the priming step, allogeneic cells are administered (e.g., intravenously) to induce an immune response that results in endogenous activated CD137+ T cells. Thus, the priming amount of allogeneic cells is preferably an amount effective to induce an immune response that includes activation of endogenous CD137+ T cells. In an exemplary embodiment, an effective priming dose is about 1x10 5 cells / kg, 0.5x10 6 cells / kg, approx. 1x10 6 cells / kg, approx. 1x10 7 cells / kg, approx. 1x10 8 Allogeneic cells / kg, approximately 1x10 9 Allogeneic cells / kg, approximately 1x10 10 Allogeneic cells / kg, approx. 0.5x10 6 ~ approx. 1x10 10 cells / kg, approx. 0.5x10 6 ~ approx. 1x10 8 cells / kg, approx. 1x10 7 ~ approx. 1x10 10 cells / kg, approx. 1x10 8 ~ approx. 1x10 10 cells / kg, or approximately 1x10 9 ~ approx. 1x10 10 In another exemplary embodiment, the priming dose is about 1x10 cells / kg. 5 ~1x10 12 Allogeneic cells / kg, eg, 1x10 5 ~1x10 6 cells / kg, 1x10 5 ~1x10 7 cells / kg, 1x10 5 ~1x10 8 cells / kg, 1x10 5 ~1x10 9 cells / kg, 1x10 5 ~1x10 10 cells / kg, 1x10 5 ~1x10 11 Cells / kg, or 1x10 5 ~1x10 12In another exemplary embodiment, the priming dose of allogeneic cells comprises about 1x10 cells / kg. 6 ~1x10 12 cells / kg, 1x10 7 ~1x10 12 cells / kg, 1x10 8 ~1x10 12 cells / kg, 1x10 9 ~1x10 12 cells / kg, 1x10 10 ~1x10 12 Cells / kg, or 1x10 11 ~1x10 12 Doses intermediate to the above ranges are also contemplated for use in the methods of the invention. Doses of allogeneic cells outside these ranges that are capable of eliciting an immune response are also within the scope of the invention.

[0087] In certain embodiments, the allogeneic cells include, but are not limited to, allogeneic T cells or allogeneic NK cells. In exemplary embodiments, the allogeneic cells express a chimeric antigen receptor, as described herein.

[0088] After administering a priming amount of allogeneic cells, an anti-CD137 antibody-drug conjugate (ADC) is administered to the human subject. CD137 is expressed on activated T cells. Thus, anti-CD137 ADCs are used to reduce the number of activated T cells in the human subject prior to administering allogeneic cell therapy. Exemplary anti-CD137 antibodies, cytotoxins, and linkers suitable for use in the methods described herein are described in detail below.

[0089] After administration of the CD137 ADC, a therapeutically effective amount of allogeneic cells is administered to the human subject. Selectively depleting the activated T cell population prior to administration of the allogeneic cells (e.g., allogeneic cells expressing a CAR) improves the overall efficacy of the allogeneic cell therapy by treating or preventing rejection of the allogeneic cells. A therapeutically effective amount of allogeneic cells can be an amount effective to achieve a therapeutic benefit in a patient. For example, when allogeneic cells are used in a therapy for a subject having cancer, a therapeutically effective amount can be an amount effective to treat the cancer, reduce the size or volume of the cancer, reduce one or more symptoms associated with the cancer, induce remission of the cancer, and / or extend the survival or disease-free survival of a subject having cancer. In one embodiment, the therapeutically effective amount of allogeneic cells is greater than the number of cells administered to the subject during the priming 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 priming step. In another embodiment, the therapeutically effective amount of allogeneic cells is less than the number of cells administered to said subject during the priming step.

[0090] In an exemplary embodiment, the therapeutically effective amount of allogeneic cells is at least 1×10 5 Cells / kg, eg, at least 1x10 6 Cells / kg, at least 1x10 7 Cells / kg, at least 1x10 8 Cells / kg, at least 1x10 9 Cells / kg, at least 1x10 10 Cells / kg, at least 1x10 11 Cells / kg, at least 1x10 12 Cells / kg, at least 1x10 13 Cells / kg, at least 1x10 14 Cells / kg, at least 1x10 15 Cells / kg, at least 1x10 16 Cells / kg, at least 1x10 17In another exemplary embodiment, the therapeutically effective amount of allogeneic cells is up to 1x10 cells / kg, or more. 5 cells / kg, eg, up to 1x10 6 cells / kg, up to 1x10 7 cells / kg, up to 1x10 8 cells / kg, up to 1x10 9 cells / kg, up to 1x10 10 cells / kg, up to 1x10 11 cells / kg, up to 1x10 12 cells / kg, up to 1x10 13 cells / kg, up to 1x10 14 cells / kg, up to 1x10 15 cells / kg, up to 1x10 16 cells / kg, or up to 1x10 17 In another exemplary embodiment, the therapeutically effective amount of allogeneic cells is 1x10 5 ~1x10 17 cells / kg, eg, 1x10 6 ~1x10 17 cells / kg, 1x10 7 ~1x10 17 cells / kg, 1x10 8 ~1x10 17 cells / kg, 1x10 9 ~1x10 17 cells / kg, 1x10 10 ~1x10 17 cells / kg, 1x10 11 ~1x10 17 cells / kg, 1x10 12 ~1x10 17 cells / kg, 1x10 13 ~1x10 17 cells / kg, 1x10 14 ~1x10 17 cells / kg, 1x10 15 ~1x10 17 Cells / kg, or 1x10 16 ~1x10 17 In another exemplary embodiment, the therapeutically effective amount of allogeneic cells is 1x10 5 ~1x10 17cells / kg, eg, 1x10 5 ~1x10 16 cells / kg, 1x10 5 ~1x10 15 cells / kg, 1x10 5 ~1x10 14 cells / kg, 1x10 5 ~1x10 13 cells / kg, 1x10 5 ~1x10 12 cells / kg, 1x10 5 ~1x10 11 cells / kg, 1x10 5 ~1x10 10 cells / kg, 1x10 5 ~1x10 9 cells / kg, 1x10 5 ~1x10 8 cells / kg, 1x10 5 ~1x10 7 Cells / kg, or 1x10 5 ~1x10 6 In another exemplary embodiment, the therapeutically effective amount of allogeneic cells comprises 1×10 8 ~1×10 10 In another exemplary embodiment, the therapeutically effective amount of allogeneic cells is 1×10 10 ~1×10 12 In another exemplary embodiment, the therapeutically effective amount of allogeneic cells is 1×10 12 ~1×10 14 Contains cells / kg.

[0091] The allogeneic cells used in the initial priming step of treatment may be of the same type (or similar) to the allogeneic cells administered to the human subject as part of the allogeneic cell therapy. For example, the allogeneic cells used in the priming step may be derived from the same donor as the cells used in the allogeneic cell therapy. In another embodiment, the allogeneic cells used in the priming step may express the same or similar complement MHC molecules as the cells used in the allogeneic cell therapy. The complement MHC molecules expressed by the priming and / or therapeutic cells may be naturally occurring or may be modified, for example, using genetic engineering. In an embodiment, in an allogeneic CAR cell therapy, allogeneic cells that do not express a CAR are administered to the human subject to elicit a priming response that is the same type as the allogeneic cells used in the CAR cell therapy. Alternatively, the priming response may be elicited with CAR-expressing allogeneic cells that are the same type as the CAR-expressing cells that will be used to treat the patient.

[0092] The risk of rejection of allogeneic cells expressing CAR may be high after CAR cell therapy is prescribed. Anti-CD137 ADC may be used to selectively target activated T cells in patients who are about to receive allogeneic CAR cell therapy. In certain embodiments, the methods disclosed herein may be used to inhibit or prevent rejection of allogeneic cells expressing CAR in human patients.

[0093] The anti-CD137 ADC may be administered to a human patient in need thereof prior to, concurrently with, or after one or more priming doses of allogeneic cell therapy are administered. In certain embodiments, the anti-CD137 ADC is administered to a human patient in need thereof prior to prescribing a priming dose of allogeneic cells. For example, in some embodiments, the anti-CD137 ADC may be administered up to about 10 days prior to prescribing a priming dose of allogeneic cells, e.g., 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 prior to administering the priming dose of allogeneic cells. In some embodiments, an anti-CD137 ADC is administered to a human patient in need thereof about 1 to about 48 hours; about 1 to about 24 hours; about 1 to about 18 hours; about 1 to about 12 hours; about 1 to about 10 hours; about 1 to about 6 hours; about 1 to about 3 hours; about 1 to about 2 hours; about 1 to 2 days; about 1 to 3 days; about 1 to 4 days; about 1 to 7 days; about 1 to 10 days; about 3 to 5 days; about 3 to 7 days; about 3 to 10 days; about 12 hours to about 48 hours; or about 12 hours to about 36 hours prior to administering a priming dose of allogeneic cells.

[0094] In another embodiment, the anti-CD137 ADC is administered to a human patient in need thereof simultaneously with administration of a priming dose of allogeneic cells.

[0095] In other embodiments, the anti-CD137 ADC is administered to a human patient in need thereof after a priming dose of allogeneic cells has been administered. For example, in some embodiments, the anti-CD137 ADC may be administered to a human patient in need thereof 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 after a priming dose of allogeneic cells has been administered. In other embodiments, the anti-CD137 ADC may be administered to a human patient in need thereof about 1 hour to about 10 days, e.g., 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 to about 10 days; about 1 to about 9 days; about 1 to about 8 days; about 1 to about 7 days; about 1 to about 6 days; about 1 to about 5 days; about 1 to about 4 days; about 1 to about 3 days; about 1 to about 2 days, about 3 days to about 10 days; about 5 days to about 7 days; etc., or longer, after the priming dose of allogeneic cells is administered.

[0096] In one embodiment, a single dose of an anti-CD137 ADC is administered to the human patient before, after, or simultaneously with a priming dose of allogeneic cells, where such single dose is sufficient to selectively reduce the population of T cells that are activated in response to administration of allogeneic cells.

[0097] An anti-CD137 ADC is administered to a human patient in need thereof prior to, concurrently with, or after administration of a therapeutically effective amount of allogeneic cells (e.g., allogeneic cells expressing a CAR). In some embodiments, the anti-CD137 ADC is administered prior to administration of allogeneic cells expressing a CAR to a human patient in need thereof. In some embodiments, the allogeneic cell therapy is administered to the patient after the anti-CD137 ADC has been cleared or substantially cleared from the patient's blood. In some embodiments, an anti-CD137 ADC is administered to a human patient in need thereof prior to (e.g., about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 2 days, or about 1 day) administering an allogeneic cell therapy (e.g., CAR cell therapy). In certain embodiments, the anti-CD137 ADC is administered to a human patient in need thereof about 1 to about 14 days; about 2 to about 13 days; about 3 to about 12 days; about 3 to about 11 days; about 4 to about 10 days; about 5 to about 9 days; about 6 to about 8 days; about 1 to about 13 days; about 1 to about 12 days; about 1 to about 11 days; about 1 to about 10 days; about 1 to about 9 days; about 1 to about 8 days; about 1 to about 7 days; about 1 to about 6 days; about 1 to about 5 days; about 1 to about 4 days; about 1 to about 3 days; or about 1 to about 2 days prior to administering the allogeneic cell therapy.

[0098] In some embodiments, the anti-CD137 ADC is administered to the human patient prior to the allogeneic cells expressing a CAR, wherein the anti-CD137 ADC is administered about 12 hours to about 14 days prior to administering the allogeneic cells expressing a CAR to the human subject. In some embodiments, the anti-CD137 ADC is administered to the human patient prior to the allogeneic cells expressing a CAR, wherein the anti-CD137 ADC is administered about 24 hours to about 12 days prior to administering the allogeneic cells expressing a CAR to the human subject. In some embodiments, the anti-CD137 ADC is administered to the human patient prior to the allogeneic cells expressing a CAR, wherein the anti-CD137 ADC is administered about 1 day to about 10 days prior to administering the allogeneic cells expressing a CAR to the human subject. In one embodiment, an anti-CD137 ADC is administered to the human patient prior to the allogeneic cells expressing a CAR, wherein the anti-CD137 ADC is administered to the human subject about 2 days to about 8 days prior to administration of the allogeneic cells expressing a CAR.

[0099] In one embodiment, the anti-CD137 ADC is administered simultaneously with administration of allogeneic cells expressing a CAR to a human patient in need thereof.

[0100] After administration of the anti-CD137 ADC, the human patient may be tested to ensure that CD137+ activated T cells are substantially depleted from the patient before initiating allogeneic CAR therapy. The level of CD137+ activated T cells may be assayed in a biological sample (e.g., blood) from the human patient after administration of the CD137 ADC, and compared to the level of CD137+ activated T cells from a biological sample (e.g., the same source, e.g., blood) before administration of the anti-CD137 ADC (but after allogeneic cell priming). A reduction in the number of endogenous CD137+ activated T cells indicates that the anti-CD137 ADC was effective in reducing the risk of rejection of allogeneic (using the same or substantially similar allogeneic cells) CAR therapy. In some embodiments, the level of endogenous CD137+ activated T cells in a biological sample from the 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%, compared to the level of CD137+ activated T cells in a biological sample (of the same type, e.g., blood) from the human patient immediately prior to administration of the anti-CD137 ADC. In some embodiments, the level of endogenous CD137+ activated T cells is measured no more than 1 day prior to administration of the anti-CD137 ADC.

[0101] Alternatively, the overall level of T cells in a biological sample from a human patient may be assessed after administration of an anti-CD137 ADC (after allogeneic cell priming), where a decrease in the overall number of T cells in the human patient after administration of an anti-CD137 ADC compared to pre-administration levels indicates the effectiveness of the anti-CD137 ADC to prevent rejection of the allogeneic cell therapy. In certain embodiments, the level of endogenous T cells in the biological sample from the human patient is reduced by at least about 5%, at least about 10%, at least about 15%, or at least about 20% compared to the level of T cells in a biological sample (e.g., of the same type, blood) from the human patient immediately prior to administration of the anti-CD137 ADC. In certain embodiments, the level of endogenous T cells in the biological sample from the human patient is reduced by about 5% to 25%, about 5% to 20%, about 5% to 15%, or about 5% to 10% compared to the level of T cells in a biological sample (e.g., blood of the same type) from the human patient immediately prior to administration of the anti-CD137 ADC. In certain embodiments, the level of endogenous T cells is measured no more than one day prior to administration of the anti-CD137 ADC.

[0102] Levels of T cells, including CD137+ activated T cells, may be measured according to standard methods known in the art, including but not limited to in vitro antigen stimulation assays, in which the number of activated alloantigen T cells following exposure to allogeneic cells is compared to the number of activated alloantigen T cells following exposure to allogeneic cells in the presence of anti-CD137 ADC.

[0103] The method disclosed herein is used to deliver allogeneic cells expressing CAR to a subject.Importantly, the anti-CD137 ADC conditioning method described herein is useful for expanding the types of immune cells that can be used in CAR therapy by providing a means by which tolerance of allogeneic cells can be provided.In some embodiments, the allogeneic cells expressing CAR include, but are not limited to, allogeneic T cells or allogeneic NK cells.

[0104] In one embodiment, an anti-CD137 antibody-drug conjugate is used to deplete CD137-expressing T cells (e.g., activated T cells expressing CD137) by administering the anti-CD137 antibody-drug conjugate prior to, simultaneously with, or after administering a priming dose of allogeneic cells.

[0105] The methods disclosed herein are particularly useful for treating cancer or an autoimmune disease in a human subject having one of these conditions.

[0106] In some embodiments, the methods disclosed herein are used to treat cancer. Examples of types of cancer that can be treated using the methods disclosed herein include, but are not limited to, advanced adult cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple-negative breast cancer, hematopoietic / lymphatic 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 intestine, and pulmonary arterial cancer. large cell lymphoma, relapsed or refractory diffuse large cell lymphoma, anaplastic large cell lymphoma, primary mediastinal B cell lymphoma, relapsed mediastinal, refractory mediastinal large B cell lymphoma, large B cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, relapsed or refractory non-Hodgkin's lymphoma, refractory progressive non-Hodgkin's lymphoma, B cell non-Hodgkin's lymphoma, refractory non-Hodgkin's lymphoma, colorectal carcinoma carcinoma, gastric carcinoma, pancreatic carcinoma, triple-negative invasive breast carcinoma, renal cell carcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphocytic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia refractory childhood acute lymphoblastic leukemialeukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, recurrent 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, neoplasm, blastic plasmacytoid dendritic cell neoplasm, liver metastasis, solid tumor, advanced solid tumor, mesothelin positive tumor These include advanced malignancies, hematologic malignancies, and other advanced malignancies.

[0107] In some embodiments, the methods disclosed herein are used to treat autoimmune diseases. More specifically, anti-CD137 ADCs are administered in combination with allogeneic cell therapy (e.g., allogeneic cells expressing CAR) to human subjects with autoimmune diseases. Examples of autoimmune diseases that may be treated using the combination methods disclosed herein include, but are not limited to, multiple sclerosis, Crohn's disease, ulcerative colitis, rheumatoid arthritis, type I diabetes, lupus, and psoriasis.

[0108] In certain embodiments, an anti-CD137 ADC is administered to a human patient in combination with a CAR-T cell therapy, hi some embodiments, the anti-CD137 ADC is administered to the human patient prior to administering the CAR-T therapy.

[0109] Examples of CAR-T constructs that may be expressed in allogeneic cells and used with the methods described 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 Biomedicine Technology Co.); Genechem / NCT02672501 (Shanghai GeneChem Co.); SenL_19 (Hebei Senlang Biotechnology Inc.); PCAR-019 IM19-CD28 and IM19-41BB CAR-T cells (Beijing Immunochina Medical Science & CTL019 / IT1601-CART19 (Beijing Sanwater Biological Technology Co.); 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.); iPD1 CD19 eCAR T cells (Peking University / Marino Biotechnology Co.); 2013-1018 / NCT02529813 (M.D. Anderson Cancer Center / Ziopharm / Intrexon Corp.); HenanCH CAR 2-1 (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 (e.g., 2016040 / NCT03121625 (Hebei Senlang Biotechnology Inc.)); CD22 (e.g., Ruijin-CAR-01 (Ruijin Hospital / Shanghai Unicar-Therapy Bio-medicine Technology Co.); AUTO-PA1,DB1 (Autolus Limited)), CD20 (e.g., Doing-006 (Beijing Doing Biomedical Co.)); or CD20 / CD22 / CD30 (e.g., SZ5601 (The First Affiliated Hospital of Soochow University Shanghai / Unicar-Therapy Bio-medicine Technology Co.)). 。

[0110] Chimeric antigen receptor (CAR) The present invention includes the use of allogeneic cell therapy (e.g., allogeneic CAR cell therapy) in combination with anti-CD137 immunosuppressive ADCs to treat or prevent rejection of allogeneic cells required for therapy. The present invention is based, at least in part, on the discovery that anti-CD137 ADCs may act as conditioning agents for CAR therapy by promoting acceptance of CAR-expressing cells by removing endogenous CD137+ immune cells (e.g., endogenous T cells, etc.), and therefore the present invention is generally not limited to a particular CAR construct (e.g., a particular antigen binding site or intracellular signaling domain). Specific CARs (e.g., CD19-specific CARs) are contemplated herein and included in the methods disclosed herein, but are not meant to be limiting.

[0111] CAR constructs are known in the art and generally comprise (a) an extracellular region comprising an antigen-binding domain, (b) a transmembrane domain, and (c) an intracellular signaling domain. Exemplary CAR configurations are known in the art, and any suitable configuration may be used in the methods described herein. For example, the CAR may be a first-generation, second-generation, or third-generation CAR, as described, for example, 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. Briefly, a "first-generation" CAR may comprise (a) an extracellular antigen-binding domain, (b) a transmembrane domain, (c) one or more intracellular signaling domains, and optionally (d) a hinge region connecting the antigen-binding domain to the transmembrane domain. A "second generation" CAR may include elements (a), (b), (c), and optionally (d), and may further include a costimulatory domain (e.g., a CD28 or 4-1BB costimulatory domain). A "third generation" CAR may include elements (a), (b), (c), and optionally (d), and may further include multiple costimulatory domains (e.g., CD28 and 4-1BB costimulatory domains, or CD28 and OX40 costimulatory domains). Each of the above elements is described in detail below. In some embodiments, it should be understood that the CAR molecule described by the following exemplary non-limiting arrangement is from the N-terminal to the C-terminal of the CAR, from left to right. The CAR described in this disclosure may include or further include any other combination of elements 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 Application Publication No. 2018 / 0256712A1; U.S. Patent Application Publication No. 2018 / 0271907A1; U.S. Patent Application Publication No. 2016 / 0046724A1; U.S. Patent Application Publication No. 2018 / 0044424A1; U.S. Patent Application Publication No. No. 2018 / 0258149A1; U.S. Patent Application Publication No. 2019 / 0151363A1; and U.S. Patent Application Publication No. 2018 / 0273601A1; the contents of each of the foregoing patents and patent application publications are incorporated herein by reference in their entirety.

[0112] The CAR used in the methods disclosed herein may comprise an extracellular antigen binding domain. The extracellular antigen binding domain may be any molecule that binds to an antigen, including, but not limited to, a human antibody, a humanized antibody, or any functional fragment thereof. In certain embodiments, the antigen binding domain is an scFv. In other embodiments, the extracellular antigen binding domain is a non-immunoglobulin scaffold protein. In other embodiments, the extracellular binding domain of the CAR comprises a single chain T cell receptor (scTCR). The extracellular domain may be derived from any of a wide variety of extracellular domains associated with ligand binding and / or signal transduction or secreted proteins, as described in U.S. Patents 5,359,046, 5,686,281, and 6,103,521.

[0113] The selection of the molecular target (antigen) for the extracellular binding domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen binding domain may be selected to recognize a ligand that acts as a cell surface marker for target cells associated with a particular pathology. Thus, in some embodiments, an extracellular antigen binding domain that specifically binds to a desired antigen may be engineered into the CAR to direct the CAR-mediated immune cell (e.g., T-cell) response to the antigen of interest. For example, the antigen binding domain may be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular pathology, such as cancer or autoimmune disease. Thus, examples of cell surface markers that may act as ligands for the antigen binding domain in the CAR include those associated with cancer cells and other forms of diseased cells (e.g., autoimmune disease cells and pathogen-infected cells). In some embodiments, the CAR is engineered to target a tumor antigen of interest by engineering a desired antigen binding domain that specifically binds to an antigen on tumor cells. In the context of the present invention, "tumor antigen" refers to an antigen common to a particular hyperproliferative disorder, 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 an 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.

[0114] In another embodiment, the extracellular binding region of the CAR binds to AFP (eg, ETCH17AFPCAR01 (Aeon Therapeutics (Shanghai) Co. / Eureka Therapeutics Inc.)), GPC3 (eg, GeneChem GPC-3 CART (Shanghai GeneChem Co.); 302 GPC3-CART (Shanghai GeneChem Co.); CAR-T for liver cancer (Shanghai GeneChem Co.); CAR-GPC3 T cells (Carsgen Therapeutics)), MUC1 (eg, PG-021-001,002 (PersonGen BioTherapeutics (Suzhou) Co.)), mesothelin (eg, H2017-01-P01 (Ningbo Cancer Hospital); TAI-meso-CART (Shanghai GeneChem Co.); K16-4 / NCT02930993 (China Meitan General Hospital / Marino Biotechnology Co.)), CD38 (eg, anti-CD38 A2 CAR-T / SOR-CART-MM-001 (Sorrento Therapeutics, Inc.)), herinCAR-PD1 (eg, herinCAR-PD1 / NBWYKY2016-06-001,002,003 (Ningbo Cancer Hospital); LCAR-B38M CAR-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 (bluebird bio); bb21217 (Celgene); JCARH125 (Juno Therapeutics, Inc.)), CD30 (e.g., ICAR30 T cells (Immune Cell, Inc.)), EGFR (e.g., EGFR:4-1BB:CD28:CD3 modified T cells / First Shenzhen02 (Shenzhen Sceond People’s Hospital / The Beijing Pregene Science and Technology Company); EGFR-IL12-CART (Shenzhen Second People’s Hospital / The Pregene (ShenZhen) Biotechnology Co.); SBNK-2016-015-01 (Beijing Sanbo Brain Hospital / 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 (eg, 2016-0341 / NCT03126864 (MD Anderson Cancer Center / Intrexon Corp. / Ziopharm)), EGFR / BCMA (eg, EGFRt / BCMA-41BBz CAR T cells (Memorial Sloan Kettering Cancer Center / Juno Therapeutics, Inc.)), ROR2 (eg, autologous CCT301-38 or CCT301-59 T cells (Shanghai Sinobioway Sunterra Biotech)), NKR-2 (eg, CYAD-N2T-002,003,004 (Celyad)), PSCA (eg, BP-012 (Bellicum Pharmaceuticals)), CD28 (eg, autologous CSR T Cells(Beijing Sanbo Brain Hospital / Marino Biotechnology Co.), TAA (eg, AMG 119 (Amgen)), NKG2D (eg, CM-CS1 (Celyad)), or CD123 (eg, UCART123 (Cellectis SA)). The preceding sentence further provides examples of CARs that bind to the antigens (e.g., AMG 119 (Amgen)). These CAR constructs may be used in the methods disclosed herein with anti-CD137 ADCs.

[0115] The CAR construct may further comprise a transmembrane domain that links the extracellular antigen binding domain to the intracellular signaling domain (either literally or in close proximity, e.g., using a spacer). In some embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv, Fab, or other antigen binding moiety) may be linked 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 various directions, thereby facilitating antigen recognition and binding. The cytoplasmic side of the transmembrane domain may be attached to an intracellular signaling domain, such as the intracellular signaling domain of CD28 or CD3 zeta (CD3-ζ), and may further comprise one or more co-stimulatory domains, as discussed below.

[0116] Thus, in certain embodiments, the CAR may further comprise a hinge region located between the extracellular antigen binding domain and the transmembrane domain. For example, the hinge region may be derived from an IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha hinge region. In certain embodiments, the hinge region is derived from an IgG4 hinge region. In another embodiment, the hinge region is the CD8 hinge domain (see SwissProt / GenBank Acc. NoP01732).

[0117] In one embodiment, the CAR comprises an extracellular antigen-binding domain and a transmembrane domain linked via a CD8 hinge: AKPTTTPAPR PPTPAPTIAS QPLSLRPEAC RPAAGGAVHT RGLDFA (SEQ ID NO: 2).

[0118] In one embodiment, the CAR comprises an extracellular antigen binding domain and a transmembrane domain linked via a hybrid CD8-CD28 hinge: AKPTTTPAPR PPTPAPTIAS QPLSLRPEAC RPAAGGAVHT RGLDFAPRKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSP LFPGPSKP (SEQ ID NO: 3).

[0119] The transmembrane domain may be derived from the sequence of a protein that contributes an extracellular antigen-binding domain, a protein that contributes an effector function signaling domain, a protein that contributes a proliferation signaling portion, or a completely different protein. In some embodiments, the transmembrane domain is naturally associated with one of the other domains of the CAR. For example, the transmembrane domain and the intracellular domain may be derived from the transmembrane and cytoplasmic regions of the same protein. In some embodiments, the transmembrane domain and the intracellular domain of the CAR comprise a contiguous 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, including the antigen-binding domain, to the cell membrane.

[0120] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains that may be used in the methods provided herein may be derived from (e.g., including at least the transmembrane domains described above) the alpha, beta, or zeta chains of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, LFA-1 T-cell co-receptor, CD2 T-cell co-receptor / adhesion molecule, CD8 alpha, and fragments thereof. The transmembrane domain of a protein may be identified using any method known in the art (e.g., hydrophobicity analysis, structural analysis, etc.) or by using public databases (e.g., the UniProt database).

[0121] In some embodiments, the transmembrane domain may be synthetic. In exemplary embodiments, the transmembrane domain may comprise predominantly hydrophobic residues such as leucine and valine. In certain embodiments, triplets of phenylalanine, tryptophan and valine may be located at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker (preferably 2-10 amino acids in length) may form the link between the transmembrane domain and the intracellular signaling domain of a CAR. A glycine-serine doublet provides a particularly suitable linker.

[0122] In some embodiments, the transmembrane domain in a CAR as used herein is the transmembrane domain of CD8 or a portion thereof. The sequence of CD8 for this purpose is taught in PCT Publication No. WO2014 / 055771A1.

[0123] In some embodiments, the transmembrane domain in the CAR is the transmembrane domain of CD8 or a functional portion thereof. For example, a CAR may comprise the transmembrane domain of CD3 having the amino acid sequence LDPKLCYLLD GILFIYGVIL TALFLRVK (SEQ ID NO: 4), or a functional portion thereof (e.g., LCYLLDGILF IYGVILTALFL (SEQ ID NO: 5)).

[0124] In some embodiments, the transmembrane domain in the CAR of the invention is the transmembrane domain of CD28. Exemplary sequences of CD28 are provided below, as well as exemplary transmembrane domain sequences. In some embodiments, the transmembrane domain of CD28 comprises the following exemplary transmembrane domain sequences, or fragments or variants thereof that are capable of anchoring a CAR comprising the sequence to a cell membrane. Thus, in some embodiments, the transmembrane domain of the CAR is the transmembrane domain of CD28 comprising the following amino acid sequence: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 6). In one embodiment, the transmembrane domain of the CAR is the transmembrane domain of CD28 comprising the following amino acid sequence: IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWV (SEQ ID NO: 7), or a functional fragment thereof, such as SEQ ID NO: 6.

[0125] In addition to the extracellular antigen-binding domain and the transmembrane domain, CARs further comprise an intracellular (or intracellular (cytoplasmic)) signaling domain.

[0126] It is known that signals generated through the endogenous TCR alone are insufficient to fully activate T cells, and secondary or costimulatory signals may also be required. Thus, T cell activation may be mediated by two different types of intracellular signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary intracellular signaling sequences).

[0127] The term "intracellular signaling domain" or "cytoplasmic signaling domain" as used herein refers to the intracellular portion of the CAR. The intracellular signaling domain can generate a signal that promotes immune effector function of an immune allogeneic cell (e.g., a CAR-T cell or a CAR-expressing NK cell) that contains a CAR. Examples of immune effector functions, for example in a CAR T cell or a CAR-expressing NK cell, include helper activity, including cytolytic activity and secretion of cytokines. In certain embodiments, the intracellular signaling domain transmits an effector function signal, instructing the cell to perform a specialized function. Although the entire intracellular signaling domain can be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain that is sufficient to transmit the effector function signal.

[0128] In one embodiment, the intracellular signaling domain of the CAR comprises the signaling region of CD3 zeta as set forth in SEQ ID NO: 8, or a signaling portion thereof: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 8).

[0129] Intracellular signaling domains may further include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD278 ("ICOS"), Fc epsilon RI, CD66d, DAP10, and DAP12.

[0130] CARs may further comprise an "intracellular costimulatory domain," which is a polypeptide chain derived from the intracellular signaling domain of a costimulatory protein or proteins that enhance cytokine production (e.g., CD28 and 4-1BB, etc.). Exemplary costimulatory signaling regions include 4-1BB, CD21, CD28, CD27, CD127, ICOS, IL-15Rα, and OX40.

[0131] In certain embodiments, the intracellular costimulatory domain of a CAR comprises a 4-1BB signaling domain alone or in combination with any other desired intracellular domains beneficial in the context of a CAR. 4-1BB is a member of the TNFR superfamily having the amino acid sequence provided as GenBank Acc No. AAA62478.2, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.); and a "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Acc No. AAA62478.2, or the equivalent residues from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

[0132] In one embodiment, the intracellular costimulatory domain of the CAR is the costimulatory signaling region of 4-1BB (CD137), or a signaling portion thereof: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 9).

[0133] In one embodiment, the costimulatory signaling domain of the CAR is a costimulatory signaling region sequence of CD28. For example, the costimulatory signaling domain can include the following CD28 costimulatory signaling moiety, or a signaling moiety thereof: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 10).

[0134] In exemplary embodiments, the intracellular domain of the CAR may contain a signaling domain of CD3-zeta in combination with any other desired intracellular domain useful in the context of the CAR of the invention. In certain embodiments, the intracellular domain of the CAR may include a domain of CD3-zeta and a costimulatory signaling region (such as, but not limited to, the costimulatory signaling region of 4-1BB, CD28, and / or CD27).

[0135] The intracellular signaling sequences within the intracellular signaling portion of the CAR of the invention may be linked to each other in a random or specific order. Optionally, short oligo- or polypeptide linkers or spacers (preferably 5-20 amino acids in length) may be inserted between the intracellular domains. GGGGS (SEQ ID NO: 27) or (GGGGS)x3 (SEQ ID NO: 28) provide particularly suitable linkers.

[0136] In one embodiment, a CAR as used herein comprises an extracellular domain containing a single chain variable domain of an anti-CD19 monoclonal antibody, a transmembrane domain containing the hinge and transmembrane domains of CD8α, and an intracellular domain containing the signaling domain of CD3ζ and the signaling domain of 4-1BB. An exemplary CAR comprises an anti-CD19 extracellular domain comprising an anti-CD19 monoclonal antibody as described in Nicholson IC, et al., Mol Immunol 34:1157-1165 (1997), as well as a 21 amino acid signal peptide of CD8α (translated from 63 nucleotides 26-88 of GenBank Accession No. NM_001768). The CD8α hinge and transmembrane domain consists of 69 amino acids translated from 207 nucleotides 815-1021 of GenBank Accession No. NM_001768. In a preferred embodiment, the CD3 zeta signaling domain comprises 112 amino acids translated from 339 nucleotides 1022 to 1360 of GenBank Accession No. NM_000734.

[0137] A spacer or hinge domain may be incorporated between the extracellular domain (including the antigen binding domain) and the transmembrane domain of the CAR (described above), or between the intracellular domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally refers to any oligo or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or the intracellular domain in a polypeptide chain. As used herein, a hinge domain generally refers to any oligo or polypeptide that functions to provide flexibility to the CAR, or a domain thereof, and / or to prevent steric hindrance of the CAR, or a domain thereof. In some embodiments, a spacer or hinge domain may comprise up to 300 amino acids, preferably 10-100 amino acids, and most preferably 5-20 amino acids. Often, a spacer or hinge is introduced between the extracellular antigen binding domain and the transmembrane domain to provide flexibility that allows the antigen binding domain to orient in various directions to facilitate antigen recognition and binding. It should also be understood that one or more spacer domains may be included in other regions of a CAR, as aspects of the present disclosure are not limited in this respect.

[0138] It should be understood that a CAR can include a region having a sequence provided herein (e.g., an antigen binding domain, a transmembrane domain, an intracellular domain, a signaling domain, a safety domain, and / or a linker, or any combination thereof), or a variant thereof, or a fragment of any thereof (e.g., a variant and / or fragment that retains a function required for CAR activity) can be included in the CAR proteins described herein. In some embodiments, a variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes relative to the sequence shown. In some embodiments, a 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, a 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, fragments are shorter at the N-terminus, C-terminus, or both terminal regions of the provided sequences, hi some embodiments, fragments comprise 80%-85%, 85%-90%, 90%-95%, or 95%-99% of the number of amino acids in the sequences provided herein.

[0139] In other embodiments, the invention includes nucleic acid sequences that encode the amino acid sequences disclosed herein.

[0140] In some embodiments, the above exemplary non-limiting configurations are from left to right, N-terminal to C-terminal of the CAR, The CAR may comprise or further comprise any other combination of elements as described herein.

[0141] Once the CAR construct is identified in its various portions, immune cells expressing the CAR are produced, whereby the immune cells express the CAR. The method includes introducing (e.g., transducing) the immune cells with a nucleic acid molecule described herein (e.g., an RNA molecule (e.g., mRNA)) or a vector comprising a nucleic acid molecule encoding a CAR (e.g., a CAR described herein). The invention also provides a method of making a cell population (e.g., an RNA engineered cell that transiently expresses an exogenous RNA). The method includes introducing an RNA as described herein (e.g., an in vitro transcribed RNA or a synthetic RNA; an mRNA sequence encoding a CAR polypeptide as described herein) into a cell. In an embodiment, the RNA transiently expresses a CAR polypeptide. In an embodiment, the cell is a cell described herein (e.g., an immune effector cell (e.g., a T cell or NK cell, or cell population)).

[0142] Anti-CD137 Antibody Drug Conjugates (ADCs) As described herein, anti-CD137 ADCs are used in combination with allogeneic cell therapy to treat cancer or autoimmune disease in human patients. More specifically, anti-CD137 ADCs can be used to deplete CD137+ cells (e.g., CD137+ T cells) in human subjects who are also receiving allogeneic cell therapy. Anti-CD137 ADCs target endogenous T cells and damage these cells, thereby preventing the patient's immune system from attacking allogeneic cells (e.g., allogeneic cells expressing a CAR) administered to the subject. Thus, anti-CD137 ADCs are used in combination with CAR therapy to prevent or reduce the risk of rejection of the cell therapy in the recipient patient. One advantage of using anti-CD137 ADCs as a conditioning regimen is that it specifically targets and depletes activated T cells that express CD137.

[0143] Anti-CD137 antibody The present invention is based, in part, on the discovery that antibodies and antigen-binding fragments thereof 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.

[0144] T cells have been shown to express CD137, an antigen that is a transmembrane TNF receptor superfamily of costimulatory molecules that is expressed on a variety of hematopoietic cells and promotes T cell activation as well as regulates T cell proliferation and survival (see, e.g., Cannons et al., J. Immunol. 167:1313-1324, 2001, the disclosure of which is incorporated herein by reference as it relates to expression of CD137 by T cells). The amino acid sequence of human CD137 is shown below: MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN RNQICSPCPP NSFSSAGGQR TCDICRQCKG VFRTRKECSS TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP SPADLSPGAS SVTPPAPARE PGHSPQIISF FLALTSTALL FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL (GenBank No. AAX42660.1; SEQ ID NO: 1).

[0145] Antibodies and antigen-binding fragments thereof specific for human CD137 can be identified by using techniques known in the art and described herein, such as immunization, computer modeling techniques, and in vitro selection methods (e.g., phage display and cell-based display platforms described below).

[0146] In certain embodiments, an anti-CD137 antibody that may be used in the methods and compositions (including ADCs) described herein is the murine anti-CD137 antibody BBK2 (Thermo Fisher; MS621PABX), or an anti-CD137 antibody that comprises an antigen-binding region corresponding to the BBK2 antibody. The BBK2 antibody (sometimes referred to as the BBK-2 antibody or anti-4-1BB antibody) is a murine monoclonal antibody (IgG1, κ) that binds to the ectodomain of human 4-1BB recombinant protein (4-1BB is also known as CD137). In certain embodiments, the methods and compositions of the disclosure include an anti-CD137 antibody that comprises the binding region (e.g., CDRs) of the BBK2 antibody. In another embodiment, the methods and compositions of the disclosure include an antibody that competitively inhibits binding of the BBK2 antibody to its epitope on CD137. In certain embodiments, the anti-CD137 antibody is humanized BBK2 or chimeric BBK2.

[0147] In certain embodiments, the methods and compositions described herein include a chimeric anti-CD137 (ch-BBK2) antibody comprising the 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 set forth in SEQ ID NO: 11, and the light chain amino acid sequence of ch-BBK2 is set forth in SEQ ID NO: 12. The CDR regions (CDR1, CDR2, and CDR3) of each of the heavy and light chain sequences are set forth below in bold. The variable regions are in italics.

[0148] [ka] (SEQ ID NO: 11)

[0149] [ka] (SEQ ID NO: 12)

[0150] The above CDR regions (and the BBK2 antibody) 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 amino acid sequences of the VL CDRs 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).

[0151] Alternatively, the CDR regions of BBK2 may be defined by Kabat numbering. The CDRs defined by Kabat numbering are set forth below (in bold below) for each of the heavy and light chain sequences. The variable regions of BBK2 are in italics.

[0152] [ka]

[0153] [ka]

[0154] Thus, in one embodiment, the VH CDR amino acid sequence of the anti-CD137 antibody BBK2 (including ch-BBK2) is 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 sequence of the anti-CD137 antibody BBK2 (including ch-BBK2) is as follows: RASQDLSNHLY (VL CDR1; SEQ ID NO:23); YTSRLHS (VL CDR2; SEQ ID NO:24) and QQGYTLPYT (VL CDR3; SEQ ID NO:25).

[0155] The heavy chain variable region of BBK2 is set forth in SEQ ID NO:22 as follows: QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSNTVYMQLNSPTSEDSAVYYCTRNGVEGYPHYYAMEYWGQGTSVTVSS . The light chain variable region of BBK2 is set forth in SEQ ID NO:26 as follows: DIQMTQTTSALSASLGDRVTIGCRASQDLSNHLYWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTIRNLEQEDVATYFCQQGYTLPYTFGGGTKLEIK . Anti-CD137 antibodies (including anti-CD137 ADCs) may comprise heavy and light chain variable region amino acid sequences as set forth in SEQ ID Nos: 22 and 26, respectively.

[0156] 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.

[0157] 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.

[0158] Thus, BBK2, humanized BBK2, or chimeric BBK2 antibodies may be used in the anti-CD137 ADCs and methods described herein. Each of these antibodies may be conjugated to any of the cytotoxins described below using methods known in the art and described herein.

[0159] A variety of other anti-CD137 antibodies are known in the art and are useful in the methods described herein. For example, in certain embodiments, the anti-CD137 antibody is selected from the group consisting of ADG106 (described below, e.g., WO2019105468, US20190055314, WO2019037711, WO2019036855); AGEN2373 (described below, e.g., WO2018191502, US20180344870); ATOR-1017 (described below, e.g., WO2018091740, US20180118841) PE0166 (described below, e.g., Song et al. AACR 2019, Abstract 2397 / 21), urelumab (also known as BMS-663513; described below, 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 below, eg, 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.

[0160] Other anti-CD137 antibodies are described, for example, in WO2019020774, WO2017077085, US20180327504, US20190099488, US2019006045, US20190015508, WO2019014328, US20190071510, WO2018127787, US20180258177, US10174122, WO2016110584, WO2018017761, WO2018098370, US20130149301, WO2019027754, WO2018156740, US20160244528, WO2016134358, US10233251, US20170226215, US20160083474, WO2017049452, US20180282422, WO2015188047, WO2010132389, US20120076722, US20110177104, WO2011031063, US20080305113, US20080008716, US7829088, US20090041763, WO2006126835, US20030096976, US20030223989, US20060182744, WO1996029348, Soderstromet 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.

[0161] In one embodiment, the anti-CD137 antibody comprises a heavy chain of an anti-CD137 antibody described herein, and a light chain variable region of an anti-CD137 antibody described herein. In one embodiment, the anti-CD137 antibody comprises a heavy chain comprising CDR1, CDR2, and CDR3 of an anti-CD137 antibody described herein, and a light chain variable region comprising CDR1, CDR2, and CDR3 of an anti-CD137 antibody described herein.

[0162] In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence having at least 95% identity to an anti-CD137 antibody herein, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identity to an anti-CD137 antibody herein. In certain embodiments, the antibody comprises a modified heavy chain (HC) variable region comprising the HC variable domain of an anti-CD137 antibody herein, or a variant thereof, wherein the variant (i) differs from the anti-CD137 antibody in that 1, 2, 3, 4, or 5 amino acids are substituted, added, or deleted; (ii) differs from the anti-CD137 antibody in that at most 5, 4, 3, 2, or 1 amino acid is substituted, added, or deleted; (iii) differs from the anti-CD137 antibody in that 1-5, 1-3, 1-2, 2-5, or 3-5 amino acids are substituted, added, or deleted. 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 said anti-CD137 antibody, where in any of (i)-(iv) the amino acid substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions; and where the modified heavy chain variable region may have enhanced biological activity compared to the heavy chain variable region of said anti-CD137 antibody while retaining the CD137 binding specificity of said anti-CD137 antibody.

[0163] Other anti-CD137 antibodies that may be used in conjunction with the cytotoxins described herein may be identified using techniques known in the art (e.g., hybridoma production). Hybridomas may be prepared using the mouse system. Protocols for isolating splenocytes for immunization and subsequent fusion are known in the art. Fusion partners and procedures for producing hybridomas are also known. HuMAb-Mouse 登録商標 or XenoMouse TMHuman anti-CD137 antibodies may be produced by the method of the present invention. In producing anti-CD137 antibodies, the CD137 antigen is isolated and / or purified. The CD137 antigen may be a fragment of CD137 derived from the extracellular domain of CD137. Immunization of animals may be performed 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. Pat. No. 5,994,619. The CD137 antigen may be administered with an adjuvant to stimulate the immune response. Adjuvants known in the art include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulating complex). After immunization of an animal with CD137 antigen, antibody-producing immortalized cell lines are prepared from cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph node and / or splenic B cells are immortalized by methods known in the art (e.g., oncogene transduction, oncogenic virus transduction, exposure to carcinogenic or mutagenic compounds, fusion with immortalized cells (e.g., myeloma cells), and inactivation of tumor suppressor genes). See, e.g., Harlow and Lane, supra. Hybridomas may be selected, cloned, and further screened for desired properties, including, e.g., robust growth, high antibody production, and desired antibody properties.

[0164] Anti-CD137 antibodies may be generated from isolated nucleic acid molecules comprising a nucleotide sequence encoding the amino acid sequence of a CD137 binding molecule provided by the present invention. The amino acid sequence encoded by the nucleotide sequence may be any portion of an antibody (e.g., a CDR, a sequence comprising one, two or three CDRs, a heavy chain variable region, a light chain variable region), or a full-length heavy chain or full-length light chain. Nucleic acids of the present disclosure may be, for example, DNA or RNA, and may or may not contain intron sequences. Typically, the nucleic acid is a cDNA molecule.

[0165] In some embodiments, the anti-CD137 antibody (or fragment thereof) has a defined serum half-life. For example, an anti-CD137 antibody (or fragment thereof) may have a serum half-life of about 1-24 hours in a human patient. An ADC comprising such an anti-CD137 antibody may also have a serum half-life of about 1-24 hours in a human patient, for example. Pharmacokinetic analysis by measuring serum levels can be performed by assays known in the art.

[0166] Antibodies for use in conjunction with the ADCs described herein include variants of those antibodies described above (e.g., antibody fragments that contain or lack an Fc domain, including one or more or all of the CDRs or equivalent regions of the antibodies, antibody fragments described herein, etc.).

[0167] Methods for identifying antibodies Methods for high-throughput screening of libraries of antibodies or antibody fragments capable of binding to CD137 may be used to identify and affinity mature agents useful, for example, for treating or preventing allogeneic cell therapy rejection. Such methods include in vitro display techniques known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display, among others. The use of phage display to isolate antibodies or antigen-binding fragments that bind biologically relevant molecules has been reviewed, for example, in 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 disclosures of each of which are incorporated herein by reference as they relate to in vitro display techniques. Randomized combinatorial peptide libraries have also been constructed to select polypeptides that bind to cell surface antigens, as described in Kay, Perspect. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, 1996, the disclosures of each of which are incorporated by reference herein as they relate to the discovery of antigen-binding molecules.Phage display of proteins (e.g., multimeric proteins) as functional molecules has been successfully achieved (see, e.g., EP 0349578; EP 4527839; and EP 0589877, as well as Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992, the disclosures of each of which are incorporated herein by reference as they relate to the use of in vitro display techniques for the discovery of antigen-binding molecules). Additionally, functional antibody fragments (e.g., 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 be expressed, for example, in HuMAb-mice. 登録商標 or XenoMouse TM These techniques may be used, inter alia, to identify and improve the affinity of antibodies or antibody fragments that bind to CD137, which may then be used to deplete hematopoietic cells in a patient.

[0168] In addition to in vitro display techniques, computational modeling techniques may be used to design and identify anti-CD137 antibodies or antibody fragments in silico, for example using the procedures described in US2013 / 0288373, the disclosure of which is incorporated herein as it relates to molecular modeling methods for identifying anti-CD137 antibodies. For example, using computational modeling techniques, one skilled in the art may screen a library of antibodies or antibody fragments in silico for molecules capable of binding to a specific epitope on CD137, such as an extracellular epitope of CD137.

[0169] Further techniques may be used to identify antibodies or antibody fragments that bind to CD137 on the surface of cells (e.g., T cells) and can be internalized by the cells, for example, by receptor-mediated endocytosis. For example, the in vitro display techniques described above may be adapted to screen for antibodies or antibody fragments thereof that bind to CD137 on the surface of hematopoietic stem cells and are subsequently internalized. Phage display represents one such technique that may be used in combination with this screening paradigm. To identify anti-CD137 antibodies or antibody fragments that bind to CD137 and are subsequently internalized by hematopoietic stem cells, one of skill in the art may use the phage display technique described in Williams et al., Leukemia 19:1432-1438, 2005, the disclosure of which is incorporated herein by reference in its entirety. For example, mutagenesis methods known in the art may be used to identify antibodies, antibody fragments (e.g., scFv fragments, Fab fragments, diabodies, triabodies, and antibodies, among others). 10Recombinant phage libraries can be produced that encode antibodies, polypeptides, or ligands (which contain randomized amino acid cassettes (e.g., in one or more or all of the CDRs or equivalent regions, or in an antibody or antibody fragment). The framework regions, hinges, Fc domains, and other regions of the antibodies or antibody fragments may be designed to be non-immunogenic in humans, for example, by having human germline antibody sequences or sequences that differ slightly compared to human germline antibodies.

[0170] Using phage display techniques described herein or known in the art, a phage library comprising randomized antibodies or antibody fragments covalently attached to phage particles can be incubated, for example, by first incubating the phage library with a blocking agent (e.g., milk protein, bovine serum albumin, and / or IgG, etc., to remove phage encoding antibodies or antibody fragments that exhibit non-specific protein binding and to remove phage encoding antibodies or fragments thereof that bind to Fc domains) and then incubating with CD137 (incubating the phage library with a population of hematopoietic stem cells that are CD137+). The phage library is incubated with the hematopoietic stem cells for a period of time (e.g., 30 minutes to 6 hours, e.g., 1 hour at 4°C) sufficient for CD137-specific antibodies or antibody fragments to bind to cell surface CD137 and subsequently be internalized by the hematopoietic stem cells. Phage containing antibodies or antibody fragments that do not exhibit sufficient affinity for CD137 to allow binding to and internalization by hematopoietic stem cells may then be removed, for example by washing with cold (4° C.) 0.1 M glycine buffer, pH 2.8. Phage that bound to the hematopoietic stem cell-internalized antibodies or antibody fragments may then be identified, for example by lysing the cells and recovering the internalized phage from the cell culture medium. The phage may then be amplified in bacterial cells, for example by incubating the bacterial cells with the recovered phage in 2×YT medium using methods known in the art. Phage recovered from this medium may then be identified, for example by determining the nucleic acid sequence of the antibody or antibody fragment-encoding gene inserted into the phage genome. The encoded antibody or antibody fragment may then be prepared de novo, for example by chemical synthesis (for example, an antibody fragment, e.g., an scFv fragment) or recombinant expression (for example, a full-length antibody).

[0171] The capacity of the prepared antibody or antibody fragment to be incorporated may be assessed, for example, using radionuclide internalization assays known in the art. For example, an anti-CD137 antibody or antibody fragment identified using in vitro display techniques described herein or known in the art may be immunized with a radioisotope (e.g., 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 Bi, or 225 Ac) to make it functional. For example, radioactive halogens (e.g. 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211Radiolabeled antibodies, fragments thereof, or ADCs may be incorporated into the antibodies or antibody fragments using beads (e.g., polystyrene beads) containing an electrophilic halogenated reagent (e.g., iodized beads, Thermo Fisher Scientific, Inc., Cambridge, MA). The radiolabeled antibodies, fragments thereof, or ADCs may be incubated with hematopoietic stem cells for a time sufficient for incorporation (e.g., 30 minutes to 6 hours at 4°C, 1 hour at 4°C, etc.). The cells are then washed (e.g., with cold (4°C) 0.1 M glycine buffer, pH 2.8) to remove any antibodies or fragments thereof that have not been incorporated. Incorporated antibodies or antibody fragments may be identified by detecting radiation (e.g., gamma radiation) emitted from the resulting hematopoietic stem cells and comparing it to radiation (e.g., gamma radiation) emitted from the recovered wash buffer. The internalization assay described above may be used to characterize ADCs.

[0172] The antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent 4,816,567. In one embodiment, an isolated nucleic acid is provided that encodes an anti-CD137 antibody described herein. Such a nucleic acid molecule may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In a further embodiment, a host cell comprising such a nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed by): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody, and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method of producing an anti-CLL-1 antibody is provided, the method comprising culturing a host cell comprising an antibody-encoding nucleic acid as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or the culture medium of the host cell).

[0173] To recombinantly produce an anti-CD137 antibody, nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid molecules can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0174] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly where glycosylation and Fc effector functions are not required. See, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 for expression of antibody fragments and polypeptides in bacteria (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody may be isolated as a soluble fraction from the bacterial cell paste and further purified.

[0175] Vertebrate cells can also be used as hosts, for example mammalian cell lines adapted to grow in suspension can be beneficial. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, e.g., as described 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, e.g., as described in Mather et al., Annals NY 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, for example, DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines (e.g., Y0, NS0, and Sp2 / 0). For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In certain embodiments, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).

[0176] cytotoxin A variety of cytotoxins may be conjugated to anti-CD137 antibodies via linkers for use in the combination therapies described herein. In particular, anti-CD137 ADCs include an antibody (or antigen-binding fragment thereof) conjugated (i.e., covalently attached by a linker) to a cytotoxic moiety (or cytotoxin). As used herein, the terms "cytotoxin," "cytotoxic moiety," and "drug" are used interchangeably. In various embodiments, the cytotoxic moiety has reduced or no cytotoxicity when attached as a conjugate. However, after cleavage from the linker, it regains cytotoxicity. In various embodiments, the cytotoxic moiety retains cytotoxicity without cleavage from the linker. In some embodiments, the cytotoxic molecule is conjugated to an antibody or antigen-binding fragment thereof that is internalized into a cell, as described herein, such that after the antibody or fragment thereof is internalized into a cell, the cytotoxin may have access to its intracellular target and, for example, mediate T cell death.

[0177] Thus, the ADCs of the invention may be of the general formula I, where an antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently attached) to a linker (L) and, through a chemical moiety (Z), to a cytotoxic moiety ("drug", D). Ab-(ZLD) n (I)

[0178] Thus, the antibody or antigen-binding fragment thereof may be conjugated to multiple drug moieties as indicated by the integer n, where the integer n represents the average number of cytotoxins per antibody, where the integer n may range, for example, from about 1 to about 20. Any number of cytotoxins (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8) may be conjugated to the antibody. 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 a preparation of ADC from a conjugation reaction can be determined by conventional means such as mass spectroscopy, ELISA assays, and HPLC. The quantitative distribution of the ADC with respect to n can also be determined. In some cases, homogenous ADCs with a particular value of n can be separated, purified, and characterized from ADCs with other drug loads by means such as reverse phase HPLC or electrophoresis.

[0179] For some anti-CD137 ADCs, n may be limited by the number of binding sites on the antibody. For example, if the linkage is a cysteine ​​thiol, the antibody may have only one or a few cysteine ​​thiol groups, or may have only one or a few thiol groups that are sufficiently reactive for the linker to be attached. In general, antibodies do not contain many free and reactive cysteine ​​groups that can be attached to a drug moiety; primarily, the cysteine ​​thiol residues in antibodies are present as disulfide bridges. In certain embodiments, antibodies can be reduced with a reducing agent, such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), to generate reactive cysteine ​​thiol groups under partial or total reducing conditions. In certain embodiments, higher drug loading (e.g., n>5) may cause aggregation, insolubility, toxicity, or loss of cell permeability in certain antibody-drug conjugates.

[0180] In certain embodiments, fewer than the theoretical maximum number of drug moieties are conjugated to the antibody during the conjugation reaction. The antibody may contain lysine residues that do not react with the drug-linker intermediate or linker reagent, for example, as discussed below. Only the most reactive lysine groups can react with amine-reactive linker reagents. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine.

[0181] The loading (drug / antibody ratio) of ADCs may be controlled in a variety of ways, such as (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the time or temperature of the conjugation reaction, (iii) using partial or limiting reducing conditions for thiol modification of cysteines, and (iv) recombinantly engineering the amino acid sequence of the antibody to alter the number and position of cysteine ​​residues to control the number and / or position of linker-drug bonds.

[0182] Cytotoxins suitable for use with the compositions and methods described herein include those known in the art, including, among others, DNA-intercalating agents (e.g., anthracyclines), agents capable of disrupting the spindle apparatus (e.g., vinca alkaloids, maytansine, maytansinoids, and derivatives thereof), RNA polymerase inhibitors (e.g., amatoxins such as α-amanitin, and derivatives thereof), and agents capable of disrupting protein biosynthesis (e.g., agents exhibiting rRNA N-glycosidase activity, such as saporin and ricin A chain).

[0183] In some embodiments, the cytotoxin is a microtubule binding agent (e.g., maytansine or a maytansinoid), an amatoxin, Pseudomonas exotoxin A, de bouganin, diphtheria toxin, saporin, auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an indolinobenzodiazepine, an indolinobenzodiazepine dimer, or a variant thereof, or other cytotoxic compound described herein or known in the art.

[0184] In some embodiments, the cytotoxin of the antibody-drug conjugate is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is an amatoxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody-drug conjugate disclosed herein is an amatoxin or a derivative thereof (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, proamanutin, or a derivative thereof, etc.).

[0185] Further details regarding cytotoxins that may be used in the anti-CD137 ADCs useful in the methods of the invention are provided below.

[0186] Amatoxin In some embodiments, the RNA polymerase inhibitor is an amatoxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody-drug conjugate disclosed herein is an amatoxin or a derivative thereof (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanurin, proamanuline, or a derivative thereof, etc.). The structures of various naturally occurring amatoxins are represented by Formula II and Table 1 presented together, and are disclosed, for example, in Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459. [ka]

[0187] [Table 1]

[0188] In one embodiment, the cytotoxin is amanitin or a derivative thereof, hi one embodiment, the cytotoxin is α-amanitin or a derivative thereof.

[0189] Many positions on an amatoxin or derivative thereof can serve as a position for covalent attachment to a linking moiety L, and therefore, can serve as a position for covalent attachment to the antibody, or antigen-binding fragment thereof. In some embodiments, the cytotoxin in an ADC of formula I is an amatoxin or derivative thereof of formula (II). In certain embodiments, the ADC has the formula Ab-ZL-Am, where 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 amatoxin. In certain embodiments, the linker-amatoxin conjugate Am-LZ has the formula (III): [ka] where: R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R 5,R6 and R7 are each independently H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , N.H.R. C , or NR C R D is; R9 is H, OH, OR C OR D is; Q is -S-, -S(O)-, or -SO2-; R C is -L-Z' or -LZ-Ab, where L 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; or comprises a dipeptide; or -((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 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 combinations thereof, where 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 alkyl, and optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl, heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0190] Formula (III) comprises an amatoxin and a linker, and in some embodiments, comprises a linker, a chemical moiety, and an antibody.

[0191] In some embodiments, the cytotoxin is an amatoxin, and the linker-amatoxin conjugate or antibody-linker-amatoxin conjugate has formula (IIIA): [ka] where: R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R 5, R6 and R7 are each independently H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , N.H.R. C , or NR C R D is; R9 is H, OH, OR C OR D is; Q is -S-, -S(O)-, or -SO2-; R C is -L-Z' or -LZ-Ab, where L is a linker and 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; or comprises a dipeptide; or -(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 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 combinations thereof, where 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 alkyl, and optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl, heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0192] In some embodiments, the conjugate comprises one R C Contains substituents.

[0193] In some embodiments, R A and R B together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl group of the formula: [ka] Here, Y is -(C=O)-, -(C=S)-, -(C=NR E) -, or -(CR E R E’ )-is; and Here, R E and R E’ are each 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 is optionally substituted with 1 to 5 substituents each independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl / heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0194] Formula (IIIA) comprises an amatoxin and a linker, and in some embodiments, comprises a linker, a chemical moiety, and an antibody.

[0195] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the amatoxin-linker conjugate has formula IIIA, wherein: R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached form: [ka] where R3 is H or R C It is.

[0196] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the conjugate has formula IIIA, wherein: R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached form: [ka] Where: R3 is H or R C is; R4 and R5 each independently represent H, OH, OR C , R C OR D is; R6 and R7 are each H; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; and Here, R C and R D is as defined above.

[0197] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the amatoxin-linker conjugate has formula IIIA, wherein: R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; R A and R B When present, together with the oxygen atom to which they are attached form: [ka] Where: R3, R4, R6, and R7 are each H; R5 is OR C is; R8 is OH or NH2; R9 is H or OH; Q is -S-, -S(O)-, or -SO2-; and Here, R C and R D Such amatoxin 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.

[0198] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the amatoxin-linker conjugate has formula IIIA, wherein: R1 and R2 are each independently H or OH; R3 is R C is; R4, R6, and R7 are each H; R5 is H, OH, or OC1-C6 alkyl; R8 is OH or NH2; R9 is H or OH; Q is -S-, -S(O)-, or -SO2-; and Here, R C and R D Such amatoxin-linker conjugates are described, for example, in U.S. Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated herein by reference in its entirety.

[0199] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the amatoxin-linker conjugate has formula IIIA, wherein: R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 each independently represent H, OH, OR C , or R C is; R8 is OH or NH2; R9 is H or OH; Q is -S-, -S(O)-, or -SO2-; and Here, R C and R D Such amatoxin-linker conjugates 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.

[0200] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the amatoxin-linker conjugate has formula IIIA, wherein: R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are each independently H or OH; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; Q is -S-, -S(O)-, or -SO2-; and Here, R C and R D Such amatoxin conjugates are described, for example, in U.S. Patent Nos. 9,233,173 and 9,399,681, the disclosures of which are incorporated herein by reference in their entireties.

[0201] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by Formula IIIB: [ka] where: R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl group; R3 is H, R C , or R D is; R4, R 5, R6 and R7 are each independently H, OH, OR C , OR D , R C , or R D is; R8 is OH, NH2, OR C , OR D , N.H.R. C , or NR C R D is; R9 is H, OH, OR C OR D is; Q is -S-, -S(O)-, or -SO2-; R Cis -L-Z' or -LZ-Ab, where L is a linker and 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, 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 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 combinations thereof, where 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 alkyl, and optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl, heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0202] Formula (IIIB) comprises an amatoxin and a linker, and in some embodiments, comprises a linker, a chemical moiety, and an antibody.

[0203] In some embodiments, R A and R B together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl of the formula: [ka] Here, Y is -(C=O)-, -(C=S)-, -(C=NR E) -, or -(CR E R E’ )-is; and Here, R E and R E’ are each 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 Cis optionally substituted with 1 to 5 substituents each independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl / heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0204] In some embodiments, the antibody or antigen-binding fragment thereof described herein is conjugated to an amatoxin-linker conjugate or derivative thereof and represented by formula IIIB, wherein: R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl of the formula: [ka] where R3 is H or R C It is.

[0205] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by Formula IIIB, wherein: R1 is H, OH, OR A OR C is; R2 is H, OH, OR B OR C is; R A and R Bwhen present, together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl group of the formula: [ka] Where: R3 is H or R C is; R4 and R5 each independently represent H, OH, OR C , R C OR D is; R6 and R7 are each H; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; and Here, R C and R D is as defined above.

[0206] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by Formula IIIB, wherein: R1 is H, OH, or OR A is; R2 is H, OH, or OR B is; R A and R B when present, together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl group of the formula: [ka] Where: R3, R4, R6, and R7 are each H; R5 is OR C is; R8 is OH or NH2; R9 is H or OH; Q is -S-, -S(O)-, or -SO2-; and Here, R Cand R D Such amatoxin-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.

[0207] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by Formula IIIB, wherein: R1 and R2 are each independently H or OH; R3 is R C is; R4, R6, and R7 are each H; R5 is H, OH, or OC1-C6 alkyl; R8 is OH or NH2; R9 is H or OH; Q is -S-, -S(O)-, or -SO2-; and Here, R C and R D Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated herein by reference in its entirety.

[0208] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by Formula IIIB, wherein: R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 each independently represent H, OH, OR C , or R C is; R8 is OH or NH2; R9 is H or OH; Q is -S-, -S(O)-, or -SO2-; and Here, RC and R D Such amatoxin-linker conjugates 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.

[0209] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by Formula IIIB, wherein: R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are each independently H or OH; R8 is OH, NH2, OR C , or NHR C is; R9 is H or OH; Q is -S-, -S(O)-, or -SO2-; and Here, R C and R D Such amatoxin-linker conjugates are described, for example, in U.S. Patent Nos. 9,233,173 and 9,399,681, the disclosures of which are incorporated herein by reference in their entireties.

[0210] Auristatins The anti-CD137 antibodies and antigen-binding fragments thereof described herein may be conjugated to cytotoxins that are auristatins (U.S. Patent Nos. 5,635,483; 5,780,588). Auristatins are antimitotic agents that interfere with microtubule dynamics, GTP hydrolysis, nuclear division and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), as well as anticancer (U.S. Patent No. 5,663,149) and antifungal (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). (U.S. Patent Nos. 5,635,483; 5,780,588). The auristatin drug moiety may be attached to the antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02 / 088172).

[0211] 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, Volume 45, Abstract Number 623, March 28, 2004, the disclosure of which is incorporated herein by reference in its entirety.

[0212] An exemplary auristatin embodiment is MMAE: [ka] Here, the wavy line indicates the position of covalent attachment to the linker of the antibody-drug or drug-linker conjugate (-LZ-Ab or -L-Z', as described herein).

[0213] Another exemplary auristatin embodiment is MMAF: [ka] where the wavy line indicates the position of covalent attachment to the linker of the antibody-linker conjugate (-LZ-Ab or -L-Z', as described herein), as disclosed in US 2005 / 0238649:

[0214] Auristatins can be prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S. Pat. 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.

[0215] Maytansinoids The antibodies and antigen-binding fragments thereof described herein may be conjugated to a cytotoxin that is a microtubule-binding agent. In some embodiments, the microtubule-binding agent is maytansine, a maytansinoid, or a maytansinoid analog. Maytansinoids are mitotic inhibitors that act by binding to microtubules and inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids (e.g., maytansinol and C-3 maytansinol esters) (U.S. Pat. No. 4,151,042). Synthetic maytansinol, as well as its derivatives and analogs, are disclosed in, for example, U.S. Patents 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; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and Maytansinoid drug moieties are attractive drug moieties for antibody-drug conjugates because they are: (i) relatively accessible to preparation by fermentation or chemical modification or derivatization of the fermentation products, (ii) amenable to derivatization 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.

[0216] Examples of suitable maytansinoids include esters of maytansinol, synthetic maytansinol, and maytansinol analogues and derivatives. Included herein as a maytansinoid, maytansinol, and maytansinol analogues and derivatives is any cytotoxin that inhibits microtubule formation and is highly toxic to mammalian cells.

[0217] Examples of suitable maytansinol esters include those with modified aromatic rings, and those with modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patents 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; 4,361,650; 4,362,663; 4,364,866; 4,424,219; 4,450,254; Nos. 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, each of which is incorporated by reference herein as relating to maytansinoids and derivatives thereof.

[0218] In some embodiments, the antibody-drug conjugates (ADCs) of the invention contain, as the cytotoxic drug, a thiol-containing maytansinoid (DM1) (formerly known as N 2 '-Deacetyl-N 2 DM1 is represented by the following structural formula IV: [ka]

[0219] In another embodiment, the conjugate of the invention comprises a thiol-containing maytansinoid N 2 '-Deacetyl-N 2 Utilizing '(4-methyl-4-mercapto-1-oxopentyl)-maytansine (e.g., DM4) as a cytotoxic drug. DM4 is represented by the following structural formula V: [ka]

[0220] Another maytansinoid that contains a side chain containing a sterically hindered thiol bond is N 2 '-Deacetyl-N- 2 '(4-mercapto-1-oxopentyl)-maytansine (referred to as DM3), which is represented by the following structural formula VI: [ka]

[0221] Each of the maytansinoids taught in U.S. Patent Nos. 5,208,020 and 7,276,497 may also be used in the conjugates of the present invention, the entire disclosures of which are incorporated herein by reference in this regard.

[0222] Many positions on the maytansinoid can serve as a position for covalent attachment to a linking moiety, and therefore to the antibody or antigen-binding fragment thereof (-LZ-Ab or -L-Z', as described herein). For example, the C-3 position bearing a hydroxyl group, the C-14 position modified with a hydroxymethyl, the C-15 position modified with a hydroxy, and the C-20 position bearing a hydroxy group are all expected to be useful. In some embodiments, the C-3 position serves as a position for covalent attachment to a linker moiety, and in some particular embodiments, the C-3 position of maytansinol serves as a position for covalent attachment to a linking moiety. There are many linking groups known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Patent Nos. 5,208,020, 6,441,163, and European Patent No. 0425235 B1; Chari et al., Cancer Research 52:127-131 (1992); and US 2005 / 0169933 A1, the disclosures of which are expressly incorporated herein by reference. Additional linking groups are described and exemplified herein.

[0223] The present invention also includes various isomers and mixtures of maytansinoids and conjugates. Certain compounds and conjugates of the present invention may exist in various stereoisomeric, enantiomeric, and diastereomeric forms. Producing such antibody-maytansinoid conjugates is described in U.S. Patents 5,208,020, 5,416,064, 6,333,410, 6,441,163, 6,716,821, and 7,368,565, each of which is incorporated herein in its entirety.

[0224] Anthracyclines In other embodiments, the antibodies and antigen-binding fragments thereof described herein may be conjugated to a cytotoxin that is an anthracycline molecule. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Studies have shown that anthracyclines can kill cells by a number of different mechanisms, including: 1) the drug molecule intercalates into the cell's DNA, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) the drug produces free radicals, which then react with cellular macromolecules and cause damage to the cell; or 3) the drug molecule interacts with the cell membrane [see, e.g., C. Peterson et al., "Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia" in Anthracycline Antibiotics In Cancer Therapy; See NR Bachur, "Free Radical Damage" id. at pp.97-102.] Because of their potential for cytotoxicity, anthracyclines are used in the treatment of a number of cancers, such as leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma [see, e.g., PH-Wiernik, in Anthracycline: Current Status And New Developments see p 11. Commonly used anthracyclines include doxorubicin, epirubicin, idarubicin and daunomycin.

[0225] Representative examples of anthracyclines include, but are not limited to, daunorubicin (Cerubidine; Bedford Laboratories), doxorubicin (Adriamycin; Bedford Laboratories; also known as doxorubicin hydrochloride, hydroxy-daunorubicin, and Rubex), epirubicin (Ellence; Pfizer), and idarubicin (Idamycin; Pfizer). The anthracycline analog doxorubicin (ADRIAMYCINO) is believed to interact with DNA by intercalation and inhibit the progression of the enzyme topoisomerase II, which unwinds DNA for transcription. Doxorubicin stabilizes the topoisomerase II complex after it has broken the DNA strand for replication, preventing the DNA double helix from reclosing, thereby halting the replication process. Doxorubicin and daunorubicin (DAUNOMYCIN) are the prototypic cytotoxic natural product anthracycline chemotherapeutic agents (Sessa et al., (2007) Cardiovasc. Toxicol. 7:75-79).

[0226] A non-limiting example of an anthracycline suitable for use herein is PNU-159682 ("PNU"). PNU is more than 3000 times more cytotoxic than its parent nemorubicin (Quintieri et al., Clinical Cancer Research 2005, 11, 1608-1617). PNU is represented by the following structural formula: [ka]

[0227] Many positions on anthracyclines, such as PNU, can serve as positions for covalent attachment to the linking moiety, and therefore to an anti-CD137 antibody or antigen-binding fragment thereof, as described herein. For example, a linker may be introduced via modification to a hydroxymethyl ketone side chain.

[0228] In some embodiments, the cytotoxin is a PNU derivative having the following structural formula: [ka] Here, the wavy line indicates the position of covalent attachment to a linker of an ADC as described herein.

[0229] In some embodiments, the cytotoxin is a PNU derivative having the following structural formula: [ka] Here, the wavy line indicates the position of covalent attachment to a linker of an ADC as described herein.

[0230] Pyrrolobenzodiazepines (PBDs) In other embodiments, the anti-CD137 antibodies or antigen-binding fragments thereof described herein may be conjugated to a cytotoxin that is or includes a pyrrolobenzodiazepine (PBD). PBDs are produced by certain actinomycetes and have been shown to be sequence-selective DNA alkylating compounds. PBD cytotoxins include, but are not limited to, anthramycin, dimeric PBDs, and those disclosed, for example, in Hartley, JA (2011). "The development of pyrrolobenzodiazepines as antitumour 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.

[0231] In some embodiments, the cytotoxin may be a pyrrolobenzodiazepine dimer having the following structural formula: [ka] Here, the wavy line indicates the position of covalent attachment to a linker of an ADC as described herein. This PBD-based ADC is disclosed, for example, in Sutherland et al., Blood 2013 122:1455-1463, which is incorporated by reference in its entirety.

[0232] In some embodiments, the cytotoxin may be a PBD dimer having the following structural formula: [ka] wherein n is 3 or 5, where the wavy line indicates the position of covalent attachment to a linker of an ADC as described herein.

[0233] In some embodiments, the cytotoxin may be a PBD dimer having the following structural formula: [ka] Here, the wavy line indicates the position of covalent attachment to a linker of an ADC as described herein.

[0234] In certain embodiments, the cytotoxin may be a PBD dimer, which, when taken together with a linker and a reactive moiety structure Z′, may be represented by the following structure: [ka]

[0235] This particular cytotoxin-linker conjugate is known as Tesilin (SG3249) and is 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.

[0236] In certain embodiments, the cytotoxin may be a PBD dimer, which, when taken together with a linker and a reactive moiety structure Z′, may be represented by the following structure: [ka]

[0237] This particular cytotoxin-linker conjugate is known as taliline and is 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.

[0238] Calicheamicin In other embodiments, the antibodies and antigen-binding fragments thereof described herein may be conjugated to a cytotoxin that is an enediyne antitumor antibiotic (e.g., calicheamicin, ozogamicin). The calicheamicin family of antibiotics is capable of generating double-stranded DNA breaks at sub-picomolar concentrations. Examples of calicheamicins suitable for use in the present invention are disclosed, for example, in U.S. Pat. No. 4,671,958; U.S. Pat. No. 4,970,198; U.S. Pat. No. 5,053,394; U.S. Pat. No. 5,037,651; and U.S. Pat. No. 5,079,233, which are incorporated herein in their entireties.

[0239] Exemplary calicheamicins have been designated γ1, also referred to herein simply as gamma, and have the following structural formula: [ka]

[0240] In some embodiments, the calicheamicin is a gamma-calicheamicin derivative or an N-acetyl-gamma-calicheamicin derivative. Structural analogs of calicheamicin that may be used include, but are not limited to, those disclosed in Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the U.S. patents cited above. Calicheamicin contains a methyl trisulfide moiety that can react with an appropriate thiol to form a disulfide, which at the same time introduces a functional group useful for coupling the calicheamicin derivative to an anti-CD137 antibody or antigen-binding fragment thereof described herein via a linker. For the preparation of conjugates of the calicheamicin family, see U.S. Patent 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 Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the above U.S. patents to American Cyanamid.

[0241] In one embodiment, the cytotoxin of the ADCs disclosed herein is a calicheamicin disulfide derivative having the following formula: [ka] Here, the wavy line indicates the position of attachment of the linker.

[0242] Additional cytotoxins In other embodiments, the antibodies and antigen-binding fragments thereof described herein may be conjugated to cytotoxins other than, or in addition to, the cytotoxins disclosed herein above. Additional cytotoxins suitable for use with the compositions and methods described herein include, but are not limited to, 5-ethynyluracil, abiraterone, acylfulvene, adecypenol, adozelesin, aldesleukin, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antarelix, anti-dorsalizing morphogenetic protein-1, protein-1, antiandrogens, prostate cancer, antiestrogens, antineoplastic agents (antineoplastons), antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III. derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorines, benzoylstaurosporine, β-lactam derivatives,beta-alethine, beta-clamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistratene A, bizelesin, breflate, bleomycin A2, bleomycin B2, bropirimine, budotitane, buthionine sulfoximine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives (e.g., 10-hydroxy-camptothecin), capecitabine, carboxamido-amino-triazoles, carboxyamidotriazoles, carzelesin, casein kinase inhibitors, castanospermine, cecropin B, cetrorelix, chlorins, chloroquinoxaline sulfonamides sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene and its analogs, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogs, conagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cycloplatam, cypemycin,Cytarabine ocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B, 2'-deoxycoformycin (DCF), deslorelin, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin, diphenylspiromustine spiromustine, discodermolide, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epothilones, epithilones, epristeride, estramustine and its analogs, etoposide, etoposide 4'-phosphate 4'-phosphate) (also called etopophos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol,Flezelastine, 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 acid, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridone, imiquimod, immune stimulating peptides, iobenguane, iododoxorubicin, ipomeanol, irinotecan, iroplact, irsogladine, isobengazole, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate sulfate, leptolstatin, letrozole, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol, lonidamine, losoxantrone,loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, masoprocol, maspin, matrix metalloproteinase inhibitors, menogaril, merbarone, meterelin, methioninase, metoclopramide, MIF inhibitors, mifepristone, miltefosine, mirimostim im), mithracin, mitoguazone, mitolactol, mitomycin and its analogues, mitonafide, mitoxantrone, mofarotene, molgramostim, mycaperoxide B, myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin (nafarelin), nagrestip, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, nilutamide, nisamycin, nitrullyn, octreotide, oxenone, onapristone ristone, ondansetron, oracin, ormaplatin, oxaliplatin, oxaunomycin, paclitaxel and its analogs, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene,parabactin, pazelliptine, pegaspargase, peldesine, sodium pentosan polysulfate, pentostatin, pentrozole, perflubron, perfosfamide, phenazinomycin, picibanil, pirarubicin, piritrexim, podophyllotoxin, Porfiromycin, purine nucleoside phosphorylase inhibitors, raltitrexed, rhizoxin, rogletimide, rohitukine, rubiginone B1, ruboxyl, safingol, saintopin, sarcophytol A, sargramostim, sobuzoxane, sonermin, sparfosic acid acid, spicamycin D, spiromustine, stipiamide, sulfinosine, tallimustine, tegafur, temozolomide, teniposide, thaliblastine, thiocoraline, tirapazamine, topotecan, topsentin, triciribine, trimetrexate, veramine, vinorelbine, vinxaltine, vorozole, zeniplatin, and zilascorb.

[0243] Linker The term "linker" as used herein means a bivalent chemical moiety, including a covalent bond or chain of atoms, that covalently attaches an anti-CD137 antibody or fragment thereof (Ab) to a drug moiety (D) to form an antibody-drug conjugate (ADC) of formula I. A suitable linker has two reactive ends, one for conjugation to an antibody and the other for conjugation to a cytotoxin. The reactive end of the linker that conjugates to an antibody (reactive moiety, Z') is typically a site that can be conjugated to the antibody via a cysteine ​​thiol or lysine amine group on the antibody, and is therefore typically a group reactive with thiols such as double bonds (e.g., maleimide, etc.), or leaving groups such as chloro, bromo, iodo, or R-sulfanyl, or a group reactive with amines such as a carboxyl group; whereas the reactive end of the linker that conjugates to a cytotoxin is typically a site that can be conjugated to a cytotoxin. Non-limiting examples of linker-cytotoxin conjugation include, for example, formation of an amide bond with a basic amine or carboxyl group on the cytotoxin (via a carboxyl or basic amine group of the linker, respectively), or formation of an ether or the like (via alkylation of an OH group of the cytotoxin, e.g., via a leaving group of the linker). In some embodiments, the cytotoxin-linker conjugation is via formation of an amide bond with an amine or carboxyl group of the cytotoxin, and in which case the reactive substituent of the linker is a carboxyl or basic amine group, respectively. When the term "linker" is used to describe a conjugated form of a linker, one or both of the reactive ends are either absent (e.g., converted to a reactive moiety Z', chemical moiety Z) or in the process of reacting (e.g., only a carboxylic acid carbonyl, etc.) as bonds are formed between the linker and / or the cytotoxin, and between the linker and / or the antibody or antigen-binding fragment thereof. Such conjugating reactions are further described herein below.

[0244] A variety of linkers can be used to conjugate the described antibodies or antibody fragments to cytotoxic molecules. In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In yet other embodiments, the linker unit is not cleavable, and the drug is released, for example, by antibody degradation. Linkers useful in the ADCs of the invention are preferably stable outside the cell, prevent the ADC molecules from aggregating, and keep the ADC soluble in aqueous media and in a monomeric form. Before being transported or delivered into the cell, the ADC is preferably stable and remains intact, i.e., the antibody remains linked to the drug moiety. The linker is stable outside the target cell and may be cleaved at an effective rate inside the cell. An effective linker: (i) maintains the specific binding properties of the antibody; (ii) allows the conjugate or drug moiety to be delivered intracellularly; (iii) remains stable and intact, i.e., is not cleaved, until the conjugate is delivered or transported to its target site; and (iv) maintains the cytotoxic, cytotoxic or cytostatic effect of the cytotoxic moiety. The stability of the ADC can be measured by standard analytical techniques, such as mass spectrometry, HPLC, and LC / MS separation / analysis techniques. Covalent attachment of the antibody and the drug moiety requires that the linker has two reactive functional groups (i.e., is bivalent in the reactive sense). 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 obtaining these conjugates have been described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, p.234-242).

[0245] Suitable cleavable linkers include, for example, those that can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al., Bioorg.Med.Chem, 20:571-582, 2012, the disclosures of which are incorporated herein by reference as they relate to linkers suitable for covalent conjugation). Suitable cleavable linkers can include, for example, chemical moieties such as hydrazines, disulfides, thioethers, or dipeptides.

[0246] Linkers that are hydrolyzable under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, and the like (see, e.g., U.S. Patent 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 for their disclosures regarding linkers suitable for covalent conjugation). Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but are unstable below pH 5.5 or 5.0, which is approximately the pH of the lysosome.

[0247] Linkers that are cleavable under reducing conditions include, for example, disulfides. A variety of disulfide linkers are known in the art, including, for example, disulfide linkers that can be formed using 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-pyridyl-dithio)toluene), SPDB and SMPT (see, e.g., 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. Patent No. 4,880,935, the disclosures of each of which are incorporated herein by reference in their entirety for purposes of linkers suitable for covalent conjugation.

[0248] Linkers that are susceptible to enzymatic hydrolysis include, for example, intracellular peptidase or protease enzymes (such as lysosomal or endosomal proteases). The peptidyl linker may be a peptide-containing linker that is cleaved by, but is not limited to, a peptide-containing linker that is cleaved by, for example, a cleavage site, such as, for example, a peptide-containing linker that is cleaved by ... Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). In some embodiments, the linker includes 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 including dipeptides such as Val-Cit or Phe-Lys are disclosed, for example, in U.S. Patent No. 6,214,345 (the disclosure of which is incorporated herein by reference in its entirety as it relates to linkers suitable for covalent conjugation). In some embodiments, the linker includes a dipeptide selected from Val-Ala and Val-Cit.

[0249] Suitable linkers for attachment to the antibodies or antibody fragments described herein include linkers capable of releasing cytotoxins via a 1,6-elimination process, such as chemical moieties p-aminobenzyl (PAB) groups, 6-maleimidohexanoic acid, pH-sensitive carbonates, and other reagents described in Jain et al., Pharm. Res. 32:3526-3540, 2015, the disclosure of which is incorporated herein by reference in its entirety as it relates to linkers suitable for covalent conjugation.

[0250] In some embodiments, the linker includes a "self-immolative" group such as PAB or PABC (para-aminobenzyloxycarbonyl) as described above, e.g., in Carl et al., J. Med. Chem. (1981) 24:479-480; Chakravarty et al (1983) J. Med. Chem. 26:638-644; US 6214345; US20030130189; US20030096743; US6759509; US20040052793; US6218519; US6835807; US6268488; US20040018194; WO98 / 13059; US20040052793; US6677435; US5621002; US20040121940; WO2004 / 032828. Other such chemical moieties ("self-immolative linkers") that enable this process include methylene carbamates and heteroaryl groups, such as aminothiazoles, aminoimidazoles, aminopyrimidines, and the like. Linkers containing such heterocyclic self-immolative groups are disclosed, for example, in U.S. Patent Application Publications 20160303254 and 20150079114, as well as 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 7,223837. In some embodiments, a dipeptide is used in combination with a self-immolative linker.

[0251] Linkers suitable for use herein may further include one or more groups selected from 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, where p is an integer from 1 to 6, independently selected for each occurrence.

[0252] 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, sulfonylcycloalkyl, aryl, or heteroaryl group is optionally substituted with 1 to 5 substituents independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl-heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0253] 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, sulfonylcycloalkyl, aryl, or heteroaryl group may be optionally interrupted by one or more heteroatoms selected from O, S, and N.

[0254] 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, sulfonylcycloalkyl, aryl, or heteroaryl group may be optionally interrupted by one or more heteroatoms selected from O, S, and N, and is optionally substituted with 1 to 5 substituents each independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkyl-heteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0255] Suitable linkers may contain groups that have solubility enhancing properties, for example (CH2CH2O). p Linkers containing units (polyethylene glycol, PEG) can enhance solubility (e.g., alkyl chains can be substituted with amino, sulfonic acid, phosphonic acid, or phosphate residues to enhance solubility). Linkers containing such moieties are disclosed, for example, in U.S. Patent Nos. 8,236,319 and 9,504,756, the disclosures of each of which are incorporated herein by reference in their entirety as they relate to linkers suitable for covalent conjugation. Further solubility enhancing groups include, for example, acyl and carbamoyl sulfamide groups having the following structures: [ka] where a is 0 or 1; and R 10 is 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 groups, 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 is selected from the group consisting of O, S, and NR 11 R 12 wherein R 11 and R 12 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; or R 10 is a cytotoxin, where the cytotoxin is optionally attached to N via a spacer moiety. Linkers containing 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 by reference in their entireties as they relate to linkers suitable for covalent conjugation to cytotoxins and antibodies or antigen-binding fragments thereof.

[0256] In some embodiments, the linker is a hydrazine, a disulfide, a thioether, a dipeptide, a p-aminobenzyl (PAB) group, a heterocyclic self-immolative 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, an acyl, -(C=O)-, or -(CH2CHO). p- groups, where p is an integer from 1 to 6. Some of ordinary skill in the art will recognize that one or more of the listed groups may exist in the form of a divalent (diradical) species (e.g., C1-C6 alkylene, etc.).

[0257] In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the p-aminobenzyl group is disposed between the cytotoxic drug and a protease cleavage site in the linker. In some embodiments, the p-aminobenzyl group is part of a p-aminobenzyloxycarbonyl unit. In some embodiments, the p-aminobenzyl group is part of a p-aminobenzylamide unit.

[0258] 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.

[0259] 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.

[0260] In some embodiments, the linker is 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.

[0261] In some embodiments, the linker is -(C=O)(CH2) p - units, where p is an integer from 1 to 6.

[0262] In some embodiments, the linker is -(CH2) n - units, where n is an integer from 2 to 6. In some embodiments, the linker comprises -((CH2) n In some embodiments, the LZ comprises: [ka] where S is a sulfur atom representing a reactive substituent present in an antibody, or antigen-binding fragment thereof, that binds to CD137 (e.g., resulting from the -SH group of a cysteine ​​residue).

[0263] In some embodiments, the linker is ((CH2) m O) n (CH2) m - group, where n and m are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and a heteroaryl group, where the heteroaryl group is a triazole. In some embodiments, ((CH2) m O) n (CH2) m The - group and the triazole together include [ka] where n is 1-10 and the wavy line indicates the position of attachment to an additional linker component, chemical substructure Z, or amatoxin. Other linkers that may be used in the methods and compositions described herein are described in U.S. Patent Application Publication No. 2019 / 0144504, which is incorporated herein by reference.

[0264] In a specific embodiment, the linker comprises PAB-Ala-Val-propionyl and is represented by the structure: [ka] Here, the wavy lines indicate the positions of attachment to the cytotoxin and reactive moiety Z'.

[0265] In another specific embodiment, the linker comprises PAB-Cit-Val-propionyl and is represented by the structure: [ka] Here, the wavy lines indicate the position of attachment to the cytotoxin and the reactive moiety Z'. Such PAB-dipeptide-propionyl linkers are disclosed, for example, in published patent application WO2017 / 149077, which is incorporated herein by reference in its entirety. Additionally, the cytotoxins disclosed in WO2017 / 149077 are incorporated herein by reference.

[0266] It will be appreciated by one of skill in the art that any one or more of the chemical groups, moieties, and features disclosed herein may be combined in a variety of ways to form linkers useful for conjugating antibodies and cytotoxins as disclosed herein. Additional linkers useful 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.

[0267] In certain embodiments, an intermediate that is a precursor of a linker is reacted with a drug moiety under suitable conditions. In certain embodiments, a reactive group is used on the drug and / or the intermediate or linker. The product of the reaction between the drug and the intermediate, or a derivatized drug, is then reacted with an antibody or antigen-binding fragment under suitable conditions. Alternatively, the linker or intermediate may be first reacted with the antibody or derivatized antibody and then reacted with the drug or derivatized drug. Such conjugation reactions are now more fully described.

[0268] Many different reactions can be used to covalently attach linkers or drug-linker conjugates to the antibody or antigen-binding fragment thereof. Suitable attachment locations on the antibody molecule include the amine groups of lysines, the free carboxylic acid groups of glutamic and aspartic acids, the sulfhydryl groups of cysteine, and various moieties of aromatic amino acids. For example, non-specific covalent attachment may be performed using a carbodiimide reaction to link a carboxy (or amino) group on a compound to an amino (or carboxy) group on the antibody moiety. Additionally, bifunctional agents such as dialdehydes or imidoesters may be used to couple amino groups on a compound to amino groups on the antibody moiety. Drugs may also be attached to the binding agent using the Schiff base reaction. This method involves periodate oxidation of a drug containing a glycol or hydroxyl group, thus forming an aldehyde that is then reacted with the binding agent. Attachment occurs via the formation of a Schiff base with the amino group of the binding agent. Isothiocyanates may also be used as coupling agents to covalently attach drugs to binding agents. Other techniques are known to those skilled in the art and are within the scope of the present invention.

[0269] As described herein, linkers useful for conjugation to an antibody or antigen-binding fragment include, but are not limited to, linkers comprising a chemical substructure Z formed by a coupling reaction, as shown below in Table 2. The curved lines indicate the positions of attachment to the antibody or antigen-binding fragment, and the cytotoxic molecule, respectively.

[0270] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0271] One of skill in the art will recognize that the linker and the reactive substituent Z' attached to the reactive substituent on the antibody or antigen-binding fragment thereof are those that participate in a covalent coupling reaction to generate chemical moiety Z, and recognize the reactive moiety Z'. Thus, antibody-drug conjugates useful in conjunction with the methods described herein may be formed by reacting an antibody or antigen-binding fragment thereof with a linker or cytotoxin-linker conjugate, as described herein, that includes a reactive substituent Z', etc., suitable for reaction with a reactive substituent on an antibody or antigen-binding fragment thereof to form chemical moiety Z.

[0272] As shown in Table 2, examples of suitable reactive substituents on the linker and antibody, or antigen-binding fragment thereof, include nucleophilic / electrophilic pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, etc.), diene / dienophile pairs (e.g., azide / alkyne pairs, or diene / α,β-unsaturated carbonyl pairs, among others). Coupling reactions between reactive substituents to form chemical moieties 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., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, among others), aromatic nucleophilic substitution, aromatic electrophilic substitution, and other reaction modes known in the art or described herein. Preferably, the linker comprises an electrophilic functional group for reaction with a nucleophilic functional group on the antibody or antigen-binding fragment thereof.

[0273] As disclosed herein, reactive substituents that may be present in an antibody, or antigen-binding fragment thereof, include nucleophilic groups such as, but are not limited to, (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 (where the antibody is glycosylated). As disclosed herein, reactive substituents that may be present in an antibody, or antigen-binding fragment thereof, include, but are not limited to, the hydroxyl moieties of serine, threonine, and tyrosine residues; the amino moieties of lysine residues; the carboxyl moieties of aspartic acid and glutamic acid residues; and the thiol moiety of cysteine ​​residues, as well as the propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of unnatural amino acids. In some embodiments, as disclosed herein, reactive substituents present in an antibody, or antigen-binding fragment thereof, include an amine or thiol moiety. Certain antibodies have reducible interchain disulfides (i.e., cysteine ​​bridges). Antibodies can be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine ​​bridge thus theoretically forms two reactive thiol nucleophiles. Additional nucleophiles can be introduced into antibodies by reaction of lysines with 2-iminothiolane (Traut's reagent) to convert amines to thiols. Reactive thiol groups may be introduced into antibodies (or fragments thereof) by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing a mutant antibody containing one or more non-existent cysteine ​​amino acid residues). U.S. Patent No. 7,521,541 teaches engineered antibodies by introducing reactive cysteine ​​amino acids.

[0274] In some embodiments, the reactive moiety Z' attached to the linker is a nucleophilic group that is reactive with an electrophilic group present on an antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of the nucleophilic group may react with an electrophilic group on an antibody to form a covalent bond with the antibody. Useful nucleophilic groups include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide.

[0275] In some embodiments, Z is the product of a reaction between reactive nucleophilic substituents (e.g., amine and thiol moieties) present in an antibody or antigen-binding fragment thereof and a reactive electrophilic substituent Z'. For example, Z' can be a Michael acceptor (e.g., maleimide), an activated ester, an electron-deficient carbonyl compound, and an aldehyde, among others. Some representative and non-limiting examples of reactive substituents Z' and the resulting chemical substructures Z are shown in Table 3.

[0276] [Table 3]

[0277] For example, suitable linkers for the synthesis of ADCs include, but are not limited to, reactive substituents Z', such as maleimide or haloalkyl groups, which may be attached to the linker by reagents such as succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among others, as 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.

[0278] 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 the non-cleavable maleimidocaproyl-based linker, which is particularly useful for conjugating microtubule disrupting agents such as auristatins. Such linkers are described in Doronina et al., Bioconjugate Chem. 17:14-24, 2006, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation.

[0279] In some embodiments, the reactive substituent Z' is -(C=O)- or -NH(C=O)-, such that the linker may be attached to the antibody or antigen-binding fragment thereof through an amide or urea moiety resulting from reaction of the -(C=O)- or -NH(C=O)- group with an amino group of the antibody or antigen-binding fragment thereof, respectively.

[0280] In some embodiments, the reactive substituent is an N-maleimidyl group, a halogenated N-alkylamide group, a sulfonyloxy N-alkylamide group, a carbonate group, a halogenated sulfonyl group, a thiol group or derivatives 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 azide group, a phosphine group, a nitrile oxide group, a nitrone group, a nitrile imine group, a diazo group, a ketone group, an (O-alkyl)hydroxylamino group, a hydrazine group, a halogenated N-maleimidyl group, a 1,1-bis(sulfonylmethyl)methylcarbonyl group or elimination derivatives thereof, a carbonyl halide group, or an allenamide group, each of which may be optionally substituted. In some embodiments, the reactive substituent comprises a cycloalkene group, a cycloalkyne group, or an optionally substituted (hetero)cycloalkynyl group.

[0281] Non-limiting examples of amatoxin-linker conjugates containing a reactive substituent Z' suitable for reaction with a reactive residue on the antibody or antigen-binding fragment thereof include, but are not limited to, 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin; 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin; 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin; cin;7'C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperazin-1-yl)-amatoxine;7'C-(4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)-amatoxine;7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine ;7'C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(3-carboxypropanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)-amatoxine C-(4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(maleimido)acetyl)piperazin-1-yl)-amatoxine;7'C-(4-(3-(maleimido)propanoyl)piperazin-1-yl)-amatoxine;7'C-(4-(4-(maleimido)butanoyl)piperazin-1-yl)-amatoxine;7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxine;7'C-(3-((6-(6-(maleimido)hexanamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxine;7'C-(3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrolidin-1-yl)- Amatoxin;7'C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7'C-(4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7'C-(4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)-amatoxin;7'C-(4-(4-(2-(aminooxy)acetamido)butanoyl)piperidine (R)-7'C-((4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazin-1-yl)-amatoxin;7'C-((4-(6-(maleimido)hexanamido)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(6-(maleimido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;(R)-7'C-((3-( (6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;(S)-7'C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl)-amatoxine 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine;7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C- ((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(3-carboxypropanamido)ethyl)piperidine 7'C-((4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(maleimido)acetyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(3-(maleimido)propanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(4-(maleimido)butanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(2-(maleimido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C- ((3-((6-(maleimido)hexanamido)methyl)azetidin-1-yl)methyl)-amatoxine;7'C-((3-(2-(6-(maleimido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxine;7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amatoxine;7'C-((3-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)azetidin-1-yl)methyl)-amatoxine;7' C-((3-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxine;7'C-(((2-(6-(maleimido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxine;7'C-(((4-(6-(maleimido)-N-methylhexanamido)butyl(methyl)amino)methyl)-amatoxine;7'C-((2-(2-(6-(maleimido)hexanamido)ethyl)aziridin-1-yl)methyl) -Amatoxin;7'C-((2-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxin;7'C-((4-(6-(6-(2-(aminooxy)acetamido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-oyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(2-(2-(aminooxy)acetamido)acetyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(3-(2-(aminooxy)acetamido)propanoyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin ;7'C-((4-(2-(2-(2-(aminooxy)acetamido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(20-(aminooxy)-4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaicosyl)piperidin-1-yl)methyl)-amatoxine;7'C-(((2-(6-(2-(aminooxy)amatoxine 7'C-(((4-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)butyl)(methyl)amino)methyl)-amatoxin;7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-S-methyl)-amatoxin;7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-S-methyl)-amatoxin 7'C-((4-(2-(2-bromoacetamido)ethyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;6'O-(6-(6-(maleimido)hexanamido)hexyl)-amatoxine;6'O-(5-(4-((maleimido)methyl)cyclohexanecarboxamido)pentyl)-amatoxine;6'O-(2-((6-(maleimido)hexyl)oxy)-2-oxoethyl)-amatoxine;6'O-((6-(maleimido)hexyl)carbamoyl)-amatoxine;6'O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexyl)carbamoyl)-amatoxine;6'O-(6-(2-bromoacetamido)hexyl)-amatoxine;7'C-(4-(6-(azido)hexanamido)piperidin-1-yl)-amatoxine;7'C-(4-(hex-5-ynoylamino)piperidin-1-yl)-amatoxine toxin;7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;7'C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;6'O-(6-(6-(11,12-didehydro-5,6-dihydro-dibenz[b,f]azocin-5-yl)-6-oxohexanamido)hexyl)-amatoxin;6'O-(6-(hex-5-ynoylamino)hexyl)-amatoxin;6'O-(6-(2-(aminooxy)acetylamido)hexyl)-amatoxin;6'O-((6-aminooxy)hexyl)-amatoxin;and 6'O-(6-(2-iodoacetamido)hexyl)-amatoxin;

[0282] In some embodiments, the chemical substructure Z is selected from Table 2 or Table 3. In some embodiments, the chemical substructure Z is: [ka] Here, S is a sulfur atom representing a reactive substituent present in an antibody, such as an anti-CD-137 antibody, or an antigen-binding fragment thereof.

[0283] In some embodiments, the amatoxins disclosed herein are conjugated to a linker-reactive moiety structure, -L-Z', having the formula: [ka] Here, the wavy line indicates the location of attachment to a substituent on the cytotoxin (e.g., an amatoxin). This linker-reactive substituent, L-Z', may alternatively be referred to as N-beta-maleimidopropionyl-Val-Ala-para-aminobenzyl (BMP-Val-Ala-PAB).

[0284] In some embodiments, the amatoxins disclosed herein are conjugated to a linker-reactive moiety, -L-Z', having the formula: [ka] Here, the wavy line indicates the location of attachment to a substituent on the cytotoxin (e.g., an amatoxin). This linker-reactive substituent, L-Z', may alternatively be referred to as N-beta-maleimidopropyl-Val-Cit-para-aminobenzyl (BMP-Val-Cit-PAB).

[0285] In some embodiments, the linker L and chemical moiety Z are taken together as LZ: [ka] where 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 amatoxin.

[0286] In some embodiments, the linker L and chemical moiety Z, together after conjugation to the antibody, as LZ-Ab, have the following structure: [ka] where 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 amatoxin.

[0287] The above linker moieties and amatoxin-linker conjugates are useful, inter alia, in connection with the compositions and methods described herein and 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 by reference herein in their entireties.

[0288] The above linker moieties and amatoxin-linker conjugates are useful, inter alia, in connection with the compositions and methods described herein and 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 by reference herein in their entireties.

[0289] In one embodiment, a CD137 antibody or antigen-binding fragment thereof as described herein may be conjugated to an amatoxin to form a conjugate represented by the formula Ab-ZL-Am, where Ab is the CD137 antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an amatoxin, each as described herein.

[0290] In some embodiments, Am-LZ-Ab is: [ka] It is.

[0291] In some embodiments, Am-LZ-Ab is: [ka] It is.

[0292] In some embodiments, Am-LZ-Ab is: [ka] It is.

[0293] In some embodiments, Am-LZ-Ab is: [ka] It is.

[0294] In some embodiments, Am-LZ-Ab is: [ka] It is.

[0295] Preparation of antibody-drug conjugates In the ADCs of formula I disclosed herein, an anti-CD137 antibody, or antigen-binding fragment thereof, is conjugated to one or more cytotoxic drug moieties (D) (e.g., about 1 to about 20 drug moieties per antibody) via a linker L and a chemical moiety Z as disclosed herein. The ADCs of the invention can be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those of skill in the art, including: (1) reacting a reactive substituent of an antibody, or antigen-binding fragment thereof, with a bivalent linker reagent to form Ab-ZL, as described herein above, followed by reaction with a drug moiety D; or (2) reacting a reactive substituent of a drug moiety with a bivalent linker reagent to form DL-Z', followed by reaction with a reactive substituent of an antibody, or antigen-binding fragment thereof, as described herein above. Additional methods for preparing ADCs are described herein.

[0296] In another embodiment, the anti-CD137 antibody, or antigen-binding fragment thereof, has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. The ADC is then formed by conjugating through the sulfur atom of the sulfhydryl group as described hereinabove. Reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).

[0297] In another embodiment, 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, and the ADC is then formed by conjugation through the sulfur atom of the sulfhydryl group, as described hereinabove.

[0298] In yet another embodiment, the anti-CD137 antibody may have one or more carbohydrate groups that may be oxidized to provide aldehyde (-CHO) groups (see, e.g., Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). The ADC is then formed by conjugation via the corresponding aldehyde as described hereinabove. Other protocols for modifying proteins to attach or associate with cytotoxins are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), incorporated herein by reference.

[0299] Methods for conjugating linker-drug moieties to cell-targeting proteins, such as antibodies, immunoglobulins or fragments thereof, are described, for example, in 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 by reference herein in their entireties.

[0300] Route of administration and dosage Alternatively, a fusion protein comprising the antibody and cytotoxic drug may be produced, for example, by recombinant techniques or peptide synthesis. The length of DNA may contain regions encoding the two portions of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0301] The ADCs described herein may be administered to a patient (e.g., a human patient suffering from an autoimmune disease or cancer) in a variety of dosage forms. For example, the ADCs described herein may be administered to a patient suffering from an autoimmune disease or cancer in the form of an aqueous solution, such as an aqueous solution containing one or more pharma- ceutically acceptable excipients. Suitable pharma-ceutically acceptable excipients for use with the compositions and methods described herein include viscosity modifiers. The aqueous solution may be sterilized using techniques known in the art.

[0302] Pharmaceutical formulations comprising anti-CD137 ADCs as described herein are prepared by mixing such ADCs with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, AEd. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages 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, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 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 sugars such as 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 non-ionic surfactants such as polyethylene glycol (PEG).

[0303] The amount of ADC administered should be sufficient to reduce cells (e.g., activated T cells) that reject allogeneic cell therapy (e.g., CAR cell therapy). While determining a therapeutically effective dose is within the ability of one of skill in the art, in embodiments of the methods described herein utilizing systemic administration of ADC, e.g., to treat immune disease or cancer, an effective human dose will be in the range of 0.1-150 mg / kg (e.g., 5 mg / kg, 10 mg / kg, 25 mg / kg, 50 mg / kg, 75 mg / kg, 100 mg / kg, 150 mg / kg, etc.). The route of administration may affect the recommended dose. Depending on the mode of administration employed, repeated systemic administration is contemplated to maintain effective levels, e.g., to reduce the risk of CAR-T cell rejection.

[0304] The anti-CD137 ADCs described herein may be administered by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular, or parenteral. The most suitable route of administration in any given case will depend on the particular ADC, the patient, the pharmaceutical formulation method, the method of administration (e.g., time and route of administration), the patient's age, body weight, sex, the severity of the disease being treated, the patient's diet, and the patient's excretion rate.

[0305] An effective dose of an anti-CD137 ADC described herein may be, for example, in the range of about 0.001 to about 100 mg / kg of body weight per single (e.g., bolus), multiple, or continuous administration, to provide an optimal blood concentration of the anti-CD137 ADC (e.g., a blood concentration of 0.0001 to 5000 μg / mL). A single dose of the anti-CD137 ADC may be administered one or more times (e.g., 2 to 10 times) per day, week, or month to a human patient who is concurrently undergoing or will be undergoing CAR therapy at a time following administration of the anti-CD137 ADC. The anti-CD137 ADC may be administered to a human patient as a single or multiple doses. In one embodiment, the anti-CD137 ADC may be administered in an amount sufficient to reduce the amount of host reactive T cells (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more compared to pre-CAR therapy). EXAMPLES

[0306] The following examples are presented to provide one of ordinary skill in the art with a description of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely exemplary 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 WO2018 / 134787 (published July 26, 2018), the entire contents of which are incorporated herein by reference.

[0307] Example 1. In vitro analysis of anti-CD137-amanitin antibody drug conjugates (ADCs) using an in vitro T cell cytotoxicity assay 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, 2 x 10 cells were stimulated per well of a 384-well plate. 4T cells were seeded and ADCs were added to the wells at various concentrations between 0.003 nM and 30 nM, then placed in an incubator at 37°C and 5% CO2. After 4 days of culture, cells were analyzed by flow cytometry. Cells were stained with the viability marker Live / Dead Yellow (Invitrogen) and measured with a volumetric flow cytometer. The number of live, activated cells (Figure 1A) and live, non-activated cells (Figure 1B) were measured by FSC vs. SSC. Non-specific human IgG conjugated to amanitin (hIgG-amanitin) was used as a negative control. Thus, controls were compared with two different ADCs: 1) the chimeric anti-CD137 antibody BBK2 conjugated to amanitin (CD137-amanitin); and 2) an ADC containing an antibody specific for a T-cell antigen conjugated to amanitin (anti-T cell-amanitin). The anti-T cell-amanitin ADC was used as a positive control because it binds to and kills both activated and non-activated T cells.

[0308] As shown in Figures 1A and 1B, the anti-CD137-amanitin ADC (i.e., "CD137-amanitin") specifically killed activated T cells and did not significantly kill non-activated (resting, steady-state) T cells. Furthermore, a positive control ADC that specifically targets both activated and non-activated T cells (i.e., "anti-T cell-amanitin" ADC) killed both activated and non-activated T cells.

[0309] Example 2. Analysis of GvHD prevention using a Xeno-GvHD mouse model The ability of anti-CD137-amanitin ADC to prevent the formation or development of GvHD was evaluated in vivo using a xeno-GvHD mouse model. Female, 6- to 8-week-old NSG mice were irradiated (200 cGy) and then administered 6 × 10 6GvHD mouse models were generated by transplantation with human peripheral blood mononuclear cells (PBMCs). One day later, animals were administered (at 3 mg / kg) anti-CD137-amanitin ADC (i.e., "CD137-amanitin") or various control reagents (i.e., buffer alone ("PBS"), anti-CD137 antibody ("CD137 naked"), or amanitin-based ADC not specific for CD137 ("isotype-amanitin")). The anti-CD137 antibody used in this example of ADC and naked antibody is chimeric BBK2.

[0310] After dosing, animals were closely followed daily for signs of GvHD and / or impaired physical condition, including but not limited to hunched posture, ruffled fur, weight loss, and / or reduced activity. Animals with significant physical condition issues or weight loss >20% were sacrificed and tissues analyzed for human cells. Peripheral blood and spleens of mice were stained with a cocktail of antibodies including hCD45, mCD45, hCD3, and hCD137 antibodies, and 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 to the input blood volume. Percent survival versus days after treatment is shown in Figure 2. The number of human T cells in the peripheral blood versus days after transplant is shown in Figure 3.

[0311] As shown in FIG. 2, animals treated with a single dose of the anti-CD137-amanitin ADC ("CD137-amanitin") essentially completely prevented GvHD even 80 days after transplantation. However, all animals treated with controls (i.e., PBS, anti-CD137 antibody (naked), and isotype-amanitin) died within 11-13 days after transplantation. These results also indicate that the anti-CD137-amanitin ADC was well tolerated in all animals, and that a single dose of the anti-CD137-amanitin ADC (as opposed to requiring multiple doses) was sufficient to completely prevent GvHD. As shown in FIG. 3, in mice treated with anti-CD137-amanitin, no human T cells were detectable in the peripheral blood of the mice for at least 70 days after transplantation. However, control-treated animals were identified as having detectable human T cells in the peripheral blood of mice several days after transplantation, indicating that GvHD had developed in the control-treated animals.

[0312] Example 3. Measurement of engraftment rate and steady-state T cell depletion in the hNSG-SGM3 mouse model Female, humanized NSG-SGM3 mice were assessed for baseline human hematopoietic (total and T-cell) engraftment in their peripheral blood by flow cytometry using hCD45, mCD45, hCD3, and hCD137 antibodies. Mice were randomly assigned to be treated with CD137-amanitin ADC (chimeric BBK2-amanitin ADC), isotype-amanitin ADC, or anti-T cell-amanitin ADC at a dose of 3 mg / kg each. Engraftment and T cell loss in the periphery of the mice were measured on days 5, 9, 14, 22, and 27 after treatment (Figures 4A and 4B). Peripheral blood was stained to assess changes in chimerism and T cell numbers. Equal volumes of blood were loaded onto a volumetric flow cytometer to measure absolute counts based on the number of events. Engraftment and T cell number loss were normalized to baseline values.

[0313] Results from Figures 4A and 4B show that the anti-CD137-amanitin ADC was well tolerated in this mouse model. Furthermore, engraftment and T cell frequencies (after normalization to baseline) remained near baseline levels, and the reduction in engraftment and T cell frequencies was moderate to transient for mice treated with the anti-CD137-amanitin ADC. These data indicate that T cells are generally not depleted in steady-state humanized NSG mice, indicating that T cells are largely undepleted in mice treated with the anti-CD137-amanitin ADC and therefore may retain their immune function. Conversely, engraftment and T cell numbers were completely abolished in mice treated with an ADC specifically targeting another T cell marker ("anti-T cell-amanitin").

[0314] The heavy and light chain amino acid sequences of the chimeric BBK2 described in the above Examples are set forth as SEQ ID NOs: 11 and 12, respectively.

[0315] Example 4. Administration of allogeneic CAR-T cells in a mouse model The following studies are performed to evaluate the levels of CAR-T cells present in allogeneic recipients under various conditions.

[0316] This study uses a mouse allogeneic CAR-T model. One suitable model is described in Ghosh et al., Nature Medicine (2017), 23(2): 242-249.

[0317] Mice in the first treatment group were dosed with 1 × 10 7 ~1×10 9The mice are treated with an initial dose of allogeneic T cells by intravenous injection of cells / kg. On day 3, the mice are administered anti-CD137-α-amanitin ADC at a dose of 3 mg / kg. On day 10, after the ADC has been substantially cleared from the mouse's blood, the mice are administered allogeneic CAR-T cells. The CAR-T cells are derived from the same donor as the allogeneic T cells administered on day 0.

[0318] Mice in a second treatment group are treated using the same protocol as the first treatment group, except that unconjugated anti-CD137 antibody is administered on day 3 instead of the anti-CD137 ADC.

[0319] Mice in the third treatment group are treated using the same protocol as the first treatment group, except that an isotype control antibody conjugated to α-amanitin is administered on day 3 instead of the anti-CD137 ADC.

[0320] Mice in a fourth treatment group are treated using the same protocol as the first treatment group, except that they receive an initial dose of autologous T cells on day 0 instead of allogeneic T cells.

[0321] Mice in the fifth treatment group will receive allogeneic CAR-T cells on day 10 without any pretreatment.

[0322] Mice in the sixth treatment group will receive autologous CAR-T cells on day 10 without any pretreatment.

[0323] The number of CAR-T cells present in the spleen and peripheral blood of mice from each treatment group is measured on days 14, 17, and 30. The number of CD137+ activated T cells is measured in the spleen and peripheral blood of mice from each treatment group on day 9. Mice are monitored for GVHD symptoms and the presence of CAR-T cells throughout the study.

[0324] Example 5. Administration of anti-CD137 antibody drug conjugates to human patients to prevent rejection of allogeneic cell therapy A human patient is selected to receive allogeneic cell therapy, such as allogeneic CAR cell therapy. To inhibit or prevent rejection of the allogeneic cells, an anti-CD137 antibody drug conjugate (ADC) is administered according to the methods disclosed herein. The physician performs the following treatment steps:

[0325] First, an initial dose of allogeneic cells is administered intravenously to the patient in an amount sufficient to induce a priming immune response against the allogeneic cells. In the priming phase, allogeneic cells are administered to the patient to induce an immune response generated by endogenous activated CD137+ T cells.

[0326] The patient is then administered an 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 is assessed to confirm the depletion.

[0327] A therapeutically effective amount of allogeneic cells expressing a CAR are then administered, the allogeneic cells being derived from the same donor as the cells administered to the patient during the priming step, enhancing acceptance of the allogeneic cells in the recipient patient and reducing the risk of host-versus-graft (HvG) reactions compared to patients receiving allogeneic cell therapy without priming and administration of an anti-CD137 ADC.

[0328] [Table 4-1] [Table 4-2] [Table 4-3]

[0329] Other embodiments 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.

[0330] While the invention has been described in relation to particular embodiments thereof, it will be understood that the invention is capable of further modifications, and this application is intended to cover, broadly, any variations, uses, or adaptations of the invention which come within known or customary practice in the art to which the invention pertains and which include departures therefrom as may be applied to the essential features as hereinbefore described and in accordance with the claims.

[0331] Other embodiments are within the scope of the claims.

Claims

1. 1. A composition comprising an anti-CD137 antibody drug conjugate (ADC) for use in a method 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 an amatoxin via a linker, and the method comprises: (a) administering to the human subject a first amount of allogeneic cells, wherein the first amount is sufficient to elicit a priming response in the human subject to the allogeneic cells; (b) administering to the human subject an anti-CD137 antibody drug conjugate (ADC) to deplete endogenous CD137+ activated T cells, wherein the anti-CD137 ADC comprises an anti-CD137 antibody, or an antigen-binding fragment thereof, conjugated to a cytotoxin via a linker; and (c) administering to the human subject a therapeutically effective amount of an allogeneic cell expressing a CAR, wherein the allogeneic cell is of the same type as the allogeneic cell administered in (a), and the CAR comprises an extracellular domain that binds to a tumor antigen, a transmembrane domain, and an intracellular signaling domain, wherein The composition, 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 set forth in SEQ ID NOs: 19, 20, and 21, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively, and the anti-CD137 antibody or antigen-binding fragment thereof is chimerized or humanized.

2. 2. The composition for use according to claim 1, wherein the method comprises administering to the human subject a first amount of allogeneic cells about 48 hours to about 7 days prior to step (b).

3. 2. The composition for use of claim 1, wherein the method comprises administering to the human subject a therapeutically effective amount of allogeneic cells expressing the CAR about 48 hours to about 7 days after step (b).

4. 1. A composition comprising an anti-CD137 antibody drug conjugate (ADC) for use in a method for treating or preventing rejection of transplanted allogeneic cells in a human subject, wherein the anti-CD137 ADC comprises an anti-CD137 antibody, or an antigen-binding fragment thereof, conjugated to an amatoxin via a linker, the method comprising: (a) administering to the human subject an anti-CD137 antibody drug conjugate (ADC), 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 as having activated T cells against allogeneic cells previously administered to the human subject; and (b) administering to the human subject a therapeutically effective amount of an allogeneic cell expressing a CAR, wherein the allogeneic cell is of the same type as the allogeneic cell described in (a), and the CAR comprises an extracellular domain that binds to a tumor antigen, a transmembrane domain, and an intracellular signaling domain, wherein The composition, 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 set forth in SEQ ID NOs: 19, 20, and 21, respectively, and a light chain variable region comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 23, 24, and 25, respectively, and the anti-CD137 antibody or antigen-binding fragment thereof is chimerized or humanized.

5. 5. The composition for use of claim 4, wherein the method comprises administering to the human subject a therapeutically effective amount of allogeneic cells expressing the CAR about 24 hours to about 14 days after step (a).

6. A composition for use according to claim 1 or claim 4, comprising (i) the allogeneic cells are allogeneic T cells or allogeneic NK cells; (ii) the therapeutically effective amount of allogeneic cells expressing the CAR is about 2×10 6 ~Approx. 3.0×10 8 cells / kg; and / or (iii) The composition, wherein the rejection of the allogeneic cells is characterized by cytokine release syndrome (CRS).

7. 7. The composition for use according to claim 6, wherein the human subject has cancer or an autoimmune disease, wherein the cancer is selected from the group consisting of leukemia, adult advanced cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple-negative breast cancer, hematopoietic / lymphatic 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, leukemia ... lymphoma, diffuse large cell lymphoma, relapsed or refractory diffuse large cell lymphoma, anaplastic large cell lymphoma, primary mediastinal B-cell lymphoma, relapsed mediastinal, refractory mediastinal large B-cell lymphoma, large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, relapsed or refractory non-Hodgkin's lymphoma, refractory progressive non-Hodgkin's lymphoma, B-cell non-Hodgkin's lymphoma, refractory non-Hodgkin's lymphoma, colorectal carcinoma carcinoma, gastric carcinoma, pancreatic carcinoma, triple-negative invasive breast carcinoma, renal cell carcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphocytic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia acute lymphoblastic leukemia), acute leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemialeukemia, chronic lymphocytic leukemia, acute myeloid leukemia, recurrent 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 metastases, solid tumors, advanced solid tumors, mesothelin positive tumors, hematological malignancies, and other advanced malignancies.

8. The composition for use according to claim 1 , wherein the anti-CD137 antibody or antigen-binding fragment thereof is of the IgG1 or IgG4 isotype.

9. 5. The composition for use according to claim 1 or claim 4, wherein the anti-CD137 antibody or antigen-binding fragment thereof conjugated to an amatoxin is represented by the formula Ab-ZL-Am, where Ab is an antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an amatoxin represented by formula (III): [Formula 1] Where: R 1 H, OH, OR A OR C is; R 2 H, OH, OR B OR C is; R A and R Bwhen present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R 3 , H, R C , or R D is; R 4 , R 5 , R 6 , and R 7 are independently H, OH, or OR C , OR D , R C , or R D is; R 8 OH, NH 2 , OR C , OR D , N.H.R. C , or NR C R D is; R 9 H, OH, OR C OR D is; Q is -S-, -S(O)-, or -SO 2 -is; R C is -LZ; R D is an optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 Heteroalkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 Heteroalkenyl, optionally substituted C 2 -C 6 Alkynyl, optionally substituted C 2 -C 6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a linker; and Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an anti-CD137 antibody or antigen-binding fragment thereof, where Am is exactly one R c A composition comprising a substituent.

10. 10. The composition for use according to claim 9, wherein the linker is an optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 Heteroalkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 Heteroalkenyl, optionally substituted C 2 -C 6 Alkynyl, optionally substituted C 2 -C 6 Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, dipeptide, -(C=O)-, -(CH 2 ) m O) n (CH 2 ) 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.

11. 10. The composition for use according to claim 9, wherein the linker of the ADC is N-beta-maleimidopropionyl-Val-Ala-para-aminobenzyl (BMP-Val-Ala-PAB).

12. 10. The composition for use of claim 1 or claim 4, wherein the anti-CD137 ADC is represented by any one of the following structures: [chemical 2] Or, [C3] Or, [C4] .

13. 5. A composition for use according to claim 1 or claim 4, wherein the ADC has a serum half-life of 3 days or less.

14. 5. A composition for use according to claim 1 or claim 4, wherein the extracellular domain comprises an scFv antibody or a single chain T cell receptor (scTCR).

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

16. 5. The composition for use of 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 (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or CD123.

17. 5. The composition for use of claim 1 or claim 4, wherein the intracellular signaling domain of the CAR comprises a CD28 intracellular signaling domain, a CD3 zeta intracellular signaling domain, an OX40 intracellular signaling domain, and / or a CD137 intracellular signaling domain.

18. 5. The composition for use of claim 1 or claim 4, wherein the intracellular signaling domain of the CAR comprises a CD3 zeta intracellular signaling domain.

Images