Use of anti-CD5 antibody drug conjugates (ADCs) in allogeneic cell therapy

Abstract

The present invention provides a method for consuming CD5+ cells in human patients undergoing chimeric antigen receptor (CAR) immunotherapy to promote acceptance of immune cells expressing CAR. Anti-CD5 antibody drug conjugates (ADCs) are administered as a conditioning regimen to human patients receiving autologous or allogeneic CAR-expressing immune cells so that CAR-expressing immune cells are accepted by human patients. The compositions and methods of the present invention can be used in combination with CAR therapy to treat a variety of diseases, including autoimmune diseases and cancer.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 702,296, filed on July 23, 2018, and U.S. Provisional Patent Application No. 62 / 773,047, filed on November 29, 2018. The contents of the foregoing applications are incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in ASCII format, and the sequence listing is hereby incorporated by reference in its entirety. The ASCII copy created on July 19, 2019 is named M103034_2060WO_SL.txt and is 85,119 bytes in size. Field of the Invention

[0005] The present invention generally relates to methods of promoting uptake of chimeric antigen receptor (CAR) expressing immune cells by human subjects through the use of anti-CD5 antibody drug conjugates (ADCs). Background of the Invention

[0007] Chimeric antigen receptor (CAR) therapy is an immunotherapy that uses the body's own immune system to destroy cells expressing specific antigens associated with a disease (such as cancer). For example, in cancer, CAR therapy recruits and enhances the ability of the patient's immune system to attack tumors. In the past few years, this immunotherapy has become a promising and revolutionary therapy. CAR therapy is based on immune cells such as T cells that express CAR, and CAR is generally a transmembrane fusion protein that combines extracellular antigen binding domains (such as scFv) with cytoplasmic active signaling and "co-stimulation" domains (transducing signals from surface receptors to cells). Therefore, when immune cells (such as T cells) express CAR, the immune cells can recognize and kill cells expressing antigens (e.g., tumor-associated antigens) targeted by the antigen binding domains of CAR (Geyer and Brentjens (2016) Cytotherapy 18 (11): 1393-1409).

[0008] Although CAR therapy is an extremely powerful technology, it does carry serious risks and adverse side effects (Kay and Turtle (2017) Drugs 77 (3): 237-245; Hill et al. (2018) Blood 131: 121-130). In order to minimize the rejection of CAR-expressing cells by patients receiving treatment, lymphodepleting chemotherapy is often used as a conditioning therapy in combination with CAR therapy (Wei et al. (2017) Exp Hematol Oncol. 6: 10). For example, the combination of lymphodepleting agents fludarabine and cyclophosphamide improves the duration of CAR-T cells in recipient patients (Turtle et al. (2016) J Clinic Invest 126 (6): 2123; see also US20170368101). Although conditioning therapy improves the efficacy of CAR-T cells, lymphodepleting chemotherapy often has serious negative side effects. SUMMARY OF THE INVENTION

[0010] The present invention provides a regulatory scheme that can be used with CAR therapy to promote the acceptance of CAR-expressing immune cells. The methods described herein can be used to promote the acceptance of autologous immune cells expressing CAR or allogeneic immune cells expressing CAR. Traditionally, the acceptance of such cells is achieved by treatment with lympho-depleting chemotherapeutic agents. This article describes an improved method for promoting the acceptance of CAR-expressing cells in recipient patients.

[0011] The present invention includes a method for promoting acceptance of immune cells expressing a chimeric antigen receptor (CAR) by human subjects suffering from cancer or autoimmune diseases, the method comprising administering an anti-CD5 antibody drug conjugate (ADC) to a human subject suffering from cancer or autoimmune diseases, wherein the anti-CD5 ADC comprises an anti-CD5 antibody or an antigen-binding fragment thereof conjugated to a cytotoxin via a linker; and administering a therapeutically effective amount of immune cells expressing the CAR to the human subject, wherein the CAR comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain that bind to a tumor antigen or an antigen associated with an autoimmune disease.

[0012] In one embodiment, human subjects are not administered alemtuzumab before the administration of CAR-expressing immune cells, simultaneously with the administration of CAR-expressing immune cells, or after the administration of CAR-expressing immune cells. In another embodiment, human subjects are not administered lymphatic depletion chemotherapeutics before the administration of CAR-expressing immune cells, simultaneously with the administration of CAR-expressing immune cells, or after the administration of CAR-expressing immune cells. In one embodiment, lymphatic depletion chemotherapeutics are fludarabine, cyclophosphamide, bendamustine and / or pentostatin.

[0013] In one embodiment, the method further comprises administering an anti-CD5 ADC to the human subject prior to administering the CAR-expressing immune cell.

[0014] In one embodiment, the method further comprises administering the anti-CD5 ADC to the human subject about 12 hours to about 21 days prior to administering the CAR-expressing immune cells.

[0015] In one embodiment, the immune cell is an allogeneic cell or an autologous cell. In one embodiment, the allogeneic cell is an allogeneic T cell or an allogeneic NK cell.

[0016] In certain embodiments, the therapeutically effective amount of allogeneic cells expressing CAR is about 1×10 4 cells / kg to about 1.0×10 8 cells / kg.

[0017] The invention also features a method of treating a patient suffering from a tumor, the method comprising administering an anti-CD5 ADC to a patient in need thereof, wherein the anti-CD5 ADC comprises an anti-CD5 antibody or antigen-binding fragment thereof conjugated to a cytotoxin via a linker, and administering a therapeutically effective amount of about 1×10 6 Engineered CAR T cells / kg to about 1×10 8 In one embodiment, the therapeutically effective amount of engineered CAR T cells is about 1×10 6 cells / kg or about 2×10 6 cells / kg.

[0018] In certain embodiments of the invention, the anti-CD5 ADC is administered to the patient as a single dose or as multiple doses.

[0019] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively, and the anti-CD5 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences set forth in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0020] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof is chimeric or humanized.

[0021] In another embodiment, the anti-CD5 antibody or antigen-binding fragment thereof is of the IgG1 isotype or the IgG4 isotype.

[0022] In yet another embodiment, the cytotoxin is an antimitotic agent or an RNA polymerase inhibitor.

[0023] In other embodiments, the cytotoxin is maytansine, calicheamicin, pyrrolobenzodiazepine, indolebenzodiazepine, or auristatin. In one embodiment, the auristatin is monomethyl auristatin F (MMAF) or monomethyl auristatin E (MMAE). In one embodiment, the cytotoxin is maytansine. In one embodiment, the cytotoxin is a pyrrolobenzodiazepine (PBD). For example, in some embodiments, the PBD can be selected from tesirine or talirine. In some embodiments, the cytotoxin can be calicheamicin. For example, in some embodiments, the calicheamicin can be ozogamicin.

[0024] In one embodiment, the RNA polymerase inhibitor is an amanitin. In another embodiment, the RNA polymerase inhibitor is an amanitin (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, amanullinic acid, or proamanullin).

[0025] In one embodiment, the antibody drug conjugate (ADC) is represented by the formula Ab-ZL-Am, wherein Ab is an antibody or antigen-binding fragment thereof that binds CD5, L is a linker, Z is a chemical moiety, and Am is an Amanita ani toxin. In certain embodiments, the linker-amanitin toxin conjugate Am-LZ is represented by the formula (III)

[0026]

[0027] Where R1 is H, OH, OR A OR C ;

[0028] R2 is H, OH, OR B OR C ;

[0029] R A and R B When present, taken together with the oxygen atom to which they are attached, form an optionally substituted 5-membered heterocycloalkyl group;

[0030] R3 is H, R C or R D ;

[0031] R4, R5, R6 and R7 are each independently H, OH, OR C , OR D , R C or R D ;

[0032] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0033] R9 is H, OH, OR C OR D ;

[0034] Q is -S-, -S(O)- or -SO2-;

[0035] R C Yes - LZ;

[0036] R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0037] L is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl or optionally substituted heteroaryl; or includes a dipeptide; or -((CH2) m O) n (CH2) m –, wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; and

[0038] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the anti-CD5 antibody or antigen-binding fragment thereof.

[0039] In this embodiment, the linker-amanitin conjugate Am-LZ is represented by formula (III)

[0040]

[0041] Where R1 is H, OH, OR A OR C ;

[0042] R2 is H, OH, OR B OR C ;

[0043] R A and R B When present, taken together with the oxygen atom to which they are attached, form an optionally substituted 5-membered heterocycloalkyl group;

[0044] R3 is H, R C or R D ;

[0045] R4, R5, R6 and R7 are each independently H, OH, OR C , OR D , R C or R D ;

[0046] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0047] R9 is H, OH, OR COR D ;

[0048] Q is -S-, -S(O)- or -SO2-;

[0049] R C Yes - LZ;

[0050] R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0051] L is a connector; and

[0052] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the anti-CD5 antibody or antigen-binding fragment thereof.

[0053] In one embodiment of Formula (III), L is a peptide comprising a linker.

[0054] In some embodiments, the linker includes one or more of the following: a dipeptide, a p-aminobenzyl (PAB) group, an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted C3-C6 cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, a solubility enhancing group, -(C=O)-, -(CH2CH2O) p - group, wherein p is an integer from 1 to 6, ((CH2) m O) n (CH2) m –, wherein n and each m are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; or a combination thereof.

[0055] In some embodiments, the linker comprises ((CH2) m O) n (CH2) m -group and a heteroaryl group, wherein the heteroaryl group is triazole. In some embodiments, ((CH2) m O) n (CH2) m – group and triazole together include

[0056] wherein n is 1 to 10 and the wavy line indicates the point of attachment to an additional linker component, chemical moiety Z, or Amanita toxin.

[0057] In some embodiments, Am contains exactly one R C Substituent.

[0058] In one embodiment, the linker of the ADC is N-β-maleimidopropionyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB). In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0059]

[0060] wherein S is a sulfur atom and represents a reactive substituent (eg, a -SH group from a cysteine ​​residue) present in the antibody or antigen-binding fragment thereof that binds CD5.

[0061] In some embodiments, LZ is

[0062]

[0063] In one embodiment, ADC is represented by any of the following structures:

[0064]

[0065] In one embodiment, ADC is represented by one of the following structures:

[0066]

[0067] In one embodiment, the ADC has a serum half-life of 3 days or less.

[0068] In one embodiment, the extracellular domain of the CAR is a scFv antibody.

[0069] In one embodiment, the extracellular domain of CAR is a single-chain T cell receptor (scTCR).In one embodiment, the extracellular domain of CAR includes a non-immunoglobulin scaffold protein.

[0070] In one embodiment, the extracellular domain of the CAR binds to a tumor antigen that 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.

[0071] In certain embodiments, the cytoplasmic domain of CAR includes a CD28 cytoplasmic signaling domain, a CD3 ζ cytoplasmic signaling domain, an OX40 cytoplasmic signaling domain and / or a CD137 (4-1BB) cytoplasmic signaling domain. In one embodiment, the cytoplasmic domain of CAR includes a CD3 ζ cytoplasmic signaling domain.

[0072] The methods and compositions disclosed herein can be used to treat human subjects with cancer, including but not limited to leukemia, advanced cancer in adults, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple negative breast cancer, hematopoietic / lymphoid cancer, colon cancer liver metastasis, small cell lung cancer, non-small cell lung cancer, B cell lymphoma, relapsed or refractory B cell lymphoma, follicular lymphoma, mantle cell lymphoma , diffuse large cell lymphoma, relapsed or refractory diffuse large cell lymphoma, anaplastic large cell lymphoma, primary mediastinal B-cell lymphoma, relapsed mediastinal large B-cell lymphoma, refractory mediastinal large B-cell lymphoma, large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, colorectal cancer carcinoma), gastric cancer, pancreatic cancer, triple-negative invasive breast cancer, renal cell carcinoma, squamous cell lung carcinoma, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, prolymphocytic leukemia Baxyblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, multiple myeloma of bone, malignant glioma of brain, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasms, blastic plasmacytoid dendritic cell neoplasm, liver metastasis, solid tumors, advanced solid tumors, mesothelin-positive tumors, hematological malignancies and other advanced malignancies.

[0073] In certain embodiments of any of the above aspects, the anti-CD5 antibody or antigen-binding fragment thereof comprises a combination of CDRs (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions) as listed in Table 1A and Table 1B below. In certain embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises a combination of heavy chain variable regions and light chain variable regions as listed in Table 1A and Table 1B. Sequence Listing <110> Migenda Therapeutics <120> Use of anti-CD5 antibody drug conjugates (ADCs) in allogeneic cell therapy <130> M103034 2060WO <140> <141> <150> 62 / 773,047 <151> 2018-11-29 <150> 62 / 702,296 <151> 2018-07-23 <160> 282 <170> PatentIn version 3.5 <210> 1 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 1 Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala His Ile Trp Trp Asp Asp Asp Val Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Ala Ser Lys Asp Gln Val 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Val Arg Arg Arg Ala Thr Gly Thr Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 2 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 2 Asn Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys His Gln Tyr Asn Ser Tyr Asn Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 3 Phe Ser Leu Ser Thr Ser Gly Met Gly 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 4 Trp Trp Asp Asp Asp 1 5 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 5 Arg Arg Ala Thr Gly Thr Gly Phe Asp Tyr 1 5 10 <210> 6 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 6 Gln Asp Val Gly Thr Ala 1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 7 Trp Thr Ser Thr Arg His Thr 1 5 <210> 8 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 8 Tyr Asn Ser Tyr Asn Thr 1 5 <210> 9 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 9 Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 1 5 10 15 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 20 25 30 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala 35 40 45 <210> 10 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 10 Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro 1 5 10 15 Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro 20 25 30 Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Pro Arg 35 40 45 Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser 50 55 60 Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro 65 70 75 80 Leu Phe Pro Gly Pro Ser Lys Pro 85 <210> 11 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 11 Leu Asp Pro Lys Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr 1 5 10 15 Gly Val Ile Leu Thr Ala Leu Phe Leu Arg Val Lys 20 25 <210> 12 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 12 Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu 1 5 10 15 Thr Ala Leu Phe Leu 20 <210> 13 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 13 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 14 <400> 14 000 <210> 15 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 15 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 16 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> Description of synthetic polypeptide <400> 16 Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 35 40 45 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 50 55 60 Trp Val 65 <210> 17 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 17 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 18 Gly Gly Gly Gly Ser 1 5 <210> 19 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide description <400> 19 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 20 <211> 495 <212> PRT <213> Homo sapiens <400> 20 Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly 1 5 10 15 Met Leu Val Ala Ser Cys Leu Gly Arg Leu Ser Trp Tyr Asp Pro Asp 20 25 30 Phe Gln Ala Arg Leu Thr Arg Ser Asn Ser Lys Cys Gln Gly Gln Leu 35 40 45 Glu Val Tyr Leu Lys Asp Gly Trp His Met Val Cys Ser Gln Ser Trp 50 55 60 Gly Arg Ser Ser Lys Gln Trp Glu Asp Pro Ser Gln Ala Ser Lys Val 65 70 75 80 Cys Gln Arg Leu Asn Cys Gly Val Pro Leu Ser Leu Gly Pro Phe Leu 85 90 95 Val Thr Tyr Thr Pro Gln Ser Ser Ile Ile Cys Tyr Gly Gln Leu Gly 100 105 110 Ser Phe Ser Asn Cys Ser His Ser Arg Asn Asp Met Cys His Ser Leu 115 120 125 Gly Leu Thr Cys Leu Glu Pro Gln Lys Thr Thr Pro Pro Thr Thr Arg 130 135 140 Pro Pro Pro Thr Thr Thr Pro Glu Pro Thr Ala Pro Pro Arg Leu Gln 145 150 155 160 Leu Val Ala Gln Ser Gly Gly Gln His Cys Ala Gly Val Val Glu Phe 165 170 175 Tyr Ser Gly Ser Leu Gly Gly Thr Ile Ser Tyr Glu Ala Gln Asp Lys 180 185 190 Thr Gln Asp Leu Glu Asn Phe Leu Cys Asn Asn Leu Gln Cys Gly Ser 195 200 205 Phe Leu Lys His Leu Pro Glu Thr Glu Ala Gly Arg Ala Gln Asp Pro 210 215 220 Gly Glu Pro Arg Glu His Gln Pro Leu Pro Ile Gln Trp Lys Ile Gln 225 230 235 240 Asn Ser Ser Cys Thr Ser Leu Glu His Cys Phe Arg Lys Ile Lys Pro 245 250 255 Gln Lys Ser Gly Arg Val Leu Ala Leu Leu Cys Ser Gly Phe Gln Pro 260 265 270 Lys Val Gln Ser Arg Leu Val Gly Gly Ser Ser Ile Cys Glu Gly Thr 275 280 285 Val Glu Val Arg Gln Gly Ala Gln Trp Ala Ala Leu Cys Asp Ser Ser 290 295 300 Ser Ala Arg Ser Ser Leu Arg Trp Glu Glu Val Cys Arg Glu Gln Gln 305 310 315 320 Cys Gly Ser Val Asn Ser Tyr Arg Val Leu Asp Ala Gly Asp Pro Thr 325 330 335 Ser Arg Gly Leu Phe Cys Pro His Gln Lys Leu Ser Gln Cys His Glu 340 345 350 Leu Trp Glu Arg Asn Ser Tyr Cys Lys Lys Val Phe Val Thr Cys Gln 355 360 365 Asp Pro Asn Pro Ala Gly Leu Ala Ala Gly Thr Val Ala Ser Ile Ile 370 375 380 Leu Ala Leu Val Leu Leu Val Val Leu Leu Val Val Cys Gly Pro Leu 385 390 395 400 Ala Tyr Lys Lys Leu Val Lys Lys Phe Arg Gln Lys Lys Gln Arg Gln 405 410 415 Trp Ile Gly Pro Thr Gly Met Asn Gln Asn Met Ser Phe His Arg Asn 420 425 430 His Thr Ala Thr Val Arg Ser His Ala Glu Asn Pro Thr Ala Ser His 435 440 445 Val Asp Asn Glu Tyr Ser Gln Pro Pro Arg Asn Ser His Leu Ser Ala 450 455 460 Tyr Pro Ala Leu Glu Gly Ala Leu His Arg Ser Ser Met Gln Pro Asp 465 470 475 480 Asn Ser Ser Asp Ser Asp Tyr Asp Leu His Gly Ala Gln Arg Leu 485 490 495 <210> twenty one <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> twenty one Gly Tyr Thr Phe Thr Asn Tyr 1 5 <210> twenty two <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> twenty two Asn Thr His Thr Gly Glu 1 5 <210> twenty three <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> twenty three Arg Gly Tyr Asp Trp Tyr Phe Asp Val 1 5 <210> twenty four <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> twenty four Arg Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser 1 5 10 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 25 Arg Ala Asn Arg Leu Val Asp 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 26 Gln Gln Tyr Asp Glu Ser Pro Trp Thr 1 5 <210> 27 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 27 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr 65 70 75 80 Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 28 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 28 Glu Ile Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr His Tyr Gly Glu Pro Thr Tyr Ala Asp Ser Phe 50 55 60 Lys Gly Thr Arg Thr Phe Ser Leu Asp Asp Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 29 Gly Tyr Thr Phe Thr Asn Tyr 1 5 <210> 30 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 30 Asn Thr His Tyr Gly Glu 1 5 <210> 31 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 31 Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val 1 5 10 <210> 32 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 32 Arg Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser 1 5 10 <210> 33 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 33 Arg Ala Asn Arg Leu Glu Ser 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 34 Gln Gln Tyr Asp Glu Ser Pro Trp Thr 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 35 Gly Tyr Ser Ile Thr Ser Gly Tyr Tyr 1 5 <210> 36 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 36 Ile Ser Tyr Ser Gly Phe Thr 1 5 <210> 37 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 37 Ala Gly Asp Arg Thr Gly Ser Trp Phe Ala Tyr 1 5 10 <210> 38 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 38 Gln Asp Ile Ser Asn Tyr 1 5 <210> 39 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 39 Ala Thr Ser 1 <210> 40 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 40 Leu Gln Tyr Ala Ser Tyr Pro Phe Thr 1 5 <210> 41 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 41 Gly Tyr Ile Phe Thr Asn Tyr Gly 1 5 <210> 42 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 42 Ile Asn Thr Tyr Asn Gly Glu Pro 1 5 <210> 43 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 43 Ala Arg Gly Asp Tyr Tyr Gly Tyr Glu Asp Tyr 1 5 10 <210> 44 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 44 Gln Gly Ile Ser Asn Tyr 1 5 <210> 45 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 45 Tyr Thr Ser 1 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 46 Gln Gln Tyr Ser Lys Leu Pro Trp Thr 1 5 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 47 Phe Ser Leu Ser Thr Ser Gly Met Gly 1 5 <210> 48 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 48 Trp Trp Asp Asp Asp 1 5 <210> 49 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 49 Arg Arg Ala Thr Gly Thr Gly Phe Asp Tyr 1 5 10 <210> 50 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 50 Gln Asp Val Gly Thr Ala 1 5 <210> 51 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 51 Trp Thr Ser Thr Arg His Thr 1 5 <210> 52 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 52 Tyr Asn Ser Tyr Asn Thr 1 5 <210> 53 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 53 Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala His Ile Trp Trp Asp Asp Asp Val Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Ala Ser Lys Asp Gln Val 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Val Arg Arg Arg Ala Thr Gly Thr Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 54 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 54 Asn Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys His Gln Tyr Asn Ser Tyr Asn Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 55 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Glu Asn Gly Ser Asp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Gly Ala Val Ser Tyr Phe Asp Val Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 56 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 56 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr 100 105 <210> 57 <211> 438 <212> PRT <213> Homo sapiens <400> 57 Met Val Cys Ser Gln Ser Trp Gly Arg Ser Ser Lys Gln Trp Glu Asp 1 5 10 15 Pro Ser Gln Ala Ser Lys Val Cys Gln Arg Leu Asn Cys Gly Val Pro 20 25 30 Leu Ser Leu Gly Pro Phe Leu Val Thr Tyr Thr Pro Gln Ser Ser Ile 35 40 45 Ile Cys Tyr Gly Gln Leu Gly Ser Phe Ser Asn Cys Ser His Ser Arg 50 55 60 Asn Asp Met Cys His Ser Leu Gly Leu Thr Cys Leu Glu Pro Gln Lys 65 70 75 80 Thr Thr Pro Pro Thr Thr Arg Pro Pro Pro Thr Thr Thr Pro Glu Pro 85 90 95 Thr Ala Pro Pro Arg Leu Gln Leu Val Ala Gln Ser Gly Gly Gln His 100 105 110 Cys Ala Gly Val Val Glu Phe Tyr Ser Gly Ser Leu Gly Gly Thr Ile 115 120 125 Ser Tyr Glu Ala Gln Asp Lys Thr Gln Asp Leu Glu Asn Phe Leu Cys 130 135 140 Asn Asn Leu Gln Cys Gly Ser Phe Leu Lys His Leu Pro Glu Thr Glu 145 150 155 160 Ala Gly Arg Ala Gln Asp Pro Gly Glu Pro Arg Glu His Gln Pro Leu 165 170 175 Pro Ile Gln Trp Lys Ile Gln Asn Ser Ser Cys Thr Ser Leu Glu His 180 185 190 Cys Phe Arg Lys Ile Lys Pro Gln Lys Ser Gly Arg Val Leu Ala Leu 195 200 205 Leu Cys Ser Gly Phe Gln Pro Lys Val Gln Ser Arg Leu Val Gly Gly 210 215 220 Ser Ser Ile Cys Glu Gly Thr Val Glu Val Arg Gln Gly Ala Gln Trp 225 230 235 240 Ala Ala Leu Cys Asp Ser Ser Ser Ala Arg Ser Ser Leu Arg Trp Glu 245 250 255 Glu Val Cys Arg Glu Gln Gln Cys Gly Ser Val Asn Ser Tyr Arg Val 260 265 270 Leu Asp Ala Gly Asp Pro Thr Ser Arg Gly Leu Phe Cys Pro His Gln 275 280 285 Lys Leu Ser Gln Cys His Glu Leu Trp Glu Arg Asn Ser Tyr Cys Lys 290 295 300 Lys Val Phe Val Thr Cys Gln Asp Pro Asn Pro Ala Gly Leu Ala Ala 305 310 315 320 Gly Thr Val Ala Ser Ile Ile Leu Ala Leu Val Leu Leu Val Val Leu 325 330 335 Leu Val Val Cys Gly Pro Leu Ala Tyr Lys Lys Leu Val Lys Lys Phe 340 345 350 Arg Gln Lys Lys Gln Arg Gln Trp Ile Gly Pro Thr Gly Met Asn Gln 355 360 365 Asn Met Ser Phe His Arg Asn His Thr Ala Thr Val Arg Ser His Ala 370 375 380 Glu Asn Pro Thr Ala Ser His Val Asp Asn Glu Tyr Ser Gln Pro Pro 385 390 395 400 Arg Asn Ser His Leu Ser Ala Tyr Pro Ala Leu Glu Gly Ala Leu His 405 410 415 Arg Ser Ser Met Gln Pro Asp Asn Ser Ser Asp Ser Asp Tyr Asp Leu 420 425 430 His Gly Ala Gln Arg Leu 435 <210> 58 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 58 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr 65 70 75 80 Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 59 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 59 Gln Ile Gln Leu Val Gln Ser Gly Pro Gly Leu Lys Lys Pro Gly Gly 1 5 10 15 Ser Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met 35 40 45 Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 60 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Description of synthetic polypeptide <400> 60 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 61 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 61 Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met 1 5 10 <210> 62 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 62 Ser Gly Tyr Ser Phe Thr Asp Tyr Thr Met 1 5 10 <210> 63 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 63 Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met 1 5 10 <210> 64 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 64 Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met 1 5 10 <210> 65 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 65 Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met 1 5 10 <210> 66 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 66 Ser Gly Phe Thr Phe Ser Asn Tyr Ala Met 1 5 10 <210> 67 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 67 Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met 1 5 10 <210> 68 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 68 Ser Gly Tyr Ser Phe Thr Ala Tyr Asn Ile 1 5 10 <210> 69 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 69 Ser Gly Tyr Ser Phe Thr Ala Tyr Ser Met 1 5 10 <210> 70 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 70 Ser Gly Tyr Thr Phe Thr Asn Phe Ala Ile 1 5 10 <210> 71 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 71 Ser Gly Tyr Thr Phe Thr Asn Phe Ala Ile 1 5 10 <210> 72 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 72 Ser Gly Tyr Thr Phe Thr Asn Phe Ala Ile 1 5 10 <210> 73 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 73 Ser Gly Tyr Thr Phe Thr Asn Phe Ala Ile 1 5 10 <210> 74 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 74 Ser Gly Phe Asn Ile Lys Asp Thr Tyr Met 1 5 10 <210> 75 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 75 Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met 1 5 10 <210> 76 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 76 Ser Gly Phe Ser Leu Thr Asn Tyr Asp Val 1 5 10 <210> 77 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 77 Ser Gly Phe Ser Leu Thr Asn Tyr Asp Val 1 5 10 <210> 78 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 78 Ser Gly Phe Thr Phe Ser Asn Tyr Gly Met 1 5 10 <210> 79 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 79 Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met 1 5 10 <210> 80 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 80 Ser Gly Tyr Ile Phe Ala Asn Tyr Gly Met 1 5 10 <210> 81 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 81 Ser Gly Tyr Asn Phe Thr Asn Tyr Gly Met 1 5 10 <210> 82 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 82 Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met 1 5 10 <210> 83 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 83 Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Ile 1 5 10 <210> 84 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 84 Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Ile 1 5 10 <210> 85 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 85 Ser Gly Asn Thr Phe Thr Asn Phe Tyr Leu 1 5 10 <210> 86 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 86 Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met 1 5 10 <210> 87 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 87 Ser Glu Phe Thr Phe Ser Asn Tyr Ala Met 1 5 10 <210> 88 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 88 Ser Gly Tyr Thr Phe Thr Ser Tyr Arg Met 1 5 10 <210> 89 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 89 Ser Gly Phe Asn Ile Lys Asp Thr Tyr Met 1 5 10 <210> 90 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 90 Ser Gly Tyr Ser Phe Thr Asp Tyr Thr Met 1 5 10 <210> 91 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 91 Ser Gly Tyr Met Phe Thr Asn His Gly Met 1 5 10 <210> 92 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 92 Ser Gly Tyr Met Phe Thr Asn Tyr Gly Met 1 5 10 <210> 93 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 93 Ser Gly Tyr Ile Phe Thr Asn Tyr Gly Met 1 5 10 <210> 94 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 94 Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Ile 1 5 10 <210> 95 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 95 Ser Gly Tyr Thr Phe Ile Asn Tyr Gly Met 1 5 10 <210> 96 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 96 Ser Gly Tyr Thr Phe Thr Asp Tyr Phe Ile 1 5 10 <210> 97 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 97 Ser Gly Tyr Ile Phe Thr Gly Tyr Asn Ile 1 5 10 <210> 98 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 98 Leu Ile Asn Pro Tyr Asn Gly Gly Thr Thr 1 5 10 <210> 99 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 99 Leu Ile Asn Pro Tyr Asn Gly Gly Thr Met 1 5 10 <210> 100 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 100 Leu Ile Asn Pro Tyr Asn Gly Gly Thr Met 1 5 10 <210> 101 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 101 Leu Ile Asn Pro Tyr Asn Gly Gly Thr Met 1 5 10 <210> 102 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 102 Leu Ile Asn Pro Tyr Asn Gly Gly Thr Thr 1 5 10 <210> 103 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 103 Ser Ile Ser Ser Gly Gly Asn Thr Phe 1 5 <210> 104 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 104 Ser Ile Ser Ser Gly Gly Ser Thr Tyr 1 5 <210> 105 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 105 Ser Ile Asp Pro Tyr Tyr Gly Asp Thr Lys 1 5 10 <210> 106 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 106 Ser Ile Asp Pro Tyr Tyr Gly Asp Thr Lys 1 5 10 <210> 107 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 107 Leu Ile Ser Ser Asn Ser Gly Asp Val Ser 1 5 10 <210> 108 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 108 Leu Ile Ser Thr Ser Ser Gly Asp Val Ser 1 5 10 <210> 109 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 109 Leu Ile Ser Ser Asn Ser Gly Asp Val Ser 1 5 10 <210> 110 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 110 Leu Ile Ser Ser Asn Ser Gly Asp Val Ser 1 5 10 <210> 111 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 111 Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys 1 5 10 <210> 112 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 112 Met Ile His Pro Ser Asp Ser Glu Thr Arg 1 5 10 <210> 113 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 113 Val Ile Trp Ser Gly Gly Asn Thr Asp 1 5 <210> 114 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 114 Val Ile Trp Ser Gly Gly Asn Thr Asp 1 5 <210> 115 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 115 Ala Ile Asn Ser Asn Gly Asp Ile Thr Tyr 1 5 10 <210> 116 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 116 Leu Ile Asn Pro Tyr Asn Gly Gly Thr Arg 1 5 10 <210> 117 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 117 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr 1 5 10 <210> 118 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 118 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr 1 5 10 <210> 119 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 119 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr 1 5 10 <210> 120 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 120 Trp Ile Tyr Pro Gly Gly Gly Asn Thr Arg 1 5 10 <210> 121 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 121 Trp Ile Tyr Pro Gly Gly Gly Asn Thr Arg 1 5 10 <210> 122 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 122 Cys Ile Tyr Pro Gly Asn Val Lys Thr Lys 1 5 10 <210> 123 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 123 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr 1 5 10 <210> 124 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 124 Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr 1 5 10 <210> 125 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 125 Arg Ile Asp Pro Tyr Asp Ser Gly Thr His 1 5 10 <210> 126 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 126 Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys 1 5 10 <210> 127 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 127 Leu Ile Asn Pro Tyr Asn Gly Gly Thr Arg 1 5 10 <210> 128 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 128 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr 1 5 10 <210> 129 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 129 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr 1 5 10 <210> 130 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 130 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr 1 5 10 <210> 131 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 131 Trp Ile Asp Pro Glu Asn Gly Arg Thr Glu 1 5 10 <210> 132 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 132 Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr 1 5 10 <210> 133 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 133 Glu Ile Tyr Pro Gly Ser Ser Asn Thr Tyr 1 5 10 <210> 134 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 134 Ala Val Tyr Pro Gly Asn Gly Asp Thr Ser 1 5 10 <210> 135 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 135 Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Asp Phe Asp Tyr Trp 1 5 10 15 <210> 136 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 136 Cys Ala Arg Asp Asn Tyr Gly Ser Ser Pro Asp Phe Asp Tyr Trp 1 5 10 15 <210> 137 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 137 Cys Ala Arg Asp Asn Tyr Gly Ser Ser Pro Tyr Phe Asp Tyr Trp 1 5 10 15 <210> 138 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 138 Cys Ala Arg Asp Asn Tyr Gly Ser Ser Pro Tyr Phe Asp Tyr Trp 1 5 10 15 <210> 139 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 139 Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Asp Phe Asp Tyr Trp 1 5 10 15 <210> 140 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 140 Cys Val Arg Tyr Tyr Tyr Gly Val Thr Tyr Trp Tyr Phe Asp Val Trp 1 5 10 15 <210> 141 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 141 Cys Val Arg Tyr Tyr Tyr Gly Ile Arg Tyr Trp Tyr Phe Asp Val Trp 1 5 10 15 <210> 142 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 142 Cys Ala Arg Arg Met Ile Thr Met Gly Asp Trp Tyr Phe Asp Val Trp 1 5 10 15 <210> 143 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 143 Cys Ala Arg Arg Met Ile Thr Thr Gly Asp Trp Tyr Phe Asp Val Trp 1 5 10 15 <210> 144 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 144 Cys Ala Arg His Tyr Gly Ala His Asn Tyr Phe Asp Tyr Trp 1 5 10 <210> 145 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 145 Cys Ala Arg His Tyr Gly Ala Asn Asn Tyr Phe Asp Tyr Trp 1 5 10 <210> 146 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 146 Cys Ala Arg His Tyr Gly Ala His Asn Tyr Phe Asp Tyr Trp 1 5 10 <210> 147 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 147 Cys Ala Arg His Tyr Gly Ala His Asn Tyr Phe Asp Tyr Trp 1 5 10 <210> 148 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 148 Cys Ala Arg Glu Glu Asn Tyr Tyr Gly Thr Tyr Tyr Tyr Phe Asp Tyr Trp 1 5 10 15 <210> 149 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 149 Cys Ala Arg Trp Gly Asp His Asp Asp Ala Met Asp Phe Trp 1 5 10 <210> 150 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 150 Cys Ala Arg Asn His Gly Asp Gly Tyr Phe Asn Trp Tyr Phe Asp Val 1 5 10 15 Trp <210> 151 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 151 Cys Ala Arg Asn His Gly Asp Gly Tyr Tyr Asn Trp Tyr Phe Asp Val 1 5 10 15 Trp <210> 152 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 152 Cys Ala Arg Gly Thr Ala Trp Phe Thr Tyr Trp 1 5 10 <210> 153 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 153 Cys Ala Arg Asp Gly Asp Asp Gly Trp Asp Ile Asp Val Trp 1 5 10 <210> 154 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 154 Cys Ala Arg Arg Gly Thr Tyr Trp His Phe Asp Val Trp 1 5 10 <210> 155 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 155 Cys Ala Arg Arg Gly Ser Tyr Trp His Phe Asp Val Trp 1 5 10 <210> 156 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 156 Cys Ala Arg Arg Ser Thr Leu Val Phe Asp Tyr Trp 1 5 10 <210> 157 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 157 Cys Ala Arg Asn Gly Tyr Trp Tyr Phe Asp Val Trp 1 5 10 <210> 158 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 158 Cys Ala Arg Asn Gly Tyr Trp Tyr Phe Asp Val Trp 1 5 10 <210> 159 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 159 Cys Ala Lys Glu Gly Asp Tyr Asp Gly Thr Ala Tyr Phe Asp Tyr Trp 1 5 10 15 <210> 160 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 160 Cys Ala Arg Arg Arg Asp Gly Asn Phe Asp Tyr Trp 1 5 10 <210> 161 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 161 Cys Val Arg His Gly Tyr Phe Asp Val Trp 1 5 10 <210> 162 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 162 Cys Ala Phe Tyr Asp Gly Ala Tyr Trp 1 5 <210> 163 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 163 Cys Ala Ser Tyr Asp Pro Asp Tyr Trp 1 5 <210> 164 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 164 Cys Ala Arg Asp Thr Thr Ala Thr Tyr Tyr Phe Asp Tyr Trp 1 5 10 <210> 165 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 165 Cys Ala Arg Arg Val Ala Thr Tyr Phe Asp Val Trp 1 5 10 <210> 166 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 166 Cys Thr Arg Arg Ser His Ile Thr Leu Asp Tyr Trp 1 5 10 <210> 167 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 167 Cys Ala Arg Arg Arg Thr Thr Ala Phe Asp Tyr Trp 1 5 10 <210> 168 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 168 Cys Asn Asn Gly Asn Tyr Val Arg His Tyr Tyr Phe Asp Tyr Trp 1 5 10 15 <210> 169 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 169 Cys Thr Arg Arg Arg Glu Ile Thr Phe Asp Tyr Trp 1 5 10 <210> 170 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 170 Cys Ala Arg Ser Gly Ile Ser Pro Phe Thr Tyr Trp 1 5 10 <210> 171 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 171 Cys Ala Lys Tyr Asp Arg Phe Phe Ala Ser Trp 1 5 10 <210> 172 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 172 Ser Gln Gly Ile Ser Asn His Leu 1 5 <210> 173 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 173 Ser Gln Gly Ile Arg Asn Tyr Leu 1 5 <210> 174 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 174 Ser Gln Gly Ile Ser Asn His Leu 1 5 <210> 175 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 175 Ser Gln Gly Ile Asn Asn Tyr Leu 1 5 <210> 176 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 176 Ser Gln Gly Ile Ser Asn His Leu 1 5 <210> 177 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 177 Ser Gln Ser Val Asp His Asp Gly Asp Ser Tyr Met 1 5 10 <210> 178 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 178 Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met 1 5 10 <210> 179 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 179 Ser Gln Asp Ile Ser Asn Tyr Leu 1 5 <210> 180 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 180 Ser Gln Asp Ile Ser Thr Tyr Leu 1 5 <210> 181 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 181 Thr Ser Ser Ile Ser Ser Ser Tyr Leu 1 5 <210> 182 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 182 Asn Ser Ser Val Ser Ser Ser Tyr Leu 1 5 <210> 183 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 183 Thr Ser Ser Ile Ser Ser Ser Tyr Leu 1 5 <210> 184 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 184 Thr Ser Ser Ile Ser Ser Ser Tyr Leu 1 5 <210> 185 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 185 Ser Glu Asn Ile Tyr Tyr Asn Leu 1 5 <210> 186 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 186 Ser Glu Asn Ile Tyr Gly Tyr Phe 1 5 <210> 187 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 187 Ser Gln Asp Ile Asn Asn Tyr Ile 1 5 <210> 188 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 188 Ser Gln Asp Ile Asn Lys Tyr Ile 1 5 <210> 189 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 189 Ser Glu Asn Ile Tyr Ser Tyr Leu 1 5 <210> 190 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 190 Ser Gln Gly Ile Arg Asn Tyr Leu 1 5 <210> 191 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 191 Ser Gln Asp Val Arg Thr Asp Val 1 5 <210> 192 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 192 Ser Gln Asp Val Ile Thr Ala Val 1 5 <210> 193 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 193 Ser Gln Ser Ile Gly Thr Ser Ile 1 5 <210> 194 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 194 Ser Ser Gln Ser Leu Leu Asn Gln Lys Asn Tyr Leu 1 5 10 <210> 195 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 195 Ser Ser Ser Val Ser Ser Ser Tyr Leu 1 5 <210> 196 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 196 Ser Glu Asn Ile Tyr Tyr Asn Leu 1 5 <210> 197 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 197 Ser Gln Thr Ile Gly Thr Ser Ile 1 5 <210> 198 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 198 Ser Gln Ser Leu Leu Tyr Ser Ser Asp Gln Lys Asn Tyr Leu 1 5 10 <210> 199 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 199 Asn Ser Ser Val Ser Tyr Met 1 5 <210> 200 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 200 Ser Glu Asn Ile Tyr Tyr Asn Leu 1 5 <210> 201 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 201 Ser Ser Ser Leu Ser Tyr Me 1 5 <210> 202 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 202 Ser Gln Arg Ile Gly Thr Ser Met 1 5 <210> 203 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 203 Ser Gln Ser Ile Gly Thr Ser Ile 1 5 <210> 204 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 204 Ser Gln Asn Ile Gly Thr Ser Ile 1 5 <210> 205 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 205 Ile Ser Ser Val Ser Tyr Met 1 5 <210> 206 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 206 Ser Gln Thr Ile Ala Thr Ser Ile 1 5 <210> 207 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 207 Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu 1 5 10 <210> 208 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 208 Asn Glu Ser Val Glu Tyr Ser Gly Thr Ser Leu Met 1 5 10 <210> 209 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 209 Tyr Phe Thr Ser Ser 1 5 <210> 210 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 210 Tyr Phe Thr Ser Ser 1 5 <210> 211 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 211 Tyr Phe Thr Ser Ser 1 5 <210> 212 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 212 Tyr Tyr Thr Ser Ser 1 5 <210> 213 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 213 Tyr Phe Thr Ser Ser 1 5 <210> 214 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 214 Tyr Ala Ala Ser Asn 1 5 <210> 215 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 215 Tyr Ala Ala Ser Asn 1 5 <210> 216 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 216 Tyr Tyr Thr Ser Arg 1 5 <210> 217 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 217 Phe Tyr Thr Ser Arg 1 5 <210> 218 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 218 Tyr Gly Thr Ser Asn 1 5 <210> 219 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 219 Tyr Gly Thr Ser Asn 1 5 <210> 220 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 220 Tyr Gly Thr Ser Asn 1 5 <210> 221 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 221 Tyr Gly Thr Ser Asn 1 5 <210> 222 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 222 Tyr Asn Ala Asn Ser 1 5 <210> 223 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 223 Tyr Asn Ala Lys Thr 1 5 <210> 224 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 224 His Tyr Thr Ser Thr 1 5 <210> 225 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 225 His Tyr Thr Ser Thr 1 5 <210> 226 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 226 Tyr Asn Ala Lys Thr 1 5 <210> 227 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 227 Tyr His Thr Ser Thr 1 5 <210> 228 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 228 Tyr Ser Ala Ser Phe 1 5 <210> 229 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 229 Tyr Ser Ala Ser Tyr 1 5 <210> 230 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 230 Lys Ser Ala Ser Glu 1 5 <210> 231 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 231 Tyr Trp Ala Ser Thr 1 5 <210> 232 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 232 Tyr Ser Thr Ser Asn 1 5 <210> 233 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 233 Tyr Asn Ala Asn Ser 1 5 <210> 234 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 234 Lys Asn Ala Ser Glu 1 5 <210> 235 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 235 Tyr Trp Ala Ser Thr 1 5 <210> 236 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 236 Tyr Asp Thr Ser Lys 1 5 <210> 237 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 237 Tyr Asn Ala Asn Ser 1 5 <210> 238 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 238 Tyr Asp Thr Ser Asn 1 5 <210> 239 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 239 Lys Ser Ala Ser Glu 1 5 <210> 240 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 240 Lys Ser Ala Ser Glu 1 5 <210> 241 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 241 Lys Asp Ala Ser Glu 1 5 <210> 242 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 242 Tyr Ala Thr Ser Asn 1 5 <210> 243 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 243 Lys Asn Ala Ser Glu 1 5 <210> 244 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 244 Tyr Lys Val Ser Asn 1 5 <210> 245 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 245 Ser Ala Ala Ser Asn 1 5 <210> 246 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 246 Cys Gln Gln Tyr Ser Asn Leu Pro Tyr Thr Phe 1 5 10 <210> 247 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 247 Cys Gln Gln Tyr Ser Asn Leu Pro Tyr Thr Phe 1 5 10 <210> 248 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 248 Cys Gln Gln Tyr Ser Asn Leu Pro Tyr Thr Phe 1 5 10 <210> 249 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 249 Cys Gln Gln Tyr Ser Lys Ile Pro Tyr Thr Cys 1 5 10 <210> 250 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 250 Cys Gln Gln Tyr Ser Asn Leu Pro Tyr Thr Phe 1 5 10 <210> 251 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 251 Cys Gln Gln Asn Tyr Glu Asp Pro Thr Phe 1 5 10 <210> 252 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 252 Cys Gln Gln Ser Asn Glu Asp Pro Thr Phe 1 5 10 <210> 253 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 253 Cys Gln Gln Gly Asp Ala Leu Pro Trp Thr Phe 1 5 10 <210> 254 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 254 Cys Gln Gln Gly Asn Ser Leu Pro Phe Thr Phe 1 5 10 <210> 255 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 255 Cys Gln Gln Trp Ser Ser Arg Pro Pro Thr Phe 1 5 10 <210> 256 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 256 Cys Gln Gln Tyr Ser Gly Tyr Pro Leu Thr Phe 1 5 10 <210> 257 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 257 Cys Gln Gln Tyr Ser Asp Tyr Pro Leu Thr Phe 1 5 10 <210> 258 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 258 Cys Gln Gln Arg Ser Tyr Phe Pro Phe Thr Phe 1 5 10 <210> 259 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 259 Cys Lys Gln Val Tyr Asp Val Pro Phe Thr Phe 1 5 10 <210> 260 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 260 Cys Gln His His Tyr Gly Thr Pro Phe Thr Phe 1 5 10 <210> 261 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 261 Cys Leu Gln Tyr Asp Asn Leu Trp Thr Phe 1 5 10 <210> 262 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 262 Cys Leu Gln Tyr Asp Asn Leu Trp Thr Phe 1 5 10 <210> 263 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 263 Cys Gln His His Tyr Gly Tyr Pro Tyr Thr Phe 1 5 10 <210> 264 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 264 Cys Gln Gln Tyr Ser Asn Leu Pro Leu Thr Phe 1 5 10 <210> 265 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 265 Cys Gln Gln His Tyr Thr Ser Pro Trp Thr Phe 1 5 10 <210> 266 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 266 Cys Gln Gln His Tyr Ser Thr Pro Trp Thr Phe 1 5 10 <210> 267 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 267 Cys Gln Gln Ser Asn Arg Trp Pro Leu Thr Phe 1 5 10 <210> 268 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 268 Cys Gln Asn Asp Tyr Asp Tyr Pro Tyr Thr Phe 1 5 10 <210> 269 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 269 Cys His Gln Tyr His Arg Ser Pro Leu Thr Phe 1 5 10 <210> 270 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 270 Cys Gln Gln Thr Phe Asp Val Pro Trp Thr Phe 1 5 10 <210> 271 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 271 Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Tyr 1 5 10 <210> 272 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 272 Cys Gln Gln Tyr Tyr Asn Tyr Pro Leu Thr Phe 1 5 10 <210> 273 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 273 Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe 1 5 10 <210> 274 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 274 Cys Lys Gln Ala Tyr Asp Val Pro Trp Thr Phe 1 5 10 <210> 275 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 275 Cys Gln Gln Trp Ser Ser Phe Pro Pro Thr Phe 1 5 10 <210> 276 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 276 Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe 1 5 10 <210> 277 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 277 Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe 1 5 10 <210> 278 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 278 Cys Gln Gln Ser Asp Ser Trp Pro Leu Thr Phe 1 5 10 <210> 279 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 279 Cys Gln Gln Trp Ser Ser Asn Pro Arg Thr Phe 1 5 10 <210> 280 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 280 Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe 1 5 10 <210> 281 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 281 Cys Trp Gln Asn Thr His Phe Pro Gln Thr Phe 1 5 10 <210> 282 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Artificial sequences: description of synthetic peptides <400> 282 Cys Gln Gln Ser Arg Gln Val Pro Leu Thr Phe 1 5 10 BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 The diagram 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 lymphoblastoid cell line) and subsequently incubated with a fluorophore-conjugated anti-IgG antibody. Signals were detected by flow cytometry and are expressed as geometric mean fluorescence intensity (y-axis) versus anti-CD5 antibody concentration (x-axis).

[0076] Figure 2 The graphic depicts the results of an in vitro primary cell binding assay in which the indicated anti-CD5 antibodies (i.e., "CD55D7") or negative controls (i.e., hIgG1) were incubated with primary human T cells and subsequently incubated with fluorophore-conjugated anti-IgG antibodies. Signals were detected by flow cytometry and are expressed as geometric mean fluorescence intensity (y-axis) versus anti-CD5 antibody concentration (x-axis).

[0077] Figure 3A and Figure 3B The graph depicts the results of an in vitro T cell killing assay comprising an anti-CD5 amanitin ADC (i.e., 5D7-AM or "CD5 5D7 AM") having an 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 ).exist Figure 3AIn Figure , an analysis of anti-CD5-ADC T cell killing compared to unconjugated anti-CD5 5D7 antibody (ie, "naked CD5 5D7") is shown. Figure 3B In the figure, an anti-CD5-ADC T cell killing analysis is shown compared to an anti-CD5 5D7 antibody (i.e., CD5 5D7 D265C.H435AAM) with a H435A mutation that reduces the half-life of the antibody (i.e., "CD5 5D7 fast half-life AM"). The results show the number of surviving T cells (y-axis) as a function of ADC (CD5 5D7 AM, CD5 5D7 D265C.H435AAM) or non-conjugated antibody (naked CD5 5D7) concentration (x-axis), as assessed using flow cytometry.

[0078] Figure 4A-4B The graph depicts the results of an in vivo T cell depletion assay showing peripheral blood ( T cell depletion in vivo) of humanized NSG mice 7 days after a single administration of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of an anti-CD55D7 amanitin ADC with an interchain DAR of 6 (i.e., CD55D7-AM). Figure 4A ) and bone marrow ( Figure 4B ) in absolute levels of T cells (CD3+ cells; y axis). For comparison, Figure 4A-4B Also shown are the levels of T cell depletion following treatment of humanized NSG mice with the indicated controls (ie, 25 mg / kg anti-CD52 antibody, 3 mg / kg hIgG1-amanitin ADC (ie, hIgG1-AM), 25 mg / kg hIgG1, or PBS).

[0079] Figures 5A-5C The graph depicts the results of an in vivo T cell depletion assay showing peripheral blood ( Figure 5A ),marrow( Figure 5B ) and thymus ( Figure 5C ) in absolute levels of T cells (CD3+ cells; y axis). For comparison, Figures 5A-5C Also shown are the levels of T cell depletion following treatment of humanized NSG mice with 3 mg / kg of an unconjugated anti-CD5 antibody (ie, CD5 5D7) or with the indicated controls (ie, 3 mg / kg of hIgG1-amanitin ADC ("hIgG1-AM") or PBS).

[0080] Details

[0081] The present invention provides a method for promoting the acceptance of CAR-expressing immune cells (autologous or allogeneic) by human subjects receiving chimeric antigen receptor (CAR) therapy, the method being performed by administering an anti-CD5 antibody drug conjugate (ADC) to a patient receiving CAR therapy. The methods disclosed herein can be used to improve the acceptance of autologous or allogeneic immune cells (e.g., T cells) without relying on (or optionally reducing the use of) lymphodepleting chemotherapy that is commonly used as a conditioning therapy to reduce rejection of CAR-expressing immune cells.

[0082] I. Definitions

[0083] As used herein, the term "about" refers to a value within 5% above or below the stated value.

[0084] As used herein, the term "allogeneic", when used in the context of transplantation, is used to define cells (or tissues or organs) that are transplanted from a donor to a recipient of the same species, wherein the donor and recipient are not the same subject.

[0085] As used herein, the term "autologous" refers to cells or transplants where the donor and recipient are the same subject.

[0086] As used herein, the term "xenogeneic" refers to cells where the donor and recipient species are different.

[0087] As used herein, the term "immune cell" is intended to include, but is not limited to, cells of hematopoietic origin and that play a role in an immune response. Immune cells include, but are not limited to, T cells and natural killer (NK) cells. Natural killer cells are well known in the art. In one embodiment, natural killer cells include cell lines, such as NK-92 cells. Additional examples of NK cell lines include NKG cells, YT cells, NK-YS cells, HANK-1 cells, YTS cells, and NKL cells. Immune cells can be allogeneic or autologous.

[0088] "Engineered cell" means any cell of any organism modified, transformed or manipulated by adding a gene, DNA or RNA sequence or protein or polypeptide or modifying a gene, DNA or RNA sequence or protein or polypeptide. The separated cells, host cells and genetically engineered cells of the present disclosure include immune cells, such as NK cells and T cells, comprising a DNA or RNA sequence encoding CAR and expressing CAR on the cell surface. The separated host cells and engineered cells can be used, for example, to enhance NK cell activity or T lymphocyte activity, treat cancer, and treat autoimmune diseases. In embodiments, engineered cells include immune cells, such as T cells or natural killer (NK) cells.

[0089] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or immunoreacts with a specific antigen. Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided that they exhibit the desired antigen-binding activity.

[0090] Typically, antibodies comprise heavy and light chains containing antigen-binding regions. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

[0091] As used herein, the term "antigen binding fragment" refers to one or more portions of an antibody that retain the ability to specifically bind to a target antigen. The antigen binding function of an antibody can be performed by a fragment of a full-length antibody. An antibody fragment can be, for example, a Fab, F(ab')2, scFv, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb comprising the VH and VL domains; (vi) a dAb fragment consisting of the VH domain (see, e.g., Ward et al., Nature 341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) isolated complementarity determining regions (CDRs); and (ix) a combination of two or more (e.g., two, three, four, five or six) isolated CDRs, which may optionally be linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be connected by a linker using recombinant methods, which enables them to become a single protein chain, wherein the VL and VH regions are paired to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of these fragments can be screened in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA technology, enzymatic or chemical cleavage of intact immunoglobulins, or in some cases, by chemical peptide synthesis procedures known in the art.

[0092] As used herein, an "intact" or "full length" antibody refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds.

[0093] As used herein, the term "anti-CD5 antibody" or "antibody that binds to CD5" or "anti-CD5 ADC" or "ADC that binds to CD5" refers to an antibody or ADC that specifically binds to human CD5 when CD5 is present on the cell surface of a cell such as a T cell. The amino acid sequence of human CD5 to which the anti-CD5 antibody (or anti-CD5 ADC) will bind is described below in SEQ ID NO: 20.

[0094] As used herein, the term "specifically binds" refers to the ability of an antibody (or ADC) to recognize and bind to a specific protein structure (epitope) rather than to proteins in general. If the antibody is specific for epitope "A", then in a reaction containing labeled "A" and the antibody, the presence of molecules containing epitope A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody. By way of example, if the antibody can be competed away from its target by the corresponding unlabeled antibody when labeled, then the antibody "specifically binds" to the target. In one embodiment, if the antibody has at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or smaller (smaller means less than 10 -12 Number, such as 10 -13 ) D , then the antibody specifically binds to the target, such as CD5. In one embodiment, as used herein, the term "specific binding to CD5" or "specific binding to CD5" refers to binding to CD5 and having a specific binding affinity of 1.0×10 -7 M or less dissociation constant (K D ) as determined by surface plasmon resonance. In one embodiment, K D Determined by standard biolayer interferometry (BLI). However, it will be appreciated that an antibody may be able to specifically bind to two or more sequence-related antigens. For example, in one embodiment, an antibody may specifically bind to both a human ortholog and a non-human (e.g., mouse or non-human primate) ortholog of CD5.

[0095] The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies are obtained from a single clone, including any eukaryotic clone, prokaryotic clone, or phage clone, by any available or known means in the art. Monoclonal antibodies useful in the present disclosure can be prepared using a variety of techniques known in the art, including the use of hybridoma technology, recombinant technology, and phage display technology, or a combination thereof.

[0096] As used herein, the term "chimeric" antibody refers to an antibody having a variable sequence derived from a non-human immunoglobulin (such as a rat or mouse antibody) and a human immunoglobulin constant region (usually selected from a human immunoglobulin template). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719): 1202-7; Oi et al., 1986, BioTechniques 4: 214-221; Gillies et al., 1985, J. Immunol. Methods 125: 191-202; U.S. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397.

[0097] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins that contain minimal sequence derived from a non-human immunoglobulin. Typically, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are FR regions of human immunoglobulin sequences. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370 to Queen et al.; EP 239400; PCT Publication No. WO 91 / 09967; U.S. Patent No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Patent No. 5,565,332.

[0098] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide comprising at least one extracellular domain, a transmembrane domain and at least one intracellular signaling domain that can specifically bind to an antigen. Typically, CAR is a genetically engineered receptor that redirects the cytotoxicity of immune effector cells toward cells presenting a given antigen. CAR is a molecule that combines the specificity of antibodies based on the desired antigen (eg, tumor antigen) with the intracellular domain of the activated T cell receptor to produce a chimeric protein that exhibits specific cellular immune activity. In a specific embodiment, CAR comprises an extracellular domain (also referred to as a binding domain or an antigen-specific binding domain), a transmembrane domain and an intracellular (cytoplasmic) signaling domain. The engagement of the antigen binding domain of CAR with the target antigen on the surface of the target cell results in the aggregation of CAR, and the activation stimulus is delivered to the cell comprising CAR. The main feature of CAR is that it uses the cell-specific targeting ability of monoclonal antibodies, soluble ligands or cell-specific co-receptors to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or molecular production (capable of mediating cell death of cells expressing target antigens in a manner independent of major histocompatibility (MHC)). In some embodiments, CAR comprises an extracellular binding domain that specifically binds to a tumor antigen; a transmembrane domain and one or more intracellular signaling domains. In various embodiments, CAR comprises an extracellular binding domain, a transmembrane domain and one or more intracellular signaling domains that specifically bind to human CD5.

[0099] As used herein, the term "CAR therapy" refers to the administration of immune cells engineered to express CAR to human subjects to treat a given disease, such as cancer or autoimmune disease. CAR therapy refers to the specific treatment of patients with engineered immune cells, and is not intended to include therapies commonly used in conjunction with CAR cell therapy, such as lymphodepleting chemotherapy. It is worth noting that, unless otherwise stated, in the case of the term "cell" used throughout the text, the term also includes cell populations. For example, because CAR therapy requires the administration of engineered cell populations.

[0100] As used herein, the term "combination" or "combination therapy" refers to the use of two (or more) therapies in a single human patient. These terms are not intended to refer to the combined components. For example, a combination therapy comprising the administration of an anti-CD5 ADC and a CAR therapy is described herein.

[0101] The term "regulation" refers to preparing the patient who needs CAR therapy as a suitable condition. Regulation as used herein includes, but is not limited to, reducing the number of endogenous lymphocytes before T cell therapy, removing cytokine pools (sink), increasing the serum levels of one or more steady-state cytokines or proinflammatory factors, enhancing the effector function of the T cells used after regulation, enhancing the activation and / or availability of antigen presenting cells or any combination thereof.

[0102] In the context of the effect of an anti-CD5 antibody or ADC on cells expressing CD5, the term "depletion" refers to a decrease or elimination in the number of cells expressing CD5.

[0103] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to achieve a desired result or have an effect on an autoimmune disease or cancer.

[0104] As used herein, the terms "subject" and "patient" refer to an organism, such as a human, who is receiving treatment for a particular disease or condition as described herein.

[0105] As used herein, "to treat" or "treatment" refers to any improvement in disease outcomes, such as prolonged survival, less morbidity, and / or mitigation of side effects as a byproduct of alternative treatment modalities; as is readily understood in the art, complete elimination of the disease is preferred, but not a requirement for therapeutic behavior. Beneficial or desired clinical outcomes include, but are not limited to, promoting the acceptance of immune cells expressing CAR (allogeneic or autologous—both can cause an immune response in patients receiving CAR therapy). Insofar as the methods of the present invention are intended to prevent disorders, it should be understood that the term "prevention" does not require that the disease state be completely prevented. Rather, as used herein, the term prevention refers to the ability of a technician to identify a population susceptible to the disorder so that administration of the compounds of the present invention can be performed before the onset of the disease. The term does not mean that the disease state is completely avoided.

[0106] As used herein, the term "vector" includes nucleic acid vectors, such as plasmids, DNA vectors, plasmids, RNA vectors, viruses or other suitable replicons. The expression vectors described herein may contain polynucleotide sequences and, for example, other sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Some vectors that may be used to express CAR include plasmids containing regulatory sequences (such as promoters and enhancer regions) for guiding gene transcription. Other useful vectors for antibody or CAR expression include polynucleotide sequences that enhance the translation rate of these genes or improve the stability of mRNA produced by gene transcription or nuclear export. These sequence elements may include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to guide the effective transcription of genes carried on the expression vector. The expression vectors described herein may also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin and nourseothricin.

[0107] As used herein, the term "antibody drug conjugate" or "ADC" refers to an antibody connected to a cytotoxin. ADC is formed by chemical bonding of a reactive functional group of a molecule (such as an antibody or its antigen-binding fragment) to an appropriate reactive functional group of another molecule (such as a cytotoxin described herein). The conjugate can include a joint between two molecules bound to each other (e.g., between an antibody and a cytotoxin). It is worth noting that the term "conjugate" (when referring to a compound) is also referred to interchangeably herein as "drug conjugate" or "antibody drug conjugate" or "ADC". Examples of joints that can be used to form conjugates include peptide-containing joints, such as joints containing naturally occurring or non-naturally occurring amino acids such as D-amino acids. Joints can be prepared using a variety of strategies described herein and known in the art. Depending on the reactive components therein, the linker can be cleaved by, for example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0108] As used herein, the term "coupling reaction" refers to a chemical reaction in which two or more substituents suitable for reacting with each other react to form a chemical moiety that links together (e.g., covalently) the molecular fragments to which each substituent is bound. Coupling reactions include those in which a reactive substituent bound to a fragment that is a cytotoxin (such as a cytotoxin known in the art or described herein) reacts with a suitable reactive substituent bound to a fragment that is an antibody or antigen-binding fragment thereof (such as an antibody, an antigen-binding fragment thereof, or a specific anti-CD5 antibody that binds CD5 known in the art or described herein). Examples of suitable reactive substituents include nucleophiles / electrophiles pairs (e.g., especially thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs), dienes / dienophiles pairs (e.g., especially azide / alkyne pairs), and the like. Coupling reactions include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine condensation, amidation, esterification, disulfide formation, cycloadditions (such as, inter alia, [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reactive modes known in the art or described herein.

[0109] As used herein, the term "microtubule-binding agent" refers to a compound that acts by disrupting the microtubule network, which is essential for mitotic and interphase cell function in cells. Examples of microtubule-binding agents include, but are not limited to, maytansine, maytansine alkaloids and derivatives thereof, such as those described herein or known in the art, vinca alkaloids, such as vinblastine, vinblastine sulfate, vincristine, vincristine sulfate, vindesine and vinorelbine, taxanes, such as docetaxel and paclitaxel, macrolides, such as discodermolide, colchicine and epothilones and derivatives thereof, such as epothilone B or its derivatives.

[0110] As used herein, the term "amanitin" refers to a member of the amanitatoxin family of peptides produced by the Amanita phalloides mushroom or a derivative thereof, such as a variant or derivative thereof that is capable of inhibiting RNA polymerase II activity. Amanitatoxins useful for use in conjunction with the compositions and methods described herein include compounds such as, but not limited to, compounds of formula (II), for example, α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitinamide, amanitin, monohydroxyamanitin carboxylic acid, and proamanitin. Amanitin toxins can be isolated from a variety of mushroom species (e.g., Amanita phalloides, Galerina marginata, Lepiotabrunneo-incarnata), or can be prepared semisynthetically or synthetically. A member of this family, α-amanitin, is described in Wieland, Int. J. Pept. Protein Res. 1983, 22 (3): 257-276. Derivatives of amanita phalloides toxins can be obtained by chemical modification of naturally occurring compounds ("semi-synthesis"), or can be obtained from completely synthetic sources. Synthetic pathways for a variety of amanita phalloides toxin derivatives are disclosed in, for example, U.S. Patent No. 9,676,702 and Perrin et al., J. Am. Chem. Soc. 2018, 140, pp. 6513-6517, each of which is incorporated herein by reference in its entirety for the synthetic methods for preparing and deriving amanita phalloides toxins.

[0111] As described herein, an amanita toxin can be conjugated to an antibody or antigen-binding fragment thereof (thereby forming an ADC) via, for example, a linker moiety (L). The structures of exemplary amanita toxin-linker conjugates are represented by formula (III), formula (IIIA), and formula (IIIB). Exemplary methods of amanita toxin conjugation and linkers useful for such methods are described below. Also described herein are exemplary amanita toxins containing linkers that can be used for conjugation to antibodies or antigen-binding fragments according to the compositions and methods.

[0112] As used herein, the term "acyl" refers to -C(=O)R, wherein R is hydrogen ("aldehyde"), C1-C 12 Alkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, C3-C7 carbocyclic group, C6-C 20 Aryl, 5-10 membered heteroaryl or 5-10 membered heterocyclyl. Non-limiting examples include formyl, acetyl, propionyl, benzoyl and acryloyl.

[0113] As used herein, the term "C1-C 12"Alkyl" refers to a straight or branched chain saturated hydrocarbon having from 1 to 12 carbon atoms. Representative C1-C 12 Alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; and branched C1-C 12 Alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl and 2-methylbutyl. 12 Alkyl groups can be unsubstituted or substituted.

[0114] As used herein, the term "alkenyl" refers to a group comprising a hydrocarbon having at least one site of unsaturation (ie, a carbon-carbon sp 2 double bond) of a normal, secondary or tertiary carbon atom 12 Examples include, but are not limited to, ethylene or vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, etc. The alkenyl group may be unsubstituted or substituted.

[0115] As used herein, "alkynyl" refers to a C2-C6 group containing a normal, secondary, or tertiary carbon atom with at least one site of unsaturation (i.e., a carbon-carbon sp triple bond). 12 Examples include, but are not limited to, acetylenic and propargyl. Alkynyl groups can be unsubstituted or substituted.

[0116] As used herein, "aryl" refers to a C6-C 20 Carbocyclic aromatic groups. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. Aryl groups may be unsubstituted or substituted.

[0117] As used herein, "arylalkyl" refers to a group in which the carbon atom (typically a terminal carbon atom or sp 3 The alkyl radical of aryl group is substituted with 2- phenylethyl-1-yl, 2-phenylethane-1-yl, 2-naphthylethene-1-yl, naphthylbenzyl, 2-naphthylphenylethane-1-yl, etc. The arylalkyl group comprises 6 to 20 carbon atoms, and the alkyl moiety (comprising alkyl (alkanyl), alkenyl or alkynyl group) of arylalkyl group is 1 to 6 carbon atoms, and the aryl moiety is 5 to 14 carbon atoms. The alkaryl group can be unsubstituted or substituted.

[0118] As used herein, "cycloalkyl" refers to a saturated carbocyclic group, which may be monocyclic or bicyclic. Cycloalkyl groups include rings having 3 to 7 carbon atoms as monocyclic or 7 to 12 carbon atoms as bicyclic. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be unsubstituted or substituted.

[0119] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic group, which may be monocyclic or bicyclic. The cycloalkenyl group includes a ring having 3 to 6 carbon atoms as a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. Examples of monocyclic cycloalkenyl groups include 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl and 1-cyclohex-3-enyl. The cycloalkenyl group may be unsubstituted or substituted.

[0120] As used herein, "heteroaralkyl" refers to an alkyl radical in which the carbon atom (typically a terminal carbon atom or sp 3 Acyclic alkyl groups in which one hydrogen atom bonded to a heteroaryl group (carbon atom) is replaced by a heteroaryl group. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, etc. The heteroarylalkyl group contains 6 to 20 carbon atoms, for example, the alkyl portion of the heteroarylalkyl group (including alkyl, alkenyl or alkynyl groups) is 1 to 6 carbon atoms, and the heteroaryl portion is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S. The heteroaryl portion of the heteroarylalkyl group can be a monocyclic ring with 3 to 7 ring members (2 to 6 carbon atoms) or a bicyclic ring with 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example: bicyclic [4,5], [5,5], [5,6] or [6,6] systems.

[0121] As used herein, "heteroaryl" and "heterocycloalkyl" refer to an aromatic ring system or a non-aromatic ring system, respectively, in which one or more ring atoms are heteroatoms, such as nitrogen, oxygen and sulfur. The heteroaryl or heterocycloalkyl group contains 2 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S. The heteroaryl or heterocycloalkyl group can be a monocyclic ring with 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S) or a bicyclic ring with 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example: a bicyclic [4,5], [5,5], [5,6] or [6,6] system. Heteroaryl and heterocycloalkyl can be unsubstituted or substituted.

[0122] Heteroaryl groups and heterocycloalkyl groups are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, Aseries 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.

[0123] For example, examples of heteroaryl groups include, but are not limited to, pyridyl, thiazolyl, tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolinyl, quinolyl, isoquinolyl, benzimidazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl (4H-quinolizinyl), 2H-dole-2-yl, 2H-dole-3-yl, 2H-dole-4-yl, 2H-dole-5-yl, 2H-dole-6-yl, 2H-dole-7-yl, 2H-dole-8-yl, 2H-dole-9-yl, 2H-dole-10-yl, 2H-dole-21-yl, 2H-dole-12-yl, 2H-dole-13-yl, 2H-dole-14-yl, 2H-dole-15-yl

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

[0124] For instance, examples of heterocycloalkyl include, but are not limited to, dihydropyridinyl, tetrahydropyridinyl (piperidyl), tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidinonyl, tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, piperazinyl, quinuclidinyl, and morpholinyl.

[0125] For example, but not limited to, carbon-bonded heteroaryl and heterocycloalkyl are bonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or 6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2, 3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, position 2, 4, or 5 of an oxazole, imidazole, or thiazole, position 3, 4, or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of an aziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6, 7, or 8 of a quinoline, or position 1, 3, 4, 5, 6, 7, or 8 of an isoquinoline. Still more typically, the carbon-bonded heterocycle includes 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl or 5-thiazolyl.

[0126] For example, but not limited to, nitrogen-bonded heteroaryl and heterocycloalkyl are bonded at position 1 of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, dihydroindoline, 1H-indazole, position 2 of isoindole or isoindoline, position 4 of morpholine, and position 9 of carbazole or β-carboline. Still more typically, nitrogen-bonded heterocycles include 1-aziridinyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0127] "Substituted" as used herein and applied to any of the above alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, etc. means that one or more hydrogen atoms are each independently replaced by a substituent. Unless otherwise limited by the definition of an individual substituent, the aforementioned chemical moieties, such as "alkyl", "alkylene", "heteroalkyl", "heteroalkylene", "alkenyl", "alkenylene", "heteroalkenyl", "heteroalkenylene", "alkynyl", "alkynylene", "heteroalkynyl", "heteroalkynylene", "cycloalkyl", "cycloalkylene", "heterocycloalkyl", "heterocycloalkylene", "aryl", "arylene", "heteroaryl" and "heteroarylene" groups may be optionally substituted.

[0128] Typical substituents include, but are not limited to, -X, -R, -OH, -OR, -SH, -SR, NH2, -NHR, -N(R)2, -N +(R)3, -CX3, -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO2, -N3, -NC(=O)H, -NC(=O)R, -C(=O)H, -C(=O)R, -C(=O)NH2, -C(=O)N (R)2, -SO3-, -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NH2, -S(=O)2N(R)2, -S(=O)R, -OP(=O)(OH)2, -OP(=O)(OR)2, -P( =O)(OR)2, -PO3, -PO3H2, -C(=O)X, -C(=S)R, -CO2H, -CO2R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NH2, -C(=O)N(R)2, -C(=S)NH2, -C(=S)N(R)2, -C(=NH)NH2, and -C(=NR)N(R)2; wherein for each occurrence each X is independently selected from F, Cl, Br, and I; and for each occurrence each R is independently selected from Cl-C 12 Alkyl, C6-C 20 Aryl, C3-C 14 Heterocycloalkyl or heteroaryl, protecting groups and prodrug moieties. In all cases where a group is described as "optionally substituted", the group may, independently for each occurrence, be substituted with one or more of the above substituents.

[0129] It is understood that certain group naming conventions may include monovalent groups or divalent groups, depending on the context. For example, where a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a divalent group. For example, a substituent of an alkyl group identified as requiring two points of attachment includes a divalent group, such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc. Other group naming conventions explicitly indicate that the group is a divalent group, such as "alkylene", "alkenylene", "arylene", "heterocycloalkylene", etc.

[0130] In all cases where a substituent is described as a diradical (ie, having two points of attachment to the rest of the molecule), it is understood that the substituent may be attached in any orientation configuration unless otherwise specified.

[0131] "Isomeric" means compounds that have the same molecular formula but differ in the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed "stereoisomers." Stereoisomers that are not mirror images of one another are termed "diastereoisomers," and stereoisomers that are non-superimposable mirror images of one another are termed "enantiomers" or sometimes "optical isomers."

[0132] A carbon atom bonded to four non-identical substituents is called a "chiral center." A "chiral isomer" means a compound having at least one chiral center. Compounds having more than one chiral center may exist as individual diastereomers or as a mixture of diastereomers (called a "diastereomeric mixture"). When one chiral center is present, the stereoisomers may be characterized by the absolute configuration (R or S) of that chiral center. The absolute configuration refers to the spatial arrangement of the substituents attached to the chiral center in question. The substituents attached to the chiral center in question are ordered according to the sequence rules of Cahn, Ingold, and Prelog. (Cahn et al., Angew. Chem. Inter. ed. 1966, 5, 385; corrigendum 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116.) A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is called a "racemic mixture".

[0133] The compounds disclosed in this specification and claims may contain one or more asymmetric centers, and different diastereomers and / or enantiomers may exist for each compound. Unless otherwise stated, the description of any compound in this specification and claims is intended to include all enantiomers, diastereomers and mixtures thereof. In addition, unless otherwise stated, the description of any compound in this specification and claims is intended to include both a single enantiomer and any racemic or other mixtures of enantiomers. When the structure of a compound is described as a specific enantiomer, it should be understood that the invention of the present application is not limited to the specific enantiomer. Accordingly, enantiomers, optical isomers and diastereomers of each structural formula of the present disclosure are considered herein. In this specification, for convenience, the structural formula of a compound represents a certain isomer in some cases, but the present disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, etc., and it should be understood that not all isomers can have the same activity level. Compounds may exist in different tautomeric forms. Unless otherwise stated, compounds according to the present disclosure are intended to include all tautomeric forms. When the structure of a compound is described as a specific tautomer, it should be understood that the invention of the present application is not limited to this specific tautomer.

[0134] Compounds of any formula described herein include the compounds themselves, and if applicable, include their salts and their solvates. For example, salts can be formed between anions and positively charged groups (e.g., amino) on the compounds of the present disclosure. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, mesylate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, toluenesulfonate, salicylate, lactate, naphthylidenesulfonate and acetate (e.g., trifluoroacetate). The term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Similarly, salts can also be formed between negatively charged groups (e.g., carboxylates) on cations and compounds of the present disclosure. Suitable cations include sodium, potassium, magnesium, calcium and ammonium cations, such as tetramethylammonium ions. Examples of some suitable substituted ammonium ions are those derived from ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and amino acids such as lysine and arginine. Compounds of the present disclosure also include those salts containing quaternary nitrogen atoms.

[0135] Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid and phosphorous acid. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0136] Additionally, the compounds of the present disclosure, for example, salts of the compounds, may exist in hydrated or non-hydrated (anhydrous) forms, or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc. "Solvate" means a solvent addition form containing a stoichiometric or non-stoichiometric amount of solvent. Some compounds have a tendency to capture a fixed molar ratio of solvent molecules in a crystalline solid state, thereby forming a solvate. If the solvent is water, the solvate formed is a hydrate; and if the solvent is an alcohol, the solvate formed is an alcoholate. Hydrates are formed by combining one or more water molecules with a substance molecule, wherein the water maintains its molecular state as H2O. Hydrates refer to, for example, monohydrates, dihydrates, trihydrates, etc.

[0137] In addition, the compounds represented by the formula disclosed herein or salts thereof may exist in crystal polymorphism. It should be noted that any crystal form, mixture of crystal forms or anhydrides or hydrates thereof are included within the scope of the present disclosure.

[0138] The following sections provide a description of methods based on administering an anti-CD5 ADC to a human patient to promote uptake of CAR-expressing immune cells in CAR therapy.

[0139] II. Therapeutic approaches with anti-CD5 ADC and CAR

[0140] One challenge of chimeric antigen receptor (CAR) therapy is to determine the means by which engineered cells expressing CAR, such as CAR-T cells, can be accepted by human recipients. Such acceptance of engineered immune cells can affect the efficacy of the treatment and the outcome of adverse side effects to the patient.

[0141] Lympho-depleting chemotherapy is a traditional method of suppressing the recipient's immune system to improve acceptance, but it often has adverse side effects. Methods for promoting acceptance of immune cells expressing (CAR) in human patients undergoing CAR therapy are described herein. The methods described herein specifically target CD5+ cells such as CD5+T cells in human patients undergoing CAR therapy and eliminate CD5+ cells. The methods disclosed herein are more targeted than lympho-depleting chemotherapy and provide a means by which autologous cells or allogeneic cells can be used.

[0142] Described herein is the use of anti-CD5 antibody drug conjugates (ADC) to deplete CD5-specific immune cell populations in patients receiving CAR therapy in order to promote the acceptance and efficacy of CAR-expressing immune cells. This selective consumption of cells that specifically express CD5 of the immune system improves the overall survival rate and recurrence-free survival rate of patients, while reducing the risk of rejection of CAR-expressing immune cells for the treatment of autoimmune disorders or cancer.

[0143] After administering CAR cell therapy, the risk of rejection of CAR-expressing immune cells is still high. The methods and compositions disclosed herein can be used to inhibit or prevent rejection of CAR cells in human patients. Anti-CD5 ADC can be used to selectively target activated T cells in patients who will receive CAR cell therapy. Anti-CD5 ADC as described herein can also be used to reduce the risk of CAR cell rejection by targeting and depleting CD5-positive cells in human patients who have received CAR therapy.

[0144] The compositions and methods described herein can be used to deplete CD5+ cells, such as T cells, associated with CAR cell therapy rejection. The method of the present invention promotes acceptance of CAR-expressing immune cells by human subjects (e.g., human subjects with cancer or autoimmune diseases). In one embodiment, the method includes administering an anti-CD5 antibody drug conjugate (ADC) to a human subject who will or has undergone CAR therapy, and administering a therapeutically effective amount of CAR-expressing immune cells to a human subject.

[0145] Anti-CD5 ADC can be applied to human patients with corresponding needs before the administration of one or more CAR cell therapies, simultaneously with the administration of one or more CAR cell therapies, or after the administration of one or more CAR cell therapies. In one embodiment, anti-CD5 ADC is applied to human patients with corresponding needs before the administration of CAR cell therapy (for example, about 3 days before the administration of CAR cell therapy, about 2 days before the administration of CAR cell therapy, about 12 hours before the administration of CAR cell therapy). Single doses of anti-CD5 ADC can be applied to human patients before the administration of CAR cell therapy, after the administration of CAR cell therapy, or simultaneously with the administration of CAR cell therapy, wherein such a single dose is sufficient to prevent the consumption of immune cells expressing CAR or reduce the risk of immune cell consumption expressing CAR. In one embodiment, anti-CD5 ADC is applied to human patients with corresponding needs about 3 days before the administration of CAR cell therapy. In one embodiment, anti-CD5 ADC is applied to human patients with corresponding needs about 2 days before the administration of CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need about 1 day before the administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need about 20 hours before the administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need about 18 hours before the administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need 15 hours before the administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need about 12 hours before the administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need about 6 hours before the administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need about 4 hours before the administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need about 2 hours before the administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need simultaneously with the administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need about 2 hours after the administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need about 4 hours after the administration of the CAR cell therapy. In one embodiment, the anti-CD5 ADC is administered to a human patient in need about 6 hours after the administration of the CAR cell therapy.In one embodiment, the anti-CD5 ADC is administered to a human patient in need thereof about 12 hours after administration of the CAR cell therapy.

[0146] In some embodiments, the anti-CD5 ADC can be administered up to about 21 days prior to administration of one or more CAR cell therapies, such as about 21 days, about 20 days, about 19 days, about 18 days, about 17 days, about 16 days, about 15 days, about 14 days, about 13 days, about 12 days, about 11 days, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day, about 24 hours, about 12 hours, about 6 hours, about 3 hours, about 2 hours, or about 1 hour prior to administration of one or more CAR cell therapies.

[0147] In some embodiments, the anti-CD5 ADC can be administered about 12 hours after the administration of the CAR cell therapy, for example, about 12 hours, about 11 hours, about 10 hours, about 9 hours, about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour after the administration of one or more CAR cell therapies. In some embodiments, the anti-CD5 ADC can be administered about 10 days after the administration of the CAR cell therapy, for example, about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day after the administration of one or more CAR cell therapies.

[0148] In one embodiment, the anti-CD5 ADC is administered before the immune cells expressing CAR are administered to human patients in need thereof. In one embodiment, the anti-CD5 ADC is administered to human patients in combination with CAR therapy, wherein the anti-CD5 ADC is administered to human subjects about 12 hours to about 21 days before the administration of the immune cells expressing CAR. In one embodiment, the anti-CD5 ADC is administered to human patients in combination with CAR therapy, wherein the anti-CD5 ADC is administered to human subjects about 18 hours to about 20 days before the administration of the immune cells expressing CAR. In one embodiment, the anti-CD5 ADC is administered to human patients in combination with CAR therapy, wherein the anti-CD5 ADC is administered to human subjects about 20 hours to about 18 days before the administration of the immune cells expressing CAR. In one embodiment, the anti-CD5 ADC is administered to human patients in combination with CAR therapy, wherein the anti-CD5 ADC is administered to human subjects about 1 day to about 15 days before the administration of the immune cells expressing CAR. In one embodiment, the anti-CD5 ADC is administered to a human patient in combination with a CAR therapy, wherein the anti-CD5 ADC is administered to the human subject about 1 day to about 10 days prior to the administration of the CAR-expressing immune cells. In one embodiment, the anti-CD5 ADC is administered to a human patient in combination with a CAR therapy, wherein the anti-CD5 ADC is administered to the human subject about 2 days to about 8 days prior to the administration of the CAR-expressing immune cells. In one embodiment, the anti-CD5 ADC is administered to a human patient in combination with a CAR therapy, wherein the anti-CD5 ADC is administered to the human subject about 3 days to about 6 days prior to the administration of the CAR-expressing immune cells.

[0149] The total level of T cells in biological samples from human patients can be tested after the administration of anti-CD5 ADC, wherein the total number of T cells after the administration of anti-CD5 ADC in human patients is relative to the reduction of the level before the administration, indicating the efficacy of anti-CD5 ADC for preventing CAR cell therapy rejection. In one embodiment, the level of endogenous T cells in biological samples from human patients is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20% relative to the level of T cells in biological samples (same type, such as blood) from human patients before the administration of anti-CD5 ADC. In one embodiment, the level of endogenous T cells in biological samples from human patients is reduced by about 5% to 25%, about 5% to 20%, about 5% to 15% or about 5% to 10% relative to the level of T cells in biological samples (same type, such as blood) from human patients before the administration of anti-CD5 ADC. In one embodiment, the level of endogenous T cells is determined one day or less before the administration of anti-CD5 ADC.

[0150] The level of T cells can be determined according to standard methods known in the art including, but not limited to, fluorescence activated cell sorting (FAC) analysis or a blood analyzer.

[0151] As described above, one advantage of the method described herein is that the amount of lymphatic depletion chemotherapeutic agents can be reduced or lymphatic depletion chemotherapeutic agents are not included in the regulation regimen for human patients receiving or planning to receive CAR therapy. Lymphatic depletion chemotherapeutic agents, such as but not limited to fludarabine, cyclophosphamide, bendamustine and / or pentostatin, are generally used as anti-rejection agents to promote acceptance of CAR-expressing cells by humans receiving CAR therapy. In certain embodiments, anti-CD5 ADC is administered to human patients in a manner combined with the administration of CAR-expressing immune cells (e.g., T cells) (e.g., before the administration of CAR-expressing immune cells (e.g., T cells)), so that human patients do not receive lymphatic depletion chemotherapeutic agents, e.g., fludarabine and / or cyclophosphamide, before the administration of CAR-expressing immune cells, simultaneously with the administration of CAR-expressing immune cells, or after the administration of CAR-expressing immune cells.

[0152] By using anti-CD5 ADC as an agent for consuming endogenous immune cells in human subjects and reducing the risk of rejection of immune cells expressing CAR, the use of other immune depletion agents can also be avoided or reduced. For example, alemtuzumab is generally used as an anti-rejection agent in combination with CAR therapy to promote acceptance of CAR-expressing cells by humans receiving CAR therapy. In certain embodiments, anti-CD5 ADC is administered to human patients in a manner combined with the administration of immune cells (e.g., T cells) expressing CAR (e.g., before the administration of immune cells (e.g., T cells) expressing CAR), so that human patients do not receive alemtuzumab before the administration of immune cells expressing CAR, simultaneously with the administration of immune cells expressing CAR, or after the administration of immune cells expressing CAR.

[0153] In certain embodiments, anti-CD5 ADC is used in combination with another therapy to promote tolerance of immune cells expressing CAR. For example, anti-CD2 ADC can also be administered to human patients before they receive CAR therapy. Anti-CD2 ADC can be administered before, simultaneously with, or after anti-CD5 ADC, anti-CD2 ADC and anti-CD5 ADC, wherein both anti-CD2 ADC and anti-CD5 ADC are administered to human patients before CAR therapy.

[0154] The methods disclosed herein can be used for both autologous cells and allogeneic cells expressing CAR. Importantly, the anti-CD5 ADC regulation methods described herein can be used to expand the types of immune cells that can be used in CAR therapy by providing a method by which allogeneic cell tolerance can be provided. In one embodiment, the immune cells expressing CAR are allogeneic cells or autologous cells. Examples of immune cell types that can be engineered to express CAR include, but are not limited to, allogeneic T cells, autologous T cells, autologous NK cells, or allogeneic NK cells.

[0155] In one embodiment, the anti-CD5 antibody drug conjugate is used to deplete donor cells expressing CD5, e.g., activated T cells expressing CD5, by administering the anti-CD5 antibody drug conjugate after administration of the CAR cell therapy. In one embodiment, the CAR cell therapy includes allogeneic cells.

[0156] The methods disclosed herein are particularly useful for treating cancer or an autoimmune disease in a human subject having one of these disorders.

[0157] In one embodiment, the methods disclosed herein are used to treat cancer. More particularly, an anti-CD5 ADC is administered in combination with a CAR therapy to a human subject with cancer. Examples of cancer types that can be treated using the methods disclosed herein include, but are not limited to, adult advanced cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple-negative breast cancer, hematopoietic / lymphoid cancer, colon cancer liver metastasis, small cell lung cancer, non-small cell lung cancer, B cell lymphoma, relapsed or refractory B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, relapsed or refractory diffuse large cell lymphoma B-cell lymphoma, anaplastic large cell lymphoma, primary mediastinal B-cell lymphoma, relapsed mediastinal large B-cell lymphoma, refractory mediastinal large B-cell lymphoma, large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, colorectal epithelial carcinoma, gastric cancer, pancreatic cancer, triple-negative invasive breast cancer, renal cell carcinoma , squamous cell carcinoma of the lung, hepatocellular carcinoma, urothelial carcinoma, leukemia, B-cell leukemia, B-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, multiple myeloma of the bone, malignant glioma of the brain, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasms, blastic plasmacytoid dendritic cell tumor, liver metastasis, solid tumors, advanced solid tumors, mesothelin-positive tumors, hematological malignancies, and other advanced malignancies.

[0158] In one embodiment, the methods disclosed herein are used to treat autoimmune diseases. More particularly, anti-CD5 ADCs are administered in combination with CAR therapy to human subjects suffering from autoimmune diseases. Examples of autoimmune diseases that can be treated using the combined methods disclosed herein include, but are not limited to, multiple sclerosis, Crohn's disease, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, lupus, and psoriasis.

[0159] In certain embodiments, an anti-CD5 ADC is administered to a human patient in combination with a CAR-T cell therapy. In one embodiment, an anti-CD5 ADC is administered to a human patient prior to administration of the CAR-T therapy. Examples of CAR-T cells that can be used in combination with the anti-CD5 ADC therapies described herein include, but are not limited to, CD19 CAR-T (e.g., CART-19-01, 02, 03 (Fujian Medical University); daopeicart (Hebei Senlang Biotechnology Inc.); IM19CART / 001, YMCART201702 (Beijing Immunochina Medical Science & Technology Co.); CART-CD19-02, 03 (Wuhan Sian Medical Technology Co.); Universal CD19-CART / SHBYCL001, 002 (Shanghai Bioray Laboratory Inc.); UnicarTherapy201701 (Shanghai Unicar-Therapy Biomedicine Technology Co.); Genechem / NCT02672501 (Shanghai GeneChem Co.); SenL-19 (Hebei Senlang Biotechnology Inc.); PCAR-019 (PersonGenBioTherapeutics (Suzhou); ICAR19 (Immune Cell, Inc.); WM-CART-02 (Sinobioway CellTherapy Co.); HenanCH080,109,152 (Henan Cancer Hospital / The Pregene (ShenZhen) Biotechnology Co.); IM19-CD28 and IM19-41BB CAR-T cells (Beijing Immunochina Medical Science & Technology Company); CTL019 / IT1601-CART19 (Beijing Sanwater Biological Technology Co.);CTL019 / CCTL019C2201 (Novartis Pharmaceuticals); CD19:4-1BB:CD28:CD3 / FirstShenzhen01 (Shenzhen Second People’s Hospital / The Beijing Pregene Science and Technology Company); MB-CART19.1 (Shanghai Children’s Medical Center / Miltenyi Biotec GmbH); PZ01 CAR-T cells (Pinze Lifetechnology Co.); YMCART201701 (Beijing Immunochina Medical Science & Technology Co.); 2016YJZ12 (Peking University / Marino Biotechnology Co.); EGFRt / 19-28z / 4-1BBL CAR T cells (Memorial Sloan Kettering Cancer Center / Juno Therapeutics, Inc.); Doing-002 (Beijing Doing Biomedical Co.); PCAR-019 (PersonGenBioTherapeutics(Suzhou)Co.); C-CAR011 (Peking Union Medical College Hospital / Cellular Biomedicine Group Ltd.); iPD1 CD19 eCAR T cells (Peking University / Marino Biotechnology Co.); 2013-1018 / NCT02529813 (M.D. Anderson Cancer Center / Ziopharm / Intrexon Corp.); HenanCH CAR 2-1 (Henan Cancer Hospital / The Pregene(ShenZhen)Biotechnology Co.); JCAR015 (Juno Therapeutics, Inc.); JCAR017 / 017001,004,006 (Juno Therapeutics, Inc.); JCAR017 (Celgene); TBI-1501 (Takara Bio Inc.); JMU-CD19CAR (Jichi Medical University); KTE-C19 (Kite, A Gilead Company); TriCAR-T-CD19 (Timmune Biotech Inc.); PF-05175157 (Fred Hutchinson Cancer ResearchCenter)); CD22 / CD30 / CD7 / BCMA / CD123 (for example, 2016040 / NCT03121625 (Hebei Senlang Biotechnology Inc.)); CD22 (for example, Ruijin-CAR-01 (Ruijin Hospital / Shanghai Unicar-Therapy Bio-medicine Technology Co.); AUTO-PA1, DB1 (Autolus Limited)), CD20 (e.g., Doing-006 (Beijing Doing Biomedical Co.)) or CD20 / CD22 / CD30 (e.g., SZ5601 (TheFirst Affiliated Hospital of Soochow University Shanghai / Unicar-Therapy Bio-medicine Technology Co.)). .

[0160] Chimeric Antigen Receptor (CAR)

[0161] The present invention includes the use of CAR therapy in combination with anti-CD5 immunosuppressive ADC. The present invention is generally not limited to a specific CAR construct, such as a specific antigen binding region or intracellular signaling domain, because the present invention is based at least in part on the discovery that anti-CD5 ADC can promote the acceptance of CAR-expressing cells by eliminating endogenous CD5+ immune cells, such as endogenous T cells, thereby serving as a regulator of CAR therapy. Specific CARs, such as CD19-specific CARs, are contemplated herein and are included in the methods disclosed herein, but are not intended to be limited.

[0162] CAR constructs are known in the art and typically include (a) an extracellular region including an antigen binding domain, (b) a transmembrane domain, and (c) a cytoplasmic signaling domain. Exemplary CAR configurations are known in the art, and any suitable configuration can be used in the methods described herein. For example, CAR can be a first generation CAR, a second generation CAR, or a third generation CAR, for example, as described in: Guedan et al. Molecular Therapy-Methods & Clinical Development. 12: 145-156 (2019) or Sadelain et al. Cancer discovery 3.4: 388-398 (2013), the entire contents of which are incorporated herein by reference. In short, a "first generation" CAR may include (a) an extracellular antigen binding domain, (b) a transmembrane domain, (c) one or more intracellular signaling domains, and optionally (d) a hinge region connecting an antigen binding domain to a transmembrane domain. "Second generation" CAR may include elements (a), (b), (c) and optionally (d), and also include a costimulatory domain, such as a costimulatory domain of CD28 or 4-1BB. "Third generation" CAR may include elements (a), (b), (c) and optionally (d), and also include more than one costimulatory domain, such as a costimulatory domain of CD28 and 4-1BB, or a costimulatory domain of CD28 and OX40. Each of the above elements is described in detail below. It should be understood that in some embodiments, the CAR molecules described by the following exemplary non-limiting arrangements are from left to right, N-terminal to C-terminal of CAR. CAR as described in the present disclosure may include or also include any other combination of elements as described herein.

[0163] The CAR used in the methods disclosed herein may include an extracellular antigen binding domain. The extracellular antigen binding domain may be any molecule that binds to an antigen, including but not limited to human antibodies, humanized antibodies, or any functional fragments thereof. In certain embodiments, the antigen binding domain is a scFv. In other embodiments, the extracellular antigen binding domain is a non-immunoglobulin scaffold protein. In other embodiments, the extracellular binding domain of CAR includes a single-chain T cell receptor (scTCR). As described in U.S. Patents Nos. 5,359,046, 5,686,281, and 6,103,521, the extracellular domain may also be obtained from any of a variety of extracellular domains or secreted proteins associated with ligand binding and / or signal transduction.

[0164] The selection of the molecular target (antigen) of the extracellular binding domain depends on the type and number of the ligands defining the target cell surface. For example, antigen binding domains can be selected to identify the ligands acting as cell surface markers on target cells related to a specific disease state. Therefore, on the one hand, CAR-mediated immune cells (such as T cells) can be directed to the antigen of interest by engineering the extracellular antigen binding domains specifically binding to the desired antigen into the CAR. For example, antigen binding domains can be selected to identify the ligands acting as cell surface markers on target cells related to a specific disease state (such as cancer or autoimmune diseases). Therefore, examples of cell surface markers that can serve as ligands of the antigen binding domains in CAR include those associated with cancer cells and other forms of diseased cells (such as autoimmune disease cells and pathogen-infected cells). In some embodiments, by engineering the desired antigen binding domains specifically bound to the antigen on tumor cells, CAR is engineered to target tumor antigens of interest. In the context of the present invention, "tumor antigens" refer to antigens common to specific hyperproliferative disorders such as cancer. In one embodiment, the antigen is a tumor antigen, examples of which include, but are not limited to, CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or CD123. In one embodiment, the CAR comprises a scFv that binds to CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or CD123.

[0165] On the other hand, the extracellular binding domain of CAR binds to an antigen that is: AFP (e.g., ETCH17AFPCAR01 (Aeon Therapeutics (Shanghai) Co. / Eureka Therapeutics Inc.)), GPC3 (e.g., GeneChem GPC-3CART (Shanghai GeneChem Co.); 302GPC3-CART (ShanghaiGeneChem Co.); CAR-T for liver cancer (Shanghai GeneChem Co.); CAR-GPC3 T cells (CarsgenTherapeutics)), MUC1 (e.g., PG-021-001,002 (PersonGen BioTherapeutics (Suzhou) Co.)), mesothelin (e.g., H2017-01-P01 (Ningbo Cancer Hospital); TAI-meso-CART (ShanghaiGeneChem Co.); K16-4 / NCT02930993 (China Meitan General Hospital / MarinoBiotechnology Co.)), CD38 (e.g., Anti-CD38 A2 CAR-T / SOR-CART-MM-001 (SorrentoTherapeutics, Inc.)), herinCAR-PD1 (e.g., herinCAR-PD1 / NBWYKY2016-06-001, 002, 003 (Ningbo Cancer Hospital); SIMC-20160101, 02, 03 (Shanghai International Medical Center)), BCMA (e.g., P-BCMA-101 autologous memory T stem cell (Tscm) CAR-T cells / P-BCMA-101-001 (Poseida Therapeutics, Inc.); HenanCH284 (Henan Cancer Hospital / The Pregene (ShenZhen) Biotechnology Company); LCAR-B38MCAR-T cells (Nanjing Legend BiotechCo.); 9762 / NCT03338972 (Fred Hutchinson Cancer Research Center / JunoTherapeutics, Inc.); Descartes-08 (Cartesian Therapeutics); KITE-585 (Kite, AGilead Company); bb21217 (bluebird bio); bb21217 (Celgene); JCARH125 (JunoTherapeutics, Inc.)), CD30 (e.g., ICAR30 T cells (Immune) Cell, Inc.)), EGFR (for example, EGFR:4-1BB:CD28:CD3 modified T cells / First Shenzhen02 (Shenzhen Sceond People's Hospital / The Beijing Pregene Science and Technology Company); EGFR-IL12-CART (ShenzhenSecond People's Hospital / The Pregene (ShenZhen) Biotechnology Co.); SBNK-2016-015-01 (Beijing Sanbo Brain Hospital / Marino Biotechnology Co.)), MG7 (e.g., MG7-CART (Xijing Hospital / Shanghai GeneChem Co.)), BCMA / TACI (e.g., AUTO2-MM1 (Autolus Limited)), CEA (e.g., 383-74 / NCT02416466 (Roger Williams Medical Center / Sirtex Medical)), mesothelin / PSCA / CEA / HER2 / MUC1 / EGFRvIII (e.g., NCT03267173 (First Affiliated Hospital of Harbin Medical University / Shanghai Unicar-Therapy Bio-medicine Technology Co.)), CD20 (e.g., EY201605-19 (Beijing Biohealthcare Biotechnology Co.)), CD33 (e.g., 2016-0341 / NCT03126864 (MD Anderson Cancer Center / Intrexon Corp. / Ziopharm)), EGFR / BCMA (e.g., EGFRt / BCMA-41BBz CAR T cells (Memorial Sloan Kettering Cancer Center / Juno Therapeutics, Inc.)), ROR2 (e.g., autologous CCT301-38 or CCT301-59 T cells (Shanghai Sinobioway Sunterra Biotech)), NKR-2 (e.g., CYAD-N2T-002, 003, 004 (Celyad)), PSCA (e.g., BP-012 (Bellicum Pharmaceuticals)), CD28 (e.g., autologous CSR T cells (Beijing Sanbo Brain Hospital / Marino Biotechnology Co.)), TAA (e.g., AMG 119 (Amgen)), NKG2D (e.g., CM-CS1 (Celyad)) or CD123 (e.g., UCART123 (Cellectis SA)). The preceding sentence also provides examples of CARs that bind to the antigen (e.g., AMG119 (Amgen)). These CAR constructs can be used with anti-CD5 ADCs in the modulation methods disclosed herein. .

[0166] The CAR construct may also include a transmembrane domain that connects the extracellular antigen binding domain to the signaling domain (either literally or generally close, such as with a spacer). In some embodiments, the extracellular antigen binding domain of CAR (e.g., scFv, Fab or other antigen binding moieties) can be connected to the transmembrane domain using a hinge or other linker. A spacer, linker or hinge may be introduced between the extracellular antigen binding domain and the transmembrane domain to provide flexibility that allows the antigen binding domain to be oriented in different directions, thereby promoting antigen recognition and binding. As discussed below, the cytoplasmic side of the transmembrane domain may be attached to an intracellular signaling domain, such as an intracellular signaling domain of CD28 or CD3 ζ (CD3-ζ), and may additionally include one or more costimulatory domains.

[0167] Therefore, in certain embodiments, CAR may also include a hinge region between the extracellular antigen binding domain and the transmembrane domain. For example, the hinge region may be derived from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28 or CD8α. In a specific embodiment, the hinge region is derived from the hinge region of IgG4. In another embodiment, the hinge region is a CD8 hinge domain (see SwissProt / GenBank accession number P01732).

[0168] In one embodiment, the CAR comprises an extracellular antigen binding domain and a transmembrane domain connected via a CD8 hinge: AKPTTTPAPR PPTPAPTIAS QPLSLRPEAC RPAAGGAVHT RGLDFA (SEQ ID NO: 9).

[0169] In one embodiment, the CAR comprises an extracellular antigen binding domain and a transmembrane domain connected via a hybrid CD8-CD28 hinge: AKPTTTPAPR PPTPAPTIAS QPLSLRPEAC RPAAGGAVHT RGLDFAPRKIEVMYPPPYLD NEKSNGTIIH VKGKHLCPSP LFPGPSKP (SEQ ID NO: 10).

[0170] The transmembrane domain can be derived from the sequence of the protein that contributes the extracellular antigen binding domain, the sequence of the protein that contributes the effector function signaling domain, the sequence of the protein that contributes the proliferation signaling part, or the sequence of a completely different protein. In some embodiments, the transmembrane domain is naturally associated with one of the other domains of CAR. For example, the transmembrane domain and the cytoplasmic domain can be derived from the transmembrane region and the cytoplasmic region of the same protein. In one embodiment, the transmembrane domain and the cytoplasmic domain of CAR include a continuous portion of the CD28 sequence. Any transmembrane domain can be used in the CAR constructs described herein, provided that the domain is capable of anchoring the CAR comprising the antigen binding domain to the cell membrane.

[0171] Transmembrane domains are derived from natural sources or from synthetic sources. When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. The exemplary transmembrane domains that can be used in the methods provided herein can be derived from (for example, at least comprising the following transmembrane domains): α, β or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, LFA-1T cell co-receptors, CD2 T cell co-receptors / adhesion molecules, CD8α and fragments thereof. Any method known in the art (e.g., hydrophobicity analysis, structural analysis, etc.) or by using a public database such as the UniProt database to identify the transmembrane domain of the protein can be used.

[0172] In some embodiments, the transmembrane domain can be synthetic.In an exemplary embodiment, the transmembrane domain can mainly include hydrophobic residues, such as leucine and valine.In one embodiment, the triplets of phenylalanine, tryptophan and valine can be located at each end of the synthetic transmembrane domain.Optionally, a short oligopeptide or polypeptide linker (preferably, a length of 2 to 10 amino acids) can form a connection between the transmembrane domain and the cytoplasmic signaling domain of CAR.Glycine-serine doublets provide particularly suitable linkers.

[0173] In some embodiments, the transmembrane domain in the CAR used herein is a CD8 transmembrane domain or a portion thereof. The sequence of CD8 for this purpose is taught in PCT Publication No. WO2014 / 055771A1.

[0174] In some embodiments, the transmembrane domain in CAR is a CD8 transmembrane domain or a functional portion thereof. For example, CAR can include a CD3 transmembrane domain or a functional portion thereof having an amino acid sequence LDPKLCYLLD GILFIYGVIL TALFLRVK (SEQ ID NO: 11), such as LCYLLDGILF IYGVILTALFL (SEQ ID NO: 12).

[0175] In some embodiments, the transmembrane domain of the CAR of the present invention is a CD28 transmembrane domain. Exemplary sequences of CD28 and exemplary transmembrane domain sequences are provided below. In some embodiments, the CD28 transmembrane domain comprises the following exemplary transmembrane domain sequence or a fragment or variant thereof, and the CAR comprising the sequence can be anchored to the cell membrane. Therefore, in some embodiments, the transmembrane domain of CAR is a CD28 transmembrane domain comprising the following amino acid sequence: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 13). In one embodiment, the transmembrane domain of CAR is a CD28 transmembrane domain comprising the following amino acid sequence: IEVMYPPPYL DNEKSNGTII HVKGKHLCPSPLFPGPSKPF WVLVVVGGVLACYSLLVTVA FIIFWV (SEQ ID NO: 16) or a functional fragment thereof, such as SEQ ID NO: 14.

[0176] In addition to the extracellular antigen-binding domain and the transmembrane domain, CARs also contain an intracellular (or cytoplasmic) signaling domain.

[0177] It is known that the signal generated by the endogenous TCR alone is insufficient to fully activate T cells, and that secondary or co-stimulatory signals may also be required. Therefore, T cell activation can be mediated by two different types of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0178] As used herein, the term "intracellular signaling domain" or "cytoplasmic signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can generate signals that promote the immune effector function of immune cells (e.g., CAR-T cells or NK cells expressing CAR) comprising CAR. Examples of immune effector functions, such as in CART cells or NK cells expressing CAR, include cytolytic activity and auxiliary activity, including the secretion of cytokines. In an embodiment, the intracellular signaling domain transduces effector function signals and instructs cells to perform specific functions. Although the entire intracellular signaling domain can be used, in many cases, it is not necessary to use the entire chain. To the extent that a truncated portion of an intracellular signaling domain is used, such a truncated portion can be used to replace a complete chain, provided that the truncated portion transduces an effector function signal. Therefore, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce an effector function signal.

[0179] In one embodiment, the intracellular signaling domain of the CAR comprises a CD3 zeta signaling region or a signaling portion thereof as described in SEQ ID NO: 15.

[0180]

[0181] The cytoplasmic signaling domain may also include, but is not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, CD278 ("ICOS"), FcεRI, CD66d, DAP10, and DAP12.

[0182] The CAR may also comprise an "intracellular co-stimulatory domain," which is a polypeptide chain derived from the intracellular signaling domain of one or more co-stimulatory proteins (such as CD28 and 4-1BB) that enhances cytokine production.

[0183] Exemplary co-stimulatory signaling regions include 4-1BB, CD21, CD28, CD27, CD127, ICOS, IL-15Rα, and OX40.

[0184] In certain embodiments, the cytoplasmic costimulatory domain of CAR comprises the 4-1BB signaling domain itself or in combination with any other desired cytoplasmic domain that can be used in the context of CAR. 4-1BB is a member of the TNFR superfamily, having an amino acid sequence provided with GenBank accession number AAA62478.2 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.); and "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank accession number AAA62478.2 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.).

[0185] In one embodiment, the intracellular co-stimulatory signaling domain of the CAR is a 4-1BB (CD137) co-stimulatory signaling region, or a signaling portion thereof:

[0186]

[0187] In one embodiment, the costimulatory signaling domain of the CAR is a CD28 costimulatory signaling region sequence. For example, the costimulatory signaling domain can include the following CD28 costimulatory signaling region or a signaling portion thereof:

[0188]

[0189] In an exemplary embodiment, the cytoplasmic domain of CAR can include a CD3 ζ signaling domain in combination with any other desired cytoplasmic domain that can be used in the context of the CAR of the present invention. In certain embodiments, the cytoplasmic domain of CAR can include a CD3 ζ domain and a costimulatory signaling region, including but not limited to the costimulatory signaling regions of 4-1BB, CD28 and CD27.

[0190] The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR of the present invention can be connected to each other in a random or specific order. Optionally, a short oligopeptide or polypeptide linker or spacer preferably having a length between 5 and 20 amino acids can be inserted between the cytoplasmic domains. A GGGGS (SEQ ID NO: 18) or (GGGGS) × 3 (SEQ ID NO: 19) provides a particularly suitable linker.

[0191] In one embodiment, the CAR used herein comprises an extracellular domain containing a single-chain variable domain of an anti-CD19 monoclonal antibody, a transmembrane domain containing a hinge and a transmembrane domain of CD8α, and a cytoplasmic domain containing a signaling domain of CD3ζ and a signaling domain of 4-1BB. An exemplary CAR comprises an extracellular domain, which is included in Nicholson IC et al., Mol Immunol 34: 1157-1165 (1997) described in the anti-CD19 monoclonal antibody plus CD8α 21 amino acid signal peptide (translated from 63 nucleotides at positions 26-88 of GenBank accession number NM_001768). The CD8α hinge and transmembrane domain consist of 69 amino acids translated from 207 nucleotides at positions 815-1021 of GenBank accession number NM_001768. The CD3 zeta signaling domain of a preferred embodiment comprises 112 amino acids translated from 339 nucleotides at positions 1022-1360 of GenBank Accession No. NM_000734.

[0192] Between the extracellular domain (including antigen binding domain) and the transmembrane domain (as described above) of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a spacer or hinge domain can be incorporated. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide whose function is to connect the transmembrane domain in the polypeptide chain to the extracellular domain and / or the cytoplasmic domain. As used herein, the hinge domain generally means any oligopeptide or polypeptide whose function is to provide flexibility and / or prevent the steric hindrance of CAR or its domain for CAR or its domain. In some embodiments, the spacer or hinge domain may include up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 5 to 20 amino acids. It should also be understood that one or more spacer domains may be included in other regions of CAR, because aspects of the present disclosure are not limited to this aspect.

[0193] It should be understood that CAR may include a region having a sequence provided herein (e.g., an antigen binding domain, a transmembrane domain, a cytoplasmic domain, a signaling domain, a safety domain and / or a joint or any combination thereof), or a variant thereof or a fragment of any of them (e.g., a variant and / or a fragment that maintains the required function of CAR activity) may be included in the CAR protein described herein. In some embodiments, the variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes relative to the sequence shown. In some embodiments, the variant has a sequence that is at least 80%, at least 85%, at least 90%, 90%-95%, at least 95%, or at least 99% identical to the sequence shown. In some embodiments, the fragment is 1-5, 5-10, 10-20, 20-30, 30-40, or 40-50 amino acids shorter than the sequence provided herein. In some embodiments, the fragment is shorter at the N-terminus, C-terminus, or two terminal regions of the provided sequence. In some embodiments, fragments comprise 80%-85%, 85%-90%, 90%-95%, or 95%-99% of the number of amino acids in the sequences provided herein.

[0194] In some embodiments, the above exemplary, non-limiting arrangement is from left to right, N-terminal to C-terminal of CAR.CAR can include or also include any other combination of elements as described herein.

[0195] After identifying each part of the CAR construct, immune cells expressing CAR are produced, and thus immune cells express CAR. The method includes introducing immune cells with nucleic acid molecules (e.g., RNA molecules, e.g., mRNA) described herein or vectors comprising nucleic acid molecules encoding CAR (e.g., CAR described herein), e.g., transducing immune cells. It is noteworthy that the present invention includes nucleic acids encoding amino acid sequences disclosed herein. The present invention also provides a method for producing a cell colony (e.g., RNA engineered cells transiently expressing exogenous RNA). The method includes introducing RNA (e.g., RNA transcribed in vitro or synthetic RNA; mRNA sequences encoding CAR polypeptides as described herein) as described herein into cells. In embodiments, RNA transiently expresses CAR polypeptides. In one embodiment, the cell is a cell as described herein, such as an immune effector cell (e.g., T cell or NK cell, or cell colony).

[0196] Other exemplary chimeric antigen receptor constructs are disclosed in: U.S. Patent No. 9,328,156; U.S. Patent No. 9,783,591; U.S. Patent No. 9,714,278; U.S. Patent No. 9,765,156; U.S. Patent No. 10,117,896; U.S. Patent No. 9,573,988; U.S. Patent No. 10,308,717; U.S. Patent No. 10,221,245; U.S. Patent No. 10,040,865; U.S. Patent No. 2018 / 0256712A1; U.S. Patent Publication No. 2018 / 0271907A1; U.S. Patent Publication No. 2016 / 0046724A1; U.S. Patent Publication No. 2018 / 0044424A1; U.S. Patent Publication No. 2018 / 0258149A1; U.S. Patent Publication No. 2019 / 0151363A1 and U.S. Patent Publication No. 2018 / 0273601A1; the contents of each of the foregoing patents and patent publications are incorporated herein by reference in their entirety.

[0197] III. Anti-CD5 Antibody Drug Conjugate (ADC)

[0198] As described herein, anti-CD5 ADC can be used in combination with CAR therapy to treat cancer or autoimmune diseases in human patients. More specifically, anti-CD5 ADC can be used to consume CD5+ cells (e.g., CD5+ T cells) in human subjects who also receive CAR therapy. Anti-CD5 ADC targets endogenous T cells and kills these cells so that the patient's immune system will not attack the cells (e.g., autologous or allogeneic) expressing CAR applied to the subject. Therefore, anti-CD5 ADC is used as a regulatory step combined with CAR therapy to promote the acceptance of engineered immune cells expressing CAR in recipient patients. Compared to the more traditional regulatory methods for CAR therapy in which general lympho-depleting chemotherapeutic agents are applied to subjects, an advantage of using anti-CD5 ADC as a regulatory scheme is that endogenous T cells expressing CD5 can be specifically targeted for consumption.

[0199] Anti-CD5 antibody

[0200] ADCs capable of binding to CD5 can be used as therapeutic agents to promote acceptance of CAR-expressing immune cells in human patients by preventing or reducing the risk of rejection of CAR-expressing immune cells.

[0201] The anti-CD5 ADCs described herein include an anti-CD5 antibody, or an antigen-binding portion thereof, linked to a cytotoxin.

[0202] Human CD5 is also known as 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 contains 495 amino acids and is described in Gladkikh et al. (2017) Cancer Med. 6(12):2984 and Jones et al. (1986) Nature 323(6086):346). The amino acid sequence of CD5 (isoform 1) is provided below (NCBI reference sequence: NP_055022.2):

[0203]

[0204] The second isoform of human CD5 is 438 amino acids and is identified below as the NCBI reference sequence: NP_001333385.1. Unlike isoform 1, CD5 isoform 2 is an intracellular protein. Compared to isoform 1, isoform 2 contains a different 5'UTR and lacks the in-frame portion of the 5' coding region. Compared to isoform 1, the resulting isoform 2 has a shorter N-terminus. Compared to isoform 1, CD5 isoform 2 lacks a leader peptide and represents an intracellular isoform present in a subset of B lymphocytes. The ADC described herein is specific for human CD5 isoform 1, which represents an extracellular form of human CD5.

[0205] In one embodiment, an anti-CD5 antibody that can be used in the methods and compositions described herein is antibody 5D7v (Ab5D7v). The heavy chain variable region (VH) amino acid sequence of Ab5D7v is provided below as SEQ ID NO: 1.

[0206]

[0207] The VH CDR amino acid sequences of Ab5D7v above are underlined and are as follows: FSLSTSGMG (VH CDR1; SEQ ID NO:3); WWDDD (VH CDR2; SEQ ID NO:4); and RRATGTGFDY (VH CDR3; SEQ ID NO:5).

[0208] The light chain variable region (VL) amino acid sequence of Ab5D7v is provided below as SEQ ID NO:2.

[0209]

[0210] The VL CDR amino acid sequences of Ab5D7v above are underlined and are as follows: QDVGTA (VL CDR1; SEQ ID NO:6); WTSTRHT (VL CDR2; SEQ ID NO:7); and YNSYNT (VL CDR3; SEQ ID NO:8).

[0211] In one embodiment, the anti-CD5 ADC comprises an anti-CD5 antibody comprising a heavy chain comprising a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:3, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:4, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:5, and the anti-CD5 antibody comprises a light chain comprising a CDR1 domain comprising the amino acid sequence set forth in SEQ ID NO:6, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID NO:7, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID NO:8, wherein the antibody is conjugated to a cytotoxin via a linker.

[0212] In one embodiment, the anti-CD5 ADC comprises an anti-CD5 antibody comprising a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 2, wherein the antibody is conjugated to a cytotoxin via a linker.

[0213] In another embodiment, the anti-CD5 antibody used in the ADC described herein is the 5D7 antibody (see, e.g., US20080254027, the disclosure of which is incorporated herein by reference). In another embodiment, the anti-CD5 antibody that can be used in the methods and compositions described herein (including ADC) is a variant of the 5D7 antibody (see, e.g., US 20080254027, the disclosure of which is incorporated herein by reference).

[0214] Furthermore, in certain embodiments, the anti-CD5 ADC has a serum half-life of 3 days or less in human subjects.

[0215] Additional anti-CD5 antibodies that can be used in the ADCs described herein can be identified using techniques known in the art, such as hybridoma production. Hybridomas can be prepared using a murine system. Protocols for immunization and subsequent isolation of splenocytes for fusion are known in the art. Fusion partners and procedures for hybridoma production are also known. Alternatively, anti-CD5 antibodies can be produced using or XenoMouse TMProduced. When preparing additional anti-CD5 antibodies, the CD5 antigen is separated and / or purified. The CD5 antigen can be a CD5 fragment from the extracellular domain of CD5. Immunization of animals can be performed by any method known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods for immunizing animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, for example, Harlow and Lane, supra, and U.S. Patent No. 5,994,619. The CD5 antigen can be administered with an adjuvant to stimulate an immune response. Adjuvants known in the art include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide) or ISCOM (immunostimulatory complex). After immunizing an animal with a CD5 antigen, an immortalized cell line producing antibodies is prepared by cells isolated from the immunized animal. After immunization, the animal is sacrificed and lymph node and / or spleen B cells are immortalized by methods known in the art (e.g., oncogene transfer, oncogenic viral transduction, exposure to carcinogenic or mutagenic compounds, fusion with immortalized cells such as myeloma cells, and inactivation of tumor suppressor genes). See, e.g., Harlow and Lane, supra. Hybridomas can be selected, cloned, and further screened for desired properties, including robust growth, high antibody production, and desired antibody properties.

[0216] Anti-CD5 antibodies for use in the anti-CD5 ADC described herein can also be identified using high-throughput screening of molecules capable of binding to CD5 in antibody libraries or antibody fragment libraries. Such methods include in vitro display techniques known in the art, such as, in particular, phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display. The use of phage display to separate antibodies, antigen-binding fragments, or ligands that bind to biologically relevant molecules has been reviewed in, for example, Felici et al., Biotechnol. Annual Rev. 1: 149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26: 27-45, 1997; and Hoogenboom et al., Immunotechnology 4: 1-20, 1998, the disclosure of each of which is incorporated herein by reference when it relates to in vitro display techniques. Randomized combinatorial peptide libraries have been constructed to select polypeptides that bind to cell surface antigens, such as Kay, Perspect. Drug Discovery Des. 2: 251-268, 1995 and Kay et al., Mol. Divers. 1: 139-140, 1996, the disclosure of each of which is incorporated herein by reference when it relates to the discovery of antigen binding molecules. Proteins, such as multimeric proteins, have been successfully displayed on phages as functional molecules (see, e.g., EP 0349578, EP 4527839 and EP 0589877 and Chiswell and McCafferty, Trends Biotechnol. 10: 80-84 1992, the disclosure of each of which is incorporated herein by reference when it relates to the use of in vitro display techniques for the discovery of antigen binding molecules). In addition, functional antibody fragments such as Fab and scFv fragments have been expressed in in vitro display formats (see, e.g., McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, the disclosures of each of which are incorporated herein by reference as they relate to in vitro display platforms for discovering antigen-binding molecules).

[0217] In addition to in vitro display techniques, computer modeling techniques can be used to design and identify anti-CD5 antibodies or antibody fragments in silico, for example, using the program described in US 2013 / 0288373, the disclosure of which is incorporated herein when it relates to molecular modeling methods for identifying anti-CD5 antibodies. For example, using computer modeling techniques, one skilled in the art can screen an antibody library or antibody fragment library in a computer for molecules that can bind to a specific epitope on CD5, such as an extracellular epitope of CD5.

[0218] In one embodiment, the anti-CD5 antibody used in the ADC described herein can be internalized into a cell. When identifying an anti-CD5 antibody (or a fragment thereof), additional techniques can be used to identify antibodies or original binding fragments that bind to CD5 on the surface of a cell (e.g., a T cell) and can also be internalized by a cell, for example, through receptor-mediated endocytosis. For example, the in vitro display technology described above can be modified to screen for antibodies or antigen-binding fragments that bind to CD5 on the surface of a hematopoietic stem cell and are subsequently internalized. Phage display represents such a technology that can be used in conjunction with this screening paradigm. In order to identify an anti-CD5 antibody or a fragment thereof that binds to CD5 and is subsequently internalized by a CD5+ cell, those skilled in the art can use Williams et al., Leukemia 19: 1432-1438, 2005, the phage display technology described, the disclosure of which is incorporated herein by reference in its entirety.

[0219] The internalization ability of an anti-CD5 antibody or fragment thereof can be assessed, for example, using a radionuclide internalization assay known in the art. For example, an anti-CD5 antibody or fragment thereof identified using an in vitro display technique described herein or known in the art can be functionalized by incorporating a radioisotope such as 18 F. 75 Br, 77 Br, 122 I. 123 I. 124 I. 125 I. 129 I. 131 I. 211 At 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu, 177 Lu, 77 As, 72 As, 86 Y. 90 Y. 89 Zr,212 Bi, 213 Bior 225 Ac. For example, a radioactive halogen, such as Ac, can be introduced into the cell using beads containing an electrophilic halogen reagent, such as polystyrene beads (e.g., Iodination Beads, Thermo Fisher Scientific, Inc., Cambridge, MA). 18 F. 75 Br, 77 Br, 122 I. 123 I. 124 I. 125 I. 129 I. 131 I. 211 At is incorporated into an antibody, a fragment thereof or a ligand. Radiolabeled antibodies or fragments thereof can be incubated with hematopoietic stem cells for a time sufficient to allow internalization. By detecting the emitted radiation (e.g., γ-radiation) of the resulting hematopoietic stem cells compared with the emitted radiation (e.g., γ-radiation) of the recovered wash buffer, the internalized antibody or fragment thereof can be identified. The aforementioned internalization assay can also be used to characterize ADC.

[0220] In some embodiments, the anti-CD5 antibody (or its fragment) has a determined serum half-life. For example, in human patients, the anti-CD5 antibody (or its fragment) can have a serum half-life of about 1-24 hours. For example, in human patients, an ADC comprising such an anti-CD5 antibody can also have a serum half-life of about 1-24 hours. Pharmacokinetic analysis by measuring serum levels can be performed by assays known in the art.

[0221] In order to recombinantly produce anti-CD5 antibodies, nucleic acids encoding antibodies such as those described above are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding antibody heavy and light chains).

[0222] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. Regarding the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents No. 5,648,237, No. 5,789,199 and No. 5,840,523. (See also: Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in Escherichia coli (E. coli.). After expression, antibodies can be separated from the soluble fraction of bacterial cell mass (paste), and can be further purified.

[0223] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7), human embryonic kidney cell line (293 or 293 cells, as described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK, Buffalo rat liver cells (BRL 3A); human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells (as described, for example, in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)), MRC 5 cells and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)) and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of some mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., Y0, NS0, Sp20 cell).

[0224] In some embodiments, anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those antibodies that comprise a combination of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions listed in Table 1A and Table 1B below.

[0225] Table 1A.

[0226]

[0227]

[0228] Table 1B.

[0229]

[0230]

[0231] Cytotoxins

[0232] Various cytotoxins can be conjugated to anti-CD5 antibodies via a linker for use in the combination therapy described herein. In particular, anti-CD5 ADCs include antibodies (or antigen-binding fragments thereof) conjugated to a cytotoxic moiety (or cytotoxin) (i.e., covalently attached via a linker). As used herein, the terms "cytotoxin," "cytotoxic moiety," and "drug" are used interchangeably. In various embodiments, the cytotoxic moiety exhibits reduced cytotoxicity or no cytotoxicity when incorporated into the conjugate, but recovers cytotoxicity after cleavage from the linker. In various embodiments, the cytotoxic moiety maintains cytotoxicity without cleavage from the linker. In some embodiments, a cytotoxic molecule is conjugated to a cell-internalizing antibody or its antigen-binding fragment as disclosed herein, so that after the cell takes up the antibody or its fragment, the cytotoxin can approach its intracellular target and, for example, mediate T cell death.

[0233] Thus, the ADCs of the invention may have the general formula I, wherein an antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently linked) to a cytotoxic moiety ("drug", D) via a chemical moiety (Z) and a linker (L).

[0234] Ab-(ZLD) n (I)

[0235] Accordingly, the antibody or its antigen-binding fragment can be conjugated with a plurality of drug moieties as indicated by an integer n, the integer n representing the average number of cytotoxins per antibody, and the integer n range can be, for example, from about 1 to about 20. Any number of cytotoxins can be conjugated to the antibody, for example, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In some embodiments, n is 1 to 4. In some embodiments, n is 1 to 3. In some embodiments, n is about 2. In some embodiments, n is about 1. The average number of drug moieties per antibody in the ADC prepared by the conjugation reaction can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of ADC in terms of n can also be determined. In some cases, separation, purification, and characterization of homogeneous ADCs with a certain value of n from ADCs with other drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis.

[0236] For some anti-CD5 ADCs, n can be limited by the number of attachment sites on the antibody. For example, in the case where the attachment is a cysteine ​​thiol, the antibody may have only one or several cysteine ​​thiol groups, or may have only one or several sufficiently reactive thiol groups through which the linker can be attached. Typically, antibodies do not contain many free and reactive cysteine ​​thiol groups that can be connected to the drug moiety; the cysteine ​​thiol residues in the antibody are mainly present in the form of disulfide bridges. In certain embodiments, the antibody can be reduced with a reducing agent such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP) under partial or full reducing conditions to generate reactive cysteine ​​thiol groups. In certain embodiments, higher drug loadings, such as n>5, can cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody drug conjugates.

[0237] In certain embodiments, during the conjugation reaction, less than the theoretical maximum drug moiety is conjugated to the antibody. The antibody may contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as described below. Only the most reactive lysine groups can react with amine-reactive linker reagents. In certain embodiments, the antibody undergoes denaturation conditions to expose reactive nucleophilic groups, such as lysine or cysteine.

[0238] The loading capacity (drug / antibody ratio) of the ADC can be controlled in different ways, for example by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limited reducing conditions for modification of cysteine ​​thiol groups, (iv) engineering the amino acid sequence of the antibody by recombinant technology so that the number and position of cysteine ​​residues are modified to control the number and / or position of linker-drug attachments.

[0239] Cytotoxins suitable for use with the compositions and methods described herein include, among others known in the art, DNA intercalators (e.g., anthracyclines), agents capable of disrupting the mitotic spindle (e.g., vinca alkaloids, maytansine, maytansine alkaloids, and derivatives thereof), RNA polymerase inhibitors (e.g., amanita toxins such as α-amanitin and its derivatives), and agents capable of disrupting protein biosynthesis (e.g., agents exhibiting rRNA N-glycosidase activity such as saporin and ricin A chain).

[0240] In some embodiments, the cytotoxin is a microtubule-binding agent (e.g., maytansine or a maytansine alkaloid), amanita anitoxin, Pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolebenzodiazepines, and indolebenzodiazepine dimers, or variants thereof, or another cytotoxic compound described herein or known in the art.

[0241] In some embodiments, the cytotoxin of the antibody drug conjugate is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is an amanita toxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody drug conjugate as disclosed herein is an amanita toxin or a derivative thereof, such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin non-toxic cyclic peptide, monohydroxyamanitin carboxylic acid, proamanitin non-toxic cyclic peptide or a derivative thereof.

[0242] Additional details regarding cytotoxins that may be used in anti-CD5 ADCs useful in the methods of the invention are described below.

[0243] Amanita phalloides

[0244] In some embodiments, the RNA polymerase inhibitor is an amanita toxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody drug conjugate as disclosed herein is an amanita toxin or a derivative thereof, such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin non-toxic cyclic peptide, monohydroxyamanitin carboxylic acid, proamanitin non-toxic cyclic peptide or a derivative thereof. The structures of a variety of naturally occurring amanitin toxins are represented by formula II and the accompanying Table 2, and are disclosed in, for example, Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459.

[0245]

[0246] Table 2. Table of structures of Amanita phalloides toxins.

[0247]

[0248]

[0249] In one embodiment, the cytotoxin is amanitin or a derivative thereof. In one embodiment, the cytotoxin is alpha-amanitin or a derivative thereof.

[0250] Many positions on the Amanita toxin or derivative thereof can serve as locations for covalent bonding to the linking moiety L and thereby to the antibody or antigen-binding fragment thereof. In some embodiments, the cytotoxin in the ADC of Formula I is an Amanita toxin or derivative thereof according to Formula (II).

[0251] In one embodiment, the ADC is represented by the formula Ab-ZL-Am, wherein Ab is an antibody or antigen-binding fragment thereof that binds to CD5, L is a linker, Z is a chemical moiety, and Am is an Amanita ani toxin. In this embodiment, the linker-amanitin toxin conjugate Am-LZ is represented by formula (III):

[0252]

[0253] in:

[0254] R1 is H, OH, OR A OR C ;

[0255] R2 is H, OH, OR B OR C ;

[0256] R A and R B When present, together with the oxygen atoms to which they are attached, they combine to form a 5-membered heterocycloalkyl group;

[0257] R3 is H, R C or R D ;

[0258] Each of R4, R5, R6 and R7 is independently H, OH, OR C , OR D , R C or R D ;

[0259] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0260] R9 is H, OH, OR C OR D ;

[0261] Q is -S-, -S(O)- or -SO2-;

[0262] R Cis -LZ' or -LZ-Ab, wherein L is a linker and is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl; or includes a dipeptide; or -((CH2) m O) n (CH2) m –, wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Z′ is a reactive moiety, and Z is a chemical moiety resulting from a coupling reaction between Z′ and a functional group on Ab; and

[0263] R D is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or a combination thereof, wherein each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally substituted by 1 to 5 Substituents are selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

[0264] Formula (III) comprises an amanita ani toxin and a linker, and in some embodiments, comprises a linker, a chemical moiety, and an antibody.

[0265] In some embodiments, the cytotoxin is Amanita toxin, and the linker-Amanita toxin conjugate or antibody-linker-Amanita toxin conjugate is represented by formula (IIIA):

[0266]

[0267] in:

[0268] R1 is H, OH, OR A OR C ;

[0269] R2 is H, OH, OR B OR C ;

[0270] R A and R B When present, together with the oxygen atoms to which they are attached, they combine to form a 5-membered heterocycloalkyl group;

[0271] R3 is H, R C or R D ;

[0272] Each of R4, R5, R6 and R7 is independently H, OH, OR C , OR D , R C or R D ;

[0273] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0274] R9 is H, OH, OR C OR D ;

[0275] Q is -S-, -S(O)- or -SO2-;

[0276] R C is -LZ' or -LZ-Ab, wherein L is a linker and is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl; or includes a dipeptide; or -((CH2) m O) n (CH2) m –, wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Z′ is a reactive moiety, and Z is a chemical moiety resulting from a coupling reaction between Z′ and a functional group on Ab; and

[0277] R Dis C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or a combination thereof, wherein each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally substituted by 1 The invention further comprises a cycloalkyl radical, a cycloalkyl radical, a heterocycloalkyl radical, a alkylaryl radical, a alkylheteroaryl radical, a cycloalkyl ...

[0278] In some embodiments, the amanita ani toxin comprises an R C Substituent.

[0279] In some embodiments, R A and R B Together with the oxygen atom to which they are attached, they combine to form a 5-membered heterocycloalkyl group of the formula:

[0280]

[0281] wherein Y is -(C=O)-, -(C=S)-, -(C=NR E) -or-(CR E R E’ )-;and

[0282] Where R E and R E’ are independently H, C1-C6 alkylene-R C 、C1-C6 heteroalkylene-R C 、C2-C6 alkenylene-R C 、C2-C6 heteroalkenylene-R C 、C2-C6 alkynylene-R C 、C2-C6 heteroalkynylene-R C , cycloalkylene-R C , heterocycloalkylene-R C , arylene-R C or heteroarylene-R C , or a combination thereof; wherein each C1-C6 alkylene-R C 、C1-C6 heteroalkylene-R C 、C2-C6 alkenylene-R C 、C2-C6 heteroalkenylene-R C 、C2-C6 alkynylene-RC 、C2-C6 heteroalkynylene-R C , cycloalkylene-R C , heterocycloalkylene-R C , arylene-R C or heteroarylene-R C Optionally substituted with 1 to 5 substituents, the substituents being independently selected for each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxyl, thiol, and nitro. Formula (IIIA) includes an amanita ani toxin and a linker, and in some embodiments, includes a linker, a chemical moiety, and an antibody.

[0283] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIA, wherein

[0284] R1 is H, OH, OR A OR C ;

[0285] R2 is H, OH, OR B OR C ;

[0286] R A and R B When present, they combine with the oxygen atoms to which they are bound to form:

[0287]

[0288] Where R3 is H or R C .

[0289] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIA, wherein

[0290] R1 is H, OH, OR A OR C ;

[0291] R2 is H, OH, OR B OR C ;

[0292] R A and R B When present, they combine with the oxygen atoms to which they are bound to form:

[0293]

[0294] in

[0295] R3 is H or R C ;

[0296] R4 and R5 are each independently H, OH, OR C , R C OR D ;

[0297] R6 and R7 are each H;

[0298] R8 is OH, NH2, OR C or NHR C ;

[0299] R9 is H or OH; and

[0300] Where R C and R D As defined above.

[0301] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIA, wherein:

[0302] R1 is H, OH or OR A ;

[0303] R2 is H, OH or OR B ;

[0304] R A and R B When present, they combine with the oxygen atoms to which they are bound to form:

[0305]

[0306] in

[0307] R3, R4, R6 and R7 are each H;

[0308] R5 is OR C ;

[0309] R8 is OH or NH2;

[0310] R9 is H or OH;

[0311] Q is -S-, -S(O)- or -SO2-; and

[0312] Where R C and R DAs defined above. Such Amanita anitoxin-linker conjugates are described, for example, in U.S. Patent Application Publication No. 2016 / 0002298, the disclosure of which is incorporated herein by reference in its entirety.

[0313] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIA, wherein:

[0314] R1 and R2 are each independently H or OH;

[0315] R3 is R C ;

[0316] R4, R6 and R7 are each H;

[0317] R5 is H, OH or OC1-C6 alkyl;

[0318] R8 is OH or NH2;

[0319] R9 is H or OH;

[0320] Q is -S-, -S(O)- or -SO2-; and

[0321] Where R C and R D As defined above. Such Amanita anitoxin-linker conjugates are described, for example, in US Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated herein by reference in its entirety.

[0322] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIA, wherein:

[0323] R1 and R2 are each independently H or OH;

[0324] R3, R6 and R7 are each H;

[0325] R4 and R5 are each independently H, OH, OR C or R C ;

[0326] R8 is OH or NH2;

[0327] R9 is H or OH;

[0328] Q is -S-, -S(O)- or -SO2-; and

[0329] Where R C and R DAs defined above. Such amanita anitoxin-linker conjugates are described, for example, in US Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0330] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIA, wherein:

[0331] R1 and R2 are each independently H or OH;

[0332] R3, R6 and R7 are each H;

[0333] R4 and R5 are each independently H or OH;

[0334] R8 is OH, NH2, OR C or NHR C ;

[0335] R9 is H or OH;

[0336] Q is -S-, -S(O)- or -SO2-; and

[0337] Where R C and R D As defined above. Such amanita anitoxin-linker conjugates are described, for example, in U.S. Patent Nos. 9,233,173 and 9,399,681, the disclosures of each of which are incorporated herein by reference in their entirety.

[0338] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein:

[0339]

[0340] in:

[0341] R1 is H, OH, OR A OR C ;

[0342] R2 is H, OH, OR B OR C ;

[0343] R A and R B When present, together with the oxygen atoms to which they are attached, they combine to form a 5-membered heterocycloalkyl group;

[0344] R3 is H, R C or R D ;

[0345] Each of R4, R5, R6 and R7 is independently H, OH, OR C , OR D , R C or R D ;

[0346] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0347] R9 is H, OH, OR C OR D ;

[0348] Q is -S-, -S(O)- or -SO2-;

[0349] R C is -LZ' or -LZ-Ab, wherein L is a linker and is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl; or includes a dipeptide; or -((CH2) m O) n (CH2) m –, wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; Z′ is a reactive moiety, and Z is a chemical moiety resulting from a coupling reaction between Z′ and a functional group on Ab; and

[0350] R D is C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl or a combination thereof, wherein each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl is optionally substituted by 1 The invention further comprises a cycloalkyl radical, a cycloalkyl radical, a heterocycloalkyl radical, a alkylaryl radical, a alkylheteroaryl radical, a cycloalkyl ...

[0351] Formula (IIIA) comprises an amanita ani toxin and a linker, and in some embodiments, comprises a linker, a chemical moiety, and an antibody.

[0352] In some embodiments, R A and R B Together with the oxygen atom to which they are attached, they form a 5-membered heterocycloalkyl group of the following formula:

[0353]

[0354] wherein Y is -(C=O)-, -(C=S)-, -(C=NR E) -or-(CR E R E’ )-;and

[0355] Where R E and R E’ are independently H, C1-C6 alkylene-R C 、C1-C6 heteroalkylene-R C 、C2-C6 alkenylene-R C 、C2-C6 heteroalkenylene-R C 、C2-C6 alkynylene-R C 、C2-C6 heteroalkynylene-R C , cycloalkylene-R C , heterocycloalkylene-R C , arylene-R C or heteroarylene-R C , or a combination thereof; wherein each C1-C6 alkylene-R C 、C1-C6 heteroalkylene-R C 、C2-C6 alkenylene-R C 、C2-C6 heteroalkenylene-R C 、C2-C6 alkynylene-R C 、C2-C6 heteroalkynylene-R C , cycloalkylene-R C , heterocycloalkylene-R C , arylene-R C or heteroarylene-R C Optionally substituted with 1 to 5 substituents, the substituents being independently selected for each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxy, mercapto and nitro.

[0356] In some embodiments, an antibody or antigen-binding fragment thereof as described herein is conjugated to an amanita ani toxin-linker conjugate represented by Formula IIIB or a derivative thereof, wherein

[0357] R1 is H, OH, OR A OR C ;

[0358] R2 is H, OH, OR B OR C ;

[0359] R A and R B When present, together with the oxygen atom to which they are attached, they combine to form a 5-membered heterocycloalkyl group of the formula:

[0360]

[0361] Where R3 is H or R C .

[0362] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein

[0363] R1 is H, OH, OR A OR C ;

[0364] R2 is H, OH, OR B OR C ;

[0365] R A and R B When present, together with the oxygen atom to which they are attached, they combine to form a 5-membered heterocycloalkyl group of the formula:

[0366]

[0367] in

[0368] R3 is H or R C ;

[0369] R4 and R5 are each independently H, OH, OR C , R C OR D ;

[0370] R6 and R7 are each H;

[0371] R8 is OH, NH2, OR C or NHR C ;

[0372] R9 is H or OH; and

[0373] Where R C and R D As defined above.

[0374] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein:

[0375] R1 is H, OH or OR A ;

[0376] R2 is H, OH or OR B ;

[0377] R A and R B When present, together with the oxygen atom to which they are attached, they combine to form a 5-membered heterocycloalkyl group of the formula:

[0378]

[0379] in

[0380] R3, R4, R6 and R7 are each H;

[0381] R5 is OR C ;

[0382] R8 is OH or NH2;

[0383] R9 is H or OH;

[0384] Q is -S-, -S(O)- or -SO2-; and

[0385] Where R C and R D As defined above. Such Amanita anitoxin-linker conjugates are described, for example, in U.S. Patent Application Publication No. 2016 / 0002298, the disclosure of which is incorporated herein by reference in its entirety.

[0386] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein:

[0387] R1 and R2 are each independently H or OH;

[0388] R3 is R C ;

[0389] R4, R6 and R7 are each H;

[0390] R5 is H, OH or OC1-C6 alkyl;

[0391] R8 is OH or NH2;

[0392] R9 is H or OH;

[0393] Q is -S-, -S(O)- or -SO2-; and

[0394] Where R C and R D As defined above. Such Amanita anitoxin-linker conjugates are described, for example, in US Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated herein by reference in its entirety.

[0395] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein:

[0396] R1 and R2 are each independently H or OH;

[0397] R3, R6 and R7 are each H;

[0398] R4 and R5 are each independently H, OH, OR C or R C ;

[0399] R8 is OH or NH2;

[0400] R9 is H or OH;

[0401] Q is -S-, -S(O)- or -SO2-; and

[0402] Where R C and R D As defined above. Such amanita anitoxin-linker conjugates are described, for example, in US Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0403] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, and the amanita toxin-linker conjugate is represented by Formula IIIB, wherein:

[0404] R1 and R2 are each independently H or OH;

[0405] R3, R6 and R7 are each H;

[0406] R4 and R5 are each independently H or OH;

[0407] R8 is OH, NH2, OR C or NHR C ;

[0408] R9 is H or OH;

[0409] Q is -S-, -S(O)- or -SO2-; and

[0410] Where R C and R D As defined above. Such amanita anitoxin-linker conjugates are described, for example, in U.S. Patent Nos. 9,233,173 and 9,399,681, the disclosures of each of which are incorporated herein by reference in their entirety.

[0411] Auristatin

[0412] The anti-CD5 antibodies and antigen-binding fragments thereof described herein can be conjugated to a cytotoxin that is auristatin (U.S. Pat. Nos. 5,635,483 and 5,780,588). Auristatins are antimitotic agents that interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584), and have anticancer activity (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42: 2961-2965). (U.S. Pat. Nos. 5,635,483 and 5,780,588). Auristatin drug moieties can be attached to antibodies via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172).

[0413] Exemplary auristatin embodiments include the N-terminally linked monomethyl auristatin drug moieties DE and DF (MMAE and MMAF, respectively), which are disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Vol. 45, Abstract No. 623, filed March 28, 2004, the disclosure of which is expressly incorporated by reference in its entirety.

[0414] An exemplary auristatin embodiment is MMAE:

[0415]

[0416] Therein the wavy line indicates the point of covalent attachment of the linker of the antibody-drug or drug-linker conjugate (-LZ-Ab or -LZ', as described herein).

[0417] Another exemplary auristatin embodiment is MMAF,

[0418]

[0419] Wherein the wavy line indicates the point of covalent attachment of the linker of the antibody-linker conjugate (-LZ-Ab or -LZ', as described herein), as disclosed in US 2005 / 0238649.

[0420] Auristatins can be prepared according to the following methods: U.S. Pat. No. 5,635,483; U.S. Pat. No. 5,780,588; Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277; Pettit, GR, et al. Synthesis, 1996, 719-725; Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 15:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784. U.S. Pat.

[0421] Maytansine alkaloids

[0422] Antibodies and Fabs thereof described herein can be conjugated with cytotoxins that are microtubule binding agents. In some embodiments, the microtubule binding agent is an analog of maytansine, maytansine alkaloids or maytansine alkaloids. Maytansine alkaloids are mitotic inhibitors that bind microtubules and play a role by inhibiting tubulin polymerization. For the first time, maytansine (U.S. Patent No. 3,896,111) was isolated from the East African shrub Maytenus serrata. Subsequently, it was found that some microorganisms also produce maytansine alkaloids, such as maytansinol and C-3 maytansine alcohol esters (U.S. Patent No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed in, for example, U.S. Pat. Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,3 No. 09,428, No. 4,313,946, No. 4,315,929, No. 4,317,821, No. 4,322,348, No. 4,331,598, No. 4,361,650, No. 4,364,866, No. 4,424,219, No. 4,450,254, No. 4,362,663 and No. 4,371,533. Maytansine alkaloid drug moieties are attractive drug moieties in antibody drug conjugates because they are: (i) relatively easy to prepare by fermentation or chemical modification, derivatization of fermentation products, (ii) easily derivatized with functional groups suitable for conjugation to antibodies via non-disulfide linkers, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.

[0423] Examples of suitable maytansine alkaloids include esters of maytansinol, synthetic maytansinol, and maytansinol analogs and derivatives. Included herein are any cytotoxins that inhibit microtubule formation and are highly toxic to mammalian cells, such as maytansine alkaloids, maytansinol, and maytansinol analogs and derivatives.

[0424] The example of suitable maytansinol ester includes those with modified aromatic ring and those with modified maytansinol ester at other positions. Such suitable maytansine alkaloids are disclosed in the following: U.S. Patent Nos. 4,137,230, 4,151,042, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,36 No. 1,650, 4,362,663, 4,364,866, 4,424,219, 4,450,254, 4,322,348, 4,362,663, 4,371,533, 5,208,020, 5,416,064, 5,475,092, 5,585,499, 5,846,545, 6,333,410, 7,276,497, and 7,473,796, the disclosures of each of which are incorporated herein by reference as they relate to maytansine alkaloids and derivatives thereof.

[0425] In some embodiments, the antibody drug conjugates (ADCs) of the present disclosure utilize a thiol-containing maytansine alkaloid (DM1) as a cytotoxic agent, which maytansine alkaloid is formally known as N 2 ′-Deacetyl-N 2 '-(3-mercapto-1-oxopropyl)-maytansine. DM1 is represented by the following structural formula IV:

[0426]

[0427] In another embodiment, the conjugates of the invention utilize thiol-containing maytansine alkaloids N 2 ′-Deacetyl-N 2 '(4-methyl-4-mercapto-1-oxopentyl)-maytansine (e.g., DM4) as a cytotoxic agent. DM4 is represented by the following structural formula V:

[0428]

[0429] Another maytansine alkaloid containing a side chain containing a sterically hindered thiol bond is N 2 ′-Deacetyl-N- 2 '(4-mercapto-1-oxopentyl)-maytansine (referred to as DM3) is represented by the following structural formula VI:

[0430]

[0431] Each of the maytansine alkaloids taught in U.S. Pat. Nos. 5,208,020 and 7,276,497 may also be used in the conjugates of the present disclosure. In this regard, the entire disclosures of 5,208,020 and 7,276,697 are incorporated herein by reference.

[0432] Many positions on the maytansine alkaloid can be used as positions for covalent bonding to the linking moiety, and thus covalent bonding to the antibody or its antigen-binding fragment (-LZ-Ab or -LZ ', as described herein). For example, the C-3 position with a hydroxyl group, the C-14 position modified with a hydroxymethyl group, the C-15 position modified with a hydroxyl group, and the C-20 position with a hydroxyl group are all expected to be useful. In some embodiments, the C-3 position is used as a position for covalent bonding to the linking moiety, and in some specific embodiments, the C-3 position of maytansine is used as a position for covalent bonding to the linking moiety. There are many known linker groups in the art for preparing antibody-maytansine alkaloid conjugates, including, for example, those disclosed in U.S. Pat. Nos. 5,208,020, 6,441,163, and EP Patent No. 0425235B1; Chari et al., Cancer Research 52:127-131 (1992); and US2005 / 0169933A, the disclosure of which is expressly incorporated by reference herein. Additional linker groups are described and exemplified herein.

[0433] The present invention also includes multiple isomers and mixtures of maytansine alkaloids and conjugates. Certain compounds of the present invention and conjugates can exist in the form of multiple stereoisomers, enantiomers and diastereomers. Some descriptions for producing such antibody-maytansine alkaloid conjugates are provided below: U.S. Patent No. 5,208,020, No. 5,416,064, No. 6,333,410, No. 6,441,163, No. 6,716,821 and No. 7,368,565, each of which is incorporated herein in its entirety.

[0434] Anthracyclines

[0435] In other embodiments, the antibodies and antigen-binding fragments thereof described herein can be conjugated to a cytotoxin that is an anthracycline molecule. Anthracyclines are antibiotic compounds that exhibit cytotoxicity. Studies have shown that anthracyclines can operate to kill cells through a number of different mechanisms, including: 1) intercalation of drug molecules into the DNA of cells, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) generation of free radicals by the drug, which then react with cellular macromolecules to cause damage to the cell, or 3) interaction of drug molecules with cell membranes [see, e.g., Anthracycline Antibiotics In Cancer Therapy

[0013] C. Peterson et al., "Transport And Storage Of Anthracycline In Experimental Systems And Human Leukemia" in; N.R. Bachur, "Free Radical Damage" supra, see pages 97-102]. Due to their cytotoxic potential, anthracyclines have been used to treat many cancers, such as leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma. [See, e.g., Anthracycline:Current Status And New Developments PH-Wiernik, p. 11]. Commonly used anthracyclines include doxorubicin, epirubicin, idarubicin and daunomycin.

[0436] Representative examples of anthracyclines include, but are not limited to, daunorubicin (Cerubidine; Bedford Laboratories), doxorubicin (Adriamycin; Bedford Laboratories; also known as doxorubicin hydrochloride, hydroxydaunorubicin and Rubex), epirubicin (Ellence; Pfizer) and idarubicin (Idamycin; Pfizer Inc.). The anthracycline analog doxorubicin (ADRIAMYCINO) is believed to interact with DNA by intercalation and inhibit the process of topoisomerase II, which unwinds DNA for transcription. After the topoisomerase II complex breaks the DNA strand for replication, doxorubicin stabilizes the topoisomerase II complex, preventing the DNA double helix from being resealed, thereby stopping the replication process. Doxorubicin and daunorubicin (DAUNOMYCIN) are the prototypical cytotoxic natural product anthracycline chemotherapeutic agents (Sessa et al., (2007) Cardiovasc. Toxicol. 7:75-79).

[0437] A non-limiting example of a suitable anthracycline for use herein is PNU-159682 ("PNU"), a highly potent major metabolite of nemorubicin. PNU exhibits greater than 3000-fold cytotoxicity relative to the parent nemorubicin (Quintieri et al., Clinical Cancer Research 2005, 11, 1608-1617). PNU is represented by the following structural formula:

[0438]

[0439] More than one position on an anthracycline such as PNU can serve as a location for covalent bonding to a linking moiety L and thereby to an anti-CD5 antibody or antigen-binding fragment thereof as described herein. For example, a linker can be introduced by modification of the hydroxymethylketone side chain.

[0440] In some embodiments, the cytotoxin is a PNU derivative represented by the following structural formula:

[0441]

[0442] Therein the wavy line indicates the point of covalent attachment of a linker of an ADC as described herein.

[0443] In some embodiments, the cytotoxin is a PNU derivative represented by the following structural formula:

[0444]

[0445] Therein the wavy line indicates the point of covalent attachment of a linker of an ADC as described herein.

[0446] Pyrrolobenzodiazepines (PBDs)

[0447] In other embodiments, the anti-CD5 antibodies or antigen-binding fragments thereof described herein can be conjugated to cytotoxins that are pyrrolobenzodiazepines (PBDs) or to cytotoxins that contain PBDs. PBDs can be produced by certain actinomycetes and have been shown to be sequence-selective DNA alkylating compounds. PBD cytotoxins include, but are not limited to, anthramycin, dimeric PBDs, and those disclosed in, for example, Hartley, JA (2011) The development of pyrrolobenzodiazepines as antitumor agents. Expert Opin Inv Drug, 20 (6), 733-744 and Antonow D, Thurston DE (2011) Synthesis of DNA-interactive pyrrolo [2, 1-c] [1, 4] benzodiazepines (PBDs). Chem Rev 111: 2815-2864.

[0448] In some embodiments, the cytotoxin may be a pyrrolobenzodiazepine dimer represented by the following structural formula:

[0449]

[0450] Wherein the wavy line indicates the covalent attachment point of the linker of the ADC as described herein. ADCs based on this PBD are disclosed in, for example, Sutherland et al., Blood 2013 122: 1455-1463, which is incorporated herein by reference in its entirety.

[0451] In some embodiments, the cytotoxin may be a PBD dimer represented by the following structural formula:

[0452]

[0453] wherein n is 3 or 5, and wherein the wavy line indicates the point of covalent attachment of a linker of an ADC as described herein.

[0454] In some embodiments, the cytotoxin may be a PBD dimer represented by the following structural formula:

[0455]

[0456] Therein the wavy line indicates the point of covalent attachment of a linker of an ADC as described herein.

[0457] In certain embodiments, the cytotoxin may be a PBD dimer, which, when taken together with a linker and a reactive moiety Z', as described herein, each may be represented by the following structure:

[0458]

[0459] This particular cytotoxin-linker conjugate is called tesirine (SG3249) and has been described, for example, in Howard et al., ACS Med. Chem. Lett. 2016, 7(11), 983-987, the disclosure of which is incorporated herein by reference in its entirety.

[0460] In certain embodiments, the cytotoxin may be a PBD dimer, which, when taken together with a linker and a reactive moiety Z', as described herein, each may be represented by the following structure:

[0461]

[0462] This particular cytotoxic linker conjugate is called talirine and has been described, for example, in Mantaj et al., Angewandte Chemie International Edition English 2017, 56, 462-488, the disclosure of which is incorporated herein by reference in its entirety.

[0463] Calicheamicin

[0464] In other embodiments, the antibodies and antigen-binding fragments thereof described herein can be conjugated to cytotoxins that are enediyne antitumor antibiotics (e.g., calicheamicin, ozogamicin). Antibiotics of the calicheamicin family can produce double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates, see U.S. Patents Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all belonging to American Cyanamid Company). Structural analogs of calicheamicin that may be used include, but are not limited to, those disclosed in, for example, Hinman et al., Cancer Research 53:3336-3342 (1993); Lode et al., Cancer Research 58:2925-2928 (1998); and the aforementioned U.S. patents to American Cyanamid.

[0465] Examples of calicheamicins suitable for use in the present invention are disclosed, for example, in U.S. Pat. No. 4,671,958, U.S. Pat. No. 4,970,198, U.S. Pat. No. 5,053,394, U.S. Pat. No. 5,037,651, and U.S. Pat. No. 5,079,233, which are incorporated herein in their entireties.

[0466] An exemplary calicheamicin is designated γ1, which is referred to herein as γ, and has the structural formula:

[0467]

[0468] In some embodiments, calicheamicin is a gamma calicheamicin derivative or an N-acetyl gamma calicheamicin derivative. Structural analogs of calicheamicin that can be used include, but are not limited to, those disclosed in, for example, Hinman et al., Cancer Research 53:3336-3342 (1993); Lode et al., Cancer Research 58:2925-2928 (1998); and the aforementioned U.S. patents. Calicheamicin contains a methyl trisulfide moiety that can react with an appropriate thiol to form a disulfide while introducing a functional group that can be used to attach a calicheamicin derivative to an anti-CD5 antibody or antigen-binding fragment thereof as described herein via a linker.

[0469] In one embodiment, the cytotoxin of the ADC as disclosed herein is a calicheamicin disulfide derivative represented by the formula:

[0470]

[0471] The wavy lines indicate the attachment points of the connectors.

[0472] Other cytotoxins

[0473] In other embodiments, the antibodies and antigen-binding fragments thereof described herein can be conjugated to cytotoxins other than or in addition to those disclosed herein above. Other cytotoxins suitable for use with the compositions and methods described herein include, but are not limited to, 5-ethynyluracil, abiraterone, acylfulvene, adecypenol, adozelesin, aldesleukin, hexamethylmelamine, aminostine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antarelix, anti-dorsalizing morphogenetic protein-1, antiandrogens, prostate cancer, antiestrogens, antineoplastons, antisense oligonucleotides, glycine aphidicolin glycinate), apoptosis gene regulators, apoptosis regulators, apurinic nucleic acids, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, diazotyrosine, baccatin III derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorins, benzoylstaurosporine, beta-lactam derivatives, beta-alethine, beta-betamycin B B), betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, bisaziridinylspermine, binasafide, bistrateneA, bizelesin, breflate, bleomycin A2, bleomycin B2, bropirimine, budotitane, buthionine sulfoxide, calcipotriol, calphostin C, camptothecin derivatives (e.g., 10-hydroxy-camptothecin), capecitabine, carboxamide-amino-triazole, carboxyamidotriazole, carzelesin, casein kinase inhibitors, castanespermine, cecropin B, cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene and its analogs, clotrimazole, collismycin A, collismycin B B), combretastatin A4, combretastatin analogs, conagenin, crambescidin 816, crisnatol, scutellariae cyclotide 8, scutellariae cyclotide A derivatives, curacin A, cyclopentanthraquinone, cycloplatam, cypemycin, cytarabineocfosfate, cytolytic factor, cytostatin, dacximab, decitabine, dehydrogenated ecteinascidin B, 2'deoxycoformycin (DCF), deslorelin, dexifosfamide, dexrazoxane, dexverapamil exverapamil), diaziquone, tunicain B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin, diphenylspiromustine, spondylolipidol, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocannycin SASA), ebselen, ecomustine, edilfosine, edrecolomab, eflornithine, elemene, ethiflu, epothilones, epithilones, epristeride, estramustine and its analogs, etoposide, etoposide 4'-phosphate (also known as etopofos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezellastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, homoharringtonine (HHT), hypericin, ibandronic acid, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridones, imiquimod, immunostimulatory peptides, iodobenzylguanidine, iodoxorubicin, irinotecan, iroplact, irsogladine, isobengazole, jasplakinolide, kahalalide F, lamellarin-N triacetate triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate, leptolstatin, letrozole, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol, lonidamine, losoxantrone, loxoribine, lurtotecan, lutetiumtexaphyrin, lysofylline, masorofol, maspin, matrix metalloproteinase inhibitors, menogaril, rnerbarone, meterelin, methioninease, metoclopramide, MIF inhibitors, ifepristone, miltefosine, mirimostim, mithracin, mitoguazone, dibromodulanol, mitomycin and its analogs, mitonafide, mitoxantrone, mofarotene, molgramostim, myloroxideB), myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagrestip, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, nilutamide, nisamycin, nitrullyn, octreotide, okicenone, onapristone, ondansetron, oracin, ormaplatin, oxaliplatin, oxaunomycin, paclitaxel and its analogs, palauamine, hexadecene palmitoylrhizoxin, pamidronate, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, sodium pentosan polysulfate, pentostatin, pentrozole, perflubron, perphosphamide, phenazinomycin, picibanil, pirarubicin, piritrexim, podophyllotoxin, porfiromycin, purine nucleoside phosphorylase inhibitors, raltitrexed, rhizotomycin, rogletimide, roxitocin, rubiginone B1 B1), ruboxyl, safingol, saintopin, sarcophytol A, sargramostim, sobuzoxane, sonermin, sparlfosic acid, spicamycin DD), spiromustine, stipiamide, sulfinosine, tallimustine, tegafur, temozolomide, teniposide, thaliblastine, thiocoraline, tirapazamine, topotecan, topsentin, triciribine, trimetrexate, veramine, vinorelbine, vinxaltine, vorozole, zeniplatin, and zilascorb.

[0474] Connectors

[0475] As used herein, the term "linker" means a bivalent chemical moiety comprising a chain of covalent bonds or atoms that covalently attaches an anti-CD5 antibody or fragment thereof (Ab) to a drug moiety (D) to form an antibody drug conjugate (ADC) of Formula I. Suitable linkers have two reactive ends, one for conjugation to an antibody and the other for conjugation to a cytotoxin. The antibody conjugation reactive end of the linker (reactive moiety, Z') is typically a site that can be conjugated to an antibody through a cysteine ​​thiol or lysine amine group on the antibody, and is therefore typically a thiol reactive group such as a double bond (as in maleimide) or a leaving group such as a chloro, bromo, iodo or R-sulfonyl group, or an amine reactive group such as a carboxyl group; while the cytotoxin conjugation reactive end of the linker is typically a site that can be conjugated to a cytotoxin. Non-limiting examples of linker-cytotoxin conjugation include, for example, respectively via a carboxyl or basic amine group on a linker and a basic amine or carboxyl group on a cytotoxin to form an amide bond, or via the alkylation of an OH group on a cytotoxin (e.g., via a leaving group on a linker) to form an ether, etc. In some embodiments, the cytotoxin linker conjugation is by forming an amide bond with a basic amine or carboxyl group on a cytotoxin, and thus the reactive substituent on the linker is a carboxyl or basic amine group, respectively. When the term "linker" is used to describe a linker in a conjugated form, due to the formation of a bond between a linker and / or a cytotoxin and between a linker and / or an antibody or its antigen-binding fragment, one or two reactive ends will be non-existent (such as reactive moiety Z', which has been converted into a chemical moiety Z) or incomplete (such as only a carbonyl group of a carboxylic acid). Such a conjugation reaction is further described below.

[0476] A variety of joints can be used to conjugate the described antibodies, antigen binding fragments and ligands with cytotoxic molecules. In some embodiments, the joint is cleavable under intracellular conditions, so that the cleavage of the joint in the intracellular environment releases the drug unit from the antibody. In yet other embodiments, the joint unit is non-cleavable, and the drug is released by, for example, antibody degradation. The joints that can be used for the ADC of the present invention are preferably stable outside the cell, prevent ADC molecules from aggregating, and keep ADC easily soluble in aqueous media and in a monomeric state. Before transport or delivery to the cell, preferably ADC is stable and remains intact, that is, the antibody remains connected to the drug portion. The joint is stable outside the target cell and can be cleaved at a certain effective rate within the cell. An effective joint will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug portion; (iii) remain stable and intact until the conjugate has been delivered or transported to its targeted site, that is, not cleaved; and (iv) maintain the cytotoxicity, cell killing or cell inhibition of the cytotoxic portion. The stability of ADC can be measured by standard analytical techniques such as mass spectrometry, HPLC and separation / analysis techniques LC / MS. Covalent attachment of the antibody and drug moiety requires the linker to have two reactive functional groups, i.e., bivalence in the sense of reactivity. Bivalent linker reagents that can be used to attach two or more functional or biologically active moieties (such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups) are known, and methods for their production of conjugates have been described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, pp. 234-242).

[0477] Suitable cleavable linkers include linkers that can be cleaved by, for example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference when it relates to linkers suitable for covalent conjugation). Suitable cleavable linkers may include, for example, chemical moieties such as hydrazines, disulfides, thioethers, or dipeptides.

[0478] Linkers that can be hydrolyzed under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, and the like. (See, for example, U.S. Pat. Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661, the disclosures of each of which are incorporated herein by reference in their entirety as they relate to linkers suitable for covalent conjugation). Such linkers are relatively stable under neutral pH conditions (such as under neutral pH conditions in blood), but are unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes).

[0479] Linkers that can be cleaved under reducing conditions include, for example, disulfides. A variety of disulfide linkers are known in the art, including, for example, disulfide linkers formed by: SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate) and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio) toluene), SPDB and SMPT (see, for example, Thorpe et al., 1987, Cancer Res. 47: 5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel, ed., Oxford U. Press, 1987). See also U.S. Pat. No. 4,880,935, the disclosure of each of which is incorporated herein by reference in its entirety as it relates to linkers suitable for covalent conjugation).

[0480] The linker susceptible to enzymatic hydrolysis can be, for example, a peptide-containing linker that is cleaved by an intracellular peptidase or protease (including but not limited to a lysosomal or endosomal protease). An advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is usually weakened when conjugated, and the serum stability of the conjugate is usually high. In some embodiments, the length of the peptidyl linker is at least two amino acids or at least three amino acids. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides or pentapeptides. Examples of suitable peptides include peptides containing the following amino acids: such as valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine and glycine. The amino acid residues constituting the amino acid linker component include naturally occurring amino acid residues, as well as a small amount of amino acids and non-naturally occurring amino acid analogs, such as citrulline. Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). In some embodiments, the linker comprises a dipeptide such as Val-Cit, Ala-Val or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg or Trp-Cit. Linkers containing dipeptides such as Val-Cit or Phe-Lys are disclosed in, for example, U.S. Pat. No. 6,214,345, the disclosure of which is incorporated herein by reference in its entirety when it relates to linkers suitable for covalent conjugation. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit.

[0481] Suitable linkers for conjugating the antibodies, antigen-binding fragments and ligands described herein to cytotoxic molecules include those capable of releasing cytotoxins by a 1,6-elimination process. Chemical moieties capable of such an elimination process include p-aminobenzyl (PAB) groups, 6-maleimidocaproic acid, pH-sensitive carbonates, and other reagents as described in Jain et al. Pharm. Res. 32: 3526-3540, 2015, the disclosure of which is incorporated herein by reference in its entirety when it relates to linkers suitable for covalent conjugation.

[0482] In some embodiments, the linker includes a "self-immolative" group, such as the aforementioned PAB or PABC (p-aminobenzyloxycarbonyl), which is disclosed in, for example, Carl et al., J. Med. Chem. (1981) 24: 479-480; Chakravarty et al. (1983) J. Med. Chem. 26: 638-644; US Other such chemical moieties capable of undergoing this process ("self-immolative linkers") include methylene carbamates and heteroaryl groups such as aminothiazoles, aminoimidazoles, aminopyrimidines, and the like. Linkers containing such heterocyclic self-immolative groups are disclosed in, for example, U.S. Patent Publication Nos. 20160303254 and 20150079114 and U.S. Patent No. 7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237; US 2005 / 0256030; de Groot et al. (2001) J. Org. Chem. 66:8815-8830; and US 7223837. In some embodiments, a dipeptide is used in combination with a self-immolative linker.

[0483] The linker suitable for use herein may also include one or more groups selected from the following: C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene and combinations thereof, each of which may be optionally substituted. Non-limiting examples of such groups include (CH2) p 、(CH2CH2O) p and -(C=O)(CH2) p- Units, wherein for each occurrence p is an independently selected integer from 1 to 6.

[0484] In some embodiments, each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocycloalkyl, aryl or heteroaryl group can be optionally substituted with 1 to 5 substituents independently selected for each occurrence from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxyl, thiol and nitro.

[0485] In some embodiments, each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocycloalkyl, aryl or heteroaryl group can be optionally interrupted by one or more heteroatoms selected from O, S and N.

[0486] In some embodiments, each C1-C6 alkyl, C1-C6 heteroalkyl, C2-C6 alkenyl, C2-C6 heteroalkenyl, C2-C6 alkynyl, C2-C6 heteroalkynyl, C3-C6 cycloalkyl, heterocycloalkyl, aryl or heteroaryl group may be optionally interrupted by one or more heteroatoms selected from O, S and N and may be optionally substituted by 1 to 5 substituents, the substituents being independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxyl, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxyl, sulfhydryl and nitro. Suitable linkers may include groups having properties that enhance solubility. For example, (CH2CH2O) p Unit (polyethylene glycol, PEG) linker can enhance solubility, as can alkyl chains substituted with amino, sulfonic acid, phosphonic acid or phosphoric acid residues. Linkers containing such moieties are disclosed, for example, in U.S. Pat. Nos. 8,236,319 and 9,504,756, the disclosure of each of which is incorporated herein by reference in its entirety when it relates to linkers suitable for covalent conjugation. Other groups that enhance solubility include, for example, acyl and carbamoylsulfonamide groups, which have the following structure:

[0487]

[0488] where a is 0 or 1; and

[0489] R 10Selected from the group consisting of: hydrogen, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C1-C 24 (Hetero)aryl groups, C1-C 24 Alkyl (hetero) aryl groups and C1-C 24 (Hetero)arylalkyl group, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero) aryl groups and C3-C 24 (Hetero)arylalkyl groups, each of which may be optionally substituted and / or optionally interrupted by one or more heteroatoms selected from O, S and NR 11 R 12 , where R 11 and R 12 are independently selected from the group consisting of: hydrogen and a C1-C4 alkyl group; or R 10 is a cytotoxin, wherein the cytotoxin is optionally linked to N via a spacer moiety. Linkers comprising such groups are described, for example, in U.S. Pat. No. 9,636,421 and U.S. Patent Application Publication No. 2017 / 0298145, the disclosures of which are incorporated herein by reference in their entireties as they relate to linkers suitable for covalent conjugation to cytotoxins and antibodies or antigen-binding fragments thereof.

[0490] In some embodiments, the linker may include one or more of the following: hydrazine, disulfide, thioether, dipeptide, p-aminobenzyl (PAB) group, heterocyclic self-immolative group, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, a solubility enhancing group, acyl, -(C=O)- or -(CH2CH2O) p -group, wherein p is an integer from 1 to 6. Those skilled in the art will recognize that one or more of the groups listed may exist in the form of a divalent (diradical) species, such as C1-C6 alkylene, etc.

[0491] In some embodiments, the linker includes a p-aminobenzyl group (PAB). In one embodiment, the p-aminobenzyl group is disposed between the cytotoxic drug and the protease cleavage site in the linker. In one embodiment, the p-aminobenzyl group is part of a p-aminobenzyloxycarbonyl unit. In one embodiment, the p-aminobenzyl group is part of a p-aminobenzylamido unit.

[0492] In some embodiments, the linker comprises a dipeptide selected from the group consisting of: Phe-Lys, Val-Lys, Phe-Ala, Phe-Cit, Val-Ala, Val-Cit, and Val-Arg. In some embodiments, the linker comprises one or more of: PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0493] In some embodiments, the linker comprises PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0494] In some embodiments, the linker comprises a combination of one or more of the following: a peptide, an oligosaccharide, -(CH2) p -、-(CH2CH2O) p -, PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB or Ala-PAB.

[0495] In some embodiments, the linker comprises -(C=O)(CH2) p -unit, wherein p is an integer from 1 to 6.

[0496] In some embodiments, the linker comprises -(CH2) n -unit, wherein n is an integer from 2 to 6. In some embodiments, the linker comprises -((CH2) n , wherein n is 6. In some embodiments, LZ is

[0497]

[0498] wherein S is a sulfur atom and represents a reactive substituent (eg, a -SH group from a cysteine ​​residue) present in the antibody or antigen-binding fragment thereof that binds CD5.

[0499] In some embodiments, the linker comprises ((CH2) m O) n (CH2) m -group and heteroaryl group, wherein n and m are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, wherein the heteroaryl group is triazole. In some embodiments, ((CH2) m O) n (CH2) m – group and triazole together include

[0500] Wherein n is 1 to 10, and the wavy line indicates the point of attachment to an additional linker component, chemical moiety Z, or amanita phalloidin. Other linkers that can be used in the methods and compositions described herein are described in US2019 / 0144504, which is incorporated herein by reference.

[0501] In one specific embodiment, the linker comprises PAB-Ala-Val-propionyl represented by the structure

[0502]

[0503] The wavy lines indicate the points of attachment of the cytotoxin and the reactive moiety Z'.

[0504] In another specific embodiment, the linker comprises PAB-Cit-Val-propionyl represented by the structure

[0505]

[0506] Wherein the wavy line indicates the attachment point of the cytotoxin and the reactive moiety Z'. Such a PAB-dipeptide-propionyl linker is disclosed, for example, in Patent Application Publication No. WO2017 / 149077, which is incorporated herein by reference in its entirety. In addition, the cytotoxins disclosed in WO2017 / 149077 are incorporated herein by reference.

[0507] Those skilled in the art will recognize that any one or more of the chemical groups, moieties, and features disclosed herein can be combined in a variety of ways to form linkers useful for conjugation of antibodies and cytotoxins as disclosed herein. Other linkers that can be used in conjunction with the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0508] In certain embodiments, the intermediate as the joint precursor reacts with the drug moiety under appropriate conditions. In certain embodiments, a reactive group is used on the drug and / or intermediate or joint. The reaction product between the drug and the intermediate or the derivatized drug reacts with the antibody or Fab under appropriate conditions subsequently. Alternatively, the joint or intermediate can react with the antibody or the derivatized antibody at first, and then react with the drug or the derivatized drug. Such conjugation reaction will now be described more fully.

[0509] Many different reactions can be used to covalently attach a joint or drug-joint conjugate to an antibody or its Fab. Suitable attachment points on the antibody molecule include the amine group of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the thiol group of cysteine, and various parts of aromatic amino acids. For example, a carbodiimide reaction can be used to connect the carboxyl (or amino) group on the compound to the amino (or carboxyl) group on the antibody portion for non-specific covalent attachment. In addition, bifunctional agents such as dialdehydes or imidoesters can also be used to connect the amino group on the compound to the amino group on the antibody portion. Schiff base reaction can also be used for the attachment of drugs to binding agents. This method includes periodic acid oxidation of drugs containing ethylene glycol or hydroxyl groups to form aldehydes, which then react with the binding agent. Attachment occurs via the formation of Schiff bases with the amino groups of the binding agent. Isothiocyanates can also be used as coupling agents for covalently attaching drugs to binding agents. Other techniques are known to the technician and are within the scope of the present disclosure.

[0510] Linkers useful for conjugation to the antibodies or antigen-binding fragments described herein include, but are not limited to, linkers comprising a chemical moiety Z formed by a coupling reaction as depicted below in Table 3. The curved lines represent the points of attachment to the antibody or antigen-binding fragment and the cytotoxic molecule, respectively.

[0511] Table 3. Exemplary chemical moieties Z formed by coupling reactions in the formation of antibody drug conjugates

[0512]

[0513]

[0514]

[0515]

[0516] Those skilled in the art will recognize that the reactive substituent Z' attached to the linker and the reactive substituent on the antibody or antigen-binding fragment thereof participate in a covalent coupling reaction to produce the chemical moiety Z, and will recognize the reactive substituent Z'. Thus, an antibody drug conjugate useful in conjunction with the methods described herein can be formed by reacting the antibody or antigen-binding fragment thereof with a linker or cytotoxin-linker conjugate as described herein, the linker or cytotoxin-linker conjugate comprising the reactive substituent Z', which is suitable for reacting with the reactive substituent on the antibody or antigen-binding fragment thereof to form the chemical moiety Z.

[0517] As depicted in Table 3, examples of suitable reactive substituents on the linker and the antibody or antigen-binding fragment thereof include nucleophile / electrophile pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, etc.), diene / dienophile pairs (e.g., especially, azide / alkyne pairs or diene / α,β-unsaturated carbonyl pairs), etc. Coupling reactions between reactive substituents to form the chemical moiety Z include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine or hydroxylamine condensation, hydrazine formation, amidation, esterification, disulfide formation, cycloaddition (e.g., especially, [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reaction forms known in the art or described herein. Preferably, the linker comprises an electrophilic functional group to react with a nucleophilic functional group on the antibody or antigen-binding fragment thereof.

[0518] Reactive substituents that may be present in an antibody or antigen-binding fragment thereof as disclosed herein include, but are not limited to, nucleophilic groups such as (i) an N-terminal amine group, (ii) a side chain amine group, e.g., lysine, (iii) a side chain thiol group, e.g., cysteine, and (iv) a sugar hydroxyl or amino group, wherein the antibody is glycosylated. Reactive substituents that may be present in an antibody or antigen-binding fragment thereof as disclosed herein include, but are not limited to, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine ​​residues, as well as propargyl, azido, halogenated aryl (e.g., fluoroaryl), halogenated heteroaryl (e.g., fluoroheteroaryl), halogenated alkyl, and halogenated heteroalkyl moieties of non-naturally occurring amino acids. In some embodiments, reactive substituents present in an antibody or antigen-binding fragment thereof as disclosed herein include amine or thiol moieties, are amine or thiol moieties. Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive to be conjugated with linker reagents by treating with a reducing agent such as DTT (dithiothreitol). Therefore, in theory, each cysteine ​​bridge will form two reactive thiol nucleophiles. Other nucleophilic groups can be introduced into antibodies by the reaction of lysine and 2-iminothiolane (2-iminothiolane) (Traut reagent), resulting in amine conversion into thiol. Reactive thiol groups can be introduced into antibodies (or their fragments) by introducing one, two, three, four or more cysteine ​​residues (for example, preparing mutant antibodies comprising one or more non-natural cysteine ​​amino acid residues). U.S. Patent No. 7,521,541 teaches antibody engineering by introducing reactive cysteine ​​amino acids.

[0519] In some embodiments, the reactive moiety Z' attached to the linker is a nucleophilic group that reacts with an electrophilic group present on the antibody. Useful electrophilic groups on the antibody include, but are not limited to, aldehyde and keto carbonyl groups. The heteroatom of the nucleophilic group can react with the electrophilic group on the antibody and form a covalent bond with the antibody. Useful nucleophilic groups include, but are not limited to, hydrazides, oximes, amino groups, hydroxyls, hydrazines, thiosemicarbazones, hydrazide carboxylates, and aryl hydrazides.

[0520] In some embodiments, Z is a reaction product between a reactive nucleophilic substituent (such as an amine and thiol moiety) present in an antibody or antigen-binding fragment thereof and a reactive electrophilic substituent Z'. For example, Z' can be, among others, a Michael acceptor (e.g., maleimide), an activated ester, an electron-deficient carbonyl compound, or an aldehyde. Several representative and non-limiting examples of reactive substituents Z' and resulting chemical moieties Z are provided in Table 4.

[0521] Table 4. Complementary reactive substituents and chemical moieties

[0522]

[0523] For example, suitable linkers for synthesizing drug-antibody conjugates and drug-ligand conjugates include, but are not limited to, reactive substituents Z', such as maleimide or haloalkyl groups. These can be attached to linkers by reagents such as, inter alia, succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, which are described, for example, in Liu et al., 18: 690-697, 1979, the disclosure of which is incorporated herein by reference when it relates to linkers for chemical conjugation.

[0524] In some embodiments, the reactive substituent Z' attached to the linker L is a maleimide, an azide, or an alkyne. An example of a maleimide-containing linker is a non-cleavable maleimidocaproyl-based linker, which is particularly useful for conjugation of microtubule disrupting agents such as auristatins. Such a linker is described by Doronina et al. Bioconjugate Chem. 17: 14-24, 2006, the disclosure of which is incorporated herein by reference when it relates to linkers for chemical conjugation.

[0525] In some embodiments, the reactive substituent Z′ is -(C=O)- or -NH(C=O)-, such that the linker can be attached to the antibody or its antigen-binding fragment via an amide or urea moiety, respectively, resulting from the reaction of the -(C=O)- or -NH(C=O)- group with an amino group of the antibody or its antigen-binding fragment.

[0526] In some embodiments, the reactive substituent is an N-maleimido group, a halo-substituted N-alkylamido group, a sulfonyloxy N-alkylamido group, a carbonate group, a sulfonyl halide group, a thiol group or a derivative thereof, an alkynyl group containing an internal carbon-carbon triple bond, a (hetero)cycloalkynyl group, a bicyclo[6.1.0]non-4-yn-9-yl group, an alkenyl group containing an internal carbon-carbon double bond, a cycloalkenyl group, a tetrazinyl group, an azido group, a phosphine group, a nitrile oxide group, a nitrone group, a nitrile imine group, a diazo group, a ketone group, an (O-alkyl)hydroxyamino group, a hydrazine group, a halo-substituted N-maleimido group, a 1,1-bis(sulfonylmethyl)methylcarbonyl group or an elimination derivative thereof, a carbonyl halide group, or an allenamide group, each of which can be optionally substituted. In some embodiments, the reactive substituents include a cycloalkene group, a cycloalkynyl group, or an optionally substituted (hetero)cycloalkynyl group.

[0527] Non-limiting examples of Amanita toxin-linker conjugates comprising a reactive substituent Z' suitable for reacting with a reactive residue on an antibody or antigen-binding fragment thereof include, but are not limited to, 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-Amanita toxin, 7'C-(4-(6-(maleimido)hexanoylamino)piperidin-1-yl)-Amanita toxin, 7'C-(4-(6-(6-(maleimido)hexanoylamino)hexanoyl)piperazin-1-yl)-Amanita toxin, 7'C-(4-(6-(6-(maleimido)hexanoylamino)hexanoyl)piperazin-1-yl)-Amanita toxin, 7'C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperazin-1-yl)-Amanita toxin, 7'C-(4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)piperazin-1-yl)- 'C-(4-(2-(6-(maleimido)hexanoylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(6-(maleimido)hexanoylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin, yl)-amanitin toxin, 7'C-(4-(2-(2-bromoacetylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(3-(pyridin-2-yldisulfanyl)propionylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(4-(maleimido)butyrylamino)ethyl)piperidin-1-yl)-amanitin toxin 1-yl)-amanitin toxin, 7'C-(4-(2-(maleimido)acetyl)piperazin-1-yl)-amanitin toxin, 7'C-(4-(3-(maleimido)propionyl)piperazin-1-yl)-amanitin toxin, 7'C-(4-(4-(maleimido)butyryl)piperazin-1-yl)-amanitin toxin, 7'C-(4 -(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(3-((6-(maleimido)hexanoylamino)methyl)pyrrolidin-1-yl)-amanitin toxin, 7'C-(3-((6-(6-(maleimido)hexanoylamino)hexanoylamino)methyl)pyrrolidin-1-yl)-amanitin toxin, 7'C-(3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrolidin-1-yl)-amanitin toxin, 7'C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)methyl)pyrrolidin-1-yl)-amanitin toxin,7'C-(4-(2-(6-(2-(aminooxy)acetylamino)hexanoylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(4-(2-(aminooxy)acetylamino)butyrylamino)ethyl)piperidin-1-yl)-amanitin toxin, 7'C-(4-(4-(2-(aminooxy)acetylamino)butyryl)piperazin-1-yl)-amanitin toxin, 7'C-(4-(6-(2-(aminooxy)acetylamino)hexanoyl)piperazin-1-yl)-amanitin toxin, 7'C-((4-(6-(maleimido)hexanoylamino)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(maleimido)hexanoylamino)piperidin-1-yl)methyl)-amanitin toxin methyl)-amanitin toxin, (S)-7'C-((3-((6-(maleimido)hexanoylamino)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(6-(maleimido)hexanoylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(maleimido)hexanoylamino)hexanoylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, (R)-7'C-((3-((6-(maleimido)hexanoylamino)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, (S)-7'C-((3-((6-(maleimido)hexanoylamino)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(6-(maleimido)hexanoylamino)hexanoylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(6-(maleimido)hexanoylamino)ethyl)piperazin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(6-(6-(maleimido)hexanoylamino)ethyl)piperazin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(6-(6-(maleimido)hexanoylamino)hexanoylamino)ethyl)piperazin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl)-Amanita toxin ) methyl)-amanitin toxin, 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)ethyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((3-((6-(6-(maleimido)hexanoylamino)hexanoylamino)-S-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((6-(6-(maleimido)hexanoylamino)hexanoylamino)-R-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin,7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-R-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(3-carboxypropionamido)ethyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(6-(6-(maleimido)hexanoylamino)hexanoyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin )hexanoyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(maleimido)acetyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(3-(maleimido)propionyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(4-(maleimido)butyryl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(2-(maleimido)acetylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(4-(maleimido)butyrylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C- ((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((6-(maleimido)hexanoylamino)methyl)azetidin-1-yl)methyl)-amanitin toxin, 7'C-((3-(2-(6-(maleimido)hexanoylamino)ethyl)azetidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amanitin toxin, 7'C-((3-((2-(6-(maleimido)hexanoylamino)ethyl)azetidin-1-yl)methyl)-amanitin toxin toxin, 7'C-(((4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)azetidin-1-yl)methyl)-amanitin toxin, 7'C-(((2-(6-(maleimido)-N-methylhexanoylamino)ethyl)(methyl)amino)methyl)-amanitin toxin, 7'C-(((4-(6-(maleimido)-N-methylhexanoylamino)butyl(methyl)amino)methyl)-amanitin toxin, 7'C-((2-(2-(6-(maleimido)hexanoylamino)ethyl)aziridine-1-yl)methyl)-amanitin toxin,7'C-((2-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)ethyl)azetidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(6-(6-(2-(aminooxy)acetylamino)hexanoylamino)hexanoyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-oyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(2-(aminooxy)acetylamino)acetyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(3-(2-(aminooxy)acetylamino)acetyl)piperazin-1-yl)methyl)-amanitin toxin oxy)acetylamino)propionyl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(4-(2-(aminooxy)acetylamino)butyryl)piperazin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(6-(2-(aminooxy)acetylamino)hexanoylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(2-(2-(aminooxy)acetylamino)acetylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(2-(4-(2-(aminooxy)acetylamino)butyrylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin, 7'C-((4-(20-(aminooxy)acetylamino)butyrylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin methyl)-amanitin toxin, 7'C-(((2-(6-(2-(aminooxy)acetylamino)-N-methylhexanoylamino)ethyl)(methyl)amino)methyl)-amanitin toxin, 7'C-(((4-(6-(2-(aminooxy)acetylamino)-N-methylhexanoylamino)butyl)(methyl)amino)methyl)-amanitin toxin, 7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoylamino)methyl)pyrrolidin-1-yl)-S-methyl)-amanitin toxin, 7'C-((3-((6-(4-(( 7'C-((4-(2-(2-bromoacetylamino)ethyl)piperazin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(2-bromoacetylamino)ethyl)piperidin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(2-bromoacetylamino)ethyl)piperidin-1-yl)methyl)-Amanita toxin, 7'C-((4-(2-(2-bromoacetylamino)ethyl)piperidin-1-yl)methyl)-Amanita toxin, 6'O-(6-(6-(maleimido)hexanoylamino)hexyl)-Amanita toxin, 6'O-(5-(4-((maleimido)methyl)cyclohexanecarboxamido)pentyl)-Amanita toxin,6'O-(2-((6-(maleimido)hexyl)oxy)-2-oxoethyl)-amanitin toxin, 6'O-((6-(maleimido)hexyl)carbamoyl)-amanitin toxin, 6'O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexyl)carbamoyl)-amanitin toxin, 6'O-(6-(2-bromoacetamido)hexyl)-amanitin toxin, 7'C-(4-(6-(azido)hexanoylamino)piperidin-1-yl)-amanitin toxin, 7'C-(4-(hex-5-ynoylamino)piperidin-1-yl)-amanitin toxin, 7'C-(4-(2-(6-(maleimido)hexanoylamino)piperidin-1-yl)-amanitin toxin The toxins of the present invention are 6'O-(6-((6-(11,12-didehydro-5,6-dihydro-dibenzo[b,f]azocin-5-yl)-6-oxohexanoylamino)hexyl)-amanitin toxin, 6'O-(6-(hex-5-ynylamino)hexyl)-amanitin toxin, 6'O-(6-(2-(aminooxy)acetylamido)hexyl)-amanitin toxin, 6'O-((6-aminooxy)hexyl)-amanitin toxin and 6'O-(6-(2-iodoacetylamido)hexyl)-amanitin toxin. ,

[0528] In some embodiments, the chemical moiety Z is selected from Table 3 or Table 4. In some embodiments, the chemical moiety Z is:

[0529]

[0530] wherein S is a sulfur atom and represents a reactive substituent present in the antibody or antigen-binding fragment thereof (such as an anti-CD-5 antibody).

[0531] In some embodiments, an Amanita toxin as disclosed herein is conjugated to a linker-reactive moiety-LZ′ having the formula:

[0532]

[0533] Wherein the wavy line indicates the point of attachment to the substituent on the cytotoxin (eg, amanita toxin).The linker-reactive substituent group LZ' may alternatively be referred to as N-β-maleimidopropionyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB).

[0534] In some embodiments, an Amanita toxin as disclosed herein is conjugated to a linker-reactive moiety-LZ′ having the formula:

[0535]

[0536] Wherein the wavy line indicates the point of attachment to the substituent on the cytotoxin (eg, amanita toxin).The linker reactive substituent group LZ' may alternatively be referred to as N-β-maleimidopropionyl-Val-Cit-p-aminobenzyl (BMP-Val-Cit-PAB).

[0537] In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0538]

[0539] Wherein S is a sulfur atom, representing a reactive substituent present in an antibody or antigen-binding fragment thereof (such as an anti-CD-5 antibody). The wavy line at the end of the linker indicates the point of attachment to the Amanita phalloidin toxin.

[0540] In some embodiments, the linker L and chemical moiety Z after conjugation to the antibody (collectively referred to as LZ-Ab) have the structure:

[0541]

[0542] The foregoing linker moieties and amanita toxin-linker conjugates, as well as other linker moieties and amanita toxin-linker conjugates that can be used in conjunction with the compositions and methods described herein, are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220 and Patent Application Publication No. WO2017 / 149077, the disclosures of each of which are incorporated herein by reference in their entirety.

[0543] The foregoing linker moieties and amanita toxin-linker conjugates, as well as other linker moieties and amanita toxin-linker conjugates that can be used in conjunction with the compositions and methods described herein, are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220 and Patent Application Publication No. WO2017 / 149077, the disclosures of each of which are incorporated herein by reference in their entirety.

[0544] In one embodiment, a CD5 antibody or antigen-binding fragment described herein can be conjugated to an Amanita ani toxin to form a conjugate represented by the formula Ab-ZL-Am, wherein Ab is a CD5 antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an Amanita ani toxin, each as described herein.

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

[0546]

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

[0548]

[0549] In some embodiments, Am-LZ-Ab is:

[0550]

[0551] In some embodiments, Am-LZ-Ab is:

[0552]

[0553] In some embodiments, Am-LZ-Ab is:

[0554]

[0555] Preparation of Antibody Drug Conjugates

[0556] In the ADC of Formula I as disclosed herein, the anti-CD5 antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxic drug moieties (D) via a linker L and a chemical moiety Z as disclosed herein, for example, about 1 to about 20 drug moieties per antibody. The ADC of the present disclosure can be prepared by several routes using organic chemical reactions, conditions and reagents known to those skilled in the art, including: (1) reacting a reactive substituent of an antibody or antigen-binding fragment thereof with a divalent linker reagent to form Ab-ZL as described above, followed by reaction with a drug moiety D; or (2) reacting a reactive substituent of a drug moiety with a divalent linker reagent to form DLZ′, followed by reaction with a reactive substituent of an antibody or antigen-binding fragment thereof as described above. Additional methods for preparing ADCs are described herein.

[0557] In another aspect, the anti-CD5 antibody or antigen-binding fragment thereof has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. The ADC is then formed by conjugation via the sulfur atom of the sulfhydryl group as described above. Reagents that can be used to modify lysine include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).

[0558] In another aspect, the anti-CD5 antibody or antigen-binding fragment thereof may have one or more carbohydrate groups that may be chemically modified to have one or more sulfhydryl groups. The ADC is then formed by conjugation via the sulfur atom of the sulfhydryl group as described above.

[0559] In yet another aspect, the anti-CD5 antibody may have one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) group (see, e.g., Laguzza et al., J. Med. Chem. 1989, 32(3), 548-55). The ADC is then formed by conjugation with the corresponding aldehyde as described above. Other protocols for modifying proteins to attach or associate cytotoxins are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), incorporated herein by reference.

[0560] Methods for conjugating linker-drug moieties to cell-targeting proteins such as antibodies, immunoglobulins, or fragments thereof are found in, e.g., U.S. Pat. No. 5,208,020; U.S. Pat. No. 6,441,163; WO2005037992; WO2005081711; and WO2006 / 034488, all of which are expressly incorporated herein by reference in their entirety.

[0561] Route of administration and dosage

[0562] Alternatively, a fusion protein comprising the antibody and the cytotoxic agent may be prepared, for example, by recombinant techniques or peptide synthesis. The length of DNA may comprise the corresponding regions encoding the two parts of the conjugate, adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0563] The ADC described herein can be administered to a patient (e.g., a human patient suffering from an immune disease or cancer) in a variety of dosage forms. For example, the ADC described herein can be administered to a patient suffering from an immune disease or cancer in the form of an aqueous solution (such as an aqueous solution containing one or more pharmaceutically acceptable excipients). Suitable pharmaceutically acceptable excipients for use with the compositions and methods described herein include viscosity modifiers. The aqueous solution can be sterilized using techniques known in the art.

[0564] Pharmaceutical formulations containing anti-CD5 ADCs as described herein are prepared by mixing such ADCs with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the doses and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 1 0 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0565] The amount of the ADC administered should be sufficient to consume cells, such as activated T cells, which reject CAR cell therapy. The determination of the therapeutic effective dose is within the ability of practitioners in the art, however, as an example, in the embodiments of the method for treating immune diseases or cancer using systemic administration of ADC described herein, effective human doses will be within the range of 0.1mg / kg-150mg / kg (e.g., 5mg / kg, 10mg / kg, 25mg / kg, 50mg / kg, 75mg / kg, 100mg / kg, 150mg / kg, etc.). The route of administration can affect the recommended dose. Depending on the mode of administration adopted, it is envisioned that repeated systemic doses are in order to maintain effective levels, for example, in order to reduce the risk of CAR-T cell rejection.

[0566] The anti-CD5 ADC described herein can be administered by a variety of routes, such as oral administration, transdermal administration, subcutaneous administration, intranasal administration, intravenous administration, intramuscular administration, intraocular administration, or parenteral administration. In any given case, the most suitable route of administration will depend on the specific ADC, the patient, the pharmaceutical formulation method, the administration method (e.g., administration time and administration route), the patient's age, weight, sex, severity of the disease being treated, the patient's diet, and the patient's excretion rate.

[0567] The effective dose of the anti-CD5 ADC described herein can range from about 0.001 mg / kg body weight to about 100 mg / kg body weight per single (e.g., bolus) administration, multiple administrations, or continuous administrations, or can be in the range of reaching the optimal serum concentration of anti-CD5 ADC (e.g., 0.0001 μg / mL-5000 μg / mL serum concentration). The dose of anti-CD5 ADC can be administered once or more (e.g., 2-10 times) per day, week, or month to human subjects who have received CAR therapy, are receiving CAR therapy at the same time, or will receive CAR therapy at a time point after delivery of anti-CD5 ADC. Anti-CD5 ADC can be administered to human patients at one or more doses. In one embodiment, anti-CD5 ADC can be administered before CAR therapy in an amount sufficient to reduce the amount of host reactive T cells, for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more. Example

[0568] The following examples are presented to provide one of ordinary skill in the art with a description of how the compositions and methods described herein may be used, prepared and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0569] Example 1: In vitro binding analysis of anti-CD5 antibodies.

[0570] To determine the binding characteristics of anti-CD5 antibody 5D7 hIgG1, antibody binding studies were performed using biolayer interferometry (BLI) with Pall ForteBio Octet Red96 at 25 degrees Celsius in 1×PBS supplemented with 0.1% w / v bovine serum albumin. Purified human anti-CD5 antibody (5D7) was immobilized on an anti-human Fc biosensor (AHC; PallForteBio 18-5063) and incubated with 50 nM purified human CD5 extracellular domain. The binding characteristics of anti-CD5 antibody 5D7 are shown in Table 5. The anti-human CD5 antibody 5D7 used in Examples 1 to 5 is a humanized form of the mouse antibody 5D7 (see US 2008 / 0254027). The sequence of antibody 5D7 as used herein is described in SEQ ID No: 53 and SEQ ID No: 54 (heavy chain and light chain variable region amino acid sequences) and SEQ ID No: 47 to SEQ ID No: 52 (heavy chain and light chain CDRs).

[0571] Table 5: Binding kinetics of 5D7 to human CD5 extracellular domain

[0572]

[0573] Example 2: In vitro cell line binding analysis of anti-CD5 antibodies

[0574] MOLT-4 cells (i.e., immortalized human T lymphoblastoid cell line) were plated at 20,000 cells / well and stained with titers of indicated mouse anti-CD5 antibodies (i.e., L17F12, UCHT2, 205919, and CRIS-1) at 4°C for 2 hours. A constant amount of secondary anti-mouse AF488 dye was added for 30 minutes at 4°C. After washing, the plates were run on a flow cytometer, and binding of the indicated antibodies (and negative controls, i.e., mIgG1) was determined based on the geometric mean fluorescence intensity in the AF488 channel. Results from these assays are presented in Figure 1 Available in.

[0575] like Figure 1 As shown in Figure 2, murine anti-CD5 antibodies L17F12 (Thermo Fisher), UCHT2 (BioLegend), 205919 (Novus Biologicals), and CRIS-1 (Novus Biologicals) bind to human T lymphoblastoid cells (i.e., MOLT-4 cells), where EC 50 =207pM (L17), 354pM (UCH), 1350pM (205) and 43pM (CRIS).

[0576] Example 3: In vitro primary cell binding analysis of anti-CD5 antibodies

[0577] Primary human T cells were cultured at 8×10 4 Cells / well were plated and stained with a titer of human anti-CD5 antibody 5D7 for 2 hours at 37°C. A constant amount of secondary anti-mouse AF488 dye was added for 30 minutes at 4°C. After washing, the plates were run on a flow cytometer and binding of the anti-CD5 5D7 antibody (and negative control, i.e., hIgG1) was determined based on the geometric mean fluorescence intensity in the AF488 channel. Results from these assays are presented in Figure 2 Available in.

[0578] like Figure 2 As shown in Figure 2, the anti-CD5 antibody 5D7 binds to primary human T cells, where EC 50 =3.0pM.

[0579] Example 4. In vitro analysis of anti-CD5-amanitin antibody drug conjugates (ADCs) using an in vitro T cell killing assay

[0580] The anti-CD5 antibody 5D7 was conjugated to amanita toxin (amanitin) with a cleavable linker to form an anti-CD5 5D7 ADC. Anti-CD5 5D7-ADCs with a drug-to-antibody ratio (DAR) of about 6 (interchain DAR6) and anti-CD5 5D7-ADCs with a DAR of about 2 (prepared using site-specific conjugation via a D265C mutation) were tested. In addition, a fast half-life variant of the anti-CD5 5D7-ADC was generated by introducing a H435A mutation in the Fc region.

[0581] Each anti-CD5 5D7-ADC was evaluated using an in vitro human T cell killing assay.

[0582] Cryopreserved negatively selected primary human T cells were thawed and stimulated with anti-CD3 antibody and IL-2. At the start of the assay, 2 × 10 4 T cells, and the indicated ADC or non-conjugated anti-CD5 antibody was added to the wells at different concentrations between 0.003nM and 30nM, and then placed in an incubator at 37°C and 5% CO2. After five days of culture, the cells were analyzed by flow cytometry. The cells were stained with the viability marker 7-AAD and run on a volume flow cytometer.

[0583] The number of surviving T cells ( Figure 3A and Figure 3B ) was determined by FSC and SSC and 7-AAD. Non-conjugated anti-CD55D7 antibody was used as a comparison object ( Figure 3A ).

[0584] like Figure 3A As shown in , anti-CD5 5D7-ADC with a DAR of about 6 exhibited potent and specific killing of human T cells (IC50=3.7 pM), while T cells remained viable in the presence of non-conjugated ("naked") anti-CD5 5D7 antibody. Figure 3B As shown in Figure 2, the ADC with a site-specific (D265C) DAR of about 2 maintained an effective T cell killing level similar to that of the DAR 6 ADC (IC50 = 5.0 pM). The fast half-life variant of the anti-CD5 5D7-ADC (H435A) exhibited similar T cell killing levels (IC50 = 4.9 pM; Figure 3B ).

[0585] Example 5. Analysis of T cell depletion using the hNSG mouse model

[0586] In vivo T cell depletion assays were performed using humanized NSG mice (Jackson Laboratories). The anti-CD5 antibody 5D7 was conjugated to amanita phalloides toxin (amanitin) with a cleavable linker to form an anti-CD5 5D7-ADC. As described above, the anti-CD5 5D7-ADC was prepared to have a DAR of about 6 or a DAR of about 2. Each anti-CD5 5D7 ADC (DAR6 or DAR2) was administered to humanized mice as a single intravenous injection (0.3 mg / kg, 1 mg / kg or 3 mg / kg for DAR6 ADC, or 1 mg / kg or 3 mg / kg for DAR2 ADC). Peripheral blood cells, bone marrow or thymus samples were collected on day 7, and the absolute number of CD3+ T cells was determined by flow cytometry (for DAR6 ADC see Figure 4A-4B , and for DAR2 ADC see Figures 5A-5C ).

[0587] like Figure 4A-4B As shown in , humanized NSG mice treated with 0.3 mg / kg, 1 mg / kg or 3 mg / kg DAR6 anti-CD55D7-ADC showed effective T cell depletion in peripheral blood or bone marrow, while thymic T cells were depleted after treatment with 1 mg / kg or 3 mg / kg of DAR6 anti-CD55D7-ADC. Negative controls used in this in vivo experiment included human IgG1 non-specific for CD5 (as naked antibody (huIgG1), and conjugated with Amanita phalloides toxin (huIgG1-AM)). FIG. 4A to FIG. 4B As described in , naked huIgG1 control and conjugated control were used for peripheral blood ( Figure 4A ) and bone marrow ( Figure 4B ) had no effect on T cell depletion in the control group, as these controls were comparable to the PBS control. Anti-CD52 antibody (antibody YTH34.5) was also used as a control and was also able to deplete peripheral and bone marrow T cells at a dose of 25 mg / kg.

[0588] like Figures 5A-5C As shown in , humanized NSG mice treated with 1 mg / kg or 3 mg / kg of the site-specific DAR2 anti-CD55D7-ADC exhibited effective T cell depletion in peripheral blood, bone marrow, and thymic T cells. FIG. 5A to FIG. 5C In each of the experiments, naked antibody 5D7 was also used as a control. Figure 5A As described in , antibody 5D7 is able to deplete peripheral T cells (relative to a nonspecific human IgG1 control or PBS), but not myeloid T cells or thymic T cells, whereas 5D7-AM ADC is effective in depleting both the bone marrow and the thymus, as shown in Figure 5B and Figure 5C Described in .

[0589] Example 6. Administration of allogeneic CAR-T cells in a mouse model

[0590] The following studies were performed to evaluate the levels of CAR-T cells present in allogeneic recipients under different conditions.

[0591] This study used a murine allogeneic CAR-T model.

[0592] On day 0, mice in the first treatment group were administered 1×10 7 cells / kg to 1×10 9 The mice were treated with a priming dose of allogeneic T cells at 100 cells / kg of allogeneic T cells. On day 3, the mice were administered anti-CD5-α-amanitin ADC at a dose of 3 mg / kg. On day 10, after the ADC was substantially cleared from the mouse blood, the mice were administered allogeneic CAR-T cells. The CAR-T cells were from the same donor as the allogeneic T cells administered on day 0.

[0593] Mice in the second treatment group were treated using the same regimen as the first treatment group, but non-conjugated anti-CD5 antibody was administered on day 3 instead of the anti-CD5 ADC.

[0594] Mice in the third treatment group were treated using the same regimen as the first treatment group, but an isotype control antibody conjugated to α-amanitin was administered on day 3 instead of the anti-CD5 ADC.

[0595] Mice in the fourth treatment group were treated using the same regimen as the first treatment group, but a priming dose of autologous T cells was administered on day 0 instead of allogeneic T cells.

[0596] Mice in the fifth treatment group were administered allogeneic CAR-T cells on day 10 without pre-treatment.

[0597] Mice in the sixth treatment group were administered autologous CAR-T cells on day 10 without pre-treatment.

[0598] The number of CAR-T cells present in the spleen and peripheral blood of mice from each treatment group was determined on days 14, 17, and 30. The number of CD5+ activated T cells in the spleen and peripheral blood of mice from each treatment group was determined on day 9. Throughout the study, mice were monitored for rejection symptoms.

[0599] Example 7. Administration of anti-CD5 antibody drug conjugates to human patients to prevent rejection of allogeneic cell therapy

[0600] Human patients are selected to receive allogeneic cell therapy, such as allogeneic CAR cell therapy. In order to inhibit or prevent rejection of allogeneic cells, anti-CD5 antibody drug conjugates (ADCs) are administered according to the methods disclosed herein. The doctor performs the following treatment steps.

[0601] First, an initial amount of allogeneic cells is intravenously administered to the patient in an amount sufficient to elicit a sensitizing immune response to the allogeneic cells. In the sensitization step, the allogeneic cells are administered to the patient in order to elicit an immune response, thereby generating endogenously activated CD5+ T cells.

[0602] Subsequently, an anti-CD5 ADC is administered to the patient, the anti-CD5 ADC comprising an anti-CD5 antibody conjugated to a cytotoxin via a linker. The anti-CD5 ADC is administered in an amount effective to deplete endogenous CD5+ activated T cells. After administration of the anti-CD5 ADC, the level of CD5+ activated T cells in the patient is assessed to confirm depletion.

[0603] Next, a therapeutically effective amount of allogeneic cells expressing CAR is administered to the patient. The allogeneic cells are derived from the same donor as the cells administered to the patient during the sensitization step. Relative to patients receiving allogeneic cell therapy without sensitization and administration of anti-CD5 ADC, the acceptance of allogeneic cells by the recipient patient is promoted and the risk of rejection is reduced.

[0604] Table 6. Sequence overview

[0605]

[0606]

[0607] [0...

Claims

1. Use of an anti-CD5 antibody drug conjugate (ADC) in the preparation of a composition for promoting acceptance of immune cells expressing a chimeric antigen receptor (CAR) by human subjects suffering from cancer or autoimmune diseases, wherein the anti-CD5 ADC comprises an anti-CD5 antibody or an antigen-binding fragment thereof conjugated to a cytotoxin via a linker, wherein when the composition is used, (a) the anti-CD5 ADC is administered to the human subject suffering from cancer or autoimmune disease; and (b) a therapeutically effective amount of immune cells expressing the CAR is administered to the human subject, The CAR comprises an extracellular domain, a transmembrane domain and a cytoplasmic domain that bind to a tumor antigen or an antigen associated with an autoimmune disease, and The anti-CD5 antibody or antigen-binding fragment thereof conjugated to a cytotoxin is represented by the formula Ab-ZL-Am, wherein Ab is the antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an Amanita phalloidin toxin represented by formula (III): Where R1 is H, OH, OR A OR C ; R2 is H, OH, OR B OR C ; R A and R B When present, taken together with the oxygen atom to which they are attached, form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C or R D ; R4, R5, R6 and R7 are each independently H, OH, OR C , OR D , R C or R D ; R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ; R9 is H, OH, OR C OR D ; Q is -S-, -S(O)- or -SO2-; R C Yes - LZ; R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a connector; and Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the anti-CD5 antibody or antigen-binding fragment thereof, wherein Am comprises exactly one R c Substituent.

2. The use according to claim 1, wherein the human subject has not been administered alemtuzumab before, simultaneously with, or after step (b).

3. The use according to claim 1 or 2, wherein the human subject has not been administered a lymphodepleting chemotherapeutic agent prior to step (b), simultaneously with step (b), or after step (b).

4. The use according to claim 3, wherein the lymphodepleting chemotherapeutic agent is fludarabine, cyclophosphamide, bendamustine and / or pentostatin.

5. The use according to claim 1, wherein the anti-CD5 ADC is administered to the human subject prior to step (b).

6. The use according to claim 1, wherein the anti-CD5 ADC is administered to the human subject 12 hours to 21 days before step (b). The use according to claim 1 , wherein the immune cells are allogeneic cells or autologous cells.

8. The use according to claim 7, wherein the allogeneic cells are allogeneic T cells or allogeneic NK cells.

9. The use according to claim 1, wherein the therapeutically effective amount of allogeneic cells expressing the CAR is 1×10 4 cells / kg to 1.0×10 8 cells / kg.

10. Use of an anti-CD5 antibody drug conjugate (ADC) in the preparation of a composition for treating a human subject with a tumor, wherein the anti-CD5 ADC comprises an anti-CD5 antibody or an antigen-binding fragment thereof conjugated to a cytotoxin via a linker, wherein when the composition is used, (i) the anti-CD5 ADC is administered to the human subject in need thereof, and (ii) a therapeutically effective amount of 1×10 6 Engineered CAR T cells / kg to 1×10 8 The invention relates to a method for treating a human subject wherein the anti-CD5 antibody or antigen-binding fragment thereof conjugated to a cytotoxin is represented by the formula Ab-ZL-Am, wherein Ab is the antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an Amanita phalloidin toxin represented by the formula (III): Where R1 is H, OH, OR A OR C ; R2 is H, OH, OR B OR C ; R A and R B When present, taken together with the oxygen atom to which they are attached, form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C or R D ; R4, R5, R6 and R7 are each independently H, OH, OR C , OR D , R C or R D ; R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ; R9 is H, OH, OR C OR D ; Q is -S-, -S(O)- or -SO2-; R C Yes - LZ; R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a connector; and Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the anti-CD5 antibody or antigen-binding fragment thereof, wherein Am comprises exactly one R c Substituent.

11. The use according to claim 10, wherein the therapeutically effective amount of the engineered CAR T cells is 1×10 6 cells / kg or 2×10 6 cells / kg.

12. The use according to claim 1 or claim 10, wherein the anti-CD5 ADC is administered to the human subject as a single dose or as multiple doses.

13. The use according to claim 1 or claim 10, wherein the anti-CD5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences listed in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and the anti-CD5 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising CDR1, CDR2 and CDR3 having the amino acid sequences listed in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

14. The use according to claim 13, wherein the anti-CD5 antibody or antigen-binding fragment thereof is chimeric or humanized.

15. The use according to claim 14, wherein the anti-CD5 antibody or antigen-binding fragment thereof is of IgG1 isotype or IgG4 isotype.

16. The method of claim 1 or claim 10, wherein the linker (L) is an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl; or comprises a dipeptide or -((CH2) m O) n (CH2) m -, wherein m and n are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

17. The use according to claim 1 or claim 10, wherein the linker of the anti-CD5 ADC is N-β-maleimidopropionyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB).

18. The use according to claim 1 or 10, wherein the anti-CD5 ADC is represented by any one of the following structures:

19. The use according to claim 1 or 10, wherein the anti-CD5 ADC is represented by:

20. The use of claim 1 or claim 10, wherein the anti-CD5 ADC has a serum half-life of 3 days or less.

21. The use according to claim 1 or claim 10, wherein the extracellular domain of the CAR comprises a single-chain Fv antibody or a single-chain T cell receptor.

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

23. The use according to claim 1 or claim 10, wherein the tumor antigen is an antigen selected from the group consisting of: CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR, MG7, TACI, CEA, PSCA, HER2, CD33, ROR2, NKR-2, CD28, TAA, NKG2D or CD123.

24. The use according to claim 23, wherein the tumor antigen is EGFRvIII.

25. The use according to claim 1 or claim 10, wherein the cytoplasmic domain of the CAR comprises a CD28 cytoplasmic signaling domain, a CD3ζ cytoplasmic signaling domain, an OX40 cytoplasmic signaling domain, and / or a CD137 or 4-1BB cytoplasmic signaling domain.

26. The use according to claim 25, wherein the cytoplasmic domain of the CAR comprises a CD3ζ cytoplasmic signaling domain.

27. The use of claim 1 or claim 10, wherein the human subject suffering from cancer suffers from a cancer selected from the group consisting of: adult advanced cancer, neoplasms, liver metastases, solid tumors, mesothelin-positive tumors, and hematological malignancies.

28. The use of claim 1 or claim 10, wherein the human subject suffering from cancer suffers from a cancer selected from the group consisting of: advanced solid tumors and hematopoietic / lymphoid cancers.

29. The method of claim 1 or claim 10, wherein the human subject having cancer has a cancer selected from the group consisting of leukemia, pancreatic cancer, colorectal cancer, ovarian cancer, triple negative breast cancer, colorectal epithelial cancer, gastric cancer, renal cell carcinoma, squamous cell carcinoma of the lung, hepatocellular carcinoma, urothelial carcinoma, small cell lung cancer, non-small cell lung cancer, B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, anaplastic large cell lymphoma, large B cell lymphoma, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, malignant glioma of the brain, myelodysplastic syndrome, and glioblastoma multiforme.

30. The use of claim 1 or claim 10, wherein the human subject having cancer has a cancer selected from the group consisting of: unresectable pancreatic cancer, metastatic colorectal cancer, liver metastasis of colon cancer, relapsed or refractory B-cell lymphoma, relapsed or refractory diffuse large cell lymphoma, primary mediastinal B-cell lymphoma, relapsed mediastinal large B-cell lymphoma, refractory mediastinal large B-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, triple-negative invasive breast cancer, B-cell leukemia, acute leukemia, relapsed or refractory multiple myeloma, multiple myeloma of bone, EGFR-positive colorectal cancer, and blastic plasmacytoid dendritic cell neoplasm.

31. The use of claim 1 or claim 10, wherein the human subject suffering from cancer suffers from a cancer selected from the group consisting of: B-cell non-Hodgkin's lymphoma, refractory non-Hodgkin's lymphoma, acute lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, and acute myeloid leukemia.

32. The use of claim 1 or claim 10, wherein the human subject suffering from cancer has a cancer selected from the group consisting of relapsed or refractory non-Hodgkin's lymphoma, refractory aggressive non-Hodgkin's lymphoma, adult acute lymphoblastic leukemia, childhood acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, and B-cell prolymphocytic leukemia.

33. The use of claim 1 or claim 10, wherein the human subject suffering from cancer suffers from refractory childhood acute lymphoblastic leukemia.

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