Use of Anti-CD5 antibody-drug conjugates in allogeneic cell therapy

Abstract

To provide conditioning regimen usable along with CAR therapy to promote the acceptance of immune cells expressing chimeric antigen receptor (CAR).SOLUTION: The invention provides a method for depleting CD5+ cells to promote the acceptance of CAR expressing immune cells in a human patient receiving CAR immune therapy. An anti-CD5 antibody-drug conjugate (ADC) is administered as a conditioning regimen to a human patient receiving immune cells expressing an autologous or allogeneic CAR so that the human patient can accept the CAR expressing immune cells. The compositions and methods of the invention can be used in combination with CAR therapy to treat various pathologies including autoimmune diseases and cancers.SELECTED DRAWING: None

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application is a joint venture of U.S. Provisional Patent Application No. 62 / 702,296, filed July 23, 2018, and U.S. Provisional Patent Application No. 62 / 702,296, filed November 29, 2018. This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 773,047, filed on the same day. The contents of this application are incorporated herein by reference.

[0002] [Array list] This application contains a Sequence Listing, which has been submitted electronically in ASCII format, and which is incorporated herein by reference in its entirety. The ASCII copy of this document, created on July 19, 2019, is incorporated by reference in its entirety hereof. It will be named 034_2060WO_SL.txt and will be 85,119 bytes in size.

[0003] [Technical field] The present invention broadly relates to the use of anti-CD5 antibody-drug conjugates (ADCs) to induce the development of human Immunoglobulins expressing chimeric antigen receptors (CARs) in a subject The present invention relates to a method for enhancing cell reception. [Background technology]

[0004] Chimeric antigen receptor (CAR) therapy is a treatment that stimulates the body's own immune system. The system is used to destroy cells that express specific antigens associated with certain diseases, such as cancer. For example, in cancer, CAR therapy is a type of immunotherapy that uses the patient's immune system to attack the cancer. Over the past few years, this immunotherapy has shown promising results. It has emerged as a revolutionary treatment. CAR therapy is based on immune cells such as T cells, Immune cells are composed of extracellular antigen-binding domains (e.g., scFvs) and surface receptors that bind to the cells. A generalized cytoplasmic active signaling domain that mediates signal transduction and a "co-stimulatory" domain are combined. The CAR is a transmembrane fusion protein expressed in the nucleus of the nucleus. Thus, immune cells such as T-cells can express the CAR. Upon expression, the immune cell binds to the antigen that the antigen-binding domain of the CAR targets (e.g., a tumor They can recognize and kill cells expressing tumor-associated antigens (Geyer and Brentjens (2 016) Cytotherapy 18(11): 1393-1409).

[0005] CAR therapy is an incredibly powerful technology, but it comes with serious risks and adverse side effects. (Kay and Turtle (2017) Drugs 77(3):237-245; Hill et al. (2018) Blood 131:121-1 30) Lymphocyte-depleting chemotherapy is a treatment that is used to minimize the rejection of CAR-expressing cells by patients receiving CAR therapy. To minimize the risk of developing leukemia, it is commonly used as a conditioning treatment in combination with CAR therapy. (Wei et al. (2017) Exp Hematol Oncol. 6: 10). For example, lymphocyte elimination The combination of the expeller fludarabine and cyclophosphamide The maintenance time of CAR-T cells was improved in this study (Turtle et al. (2016) J Clinic Invest 126(6):2123; see also US 20170368101. Conditioning therapy improves the efficacy of CAR-T cells. Although lymphodepleting chemotherapy has improved disease progression, it often has severe negative side effects. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention can be used in conjunction with CAR therapy to promote the reception of immune cells expressing CARs. The methods described herein provide a conditioning regimen for the induction of autologous CAR. To promote the reception of either immune cells expressing a CAR or immune cells expressing an allogeneic CAR Up until now, the reception of such cells has been achieved using lymphocyte-depleting chemotherapy. Promoting the acceptance of cells expressing CAR in recipient patients An improved method is described herein. [Means for solving the problem]

[0007] In human subjects with cancer or autoimmune disease, The present invention includes methods for enhancing the reception of immune cells expressing a CAR (Carcinoma Immunoglobulin Receptor). The method comprises administering an anti-CD5 antibody drug conjugate (ADC) to a human subject suffering from cancer or an autoimmune disease. wherein the anti-CD5 ADC is conjugated to a cytotoxin via a linker. and a CAR-expressing immune cell comprising a gated anti-CD5 antibody or an antigen-binding fragment thereof. and administering to the human subject a therapeutically effective amount of a CAR, wherein the CAR binds to a tumor antigen or It has an extracellular domain that binds to antigens associated with autoimmune diseases, a transmembrane domain, and an intracellular domain. Includes the domain.

[0008] In one embodiment, the human subject is administered the same prior to and in combination with the CAR-expressing immune cells. In another embodiment, the human subject is administered alemtuzumab at or after the administration of alemtuzumab. Elephants were treated with lymphocyte cytometry before, at the same time as, or after administration of CAR-expressing immune cells. In one embodiment, the lymphodepleting chemotherapy agent is administered to the patient. These are darabine, cyclophosphamide, bendamustine and / or pentostatin.

[0009] In one embodiment, the method includes administering an anti-CD5 ADC prior to administering the immune cells expressing a CAR. to said human subject.

[0010] In one embodiment, the method includes administering the CAR-expressing immune cells for about 12 hours to about 21 days. administering an anti-CD5 ADC to the human subject prior to said administration.

[0011] In some embodiments, the immune cells are allogeneic or autologous cells. wherein said allogeneic cells are allogeneic T cells or allogeneic NK cells.

[0012] In certain embodiments, the therapeutically effective amount of allogeneic cells expressing the CAR is about 1×10 4 ~Approx. 1.0×10 8 cells / kg.

[0013] The invention further features a method of treating a patient having a tumor, the method comprising administering an anti-CD5 administering the ADC to a patient in need thereof (wherein the anti-CD5 ADC is linked via a linker); and anti-CD5 antibodies or antigen-binding fragments thereof conjugated to a cytotoxin, and about 1×10 6 ~Approx. 1×10 8 administering to the patient a therapeutically effective amount of the engineered CAR T cells / kg; In one embodiment, the therapeutically effective amount of engineered CAR T cells is about 1×10 6 Or about 2 x 106 cells / kg.

[0014] In certain embodiments of the invention, the anti-CD5 ADCs are administered as a single dose or multiple doses. and administering to the patient.

[0015] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof, respectively, has the sequence and CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5. and a heavy chain variable region comprising the sequences set forth in SEQ ID NOs: 6, 7, and 8, respectively. The light chain variable region comprises CDR1, CDR2, and CDR3 having the amino acid sequence

[0016] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof is chimeric or humanized. It has been standardized.

[0017] In another embodiment, the anti-CD5 antibody or antigen-binding fragment thereof is an IgG1 isotype. The antibody is of the IgG1 or IgG2 isotype.

[0018] In yet another embodiment, the cytotoxin is an antimitotic drug or an RNA polymerase inhibitor. It is.

[0019] In another embodiment, the cytotoxin is a maytansine, a calicheamicin, or a pyrrolobenzoxin. In some embodiments, the compound is a diazepine, an indolinobenzodiazepine, or an auristatin. The auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin F (MMAF). Lystansine E (MMAE).In one embodiment, the cytotoxin is maytansine. In one embodiment, the cytotoxin is a pyrrolobenzodiazepine (PBD). In some embodiments, the PBD may be selected from tesirine or taliline. In some embodiments, the cytotoxin may be calicheamicin. In some embodiments, the calicheamicin can be ozogamicin.

[0020] In one embodiment, the RNA polymerase inhibitor is an amatoxin. In one embodiment, the RNA polymerase inhibitor is amanitin (e.g., α-amanitin, β-amanitin, Amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, amanu phosphate, or proamunulin).

[0021] In one embodiment, the antibody-drug conjugate (ADC) is represented by the formula Ab-ZL-Am. where Ab is an antibody or antigen-binding fragment thereof that binds to CD5, and L is a linker. Z is a chemical moiety and Am is an amatoxin. 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, optionally forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or RD 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 hetero Alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heptyl alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heptyl Heterocycloalkyl, optionally substituted aryl, or optionally substituted hexacycloalkyl. It is a teloaryl; L is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 hetero Alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heptyl alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heptyl cycloalkyl, optionally substituted aryl, or optionally substituted or -(CH2) m O) n (CH2) m - Including wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. and Z is a reactive substituent present on L and a substituted or unsubstituted aryl group within an anti-CD5 antibody, or antigen-binding fragment thereof. It is a chemical substructure formed from a coupling reaction between reactive substituents present in do.

[0022] In this embodiment, the linker-amatoxin conjugate Am-LZ has the formula (III): Represented [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, optionally forming 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, ORC 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 hetero Alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heptyl alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heptyl Heterocycloalkyl, optionally substituted aryl, or optionally substituted hexacycloalkyl. It is a teloaryl; L is a linker; and Z is a reactive substituent present on L and a substituted or unsubstituted aryl group within an anti-CD5 antibody, or antigen-binding fragment thereof. It is a chemical substructure formed from a coupling reaction between reactive substituents present in do.

[0023] In certain embodiments of Formula (III), L is a linker that comprises a peptide.

[0024] In some embodiments, the linker is one or more dipeptides, p-aminobenzyl (PAB) group, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 hetero Alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heptyl alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 Heteroalkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted Heteroaryl, solubility enhancing groups, -(C=O)-, -(CH2CH2O) p - group, where p is from 1 to 6 ((CH2) m O) n (CH2) m - (wherein n and m are each independently 1, 2, 3, 4 , 5, 6, 7, 8, 9 and 10); or combinations thereof.

[0025] In some embodiments, the linker is ((CH2) m O) n (CH2) m -group and heteroaryl group, where the heteroaryl group is triazole. So, ((CH2) m O) n (CH2) m The - group and the triazole together include [ka] where n is 1 to 10, and the wavy lines represent further linker moieties, chemical substructures Z, or indicates the position of binding to amatoxin.

[0026] In some embodiments, Am is exactly one R C Contains substituents.

[0027] In one embodiment, the linker of the ADC is N-beta-maleimidopropionyl-Val-Ala -para-aminobenzyl (BMP-Val-Ala-PAB). In some embodiments, the linker L and said chemical substructure Z taken together as LZ are: [ka] where S is a reactive site present in an antibody or antigen-binding fragment thereof that binds to CD5. It is a sulfur atom representing a substituent (eg, derived from the -SH group of a cysteine ​​residue).

[0028] In some embodiments, the LZ is [ka] It is.

[0029] In one embodiment, the ADC is represented by any one of the following structures: [ka]

[0030] In one embodiment, the ADC is represented by any one of the following structures: [ka] or [ka] .

[0031] In one embodiment, the ADC has a serum half-life of 3 days or less.

[0032] In one embodiment, the extracellular domain of the CAR is an scFv antibody. In one embodiment, the extracellular domain of the CAR is a single chain T cell receptor (scTCR). The extracellular domain of the CAR is a non-immunoglobulin scaffold protein (sca ffold protein).

[0033] In one embodiment, the extracellular domain of the CAR is selected from the group consisting of CD19, CD22, CD30, CD7, BCMA, CD13 7, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR (e.g., EGFRvIII), M G7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, It binds to tumor antigens, such as NKG2D or CD123.

[0034] In certain embodiments, the intracellular domain of the CAR is a CD28 intracellular signaling domain. Main, CD3 zeta intracellular signaling domain, OX40 intracellular signaling domain , and / or the CD137 (4-1BB) intracellular signaling domain. The intracellular domain of the CAR contains the CD3 zeta intracellular signaling domain.

[0035] The methods and compositions disclosed herein can be used to treat human subjects with cancer. In this case, the cancer may include, but is not limited to, leukemia, adult cancer, 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, diffuse large cell lymphoma, relapsed or refractory diffuse large cell lymphoma Lymphoma, anaplastic large cell lymphoma, primary mediastinal B-cell lymphoma, recurrent mediastinum, refractory mediastinal large cell lymphoma B-cell lymphoma, large B-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma lymphoma, recurrent or refractory non-Hodgkin's lymphoma, refractory progressive non-Hodgkin's lymphoma, B-cell lymphoma Follicular non-Hodgkin's lymphoma, refractory non-Hodgkin's lymphoma, colorectal carcinoma carcinoma, gastric carcinoma, pancreatic carcinoma, triple ne Triple-negative invasive breast carcinoma, renal cell carcinoma l cell carcinoma), lung squamous cell carcinoma, hepatocellular carcinoma tocellularcarcinoma), urothelial carcinoma, leukemia, B-cell leukemia , B-cell acute lymphocytic leukemia, B-cell acute lymphocytic leukemia Lymphoblastic leukemia (B-cell acute lymphoblastic leukemia), adult acute lymphoblastic Leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia refractory childhood acute lymphoblastic leukemia, acute leukemia, acute Lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia emia, chronic lymphocytic leukemia, acute myeloid leukemia, recurrent plasma cell myeloma ent plasma cell myeloma), refractory plasma cell myeloma (refractory plasma cell myeloma) oma), multiple myeloma, relapsed or refractory multiple myeloma, multiple myeloma of bone, malignant neurodegenerative diseases of the brain glioma (malignant glioma of brain), myelodysplastic syndrome, EGFR-positive colorectal cancer, pleomorphism Glioblastoma multiforme, neoplasm, blastic plasmacytoid dendritic cell neoplasm asmacytoid dendritic cell neoplasm, liver metastasis, solid tumor, advanced solid tumor solid tumors, mesothelin positive tumors, hematological malignancies, and and other advanced malignant tumors.

[0036] In certain embodiments of any of the above aspects, the anti-CD5 antibody or antigen-binding fragment thereof The fragments are selected from the group consisting of CDR combinations (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-H4, CDR-H5, CDR-H6, ​​CDR-H7, CDR-H8, CDR-H9, CDR-H10, CDR-H11, CDR-H12, CDR-H13, CDR-H14, CDR-H15, CDR-H16, CDR-H In certain embodiments, the anti-CD31A antibody comprises an anti-CD31A antibody, ... 5 The antibody, or antigen-binding fragment thereof, may comprise a heavy chain variable region as described in Tables 1A and 1B. The antibody comprises a combination of a primary and a secondary chain variable region. [Brief description of the drawings]

[0037] [Figure 1] Figure 1 graphically depicts the results of an in vitro cell line binding assay in which each of the indicated anti-CD5 antibodies or a negative control (i.e., mIgG1) was incubated with MOLT-4 cells (i.e., a human T lymphoblast cell line) followed by incubation with a fluorochrome-conjugated anti-IgG antibody. Signals were detected by flow cytometry and are shown as the geometric mean of fluorescence intensity (y-axis) versus anti-CD5 antibody concentration (x-axis). [Diagram 2]Figure 2 graphically depicts the results of an in vitro primary cell binding assay in which the indicated anti-CD5 antibody (i.e., "CD5 5D7") or negative control (i.e., hIgG1) was incubated with primary human T-cells, followed by incubation with a fluorochrome-conjugated anti-IgG antibody. Signal was detected by flow cytometry and is shown as the geometric mean of fluorescence intensity (y-axis) versus the concentration of anti-CD5 antibody (x-axis). [Diagram 3] Figures 3A and 3B graphically depict the results of in vitro T cell toxicity assays involving anti-CD5-amanitin ADCs (i.e., 5D7-AM or "CD5 5D7 AM") with interchain-conjugated amanitin with an average drug-to-antibody ratio (DAR) of 6 (Figure 3A), or site-specifically conjugated amanitin with a DAR of 2 (Figure 3B). In Figure 3A, the anti-CD5 ADC T cell toxicity analysis is shown in comparison to an unconjugated anti-CD5 5D7 antibody (i.e., "CD5 5D7 NAKED"). In Figure 3B, the anti-CD5 ADC T cell toxicity analysis is shown in comparison to an anti-CD5 5D7 antibody with an H435A mutation (i.e., CD5 5D7 D265C.H435A AM) that reduces the half-life of the antibody (i.e., "CD5 Fast 1 / 2 Life AM"). The results are presented as the number of viable T-cells (y-axis) versus the concentration of ADC (CD5 5D7 AM, CD5 5D7 D265C.H435A AM) or unconjugated antibody (CD5 5D7 NAKED) (x-axis), as assessed by flow cytometry. [Figure 4]Figures 4A-4B graphically depict the results of an in vivo T-cell depletion assay showing the absolute concentrations of T-cells (CD3+ cells; y-axis) in the peripheral blood (Figure 4A) and bone marrow (Figure 4B) of humanized NSG mice 7 days after a single dose of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of an anti-CD5 5D7 amanitin ADC with an interchain DAR of 6 (i.e., CD5 5D7-AM). For comparison, Figures 4A-4B also show the levels of T-cell depletion following treatment of humanized NSG mice with the indicated controls (i.e., 25 mg / kg anti-CD5 antibody; 3 mg / kg hIgG1-amanitin ADC (i.e., hIgG1-AM), 25 mg / kg hIgG1, or PBS). [Diagram 5] Figures 5A-5C graphically depict the results of an in vivo T-cell depletion assay showing absolute levels of T-cells (CD3+ cells; y-axis) in peripheral blood (Figure 5A), bone marrow (Figure 5B), and thymus (Figure 5C) of humanized NSG mice 7 days after administration of a single dose of 1 mg / kg or 3 mg / kg of an anti-CD5 5D7-amanitin ADC (i.e., CD5 5D7-AM) with a site-specific DAR of 2. For comparison, Figures 5A-5C also show the levels of T-cell depletion following treatment of humanized NSG mice with 3 mg / kg of unconjugated anti-CD5 antibody (i.e., CD5 5D7) or the indicated controls (i.e., 3 mg / kg hIgG1-amanitin-ADC ("hIgG1-AM") or PBS). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] [Detailed Description] The present invention provides a method for administering anti-CD5 antibody drug conjugates (ADCs) to patients undergoing CAR therapy. This allows the expression of chimeric antigen receptors (CAARs) in human subjects undergoing CAR therapy. Methods for enhancing the acceptance of immune cells (autologous or allogeneic) expressing antigen receptor (CAR) The methods disclosed herein can be used to reduce rejection of immune cells expressing a CAR. The reliance on lymphodepleting chemotherapy, which is commonly used as a conditioning regimen for Instead of (or instead with reduced use of) autologous or allogeneic immune cells (e.g., T cells), may be used to improve acceptance of

[0039] 1.Definition As used herein, the term "about" refers to a value that is 5% higher or lower than the stated value. say.

[0040] As used herein, the term "allogeneic" when used in the context of transplantation The cells (or tissue or tissues) to be transplanted from a donor to a recipient of the same species. is used to define the organ, where the donor and the recipient ecipient) are not the same subject.

[0041] As used herein, the term "autologous" refers to both a donor and a recipient. The term "pient" refers to a cell or graft that is the same subject.

[0042] As used herein, the term "xenogeneic" refers to a donor and recipient This refers to cells that are of a different species than the other (pient).

[0043] As used herein, the term "immune cells" includes, but is not limited to, hematopoietic It is intended to include cells that are of origin and that play a role in the immune response. Examples of such cells include, but are not limited to, T cells and natural killer (NK) cells. Natural killer cells are well known in the art. Examples of NK cell lines include NK-92 cells. Examples of immune cells include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells. It may be allogeneic or autologous.

[0044] An "engineered cell" is a cell that has been modified to contain a gene, DNA or RNA sequence, or a protein or polypeptide. Any organism that has been modified, transformed, or engineered by the addition or modification of a polypeptide. The isolated cells, host cells, and genetically engineered cells of the present disclosure are intended to mean any cell of the present invention. The present invention relates to 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 include isolated immune cells, such as cells. For example, enhancing NK cell activity or T lymphocyte activity, treating cancer, and autoimmune diseases. In one embodiment, the engineered cells may be used to treat an immune disorder. immune cells, such as T-cells or natural killer (NK) cells.

[0045] As used herein, the term "antibody" refers to an antibody that specifically binds to a particular antigen or An immunoglobulin molecule that is immunologically reactive with a specific antigen. However, the present invention is not limited to the above, and may be applied to any of a variety of antibodies, including monoclonal antibodies, polyclonal antibodies, and the like, as long as they exhibit the desired antigen-binding activity. Reclonal antibodies, multispecific antibodies (e.g., Examples of antibodies include specific antibodies (bispecific antibodies) and antibody fragments.

[0046] Generally, an antibody comprises a heavy chain that contains an antigen-binding region and a light chain. Each heavy chain comprises a heavy chain variable region. (herein, abbreviated as HCVR or VH) and a heavy chain constant region. Each light chain is composed of three domains (CH1, CH2, and CH3). In the specification, it is composed of a light chain constant region (abbreviated as LCVR or VL) and a light chain constant region. The VH and VL regions are composed of two domains (CL). The VH and VL regions are composed of two domains (CL) that are hypervariable (complementarity determining These regions can be further subdivided into more conserved regions (called framework regions (FRs)) and more conserved regions (called central determinants (CDRs)). Each VH and VL is composed of three CDRs and four FRs, and the amino terminus is From the carboxyl terminus, the sequences are arranged in the following order: FR1, CDR1, FR2, CDR2, FR 3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody is a region that determines whether the immunoglobulin binds to host tissues or factors (e.g., the immune system). various cells of the complement system (e.g., effector cells and the first component of the classical complement system (C1q, etc.) It can act as a mediator between the bonding of

[0047] As used herein, the term "antigen-binding fragment" refers to a fragment that is specific to a target antigen. The term "antigen-binding portion" refers to one or more portions of an antibody that retain the antigen-binding function of the antibody. This can be achieved by fragments of full-length antibodies. Such antibody fragments can be, for example, , Fab, F(ab')2, scFv, diabody, triabody, affibody, nanobody, aptamer The term "antigen-binding fragment" of an antibody may be a tamers or domain antibodies. Examples of combined fragments encompassed by the term include, but are not limited to, the following: (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; ii) F(ab')2 fragment, which consists of two Fab fragments linked by a disulfide bridge at the hinge region. (iii) a bivalent fragment comprising the VH and CH1 domains; and (iv) an Fd fragment comprising 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 VH and VL domain (vi) a dAb fragment consisting of a VH domain (e.g., Ward et al., Nature e 341:544-546, 1989); (vii) dAbs consisting of a VH or VL domain; (viii) isolated and (ix) optionally linked by a synthetic linker. A combination of two or more (e.g., two, three, four, five, or six) isolated CDRs. In addition, the two domains of the Fv fragment (VL and VH) are encoded by separate genes. However, they can be linked together by a linker using recombinant methods to form a single tag. A single-chain F protein (wherein the VL and VH domains pair to form a monovalent molecule) can be used. v(scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Husto (See, e.g., Nelson et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). The somatic fragments can be obtained using conventional techniques known to those skilled 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, or by the synthesis of intact immunoglobulins. By enzymatic or chemical cleavage, or in some cases by chemical methods known in the art. It can be produced by peptide synthesis procedures.

[0048] As used herein, an "intact" or "full length" antibody is one that is It has two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by This refers to an antibody that

[0049] As used herein, "anti-CD5 antibody" or "antibody that binds to CD5" or "anti-CD5 antibody" refers to The term "CD5 ADC" or "CD5-binding ADC" refers to an ADC that binds CD5 to a T cell or other cell surface that is associated with CD5. When found, it refers to an antibody or ADC that specifically binds to human CD5. The amino acid sequence of human CD5 to which the 5 ADC binds is set forth below as SEQ ID NO: 20. Will be posted.

[0050] The term "specifically binds" as used herein means that an antibody (or ADC) specifically binds to a protein. It has the ability to recognize and bind to specific protein structures (epitopes) rather than to proteins in general. If an antibody is specific for epitope "A", a reaction mixture containing labeled "A" and the antibody is In the reaction, the presence of a molecule containing epitope A (or free, unlabeled A) binds to the antibody. The amount of labeled A that binds is reduced. For example, an antibody (if labeled) binds to the corresponding A target is identified if it can be competed away from its target by an unlabeled antibody. "Specifically binds." In certain embodiments, an antibody specifically binds to a target (e.g., CD5). specifically binds to the antibody (the antibody binds to the antibody at least about -4 M, 10 -5 M, 10 -6M, 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, "CD The terms "specific binding to CD5" or "specifically binds to CD5" are used herein. or binds to CD5 and as determined by surface plasmon resonance , 1.0×10 -7 Acid dissociation constant (K D In one embodiment, the antibody has the following structure: D of, Measure according to standard Bio-Layer Interferometry (BLI). However, the antibody can specifically bind to two or more antigens that are related in sequence. For example, in certain embodiments, the antibody may be a human or non-human (e.g., The antibody may specifically bind to both the mouse or non-human primate (mouse or non-human primate) orthologs.

[0051] As used herein, the term "monoclonal antibody" refers to an antibody produced by hybridoma technology. Monoclonal antibodies are not limited to antibodies produced from any eukaryotic, prokaryotic, or From a single clone, including a phage clone, any method available or known in the art may be used. The monoclonal antibodies useful in connection with the present disclosure can be obtained by means of hybridomas. This includes using recombinant, and phage display technologies, or a combination thereof. , can be prepared using a variety of techniques known in the art.

[0052] As used herein, the term "chimeric" antibody refers to a non-human antibody, such as a rat or mouse antibody. Variable sequences derived from immunoglobulins, and typically from human immunoglobulin templates The term "chimeric antibody" refers to an antibody having a human immunoglobulin constant region selected from the group consisting of: Methods for this purpose are known in the art. See, e.g., Morrison, 1985, Science 229(4719):12 02-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol See Methods 125:191-202; US Pat. Nos. 5,807,715; 4,816,567; and 4,816,397. This is what I mean.

[0053] "Humanized" forms of non-human (e.g., murine) antibodies are derived from non-human immunoglobulins. A humanized antibody is a chimeric immunoglobulin that contains minimal sequences. Generally, a humanized antibody contains at least one , typically comprising substantially all of the two variable domains, where all or substantially all of the CDR regions Generally, all of the FR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are human. The humanized antibody also has an immunoglobulin constant region (F c) at least a portion of, typically at least a portion of, a human immunoglobulin consensus sequence Methods for antibody humanization are known in the art. See, for example, Riechmann et al. 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 91 / 09967; US Pat. No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:48 9-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. See Natl. Acad. Sci. 91:969-973; and US Pat. No. 5,565,332.

[0054] As used herein, the term "chimeric antigen receptor" refers to a "CAR" or "CAR" refers to a nucleic acid molecule that comprises at least one extracellular domain, a transmembrane domain, and a cellular antigen (CAA) capable of specifically binding to an antigen. A recombinant polypeptide comprising a polypeptide chain, a polypeptide domain, and at least one intracellular signaling domain. In general, CARs are used to direct the cytotoxicity of immune effector cells to target a given antigen. CARs are receptors engineered to target cells that express a desired antigen (e.g., Antibody-based specificity for tumor antigens (TCRs) combined with T cell receptor-activating intracellular domains Together, these molecules generate chimeric proteins that exhibit specific cellular immune activity. In certain embodiments, a CAR comprises an extracellular domain (a binding domain or an antigen-specific binding domain) It contains a transmembrane 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 CARs cluster and deliver activation stimuli to the CAR-containing cells. The ability to switch effector cell specificity, thereby Taking advantage of the cell-specific targeting potential of soluble ligands or cell-specific co-receptors Using this method, proliferation, cytokine production, phagocytosis, or major histocompatibility complex (MHC) binding was observed. In a CHO-dependent manner, the production of molecules capable of mediating cell death of cells expressing the target antigen. In some embodiments, the CAR is an extracellular binding site that specifically binds to a tumor antigen. a binding domain; a transmembrane domain; and one or more intracellular signaling domains. In various embodiments, the CAR comprises an extracellular binding domain that specifically binds human CD5; a transmembrane domain; and one or more intracellular signaling domains.

[0055] As used herein, the term "CAR therapy" refers to a method for treating a cancer cell that has been engineered to express a CAR. The cells are administered to a human subject to treat a given disease (e.g., cancer or an autoimmune disease). CAR therapy uses engineered immune cells to specifically treat patients. and therapies commonly used in conjunction with CAR cell therapy (e.g., lymphocyte depletion In particular, where the term "cells" is used throughout, In such cases, the population of cells is also included in the term unless otherwise specified. requires the administration of a population of cells to be investigated.

[0056] As used herein, the term "combination" or "combination therapy" refers to combination therapy in a single human patient. These terms refer to the use of two (or more) treatments in combination. For example, nothing described herein is intended to refer to the administration of anti-CD5 ADCs. and CAR therapy.

[0057] The term "conditioning" refers to the process of treating a patient in need of CAR therapy with a As used herein, conditioning refers to preparing a material. Although it does not affect the immune system, it does reduce the number of endogenous lymphocytes and act as a cytokine sink. Remove one or more homeostatic cytokines or pro-inflammatory factors. Increase serum levels of T cells in mice after conditioning enhancing target function; enhancing activation and / or availability of antigen-presenting cells; or any combination thereof prior to T cell therapy.

[0058] The term "deplete" refers to the effect of an anti-CD5 antibody or ADC on CD5-expressing cells. In this context, it refers to reducing in number or eliminating CD5 expressing cells.

[0059] 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. say.

[0060] As used herein, the terms "subject" and "patient" refer to a subject as defined herein. "Subject matter" refers to an organism, such as a human, receiving treatment for a specific disease or condition.

[0061] As used herein, "treat" or "treatment" refers to any outcome of a disease. Any improvement (e.g., longer survival, less morbidity, and / or alternative treatment modalities) in This refers to the reduction of side effects that are a by-product of the efficacy of the compound; As such, complete eradication of the disease, although preferred, is not a requirement for therapeutic intervention. Desired clinical outcomes include, but are not limited to, the development of immune cells (allogeneic or The researchers found that CAR inhibits the receptor for self-receptor proteins (both of which can trigger immune responses in patients undergoing CAR therapy). Insofar as the method of the invention is directed to preventing disorders, the term It is understood that "preventing" does not require that the disease state be completely averted. Rather, as used herein, the term preventing refers to reducing susceptibility to a disorder. This refers to the ability of one of skill in the art to identify populations with a disease that may be at high risk for disease, and therefore to be able to use the compounds of the present invention before the disease develops. The term does not mean to completely avoid the disease state. It is not.

[0062] As used herein, the term "vector" includes plasmids, DNA vectors, Nucleic acid vectors, such as smids, RNA vectors, viruses, or other suitable replicons. The expression vectors described herein contain polynucleotide sequences as well as, for example, and / or by locating these polynucleotides in the genome of a mammalian cell. The sequence may contain additional sequence elements that are used to integrate the CAR. Particular vectors that can be used to express the gene include those that contain regulatory sequences ( For example, a plasmid containing a promoter region and an enhancer region is included. or other useful vectors for expressing CARs that increase the translation efficiency of these genes. or a polynucleotide that improves the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements, for example, allow efficient transcription of genes present on an expression vector. In order to ensure transcription, it is necessary to include 5' and 3' untranslated regions and a polyadenylation signal site. The expression vectors described herein also include the use of cells containing such vectors. The polynucleotide may include a polynucleotide that encodes a marker for selection. Examples of antibiotics include ampicillin, chloramphenicol, kanamycin, and nource. These include genes that code for resistance to antibiotics such as nourseothricin. .

[0063] As used herein, the term "antibody drug conjugate" or "ADC" refers to a drug that is capable of transporting an antibody to a cell. ADC refers to an antibody linked to a toxin. and reacting the reactive functional group of the compound with another molecule (e.g., a cytotoxin as described herein). A conjugate is formed by chemical bonding with a functional group on the molecule. In particular, the term "conjugate" may include a linker between the conjugate and the cytotoxin. "Gate" (when referring to a compound) is also used herein as a "drug conjugate," "antibody drug conjugate," The compound used to form the conjugate is also referred to interchangeably as an "antibody conjugate" or "ADC." Examples of linkers that can be used include peptide-containing linkers (e.g., naturally occurring or linkers containing non-naturally occurring amino acids, e.g., D-amino acids. Linkers can be synthesized using a variety of strategies, as described herein and known in the art. Depending on the reactive moiety therein, the linker may be prepared using, for example, For example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, acid They may be cleaved by oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (e.g. See, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0064] As used herein, the term "coupling reaction" refers to the process of combining two or more compounds that are suitable for reacting with each other. Two or more substituents react to form molecular fragments (e.g. Coupling reaction refers to a chemical reaction that forms chemical substructures that are bonded (covalently or Optionally, a cytotoxin (e.g., a cytotoxin known in the art or described herein) may be used. The reactive substituent attached to the fragment is an antibody, or an antigen-binding fragment thereof (e.g., For example, antibodies that bind to CD5, as known in the art or described herein. , an antigen-binding fragment thereof, or a specific anti-CD5 antibody) bound to a fragment thereof. Reactions involving reaction with suitable reactive substituents are included. Examples of suitable reactive substituents include nucleophiles. / electrophilic pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / halophilic pairs, among others) ol / α,β-unsaturated carbonyl pairs, etc.), diene / dienophile pairs (e.g., among others, Coupling reactions include, but are not limited to, However, thiol alkylation, hydroxyl alkylation, amine alkylation, and amine alkylation are not possible. condensation, amidation, esterification, disulfide formation, cycloaddition (e.g., especially [4+2]Dimethylphenyl s-Alder cycloaddition, [3+2]Huisgen cycloaddition, aromatic nucleophilic substitution, aromatic electrophilic substitution, and the like. Other reaction schemes known in the art or described herein are included.

[0065] As used herein, the term "microtubule binding agent" refers to an agent that inhibits mitosis and Compounds that act by disrupting the microtubule network essential for cell function during and after interphase Examples of microtubule binding agents include, but are not limited to, maytansin, maytansin, Synoids, and derivatives thereof (as described herein or known in the art) etc.), vinblastine, vinblastine sulfate, vincristine, vincristine sulfate, Vinca alkaloids such as vindesine and vinorelbine, docetaxel and paclitaxel Taxanes such as ceramide, discodermolide, colchicine, and epothilones chlorides, and derivatives thereof, such as epothilone B or derivatives thereof. .

[0066] As used herein, the term "amatoxin" refers to the amatoxin produced by Amanita phalloides mushroo A member of the amatoxin family of peptides produced by ms, or a derivative thereof (e.g., a variant or derivative thereof capable of inhibiting RNA polymerase II activity) Amatoxins useful in conjunction with the compositions and methods described herein include, but are not limited to, Although not limited thereto, the compounds of formula (II) (e.g., α-amanitin, β-amanitin, γ-amanitin, -Amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, Amatoxins can be found in various mushroom species, such as Amanita phalloides, Galerina marginata, and Lepiota brunneo-incarnata. The members of this family (α -amanitin) is described in Wieland, Int. J. Pept. Protein Res. 1983, 22(3):257-276. Derivatives of amatoxins are produced by chemically modifying naturally occurring compounds (" They may be obtained by "semi-synthesis" or from fully synthetic sources. The synthetic route to amatoxin derivatives is described, for example, in U.S. Pat. No. 9,676,702 and Perrin et al., J. Am. Chem. Soc. 2018, 140, pp. 6513-6517 (each of which is incorporated herein by reference in its entirety). The present invention is not limited to the above-mentioned examples, and is not intended to be limiting unless otherwise specified. (taken in by illumination).

[0067] As described herein, the amatoxin can be linked, for example, via a linker moiety (L): It may be conjugated to an antibody, or an antigen-binding fragment thereof (whereby An exemplary amatoxin-linker conjugate structure has the formula (III): (IIIA), and (IIIB). Exemplary methods for conjugating amatoxins. and linkers useful in such processes are described below. Exemplary linker-containing amines useful for conjugating to an antibody or antigen-binding fragment include Amatoxins are also described herein.

[0068] The term "acyl" as used herein refers to -C(=O)R, as defined herein. As shown, R is H ("aldehyde"), C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alki Nyl, C3-C7 carbocyclyl, C6-C 20 Aryl, 5-10 membered heteroaryl, or 5-10 membered hete Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, and the like. acryloyl, and acryloyl.

[0069] As used herein, the term "C 12 "Alkyl" means a straight-chain or refers to branched saturated hydrocarbons. Representative C1-C 12 The alkyl group is not limited to the following: However, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl are On the other hand, the branch C1-C 12 Alkyl includes, but is not limited to, -isopropyl. -propyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl Examples include C1-C 12 The alkyl group may be unsubstituted or substituted. be.

[0070] 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 Refers to hydrocarbons. 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 The alkenyl group may be unsubstituted or substituted. There is.

[0071] As used herein, "alkynyl" refers to an alkynyl group having at least one site of unsaturation, i.e., a carbon- Carbon, C2-C containing normal, secondary, or tertiary carbon atoms with sp triple bonds 12 Carbonated water Examples include, but are not limited to, acetylene and propargyl. An alkynyl group can be unsubstituted or substituted.

[0072] As used herein, "aryl" refers to any group having a C6-C 20 Refers to carbocyclic aromatic groups. Examples of aryl groups Examples include, but are not limited to, phenyl, naphthyl, and anthracenyl. The aryl group can be unsubstituted or substituted.

[0073] As used herein, "arylalkyl" refers to an alkyl group having a carbon atom (typically a terminal or sp 3 Charcoal Acyclic aryl groups in which one of the hydrogen atoms bonded to the aryl atom is replaced by an aryl radical. Representative 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-naphthopheny The aryl alkyl group may include 6 to 20 carbon atoms. For example, the alkyl portion of an arylalkyl group (e.g., alkanyl, alkenyl or alkenyl) may be The alkyl group is an alkyl group having 1 to 6 carbon atoms, and the aryl portion is an alkyl group having 5 to 14 carbon atoms. The alkaryl group may be unsubstituted or substituted.

[0074] As used herein, "cycloalkyl" refers to a saturated carbocyclic radical which is a monocyclic ring. Cycloalkyl groups may be monocyclic or bicyclic. A ring having 7 to 12 carbon atoms as a bicyclic ring is an example of a monocyclic ring. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, Cycloalkyl groups include cycloalkyl, cyclohexyl, cycloheptyl, and cyclooctyl. may not have been included or may have been replaced.

[0075] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic radical, which is The cycloalkenyl group may be a cyclic or bicyclic group. A ring having 7 to 12 carbon atoms as a bicyclic ring is an example of a monocyclic cycloa Examples of alkenyl groups are 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent- 1-cyclohex-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl. Cycloalkenyl groups can be unsubstituted or substituted. This has sometimes been done.

[0076] As used herein, a "heteroaralkyl" refers to an aryl group having a carbon atom (typically a terminal or sp 3 Charcoal A non-cyclic compound in which one of the hydrogen atoms bonded to the heteroaryl group is replaced by a heteroaryl radical. Heteroarylalkyl groups are limited to the following: Examples of the aryl group include, but are not limited to, 2-benzimidazolylmethyl and 2-furylethyl. Heteroarylalkyl groups contain 6 to 20 carbon atoms, e.g., heteroarylalkyl The alkyl portion of the group (including alkanyl, alkenyl, or alkynyl groups) has 1 to 6 The heteroaryl moiety is 5 to 14 carbon atoms and N, O, P, and S. The heteroarylalkyl group has 1 to 3 heteroatoms selected from the group consisting of aryl, aryl, aryl and heteroaryl. The aryl moiety may be a monocyclic ring (2-6 carbon atoms) having 3-7 ring members; is a bicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms and one ring selected from N, O, P, and S); up to 3 heteroatoms), for example: bicyclic [4,5], [5,5], [5,6], or [6,6] Stem.

[0077] As used herein, "heteroaryl" and "heterocycloalkyl" refer to, respectively, refers to an aromatic or non-aromatic ring system in which one or more of the ring atoms may be a heteroatom, e.g. Nitrogen, oxygen, and sulfur. Heteroaryl or heterocycloalkyl radicals are It contains 2 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. Heteroaryl or heterocycloalkyl is a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms). and 1 to 3 heteroatoms selected from N, O, P, and S), or 7 to 10 Bicyclic rings having 4 to 9 carbon atoms and 1 to 3 ring members selected from N, O, P, and S Heteroatoms), for example: bicyclic [4,5], [5,5], [5,6], or [6,6] systems Heteroaryl and heterocycloalkyl are unsubstituted or substituted. There are times when this happens.

[0078] Heteroaryl and heterocycloalkyl groups are described in Paquette, Leo A.; "Principles of "Old Heterocyclic Chemistry" (W. A. ​​Benjamin, New York, 1968), especially 1, 3, 4, 6, Chapters 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.

[0079] Examples of heteroaryl groups include, but are not limited to, pyridyl, thiazol-1-yl, aryl, aryl-1-methylphenyl ... Zolyl, tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrimidine Lazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl , indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, isoxazolyl , pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-isoindolyl Ndazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalyl nyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, Phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl , phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, iso Midazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, benzotriazolyl , benzisoxazolyl, and isatinoyl.

[0080] Examples of heterocycloalkyl include, but are not limited to, dihydropyridyl. Lysyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, piperidinyl , 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydrofuranyl tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroiso Quinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinine Examples include clidinyl, and morpholinyl.

[0081] By way of example and not limitation, carbon-bonded heteroaryls and heterocycloalkyls include 2, 3, 4, 5, or 6 position of lysine, 3, 4, 5, or 6 position of pyridazine, 2 position of pyrimidine , 4, 5 or 6 position, 2, 3, 5 or 6 position of pyrazine, furan, tetrahydrofuran , thiofuran, thiophene, pyrrole or tetrahydropyrrole, is at the 5-position; oxazole, imidazole or thiazole at the 2-, 4- or 5-position; isooxazole the 3-, 4-, or 5-position of a sazole, pyrazole, or isothiazole; the 2-position of an aziridine; or 3-position, 2-, 3-, or 4-position of azetidine, 2-, 3-, 4-, 5-, 6-, 7-, or 8-position of quinoline or at the 1, 3, 4, 5, 6, 7, or 8 position of the isoquinoline. and carbon-bonded heterocyclyls include 2-pyridyl, 3-pyridyl, 4-pyridyl, and 5-pyridyl. , 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2- Pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrimidinyl Razinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl Examples include:

[0082] By way of example and not limitation, nitrogen-linked heteroaryls and heterocycloalkyls include Dimethyl ether, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole , imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyra Zoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indane The 1st position of the azole, the 2nd position of the isoindole or isoindoline, the 4th position of the morpholine, and the 1st position of the aryl group The 9-position of the beta-carboline or beta-bazole is typically linked to the nitrogen bond. The heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, and 1-imidazolyl. Examples of such radicals include 1-pyrazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0083] As used herein, alkyl, alkenyl, alkynyl, aryl, Suitable for any of arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, etc. As used herein, "substituted" means that one or more hydrogen atoms are each independently replaced with a substituent. Unless otherwise specified, the above definitions of the individual substituents are used. Chemical substructures, such as "alkyl", "alkylene", "heteroalkyl", "heteroalkyl", "Alkylene", "alkenyl", "alkenylene", "heteroalkenyl", "hetero "Alkenylene", "alkynyl", "alkynylene", "heteroalkynyl", "hetero "Alkynylene", "cycloalkyl", "cycloalkylene", "heterocycloalkyl" ", "heterocycloalkylene", "aryl", "arylene", "heteroaryl" and "heteroarylene" groups may be optionally substituted.

[0084] 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)2 R, -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 selected from F, Cl, Br, and I, in each case. and each R is independently selected from C1-C 12 Alkyl, C6-C 20 Aryl, C3-C 14 Hetero a cycloalkyl or heteroaryl, a protecting group and a prodrug moiety, in each case are independently selected. When a group is described as being "optionally substituted," In such cases, the group may be substituted, independently in each occurrence, with one or more of the above described substituents. It may be exchanged.

[0085] The naming convention for a given radical includes whether it is a mono-radical or a di-radical, depending on the context. For example, it is important to understand that a substituent may have a 2-membered bond to the remainder of the molecule. If two bond positions are required, the substituent is understood to be a di-radical. For example, substituents that are identified as alkyl, requiring two points of attachment, include -CH2-, -CH2CH Other radical naming conventions include di-radicals such as -CH2CH(CH3)CH2-, -CH2CH(CH3)CH2-, etc. The alkylene, alkenylene, arylene, and heterocycloalkylene are " etc., clearly indicating that it is a di-radical.

[0086] The substituent is shown as a di-radical (i.e., the points of both bonds to the rest of the molecule are Wherever possible, said substituents may be bonded in any directional configuration unless otherwise indicated. It should be understood that the above-mentioned configurations may be combined.

[0087] "Isomerism" means two or more molecules that have the same molecular formula but differ in the arrangement of bonds of their atoms or in their structure. The spatial arrangement of atoms is called an isomer. Stereoisomers that are not mirror images of each other are called "diastereomers", and Stereoisomers that are mirror images and cannot be superimposed on each other are called "enantiomers." and are sometimes called "optical isomers".

[0088] A carbon atom bonded to four non-identical substituents is called a "chiral center." means a compound having at least one chiral center. The compounds may be presented as individual diastereomers or as "diastereomeric mixtures." It may exist as a mixture of diastereomers called a "diastereomeric mixture." When chiral centers are present, stereoisomers are distinguished according to the absolute configuration (R or S) of the chiral centers. Absolute configuration refers to the spatial arrangement of the substituents attached to the chiral center. The substituents attached to the chiral center under consideration are arranged according to the Cahn, Ingold and Prelog arrangement rules. (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; erra ta 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. This substance is called a "racemic mixture."

[0089] The compounds disclosed and claimed herein may contain one or more asymmetric centers. and that different diastereomers and / or enantiomers of each compound exist. The description of compounds in this specification and claims is subject to change without notice. , is meant to include all enantiomers, diastereomers and mixtures thereof. Furthermore, any description of a compound in this specification and claims includes, unless otherwise stated, It is understood that the individual enantiomers as well as any mixture of said enantiomers, racemic or otherwise, may be used. When the structure of a compound is depicted as a particular enantiomer, It is to be understood that the invention of this application is not limited to that particular enantiomer. The enantiomers, optical isomers, and diastereomers of each structural formula of the present disclosure are intended to be understood as being within the scope of the present disclosure. In the present specification, the structural formulas of the compounds are shown in some cases for convenience. In the above cases, a specific isomer is represented, but the present disclosure does not include geometric isomers, optical isomers based on asymmetric carbons, etc. This includes all isomers such as isomers, stereoisomers, tautomers, etc., and all isomers are present at the same level. It is understood that the activity of these compounds does not necessarily correspond to that of the tautomers. Compounds according to the present disclosure may exist in all tautomeric forms unless otherwise stated. When a compound structure is depicted as a particular tautomer, the present application It should be understood that the invention is not limited to that particular tautomer.

[0090] The compounds of any formula described herein may be used in combination with the compounds themselves, as well as with any of their derivatives, if applicable. For example, salts include those formed by combining an anion and a compound of the present disclosure, and solvates thereof. A suitable anion may be formed between a positively charged group (e.g., amino). are chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate Salt, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronide Phosphate, glutarate, malate, maleate, succinate, fumarate, tartrate , tosylates, salicylates, lactates, naphthalenesulfonates and acetates (e.g., trifluoroacetates, The term "pharmaceutical acceptable anion" refers to a pharmaceutical acceptable anion. Similarly, a salt refers to a combination of a cation and a compound of the present disclosure. It may be formed between negatively charged groups (e.g., carboxylates) on the substrate. Examples of suitable cations include sodium ions, potassium ions, magnesium ions, Calcium ion, and ammonium cations such as tetramethylammonium ion Some suitable substituted ammonium ions include: Derivatives include: ethylamine, diethylamine, dicyclohexylamine, Triethylamine, butylamine, ethylenediamine, ethanolamine, diethanol Amines, piperazines, benzylamines, phenylbenzylamines, choline, meglumine, and amino acids such as tromethamine, lysine and arginine. The compounds of the present disclosure also include quaternary This includes salts containing nitrogen atoms.

[0091] Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: Examples of acids that are used include: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, and nitrous acid. Examples of suitable organic anions include, but are not limited to, Examples of acetoxyethyl esters include, but are not limited to, those derived from the following organic acids: 2-acetoxyethyl esters. Benzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, Citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoside Propionic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxy Sinaphthalene carboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid 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, toluene sulfone Examples of suitable polymeric organic anions include, but are not limited to, Examples of polymeric acids include those derived from the following: tannic acid, carboxymethyl cellulose.

[0092] Additionally, the compounds of the present disclosure (e.g., salts of the compounds) may be present in hydrous or non-hydrous (anhydrous) form. Non-limiting examples of hydrates include hydrates of 1,2,3,4,5,6,7,8,9,9,10,11,12,13,14,15,16,17,18,19 ...20,21,22,23,24,25,26,27,28 Examples of solvates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol, ethanolamine ... Examples of solvates include acetone solvates and acetone solvates. or non-stoichiometric amounts of any solvent. Some compounds have a tendency to trap a fixed molar proportion of solvent molecules in the crystalline solid phase. When the solvent is water, the solvate formed is and when the solvent is an alcohol, the solvate formed is an alcohol. A hydrate is a compound formed by the combination of one molecule of a substance with one or more molecules of water. In this case, water maintains its molecular state as H2O. Hydrates are, for example, monohydrates and dihydrates. , trihydrate, etc.

[0093] Furthermore, the compounds represented by the formulas disclosed herein or salts thereof may have polymorphic forms. Any crystalline form, mixture of crystalline forms, or anhydrates or hydrates thereof may be used. , please note that they are within the scope of this disclosure.

[0094] In the following sections, we will discuss how to use human CAR-expressing immune cells to promote their acceptance in CAR therapy. The present invention describes methods based on administering anti-CD5 ADCs to mice.

[0095] II. Anti-CD5 Antibodies and CAR Therapeutic Methods The challenge of chimeric antigen receptor (CAR) therapy is to engineer The CAR-expressing cells (e.g., CAR-T cells) are then received by a human recipient. The aim of this study is to determine the means by which such engineered immune cells can be received. , which may affect the effectiveness of treatment and may also result in adverse side effects for the patient.

[0096] Lymphocyte-depleting chemotherapy suppresses the recipient's immune system and reduces receptivity. CAR therapy is a traditional method to improve the condition, but it generally has adverse side effects. Described herein is a method for promoting the uptake of immune cells expressing a CAR in a human patient. The methods described herein are directed to the detection of CD5+ cells in human patients undergoing CAR therapy. The present invention relates to a method for specifically targeting CD5+ T cells (e.g., CD5+ T cells) and depleting CD5+ cells. The methods disclosed herein are more targeted than lymphodepleting chemotherapy, and and provides a means by which either autologous or allogeneic cells can be used.

[0097] CD4+ in patients undergoing CAR therapy to promote the uptake and efficacy of CAR-expressing immune cells 5. Administering Anti-CD5 Antibody Drug Conjugates (ADCs) to Deplete Specific Immune Cell Populations A method for specifically expressing CD5 cells of the immune system is described herein. To treat autoimmune diseases or cancer by selectively reducing CAR-expressing immune cells. Improved overall and recurrence-free survival while reducing the risk of cell rejection .

[0098] The risk of rejection of CAR-expressing immune cells remains even after CAR cell therapy is administered The methods and compositions disclosed herein are directed to the induction of CAR cell proliferation in human patients. Anti-CD5 ADCs may be used to inhibit or prevent rejection. Selective targeting of activated T cells in patients about to undergo chemotherapy As described herein, anti-CD5 ADCs can be used to treat patients who have already received CAR cell therapy. Targeting and depleting CD5+ cells in human patients with This may also reduce the risk of the CAR cells being rejected.

[0099] The compositions and methods described herein can be used to detect and treat CD5+ tumors associated with CAR cell therapy rejection. The methods of the invention may be used to reduce the number of cancer cells (e.g., T cells). or a human subject with an autoimmune disease) to promote reception of immune cells expressing a CAR. In one embodiment, the method includes administering to a subject who is scheduled to undergo or has undergone CAR therapy. administering to a human subject an anti-CD5 antibody drug conjugate (ADC); and administering to the human subject a administering a therapeutically effective amount of immune cells expressing AR.

[0100] The anti-CD5 ADC is administered to a human patient in need thereof prior to administering one or more CAR cell therapies. In one embodiment, the anti-CD5 ADC may be administered simultaneously with or after administration of the anti-CD5 ADC. before administering the CAR cell therapy to a human patient in need thereof (e.g., about 3 days before, about 2 days before, A single dose of anti-CD5 ADC is administered to a subject that expresses a CAR. If the effect is sufficient to prevent or reduce the risk of immune cell depletion associated with CAR cell therapy, The method may be administered to the human patient before, after, or simultaneously with the administration of the method. The authors administered the anti-CD5 ADC to a human patient in need thereof approximately three days prior to administering the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need thereof as a CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered about 2 days prior to administration of the anti-CD5 ADC to a patient in need thereof. The human patient is administered about one day prior to administering the CAR cell therapy. The DCs are administered to a human patient in need thereof approximately 20 hours prior to administering the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need thereof for administration of a CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need thereof about 18 hours prior to administration. In one embodiment, the anti-CD5 ADC is administered to a subject about 15 hours prior to administering the CAR cell therapy. The antibody is administered to a human patient in need thereof approximately 12 hours prior to administering the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need thereof to administer CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need thereof about 6 hours before the administration of the anti-CD5 ADC. In one embodiment, the anti-CD5 ADC is administered to a patient in need thereof, about 4 hours prior to administering the CAR cell therapy. The antibody is administered approximately two hours prior to administering the CAR cell therapy to a human patient in need thereof. In one embodiment, the anti-CD5 ADC is administered to a human patient in need thereof in combination with a CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need thereof via administration of CAR cells. In one embodiment, the anti-CD5 ADC is administered approximately 2 hours after the prescribed cell therapy. The CAR cell therapy is administered to a human patient in need thereof about 4 hours after it is prescribed. The anti-CD5 ADC is administered to a human patient in need thereof approximately 6 hours after administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need thereof as a CAR cell therapy. is administered approximately 12 hours after prescription.

[0101] In some embodiments, the anti-CD5 ADC is administered within about 21 days of administering one or more CAR cell therapies. (e.g., about 21 days, e.g., about 21 days, about 20 days, before, about 19 days before, about 18 days before, about 17 days before, about 16 days before, about 15 days before, about 14 days before, about 13 days before, about 12 days before, about 11 days before, about 10 days before, about 9 days before, about 8 days before, about 7 days before, about 6 days before, about 5 days before, about 4 days before, about 3 days ago, about 2 days ago, about 1 day ago, about 24 hours ago, about 12 hours ago, about 6 hours ago, about 3 hours ago, about 2 hours ago It may be administered at or about 1 hour before the

[0102] In some embodiments, the anti-CD5 ADC is administered about 12 hours (e.g., 12 hours after administering the CAR cell therapy). For example, about 12 hours, about 11 hours, about 10 hours, about 9 hours after prescribing one or more CAR cell therapies. After about 8 hours, after about 7 hours, after about 6 hours, after about 5 hours, after about 4 hours, after about 3 hours, after about 2 hours In some embodiments, the anti-CD5 ADC is administered to a subject in need of treatment with a CAR About 10 days after prescribing a cell therapy (e.g., about 10 days after prescribing one or more CAR cell therapies, about 9 days later, about 8 days later, about 7 days later, about 6 days later, about 5 days later, about 4 days later, about 3 days later, about 2 days later, about 1 day later It may be administered.

[0103] In one embodiment, the anti-CD5 ADC is administered to a human patient in need thereof by administering a CAR-expressing immune cell. In one embodiment, the anti-CD5 ADC is administered in combination with CAR therapy. and administering the anti-CD5 ADC to the human patient, wherein the anti-CD5 ADC is administered to the human patient in combination with the CAR-expressing immune cells. In one embodiment, the anti-CD5 A antibody is administered to the human subject about 12 hours to about 21 days prior to administration of the antibody to the human subject. DCs are administered to the human patient in combination with CAR therapy, wherein the anti-CD5 ADCs are administered to the human patient in combination with CAR therapy. The antibody is administered to the human subject about 18 hours to about 20 days prior to administration of the expressing immune cells. Then, the anti-CD5 ADC is administered to the human patient in combination with CAR therapy, wherein the anti- A CD5 ADC is administered to the human subject about 20 hours to about 18 days prior to administration of the CAR-expressing immune cells. In one embodiment, the anti-CD5 ADC is administered to the human patient in combination with CAR therapy. wherein the anti-CD5 ADC is administered about 1 day to about 15 days before administration of the CAR-expressing immune cells. In one embodiment, the anti-CD5 ADC is administered to the human subject in combination with CAR therapy. and administering the anti-CD5 ADC to the human patient about 1 hour prior to administration of the CAR-expressing immune cells. In one embodiment, the anti-CD5 ADC is administered to the human subject about 10 to 20 days prior to administration of the anti-CD5 ADC. R therapy, wherein the anti-CD5 ADC is administered to the human patient in combination with the CAR-expressing immune cell. The human subject is administered about 2 to about 8 days prior to administration of the cells. The anti-CD5 ADC is administered to the human patient in combination with CAR therapy, wherein the anti-CD5 ADC The antibody is administered to the human subject about 3 to about 6 days prior to administration of the CAR-expressing immune cells.

[0104] Overall levels of T cells in biological samples from human patients were assessed by measuring the overall levels of T cells in biological samples from human patients treated with anti-CD5 ADCs. where the levels after administration of the anti-CD5 ADC are compared to pre-administration levels. Reducing the overall number of T cells in human patients prevents the CAR cell therapy from being rejected In one embodiment, the anti-CD5 ADC is administered to a subject in need of treatment with a culture medium derived from the human patient. The level of endogenous T cells in a biological sample is measured immediately before administration of the anti-CD5 ADC. The level of T cells in a biological sample (e.g., blood) from the human patient is of the same type. At least about 5%, at least about 10%, at least about 15%, at least about 20% reduction compared to In one embodiment, the endogenous T in the biological sample from the human patient is The levels of cells were determined by measuring the levels of cells in a biological sample from the human patient immediately prior to administration of the anti-CD5 ADC. Approximately 5% to 25%, approximately 5% to 20%, compared to the levels of T cells in the same type (e.g., blood) %, about 5% to 15%, or about 5% to 10%. In one embodiment, the level of endogenous T cells is reduced. is measured no more than one day before administration of the anti-CD5 ADC.

[0105] T cell levels can be assessed using, but not limited to, fluorescence activated cell sorting (FACs) analysis or blood This can be determined according to standard methods known in the art, including liquid analyzers and the like.

[0106] As mentioned above, one advantage of the methods described herein is that it provides a method for treating cancer in patients undergoing CAR therapy or In a conditioning regimen prescribed to a human patient planning to receive Lymphocyte depleting chemotherapy may be reduced or eliminated Lymphocyte-depleting chemotherapy agents, such as, but not limited to, Fludara cyclophosphamide, bendamustine, and / or pentostatin, and CAR therapy As an anti-rejection agent to promote acceptance of cells expressing CAR in recipients In certain embodiments, a human patient is administered, for example, an immune cell expressing a CAR. The anti-CD5 ADC is administered in combination with immune cells (e.g., T cells) to generate immune cells expressing the CAR. Prior to, simultaneously with, or following administration of immune cells, the human patient may be subjected to lymphodepletion. and not receiving chemotherapy (e.g., fludarabine and / or cyclophosphamide).

[0107] To deplete endogenous immune cells in human subjects and to reject immune cells expressing CARs The use of anti-CD5 ADCs as agents to reduce the risk of infection with other immune For example, alemtuzumab is used as a CAR therapy. To promote uptake of CAR-expressing cells in humans undergoing chemotherapy, In certain embodiments, a human patient is administered CAR- and administering an anti-CD5 ADC prior to or in combination with immune cells (e.g., T cells) expressing the CD5A receptor. As a result, the human patient receives, for example, immune cells (e.g., T cells) that express the CAR. Do not receive alemtuzumab prior to, concurrently with, or following administration.

[0108] In certain embodiments, the anti-CD5 ADC is administered to promote tolerance to immune cells expressing a CAR. For example, an anti-CD2 ADC can be used in combination with another therapy to promote progression of CA The anti-CD2 ADC may be administered to the human patient prior to receiving R therapy. where the anti-CD2 ADC and the anti-C Both D5 ADCs are administered to the human patient prior to CAR therapy.

[0109] The methods disclosed herein can be used for both autologous and allogeneic cells expressing CAR. Importantly, the anti-CD5 ADC conditioning methods described herein may be used The method comprises providing a means by which tolerance to allogeneic cells can be provided. , which helps expand the types of immune cells that can be used in CAR therapy. In the present invention, the CAR-expressing immune cells are allogeneic or autologous cells. Examples of immune cell types that may be engineered to include, but are not limited to, These include allogeneic T cells, autologous T cells, autologous NK cells, or allogeneic NK cells.

[0110] In one embodiment, the anti-CD5 antibody-drug conjugate is used to treat CAR cell therapy. The anti-CD5 antibody-drug conjugate is then administered to the subject cell line, which expresses CD5. Depleting donor cells (e.g., activated T cells expressing CD5). In some embodiments, the CAR cell therapy comprises allogeneic cells.

[0111] The methods disclosed herein include administering to a subject a therapeutically effective amount of ketamine in a human subject having one of these disorders. The compounds are particularly useful for treating cancer or autoimmune diseases.

[0112] In some embodiments, the methods disclosed herein are used to treat cancer. To achieve this, anti-CD5 ADCs are administered in combination with CAR therapy to human subjects with cancer. Examples of cancer types that can be treated using the disclosed methods include, but are not limited to, However, there are cases of advanced adult cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, and metastatic colorectal cancer. Intestinal 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, recurrent 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 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 colorectal carcinoma, gastric carcinoma, pancreatic carcinoma ma), triple-negative invasive breast carcinoma, renal cell carcinoma, lung squamous cell carcinoma, Hepatocellular carcinoma, urothelial carcinoma, leukemia , B-cell acute lymphocytic leukemia (B-cell acute lymphocytic leukemia) ), B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia ), childhood acute lymphoblastic leukemia, refractory small Refractory childhood acute lymphoblastic leukemia (ARDS) leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia (prolymphocytic leukemia) mphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, recurrent plasma cell recurrent plasma cell myeloma, refractory plasma cell myeloma lasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, multiple myeloma of bone tumors, malignant glioma of the brain, myelodysplastic syndromes, EGFR-positive colon rectal cancer, glioblastoma multiforme, neoplasm, blastic plasmacytoid dendritic cell blastic plasmacytoid dendritic cell neoplasm, liver metastasis, solid tumor, progressive Advanced solid tumor, mesothelin positive tumor, Hematological malignancies, and other advanced malignancies.

[0113] In some embodiments, the methods disclosed herein are used to treat an autoimmune disease. More specifically, anti-CD5 ADCs are used in combination with CAR therapy in human subjects with autoimmune diseases. Autoimmune diseases that may be treated using the combination methods disclosed herein include Examples include, but are not limited to, multiple sclerosis, Crohn's disease, ulcerative colitis, These include rheumatoid arthritis, type I diabetes, lupus, and psoriasis.

[0114] In certain embodiments, the anti-CD5 ADCs are administered to a human patient in combination with CAR-T cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient prior to administering CAR-T therapy. Examples of CAR-T cells that may be used in combination with the anti-CD5 ADC therapies described herein include Examples of CAR-T include, but are not limited to, CD19 CAR-T (eg, CART-19-01,02, 03 (Fujian Medical University); daopeicart (Hebei Senlang Biotechnology Inc.); I M19CART / 001,YMCART201702 (Beijing Immunochina Medical Science & Technology Co.); CART-CD19-02,03 (Wuhan Sian Medical Technology Co.); Universal CD19-CART / SHBYCL 001,002 (Shanghai Bioray Laboratory Inc.); UnicarTherapy201701 (Shanghai Unicar- Therapy Biomedicine Technology Co.); Genechem / NCT02672501 (Shanghai GeneChem Co. ); SenL_19 (Hebei Senlang Biotechnology Inc.); PCAR-019 (PersonGen BioTherapeuti cs (Suzhou); ICAR19 (Immune Cell, Inc.); WM-CART-02 (Sinobioway Cell Therapy Co. ); HenanCH080,109,152 (Henan Cancer Hospital / The Pregene (ShenZhen) Biotechnol ogy Co.); IM19-CD28 and IM19-41BB CAR-T cells (Beijing Immunochina Medical Scien ce & Technology Company); CTL019 / IT1601-CART19 (Beijing Sanwater Biological Tech nology Co.); CTL019 / CCTL019C2201 (Novartis Pharmaceuticals); CD19:4-1BB:CD28:CD3 / FirstShenzhen01 (Shenzhen Second People’s Hospital / The Beijing Pregene Sci ence and Technology Company); MB-CART19.1 (Shanghai Children’s Medical Center / Miltenyi Biotec GmbH); PZ01 CAR-T cells (Pinze Lifetechnology Co.); YMCART20170 1 (Beijing Immunochina Medical Science & Technology Co.); 2016YJZ12 (Peking Univ ersity / Marino Biotechnology Co.); EGFRt / 19-28z / 4-1BBL CAR T cells (Memorial Sl oan Kettering Cancer Center / Juno Therapeutics, Inc.); Doing-002 (Beijing Doing Biomedical Co.); PCAR-019 (PersonGen BioTherapeutics (Suzhou) Co.); C-CAR011 (P eking Union Medical College Hospital / Cellular Biomedicine Group Ltd.); iPD1 CD 19 eCAR T cells (Peking University / Marino Biotechnology Co.); 2013-1018 / NCT025 29813 (M.D. Anderson Cancer Center / Ziopharm / Intrexon Corp.); HenanCH CAR 2-1 (Henan Cancer Hospital / The Pregene (ShenZhen) Biotechnology Co.); JCAR015 (Ju no Therapeutics, Inc.); JCAR017 / 017001,004,006 (Juno Therapeutics, Inc.); JCAR01 7 (Celgene); TBI-1501 (Takara Bio Inc.); JMU-CD19CAR (Jichi Medical University); KTE-C19 (Kite, A Gilead Company); TriCAR-T-CD19 (Timmune Biotech Inc.); PF-0517 5157 (Fred Hutchinson Cancer Research Center)); CD22 / CD30 / CD7 / BCMA / CD123 (eg, 2016040 / NCT03121625 (Hebei Senlang Biotechnology Inc.)); CD22 (eg, Ruijin-CAR- 01 (Ruijin Hospital / Shanghai Unicar-Therapy Bio-medicine Technology Co.); AUTO -PA1,DB1 (Autolus Limited)), CD20 (eg, Doing-006 (Beijing Doing Biomedical Co. )); or CD20 / CD22 / CD30 (eg, SZ5601 (The First Affiliated Hospital of Soochow U niversity Shanghai / Unicar-Therapy Bio-medicine Technology Co.)).

[0115] Chimeric antigen receptor (CAR) The present invention includes the use of CAR therapy in combination with anti-CD5 immunosuppressive ADCs. -CD5 ADCs inhibit C by depleting endogenous CD5+ immune cells (e.g., endogenous T cells) By promoting the reception of AR-expressing cells, it can be used as a conditioning agent for CAR therapy. Based at least in part on the discovery that oxidative stress may play a role in Generally, the specific CAR construct (e.g., a specific antigen-binding site or an intracellular signaling domain) is selected. Specific CARs (e.g., CD19-specific CARs) are contemplated herein. and the methods disclosed herein, but are not meant to be limiting.

[0116] CAR constructs are known in the art and generally comprise: (a) a cell that contains an antigen-binding domain; (b) an ectodomain, (b) a transmembrane domain, and (c) an intracellular signaling domain. The composition of the present invention is known in the art, and in the methods described herein, any suitable For example, the CAR may be prepared using the methods described in, for example, Guedan et al. Molecular Ther. apy-Methods & Clinical Development. 12: 145-156 (2019) or Sadelain et al. Cance As described in r discovery 3.4: 388-398 (2013) et al., the entire contents of which are incorporated herein by reference. (incorporated by reference herein), may be a first generation, second generation, or third generation CAR. Briefly, a “first generation” CAR consists of (a) an extracellular antigen-binding domain, (b) a transmembrane domain, and (c) one or more intracellular signaling domains, and optionally (d) an antigen-binding domain. A "second generation" CAR may contain a hinge region that connects the CAR to the transmembrane domain. The polypeptide may further comprise a co-stimulatory domain. (e.g., the costimulatory domain of CD28 or 4-1BB). "Third generation" CARs may include It may include elements (a), (b), (c), and optionally (d), and further include multiple co-stimulatory domains (e.g., the costimulatory domains of CD28 and 4-1BB, or the costimulatory domains of CD28 and OX40) Each of the above elements is described in more detail below. In the form, the CAR molecule is described by the following exemplary non-limiting configuration: It should be understood that the CAR is directed from the N-terminus to the C-terminus of the CAR. The AR may or may further include any other combination of elements described herein. There is.

[0117] The CAR used in the methods disclosed herein comprises an extracellular antigen-binding domain. The extracellular antigen-binding domain may be any molecule that binds to an antigen. The present invention includes, but is 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 one embodiment, the extracellular antigen-binding domain is a non-immunoglobulin scaffold protein. In another embodiment, the extracellular binding domain of the CAR is Single chain T cell receptors (scTCRs) are disclosed in U.S. Patents 5,359,046, 5,686,281 and 6, The extracellular domain can be coupled to ligand binding and / or signal transduction pathways as described in US Pat. No. 5,399,133, and US Pat. No. 5,321,521. from any of a wide variety of extracellular domains or secreted proteins involved in IL-1 signaling. There are also.

[0118] Selecting a molecular target (antigen) for the extracellular binding domain comprises It depends on the type and number of ligands that define the surface. For example, Selectively select target cells that act as cell surface markers for specific pathological conditions. Thus, in some embodiments, the antibody may be designed to recognize a ligand specific for a desired antigen. By engineering an extracellular antigen-binding domain that binds heterologously into the CAR, They can target immune cell (e.g., T-cell) responses to a target antigen. For example, the antigen-binding domain can be selected to treat a particular disease state, such as cancer or an autoimmune disease. To recognize ligands that act as cell surface markers on relevant target cells Thus, they may act as ligands for the antigen-binding domains in the CAR. Examples of cell surface markers that may be involved include those that are specific to cancer cells and other forms of diseased cells (e.g. , autoimmune disease cells, and pathogen-infected cells. In one embodiment, the method involves engineering a desired antigen-binding domain that specifically binds to an antigen on a tumor cell. The CAR is engineered to target a tumor antigen of interest. In this context, "tumor antigen" refers to an antigen that is common to a particular hyperproliferative disorder, such as cancer. In one embodiment, the antigen is a tumor antigen. Examples of these include, but are not limited to, However, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, and mesothelial cells Mesothelin, CD38, PD1, EGFR (eg, EGFRvIII), MG7, BCMA, TACI, CEA, PSC A, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or CD123. In one embodiment, the CAR is selected from the group consisting of CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR (eg, EGFRvIII), MG7 , BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NK The scFvs include those that bind to G2D, or CD123.

[0119] In another embodiment, the extracellular binding region of the CAR binds to an antigen that is AFP (e.g., ETCH17AFPCAR01 (Aeon Therapeutics (Shanghai) Co. / Eureka Therapeutics Inc.)), G PC3 (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 Hospi number / Marino Biotechnology Co.)), CD38 (eg, RO-CD38 A2 CAR-T / SOR-CART-MM-001 (Sorrento Therapeutics, Inc.)), herinCAR-PD1 (eg, herinCAR-PD1 / NBWYKY2016-06- 001,002,003 (Ningbo Cancer Hospital); SIMC-20160101,02,03 (Shanghai International l Medical Center)), BCMA (eg, P-BCMA-101 Transaction T Section Component(Tscm) CAR-T Transport / . P-BCMA-101-001 (Poseida Therapeutics, Inc.); HenanCH284 (Henan Cancer Hospital) The Pregene (ShenZhen) Biotechnology Company); LCAR-B38M CAR-T Convertible (Nanjing Leg and Biotech Co.); 9762 / NCT03338972 (Fred Hutchinson Cancer Research Center / Jun. 9762 / NCT03338972). o Therapeutics, Inc.); Descartes-08 (Cartesian Therapeutics); KITE-585 (Kite, A Gilead Company); bb21217 (bluebird bio); bb21217 (Celgene); JCARH125 (Juno Thera peutics, Inc.)), CD30 (eg, ICAR30 T cells (Immune Cell, Inc.)), EGFR (eg, EG FR:4-1BB:CD28:CD3 Antibodies / 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-201 6-015-01 (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)), メソテリン(mesothelin) / PSCA / CEA / HER2 / MUC1 / EGFRvIII (e.g., N CT03267173 (First Affiliated Hospital of Harbin Medical University / Shanghai Un icar-Therapy Bio-medicine Technology Co.)), CD20 (e.g., EY201605-19 (Beijing Bio healthcare Biotechnology Co.)), CD33 (e.g., 2016-0341 / NCT03126864 (M.D. Anderson Cancer Center / Intrexon Corp. / Ziopharm)), EGFR / BCMA (e.g., EGFRt / BCMA-41BBz CAR T cells (Memorial Sloan Kettering Cancer Center / Juno Therapeutics, Inc.)), R OR2 (eg, autologous CCT301-38 or CCT301-59 T cells (Shanghai Sinobioway Sunterra Biotec ch)), 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 / Mar ino Biotechnology Co.)), TAA (eg, AMG 119 (Amgen)), NKG2D (eg, CM-CS1 (Celya d)), or CD123 (eg, UCART123 (Cellectis SA)). The preceding sentence refers to the antigen (e.g., Examples of CARs that bind to anti-CD5 AD are further provided. C. .

[0120] The CAR construct links the extracellular antigen-binding domain to an intracellular signaling domain. Further, the transmembrane domain may be further separated (either literally or nearly adjacent, e.g., by using a spacer). In some embodiments, the extracellular antigen binding domain of the CAR (e.g., scFv The Fab or other antigen-binding portion is linked to the transmembrane domain using a hinge or other linker. A spacer, linker, or hinge may be attached to the extracellular antigen-binding domain. between the membrane domain and the transmembrane domain, allowing the antigen-binding domain to be oriented in various directions. providing flexibility that allows the molecule to be easily adapted to recognize and bind antigens; The cytoplasmic side of the transmembrane domain is connected to the intracellular signaling of CD28 or CD3 zeta (CD3-ζ). They may be attached to intracellular signaling domains such as RING domains, and further may contain one or more costimulatory domains, as discussed below.

[0121] Thus, in certain embodiments, the CAR comprises an extracellular antigen-binding domain and a membrane-binding domain. The polypeptide may further include a hinge region located between the spanning domain. For example, the hinge region may include The domains are IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha hinge domains. In certain embodiments, the hinge region may be derived from the hinge region of IgG4. In another embodiment, the hinge region is the hinge domain of CD8 ( SwissProt / GenBank Acc.NoP01732).

[0122] In one embodiment, the CAR comprises an extracellular antigen binding domain and a CD8 hinge linked thereto. Transmembrane domain-containing: AKPTTTPAPR PPTPAPTIAS QPLSLRPEAC RPAAGGAVHT RGLDFA (sequence Number(SEQ ID NO): 9).

[0123] In one embodiment, the CAR comprises an extracellular antigen binding domain and a hybrid CD8-CD28 antigen binding domain. Contains transmembrane domains linked via dimers: AKPTTTPAPR PPTPAPTIAS QPLSLRPEAC RPAAG GAVHT RGLDFAPRKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSP LFPGPSKP (SEQ ID NO: 10).

[0124] The transmembrane domain contributes to the extracellular antigen-binding domain of the protein, the effector Proteins that contribute to functional signaling domains, proteins that contribute to proliferation signaling domains The sequence may be derived from a sequence in one protein, or from a completely different protein. In 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 transmembrane domains of the same protein. In one embodiment, the CAR may be derived from the transmembrane domain and the cytoplasmic domain. The transmembrane and intracellular domains comprise contiguous portions of the CD28 sequence. , if that domain can anchor the CAR containing the antigen-binding domain to the cell membrane, It can be used in the CAR constructs described herein.

[0125] The transmembrane domain may be derived from either natural or synthetic sources. When the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary membrane spanning proteins that may be used in the methods provided herein The domains are 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 receptor CD8 alpha and their fragments (e.g., at least The transmembrane domain of a protein includes any of the above-mentioned transmembrane domains. Using any method (e.g., hydrophobicity analysis, structural analysis, etc.) or public databases ( For example, the identification can be performed by using the UniProt database.

[0126] In some embodiments, the transmembrane domain may be synthetic. In one embodiment, the transmembrane domain is comprised primarily of hydrophobic residues, such as leucine and valine. In some embodiments, the tryptophan, phenylalanine, and valine amino acids may be included. A replet may be placed at each end of the synthetic transmembrane domain. Generally, the linker is a short oligo or polypeptide linker (preferably 2-10 amino acids long). Thus, a link between the transmembrane domain and the intracellular signaling domain of CAR is formed. A glycine-serine doublet provides a particularly suitable linker.

[0127] In some embodiments, the transmembrane domain in a CAR as used herein is the transmembrane domain of CD8. The sequence of CD8 for this purpose is described in PCT Publication No. WO2014 / 055. This is taught in 771A1.

[0128] In some embodiments, the transmembrane domain in the CAR is the transmembrane domain of CD8 or a For example, CAR is a functional portion of LDPKLCYLLD GILFIYGVIL TALFLRVK (SEQ ID NO: ID NO: 11), or a functional portion thereof (e.g. , LCYLLDGILF IYGVILTALF L (SEQ ID NO: 12).

[0129] In some embodiments, the transmembrane domain in the CAR of the invention is the transmembrane domain of CD28. Exemplary sequences of CD28, as well as exemplary transmembrane domain sequences, are provided below. In some embodiments, the transmembrane domain of CD28 has the following exemplary transmembrane domain sequence: Immobilizing a CAR containing the sequence, or a fragment or variant thereof, on a cell membrane Thus, in some embodiments, the membrane of the CAR comprises The transmembrane domain is the transmembrane domain of CD28, which contains the following amino acid sequence: FWVLVVVGGVLA. CYSLLVTVAFIIFWV (SEQ ID NO: 13). In one embodiment, the transmembrane domain of the CAR is The in is the transmembrane domain of CD28, which contains the following amino acid sequence: IEVMYPPPYL DNEKSNGTI I HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWV (SEQ ID NO: 16), or a functional fragment thereof, such as SEQ ID NO:14.

[0130] In addition to the extracellular antigen-binding domain and the transmembrane domain, CARs contain an intracellular (or cytoplasmic) It further comprises a signaling domain.

[0131] Signals generated through endogenous TCRs alone are insufficient to fully activate T cells. It is known that the induction of stimulatory activity is a minor process and that secondary or co-stimulatory signals may also be required. Thus, T cell activation is mediated by two different types of intracellular signaling sequences. That is, antigen-dependent primary activation can be initiated via the TCR. Those that act antigen-independently to mediate secondary signals or co-stimulation These provide a stimulatory signal (secondary intracellular signaling sequences).

[0132] The term "intracellular signaling domain" or "cell As used herein, the term "cytoplasmic signaling domain" refers to a The intracellular signaling domain is an immunoglobulin that contains a CAR. Signals that promote immune effector functions of immune cells (e.g., CAR-T cells or CAR-expressing NK cells) For example, immune effectors in CART cells or CAR-expressing NK cells can be generated. Examples of the target function include cytolytic activity and helper activity including secretion of cytokines. In embodiments, the intracellular signaling domain transmits an effector function signal. Intracellular signaling You can use an entire domain, but it is often necessary to use the entire chain. To the extent that portions of the truncated intracellular signaling domain are used, Such a cleaved portion will function as an effector in so far as it transmits the effector function signal. can be used in place of intact chains. Thus, intracellular signaling domains The term "intracellular signaling pathway" refers to a pathway that is sufficient to transmit the effector function signal. - means to include any truncated portion of the domain.

[0133] In one embodiment, the intracellular signaling domain of the CAR is selected from the group consisting of SEQ ID NO: 15. A signaling region of CD3 zeta, or a signaling portion thereof, as described in : RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 15).

[0134] Intracellular signaling domains include, but are not limited to, CD3 zeta, FcR gamma , FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, CD278 ("ICOS"), Fc-epsilon-RI, CD66d, DAP10, DAP12 There are times when this happens.

[0135] CARs are designed to bind co-stimulatory proteins or proteins that enhance cytokine production (e.g., CD28 The polypeptide chains derived from the intracellular signaling domains of 4-1BB and 4-1BB are called "cell signaling domains." Exemplary costimulatory signaling regions include an "internal costimulatory domain" Examples of such receptors include 4-1BB, CD21, CD28, CD27, CD127, ICOS, IL-15Rα, and OX40.

[0136] In certain embodiments, the cytoplasmic costimulatory domain of the CAR is linked to the 4-1BB signaling domain. The CAR domain may be used alone or in combination with any other desired intracellular domain useful in the context of a CAR. 4-1BB includes the amino acid sequence provided under GenBank Accession No. AAA62478.2, or T with the equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.) The 4-1BB costimulatory domain is a member of the NFR superfamily; and the "4-1BB costimulatory domain" is a member of the GenBank Accession No. c Amino acid residues 214 to 255 of AAA62478.2, or from non-human species (e.g., mouse, rodent The term is defined as the equivalent residue from a human (e.g., monkey, ape, etc.).

[0137] In one embodiment, the intracellular costimulatory signaling domain of the CAR is 4-1BB (CD137). or a signaling portion thereof: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 60).

[0138] In one embodiment, the costimulatory signaling domain of the CAR is a CD28 costimulatory signaling domain. For example, the costimulatory signaling domain is the following CD28 costimulatory domain: A signaling moiety or a signaling moiety that may include: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 17).

[0139] In an exemplary embodiment, the intracellular domain of the CAR is a CD3-zeta signaling domain. The domain can be combined with any other desired intracellular domain that is useful in the context of the CAR of the present invention. In certain embodiments, the intracellular domain of the CAR may comprise, in combination, D3 zeta domains and costimulatory signaling regions (such as, but not limited to, These may include the co-stimulatory signaling domains of 4-1BB, CD28, and CD27.

[0140] The intracellular signaling sequence within the intracellular signaling portion of the CAR of the invention can be random or They may be linked together in a specific order. Optionally, short oligo- or polypeptide A linker or spacer (preferably 5 to 20 amino acids long) is inserted between the intracellular domains. GGGGS (SEQ ID NO: 18) or (GGGGS) x 3 (SEQ ID NO: 19) provides a particularly suitable linker.

[0141] In one embodiment, a CAR as used herein refers to a single chain variable region of an anti-CD19 monoclonal antibody. The extracellular domain containing the domain, the transmembrane domain containing the hinge and transmembrane domain of CD8α The trafficking domain, as well as the signaling domains of CD3ζ and 4-1BB An exemplary CAR comprises an intracellular domain containing Nol 34:1157-1165 (1997) In addition to the CD8α 21 amino acid signal peptide (GenBank accession no. NM_0 63 nucleotides from 26 to 88 of 01768 are translated. The insert was translated from 207 nucleotides from 815 to 1021 of GenBank accession number NM_001768. The CD3ζ signaling domain of a preferred embodiment is comprised of 69 amino acids that are known to be involved in the synthesis of CD3ζ. 112 nucleotides translated from 339 nucleotides from 1022 to 1360 of nk accession number NM_000734 Contains amino acids.

[0142] Between the extracellular domain (including the antigen-binding domain) and the transmembrane domain of the CAR (described above); or a spacer or hinge domain between the intracellular domain and the transmembrane domain of the CAR. As used herein, a "spacer domain" may include Generally, the transmembrane domain is located within the extracellular and / or intracellular domain of the polypeptide chain. In the present specification, the term "polypeptide" refers to any oligo or polypeptide that functions to link to an in If used, a hinge domain generally provides flexibility to the CAR, or a domain thereof. and / or function to prevent steric hindrance of the CAR, or a domain thereof. In some embodiments, a spacer or hyaluronic acid is used. A range domain may be up to 300 amino acids, preferably 10 to 100 amino acids, most preferably The embodiment of the present disclosure is not limited in this respect, and may contain 5 to 20 amino acids. It should also be understood that one or more spacer domains may be included in other regions of the CAR. be.

[0143] CARs can be constructed by incorporating regions (e.g., antigen-binding domains, transmembrane domains) having sequences as provided herein. domain, intracellular domain, signaling domain, safety domain, and / or linker, or any combination thereof) or variants thereof or any fragment thereof (e.g., a fragment thereof that retains a function required for CAR activity). The CAR proteins described herein may include variants and / or fragments thereof. It should be understood that in some embodiments, the variants are 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have been changed. In this embodiment, the variants have at least 80%, at least 85%, at least Some have sequences that are 90%, 90%-95%, at least 95%, or at least 99% identical. In certain embodiments, the fragment may be 1-5, 5-10, 1 In some embodiments, the fragment is 0 to 20, 20 to 30, 30 to 40, or 40 to 50 amino acids shorter. The fragment may be shorter at the N-terminus, C-terminus, or both terminal regions of the provided sequence. In one embodiment, the fragments are 80% to 80% of the number of amino acids in the sequences provided herein. Including 5%, 85%-90%, 90%-95%, or 95%-99%.

[0144] In some embodiments, the above exemplary, non-limiting configurations are from the left to the right side of the CAR. , N-terminal to C-terminal. The CAR may be any of the elements as described herein. Other combinations may be included or may further be included.

[0145] Once the CAR construct has been identified with its various parts, immune cells expressing the CAR are produced and The immune cells express the CAR by the nucleic acid sequence described herein. A nucleic acid molecule (e.g., an RNA molecule (e.g., an mRNA)) or a CAR (e.g., a CAR described herein) is and introducing (e.g., transducing) the immune cells with a vector comprising a nucleic acid molecule that encodes the In particular, nucleic acids encoding the amino acid sequences disclosed herein are The present invention also relates to a cell population (e.g., a cell line that transiently expresses an exogenous RNA, such as an RNAi gene) that is capable of expressing an exogenous RNA. The present invention provides a method for producing a cell engineered with an RNA as described herein. A (e.g., in vitro transcribed RNA or synthetic RNA; a CAR polypeptide as described herein) In one embodiment, the method includes introducing into the cell an mRNA sequence encoding the polypeptide. In one embodiment, the cell transiently expresses a CAR polypeptide as described herein. The cells (e.g., immune effector cells (e.g., T cells or NK cells, or cell populations) It is.

[0146] Other exemplary chimeric antigen receptor constructs are described in U.S. Pat. No. 9,328,156; U.S. Pat. No. 9,328,156; ,783,591; U.S. Patent No. 9,714,278; U.S. Patent No. 9,765,156; U.S. Patent No. 10,117,896 No. 9,573,988; U.S. Patent No. 10,308,717; U.S. Patent No. 10,221,245; U.S. Patent No. US Patent Publication No. 2018 / 0256712A1; US ​​Patent Publication No. 18 / 0271907A1; U.S. Patent Publication No. 2016 / 0046724A1; U.S. Patent Publication No. 2018 / 0044 424A1; U.S. Patent Publication No. 2018 / 0258149A1; U.S. Patent Publication No. 2019 / 0151363A1 and U.S. Patent Publication No. 2018 / 0273601A1; The contents of each of the publications are incorporated herein by reference in their entirety.

[0147] III. Anti-CD5 Antibody Drug Conjugates (ADCs) As described herein, anti-CD5 ADCs can be used in combination with CAR therapy to treat tumors in human patients. To treat cancer or autoimmune diseases. More specifically, anti-CD5 ADCs can be used to treat CAR therapy. In a human subject receiving the antibody, CD5+ cells (e.g., CD5+ T cells) can be decreased. Anti-CD5 ADCs target endogenous T cells and kill these cells, The patient's immune system recognizes the CAR-expressing immune cells (autologous or allogeneic) administered to the subject. Thus, anti-CD5 ADCs can be used in combination with CAR therapy to treat conditioned The engineered CARs were then used as a immunization regimen in recipient patients. Anti-CD5 ADC as a conditioning regimen to promote the reception of immune cells expressing One advantage of using the method is that it is possible to administer a common systemic lymphodepleting chemotherapy agent to the subject. This allows for more efficient conditioning of endogenous T cells expressing CD5 than traditional methods of conditioning CAR therapy. The reduction is specifically targeted.

[0148] Anti-CD5 antibody Using ADCs that can bind to CD5 as therapeutic agents, we aim to detect CAR-expressing immune cells that reject the drugs. The aim of this study is to prevent or reduce the risk of cancer death in human patients by administering CAR-expressing immune cells to a patient with cancer. This may promote acceptance in the field.

[0149] The anti-CD5 ADCs described herein comprise an anti-CD5 antibody or antigen binding site thereof linked to a cytotoxin. Includes parts.

[0150] Human CD5 is also called lymphocyte antigen T1, T1, Leu-1, and LEU1. CD5 is expressed on human T cells. Two isoforms of human CD5 have been identified. Isoform 1 is , containing 495 amino acids, Gladkikh et al (2017) Cancer Med.6(12):2984 and Jones et al (1986) Nature 323 (6086): 346. Amino acid sequence of CD5 (isoform 1) The sequence is shown below (NCBI reference sequence: NP_055022.2): [ka]

[0151] The second isoform of human CD5 is 438 amino acids and is shown below in NCBI reference sequence: NP_001333 Unlike isoform 1, CD5 isoform 2 is an intracellular protein. Isoform 2 contains a different 5' UTR compared to isoform 1, The resulting isoform lacks the in-frame portion of the 5' coding region. CD5 isoform 2 has a shorter N-terminus than isoform 1. Compared to chromatin 1, it lacks a leader peptide and is an intracellular protein found in a subset of B lymphocytes. The ADCs described herein are designated as human CD5 isoforms, which represent the extracellular version of human CD5. It is specific for CD5 isoform 1.

[0152] In some embodiments, they may be used in the methods and compositions described herein. The anti-CD5 antibody is antibody 5D7v (Ab5D7v). The heavy chain variable region (VH) amino acid sequence of Ab5D7v is shown below. The sequence ID number is shown in SEQ ID NO: 1. [ka]

[0153] The VH CDR amino acid sequence of Ab5D7v is underlined above and is as follows: FSLSTSGMG(VH CDR DR1; SEQ ID NO: 3); WWDDD (VH CDR2; SEQ ID NO: 4); and RRATGTG FDY (VH CDR3; SEQ ID NO: 5).

[0154] The light chain variable region (VL) amino acid sequence of Ab5D7v is provided below as SEQ ID NO: 2). Provide. [ka]

[0155] The VL CDR amino acid sequence of Ab5D7v is underlined above and is as follows: QDVGTA(VL CDR1 ; SEQ ID NO: 6); WTSTRHT (VL CDR2; SEQ ID NO: 7); and YNSYNT ( VL CDR3; SEQ ID NO: 8).

[0156] In one embodiment, the anti-CD5 ADC comprises the amino acid sequence set forth in SEQ ID NO:3. a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO: 4; and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 4. and a heavy chain comprising a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO: 5; CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO: 7, and a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO: 8. and a light chain comprising a CDR3 domain comprising the amino acid sequence of is conjugated to a cytotoxin via a linker.

[0157] In one embodiment, the anti-CD5 ADC comprises the amino acid sequence set forth in SEQ ID NO:1. and a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:2. and a light chain comprising a cytotoxin A domain, wherein the antibody is coupled to a cytotoxic agent via a linker. It is conjugated to the element.

[0158] In another embodiment, the anti-CD5 antibody used in the ADCs described herein is the 5D7 antibody. (See, e.g., US 20080254027, the disclosure of which is incorporated herein by reference. In another embodiment, in the methods and compositions (including ADCs) described herein The anti-CD5 antibody that may be used is a variant of the 5D7 antibody (see, e.g., US 200802540 27, the disclosure of which is incorporated herein by reference).

[0159] Moreover, in certain embodiments, the anti-CD5 ADCs are characterized by serum CD4+ levels in human subjects for 3 days or less. It has a half-life.

[0160] Additional anti-CD5 antibodies that may be used in the ADCs described herein include those that are hybridized with The hybridoma may be identified using techniques known in the art, such as doma production. For immunization and subsequent fusion, mAbs may be prepared using the mouse system. Protocols for isolating splenocytes in vitro are known in the art. Fusion partners and procedures for making HuMAb-Mouse are also known. 登録商標 or XenoMouseTM To generate additional anti-CD5 antibodies, The CD5 antigen is isolated and / or purified during the preparation of the antibody. The immunization of animals may be by any method known in the art. For example, the method of Harlow and Lane, Antibodies: A Laboratory Manual See the Y Manual, New York: Cold Spring Harbor Press, 1990. Mice, rats Methods for immunizing animals such as rabbits, 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 CD5 antigen may be administered with an adjuvant to stimulate the immune response. Adjuvants known in the art include complete and incomplete Freund's adjuvant, RI BI (muramyl dipeptide) or ISCOM (immunostimulating complex). After immunization, antibody-producing immortalized cell lines are prepared from cells isolated from the immunized animal. The animals are then sacrificed and lymph node and / or splenic B cells are isolated using methods known in the art ( For example, oncogene transduction, oncogenic viral transduction, exposure to carcinogenic or mutagenic compounds Exposure, fusion with immortalized cells (e.g., myeloma cells), and inactivation of tumor suppressor genes See, e.g., Harlow and Lane, supra. The desired properties (e.g., robust growth, high antibody production, and desired antibody properties) The selected vector may then be selected, cloned and further screened.

[0161] Anti-CD5 antibodies for use in the anti-CD5 ADCs described herein also bind to CD5. High-throughput screening of antibody or antibody fragment libraries for binding molecules. Screening may be used to identify the In vitro display techniques known in the art, such as, inter alia, phage display Bacterial display, yeast display, mammalian cell display, ribosomal These include genome display, mRNA display, and cDNA display. Isolating antibodies, antigen-binding fragments, or ligands that bind to biologically related molecules The use of phage display for gene expression has been reported, for example, by Felici et al., Biotechnol. Annual Rev. 1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-4 5, 1997; and Hoogenboom et al., Immunotechnology 4:1-20, 1998, reviewed in Each of these disclosures is incorporated herein by reference as it relates to in vitro display technology. Kay, Perspect. Drug Discovery Des. 2:251-268, 19 95 and Kay et al., Mol. Divers. 1:139-140, 1996. We constructed a combinatorial peptide library to identify polypeptides that bind to cell surface antigens. Each of these disclosures relates to the discovery of antigen-binding molecules. The present disclosure is incorporated by reference herein as it relates to proteins (e.g., poly Phage display of functional molecules (multimeric proteins) has been successfully demonstrated. (see, for example, EP 0349578; EP 4527839; and EP 0589877, as well as Chiswell and See McCafferty, Trends Biotechnol. 10:80-84 1992, each of which discloses antigen The present specification relates to the use of in vitro display techniques for the discovery of binding molecules. In addition, functional antibody fragments (e.g., Fab and scFv fragments) can be used. It has also been demonstrated that the GFP gene (a gene fragment) can be expressed in an in vitro display format. (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, each of which discloses an in vitro display platform for the discovery of antigen-binding molecules. (which are incorporated herein by reference as they relate to the platform).

[0162] In addition to in vitro display techniques, we have used the procedures described in US2013 / 0288373. For example, using computational modeling techniques, anti-CD5 antibodies or antibody fragments can be modeled in silico o may be designed and identified (the disclosure provides molecular methods for identifying anti-CD5 antibodies). (The modeling method is incorporated herein by reference.) For example, computational modeling Using genomic DNA techniques, one of skill in the art can identify specific epitopes on CD5, such as extracellular epitopes of CD5. A library of antibodies or antibody fragments is screened in search of molecules that can bind to the antibody. Screening may be performed in silico.

[0163] In certain embodiments, the anti-CD5 antibodies used in the ADCs described herein are Further investigations are needed to identify anti-CD5 antibodies (or fragments thereof). Using techniques such as these, the antibodies bind to CD5 on the surface of cells (e.g., T cells) and further bind to, for example, receptors. The antibody or antibody can be taken up by the cell by target-mediated endocytosis. For example, the in vitro display techniques described above may be used to identify the original binding fragments. The antibody or antigen-binding fragment thereof binds to CD5 on the surface of hematopoietic stem cells and is subsequently internalized therein. Phage differentiation may be adapted to screen for fragments. Splays may be used in conjunction with this screening paradigm such as One representative technique is the use of anti-CD5 antibodies or antibodies that bind to CD5 and subsequently are internalized into CD5+ cells. To identify such fragments, one of skill in the art can refer to the method described in Williams et al., Leukemia 19:1432-1438, 2005 (which is also (the disclosure of which is incorporated herein by reference in its entirety).

[0164] The amount of anti-CD5 antibody or fragment thereof that can be incorporated into a given antibody can be determined, for example, by any method known in the art. Radionuclide internalization assays may be used to assess, for example, Anti-CD5 antibodies identified using in vitro display techniques described herein or known in the art. or a fragment thereof, by the addition of 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, 7 7 As, 72 As, 86 Y, 90 Y, 89 Zr, 212 Bi, 213 Bi, or 225 By incorporating Ac), For example, radioactive halogens (e.g. 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, etc.) with electrophilic halogen reagents (e.g., beta iodide Beads (e.g., polystyrene; Thermo Fisher Scientific, Inc., Cambridge, MA) were used. Using a fluorophore (Len beads) to incorporate antibodies, their fragments, or ligands Radiolabeled antibodies or fragments thereof may be introduced into the hematopoietic stem cells. The antibody may be incubated for a period of time sufficient to allow the antibody to be incorporated into the The fragments are then subjected to detection of radiation (e.g., gamma rays) emitted from the hematopoietic stem cells obtained. and comparing it with the radiation (e.g., gamma radiation) emitted from the collected washing buffer to identify it. The internalization assays described above may also be used to characterize ADCs.

[0165] In some embodiments, the anti-CD5 antibody (or fragment thereof) is a defined serum For example, an anti-CD5 antibody (or a fragment thereof) has a half-life of about Such anti-CD5 antibody-containing ADCs may also be used, e.g., For example, it may have a serum half-life of about 1 to 24 hours in human patients. Pharmacokinetic analysis by measuring the amount of the compound may be performed by assays known in the art. .

[0166] To recombinantly produce an anti-CD5 antibody, a nucleic acid encoding the antibody (e.g., (such as the above) are isolated and further cloned and / or expressed in a host cell. For purposes of this invention, nucleic acid molecules are inserted into one or more vectors. Such nucleic acid molecules can be synthesized using conventional procedures (e.g., For example, oligonucleotides capable of specifically binding to genes encoding the heavy and light chains of the antibody can be used. can be easily isolated and sequenced (by using a tide probe) It is possible to do this.

[0167] Suitable host cells for cloning or expressing vectors encoding antibodies include Examples of such cells include the prokaryotic and eukaryotic cells described herein. , especially when glycosylation and Fc effector function are not required. Expression of antibody fragments and polypeptides in bacteria See, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (Charlton, Methods, 2003, describing the expression of antibody fragments in E. coli.) in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003 (See also, pp. 245-254.) After expression, the antibody is added to a paste of bacterial cells. It may be isolated as a soluble fraction from the paste and further purified.

[0168] Vertebrate cells can also be used as hosts. For example, cells grown in suspension can be used Other useful mammalian host cell lines include mammalian cell lines adapted for use in the Examples include the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293 cell line); cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977); infant Hamster's Star kidney cells (BHK); mouse Sertoli cells (TM4 cells, e.g., Mather, Biol. Reprod. 23:243-251 (1980); monkey kidney cells (CV1); African green monkey kidney cells Human cervical carcinoma cells (HELA); Canine kidney cells (MDCK); Buffer Row rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse breast Tumor (MMT 060562); TRI cells, e.g., Mather et al., Annals NY Acad. Sci. 383:44- 68 (1982); MRC 5 cells; and FS4 cells. The main cell line is Chinese hamster ovary (CHO) cells (e.g., DHFR-CHO cells, etc.). (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines (e.g., Y0, NS0 and Sp2 / 0). Certain mammalian host cells suitable for antibody production are For reviews of major cell lines, see, e.g., Yazaki and Wu, Methods in Molecular Biolog y, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In one embodiment, the host cell is a eukaryotic cell, e.g., Chinese ham Star ovary (CHO) cells or lymphoid cells (eg, Y0, NS0, Sp20 cells).

[0169] In some embodiments, for use in conjunction with the compositions and methods described herein The anti-CD5 antibodies include those having CDR-H1, CDR-H2, CDR-H3, and CDR-H4 as described in Tables 1A and 1B below. These include those that contain a combination of CDR-L1, CDR-L2, and CDR-L3 regions.

[0170] [Table 1-1] [Table 1-2]

[0171] [Table 2-1] [Table 2-2]

[0172] cytotoxin A variety of cytotoxins can be combined with linkers for use in the combination therapies described herein. In particular, anti-CD5 ADCs may be conjugated to anti-CD5 antibodies via a cytotoxic moiety. (or a cytotoxin) conjugated (i.e., covalently attached via a linker) to an antibody (or As used herein, the terms "cytotoxin", " The terms "cytotoxic moiety" and "drug" are used interchangeably. In various embodiments, the cytotoxic When the moiety is attached as a conjugate, the cytotoxicity is reduced or the cytotoxic However, after cleavage from the linker, the cytotoxicity is restored. In various embodiments, the cytotoxic moiety is capable of binding to the cell without being cleaved from the linker. In some embodiments, the cytotoxic molecule is a cytotoxic molecule as described herein. Such antibodies or fragments thereof are conjugated to the antibodies or fragments thereof that are taken up into cells. As a result, after the antibody or fragment thereof is taken up into the cell, the cytotoxin is These proteins may access intracellular targets and, for example, mediate T cell death.

[0173] Thus, the ADCs of the invention may be of the general formula I, where the antibody or antigen-binding fragment thereof is The fragment (Ab) is conjugated (covalently bonded) to a linker (L) and contains a chemical moiety (Z). via which it is conjugated to a cytotoxic moiety ("drug", D). Ab-(ZLD) n (I)

[0174] Thus, the antibody or antigen-binding fragment thereof may comprise multiple antibodies, each of which may be represented by an integer n. may be conjugated to a number of drug moieties, where n is the integer number of cytotoxins per antibody. where the integer n can be, for example, in the range of about 1 to about 20. (e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8) cytotoxins are 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 this embodiment, n is about 1. The number of drugs per antibody in the preparation of ADCs by conjugation reactions is The average number of individual parts can be determined by conventional techniques such as mass spectroscopy, ELISA assays, and HPLC. The quantitative distribution of ADC with respect to n can also be determined. In some cases, it may be possible to identify by means such as reverse phase HPLC or electrophoresis that n is a specific The homogeneous ADCs are separated, purified, and characterized from ADCs with other drug loads. Sexuality can be evaluated.

[0175] For some anti-CD5 ADCs, n is limited by the number of binding sites on the antibody. For example, if the bond is a cysteine ​​thiol, the antibody may bind to one or several cysteines. The linker is a thiol group that has only one thiol group, or a thiol group that is sufficiently reactive to be attached to the linker. Generally, antibodies have only one or a few free radicals that can be attached to a drug moiety. Does not contain many free and reactive cysteine ​​groups; mainly, thiol residues of cysteines in antibodies are present as disulfide bridges. In certain embodiments, the antibody is The partial reduction was performed with a reducing agent such as diethyl ether (DTT) or tricarbonylethylphosphine (TCEP). The thiol group of the reactive cysteine ​​can be generated under partial or total reducing conditions. In certain embodiments, higher drug loading (e.g., n>5) results in certain antibody-drug Conjugates may become aggregated, insoluble, toxic, or lose cell permeability This may be triggered.

[0176] In certain embodiments, during the conjugation reaction, less than the theoretical maximum The drug moiety is conjugated to the antibody. The antibody can be, for example, a drug- It may contain lysine residues that do not react with the linker intermediate or the linker reagent. Only highly acidic lysine groups can react with amine-reactive linker reagents. In an embodiment, the antibody is subjected to denaturing conditions to remove reactive nucleophilic groups, such as lysine or cysteine. Bring it to the surface.

[0177] The loading of the ADC (drug / antibody ratio) can be varied in various ways (e.g., (i) by using a drug-linker (ii) limiting the molar excess of intermediates or linker reagents during the conjugation reaction; (iii) limiting the time or temperature for cysteine ​​thiol modification; (iv) controlling the number and / or location of linker-drug bonds. To achieve this, the amino acid sequence of the antibody is modified to alter the number and position of cysteine ​​residues. It can be used to manipulate or control something.

[0178] Suitable cytotoxins for use with the compositions and methods described herein include those known in the art. In particular, DNA-intercalating agents known in the art (e.g., anthracyclines, iucrine), drugs that can disrupt the spindle apparatus (e.g., vinca alkaloids, melanoma itansine, maytansinoids, and their derivatives), RNA polymerase inhibitors (e.g., α- amatoxins such as amanitin and their derivatives) and disrupt protein biosynthesis Agents that can inhibit rRNA N-glycosidase activity (e.g., saporin and ricin A chain) Drugs) are included.

[0179] In some embodiments, the cytotoxin is a microtubule binding agent (e.g., maytansine or Maytansinoids), amatoxins, Pseudomonas exotoxin A, debouganin, diphtheria Toxins, saporin, auristatin, anthracyclines, calicheamicin, irinotecan SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine diamine indolinobenzodiazepine, indolinobenzodiazepine dimer, or its variants or other cytotoxic compounds described herein or known in the art. do.

[0180] In some embodiments, the cytotoxin of the antibody-drug conjugate inhibits an RNA polymerase In some embodiments, the RNA polymerase inhibitor is an amatoxidase inhibitor. In some embodiments, the antibody-drug complexes disclosed herein are The cytotoxin of the conjugate is amatoxin or its derivative (e.g., α-amanitin, β- Amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, proamanuline or its derivatives, etc.

[0181] Regarding cytotoxins that may be used in anti-CD5 ADCs useful in the methods of the invention Further details are provided below.

[0182] Amatoxin In some embodiments, the RNA polymerase inhibitor is an amatoxin or a derivative thereof. In some embodiments, the antibody-drug conjugates disclosed herein The cytotoxin may be an amatoxin or a derivative thereof (e.g., α-amanitin, β-amanitin, γ -Amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, Proamanulin or its derivatives, etc. The structures of various naturally occurring amatoxins. is represented by Formula II and Table 2 presented together, and is described, for example, in Zanotti et al., In t. J. Peptide Protein Res. 30, 1987, 450-459. [ka]

[0183] [Table 3]

[0184] In one embodiment, the cytotoxin is amanitin or a derivative thereof. The cytotoxin is α-amanitin or a derivative thereof.

[0185] Many positions on the amatoxin or its derivatives are available as positions for covalent attachment of the linking moiety L. and therefore, said antibody, or antigen-binding fragment thereof, In some embodiments, the compound of formula I can serve as a site for covalent bonding to The cytotoxin in the ADC is an amatoxin represented by formula (II) or a derivative thereof.

[0186] In one embodiment, the ADC has the formula Ab-ZL-Am, where Ab is an antibody that binds to CD5. L is a linker; Z is a chemical moiety. In this embodiment, the linker-amatoxin conjugate The amide-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, they form a five-membered helix. Forming a tercycloalkyl 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 optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl; optionally substituted C2-C6 heteroalkynyl; optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl; or comprising a dipeptide; or -((CH2) m O) n (CH2) m - where m and n are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Z' is a reactive moiety, and Z is a coupling reaction between Z' and a functional group on Ab. is a chemical substructure resulting from R D is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkene, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkynyl C1-C6 aryl, aryl, heteroaryl, or combinations thereof, alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or Heteroaryl is an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo. Alkyl, alkaryl, alkyl, heteroaryl, amino, ammonium, acyl, a Siloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamide Bamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, arsenic Coxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, 1 to 5 substituents independently selected from the group consisting of mercapto, nitro, Optionally substituted.

[0187] Formula (III) comprises an amatoxin and a linker, and in some embodiments, This includes anchors, chemical substructures, and antibodies.

[0188] In some embodiments, the cytotoxin is an amatoxin, and the linker-amatoxin is The amatoxin conjugate or antibody-linker-amatoxin conjugate has the formula (IIIA): Represented: [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, they form a five-membered helix. Forming a tercycloalkyl 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 RD 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 optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl; optionally substituted C2-C6 heteroalkynyl; optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl; or comprising a dipeptide; or -((CH2) m O) n (CH2) m - where m and n are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Z' is a reactive moiety, and Z is a coupling reaction between Z' and a functional group on Ab. is a chemical substructure resulting from R D is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkene, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkynyl C1-C6 aryl, aryl, heteroaryl, or combinations thereof, alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or Heteroaryl is an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo. Alkyl, alkaryl, alkyl, heteroaryl, amino, ammonium, acyl, a Siloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamide Bamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, arsenic Coxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, 1 to 5 substituents independently selected from the group consisting of mercapto, nitro, Optionally substituted.

[0189] In some embodiments, the amatoxin comprises one R C Contains substituents.

[0190] In some embodiments, R A and R B together with the oxygen atom to which they are attached. to 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 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 , Ally Ren-R C or heteroarylene-R C , or a combination thereof; where each C1 -C6 alkylene-R C , C1-C6 heteroalkylene-R C , C2-C6 alkenylene-R C , C2-C6 heteroa Rukenylene-R C , C2-C6 alkynylene-R C , C2-C6 heteroalkynylene-R C , cycloalkyl N-R C , heterocycloalkylene-R C , Arylene-R C or heteroarylene-R C Is, Al alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, Alkyl, heteroaryl, amino, ammonium, acyl, acyloxy, acylamin No, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, Tetraaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, Halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro and optionally substituted with 1 to 5 substituents each independently selected from the group consisting of Formula (IIIA) includes an amatoxin and a linker, and in some embodiments , linkers, chemical moieties, and antibodies.

[0191] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and The amatoxin-linker conjugate is represented by formula IIIA, wherein: R1 is H, OH, OR A ORC 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.

[0192] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and The amatoxin-linker conjugate is represented by 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 Dis as defined above.

[0193] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and The amatoxin-linker conjugate is represented by 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 is as defined above. -conjugates are described, for example, in U.S. Patent Application Publication No. 2016 / 0002298; The disclosure of this application is incorporated herein by reference in its entirety.

[0194] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and The amatoxin-linker conjugate is represented by 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 is as defined above. -conjugates are described, for example, in U.S. Patent Application Publication No. 2014 / 0294865; The disclosure of this application is incorporated herein by reference in its entirety.

[0195] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and The amatoxin-linker conjugate is represented by formula IIIA, wherein: R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are independently H, OH, or 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 is as defined above. -conjugates are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220; The disclosure of this application is incorporated herein by reference in its entirety.

[0196] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and The amatoxin-linker conjugate is represented by 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 is as defined above. Conjugates are described, for example, in U.S. Pat. Nos. 9,233,173 and 9,399,681. No. 6,399,623, the disclosure of which is incorporated herein by reference in its entirety.

[0197] 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, they form a five-membered helix. Forming a tercycloalkyl 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 optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl; optionally substituted C2-C6 heteroalkynyl; optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl; or comprising a dipeptide; or -((CH2) m O) n (CH2) m - where m and n are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Z' is a reactive moiety, and Z is a coupling reaction between Z' and a functional group on Ab. is a chemical substructure resulting from R D is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkene, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkynyl C1-C6 aryl, aryl, heteroaryl, or combinations thereof, alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or Heteroaryl is an alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo. Alkyl, alkaryl, alkyl, heteroaryl, amino, ammonium, acyl, a Siloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamide Bamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, arsenic Coxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, 1 to 5 substituents independently selected from the group consisting of mercapto, nitro, Optionally substituted.

[0198] Formula (IIIA) comprises an amatoxin and a linker, and in some embodiments, This includes anchors, chemical substructures, and antibodies.

[0199] In some embodiments, R A and R B together with the oxygen atom to which they are attached. to 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 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 , Arele N-R C or heteroarylene-R C or a combination thereof; where each C1-C 6Alkylene-R C , C1-C6 heteroalkylene-R C , C2-C6 alkenylene-R C , C2-C6 Heteroar Kenylen-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 A alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl , alkyl, heteroaryl, amino, ammonium, acyl, acyloxy, acyla amino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, Heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl , halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro and optionally substituted with 1 to 5 substituents independently selected from the group consisting of is.

[0200] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are The compound is conjugated to a cysteine-linker conjugate or a derivative thereof and represented by formula IIIB Here, 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 Form a 5-membered heterocycloalkyl of: [ka] where R3 is H or R C It is.

[0201] 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 B When present, together with the oxygen atom to which they are attached, form to form a 5-membered heterocycloalkyl group of: [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.

[0202] 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 to form a 5-membered heterocycloalkyl group of: [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 is as defined above. -conjugates are described, for example, in U.S. Patent Application Publication No. 2016 / 0002298; The disclosure of this application is incorporated herein by reference in its entirety.

[0203] 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 RC 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 is as defined above. -conjugates are described, for example, in U.S. Patent Application Publication No. 2014 / 0294865; The disclosure of this application is incorporated herein by reference in its entirety.

[0204] 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 independently H, OH, or 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 is as defined above. -conjugates are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220; The disclosure of this application is incorporated herein by reference in its entirety.

[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 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 is as defined above. Conjugates are described, for example, in U.S. Pat. Nos. 9,233,173 and 9,399,681. No. 6,399,623, the disclosure of which is incorporated herein by reference in its entirety.

[0206] Auristatins The anti-CD5 antibodies and antigen-binding fragments thereof described herein are auristatins. These may be conjugated to cytotoxins that are suitable for use in the treatment of cancer (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 activity (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Ant. imicrob. Agents Chemother. 42:2961-2965). (U.S. Patent Nos. 5,635,483; 5,780,588) The auristatin drug moiety may be attached at the N-terminus or C-terminus of the peptide drug moiety. The carboxyl group may be linked to an antibody via its terminal end (WO02 / 088172).

[0207] Exemplary auristatin embodiments include an N-terminally linked monomethyl auristatin drug moiety. These include DE and DF (MMAE and MMAF, respectively), which are described in Senter et al, Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, 2 No. 6,399,633, filed on Mar. 28, 2004, the disclosure of which is incorporated herein by reference in its entirety. can be).

[0208] An exemplary auristatin embodiment is MMAE: [ka] Here, the wavy line represents the antibody-drug or drug-linker conjugate (as described herein). The position of the covalent attachment of -LZ-Ab or -L-Z') to the linker is indicated.

[0209] Another exemplary auristatin embodiment is MMAF: [ka] Here, the wavy line represents the antibody-linker conjugate as disclosed in US 2005 / 0238649. The position of covalent attachment of the jugate (-LZ-Ab or -L-Z' as described herein) to the linker Show location:

[0210] Auristatins can be prepared according to the method of U.S. Pat. No. 5,635,483. ;U.S. Patent No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit it et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, GR, et al. Synthe sis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin Trans. 15:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784.

[0211] Maytansinoids The antibodies and antigen-binding fragments thereof described herein are capable of binding to cytotoxins that are microtubule-binding agents. In some embodiments, the microtubule binding agent may be conjugated to a , maytansine, a maytansinoid or a maytansinoid analogue. It is an inhibitor of mitosis that acts by binding to microtubules and inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (US (Patent No. 3,896,111). Subsequently, certain microorganisms have also been shown to secrete maytansinoids (e.g., maytansinoids). It was discovered that the compound produces maytansinol and C-3 maytansinol esters (U.S. Pat. No. 5,333,363). Synthetic maytansinol and its derivatives and analogues are disclosed in, for example, the U.S. Patent No. 4,151,042. National Patents 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,01 6; 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 4,371,533 The maytansinoid drug moiety is disclosed in the antibody drug conjugates. Maytansinoid drug moieties are attractive drug moieties in the treatment of cancer because: (i) they are They are relatively accessible to preparation by fermentation or chemical modification or derivatization of fermentation products. (ii) a functional group suitable for conjugation to an antibody via a non-disulfide linker; (iii) it is stable in plasma; and (iv) it shows activity against various tumor cell lines. This is because it is effective.

[0212] Examples of suitable maytansinoids include maytansinol, synthetic maytansinol, and and esters of maytansinol analogues and derivatives. Maytansinol and its analogs and derivatives inhibit microtubule formation. Any cytotoxin that inhibits and is highly toxic to mammalian cells is included herein. .

[0213] Examples of suitable maytansinol esters include those with modified aromatic rings, and Such suitable maytansinoids include those having modifications at the 5'-amino acid residue and other positions. Patent Nos. 4,137,230; 4,151,042; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,7 57; 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; 4, 322,348; 4,362,663; 4,371,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846 Nos. 6,333,410; 7,276,497; and 7,473,796, each of which is incorporated herein by reference. The present disclosure is incorporated by reference as it relates to itanthinoids and their derivatives. do.

[0214] In some embodiments, the antibody-drug conjugates (ADCs) of the invention are administered in combination with a cytotoxic drug. The thiol-containing maytansinoids (DM1) (formally N 2 '-Deacetyl-N 2 '-(3-mercapto DM1 is a compound represented by the following structural formula IV: Represented: [ka] .

[0215] In another embodiment, the conjugate of the invention comprises a thiol-containing maytansinoid N 2 '-Deacetyl-N 2 '(4-methyl-4-mercapto-1-oxopentyl)-maytansine (e.g., DM 4) is used as a cytotoxic drug. DM4 is represented by the following structural formula V: [ka] .

[0216] Another maytansinoid that contains a side chain containing a sterically hindered thiol bond is N 2 '-Deacetyl-N-2 '(4-mercapto-1-oxopentyl)-maytansine (called DM3), which has the following structural formula: Represented by VI: [ka] .

[0217] Each of the maytansinoids taught in U.S. Patents 5,208,020 and 7,276,497 may be used in the compositions of the present invention. In this regard, references 5,208,020 and 7,276,697 are also included. The entire disclosure is incorporated herein by reference.

[0218] Many positions on the maytansinoid serve as sites for covalent attachment of a linking moiety. and thus covalently bonded to said antibody or antigen-binding fragment thereof. can serve as a position (as described herein, -LZ-Ab or -LZ' For example, the C-3 position has a hydroxyl group, the C-14 position is modified with hydroxymethyl, The hydroxy-modified C-15 position and the hydroxy-bearing C-20 position are all expected to be useful. In some embodiments, the C-3 position is contemplated as a position for covalent attachment to a linking moiety. In some particular embodiments, the C-3 position of maytansinol serves as a linking moiety. It serves as a site for covalent attachment of the antibody-maytansinoid conjugate. There are many linking groups known in the art to create, for example, Nos. 5,208,020, 6,441,163, and EP 0425235 B1; Chari et al., Canc J. Am. Chem. Soc. 52:127-131 (1992); and those disclosed in US 2005 / 0169933 A1. (the disclosures of which are expressly incorporated herein by reference). Further linking groups include , as described and exemplified herein.

[0219] The present invention also includes various isomers and mixtures of maytansinoids and conjugates. Certain compounds and conjugates of the present invention may be used in various stereoisomers, enantiomers, and dimers. Maytansinoid complexes may exist in asteorimeric forms. Producing jugates is disclosed in U.S. Patents 5,208,020; 5,416,064; 6,333,410; 6,441,163; Nos. 6,716,821 and 7,368,565, each of which is incorporated herein in its entirety. (included).

[0220] Anthracyclines In other embodiments, the antibodies and antigen-binding fragments thereof described herein are They are sometimes conjugated to cytotoxins called anthracycline molecules. Iclin is an antibiotic compound that exhibits cytotoxic activity. Phosphorus can kill cells by a number of different mechanisms, including: It has been shown that: 1) the drug molecule intercalates into the DNA of cells, 2) inhibiting DNA-dependent nucleic acid synthesis; The radicals then react with cellular macromolecules causing damage to the cell, or 3) with drug molecules. and its interaction with the cell membrane [see, e.g., C. Peterson et al., "Transport and Storage of Anthr acycline In Experimental Systems And Human Leukemia" in Anthracycline Antibiotic s In Cancer Therapy; See NR Bachur, "Free Radical Damage" id. at pp.97-102. Due to potential cytotoxicity, the incidence of leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma has been reported. Anthracyclines are used to treat many cancers, including in Anthracycline: Current Status And New Developments See p. 11. Commonly used The anthracyclines used include doxorubicin, epirubicin, idarubicin, and doxorubicin. Contains nomycin.

[0221] Representative examples of anthracyclines include, but are not limited to, daunorubicin. Cerubidine (Bedford Laboratories), doxorubicin (Adriamycin; Bedford Laboratories), aboratories; doxorubicin hydrochloride, hydroxy-daunorubicin, and Rubex ), epirubicin (Ellence; Pfizer), and idarubicin (idamycin; Pfizer Inc. Anthracycline analogues include doxorubicin (ADRIAMYCIN INO) interacts with DNA by intercalation and unwinds DNA for transcription It is believed to inhibit the enzyme topoisomerase II from progressing. The topoisomerase II complex stabilizes the DNA strand after it is broken for replication, and the DNA duplex is then It prevents the cytoplasm from closing again, thereby stopping the replication process. Daunorubicin and daunorubicin are the prototypic cytotoxic natural product anthracyclines. (Sessa et al., (2007) Cardiovasc. Toxicol. 7:75-79).

[0222] One non-limiting example of an anthracycline suitable for use herein is nemorubicin. The highly potent major metabolite of the compound PNU-159682 ("PNU") is a major metabolite of the parent compound Nemol. It is more than 3000 times more cytotoxic than bicine (Quintieri et al., Clinical Cancer Res. Earch 2005, 11, 1608-1617). PNU is represented by the following structural formula: [ka] .

[0223] Many positions on anthracyclines, such as PNU, provide covalent attachment sites for the linking moiety. and therefore, the anti-CD137 antibodies as described herein or may serve as a site for covalent attachment to an antigen-binding fragment thereof. For example, linkers may be introduced via modifications to the hydroxymethyl ketone side chain. do.

[0224] In some embodiments, the cytotoxin is a PNU derivative having the following structural formula: [ka] where the wavy line indicates the position of covalent attachment to a linker of an ADC as described herein. show.

[0225] In some embodiments, the cytotoxin is a PNU derivative having the following structural formula: [ka] where the wavy line indicates the position of covalent attachment to a linker of an ADC as described herein. show.

[0226] Pyrrolobenzodiazepines (PBDs) In other embodiments, the anti-CD5 antibodies or antigen-binding fragments thereof described herein are Conjugated to a cytotoxin that is a pyrrolobenzodiazepine (PBD) or contains a PBD PBDs are produced by certain actinomycetes and act as sequence-selective DNA archiving agents. PBD cytotoxins have been shown to be, but are not limited to, However, anthramycin, dimeric PBD, and, for example, Hartley, JA (2011). “The evolution 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: 2815-2 864.

[0227] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine having the following structural formula: It can be a zepine dimer: [ka] where the wavy line indicates the position of covalent attachment to a linker of an ADC as described herein. This PBD-based ADC is described, for example, in Sutherland et al., Blood 2013 122:1455-1463 (which is incorporated herein by reference in its entirety).

[0228] In some embodiments, the cytotoxin is a PBD dimer having the following structural formula: There are cases where: [ka] where n is 3 or 5, and where the wavy line represents a linker of an ADC as described herein. - indicates the position of covalent bonding.

[0229] In some embodiments, the cytotoxin is a PBD dimer having the following structural formula: There are cases where: [ka] where the wavy line indicates the position of covalent attachment to a linker of an ADC as described herein. show.

[0230] In certain embodiments, the cytotoxin may be a PBD dimer, which may be a phosphorylation inhibitor. When taken together with the C-A and reactive moiety Z′, each as described herein, It may be represented by the following structure: [ka] .

[0231] This particular cytotoxin-linker conjugate is known as Tecilin (SG3249). For example, see Howard et al., ACS Med. Chem. Lett. 2016, 7(11), 983-987. No. 6,399,623, the disclosure of which is incorporated herein by reference in its entirety.

[0232] In certain embodiments, the cytotoxin may be a PBD dimer, which may be a phosphorylation inhibitor. When taken together with the C-A and reactive moiety Z′, each as described herein, It may be represented by the following structure: [ka] .

[0233] This particular cytotoxin-linker conjugate is known as talirin and is Mantaj et al., Angewandte Chemie International Edition English 2017,56, 462- 488, the disclosure of which is incorporated herein by reference in its entirety.

[0234] Calicheamicin In other embodiments, the antibodies and antigen-binding fragments thereof described herein are Nedjiin antitumor antibiotics (e.g., calicheamicin, ozogamicin) are cytotoxins The calicheamicin family of antibiotics may be pico-conjugated. Capable of forming double-stranded DNA breaks at submolar concentrations. Calicheamicin family For the preparation of conjugates of See US Patent Nos. 67,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,296 (all from American Structural analogs of calicheamicin that may be used include See, for example and without limitation, Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and American Cyanamid US Pat. No. 5,399,633, which is incorporated herein by reference.

[0235] Examples of calicheamicins suitable for use in the present invention include those described, for example, in U.S. Pat. No. 1,958. U.S. Patent No. 4,970,198. U.S. Patent No. 5,053,394; U.S. Patent No. 5,037,651; and U.S. Pat. No. 5,079,233, which are incorporated herein in their entireties. Can be enjoyed.

[0236] Exemplary calicheamicins are designated γ1, which are referred to herein simply as γ1. It is referred to as nucleophile and has the following structural formula: [ka] .

[0237] In some embodiments, the calicheamicin is a gamma-calicheamicin derivative. or N-acetylgamma-calicheamicin derivatives. Structural analogues of mycin include, but are not limited to, those described in Hinman et al., C ancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1 998), and those disclosed in the above-referenced U.S. patents. Contains a methyl trisulfide moiety that can react with thiols to form disulfides. The methyl trisulfide moiety simultaneously converts the calicheamicin derivative into a compound as described herein. Useful moieties for conjugation via a linker to an anti-CD5 antibody or antigen-binding fragment thereof. Introduce functional bases.

[0238] In one embodiment, the cytotoxin of the ADC disclosed herein is a potassium ion exchange inhibitor having the formula Caremicin is a disulfide derivative: [ka] Here, the wavy line indicates the position of attachment of the linker.

[0239] Additional cytotoxins In other embodiments, the antibodies and antigen-binding fragments thereof described herein are Conjugated cytotoxins other than, or in addition to, those disclosed above in the literature. Additional agents suitable for use with the compositions and methods described herein include Examples of cytotoxins include, but are not limited to, 5-ethynyluracil, abiraterone (a biraterone, acylfulvene, adecypenol, adzereshi adozelesin, aldesleukin, altretamine, Ambmustine, amidox, amifostine, ami Aminolevulinic acid, amrubicin, amsacrine ne), anagrelide, anastrozole, andrographol andrographolide, angiogenesis inhibitor, antarelix, anti-dorsal formation protein Anti-dorsalizing morphogenetic protein-1, antiandrogen, prostate cancer , antiestrogens, antineoplastic agents (antineoplastons), antisense oligonucleotides, Aphidicolin glycinate, an apoptosis gene module regulator, apoptosis regulator, apurinic acid, asulaculin ulacrine, atamestane, atrimustine, axinastatin axinastatin 1, axinastatin 2, axinastatin 3, azasetron ), azatoxin, azatyrosine, baccatin III III.) Derivatives, balanol, batimastat, BCR / ABL antagonists, Benzoylstaurosporine, β-lactam derivatives , beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitor Agents, bicalutamide, bisantrene, bisaziridinyl sperate Bisaziridinylspermine, bisnafide, bistratene A , bizelesin, breflate, bleomycin A2, bleomycin Buthionine sulfoxide, buthionine sulfonate ... imine (buthionine sulfoximine), calcipotriol, calphostin (c alphostin C, camptothecin derivatives (e.g., 10-hydroxy-camptothecin capecitabine, carboxamide-amino-triazole, carbo hydroxyamidotriazole, carzelesin, casein kinase inhibitor, cass Castanospermine, Cecropin B, Cetrorelix x), chlorins, chloroquinoxaline sulfonamides fonamide, cicaprost, cis-porphyrin, cladribine , clomiphene and its analogues, clotrimazole, collismycin A, collismycin B, combretastatin A4, Combretastatin analogues, conagenin, crambescidin 816 , crisnatol, cryptophycin 8, cryptophycin A Derivatives, curacin A, cyclopentanthraquinones ), cycloplatam, cypemycin, cytarabine-ocphosph cytarabine ocfosfate, cytolytic factor, cytostatin, dacryloxy Dacliximab, decitabine, dehydrodidemnin B, 2'Deoxycoformycin (DCF), deslorelin, dexphosphamide fosfamide, dexrazoxane, dexverapamil, diabetics Diaziquone, didemnin B, didox, diethylnorsper diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxane dioxamycin, diphenyl spiromustine, dis Codermolide, docosanol, dolasetron ), doxifluridine, droloxifene, dronabinol Dronabinol, Duocarmycin SA, Ebselen, Ecoms Ecomustine, edelfosine, edrecolomab, F lornithine, elemene, emitefur, epothilone (epothilones), epithilones, epristeride, est Estramustine and its analogues, etoposide, etoposide 4'- etoposide 4'-phosphate (also called etopophos), exemestane emestane, fadrozole, fazarabine, fenretinide enretinide, filgrastim, finasteride, flavopy Flavopiridol, Flezelastine, Fluasterone ), fludarabine, fluorodaunorunicin hydrochloride hloride, forfenimex, formestane, fostri Fostriecin, fotemustine, gadolinium texaphyrin dolinium texaphyrin, gallium nitrate, galocitabine, ganirelix irelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepatitis C Hepsulfam, homoharringtonine (HHT), hypericin hypericin, ibandronic acid, idoxifene, Idramantone, Ilmofosine, Ilomastat , imidazoacridone, imiquimod, immune stimulating peptides, iobenguane benguane, iododoxorubicin, ipomeanol, i Rinotecan, iroplact, irsogladine, isobengazone isobengazole, jasplakinolide, kahalalide F, Lamellarin-N triacetate, lanreotide, raynam Leinamycin, lenograstim, lentinan sulfate ), leptolstatin, letrozole, lipophilic platinum compounds, Lissoclinamide 7, lobaplatin, lometrexol exol, lonidamine, losoxantrone, loxoribine ribine, lurtotecan, lutetium texaphyrin n), lysofylline, masoprocol, maspin, Trix metalloproteinase inhibitors, menogaril, merbaron one), meterelin, methioninase, metoclopramide, M IF inhibitors, mifepristone, miltefosine, mirimosti mirimostim, mithracin, mitoguazone, mitolactol (mitolactol), mitomycin and its analogues, mitonafide, mitoxan mitoxantrone, mofarotene, molgramostim, Mycaperoxide B, myriaporone, N-acetyldinalin e), N-substituted benzamides, nafarelin, nagrestip, napa Napavin, naphterpin, nartograstim, nedap Nedaplatin, Nemorubicin, Neridronic acid, Nilutamide de), nisamycin, nitrullyn, octreotide , okicenone, onapristone, ondansetron on, oracin, ormaplatin, oxaliplatin , oxaunomycin, paclitaxel and its analogues, Palauamine, palmitoylrhizoxin, pamidronic acid , panaxytriol, panomifene, parabactin ctin), pazelliptine, pegaspargase, perdecin peldesine, sodium pentosan polysulfate, pentostatin, pentro Pentrozole, perflubron, perfosfamide, Phenazinomycin, picibanil, pirarubicin cin), piritrexim, podophyllotoxin, porf Porfiromycin, a purine nucleoside phosphorylase inhibitor, Raltito Raltitrexed, rhizoxin, rogletimide, rohituki rohitukine, rubiginone B1, ruboxyl, safingol ngol, saintopin, sarcophytol A, sargramostim (sargramostim), sobuzoxane, sonermin, sparhodinic acid parfosic acid), spicamycin D, spiromustine, sti stipiamide, sulfinosine, tallimustine, Tegafur, temozolomide, teniposide, talib Thaliblastine, thiocoraline, tirapazamine , topotecan, topsentin, triciribine, trimetrexa trimetrexate, veramine, vinorelbine, vinxalci vinxaltine, vorozole, zeniplatin, and zilascol Examples include zilascorb.

[0240] Linker As used herein, the term "linker" refers to a linker that connects an anti-CD5 antibody or fragment thereof (Ab) to a drug. Covalently bonded to moiety (D) to form an antibody-drug conjugate (ADC) of formula I A suitable linker is a bivalent chemical moiety that contains two reactive ends. The antibody has one end for conjugation to an antibody and the other for conjugation to a cytotoxin. The reactive end of the linker that is to be conjugated with the antibody (reactive portion) is The moiety Z') typically corresponds to a cysteine-thiol or lysine-amine group on the antibody. and thus typically is a group reactive with thiols, such as a double bond (e.g., maleimide), or a chloro , bromo, iodo, or other leaving groups, or R-sulfanyl groups, or carboxyl groups, a group reactive with amines; The reactive end is typically the site that can be conjugated to a cytotoxin. Non-limiting examples of anchor-cytotoxin conjugations include, for example, salts on the cytotoxin. Formation of an amide bond with a functional amine or carboxyl group (respectively, the carboxyl group of the linker) via carboxyl or basic amine groups, or formation of ethers, etc. (via the OH groups of the cytotoxins) via alkylation, e.g., via a leaving group on the linker; In some embodiments, the cytotoxin-linker conjugation comprises via the formation of an amide bond with the amine or carboxyl group of said The reactive substituents of the linker are carboxyl or basic amine groups, respectively. When the term "linker" is used to describe the conjugated form of the linker, it refers to the reactive end One or both of the termini may be between the linker and / or the cytotoxin, and between the linker and and / or the antibody or antigen-binding fragment thereof, does not exist (e.g., has been converted to reactive substructure Z', chemical substructure Z), or In the middle of a reaction (for example, the carboxylic acid is left as a carbonyl). The jugating reaction is further described herein below.

[0241] A variety of linkers can be used to link the described antibodies, antigen-binding fragments, and ligands to cells. In some embodiments, the linker can be conjugated to a cytotoxic molecule. The linker is cleavable under intracellular conditions, such that cleavage of the linker results in the formation of a cellular In yet another embodiment, the drug unit is released from the antibody in the intracellular environment. The Car unit is not cleavable and the drug is released by degradation of the antibody, for example. Linkers useful in the ADCs of the invention are preferably stable extracellularly and do not cause aggregation of the ADC molecules. This prevents the ADC from becoming soluble in aqueous media and in a monomeric form. Prior to being shipped or delivered, the ADC is preferably stable and intact. The linker remains intact, i.e., the antibody remains linked to the drug moiety. It is stable outside the target cell and is cleaved at an effective rate inside the cell. An effective linker: (i) maintains the specific binding properties of the antibody; (ii) (iii) enabling the conjugate or drug moiety to be delivered into a cell; The drug remains stable and intact until it is delivered or transported to its target site. and (iv) the cytotoxic, cytotoxic effect or cytotoxicity of the cytotoxic moiety. The stability of the ADC was evaluated using mass spectrometry, HPLC, and separation / analysis techniques. The affinity of the antibody and the drug moiety can be measured by standard analytical techniques such as C / MS. Covalent bonding requires that the linker have two reactive functional groups (i.e., reactive Peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporters. Bivalent linkers useful for linking two or more functional or biologically active moieties, such as groups. -Reagents are known and methods for obtaining these conjugates have been described (H ermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, p.234-242) .

[0242] Suitable cleavable linkers include those that can be cleaved, for example, by enzymatic hydrolysis, photolysis, or under acidic conditions. Hydrolysis of , hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organic and those that can be cleaved by metal cleavage (see, for example, Leriche et al., Bioorg. Med. Chem. See, e.g., 20:571-582, 2012, which disclose covalently conjugating (The above-mentioned linkers are incorporated herein by reference in their entirety as they relate to suitable linkers. Suitable cleavable linkers include, for example, hydrazine, disulfide, thioether or can be a chemical partial structure such as a dipeptide.

[0243] Examples of linkers that are hydrolyzable under acidic conditions include hydrazones and semicarbazones. , thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketters (e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik et al. and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Ch em. 264:14653-14661, each of which disclosure is suitable for covalent conjugation. (The entire contents of which are incorporated herein by reference with respect to the above linkers.) Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but are highly soluble in lysine. It is unstable below pH 5.5 or 5.0, which is the approximate pH of the serosome.

[0244] Linkers that are cleavable under reducing conditions include, for example, disulfides. For example, 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, which can be used to form disulfide bridges. A variety of disulfide linkers are known in the art, including disulfide linkers (e.g., For example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immu noconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW V ogel ed., Oxford U. Press, 1987). See also U.S. Patent No. 4,880,935. Each of these disclosures relates to linkers suitable for covalent conjugation. No. 6,313,635, which is incorporated by reference in its entirety herein.

[0245] Linkers that are susceptible to enzymatic hydrolysis include, for example, those that are hydrolyzed by intracellular peptidases or proteases. ase enzymes (lysosomal proteases or endosomal proteases The linker may be a peptide-containing linker that is cleaved by, for example, One advantage of using intracellular proteolysis to release therapeutic agents is that The therapeutic agent, when conjugated, is generally attenuated, and the conjugate In some embodiments, the serum stability of the protease is generally high. The peptidyl linker is at least two amino acids in length or at least three amino acids in length. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides, or peptides. Examples of suitable peptides include valine, alanine, citrulline, Contains amino acids such as phenylalanine, lysine, leucine, and glycine. The amino acid residues that comprise the amino acid linker moiety include naturally occurring and minor amino acids and non-naturally occurring amino acid analogues (e.g., Exemplary dipeptides include valine-citrulline (vc or val-citrulline). Exemplary tripeptides include alanine-phenylalanine (af or ala-phe). The amino acids include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine. In some embodiments, the linker includes Val-C it, Ala-Val, or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit. Dipeptides such as Val-Cit or Phe-Lys are included. Linkers that include covalent bonds are disclosed, for example, in U.S. Pat. No. 6,214,345 (the disclosure of which is incorporated herein by reference). The present invention relates generally to linkers suitable for synthetic conjugation. In some embodiments, the linker includes a Dipeptides selected from l-Ala and Val-Cit are included.

[0246] Conjugating the antibodies, antigen-binding fragments, and ligands described herein to cytotoxic molecules Suitable linkers for this purpose include those capable of releasing cytotoxins via a 1,6-elimination process. Chemical moieties that enable this elimination process include p- Aminobenzyl (PAB) group, 6-maleimidohexanoic acid, pH-sensitive carbonate, and Jain et al., Pharm. Res. 32:3526-3540, 2015 (the disclosure of which is suitable for covalent conjugation). (which is hereby incorporated by reference in its entirety as it relates to such linkers) Other reagents described herein include those described in the literature.

[0247] In some embodiments, the linker includes the aforementioned PAB or PABC (para-aminobenzamide). and the like. These include "self-immolative" groups such as phenyloxycarbonyl, which are described, for example, in Carl et al., J. Med. Chem. (1981) 24:479-480; Chakravarty et al (1983) J. Med. Chem. 26:638-6 44; US 6214345; US20030130189; US20030096743; US6759509; US20040052793; US621851 9; US6835807; US6268488; US20040018194; WO98 / 13059; US20040052793; US6677435; US No. 5621002; US20040121940; WO2004 / 032828. Other methods for making this process are disclosed in US Pat. Such chemical moieties ("self-immolative linkers") include methylene carbamates and Heteroaryl groups, such as aminothiazole, aminoimidazole, aminopyrimidine, etc. Such linkers containing a heterocyclic self-immolative group are described, for example, in U.S. Pat. Nos. 20160303254 and 20150079114, and U.S. Patent No. 7,754,681; Hay et al. (1999) Bio org. Med. Chem. Lett. 9:2237; US 2005 / 0256030; de Groot et al (2001) J. Org. Che m. 66:8815-8830; and US 7,223,837. In some embodiments, dipeptides are The tides are used in combination with self-immolative linkers.

[0248] Linkers suitable for use herein include C1-C6 alkylene, C1-C6 heteroalkylene, C2 -C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroaryl and combinations thereof, each of which may further include one or more groups selected from Each may be optionally substituted. Non-limiting examples of such groups include , (CH2) p , (CH2CH2O) p , and -(C=O)(CH2) p -unit, where p is an integer from 1 to 6. and is selected independently for each case.

[0249] In some embodiments, each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl , C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkenyl alkyl, heterocycloalkyl, sulfonylcycloalkyl, aryl, or heteroaryl The alkyl group may be an alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, Alkaryl, alkyl, heteroaryl, amino, ammonium, acyl, acyloxy , acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, Aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfur Sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto and nitro, has been replaced by.

[0250] In some embodiments, each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl , C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkenyl alkyl, heterocycloalkyl, sulfonylcycloalkyl, aryl, or heteroaryl The alkyl group is optionally interrupted by one or more heteroatoms selected from O, S and N. This is sometimes the case.

[0251] In some embodiments, each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl , C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkenyl alkyl, heterocycloalkyl, sulfonylcycloalkyl, aryl, or heteroaryl The alkyl group is optionally interrupted by one or more heteroatoms selected from O, S and N. and alkyl, alkenyl, alkynyl, cycloalkyl, heptyl, Cycloalkyl, alkaryl, alkyl, heteroaryl, amino, ammonium, a Cyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea do, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy aryl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy 1 to 5 substituents independently selected from the group consisting of aryloxy, ... Suitable linkers have the property of enhancing solubility. It may contain groups such as (CH2CH2O) p Contains the unit (polyethylene glycol, PEG) Linkers containing amines can enhance solubility (e.g., amino, sulfonic acid, phosphonic acid). or phosphate residues to increase the solubility of the alkyl chains. Linkers containing branched structures are disclosed, for example, in U.S. Pat. Nos. 8,236,319 and 9,504,756. (each of which disclosure relates to linkers suitable for covalent conjugation. (The entire contents of which are incorporated herein by reference as if fully set forth herein) Examples of groups that can be used include acyl and carbamoyl sulfamido groups having the following structures: Some of the issues mentioned are: [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 Group, 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 Al (Hetero)aryl groups, and C3-C 24 (hetero)arylalkyl groups, are selected, each of which is O, S, and NR 11 R 12 and one or more heteroatoms selected from may be optionally substituted and / or optionally interrupted by , where 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, wherein the cytotoxin is linked via a spacer moiety to any of N Linkers containing such groups are described, for example, in U.S. Pat. Nos. 9,636,421 and 9,422, and are incorporated herein by reference. and U.S. Patent Application Publication No. 2017 / 0298145, which disclose methods for treating cancer with cytotoxins and antibodies or their antagonists. Related to linkers suitable for covalent conjugation to the original binding fragment. (which is incorporated by reference in its entirety herein).

[0252] In some embodiments, the linker is a hydrazine, a disulfide, a thioether. , dipeptides, p-aminobenzyl (PAB) groups, heterocyclic self-immolative groups, optionally substituted C1-C6 Alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 Alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C 2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, solubility enhancing groups, acyl, -(C=O)- or -(CH2CH2O) p - groups, where p is an integer from 1 to 6. Some of skill in the art will appreciate that one or more of the listed groups may be a divalent (diradical) species (e.g., It is recognized that alkyl groups may exist in the form of alkylene, for example, C1-C6 alkylene.

[0253] In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In an embodiment, the p-aminobenzyl group is a protease cleavable linker between the cytotoxic drug and the linker. In one embodiment, the p-aminobenzyl group is In one embodiment, the p-aminobenzyl group is part of a p-aminobenzyloxycarbonyl unit. is part of the p-aminobenzylamide unit.

[0254] In some embodiments, the linker is Phe-Lys, Val-Lys, Phe-Ala, Phe-Cit, Va Some of the dipeptides include those selected from the group consisting of l-Ala, Val-Cit, and Val-Arg. In embodiments, the linker is selected from the group consisting of PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Ph e-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala -Contains one or more of the following PABs:

[0255] In some embodiments, the linker is 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- Contains PAB, or Ala-PAB.

[0256] In some embodiments, the linker is a peptide, an oligosaccharide, -(CH2) p -, -(CH2CH2 O) p -, PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-P AB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB Includes combinations.

[0257] In some embodiments, the linker is -(C=O)(CH2) p -units, where p is an integer from 1 to 6.

[0258] In some embodiments, the linker is -(CH) 2n -units, where n is an integer from 2 to 6. In some embodiments, the linker is -(( CH2) n In some embodiments, the LZ comprises: [ka] where S is present in an antibody, or antigen-binding fragment thereof, that binds to CD5. A sulfur atom represents a reactive substituent that can be easily reacted with aryl groups (e.g., from the -SH group of a cysteine ​​residue).

[0259] In some embodiments, the linker is ((CH2) m O) n (CH2) m - groups, where n and and m are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and An aryl group, where the heteroaryl group is a triazole. In the (CH2) m O) n (CH2) m The - group and the triazole together include [ka] where n is 1 to 10, and the wavy lines represent additional linker components, chemical substructures Z or the position of attachment to the amatoxin. Other linkers that may be used are described in U.S. Patent Application Publication No. 2019 / 0144504. No. 6,399,433, which is incorporated herein by reference.

[0260] In a specific embodiment, the linker comprises PAB-Ala-Val-propionyl, It is represented by the structure [ka] Here, the wavy lines indicate the positions of attachment to the cytotoxin and reactive moiety Z'.

[0261] In another specific embodiment, the linker comprises PAB-Cit-Val-propionyl, It is represented by the structure [ka] Here, the wavy lines indicate the positions of attachment to the cytotoxin and the reactive moiety Z'. Examples of suitable PAB-dipeptide-propionyl linkers include those described in Patent Application Publication WO2017 / 1490 77, which is incorporated herein by reference in its entirety. The cytotoxins disclosed in WO2017 / 149077 are incorporated herein by reference.

[0262] Any one or more of the chemical groups, moieties and features disclosed herein may be combined in a variety of ways. and for conjugating antibodies and cytotoxins as disclosed herein. It will be recognized by one of skill in the art that useful linkers can be formed. Additional linkers useful in conjunction with the compositions and methods described herein include, for example, See, for example, U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety. It is incorporated herein by reference.

[0263] In certain embodiments, an intermediate that is a precursor of the linker is converted to a drug moiety under suitable conditions. In certain embodiments, the reactive group is reacted with the drug and / or the intermediate. or on a linker. The encapsulated drug is then reacted with the antibody or antigen-binding fragment under suitable conditions. Alternatively, the linker or intermediate is first reacted with the antibody or derivatized antibody. and then reacted with the drug or a derivatized drug. The silanization reaction is now more fully described.

[0264] The antibody or antigen-binding fragment thereof is A number of different reactions are available for covalently attaching nucleotides to antibody molecules. Preferred attachment positions include the amine groups of lysine, glutamic acid and aspartic acid. Free carboxylic acid groups, sulfhydryl groups of cysteine, and various moieties of aromatic amino acids For example, a carboxy (or amino) group on a compound can be linked to an amino (or To link the carboxyl group, a non-specific covalent bond is formed using a carbodiimide reaction. In addition, a method for coupling an amino group on a compound to an amino group on an antibody moiety may be performed. Bifunctional drugs such as dialdehydes or imidoesters may also be used. The Schiff base reaction can also be used to bind to the binding agent. The method involves periodate oxidation of a drug containing a glycol or hydroxyl group, and such In this way, an aldehyde is formed, which is then reacted with a binding agent. This occurs via the formation of a Schiff base with the amino group of the isothiocyanate, which binds the drug. They may also be used as coupling agents to covalently bond the combination. Other techniques are known in the art. are known to those skilled in the art and are within the scope of the present invention.

[0265] As described herein, a conjugated antibody or antigen-binding fragment thereof is Examples of useful linkers include, but are not limited to, coupling linkers such as those shown in Table 3 below. The curved line includes a linker that includes a chemical substructure Z formed by a linking reaction. The positions for attachment of the antibody or antigen-binding fragment, and the cytotoxic molecule, respectively, are indicated.

[0266] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0267] One of skill in the art would be able to determine the linker and the antibody or antigen-binding fragment thereof. The reactive substituent Z' attached to the reactive substituent forms a covalent bond linking the two to form the chemical substructure Z. It recognizes that the reactive partial structure Z' is involved in the pulling reaction. Thus, antibody-drug conjugates useful in conjunction with the methods described herein include The antibody or antigen-binding fragment thereof may be coupled to a linker or a cytotoxin-linker conjugate. As described herein, the ribate may be formed by reacting with the ribate. The linker or cytotoxin-linker conjugate contains a reactive substituent Z', etc., and is capable of binding to an antibody or suitable for reaction with a reactive substituent on an antigen-binding fragment of Form.

[0268] As shown in Table 3, the linker and the preferred Examples of suitable reactive substituents include nucleophilic / electrophilic pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pair, or thiol / α,β-unsaturated carbonyl pair, etc.), diene / dieno Philic pairs (e.g., azide / alkyne pairs, or diene / α,β-unsaturated carbonyl pairs, among others) Coupling between reactive substituents to form chemical substructures Z. Examples of the alkylation reactions include, but are not limited to, thiol alkylation, hydroxyl alkylation, and cycloalkylation. alkylation, amine alkylation, amine or hydroxylamine condensation, hydrazine formation, Amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2]Diels-Al, among others) der cycloaddition, [3+2]Huisgen cycloaddition, aromatic nucleophilic substitution, aromatic electrophilic substitution, and Other reaction modes known in the art or described herein are also included. Alternatively, the linker is capable of reacting with a nucleophilic functional group on the antibody or antigen-binding fragment thereof. Contains electrophilic functional groups for reaction.

[0269] As disclosed herein, reactions that may exist within an antibody, or an antigen-binding fragment thereof, The functional substituents include, but are not limited to, (i) an N-terminal amine group, (ii) a side chain amine group ( (iii) side chain thiol groups (e.g., cysteine), and (iv) sugar hydroxyl or aryl groups. The antibody may be glycosylated, and the like. As disclosed herein, reactive sites that may be present in an antibody, or an antigen-binding fragment thereof, Substitutions include, but are not limited to, hyaluronan substitutions of serine, threonine, and tyrosine residues. the hydroxyl moiety; the amino moiety of the lysine residue; the carboxyl moiety of the aspartic acid and glutamic acid residues; the carboxyl moiety of cysteine ​​residues and the propionyl moiety of unnatural amino acids. aryl, azide, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., Examples include fluoroheteroaryl, haloalkyl, and haloheteroalkyl moieties. In some embodiments, the antibody, or antigen-binding fragment thereof, as disclosed herein, Reactive substituents present in the aryl segment include amine or thiol moieties. Certain antibodies have reducible interchain disulfides (i.e., cysteine ​​bridges). By treating with a reducing agent such as DTT (dithiothreitol), the linker reagent and the conjugate can be separated. Each cysteine ​​bridge can thus be made reactive to condense. Theoretically, two reactive thiol nucleophiles are formed: lysine and 2-iminothiolane (Traut et al. Additional nucleophilic groups are introduced into the antibody by reaction with a thiol-converting reagent, converting amines to thiols. One, two, three, four or more cysteine ​​residues can be introduced. (e.g., by creating a mutant antibody that contains one or more non-existent cysteine ​​amino acid residues) by preparing a thiol group on the antibody (or fragment thereof) US Patent No. 7,521,541 describes the introduction of reactive cysteine ​​amino acids. The present application teaches engineered antibodies using

[0270] In some embodiments, the reactive moiety Z' attached to the linker is present on an antibody. Useful electrophilic groups on antibodies include aldehydes and ketones. The heteroatoms of nucleophilic groups include, but are not limited to, the carbonyl groups of aryl groups and ketones. The molecule may react with an electrophilic group on an antibody and form a covalent bond with the antibody. The core group includes hydrazide, oxime, amino, hydroxyl, hydrazine, and thiosemicate. These include arylhydrazides, hydrazine carboxylates, and arylhydrazides. The present invention is not limited to these.

[0271] In some embodiments, Z is a reactive group present in an antibody or antigen-binding fragment thereof. of the reaction between a reactive nucleophilic substituent (e.g., amine and thiol moieties) and a reactive electrophilic substituent Z' For example, Z' may be, inter alia, a Michael acceptor (e.g., maleimide), an active Reactive substituents may be functionalized esters, electron-deficient carbonyl compounds, and aldehydes. Some representative and non-limiting examples of substituents Z′ and the resulting chemical substructures Z are shown in Table 4. As shown in.

[0272] [Table 5]

[0273] For example, as a suitable linker for the synthesis of drug-antibody and drug-ligand conjugates is a substituted aryl group having a reactive substituent Z', such as, but not limited to, maleimide or haloalkyl group; These include reagents such as succinimidyl 4-(N-maleimidomethyl) -Cyclohexane-L-carboxylate (SMCC), N-Succinimidyl Iodoacetate (S IA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester ( MBS), sulfo-MBS, and succinimidyl iodoacetate, etc., as described by Liu et al., 18:690-697, 1979 (the disclosure of which is not intended to limit the scope of the present invention). (which is incorporated herein by reference as relating to a linker for , may be attached to said linker.

[0274] In some embodiments, the reactive substituent Z′ attached to the linker L is a maleimide, an azide, An example of a maleimide-containing linker is the non-cleavable maleimidocaproyne. A cyclohexyl-based linker that can be used to conjugate microtubule-disrupting agents such as auristatins. Such linkers are particularly useful for transporting conjugates of cyclic saccharides. em. 17:14-24, 2006 (the disclosure of which is incorporated herein by reference for purposes of chemical conjugation). (herein incorporated by reference as relating to the linker).

[0275] In some embodiments, the reactive substituent Z' is -(C=O)- or -NH(C=O)-, such that: The linker is a -(C=O)- or -NH(C=O) group and an antibody or antigen-binding fragment thereof, respectively. The antibody or its derivatives may be modified by the reaction of the amide or urea moiety with the amino group of the It may bind to an antigen-binding fragment.

[0276] In some embodiments, the reactive substituent is an N-maleimidyl group, an N-alloylated N-alkylamide group, sulfonyloxy N-alkylamide group, carbonate group, halogenated sulfur phenyl group, thiol group or its derivative, alkynyl group containing a carbon-carbon triple bond inside , (hetero)cycloalkynyl group, bicyclo[6.1.0]non-4-yn-9-yl group (bicyclo[6.1 .0]non-4-yn-9-yl group), alkenyl group containing a carbon-carbon double bond inside, cycloalkenyl Alkenyl group, tetrazinyl group, azide group, phosphine group, nitrile oxide group, nitro amine group, nitrile / imine group, diazo group, ketone group, (O-alkyl)hydroxylamino group, Hydrazine group, halogenated N-maleimidyl group, 1,1-bis(sulfonylmethyl)methylcarbo a cycloalkyl group or its elimination derivative, a carbonyl halide group, or an allenamide group, Each of these may be optionally substituted. In some embodiments, the reactive groups are The substituent may be a cycloalkene group, a cycloalkyne group, or an optionally substituted (hetero)cycloalkenyl group. Contains an alkynyl group.

[0277] A reactive substituent suitable for reacting with a reactive residue on the antibody or antigen-binding fragment thereof. Non-limiting examples of amatoxin-linker conjugates containing Z' include those limited to Examples of suitable amines include, but are not limited to, 7'C-(4-(6-(maleimido)hexanoyl)piperazine- 7'C-(4-(6-(maleimido)hexanamido)piperidin-1-yl)-amatoxin; 7'C-(4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazine-1-yl 7'C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)pipera 7'C-(4-(6-(4-((maleimido)methyl)cyclohexanecarbo 7'C-(4-(2-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxine; 7'C-(4-(2-(6-(6-(hexanamido)ethyl)piperidin-1-yl)-amatoxine; 7'C-(4-Hexanamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine; (2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl) -Amatoxin; 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamide) 7'C-(4-(2-(3-carboxypeptide)ethyl)-hexanamido)- 7'C-(4-(2-(2-bromoacetamide)ethyl)piperidin-1-yl)-amatoxine; 7'C-(4-(2-(3-(pyridin-2-yl disulfide))-ethyl)piperidin-1-yl)-amatoxine; 7'C-(4-(2-(4-(phenyl)propanamido)ethyl)piperidin-1-yl)-amatoxine; 7'C-(4-(2-(maleimido)butanamido)ethyl)piperidin-1-yl)-amatoxine; 7'C-(4-(3-(maleimido)propanoyl)-amatoxine; 7'C-(4-(4-(maleimido)butanoyl)piperazine-1-yl)-amatoxine; 1-yl)-amatoxin;7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxylate 7'C-(3-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)-amatoxine; 7'C-(3-((6-(6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxine; Imido)hexanamide)hexanamide)methyl)pyrrolidin-1-yl)-amatoxin;7'C -(3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrolidine-1-yl 7'C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamide )Hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7'C-(4-(2-(6-(2-(amino 7'C-(oxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin; 4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)-a Matoxin;7'C-(4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl )-Amatoxin;7'C-(4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazine- 1-yl)-amatoxin;7'C-((4-(6-(maleimido)hexanamido)piperidin-1-yl)maleimide 7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperidine-1 -yl)methyl)-amatoxine;7'C-((4-(6-(maleimido)hexanoyl)piperazine-1-yl) (R)-7'C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidone; (S)-7'C-((3-((6-(maleimido)hexanamide) 7'C-((4-(2-(6-(6-(maleimido)hexyl)pyrrolidin-1-yl)methyl)-amatoxine; 7'C-((4'-Hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin; -(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl )methyl)-amatoxin;7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarbo 7'C-((4-( 2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine;7 'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazine-1-yl 7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarbo 7'C-((4-(2-(6-(4-((maleimido)ethyl)piperazin-1-yl)methyl)-amatoxine; Imido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperazine-1-iso 7'C-((3-((6-(6-(maleimido)hexanamide)hexanamide) 7'C-((3-((6-(maleimido) Hexanamide)hexanamide)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxine; 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidine 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamide) 7'C-((3-((6-(4-(( Maleimide)methyl)cyclohexanecarboxamide)hexanamide)methyl)pyrrolidine- 1-yl)methyl)-amatoxine;7'C-((4-(2-(3-carboxypropanamido)ethyl)piperidine 7'C-((4-(6-(maleimido)hexanamide))-amatoxin; 7'C-((4-(6-(4-((maleimido)methyl)-xanoyl)piperazin-1-yl)methyl)-amatoxine; ethyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)methyl)-amato amatoxin;7'C-((4-(2-(maleimido)acetyl)piperazin-1-yl)methyl)-amatoxin; 7'C-((4-(3-(maleimido)propanoyl)piperazin-1-yl)methyl)-amatoxine; ((4-(4-(maleimido)butanoyl)piperazin-1-yl)methyl)-amatoxine; 7'C-((4-(2 -(2-(maleimido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C- ((4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxin ;7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexaneamido 7'C-((3-((6-(maleimido)hexa 7'C-((3-(2-(6-(maleimido)methyl)azetidin-1-yl)methyl)-amatoxine; 7'C-((3-((4-(( Maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)- Amatoxin; 7'C-((3-(2-(4-((maleimido)methyl)cyclohexanecarboxamide) 7'C-((3-(2-(6-(4-((maleimido)methyl)azetidin-1yl)methyl)-amatoxine )Cyclohexanecarboxamide)Hexanamide)Ethyl)Azetidin-1-yl)Methyl)-A 7'C-(((2-(6-(maleimido)-N-methylhexanamido)ethyl)(methyl)amine 7'C-(((4-(6-(maleimido)-N-methylhexanamido)butyl( 7'C-((2-(2-(6-(maleimido)hexanamide)ethyl)-amatoxin 7'C-((2-(2-(6-(4-((maleimido)methyl)aziridin-1-yl)methyl)-amatoxine; Cyclohexanecarboxamide Hexanamide Ethyl Aziridin-1-yl Methyl Amatoxin; 7'C-((4-(6-(6-(2-(aminooxy)acetamido)hexanamide)hexa 7'C-((4-(1-(aminooxy)-2-octanoyl)piperazin-1-yl)methyl)-amatoxine; So-6,9,12,15-tetraoxa-3-azaheptadecan-17-oyl)piperazin-1-yl)methyl -Amatoxin; 7'C-((4-(2-(2-(aminooxy)acetamido)acetyl)piperazine-1-yl 7'C-((4-(3-(2-(aminooxy)acetamido)propanoyl)-amatoxin; Piperazin-1-yl)methyl)-amatoxine;7'C-((4-(4-(2-(aminooxy)acetamide )butanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(6-(2-(aminooxy) 7')Acetamido)Hexanamido)Ethyl)Piperidin-1-yl)Methyl)-Amatoxine; C-((4-(2-(2-(2-(aminooxy)acetamido)acetamido)ethyl)piperidin-1-yl) methyl)-amatoxin;7'C-((4-(2-(4-(2-(aminooxy)acetamido)butanamide) 7'C-((4-(20-(aminooxy)-4,19- Dioxo-6,9,12,15-tetraoxa-3,18-diazaicosyl)piperidin-1-yl)methyl)a Matoxin; 7'C-(((2-(6-(2-(aminooxy)acetamido)-N-methylhexanamide) 7'C-(((4-(6-(2-(aminooxy)acetatoxin 7'C-((3-amino)-N-methylhexanamido)butyl)(methyl)amino)methyl)-amatoxin; -((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl) Pyrrolidin-1-yl)-S-methyl)-amatoxine; 7'C-((3-((6-(4-((maleimido)methyl)silyl) Cyclohexanecarboxamide)Hexanamide)-R-methyl)pyrrolidin-1-yl)methyl)-a 7'C-((4-(2-(2-bromoacetamido)ethyl)piperazin-1-yl)methyl)-acetamide; 7'C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)methyl)-acetamide; Matoxin;7'C-((4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl) Piperidin-1-yl)methyl)-amatoxine; 6'O-(6-(6-(maleimido)hexanamide)he xyl)-amatoxin;6'O-(5-(4-((maleimido)methyl)cyclohexanecarboxamide )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)-ama Toxin;6'O-(6-(2-bromoacetamido)hexyl)-amatoxin;7'C-(4-(6-(azido )Hexanamido)piperidin-1-yl)-amatoxine;7'C-(4-(hex-5-ynoylamino) )piperidin-1-yl)-amatoxine;7'C-(4-(2-(6-(maleimido)hexanamido)ethyl )piperazin-1-yl)-amatoxine;7'C-(4-(2-(6-(6-(maleimido)hexanamide)he Xanamido)ethyl)piperazin-1-yl)-amatoxine;6'O-(6-(6-(11,12-didehydro) -5,6-dihydro-dibenz[b,f]azocin-5-yl)-6-oxohexanamido)hexyl)-a Amatoxin; 6'O-(6-(hex-5-ynoylamino)hexyl)-amatoxin; 6'O-(6-(2-( 6'O-((6-aminooxy)hexyl)-amatoxin; 6'O-(6-(2-iodoacetamido)hexyl)-amatoxin; and 6'O-(6-(2-iodoacetamido)hexyl)-amatoxin.

[0278] In some embodiments, the chemical substructure Z is selected from Table 3 or Table 4. In some embodiments, the chemical substructure Z is: [ka] where S is an antigen present in an antibody, such as an anti-CD5 antibody, or an antigen-binding fragment thereof. is a sulfur atom representing a reactive substituent.

[0279] In some embodiments, the amatoxins disclosed herein have the formula: The anchor is conjugated to a reactive moiety, -L-Z': [ka] Here, the wavy line indicates the position of attachment to a substituent on the cytotoxin (e.g., amatoxin). The linker-reactive substituent L-Z' is replaced by N-beta-maleimidopropionyl It is sometimes called nyl-Val-Ala-para-aminobenzyl (BMP-Val-Ala-PAB).

[0280] In some embodiments, the amatoxins disclosed herein have the formula: The anchor is conjugated to a reactive moiety, -L-Z': [ka] Here, the wavy line indicates the position of attachment to a substituent on the cytotoxin (e.g., amatoxin). The linker-reactive substituent L-Z' is replaced by N-beta-maleimidopropyl It is sometimes called -Val-Cit-para-aminobenzyl (BMP-Val-Cit-PAB).

[0281] In some embodiments, the linker L and chemical moiety Z are taken together as LZ. And, [ka] where S is present in an antibody or antigen-binding fragment thereof, such as an anti-CD5 antibody. The wavy line at the end of the linker represents the sulfur atom present in the The position of binding to the toxin is indicated.

[0282] In some embodiments, the linker L and the chemical moiety Z are linked to the tail antibody. After denaturation, together they form LZ-Ab, which has the following structure: [ka] .

[0283] The linker moieties and amatoxin-linker conjugates are, inter alia, Advantageous in connection with the compositions and methods described herein, see, for example, U.S. Pat. No. 2015 / 0218220 and published patent application WO2017 / 149077, each of which The disclosure of which is incorporated herein by reference in its entirety.

[0284] The linker moieties and amatoxin-linker conjugates are, inter alia, Advantageous in connection with the compositions and methods described herein, see, for example, U.S. Pat. No. 2015 / 0218220 and published patent application WO2017 / 149077, each of which The disclosure of which is incorporated herein by reference in its entirety.

[0285] In one embodiment, the CD5 antibody or antigen-binding fragment described herein is As described herein, the formula Ab-ZL-Am, where Ab is the CD5 antibody or its antigen binding site, is a mer fragment, L is a linker, Z is a chemical substructure, and Am is an amine. The conjugate represented by the formula (I) is a toxin. There are times when they match.

[0286] In some embodiments, Am-LZ-Ab is: [ka] It is.

[0287] In some embodiments, Am-LZ-Ab is: [ka] It is.

[0288] In some embodiments, Am-LZ-Ab is: [ka] It is.

[0289] In some embodiments, Am-LZ-Ab is: [ka] It is.

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

[0291] Preparation of antibody-drug conjugates In the ADCs of Formula I disclosed herein, the anti-CD5 antibody, or antigen-binding fragment thereof, via a linker L and a chemical moiety Z as disclosed herein, one or more cytotoxic drugs The antibody is conjugated to a drug moiety (D) (e.g., about 1 to about 20 drug moieties per antibody). The ADCs of the invention can be prepared using organic chemistry reactions, conditions, and reagents known to those of skill in the art, including: They can be prepared by several routes: (1) Reactivity of antibodies, or antigen-binding fragments thereof. The substituent is reacted with a bivalent linker reagent to form Ab-ZL as described herein above. and subsequently reacting with a drug moiety D; or (2) coupling a reactive substituent of the drug moiety to a bivalent linker React with a reagent to form DL-Z', followed by the antibody or and reacting the reactive substituent of the antigen-binding fragment of the ADC with the reactive substituent of the antigen-binding fragment of the ADC. is described herein.

[0292] In another embodiment, the anti-CD5 antibody, or antigen-binding fragment thereof, comprises one or more The peptide has one or more lysine residues that can be chemically modified to introduce a lysyl group. The ADC is then modified by removing the sulfhydryl sulfur atom from the sulfhydryl group as described hereinabove. It can be used to modify lysines. Examples of reagents that can achieve this include, but are not limited to, N-succinimidyl S-acetylglucosamine, acetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).

[0293] In another embodiment, the anti-CD5 antibody, or antigen-binding fragment thereof, comprises one or more One or more carbohydrate moieties that can be chemically modified to have a hydroxyl group The ADC may then be treated with a 5'-hydroxybutyric acid esterase (HBA) as described hereinabove. It is formed by conjugation through the sulfur atom of a sulfhydryl group.

[0294] In yet another embodiment, the anti-CD5 antibody can be oxidized to provide an aldehyde (-CHO) group. They may have one or more carbohydrate groups that (See, e.g., I., J. Med. Chem. 1989, 32(3), 548-55). The ADC is then reacted with the amines described herein. By conjugation via the corresponding aldehyde as described above. Other processes that modify proteins to bind or associate with cytotoxins are also used. The tocol was prepared according to Coligan et al., Current Protocols in Protein Science, vol. 2, John W iley & Sons (2002), incorporated herein by reference.

[0295] Cell-targeting proteins such as antibodies, immunoglobulins or fragments thereof Methods for conjugating linker-drug moieties are described, for example, in U.S. Pat. No. 5,208,020. No. 6,441,163; WO2005037992; WO2005081711; and WO2006 / 034488. (all of which are expressly incorporated herein by reference in their entireties).

[0296] Route of Administration and Dosage Alternatively, a fusion protein comprising the antibody and a cytotoxic drug can be produced, e.g., by recombinant techniques or They may be produced by peptide synthesis. The length of DNA may be adjacent to each other or The conjugate is provided with a region encoding a linker peptide that does not destroy the desired properties of the conjugate. and comprising regions encoding the two portions of said conjugate, each of which is separated by a single sequence. There is.

[0297] The ADCs described herein can be administered to patients (e.g., those with autoimmune diseases or cancer) in a variety of dosage forms. For example, the ADCs described herein may be administered to autologous patients. A patient suffering from an autoimmune disease or cancer is administered an aqueous solution (e.g., one or more pharma- ceutically acceptable The compositions and compositions described herein may be administered in the form of an aqueous solution containing a suitable excipient. Suitable pharma- ceutically acceptable excipients for use with the method include viscosity modifiers. The aqueous solutions may be sterilized using techniques known in the art.

[0298] Pharmaceutical formulations containing anti-CD5 ADCs as described herein can include such ADCs in one or more (Remington's Pharmaceuticals, Inc.) by mixing with an optional pharma- ceutically acceptable carrier. (1980) in the form of a lyophilized preparation or an aqueous solution. Pharmaceutically acceptable carriers are generally used in the formulations at dosages and concentrations used in the recipient. Non-toxic to insects, including but not limited to: buffers such as phosphate, citrate, and other organic acids; ascorbic acid and methionine Contains antioxidants; preservatives (octadecyldimethylbenzyl ammonium chloride; hexadecyl 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 etc.); low molecular weight (less than about 10 residues) polypeptides; serum albumin, gelatin, or immunoglobulins Proteins such as globulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine, Amino acids such as glutamine, asparagine, histidine, arginine, or lysine; Sugars, disaccharides, and other sugars such as glucose, mannose, or dextrins;ED Chelating agents such as TA; sucrose, mannitol, trehalose, or sorbitol, etc. salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes) and / or a non-ionic surfactant such as polyethylene glycol (PEG).

[0299] The amount of ADC administered reduces the number of cells (e.g., activated T cells) that reject the CAR cell therapy. It is within the capabilities of one skilled in the art to determine a therapeutically effective dose. However, ADCs can be used for systemic administration, for example to treat immune disorders or cancer. In embodiments of the methods described herein, an effective human dose ranges from 0.1 to 150 mg / kg (e.g., For example, 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 expression level, to maintain effective levels, for example, to reduce the risk of CAR-T cell rejection It is contemplated that systemic administration may be repeated to reduce the

[0300] The anti-CD5 ADCs described herein can be administered orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intraocularly, or intravenously. The drug may be administered by a variety of routes, including intravenous, intramuscular, or parenteral. The most suitable route of administration will depend on the particular ADC, the patient, the pharmaceutical formulation, and the mode of administration (e.g., administration time and route of administration), patient age, body weight, sex, severity of the disease being treated, patient The amount of caffeine consumed depends on the diet, and the patient's rate of excretion.

[0301] An effective dose of an anti-CD5 ADC described herein can be, for example, a single (e.g., bolus) administration. In a multiple dose or continuous administration, the dose should be in the range of about 0.001 to about 100 mg / kg of body weight. As a result, the anti-CD5 ADC reaches an optimal blood concentration (e.g., a blood concentration of 0.0001 to 5000 μg / mL). A dose of the anti-CD5 ADC may be administered to the CAR at a time after administration of the anti-CD5 ADC. once a day, week, or month to human patients who are or will be receiving concurrent therapy. The anti-CD5 ADC may be administered to a human patient one or more times (e.g., 2-10 times). In one embodiment, the anti-CD5 ADC is administered to a host-responsive T Decrease the amount of cells (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% of pre-CAR therapy) The therapeutic agent may be administered in an amount sufficient to achieve a therapeutically effective level of efficacy (90%, 95%, or more). EXAMPLES

[0302] How to use, make, and evaluate the compositions and methods described herein The following examples are presented to provide one of ordinary skill in the art with a description of what may be possible. As such, they are intended to be purely illustrative of the present invention and are not intended to be limiting unless otherwise stated by the inventors. It is not intended to limit the scope of the present invention.

[0303] Example 1: In vitro binding analysis of anti-CD5 antibodies To determine the binding properties of the anti-CD5 antibody 5D7 hIgG1, antibody binding assays were performed at 25°C in 37°F (Celsius). The samples were incubated in 1× PBS supplemented with 0.1% w / v bovine serum albumin for 1 h using Bio-Layer Interferometry (Bio-Layer Interferometry). Purified human hirsutum was analyzed using Pall ForteBio Octet Red 96 with Blue Ray Interferometry (BLI). Anti-CD5 antibody (5D7) was immobilized on an anti-human Fc biosensor (AHC; Pall ForteBio 18-5063). and incubated with 50 nM purified human CD5 ectodomain. Anti-CD5 antibody 5D7 The binding characteristics of the anti-CD5 antibody 5D7 used in Examples 1 to 5 are shown in Table 4. The sequence of antibody 5D7 used herein is represented by SEQ ID NO: SEQ ID Nos: 53 and 54 (heavy chain variable region amino acid sequence and light chain variable region amino acid sequence) and SEQ ID Nos: 47 to 52 (heavy chain CDRs and light chain CDRs).

[0304] [Table 6]

[0305] Example 2: In vitro cell line binding analysis of anti-CD5 antibodies MOLT-4 cells (i.e., an immortalized human T lymphoblastoid cell line) were plated at 20,000 cells / well. The cells were then plated with the indicated mouse anti-CD5 antibodies (i.e., L17F12, UCHT2, 205919, and CRIS-1). , and stained for 2 hours at 4° C. Secondary anti-mouse AF488 stain was added at a constant volume for 30 minutes at 4° C. After washing, the plates were analyzed by flow cytometry and the indicated antibodies (and negative controls) were The binding of the IgG1 vector (i.e., mIgG1) was determined based on the geometric mean fluorescence intensity in the AF488 channel. The results from these assays are shown in Figure 1.

[0306] As shown in FIG. 1, the mouse anti-CD5 antibodies L17F12 (Thermo Fisher) and UCHT2 (BioLegend) , 205919 (Novus Biologicals), and CRIS-1 (Novus Biologicals) are human T-lymphoblastoid cells. Binding to endothelial cells (i.e., MOLT-4 cells) and EC 50 = 207 pM(L17), 354 pM(UCH), 1350 pM(205), and and 43 pM (CRIS).

[0307] Example 3: In vitro primary cell binding analysis of anti-CD5 antibodies 8 x 10 primary human T-cells 4 Plate cells / well and drop the human anti-CD5 antibody 5D7. The secondary anti-mouse AF488 stain was added at a constant volume for 30 min at 4°C. After washing, the plate was analyzed using a flow cytometer to measure the concentration of anti-CD5 5D7 antibody (and negative Binding of the antibody (i.e., antibody control, i.e., hIgG1) was calculated as the geometric mean of the fluorescence intensity in the AF488 channel. The results from these assays are shown in FIG.

[0308] As shown in FIG. 2, the anti-CD5 antibody 5D7 inhibited EC 50 = 3.0 pM and binds to primary human T-cells Ta.

[0309] Example 4: Anti-CD5-amatoxin antibody drug conjugates using an in vitro T-cell cytotoxicity assay In vitro analysis of gates (ADC) The anti-CD5 antibody 5D7 was conjugated to amatoxin (amanitin) using a cleavable linker. Gating was performed to form the anti-CD5 5D7 ADC, which has a drug-to-antibody ratio (DAR) of approximately 6 (interchain DAR6). and an anti-CD5 5D7-ADC with a DAR of about 2 (site-specific 5D7-ADC via the D265C mutation). Additionally, the rapid half-life of the anti-CD5 5D7-ADC was The first variant was generated by introducing the H435A mutation in the Fc region.

[0310] Each anti-CD5 5D7-ADC was evaluated using an in vitro human T-cytotoxicity assay.

[0311] Cryopreserved negatively selected primary human T cells were thawed and incubated with anti-CD3 antibody and IL-2. At the beginning of the assay, the mice were stimulated with 2 x 10 cells per well of a 384-well plate. 4 T cells and the indicated ADC or non-conjugated anti-CD5 antibody was added at various concentrations between 0.003 nM and 30 nM. After adding the 5% CO2 to the wells, the wells were placed in an incubator at 37°C and 5% CO2 for 5 days. After incubation for 1 h, the cells were analyzed by flow cytometry. The cells were stained with 7-AAD and measured using a volumetric flow cytometer. .

[0312] The number of surviving T-cells (FIGS. 3A and 3B) was measured by FSC vs. SSC and 7-AAD. A non-conjugated anti-CD5 5D7 antibody was used as a comparative control (Figure 3A).

[0313] As shown in FIG. 3A, anti-CD5 5D7-ADC with a DAR of approximately 6 potently and specifically inhibited human T cells. On the other hand, non-conjugated ("naked") anti-CD5 5D T cells in the presence of the 7 antibody remained viable. As shown in Figure 3B, approximately two-fold The ADC with the site-specific (D265C) DAR showed similar potent levels of T-cell cytotoxicity ( The fast half-life variant of anti-CD5 5D7-ADC (H435A) maintained a similar level of activity. The antibody showed significant T-cytotoxicity (IC50 = 4.9 pm; Figure 3B).

[0314] Example 5: Analysis of T-cell depletion using the hNSG mouse model In vivo T-cell depletion assays were performed using humanized NSG mice (Jackson Laboratories). The anti-CD5 antibody 5D7 was conjugated to amatoxin (amanitin) using a cleavable linker. The anti-CD5 5D7-ADC was conjugated to form the anti-CD5 5D7-ADC. The anti-CD5 5D7-ADC was conjugated to the Each anti-CD5 5D7 ADC (DAR6 or DAR2) was formulated with either a DAR of 6 or a DAR of about 2. A single intravenous injection (0.3 mg / kg, 1 mg / kg, or 3 mg / kg for DAR6 ADC or On day 7, peripheral blood cells, bone marrow Alternatively, thymus samples were taken and absolute CD3+ T-cell counts were determined by flow cytometry. (See Figures 4A-4B for DAR2 ADC and 5A-5C for DAR6 ADC) .

[0315] As shown in Figures 4A-4B, the anti-CD5 5D7-ADC at 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of DAR6 In treated humanized NSG mice, T-cells were strongly depleted in the peripheral blood and bone marrow. On the other hand, thymic T-cell counts were decreased after treatment with 1 mg / kg or 3 mg / kg DAR6 anti-CD5 5D7-ADC. The negative control used in this in vivo study was human IgG1, which is not specific for CD5. (as naked antibody (huIgG1)) and conjugated to amatoxin (huIgG1-AM) As shown in Figures 4A to 4B, the naked and conjugated huIgG1 were Gated controls showed depletion of T cells in peripheral blood (Figure 4A) and bone marrow (Figure 4B). , had no effect, and these controls were equivalent to the PBS controls. YTH34.5) was used as a control, and the dose of 25 mg / kg significantly increased peripheral and bone marrow T cells. It was possible to reduce it.

[0316] As shown in Figures 5A-5C, the site-specific DAR2 anti-CD5 5D7-A at 1 mg / kg or 3 mg / kg DC-treated humanized NSG mice showed enhanced T-cell proliferation in peripheral blood, bone marrow, and thymic T-cells. In each of Figures 5A to 5C, the intact antibody 5D7 was also significantly reduced as a control. As shown in Figure 5A, antibody 5D7 (a non-specific human IgG1 control) Although it was possible to reduce peripheral T cells (compared to PBS or PBS), it was not possible to reduce bone marrow T cells or thymic T cells. On the other hand, as shown in Figures 5B and 5C, The 5D7-AM ADC was effective in depleting both bone marrow and thymus T cells.

[0317] Example 6: Administration of allogeneic CAR-T cells in a mouse model The following studies were carried out to determine the C present in allogeneic recipients under various conditions. The levels of AR-T cells are assessed.

[0318] The study uses a mouse allogeneic CAR-T model.

[0319] Mice in the first treatment group were immunized with 1 × 10 7 ~1×10 9 By intravenous injection of cells / kg On day 3, the mice are treated with an initial dose of allogeneic T cells. The mice were administered a 3 mg / kg dose of the ADC. On day 10, the ADC was substantially eliminated from the blood of the mice. After clearance, mice are administered allogeneic CAR-T cells. The CAR-T cells are administered at 0 The allogeneic T cells are derived from the same donor as the allogeneic T cells administered on day 1.

[0320] A second treatment group of mice is treated using the same protocol as the first treatment group, except for anti-C Instead of the D5 ADC, an unconjugated anti-CD5 antibody is administered on day 3.

[0321] A third treatment group of mice is treated using the same protocol as the first treatment group, except that anti-C Instead of the D5 ADC, isotype control conjugated to α-amanitin was administered on day 3. The antibody against the drug is administered.

[0322] A fourth treatment group of mice is treated using the same protocol as the first treatment group, but with the same Instead of allogeneic T cells, an initial dose of autologous T cells is administered on day 0.

[0323] Mice in the fifth treatment group will receive allogeneic CAR-T cells on day 10 without any pretreatment.

[0324] Mice in the sixth treatment group will receive autologous CAR-T cells on day 10 without any pretreatment.

[0325] Present in the spleen and peripheral blood of mice from each treatment group on days 14, 17, and 30 On day 9, the number of CAR-T cells was measured using spleens and peripheral blood samples from mice from each treatment group. The number of CD5+ activated T cells in the blood was measured. Throughout the study, symptoms of rejection were Monitor the mouse for

[0326] Example 7: Anti-CD5 antibody drug conjugates to prevent rejection of allogeneic cell therapy Administration of the drug to human patients Selecting a human patient to receive allogeneic cell therapy, such as allogeneic CAR cell therapy. To inhibit or prevent cell rejection, anti-CD5 antibody drug conjugates (ADCs) are administered as described herein. The drug is administered according to the methods disclosed herein. The physician performs the following steps of treatment:

[0327] First, in an amount sufficient to induce a priming immune response against the allogeneic cells, An initial dose of allogeneic cells is administered intravenously to the patient. The lineage cells are administered to the patient to elicit an immune response generated by endogenous activated CD5+ T cells. .

[0328] The patient is then administered a cytotoxin-containing anti-CD5 antibody conjugated via a linker. The anti-CD5 ADC is administered to reduce endogenous CD5+ activated T cells. After administration of the anti-CD5 ADC, the level of CD5+ activated T cells is assessed. Evaluate and confirm the decrease.

[0329] A therapeutically effective amount of allogeneic cells expressing a CAR are then administered. During the priming step, the cells were derived from the same donor as the cells administered to the patient. Compared to patients receiving allogeneic cell therapy without priming and administration of anti-CD5 ADC. In comparison, the acceptance of allogeneic cells in the recipient patient is promoted and rejection is reduced. The risk of reaction is reduced.

[0330] [Table 7-1] [Table 7-2] [Table 7-3]

[0331] Other embodiments All publications, patents, and patent applications mentioned herein are hereby incorporated by reference as if they were each independently incorporated. The same extent as if each publication or patent application was specifically and individually indicated to be incorporated by reference. , all of which are incorporated herein by reference.

[0332] While this invention has been described in connection with specific embodiments thereof, it is capable of further modifications. This application broadly describes the principles of the invention and any known or customary techniques within the art to which the invention pertains. The essential features falling within the scope of the exemplary implementations and described hereinabove and in accordance with the claims are set forth herein. Any variations, uses, or adaptations of the invention, including deviations therefrom, as may be applicable, are hereby expressly incorporated by reference in their entirety. It should be understood that inclusion is intended.

Claims

1. In human subjects with cancer or autoimmune disease, 2. A method for enhancing reception of immune cells expressing a CAR (Carbohydrate Immunoglobulin Receptor), the method comprising: Including: (a) administering anti-CD5 antibody drug conjugates (ADCs) to humans with cancer or autoimmune diseases administering to an animal, wherein the anti-CD5 ADC is conjugated to a cytotoxin via a linker. an anti-CD5 antibody or antigen-binding fragment thereof; and (b) administering to the human subject a therapeutically effective amount of immune cells expressing a CAR, wherein The CAR comprises an extracellular domain that binds to a tumor antigen or an antigen associated with an autoimmune disease, a transmembrane domain, and The polypeptide comprises a polypeptide domain, a polypeptide chain domain, and an intracellular domain.

2. 10. The method of claim 1, wherein the human subject is administered, prior to and simultaneously with step (b): or subsequently not receiving alemtuzumab.

3. 3. The method of claim 1 or 2, wherein the human subject is treated with a At the time or thereafter, no lymphocyte-depleting chemotherapy agents are administered.

4. 4. The method of claim 3, wherein the lymphocyte-depleting chemotherapy agent is fludarabine, cyclosporine, Rofosfamide, bendamustine, and / or pentostatin.

5. 5. The method of claim 1 , wherein prior to step (b), an anti-CD2 ADC is administered. further comprising administering to said human subject.

6. 6. The method according to claim 1, wherein the method comprises the step of: administering to said human subject an anti-CD5 ADC about 21 days prior to the time.

7. 7. The method according to any one of claims 1 to 6, wherein the immune cells are allogeneic or are autologous cells.

8. 8. The method of claim 7, wherein the allogeneic cells are allogeneic T cells or allogeneic NK cells. It is a cell.

9. 9. The method of any one of claims 1 to 8, wherein the allogeneic cell expressing the CAR The therapeutically effective dose of is approximately 1 × 10 4 to about 1.0×10 8 cells / kg.

10. 1. A method of treating a patient having a tumor, comprising administering to a subject in need thereof: and (i) an anti-CD5 ADC, wherein the anti-CD5 ADC is conjugated to a cytotoxin via a linker; comprising a gated anti-CD5 antibody or antigen-binding fragment thereof, and (ii) Approximately 1×10 6 From about 1×10 8 100 mg of engineered CAR T cells / kg are administered to the patient. To do so.

11. 11. The method of claim 10, wherein the therapeutically effective amount of the engineered CAR T cells is about 1×10 6 Or about 2 x 10 6 cells / kg.

12. 12. The method of any one of claims 1 to 11, wherein the anti-CD5 ADC is administered in a single dose or is administered to the patient as multiple doses.

13. The method according to any one of claims 1 to 12, wherein the anti-CD5 antibody or its antigen binding site is The combined fragments have the amino acid sequences set forth in SEQ ID NOs: 3, 4, and 5, respectively. and a heavy chain variable region comprising CDR1, CDR2, and CDR3 having the sequences represented by SEQ ID NO: A light chain comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in (SEQ ID NOs) 6, 7, and 8. Contains a variable region.

14. 14. The method of claim 13, wherein the anti-CD5 antibody or antigen-binding fragment thereof comprises: It is chimeric or humanized.

15. The method according to any one of claims 1 to 14, wherein the anti-CD5 antibody or its antigen binding site is The combined fragments are of the IgG1 or IgG4 isotype.

16. 16. The method of claim 1, wherein the cytotoxin is an antimitotic drug or an RNA polymerase. It is a reninase inhibitor.

17. 17. The method of claim 16, wherein the RNA polymerase inhibitor is an amatoxin. do.

18. 17. The method of claim 16, wherein the RNA polymerase inhibitor is amanitin. 。

19. 19. The method of claim 18, wherein the amanitin is α-amanitin, β-amanitin. , γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanulin acid, and proamanuline.

20. 18. The method of claim 17, wherein an anti-CD5 antibody conjugated to an amatoxin or The antigen-binding fragment is represented by the formula Ab-ZL-Am, where Ab is an antibody or its wherein L is a linker; Z is a chemical substructure; and Am is an amatoxin represented by formula (III); 【Chemistry 1】 Here, R 1 H, OH, OR A OR C is; R 2 H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached, any forming 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 Hetero Alkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 Haeta alkenyl, optionally substituted C 2 -C 6 Alkynyl, optionally substituted C 2 -C 6 Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heptyl Heterocycloalkyl, optionally substituted aryl, or optionally substituted hexacycloalkyl. It is a teraaryl; L is a linker; and Z is a substituent that is a reactive group present on L and is present in the anti-CD5 antibody or antigen-binding fragment thereof. It is a chemical substructure formed from a coupling reaction between an existing reactive substituent , where Am is exactly one R c Contains substituents.

21. 21. The method of claim 20, wherein the linker (L) 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; or dipeptides or -(CH 2 ) m O) n (CH 2 ) m where m and n are each independently 1, 2, 3, 4, 5, 6, 7 , 8, 9, and 10.

22. 17. The method of claim 16, wherein the antimitotic drug is maytansine or auric acid. This is Tachin.

23. 23. The method of claim 22, wherein the auristatin is monomethyl auristatin. The two agents are monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE).

24. 17. The method of claim 16, wherein the antimitotic drug is a pyrrolobenzodiazepine (P BD) or calicheamicin.

25. 25. The method of any one of claims 1 to 24, wherein the linker of the ADC is an N-base The compound is termaleimidopropionyl-Val-Ala-para-aminobenzyl (BMP-Val-Ala-PAB).

26. 11. The method of claim 1 or 10, wherein the ADC is represented by any one of the following structures: Represented: 【Chemistry 2】 。

27. 11. The method of claim 1 or 10, wherein the ADC is represented by: 【Chemistry 3】 or 【Chemistry 4】 。

28. 28. The method of any one of claims 1 to 27, wherein the ADC has a serum half-life of 3 days or less. It has a period.

29. 29. The method of any one of claims 1 to 28, wherein the extracellular domain of the CAR is Includes cFv antibodies or single chain T cell receptors (scTCRs).

30. 29. The method of any one of claims 1 to 28, wherein the extracellular domain is a non-immune Contains immunoglobulin scaffold protein.

31. 31. The method of any one of claims 1 to 30, wherein the tumor antigen is 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.

32. 31. The method of any one of claims 1 to 30, wherein the intracellular domain of the CAR is C D28 intracellular signaling domain, CD3 zeta intracellular signaling domain, OX40 cells and / or the CD137 (4-1BB) intracellular signaling domain. nothing.

33. 33. The method of any one of claims 1 to 32, wherein the intracellular domain of the CAR is CD3 Contains the zeta intracellular signaling domain.

34. 34. The method of any one of claims 1 to 33, wherein the human subject having 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, colorectal cancer liver metastasis Transplantation, 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 lymphoma 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, 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 cancer colorectal carcinoma, gastric carcinoma, pancreatic carcinoma ), triple-negative invasive breast carcinoma, kidney Renal cell carcinoma, lung squamous cell carcinoma, liver Hepatocellular carcinoma, urothelial carcinoma, leukemia, B cellular leukemia, B-cell acute lymphocytic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia Lymphoblastic leukemia, B-cell prolymphocytic leukemia, Childhood acute lymphoblastic leukemia, refractory pediatric acute refractory childhood acute lymphoblastic leukemia Leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia cytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, recurrent plasma cell osteomyelitis Myeloma (recurrent plasma cell myeloma), refractory plasma cell myeloma (refractory plasm a cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, multiple myeloma of bone, Malignant glioma of the brain, myelodysplastic syndrome, EGFR positive colorectal cancer, glioblastoma multiforme, neoplasm, blastic plasmacytoid dendritic cell tumor tumor (blastic plasmacytoid dendritic cell neoplasm), liver metastasis, solid tumor, progressive solid Advanced solid tumor, Mesothelin positive tumor, Blood malignant tumors, and other advanced malignant tumors.

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