阅读说明：本技术 抗cd2抗体药物缀合物(adc)在同种异体细胞疗法中的用途 (Use of anti-CD 2 Antibody Drug Conjugates (ADCs) in allogeneic cell therapy ) 是由 安东尼·博伊坦诺 迈克尔·库克 于 2019-07-23 设计创作，主要内容包括：本发明提供了在经历嵌合抗原受体(CAR)免疫疗法的人类患者中消耗CD2+细胞以便促进对表达CAR的免疫细胞的接受的方法。将抗CD2抗体药物缀合物(ADC)作为调节方案施用至接受自体或同种异体的表达CAR的免疫细胞的人类患者,使得表达CAR的免疫细胞被人类患者接受。本发明的组合物和方法可以与CAR疗法组合使用,以治疗多种疾病,包括自身免疫性疾病和癌症。(The present invention provides methods of depleting CD2+ cells in a human patient undergoing Chimeric Antigen Receptor (CAR) immunotherapy in order to facilitate the acceptance of CAR-expressing immune cells. Administering an anti-CD 2 Antibody Drug Conjugate (ADC) as a regulatory regimen to a human patient receiving autologous or allogeneic CAR-expressing immune cells, such that the CAR-expressing immune cells are received by the human patient. The compositions and methods of the invention can be used in combination with CAR therapy to treat a variety of diseases, including autoimmune diseases and cancer.)

1. A method of promoting acceptance of an immune cell expressing a Chimeric Antigen Receptor (CAR) by a human subject having cancer or an autoimmune disease, the method comprising

(a) Administering an anti-CD 2 Antibody Drug Conjugate (ADC) to a human subject having cancer or an autoimmune disease, wherein the anti-CD 2ADC comprises an anti-CD 2 antibody or antigen-binding fragment thereof conjugated to a cytotoxin via a linker; and

(b) administering to the human subject a therapeutically effective amount of an immune cell expressing a CAR, wherein the CAR comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain that binds to a tumor antigen or an antigen associated with an autoimmune disease.

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

3. The method of claim 1 or2, wherein the human subject is not administered a lymphodepleting chemotherapeutic agent prior to, simultaneously with, or after step (b).

4. The method of claim 3, wherein the lymphodepleting chemotherapeutic agent is fludarabine, cyclophosphamide, bendamustine, and/or pentostatin.

5. The method of any one of claims 1-4, further comprising administering an anti-CD 2ADC to the human subject prior to step (b).

6. The method of any one of claims 1-5, comprising administering anti-CD 2ADC to the human subject from about 12 hours to about 21 days prior to step (b).

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

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

9. The method of any of claims 1-8, wherein the therapeutically effective amount of allogeneic cells expressing the CAR is about 1 x 10⁴Individual cell/kg to about 1.0X 10⁸Individual cells/kg.

10. A method of treating a patient having a tumor, the method comprising (i) an anti-CD 2ADC, wherein the anti-CD 2ADC comprises an anti-CD 2 antibody or antigen-binding fragment thereof conjugated to a cytotoxin via a linker, and (ii) administering a therapeutically effective amount of a composition comprising aAbout 1X 10⁶(ii) engineered CAR T cells/kg to about 1X 10⁸(iii) administering to the patient one engineered CAR T cell/kg.

11. The method of claim 10, wherein the therapeutically effective amount of the engineered CAR T cell is about 1 x 10⁶Individual cell/kg or about 2X 10⁶Individual cells/kg.

12. The method of any one of claims 1-11, wherein the anti-CD 2ADC is administered to the patient as a single dose or as multiple doses.

13. The method of any one of claims 1-12, wherein the anti-CD 2 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. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively, and the anti-CD 2 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. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively.

14. The method of claim 13, wherein the anti-CD 2 antibody or antigen-binding fragment thereof is chimeric or humanized.

15. The method of any one of claims 1-14, wherein the anti-CD 2 antibody or antigen-binding fragment thereof is an IgG1 isotype or an IgG4 isotype.

16. The method of claims 1-15, wherein the cytotoxin is an anti-mitotic agent or an RNA polymerase inhibitor.

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

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

19. The method of claim 18, wherein the amanitin is selected from the group consisting of: alpha-amanitin, beta-amanitin, gamma-amanitin, epsilon-amanitin, amanitin amide, amanitin nontoxic cyclic peptide, amanitin carboxylic acid, and amanitin nontoxic cyclic peptide.

20. The method of claim 17, wherein the anti-CD 2 antibody or antigen-binding fragment thereof conjugated to amanitin is represented by the formula Ab-Z-L-Am, wherein Ab is the antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is amanitin represented by formula (III)

Wherein R is₁Is H, OH, OR_AOR OR_C；

R₂Is H, OH, OR_BOR OR_C；

R_AAnd R_BWhen present, combine together with the oxygen atom to which they are bound to form an optionally substituted 5-membered heterocycloalkyl group;

R₃is H, R_COr R_D；

R₄、R₅、R₆And R₇Each independently is H, OH, OR_C、OR_D、R_COr R_D；

R₈Is OH, NH₂、OR_C、OR_D、NHR_COr NR_CR_D；

R₉Is H, OH, OR_COR OR_D；

X is-S-, -S (O) -or-SO₂-；

R_Cis-L-Z;

R_Dis optionally substituted C₁-C₆Alkyl, optionally substituted C₁-C₆Heteroalkyl, optionally substituted C₂-C₆Alkenyl, optionally substituted C₂-C₆Heteroalkenyl, optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;

l is a linker; and is

Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present within the anti-CD 2 antibody or antigen-binding fragment thereof, wherein Am contains exactly one R_cAnd (4) a substituent.

21. The method of claim 20, wherein the linker is optionally substituted C₁-C₆Alkyl, optionally substituted C₁-C₆Heteroalkyl, optionally substituted C₂-C₆Alkenyl, optionally substituted C₂-C₆Heteroalkenyl, optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl; or comprises a dipeptide or- ((CH)₂)_mO)_n(CH₂)_m-, wherein m and n are each independently selected from 1,2, 3,4, 5,6, 7,8, 9 and 10.

22. The method of claim 16, wherein the anti-mitotic agent is maytansine or an auristatin.

23. The method of claim 22, wherein said auristatin is monomethyl auristatin f (mmaf) or monomethyl auristatin e (mmae).

24. The method of claim 14, wherein the antimitotic agent is Pyrrolobenzodiazepine (PBD) or calicheamicin.

25. The method of any one of claims 1-24, wherein the linker of the ADC is N- β -maleimidopropanoyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB).

26. The method of any one of claims 1-24, wherein the ADC is represented by any one of the following structures:

27. the method of any one of claims 1-24, wherein the ADC is represented by:

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

29. The method of any of claims 1-28, wherein the extracellular domain of the CAR comprises a scFv antibody or a single chain T cell receptor (scTCR).

30. The method of any one of claims 1-28, wherein the extracellular domain comprises a non-immunoglobulin scaffold protein.

31. The method of any one of claims 1-30, 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 (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or CD 123.

32. The method of any of claims 1-31, wherein the cytoplasmic domain of the CAR comprises a CD28 cytoplasmic signaling domain, a CD3 delta cytoplasmic signaling domain, an OX40 cytoplasmic signaling domain, and/or a CD137(4-1BB) cytoplasmic signaling domain.

33. The method of any of claims 1-32, wherein the cytoplasmic domain of the CAR comprises a CD3 delta cytoplasmic signaling domain.

34. The method of any one of claims 1-33, wherein the human subject having cancer has a cancer selected from the group consisting of: leukemia, adult advanced cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple negative breast cancer, hematopoietic/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, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, refractory large-cell lymphoma, small-cell lymphoma, refractory large cell lymphoma, Colorectal epithelial cancer, gastric cancer, pancreatic cancer, triple negative invasive breast cancer, renal cell cancer, lung squamous cell cancer, hepatocellular carcinoma, urothelial cancer, leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, recurrent plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, recurrent or refractory multiple myeloma, bone multiple myeloma, brain malignant glioma, myelodysplastic syndrome, EGFR positive colorectal cancer, glioblastoma multiforme, neoplasms, blastic plasmacytoid dendritic cell tumors, liver metastases, solid tumors, advanced solid tumors, mesothelin positive tumors, hematologic malignancies, and other advanced malignancies.

Technical Field

The present invention relates generally to methods of promoting the acceptance of Chimeric Antigen Receptor (CAR) -expressing immune cells by human subjects through the use of anti-CD 2 Antibody Drug Conjugates (ADCs).

Background

Chimeric Antigen Receptor (CAR) therapy is immunotherapy that uses the body's own immune system to destroy cells that express specific antigens associated with a certain disease, such as cancer. For example, in cancer, CAR therapy recruits and enhances the ability of the patient's immune system to attack the tumor. Over the past few years, this immunotherapy has become a promising and revolutionary therapy. CAR therapy is based on immune cells such as T cells expressing a CAR, which is typically a transmembrane fusion protein combining an extracellular antigen-binding domain (such as scFv) with a cytoplasmic active signaling and "co-stimulatory" domain (signaling from surface receptors to cells). Thus, when an immune cell (such as a T cell) expresses a CAR, the immune cell is able to recognize and kill cells expressing an antigen (e.g., a tumor-associated antigen) targeted by the antigen binding domain of the CAR (Geyer and Brentjens (2016) cytology 18(11): 1393-1409).

Although CAR therapy is an extremely powerful technique, it does bring about serious risks and adverse side effects (Kay and Turtle (2017) Drugs 77(3): 237-. To minimize rejection of CAR-expressing cells by treated patients, lymphodepleting chemotherapy is often used as a modulating therapy in combination with CAR therapy (Wei et al (2017) Exp Hematol oncol.6: 10). For example, the combination of a lymphodepleting agent fludarabine and cyclophosphamide improves the duration of CAR-T cells in recipient patients (Turtle et al (2016) J clinical Invest 126(6): 2123; see also US 20170368101). While regulatory therapies improve the efficacy of CAR-T cells, lymphodepleting chemotherapy often has serious negative side effects.

Summary of The Invention

The invention provides a modulation regimen that can be used with CAR therapy to facilitate acceptance of CAR-expressing immune cells. The methods described herein can be used to facilitate the acceptance of CAR-expressing autoimmune cells or CAR-expressing allogeneic immune cells. Traditionally, acceptance of such cells has been achieved with treatment with lymphodepleting chemotherapeutic agents. Described herein are improved methods of promoting the acceptance of CAR-expressing cells by a recipient patient.

In a first aspect, the invention features a method of promoting the acceptance of an immune cell expressing a Chimeric Antigen Receptor (CAR) by a human subject having cancer or an autoimmune disease by (a) administering an anti-CD 2 Antibody Drug Conjugate (ADC) to the human subject having cancer or an autoimmune disease, wherein the anti-CD 2ADC comprises an anti-CD 2 antibody or antigen-binding fragment thereof conjugated to a cytotoxin via a linker; and (b) administering a therapeutically effective amount of an immune cell expressing a CAR to the human subject, wherein the CAR comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain that binds to a tumor antigen or an antigen associated with an autoimmune disease. In one embodiment, the human subject is not administered alemtuzumab prior to, simultaneously with, or after step (b). In another embodiment, the human subject is not administered a lymphodepleting chemotherapeutic agent prior to, simultaneously with, or after step (b). In yet other embodiments, the lymphodepleting chemotherapeutic agent is fludarabine, cyclophosphamide, bendamustine, and/or pentostatin.

In certain embodiments, the method comprises administering an anti-CD 2ADC to the human subject prior to step (b).

In certain other embodiments, the method comprises administering the anti-CD 2ADC to the human subject about 12 hours to about 21 days prior to step (b).

In certain embodiments, the immune cell is an allogeneic cell or an autologous cell. In yet another embodiment, the allogeneic cells are allogeneic T cells or allogeneic NK cells.

In certain embodiments, the therapeutically effective amount of the CAR-expressing allogeneic cells is about 1 x 10⁴Individual cell/kg to about 1.0X 10⁸Individual cells/kg.

In another aspect, the invention features a method of treating a patient having a tumor by: (i) administering an anti-CD 2ADC, wherein the anti-CD 2ADC comprises an anti-CD 2 antibody or antigen-binding fragment thereof conjugated to a cytotoxin via a linker, and (ii) administering a therapeutically effective amount of about 1 x 10⁶(ii) engineered CAR T cells/kg to about 1X 10⁸(iii) administering to the patient one engineered CAR T cell/kg. In one embodiment, the therapeutically effective amount of the engineered CAR T cell is about 1 x 10⁶Individual cell/kg or about 2X 10⁶Individual cells/kg. In yet another embodiment, the anti-CD 2ADC is administered to the patient as a single dose or as multiple doses.

In certain embodiments, the anti-CD 2 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. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively, and the anti-CD 2 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. 4, SEQ ID No. 5, and SEQ ID No. 6, respectively. In another embodiment, the anti-CD 2 antibody or antigen-binding fragment thereof is chimeric or humanized.

In certain embodiments, the anti-CD 2 antibody or antigen-binding fragment thereof is an IgG1 isotype or an IgG4 isotype.

In certain embodiments, the cytotoxin is an antimitotic agent or an RNA polymerase inhibitor. In other embodiments, the cytotoxin is a maytansine (maytansine), a calicheamicin (calicheamicin), a pyrrolobenzodiazepine, an indolophenyldiazepine, or an auristatin (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 may be selected from tesiline or talirine. In some embodiments, the cytotoxin may be calicheamicin. For example, in some embodiments, the calicheamicin may be ozomicin (ozogamicin).

In other embodiments, the RNA polymerase inhibitor is amatoxin. In another embodiment, the RNA polymerase inhibitor is amanitin. In another embodiment, the amanitin is selected from the group consisting of: alpha-amanitine, beta-amanitine, gamma-amanitine, epsilon-amanitine, amanin (amanin), amanin amide (amanin amide), amanin nontoxic cyclic peptide (amanalin), amanin carboxylic acid (amanalinic acid), and amanin nontoxic cyclic peptide (proaninn).

In one embodiment, the Antibody Drug Conjugate (ADC) is represented by the formula Ab-Z-L-Am, wherein Ab is an antibody or antigen-binding fragment thereof that binds CD2, L is a linker, Z is a chemical moiety, and Am is amatoxin. In certain embodiments, linker-amanitin conjugate Am-L-Z is represented by formula (III)

Wherein R is₁Is H, OH, OR_AOR OR_C；

R₂Is H, OH, OR_BOR OR_C；

R_AAnd R_BWhen present, combine together with the oxygen atom to which they are bound to form an optionally substituted 5-membered heterocycloalkyl group;

R₃is H, R_COr R_D；

R₄、R₅、R₆And R₇Each independently is H, OH, OR_C、OR_D、R_COr R_D；

R₈Is OH, NH₂、OR_C、OR_D、NHR_COr NR_CR_D；

R₉Is H, OH, OR_COR OR_D；

Q is-S-, -S (O) -or-SO₂-；

R_Cis-L-Z;

R_Dis optionally substituted C₁-C₆Alkyl, optionally substituted C₁-C₆Heteroalkyl, optionally substituted C₂-C₆Alkenyl, optionally substituted C₂-C₆Heteroalkenyl, optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;

l is optionally substituted C₁-C₆Alkyl, optionally substituted C₁-C₆Heteroalkyl, optionally substituted C₂-C₆Alkenyl, optionally substituted C₂-C₆Heteroalkenyl, optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkylSubstituted aryl or optionally substituted heteroaryl; or comprises a dipeptide; or- ((CH₂)_mO)_n(CH₂)_m-, wherein m and n are each independently selected from 1,2, 3,4, 5,6, 7,8, 9 and 10; and is

Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within an anti-CD 2 antibody or antigen-binding fragment thereof.

In certain embodiments, linker-amanitin conjugate Am-L-Z is represented by formula (III)

Wherein R is₁Is H, OH, OR_AOR OR_C；

R₂Is H, OH, OR_BOR OR_C；

R_AAnd R_BWhen present, combine together with the oxygen atom to which they are bound to form an optionally substituted 5-membered heterocycloalkyl group;

R₃is H, R_COr R_D；

R₄、R₅、R₆And R₇Each independently is H, OH, OR_C、OR_D、R_COr R_D；

R₈Is OH, NH₂、OR_C、OR_D、NHR_COr NR_CR_D；

R₉Is H, OH, OR_COR OR_D；

Q is-S-, -S (O) -or-SO₂-；

R_Cis-L-Z;

R_Dis optionally substituted C₁-C₆Alkyl, optionally substituted C₁-C₆Heteroalkyl, optionally substituted C₂-C₆Alkenyl, optionally substituted C₂-C₆Heteroalkenyl radical, renOptionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;

l is a linker; and is

Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within an anti-CD 2 antibody or antigen-binding fragment thereof.

In one embodiment of formula (III), L is a peptide comprising a linker.

In some embodiments, the linker comprises one or more of the following: dipeptide, p-aminobenzyl (PAB) group, optionally substituted C₁-C₆Alkyl, optionally substituted C₁-C₆Heteroalkyl, optionally substituted C₂-C₆Alkenyl, optionally substituted C₂-C₆Heteroalkenyl, optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Heteroalkynyl, optionally substituted C₃-C₆Cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, solubility-enhancing group, - (C ═ O) -, - (CH)₂CH₂O)_pA group, wherein p is an integer from 1 to 6, ((CH)₂)_mO)_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.

In some embodiments, the linker comprises ((CH)₂)_mO)_n(CH₂)_m-a group and a heteroaryl group, wherein the heteroaryl group is a triazole. In some embodiments, ((CH)₂)_mO)_n(CH₂)_mThe radicals and triazoles together comprisingWherein n is 1 to 10, and the wavy line indicatesAn external linker component, chemical moiety Z or an attachment point for amatoxin.

In some embodiments, Am comprises exactly one R_CAnd (4) a substituent.

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

Wherein S is a sulfur atom, represents a reactive substituent (e.g., an-SH group from a cysteine residue) present within an antibody or antigen-binding fragment thereof that binds CD 2.

In some embodiments, L-Z is

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

in one embodiment, the ADC is represented by one of the following structures:

in certain embodiments, the ADC has a serum half-life of 3 days or less.

In certain embodiments, the extracellular domain of the CAR comprises a scFv antibody or a single chain T cell receptor (scTCR).

In certain embodiments, the extracellular domain comprises a non-immunoglobulin scaffold protein.

In certain embodiments, 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 (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or CD 123.

In certain embodiments, the cytoplasmic domain of the CAR comprises a CD28 cytoplasmic signaling domain, a CD3 δ cytoplasmic signaling domain, an OX40 cytoplasmic signaling domain, and/or a CD137(4-1BB) cytoplasmic signaling domain.

In certain embodiments, the cytoplasmic domain of the CAR comprises a CD3 delta cytoplasmic signaling domain.

In certain embodiments, the human subject having cancer has a cancer selected from the group consisting of: leukemia, adult advanced cancer, pancreatic cancer, unresectable pancreatic cancer, colorectal 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-cell lymphoma, refractory mediastinal large-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, relapsed or refractory non-Hodgkin lymphoma, refractory aggressive non-Hodgkin lymphoma, B-cell non-Hodgkin lymphoma, hematopoietic cancer, colorectal cancer, metastatic colorectal cancer, ovarian cancer, triple negative breast cancer, hematopoietic cancer, metastatic colorectal cancer, ovarian cancer, metastatic colorectal cancer, metastatic, Refractory non-hodgkin's lymphoma, colorectal epithelial cancer (colorectal carcinoma), gastric cancer, pancreatic cancer, triple negative invasive breast cancer, renal cell carcinoma, lung squamous cell carcinoma, hepatocellular carcinoma, urothelial cancer, leukemia, B-cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, multiple myeloma, Brain malignant glioma, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasms, blastic plasmacytoid dendritic cell tumors, liver metastases, solid tumors, advanced solid tumors, mesothelin-positive tumors, hematologic malignancies, and other advanced malignancies.

In certain embodiments of any of the above aspects, the anti-CD 2 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 set forth in table 4 below. In certain embodiments, the anti-CD 2 antibody or antigen-binding fragment thereof comprises a combination of heavy chain variable regions and light chain variable regions as set forth in table 4.

Brief Description of Drawings

Figure 1 graphically depicts the results of an in vitro cell line binding assay in which each of the indicated anti-CD 2 antibodies or a negative control (i.e., mIgG1) was incubated with MOLT-4 cells (i.e., a human T lymphoblast cell line) followed by incubation with fluorophore-conjugated anti-IgG antibodies. The signal was detected by flow cytometry and expressed as geometric mean fluorescence intensity (y-axis) as a function of anti-CD 2 antibody concentration (x-axis).

Fig. 2 graphically depicts the results of an in vitro primary cell binding assay in which an indicated anti-CD 2 antibody (RPA-2.10 or "CD 2 RPA-2.10") or negative control (i.e., mIgG1) was incubated with primary human T cells followed by fluorophore-conjugated anti-IgG antibodies. The signal was detected by flow cytometry and expressed as geometric mean fluorescence intensity (y-axis) as a function of anti-CD 2 antibody concentration (x-axis).

Fig. 3A and 3B graphically depict the results of an in vitro T cell killing assay comprising anti-CD 2 amanitin ADC (i.e., RPA-2.10-AM or "CD 2 RPA-2.10-AM") with interchain conjugated amanitin with an average drug-to-antibody ratio of 6 (fig. 3A) or site-specifically conjugated amanitin with a drug-to-antibody ratio of 2 (fig. 3B). In fig. 3A, an anti-CD 2-ADC T cell killing assay is shown compared to a non-conjugated anti-CD 2 antibody (i.e., "naked CD2 RPA-2.10"). In fig. 3B, an anti-CD 2-ADC T cell killing assay is shown compared to an anti-CD 2 antibody with an H435A mutation that reduces the half-life of the antibody (i.e., "CD 2 RPA-2.10d265c.h435a AM"). The results show the number of surviving T cells (y-axis) as a function of ADC (CD2 RPA-2.10AM, CD2 RPA-2.10d265c.h435a AM) or unconjugated antibody (naked CD2 RPA-2.10) concentration (x-axis) as assessed using flow cytometry.

FIG. 4 graphically depicts the results of an in vitro Natural Killer (NK) cell killing assay comprising anti-CD 2 amanitin ADC (i.e., RPA-2.10-AM or "CD 2 RPA-2.10-AM") with interchain conjugated amanitin with a drug to antibody ratio of 6. The results show the level of viable NK cells (y-axis) as a function of ADC (CD2 RPA-2.10-AM) or control antibody (i.e., hIgG1, hIgG 1-amanitin ("hIgG 1-AM") or CD45 amanitin ("CD 45 AM")) concentration (x-axis), as assessed using the CellTiter Glo assay.

FIGS. 5A and 5B graphically depict the results of an in vivo T cell depletion assay showing the absolute levels of T cells (CD3+ cells; y-axis) in the peripheral blood (FIG. 5A) and bone marrow (FIG. 5B) of humanized NSG mice 7 days after a single administration of 0.3mg/kg, 1mg/kg, or 3mg/kg of anti-CD 2 amanitine ADC (i.e., RPA-2.10-AM or "CD 2 RPA-2.10-AM") with an interchain drug-to-antibody ratio of 6. For comparison, fig. 5A and 5B also show the level of T cell depletion after treatment of humanized NSG mice with 25mg/kg unconjugated anti-CD 2 antibody (i.e., "CD 2 Ab 1"), or with the indicated control (i.e., 25mg/kg anti-CD 52 antibody (YTH34.5 clone), 3mg/kg hIgG 1-amanitine ADC ("hIgG 1-AM"), 25mg/kg hIgG1, or PBS).

FIGS. 6A-6C graphically depict the results of an in vivo T cell depletion assay showing the absolute levels of T cells (CD3+ cells; y-axis) in the peripheral blood (FIG. 6A), bone marrow (FIG. 6B), and thymus (FIG. 6C) of humanized NSG mice 7 days after a single administration of 1mg/kg or 3mg/kg of anti-CD 2 amanitine ADC (i.e., RPA-2.10-AM or "CD 2 RPA-2.10-AM") with a site-specific drug-to-antibody ratio of about 2. For comparison, figures 6A-6C also show the level of T cell depletion after treatment of humanized NSG mice with 3mg/kg of unconjugated anti-CD 2 antibody (i.e., "CD 2 RPA-2.10") or with the indicated control (i.e., 3mg/kg hIgG 1-amanitine ADC ("hIgG 1-AM") or PBS).

Detailed description of the invention

The invention provides methods of promoting the acceptance of immune cells (autologous or allogeneic) expressing a Chimeric Antigen Receptor (CAR) by a human subject receiving CAR therapy by administering an anti-CD 2 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, which is often used as a conditioning therapy to reduce rejection of CAR-expressing immune cells.

I. Definition of

As used herein, the term "about" refers to a value within 5% above or below the value described.

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, where the donor and recipient are not the same subject.

As used herein, the term "autologous" refers to a cell or graft in the case where the donor and recipient are the same subject.

As used herein, the term "xenogeneic" refers to cells in which the donor and recipient species are different.

As used herein, the term "immune cell" is intended to include, but is not limited to, cells of hematopoietic origin and which play a role in the immune response. Immune cells include, but are not limited to, T cells and Natural Killer (NK) cells. Natural killer cells are well known in the art. In 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. The immune cells may be allogeneic or autologous.

By "engineered cell" is meant any cell of any organism that is modified, transformed or manipulated by the addition or modification of a gene, DNA or RNA sequence or protein or polypeptide. Isolated cells, host cells, and genetically engineered cells of the disclosure include isolated immune cells, such as NK cells and T cells, that comprise a DNA or RNA sequence encoding a CAR and express the CAR on the cell surface. Isolated host cells and engineered cells can be used, for example, to enhance NK cell activity or T lymphocyte activity, to treat cancer, and to treat autoimmune diseases. In embodiments, the engineered cells include immune cells, such as T cells or Natural Killer (NK) cells.

As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or immunoreacts with a particular 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.

Typically, an antibody comprises a heavy chain and a light chain comprising an antigen binding region. 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 3 domains, CH1, CH2, and CH 3. 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 may be further subdivided into hypervariable regions known as Complementarity Determining Regions (CDRs) interspersed with more conserved regions known as Framework Regions (FRs). Each VH and VL comprises 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q).

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 may be performed by fragments of a full-length antibody. Antibody fragments may be, for example, Fab, F (ab')2, scFv, diabodies, triabodies, affibodies, nanobodies, aptamers or domain antibodies. Examples of binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) fab fragments, monovalent fragments 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) an Fd fragment consisting of the VH and CH1 domains; (iv) (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (see, e.g., Ward et al, Nature 341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) an isolated Complementarity Determining Region (CDR); and (ix) a combination of two or more (e.g., two, three, four, five, or six) isolated CDRs, which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a linker that enables them to be a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al, Science 242: 423-. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis procedures known in the art.

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

As used herein, the term "anti-CD 2 antibody" or "antibody that binds to CD 2" or "anti-CD 2 ADC" or "ADC that binds to CD 2" refers to an antibody or ADC that specifically binds to human CD2 when CD2 is present on the cell surface of a cell, such as a T cell. The amino acid sequence of human CD2 that will bind to the anti-CD 2 antibody (or anti-CD 2 ADC) is described below in SEQ ID NO: 7.

As used herein, the term "specifically binds" refers to the ability of an antibody (or ADC) to recognize and bind to a particular protein structure (epitope) rather than to a general protein. If the antibody is specific for epitope "A", then in the reaction of labeled "A" and antibody, the presence of the epitope A-containing molecule (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody. By way of example, an antibody "specifically binds" to a target if, when labeled, the antibody can compete away from its target by a corresponding unlabeled antibody. In one embodiment, if the antibody has at least about 10 to the target^-4M、10^-5M、10^-6M、10^-7M、10^-8M、10^-9M、10^-10M、10^-11M、10^-12M or less (less means less than 10)^-12A number of (2), e.g. 10^-13) K of_DThe antibody then specifically binds to a target such as CD 2. In one embodiment, as used herein, the term "specifically binds to CD 2" or "specifically binds to CD 2" refers to binding to CD2 and having 1.0 x 10^-7Dissociation constant (K) of M or less_D) As determined by surface plasmon resonance. In one embodiment, K_DDetermined according to standard biolayer interferometry (BLI). However, it is understood that an antibody may be capable of specifically binding to two or more antigens associated with a sequence. For example, in one embodiment, the antibody can specifically bind to both a human ortholog and a non-human (e.g., mouse or non-human primate) ortholog of CD 2.

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

The term "chimeric" antibody as used herein 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 (typically selected from a human immunoglobulin template). Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison,1985, Science 229(4719): 1202-7; oi et al, 1986, BioTechniques 4: 214-221; gillies et al, 1985, J.Immunol.methods 125: 191-202; U.S. patent nos. 5,807,715, 4,816,567, and 4,816,397.

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric immunoglobulin comprising minimal sequences 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 those of a human immunoglobulin sequence. The 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 publications WO 91/09967; U.S. Pat. nos. 5,225,539; EP 592106; EP 519596; 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. Pat. No. 5,565,332.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide comprising at least one extracellular domain capable of specifically binding an antigen, a transmembrane domain, and at least one intracellular signaling domain. Generally, CARs are genetically engineered receptors that redirect the cytotoxicity of immune effector cells towards cells presenting a given antigen. CARs are molecules that combine antibody-based specificity for a desired antigen (e.g., a tumor antigen) with the intracellular domain of an activating T cell receptor to produce a chimeric protein that exhibits specific cellular immune activity. In particular embodiments, the CAR comprises an extracellular domain (also referred to as a binding domain or antigen-specific binding domain), a transmembrane domain, and an intracellular (cytoplasmic) signaling domain. Engagement of the antigen binding domain of the CAR with a target antigen on the surface of a target cell results in aggregation of the CAR and delivers an activation stimulus to the CAR-containing cell. The main feature of CARs is their ability to redirect immune effector cell specificity using the cell-specific targeting ability of monoclonal antibodies, soluble ligands, or cell-specific co-receptors, triggering proliferation, cytokine production, phagocytosis, or molecular production capable of mediating cell death of cells expressing the target antigen in a Major Histocompatibility (MHC) -independent manner. In some embodiments, the 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, the CAR comprises an extracellular binding domain that specifically binds human CD2, a transmembrane domain, and one or more intracellular signaling domains.

As used herein, the term "CAR therapy" refers to the administration of immune cells engineered to express a CAR to a human subject to treat a given disease, e.g., cancer or an autoimmune disease. CAR therapy refers to the specific treatment of a patient with engineered immune cells and is not intended to include therapies typically used in conjunction with CAR cell therapy, such as lymphodepleting chemotherapy. It is noted that where the term "cell" is used throughout, the term also includes populations of cells, unless otherwise indicated. For example, because CAR therapy requires administration of an engineered cell population.

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 combination of components. For example, described herein are combination therapies comprising administration of anti-CD 2ADC and CAR therapies.

The term "modulate" refers to preparing a patient in need of CAR therapy for appropriate conditions. Modulation as used herein includes, but is not limited to, reducing the number of endogenous lymphocytes prior to T cell therapy, depleting a cytokine pool (sink), increasing the serum level of one or more homeostatic cytokines or pro-inflammatory factors, enhancing effector function of T cells administered after modulation, enhancing activation and/or availability of antigen presenting cells, or any combination thereof.

In the context of the effect of an anti-CD 2 antibody or ADC on CD 2-expressing cells, the term "depletion" refers to a reduction or elimination in the number of CD 2-expressing cells.

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

As used herein, the terms "subject" and "patient" refer to an organism, such as a human, that is being treated for a particular disease or condition as described herein.

As used herein, "treatment" or "treatment" refers to any improvement in disease outcome, such as extended survival, less morbidity, and/or alleviation of side effects as a by-product of alternative treatment modalities; as is readily understood in the art, complete elimination of the disease is preferred, but not a requirement for therapeutic action. Beneficial or desired clinical results include, but are not limited to, promoting the acceptance of CAR-expressing immune cells (allogeneic or autologous — both can elicit an immune response in a patient receiving CAR therapy). To the extent that the methods of the invention are directed to preventing a disorder, it is understood that the term "preventing" does not require that the disease state be completely prevented. Rather, as used herein, the term prophylaxis refers to the ability of the skilled person to identify a population susceptible to a disorder such that administration of a compound of the invention can be performed prior to onset of the disease. The term does not imply that the disease state is completely avoided.

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 as well as additional sequence elements, e.g., for expressing proteins and/or integrating these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can be used to express the CAR include plasmids that contain regulatory sequences (such as promoter and enhancer regions) that direct transcription of the gene. Other useful vectors for antibody or CAR expression comprise polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA produced by gene transcription. These sequence elements may include, for example, 5 'and 3' untranslated regions and polyadenylation signal sites to direct the efficient transcription of genes carried on expression vectors. 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.

As used herein, the term "antibody drug conjugate" or "ADC" refers to an antibody linked to a cytotoxin. ADCs are formed by chemical bonding of a reactive functional group of one molecule (such as an antibody or antigen-binding fragment thereof) with an appropriate reactive functional group of another molecule (such as a cytotoxin as described herein). The conjugate may comprise a linker between two molecules that bind to each other (e.g., between an antibody and a cytotoxin). Notably, the term "conjugate" (when referring to a compound) may also be interchangeably referred to herein as a "drug conjugate" or an "antibody drug conjugate" or an "ADC". Examples of linkers that can be used to form conjugates include peptide-containing linkers, such as linkers containing naturally occurring or non-naturally occurring amino acids, such as D-amino acids. Linkers can be prepared using a variety of strategies described herein and known in the art. Depending on the reactive components therein, the linker may be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Lerich et al, bioorg. Med. chem.,20: 571) -582, 2012).

As used herein, the term "coupling reaction" refers to a chemical reaction in which two or more substituents that are suitable for reacting with each other react so as 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) is reacted with an appropriate reactive substituent bound to a fragment that is an antibody or antigen-binding fragment thereof (such as an antibody that binds CD2, antigen-binding fragment thereof, or specific anti-CD 2 antibody known in the art or described herein). Examples of suitable reactive substituents include nucleophile/electrophile pairs (such as, inter alia, thiol/haloalkane pairs, amine/carbonyl pairs, or thiol/α, β -unsaturated carbonyl pairs), diene/dienophile pairs (such as, inter alia, azide/alkyne pairs), and the like. Coupling reactions include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine condensation, amidation, esterification, disulfide formation, cycloaddition (such as, inter alia, [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reactive means known in the art or described herein.

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

As used herein, the term "amatoxin" refers to an amatoxin family member of peptides produced by the mushroom of Amanita pharioides (Amanita pharioides) or derivatives thereof, such as variants or derivatives thereof capable of inhibiting RNA polymerase II activity. Amanitin can be isolated from various mushroom species (e.g., amanita phalloidea, gymnosporium striatum, pholiota carnea) or can be prepared semi-synthetically or synthetically. A member of this family, α -amanitine, is described in Wieland, int.J.Pept.protein Res.1983,22(3): 257-276. Derivatives of amanitin may be obtained by chemical modification ("semi-synthesis") of naturally occurring compounds, or may be obtained from entirely synthetic sources. Synthetic routes to a variety of amatoxin derivatives are disclosed, for example, in U.S. patent No. 9,676,702 and Perrin et al, j.am.chem.soc.2018,140, page 6513-6517, each of which is incorporated herein by reference in its entirety for its entirety with respect to synthetic methods for preparing and derivatizing amatoxin.

Amatoxins that may be used in conjunction with the compositions and methods described herein include amatoxins described in formula (II), e.g., alpha-amanitine, beta-amanitine, gamma-amanitine, epsilon-amanitine, amanitin, amanamide, amanitin nontoxic cyclic peptide, amanitin carboxylic acid, and pro-amanitin nontoxic cyclic peptide. As described herein, amatoxin can be conjugated to an antibody or antigen-binding fragment thereof (thereby forming an ADC) by, for example, a linker moiety (L). The structures of exemplary amatoxin-linker conjugates are represented by formula (III), formula (IIIA), and formula (IIIB). Exemplary methods of amanitin conjugation and linkers useful in such methods are described below. Also described herein are exemplary linker-containing amatoxins useful for conjugation to antibodies or antigen-binding fragments according to the compositions and methods.

The term "acyl" as used herein refers to-C (═ O) R, where R is hydrogen ("aldehyde"), C, as defined herein₁-C₁₂Alkyl radical, C₂-C₁₂Alkenyl radical, C₂-C₁₂Alkynyl, C₃-C₇Carbocyclyl, C₆-C₂₀Aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl. Non-limiting examples include formyl, acetyl, propionyl, benzylAcyl and acryloyl.

The term "C" as used herein₁-C₁₂Alkyl "refers to a straight or branched chain saturated hydrocarbon having from 1 to 12 carbon atoms. Representative C₁-C₁₂Alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and-n-hexyl; and branched C₁-C₁₂Alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl. C₁-C₁₂The alkyl group may be unsubstituted or substituted.

The term "alkenyl" as used herein is meant to encompass a compound having at least one site of unsaturation (i.e., a carbon-carbon sp)²Double bond) of a normal, secondary or tertiary carbon atom₂-C₁₂A hydrocarbon. 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, and the like. The alkenyl group may be unsubstituted or substituted.

"alkynyl" as used herein refers to a C containing a normal, secondary or tertiary carbon atom having at least one site of unsaturation (i.e., a carbon-carbon sp triple bond)₂-C₁₂A hydrocarbon. Examples include, but are not limited to, alkynyl (acetylenic) and propargyl. Alkynyl groups may be unsubstituted or substituted.

As used herein, "aryl" refers to C₆-C₂₀A carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl. The aryl group may be unsubstituted or substituted.

As used herein, "arylalkyl" refers to a radical having carbon atoms (typically terminal carbon atoms or sp) bonded thereto³Carbon atom) with one hydrogen atom bonded being substituted by an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethyl-1-yl, 2-phenylethan-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethen-1-yl, naphthylbenzyl, 2-naphthylphenylethan-1-yl, and the like. Aryl alkyl radicalGroups contain 6 to 20 carbon atoms, for example the alkyl portion of an arylalkyl group (including alkyl, alkenyl or alkynyl groups) is 1 to 6 carbon atoms and the aryl portion is 5 to 14 carbon atoms. The alkaryl group may be unsubstituted or substituted.

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 a monocyclic ring or 7 to 12 carbon atoms as a bicyclic ring. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be unsubstituted or substituted.

"cycloalkenyl" as used herein refers to an unsaturated carbocyclic group, which may be monocyclic or bicyclic. Cycloalkenyl groups include rings having from 3 to 6 carbon atoms as a monocyclic ring or from 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. Cycloalkenyl groups may be unsubstituted or substituted.

As used herein, "heteroaralkyl" refers to a group in which a carbon atom (typically a terminal carbon atom or sp) is bonded³Carbon atom) with one hydrogen atom bonded being substituted by a heteroaryl group. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furanylethyl, and the like. Heteroarylalkyl groups contain from 6 to 20 carbon atoms, e.g., the alkyl portion of a heteroarylalkyl group (including alkyl, alkenyl, or alkynyl groups) is from 1 to 6 carbon atoms, and the heteroaryl portion is from 5 to 14 carbon atoms and from 1 to 3 heteroatoms selected from N, O, P and S. The heteroaryl portion of the heteroarylalkyl group may be a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms) or a bicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P and S), for example: bicyclo [4,5]]、[5,5]、[5,6]Or [6,6]]Provided is a system.

As used herein, "heteroaryl" and "heterocycloalkyl" refer to aromatic or non-aromatic ring systems, 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 having 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 having 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], [5,6] or [6,6] systems. Heteroaryl and heterocycloalkyl groups may be unsubstituted or substituted.

Heteroaryl groups and heterocycloalkyl groups are described in the following: pattette, Leo a.; "Principles of Modern Heterocyclic Chemistry" (W.A. Benjamin, New York,1968), in particular Chapter 1, Chapter 3, Chapter 4, Chapter 6, Chapter 7 and Chapter 9; "The Chemistry of Heterocyclic Compounds, A series of monograms" (John Wiley & Sons, New York,1950 to date), particularly volume 13, volume 14, volume 16, volume 19 and volume 28; and j.am.chem.soc. (1960)82: 5566.

For example, examples of heteroaryl groups include, but are not limited to, pyridyl, thiazolyl, tetrahydrothienyl, pyrimidinyl, furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuryl, thionaphthyl (thianaphtalenyl), indolyl, indolinyl, quinolyl, isoquinolyl, benzimidazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl (4H-quinolizinyl), phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4 aH-carbazolyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl (phenanthrolinyl), phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, and the like, Pyrazolinyl, benzotriazolyl, benzisoxazolyl and isatinyl (isatinoyl).

For example, examples of heterocycloalkyl include, but are not limited to, dihydropyridinyl, tetrahydropyridinyl (piperidyl), tetrahydrothiophenyl, piperidyl (piperidinyl), 4-piperidonyl, pyrrolidinyl, 2-pyrrolidinonyl, tetrahydrofuranyl, tetrahydropyranyl, bistetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinuclidinyl, and morpholinyl.

For example, but not limited to, carbon-bonded heteroaryl and heterocycloalkyl are bonded at position 2,3, 4,5, or 6 of pyridine, position 3,4, 5, or 6 of pyridazine, position 2, 4,5, or 6 of pyrimidine, position 2,3, 5, or 6 of pyrazine, position 2,3, 4, or 5 of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, position 2, 4, or 5 of oxazole, imidazole, or thiazole, position 3,4, or 5 of isoxazole, pyrazole, isothiazole, position 2 or 3 of aziridine, position 2,3, or 4 of azetidine, position 2,3, 4,5, 6,7, or 8 of quinoline, or position 1, 3,4, 5,6, 7, or 8 of isoquinoline. Still more typically, carbon-bonded heterocycles include 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.

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, indoline, 1H-indazole, position 2 of isoindole or isoindoline, position 4 of morpholine and position 9 of carbazole or β -carboline. Still more typically, the nitrogen-bonded heterocyclic ring includes 1-aziridinyl, 1-azetidinyl (azetedyl), 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

"substituted" as used herein and applied to any of the above alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl and the like means that one or more hydrogen atoms are each independently replaced by a substituent.

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)NH₂and-C (═ NR) N (R)₂(ii) a Wherein each X is independently selected for each occurrence from F, Cl, Br, and I; and each R is independently selected for each occurrence from C₁-C₁₂Alkyl radical, C₆-C₂₀Aryl radical, C₃-C₁₄Heterocycloalkyl or heteroaryl, protecting groups, and prodrug moieties. In all cases where a group is described as "optionally substituted," the group may be independently substituted for each occurrence with one or more substituents.

It is understood that, depending on the context, certain radical naming conventions may include monovalent radicals or divalent radicals. 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 radical. For example, substituents identified as alkyl groups requiring two attachment points include divalent groups, such as-CH₂-、-CH₂CH₂-、-CH₂CH(CH₃)CH₂-and the like. Other group naming conventions specifically indicate that the group is a divalent group such as "alkylene," "alkenylene," "arylene," "heterocycloalkylene," and the like.

In all cases where a substituent is described as a divalent radical (i.e., two points of attachment to the rest of the molecule), it is understood that the substituent may be attached in any directional configuration unless otherwise specified.

"isomeric" means compounds having the same molecular formula but differing in their atomic bonding order or their spatial arrangement of atoms. Isomers that differ in their arrangement in atomic space are referred to as "stereoisomers". Stereoisomers that are not mirror images of each other are referred to as "diastereomers", while stereoisomers that are non-superimposable mirror images of each other are referred to as "enantiomers" or sometimes also "optical isomers".

The carbon atom bonded to four different substituents is referred to as a "chiral center". "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 mixtures of diastereomers (referred to as "diastereomeric mixtures"). When a chiral center is present, stereoisomers can be characterized by the absolute configuration (R or S) of the chiral center. Absolute configuration refers to the spatial arrangement of substituents attached to a chiral center. Substituents attached to the chiral center of interest are ordered according to the sequence rules of Cahn, Ingold and Prelog. (Cahn et al, Angew. chem. Inter. eds 1966,5, 385; reconnaissance 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). Mixtures of individual enantiomeric forms comprising equal amounts of opposite chirality are referred to as "racemic mixtures".

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

Compounds of any formula described herein include the compounds themselves, and if applicable their salts and their solvates. For example, a salt may be formed between an anion and a positively charged group (e.g., amino group) on a compound of the present disclosure. Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, toluenesulfonate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate). The term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Likewise, salts can also be formed between a cation and a negatively charged group (e.g., carboxylate) on a compound of the disclosure. Suitable cations include sodium, potassium, magnesium, calcium, and ammonium cations, such as tetramethylammonium. Some examples of 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. The compounds of the present disclosure also include those salts that contain quaternary nitrogen atoms.

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.

Additionally, compounds of the present disclosure, e.g., salts of compounds, may exist in hydrated or non-hydrated (anhydrous) forms, or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrate, dihydrate, and the like. Non-limiting examples of solvates include ethanol solvates, acetone solvates, and the like. By "solvate" is meant a solvent addition form comprising a stoichiometric or non-stoichiometric amount of a solvent. Some compounds have a tendency to trap a fixed molar ratio of solvent molecules in the crystalline solid state, thereby forming solvates. If the solvent is water, the solvate formed is a hydrate; and if the solvent is an alcohol, the solvate formed is an alcoholate. The hydrate is formed by the combination of one or more water molecules with a molecule of substance, wherein the water retains its molecular state as H₂And O. The hydrate isRefers to, for example, the monohydrate, dihydrate, trihydrate, and the like.

In addition, the compounds represented by the formulae disclosed herein or salts thereof may exist in crystal polymorphs. It should be noted that any crystal form, mixture of crystal forms, or anhydride or hydrate thereof is included within the scope of the present disclosure.

The following section provides a description of methods based on administration of anti-CD 2ADC to a human patient to facilitate the acceptance of CAR-expressing immune cells in CAR therapy.

anti-CD 2 and CAR treatment methods

One challenge of Chimeric Antigen Receptor (CAR) therapy is determining the means by which CAR-expressing engineered cells, such as CAR-T cells, can be accepted by a human recipient. Such acceptance of engineered immune cells can affect the efficacy of the treatment as well as the outcome of adverse side effects to the patient.

Lympho-depleting chemotherapy is a traditional method of suppressing the recipient's immune system to improve acceptance, but generally has adverse side effects. Described herein are methods of promoting the acceptance of a (CAR) -expressing immune cell by a human patient undergoing CAR therapy. The methods described herein specifically target CD2+ cells, e.g., CD2+ T cells, and eliminate CD2+ cells in human patients undergoing CAR therapy. The methods disclosed herein are more targeted than lymphodepleting chemotherapy and provide a means by which autologous or allogeneic cells can be used.

Described herein are methods of administering an anti-CD 2 Antibody Drug Conjugate (ADC) to deplete a population of CD 2-specific immune cells in a patient receiving CAR therapy in order to promote the acceptance and efficacy of CAR-expressing immune cells. This selective depletion of cells of the immune system that specifically express CD2 improves overall survival and relapse-free survival of patients while reducing the risk of CAR-expressing immune cell rejection for treating autoimmune disorders or cancer.

The risk of CAR-expressing immune cell rejection remains high after administration of CAR cell therapy. The methods and compositions disclosed herein can be used to inhibit or prevent rejection of CAR cells in a human patient. anti-CD 2 ADCs can be used to selectively target activated T cells in patients who will receive CAR cell therapy. anti-CD 2 ADCs as described herein may also be used to reduce the risk of CAR cell rejection by targeting and depleting CD2 positive cells in human patients who have received CAR therapy.

The compositions and methods described herein can be used to deplete CD2+ cells, e.g., T cells, that are associated with rejection of CAR cell therapy. The methods of the invention facilitate the acceptance of immune cells expressing a CAR by a human subject (e.g., a human subject having cancer or an autoimmune disease). In one embodiment, the method comprises administering an anti-CD 2 Antibody Drug Conjugate (ADC) to a human subject that will undergo or has undergone CAR therapy, and administering a therapeutically effective amount of immune cells expressing the CAR to the human subject.

The anti-CD 2ADC can be administered to a human patient in need thereof prior to, simultaneously with, or after administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof prior to administration of the CAR cell therapy (e.g., about 3 days prior to administration of the CAR cell therapy, about 2 days prior to administration of the CAR cell therapy, about 12 hours prior to administration of the CAR cell therapy). A single dose of anti-CD 2ADC may be administered to a human patient prior to, after, or simultaneously with the administration of the CAR cell therapy, wherein such single dose is sufficient to prevent or reduce the risk of depletion of immune cells expressing the CAR.

In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 3 days prior to administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 2 days prior to administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 1 day prior to administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 20 hours prior to administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 18 hours prior to administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered 15 hours prior to administration of the one or more CAR cell therapies to a human patient in need thereof. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 12 hours prior to administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 6 hours prior to administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 4 hours prior to administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 2 hours prior to administration of the one or more CAR cell therapies.

In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof concurrently with administration of the CAR cell therapy.

In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 2 hours after administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 4 hours after administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 6 hours after administration of the one or more CAR cell therapies. In one embodiment, the anti-CD 2ADC is administered to a human patient in need thereof about 12 hours after administration of the one or more CAR cell therapies.

In some embodiments, the anti-CD 2ADC may be administered up to about 21 days prior to administration of the one or more CAR cell therapies, e.g., 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 the one or more CAR cell therapies.

In some embodiments, the anti-CD 2ADC may be administered about 12 hours after administration of the CAR cell therapy, e.g., 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 administration of the one or more CAR cell therapies. In some embodiments, the anti-CD 2ADC may be administered about 10 days after administration of the CAR cell therapy, e.g., about 10 days, about 9 days, about 8 days, about 7 days, about 6 days, about 5 days, about 4 days, about 3 days, about 2 days, about 1 day after administration of the one or more CAR cell therapies.

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

The total level of T cells in a biological sample from a human patient can be tested after administration of the anti-CD 2ADC, wherein a decrease in the total number of T cells in the human patient after administration of the anti-CD 2ADC relative to the level prior to administration indicates the efficacy of the anti-CD 2ADC for preventing rejection of CAR cell therapy. In one embodiment, the level of endogenous T cells in the biological sample from the human patient is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20% relative to the level of T cells in a biological sample (of the same type, e.g., blood) from the human patient immediately prior to administration of the anti-CD 2 ADC. In one embodiment, the level of endogenous T cells in the biological sample from the human patient is reduced by about 5% to 25%, about 5% to 20%, about 5% to 15%, or about 5% to 10% relative to the level of T cells in the biological sample (of the same type, e.g., blood) from the human patient immediately prior to administration of the anti-CD 2 ADC. In one embodiment, the level of endogenous T cells is determined one day or less prior to administration of the anti-CD 2 ADC.

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 hematology analyzers.

As described above, one advantage of the methods described herein is that the amount of lymphodepleting chemotherapeutic agent may be reduced or the lymphodepleting chemotherapeutic agent is not included in the modulation regimen administered to a human patient receiving or scheduled to receive CAR therapy. Lymphodepleting chemotherapeutic agents, such as, but not limited to, fludarabine, cyclophosphamide, bendamustine, and/or pentostatin, are often used as anti-rejection agents to facilitate the acceptance of CAR-expressing cells by a human receiving CAR therapy. In certain embodiments, the anti-CD 2ADC is administered to the human patient in combination with (e.g., prior to) administration of an immune cell (e.g., T cell) that expresses the CAR, such that the human patient does not receive a lymphodepleting chemotherapeutic agent, e.g., fludarabine and/or cyclophosphamide, prior to, simultaneously with, or after administration of the immune cell that expresses the CAR.

The use of other immune depleting agents may also be avoided or reduced by using anti-CD 2ADC as an agent that depletes endogenous immune cells of a human subject and reduces the risk of rejection of immune cells expressing the CAR. For example, alemtuzumab is commonly used as an anti-rejection agent in combination with CAR therapy to facilitate the acceptance of CAR-expressing cells by a human receiving CAR therapy. In certain embodiments, the anti-CD 2ADC is administered to the human patient in combination with (e.g., prior to) administration of immune cells (e.g., T cells) that express the CAR, such that the human patient does not receive alemtuzumab prior to, simultaneously with, or after administration of immune cells that express the CAR.

In certain embodiments, the anti-CD 2ADC is used in combination with another therapy in order to promote tolerance of the CAR-expressing immune cells. For example, an anti-CD 2ADC may also be administered to a human patient prior to the human patient receiving CAR therapy. The anti-CD 2ADC may be administered prior to the anti-CD 2ADC, simultaneously with the anti-CD 2ADC, or after the anti-CD 2ADC, wherein both the anti-CD 2ADC and the anti-CD 2ADC are administered to the human patient prior to CAR therapy.

The methods disclosed herein can be used for both autologous and allogeneic cells expressing the CAR. Importantly, the anti-CD 2ADC modulation methods described herein can be used to expand the types of immune cells that can be used in CAR therapy by providing methods by which allogenic cell tolerance can be provided. In one embodiment, the immune cell expressing the CAR is an allogeneic cell or an autologous cell. Examples of immune cell types that can be engineered to express the CAR include, but are not limited to, allogeneic T cells, autologous NK cells, or allogeneic NK cells.

In one embodiment, the anti-CD 2 antibody drug conjugate is used to deplete CD2 expressing donor cells, e.g., activated CD2 expressing T cells, by administering the anti-CD 2 antibody drug conjugate after administration of the CAR cell therapy. In one embodiment, the CAR cell therapy comprises allogeneic cells.

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

In one embodiment, the methods disclosed herein are used to treat cancer. More particularly, anti-CD 2ADC is administered to a human subject having cancer in combination with CAR therapy. Examples of types of cancer 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 metastases, 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, hodgkin's lymphoma, non-hodgkin's lymphoma, relapsed or refractory non-hodgkin's lymphoma, refractory aggressive non-hodgkin's lymphoma, refractory large B-cell lymphoma, hematopoietic large cell lymphoma, refractory small cell lung cancer, small cell lung, B cell non-Hodgkin's lymphoma, refractory non-Hodgkin's lymphoma, colorectal epithelial cancer, gastric cancer, pancreatic cancer, triple negative invasive breast cancer, renal cell cancer, lung squamous cell cancer, hepatocellular carcinoma, urothelial cancer, leukemia, B cell acute lymphocytic leukemia, B cell acute lymphoblastic leukemia, adult acute lymphoblastic leukemia, B cell prolymphocytic leukemia, childhood acute lymphoblastic leukemia, refractory childhood acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, relapsed plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, relapsed or refractory multiple myeloma, Bone multiple myeloma, brain malignant glioma, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasms, blast cell plasmacytoid dendritic cell tumors, liver metastases, solid tumors, advanced solid tumors, mesothelin-positive tumors, hematologic malignancies, and other advanced malignancies.

In one embodiment, the methods disclosed herein are used to treat autoimmune diseases. More particularly, anti-CD 2ADC is administered to a human subject with an autoimmune disease in combination with CAR therapy. Examples of autoimmune diseases that can be treated using the combination methods disclosed herein include, but are not limited to, multiple sclerosis, crohn's disease, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, lupus, and psoriasis.

In certain embodiments, the anti-CD 2ADC is administered to a human patient in combination with CAR-T cell therapy. In one embodiment, the anti-CD 2ADC is administered to a human patient prior to administration of CAR-T therapy. Examples of CAR-T cells that can be used in combination with The anti-CD 2ADC therapies described herein include, but are not limited to, CD19 CAR-T (e.g., CART-19-01,02,03(Fujian Medical University); dapeicard (Hebei Senlang Biotechnology Inc.; IM19CART/001, YMCART201702(Beijing Immunochina Medical Science & Technology Co.); CART-CD19-02,03(Wuhan Sikang Medical Technology Co.); general CD 19-CART/SHBYBYCL 001,002(Shanghai Bionuclear Laboratory), Carcarepy 201701(Shanghai Immunopyray Biomedicine Co.); CAR-III Biotechnology Co.; Biochemical Co.; C6731 (Biochemical Co.); C080C III) (Biochemical Co.; C III Biochemical Co.; C6731 and III Biochemical Co.; C6778; C III Biochemical Co.; C6731, C III Biochemical Co.; C3, C. III Biogene Co.; C3, C. III Biogene, C. III Technology Company); CTL019/IT1601-CART19(Beijing Santa Biological Technology Co.); CTL019/CCTL019C2201(Novartis Pharmaceuticals); CD19:4-1BB: CD28: CD3/first Shenzhen01(Shenzhen Second peoples' Hospital/The Beijing Pregene Science and Technology Company); MB-CART19.1(Shanghai Children's Medical Center/Miltenyi Biotec GmbH); PZ01 CAR-T cells (Pinze Life technology Co.); YMCART201701(Beijing Immunochina Medical Science & Technology Co.); 2016YJZ12(Peking University/Marino Biotechnology Co.); EGFRT/19-28z/4-1BBL CAR T cells (molar Sloan Kettering Cancer Center/Juno Therapeutics, Inc.); doing-002(Beijing Doing Biomedical Co.); PCAR-019(PersonGen Biotherapeutics (Suzhou) Co.); C-CAR011(Peking Union Medical College Hospital/Cellular Biomedicine Group Ltd.); iPD1 CD19 eCR T cells (Peking University/Marino Biotechnology Co.); 2013-1018/NCT02529813(M.D. Anderson Cancer Center/Ziopharm/Intrexon Corp.); HenanCH CAR 2-1(Henan Cancer Hospital/The Pregene (ShenZHen) Biotechnology Co.); JCAR015(Juno Therapeutics, Inc.); JCAR017/017001,004,006(Juno Therapeutics, Inc.); JCAR017 (Celgene); TBI-1501(Takara Bio Inc.); JMU-CD19CAR (Jichi Medical University); KTE-C19(Kite, A Gilead Company); TRICAR-T-CD19(Timmune Biotech Inc.); PF-05175157(Fred Hutchinson Cancer Research Center)); CD22/CD30/CD7/BCMA/CD123 (e.g., 2016040/NCT03121625(Hebei Senlang Biotechnology Inc.)); CD22 (e.g., Ruijin-CAR-01(Ruijin Hospital/Shanghai Unicar-Therapy Bio-Therapy Technology Co.); AUTO-PA1, DB1(Autolus Limited)), CD20 (e.g., Doing-006(Beijing Biomedical Co.)) or CD20/CD22/CD30 (e.g., SZ5601(The First affected Hospital of Soochow University Shanghai/Unicar-Therapy Bio-Therapy Technology Co.)).

Chimeric Antigen Receptor (CAR)

The invention includes the use of CAR therapy in combination with anti-CD 2 immunosuppressive ADCs. The invention is generally not limited to a particular CAR construct, e.g., a particular antigen binding region or intracellular signaling domain, as the invention is based, at least in part, on the discovery that anti-CD 2 ADCs can be used as modulators of CAR therapy by promoting acceptance of CAR-expressing cells by eliminating endogenous CD2+ immune cells, such as endogenous T cells. Specific CARs, such as CD 19-specific CARs, are contemplated herein and included in the methods disclosed herein, but are not intended to be limiting.

CAR constructs are known in the art and typically comprise (a) an extracellular region comprising 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, the CAR can be a first generation CAR, a second generation CAR, or a third generation CAR, e.g., as described in: molecular Therapy-Methods & Clinical development.12:145-156(2019) by Guedan et al or Cancer discovery 3.4:388-398(2013) by Sadelain et al, the entire contents of which are incorporated herein by reference. Briefly, a "first generation" CAR can comprise (a) an extracellular antigen-binding domain, (b) a transmembrane domain, (c) one or more intracellular signaling domains, and optionally (d) a hinge region connecting the antigen-binding domain and the transmembrane domain. A "second generation" CAR may comprise elements (a), (b), (c), and optionally (d), and further comprise a co-stimulatory domain, for example, of CD28 or 4-1 BB. A "third generation" CAR may comprise elements (a), (b), (c), and optionally (d), and further comprise more than one co-stimulatory domain, for example the co-stimulatory domains of CD28 and 4-1BB, or the co-stimulatory domains of CD28 and OX 40. Each of the above elements is described in detail below. It is to be understood that in some embodiments, the CAR molecules described by the following exemplary non-limiting arrangements are from the left to right, N-terminus to C-terminus of the CAR. A CAR as described in this disclosure may include or further include any other combination of elements as described herein.

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

The choice of molecular target (antigen) for the extracellular binding domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen binding domain can be selected to recognize ligands that serve as cell surface markers on target cells associated with a particular disease state. Thus, in one aspect, a CAR-mediated immune cell (e.g., T cell) response can be directed against an antigen of interest by engineering an extracellular antigen-binding domain that specifically binds to the desired antigen into a CAR. For example, the antigen binding domain can be selected to recognize a ligand on a target cell that serves as a cell surface marker associated with a particular disease state (such as cancer or autoimmune disease). Thus, examples of cell surface markers that can serve as ligands for the antigen binding domain in the CAR include those associated with cancer cells and other forms of diseased cells (e.g., autoimmune disease cells and pathogen infected cells). In some embodiments, the CAR is engineered to target a tumor antigen of interest by engineering a desired antigen binding domain that specifically binds to the antigen on the tumor cell. In the context of the present invention, "tumor antigen" refers to an antigen that is common to certain 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 CD 123. In one embodiment, the CAR comprises an scFv that binds to: CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or CD 123.

In another aspect, the extracellular binding domain of the CAR binds to an antigen that is: AFP (e.g., ETCH17AFPCAR01(Aeon Therapeutics Co./Eureka Therapeutics Inc.)), GPC3 (e.g., GeneChem GPC-3CART (Shanghai GeneChem Co.); 302GPC3-CART (Shanghai GeneChem Co.); CAR-T for liver Cancer (Shanghai GeneChem Co.); GPC 3T cells (Carsgen Therapeutics), MUC1 (e.g., PG-021 001,865 (PersonGen Biotherapeutics (Suzhou) Co.); mesothelin (e.g., H2017-01-P01(Ningbo Cancer TAI-Homeso-CAR) (Sankhaat Thermoku Co.)), mesothelin (e.g., H2017-01-P01(Ningbo Cancer TM) CAR-2016035-WO 25-WO 3-III), Marc-WO 3631-WO 25-III, Mar Heteron TM-WO 25, III), 02,03(Shanghai International Medical Center)), BCMA (e.g., P-BCMA-101 self-memory T-stem cells (Tsccm) CAR-T cells/P-BCMA-101-; HenanCH284(Henan Cancer Hospital/The Pregene (ShenZHen) Biotechnology Company); LCAR-B38MCAR-T cells (Nanjing Legend Biotech Co.); 9762/NCT03338972(Fred Hutchinson Cancer Research Center/Juno Therapeutics, Inc.); descales-08 (Cartesian Therapeutics); KITE-585(Kite, A Gilead Company); bb21217(bluebird bio); bb21217 (Celgene); JCARH125(Juno Therapeutics, Inc.), CD30 (e.g., ICAR 30T cells (Immune Cell, Inc.), EGFR (e.g., EGFR:4-1BB: CD28: CD3 modified T cells/First Shenzhen02(Shenzhen Sceon peptide's Hospital/The Beijing Pregene Science and Technology Company); EGFR-IL12-CART (Shenzhen Second pendant's Hospital/The Pregene (Shenzhen) Biotechnology Co.); SBNK-2016-015-01(Beijing Sangbo 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 medicinal)), mesothelin/PSCA/CEA/HER 2/MUC1/EGFRvIII (e.g., the NCT03267173(First Australised Hospital of Harmed University/Shanghai py CAR-biological-EGFRICN Biotechnology), BCMA/TCA Biotechnology (e.g., BCMA/Biotechnology Co.) (BCMA/Biotechnology Co.)/BCMA/Biotechnology Co.) (BCMA/Biotechnology Co.))) (e.g., BCMA/TAC 3941/BCMA/Biotechnology Co.), etc.), ROR2 (e.g., autologous CCT301-38 or CCT 301-59T cells (Shanghai sinobiowyy 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 (beiijing sang Brain host/Marino Biotechnology Co.)), TAA (e.g., AMG 119(Amgen)), NKG2D (e.g., CM-CS1(Celyad)), or CD123 (e.g., UCART123(cell s s.a.)). The preceding sentence also provides an example of a CAR that binds the antigen (e.g., AMG 119 (Amgen)). These CAR constructs can be used with anti-CD 2 ADCs in the modulation methods disclosed herein.

The CAR construct may further comprise a transmembrane domain that connects (either literally or in general proximity, e.g. with a spacer) the extracellular antigen-binding domain to the cytoplasmic signaling domain. In some embodiments, the extracellular antigen-binding domain (e.g., scFv, Fab, or other antigen-binding portion) of the CAR can be connected to the transmembrane domain using a hinge or other linker. A spacer, linker or hinge can 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 facilitating antigen recognition and binding. As discussed below, the cytoplasmic side of the transmembrane domain may be attached to an intracellular signaling domain, such as the intracellular signaling domain of CD28 or CD3 δ (CD3- δ), and may additionally comprise one or more co-stimulatory domains.

Thus, in certain embodiments, the CAR may further comprise a hinge region located between the extracellular antigen-binding domain and the transmembrane domain. For example, the hinge region may be derived from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 α. In a particular embodiment, the hinge region is derived from the hinge region of IgG 4. In another embodiment, the hinge region is the CD8 hinge domain (see SwissProt/GenBank accession No. P01732).

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 8).

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 RGLDFAPRKI EVMYPPPYLD NEKSNGTIIH VKGKHLCPSP LFPGPSKP (SEQ ID NO: 9).

The transmembrane domain may be derived from the sequence of a protein that contributes an extracellular antigen-binding domain, a protein that contributes an effector function signaling domain, a protein that contributes a proliferation signaling moiety, or a sequence of a completely different protein. In some embodiments, the transmembrane domain is naturally associated with one of the other domains of the CAR. For example, the transmembrane domain and cytoplasmic domain may be derived from the transmembrane region and cytoplasmic region of the same protein. In one embodiment, the transmembrane domain and cytoplasmic domain of the CAR comprise contiguous portions of the CD28 sequence. Any transmembrane domain may be used in the CAR constructs described herein, provided that the domain is capable of anchoring a CAR comprising an antigen binding domain to a cell membrane.

The transmembrane domain is derived from a natural source or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains that can be used in the methods provided herein can be derived from (e.g., comprise at least the following transmembrane domains): an alpha, beta, or delta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD2, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, LFA-1T cell co-receptor, CD 2T cell co-receptor/adhesion molecule, CD8 alpha, and fragments thereof. The transmembrane domain of a protein can be identified using any method known in the art (e.g., hydrophobicity analysis, structural analysis, etc.) or by using public databases such as the UniProt database.

In some embodiments, the transmembrane domain may be synthetic. In exemplary embodiments, the transmembrane domain may comprise predominantly hydrophobic residues, such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan, and valine may be located at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker (preferably between 2 and 10 amino acids in length) may form a link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. The glycine-serine doublet provides a particularly suitable linker.

In some embodiments, the transmembrane domain of the CAR of the invention is a CD8 transmembrane domain. The sequence of CD8 for this purpose is taught in PCT publication No. W02014/055771A 1.

In some embodiments, the transmembrane domain in the CAR is a CD8 transmembrane domain or a functional portion thereof. For example, the CAR may comprise a CD3 transmembrane domain having amino acid sequence LDPKLCYLLD GILFIYGVIL TALFLRVK (SEQ ID NO:10) or a functional portion thereof, such as LCYLLDGILF IYGVILTALF L (SEQ ID NO: 11).

In some embodiments, the transmembrane domain of the CAR of the invention is a CD28 transmembrane domain. Exemplary sequences of CD28 are provided below, as well as exemplary transmembrane domain sequences. In some embodiments, the CD28 transmembrane domain comprises the following exemplary transmembrane domain sequence, or a fragment or variant thereof, capable of anchoring a CAR comprising the sequence to a cell membrane. Thus, in some embodiments, the transmembrane domain of the CAR is a CD28 transmembrane domain comprising the amino acid sequence: FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 12). In one embodiment, the transmembrane domain of the CAR is a CD28 transmembrane domain comprising the amino acid sequence: IEVMYPPPYL DNEKSNGTII HVKGKHLCPS PLFPGPSKPF WVLVVVGGVL ACYSLLVTVA FIIFWV (SEQ ID NO:13) or a functional fragment thereof, such as SEQ ID NO: 12.

In addition to the extracellular antigen-binding domain and transmembrane domain, the CAR comprises an intracellular (or cytoplasmic) signaling domain.

It is known that the signal produced by endogenous TCRs alone is not sufficient to fully activate T cells, and that secondary or costimulatory signals may also be required. Thus, T cell activation can be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation by the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences).

The term "intracellular signaling domain" or "cytoplasmic signaling domain" as used herein refers to the intracellular portion of a molecule. The intracellular signaling domain can generate a signal that promotes an immune effector function of an immune cell comprising the CAR (e.g., a CAR-T cell or a NK cell expressing the CAR). Examples of immune effector functions, such as in CART cells or CAR-expressing NK cells, include cytolytic and helper activities, including secretion of cytokines. In embodiments, the intracellular signaling domain transduces effector function signals and directs the cell to perform a specific function. While the entire intracellular signaling domain may be employed, in many cases, the use of the entire strand is not necessary. To the extent that truncated portions of intracellular signaling domains are used, such truncated portions may be used in place of the entire chain, provided that the truncated portions transduce effector function signals. Thus, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.

In one embodiment, the intracellular signaling domain of the CAR comprises a CD3 δ signaling region as described in SEQ ID No. 14, or a signaling portion thereof.

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO：14)

Cytoplasmic signaling domains can also include, but are not limited to, those derived from CD3 δ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ∈, CDs, CD22, CD79a, CD79b, CD278 ("ICOS"), Fc. ∈ RI, CD66d, DAP10, and DAP 12.

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

Exemplary co-stimulatory signaling regions include 4-1BB, CD21, CD28, CD27, CD127, ICOS, IL-15R α, and OX 40.

In certain embodiments, the cytoplasmic co-stimulatory domain of the CAR comprises the 4-1BB signaling domain by itself or in combination with any other desired cytoplasmic domain that can be used in the context of the CAR. 4-1BB is a member of the TNFR superfamily, having the amino acid sequence provided in GenBank accession No. AAA62478.2 or equivalent residues from non-human species (e.g., mouse, rodent, monkey, ape, etc.); and the "4-1 BB co-stimulatory domain" is defined as amino acid residue 214-255 of GenBank accession AAA62478.2 or an equivalent residue from a non-human species (e.g., mouse, rodent, monkey, ape, etc.).

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

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO：15)。

in one embodiment, the costimulatory signaling domain of the CAR is a CD28 costimulatory signaling region sequence. For example, the costimulatory signaling domain may comprise the following CD28 costimulatory signaling regions, or signaling portions thereof:

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO：16)。

in exemplary embodiments, the cytoplasmic domain of the CAR may comprise the CD3 δ signaling domain in combination with any other desired cytoplasmic domain that may be used in the context of the CARs of the present invention. In certain embodiments, the cytoplasmic domain of the CAR can comprise a CD3 delta domain and a costimulatory signaling region, including but not limited to the costimulatory signaling regions of 4-1BB, CD28, and CD 27.

The cytoplasmic signaling sequences in the cytoplasmic signaling portion of the CAR of the invention can be linked to each other in random or a specific order. Optionally, a short oligopeptide or polypeptide linker or spacer, preferably between 5 and 20 amino acids in length, may be inserted between cytoplasmic domains. A GGGGS (SEQ ID NO:17) or (GGGGS). times.3 (SEQ ID NO:18) provide particularly suitable linkers.

In one embodiment, the CAR used herein comprises an extracellular domain comprising a single chain variable domain of an anti-CD 19 monoclonal antibody, a transmembrane domain comprising the hinge and transmembrane domains of CD8 a, and a cytoplasmic domain comprising the signaling domain of CD3 δ and the signaling domain of 4-1 BB. An exemplary CAR comprises an extracellular domain comprising an anti-CD 19 monoclonal antibody described in Nicholson IC et al, Mol Immunol 34:1157-1165(1997) plus the 21 amino acid signal peptide of CD8 α (translated from 63 nucleotides at positions 26-88 of GenBank accession NM-001768). The CD8 a hinge and transmembrane domain consists of 69 amino acids translated from 207 nucleotides at position 815-1021 of GenBank accession NM-001768. The CD3 δ signaling domain of the preferred embodiment comprises 112 amino acids translated from 339 nucleotides at position 1022-1360 of GenBank accession NM-000734.

A spacer or hinge domain can be incorporated between the extracellular domain (including the antigen binding domain) and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "spacer domain" generally means any oligopeptide or polypeptide that functions to link a transmembrane domain with an extracellular domain and/or a cytoplasmic domain in a polypeptide chain. As used herein, a hinge domain generally means any oligopeptide or polypeptide that functions to provide flexibility to the CAR or domain thereof and/or to prevent steric hindrance of the CAR or domain thereof. In some embodiments, the spacer or hinge domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 5 to 20 amino acids. It is also understood that one or more spacer domains may be included in other regions of the CAR, as aspects of the disclosure are not limited in this respect.

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

In other embodiments, the invention includes nucleic acid sequences encoding the amino acid sequences disclosed herein.

In some embodiments, the above exemplary, non-limiting arrangements are from the left to right, N-terminus to C-terminus of the CAR. The CAR may include or further include any other combination of elements as described herein.

Expressing CAR after identification of parts of the CAR construct

An immune cell is generated, whereby the immune cell expresses the CAR. The method comprises introducing, e.g., transducing, an immune cell with a nucleic acid molecule described herein (e.g., an RNA molecule, e.g., an mRNA) or a vector comprising a nucleic acid molecule encoding a CAR (e.g., a CAR described herein). The invention also provides a method of generating a population of cells (e.g., RNA-engineered cells transiently expressing exogenous RNA). The method comprises introducing an RNA as described herein (e.g., an in vitro transcribed RNA or a synthetic RNA; an mRNA sequence encoding a CAR polypeptide as described herein) into a cell. In embodiments, the RNA transiently expresses the CAR polypeptide. In one embodiment, the cell is a cell as described herein, e.g., an immune effector cell (e.g., a T cell or NK cell, or population of cells).

Other exemplary chimeric antigen receptor constructs are disclosed in: U.S. patent nos. 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 publication No. 2018/0256712a 1; U.S. patent publication No. 2018/0271907a 1; U.S. patent publication No. 2016/0046724a 1; U.S. patent publication No. 2018/0044424a 1; U.S. patent publication No. 2018/0258149a 1; U.S. patent publication No. 2019/0151363a1 and U.S. patent publication No. 2018/0273601a 1; the contents of each of the foregoing patents and patent publications are incorporated herein by reference in their entirety.

anti-CD 2 Antibody Drug Conjugates (ADC)

As described herein, anti-CD 2 ADCs can be used in combination with CAR therapy to treat cancer or autoimmune disease in a human patient. More specifically, anti-CD 2 ADCs can be used to deplete CD2+ cells (e.g., CD2+ T cells) in a human subject who also receives CAR therapy. The anti-CD 2ADC targets endogenous T cells and kills these cells so that the patient's immune system will not attack the CAR-expressing cells (e.g., autologous or allogeneic) administered to the subject. Thus, anti-CD 2ADC is used as a regulatory step in combination with CAR therapy to facilitate the acceptance of engineered immune cells expressing the CAR by recipient patients. One advantage of using anti-CD 2 ADCs as a modulation scheme is that endogenous T cells expressing CD2 can be specifically targeted for depletion, compared to more traditional modulation approaches for CAR therapy where a general lymphodepleting chemotherapeutic agent is administered to the subject.

Examples of anti-CD 2 ADCs and modulation methods using anti-CD 2 ADCs are described in PCT publication WO 2019/108860, which is incorporated herein by reference.

anti-CD 2 antibodies

ADCs that are capable of binding CD2 can be used as therapeutic agents to facilitate acceptance of CAR-expressing immune cells by human patients by preventing or reducing the risk of CAR-expressing immune cell rejection.

The anti-CD 2 ADCs described herein include an anti-CD 2 antibody or antigen-binding portion thereof linked to a cytotoxin.

Human CD2 is also known as the T cell surface antigen T11/Leu-5, T11, CD2 antigen (p50) and sheep red blood cell receptor (SRBC). CD2 is expressed on T cells. Two isoforms of human CD2 have been identified. Isoform 1 comprises 351 amino acids, described in: seed, B.et al (1987)84:3365-69 (see also Sewell et al (1986)83:718-22) and below (NCBI reference sequence (1986)83:3365-69 (see also Sewell et al (1986)83:718-22) and below (NCBI reference sequence: NP-001758.2):

the second isoform of CD2 is 377 amino acids and is identified herein as the NCBI reference sequence: NP _ 001315538.1.

In one embodiment, anti-CD 2 antibodies that can be used in conjunction with the compositions and methods described herein include those antibodies having one or more or all of the following CDRs:

a. CDR-H1 having the amino acid sequence EyYMY (SEQ ID NO: 1);

b. CDR-H2 having amino acid sequence RIDPEDGSIDYVEKFKK (SEQ ID NO: 2);

c. CDR-H3 having amino acid sequence GKFNYRFAY (SEQ ID NO: 3);

d. CDR-L1 having amino acid sequence RSSQSLLHSSGNTYLN (SEQ ID NO: 4);

e. CDR-L2 having the amino acid sequence LVSKLES (SEQ ID NO: 5);

f. CDR-L3 having amino acid sequence MQFTHYPYT (SEQ ID NO: 6).

Antibodies and antigen-binding fragments thereof comprising the foregoing CDR sequences are described, for example, in U.S. patent No. 6,849,258, the disclosure of which is incorporated herein by reference as it relates to anti-CD 2 antibodies and antigen-binding fragments thereof.

Furthermore, in certain embodiments, the anti-CD 2ADC has a serum half-life in the human subject of 3 days or less.

Additional anti-CD 2 antibodies that may be used in the ADCs described herein may be identified using techniques known in the art, such as hybridoma production. Hybridomas can be prepared using the 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, the anti-CD 2 antibody may be used with HuMAb-Or XenoMouse^TMAnd (4) generating. In making additional anti-CD 2 antibodies, the CD2 antigen is isolated and/or purified. The CD2 antigen may be a CD2 fragment from the extracellular domain of CD 2. Immunization of animals can be carried out by any method known in the art. See, for example, Harlow and Lane, Antibodies: organic Manual, New York: Cold Spring Harbor Press, 1990. Methods of 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 CD2 antigen may 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 (immune stimulating complex). After immunization of an animal with the CD2 antigen, antibody-producing immortalized cell lines are prepared from cells isolated from the immunized animal. Following immunization, the animals are sacrificed and lymph nodes and/or splenic B cells are immortalized by methods known in the art (e.g., oncogene metastasis, oncogenic viral transduction, exposure to oncogenic or mutant 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 desirable properties, including robust growth, high antibody production, and desirable antibody properties.

anti-CD 2 antibodies for use in the anti-CD 2 ADCs described herein may also be identified using high throughput screening of antibody libraries or antibody fragment libraries for molecules capable of binding to CD 2. Such methods include in vitro display techniques known in the art, such as, inter alia, phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display and cDNA display. The use of phage display to isolate antibodies, antigen-binding fragments or ligands that bind biologically relevant molecules has been described in, for example, Felici et al, Biotechnol. Annual Rev.1:149-183, 1995; katz, Annual Rev.Biophys.Biomol.Structure.26: 27-45, 1997; and Hoogenboom et al, Immunotechnology 4:1-20,1998, the disclosure of each of which is incorporated herein by reference as it relates to in vitro display technology. Randomized combinatorial peptide libraries have been constructed to select polypeptides that bind to cell surface antigens as described in Kay, Perspect. drug Discovery Des.2:251-268,1995 and Kay et al, mol. Divers.1:139-140,1996, the disclosure of each of which is incorporated herein by reference as it relates to the Discovery of antigen binding molecules. Proteins, such as multimeric proteins, have been successfully phage displayed as functional molecules (see, e.g., EP 0349578, EP 4527839, and EP 0589877, and Chiswell and McCafferty, Trends biotechnol.10: 80-841992, the disclosure of each of which is incorporated herein by reference as it relates to the use of in vitro display techniques to discover antigen binding molecules). In addition, functional antibody fragments such as Fab and scFv fragments have been expressed in vitro display formats (see, e.g., McCafferty et al, Nature 348:552-554, 1990; Barbas et al, Proc. Natl. Acad. Sci. USA88:7978-7982, 1991; and Clackson et al, Nature 352:624-628,1991, the disclosure of each of which is incorporated herein by reference as it relates to an in vitro display platform for the discovery of antigen binding molecules).

In addition to in vitro display techniques, computer modeling techniques can be used to design and identify anti-CD 2 antibodies or antibody fragments in silico (e.g., using the procedures described in US 2013/0288373, the disclosure of which is incorporated herein as it relates to molecular modeling methods for identifying anti-CD 2 antibodies. For example, using computer modeling techniques, one skilled in the art can screen antibody libraries or antibody fragment libraries in silico for molecules capable of binding to a particular epitope on CD2, such as the extracellular epitope of CD 2.

In one embodiment, the anti-CD 2 antibodies used in the ADCs described herein are capable of internalizing into a cell. In identifying anti-CD 2 antibodies (or fragments thereof), additional techniques can be used to identify antibodies or pro-binding fragments that bind to CD2 on the surface of cells (e.g., T cells) and that are also capable of being internalized by the cell, e.g., by receptor-mediated endocytosis. For example, the in vitro display techniques described above may be modified to screen for antibodies or antigen-binding fragments thereof that bind to CD2 on the surface of hematopoietic stem cells and are subsequently internalized. Phage display represents one such technique that can be used in conjunction with this screening paradigm. To identify anti-CD 2 antibodies or fragments thereof that bind CD2 and are subsequently internalized by CD2+ cells, one of skill in the art can use the phage display technology described in Williams et al, Leukemia 19:1432-1438,2005, the disclosure of which is incorporated herein by reference in its entirety.

The internalization ability of an anti-CD 2 antibody or fragment thereof can be assessed, for example, using radionuclide internalization assays known in the art. For example, an anti-CD 2 antibody or fragment thereof identified using in vitro display techniques described herein or known in the art can be functionalized by incorporating the following radioisotopes: such as¹⁸F、⁷⁵Br、⁷⁷Br、¹²²I、¹²³I、¹²⁴I、¹²⁵I、¹²⁹I、¹³¹I、²¹¹At、⁶⁷Ga、¹¹¹In、⁹⁹Tc、¹⁶⁹Yb、¹⁸⁶Re、⁶⁴Cu、⁶⁷Cu、¹⁷⁷Lu、⁷⁷As、⁷²As、⁸⁶Y、⁹⁰Y、⁸⁹Zr、²¹²Bi、²¹³Bi or²²⁵Ac, is used. For example, radioactive halogens, such as a radioactive halogen, can be incorporated using Beads comprising electrophilic halogen reagents, such as polystyrene Beads (e.g., ionization Beads, Thermo Fisher Scientific, inc., Cambridge, MA)¹⁸F、⁷⁵Br、⁷⁷Br、¹²²I、¹²³I、¹²⁴I、¹²⁵I、¹²⁹I、¹³¹I、²¹¹At is incorporated into the antibody, fragment thereof or ligand. Can be used forTo incubate the radiolabeled antibody or fragment thereof with the hematopoietic stem cells for a time sufficient to allow internalization. Internalized antibodies or fragments thereof can be identified by detecting the resulting emission radiation (e.g., gamma-irradiation) of the hematopoietic stem cells as compared to the emission radiation (e.g., gamma-irradiation) of the recovered wash buffer. The aforementioned internalization assays can also be used to characterize ADCs.

In some embodiments, the anti-CD 2 antibody (or fragment thereof) has a defined serum half-life. For example, in a human patient, an anti-CD 2 antibody (or fragment thereof) may have a serum half-life of about 1-24 hours. For example, in a human patient, an ADC comprising such an anti-CD 2 antibody may 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.

For recombinant production of anti-CD 2 antibodies, nucleic acids encoding, for example, antibodies as 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 readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of an antibody).

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. nos. 5,648,237, 5,789,199, and 5,840,523. (see also: Charlton, Methods in Molecular Biology, Vol.248(B.K.C.Lo, eds., Humana Press, Totowa, N.J.,2003), pp.245-254, describing the expression of antibody fragments in E.coli (E.coli.). After expression, the antibody can be isolated from the soluble fraction of the bacterial cell mass (past) and can be further purified.

Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of useful mammalian host cell lines are the monkey kidney CV1 cell line (COS-7) transformed by SV40, the 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 support cells (TM4 cells, as described in, for example, Mather, biol.Reprod.23:243 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK, Buffalo rat liver cells (BRL 3A)), human lung cells (W138), human hepatocyte cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells (as described in, for example, Mather et al, Annals N.Y.Acad.Sci.383: 44), Chinese hamster ovary cells (CHO-38) (CHO-4), including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA77:4216(1980)) and myeloma cell lines such as Y0, NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248(B.K.C.Lo, eds., Humana Press, Totowa, N.J.), 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).

Cytotoxins

Various cytotoxins may be conjugated to anti-CD 2 antibodies via linkers for use in the combination therapies described herein. In particular, the anti-CD 2ADC comprises an antibody (or antigen-binding fragment thereof) conjugated (i.e., covalently attached by a linker) to a cytotoxic moiety (or cytotoxin). In various embodiments, the cytotoxic moiety exhibits reduced or no cytotoxicity upon binding in the conjugate, but restores cytotoxicity after cleavage from the linker. In various embodiments, the cytotoxic moiety maintains cytotoxicity without cleavage from the linker. In some embodiments, the cytotoxic molecule is conjugated to a cell internalizing antibody or antigen binding fragment thereof as disclosed herein, such that upon uptake of the antibody or fragment thereof by a cell, the cytotoxin can access its intracellular target and mediate, for example, T cell death.

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

Ab-(Z-L-D)_n (I)

Accordingly, the antibody or antigen-binding fragment thereof can be conjugated to a plurality of drug moieties as indicated by the integer n, which represents the average number of cytotoxins per antibody, which can range, for example, from about 1 to about 20. Any number of cytotoxins may 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 the ADC in n can also be determined. In some cases, separation, purification, and characterization of homogeneous ADCs where n is a certain value from ADCs with other drug loadings may be accomplished by means such as reverse phase HPLC or electrophoresis.

For some anti-CD 2 ADCs, n may be limited by the number of attachment sites on the antibody. For example, 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 a linker may be attached. Generally, antibodies do not contain many free and reactive cysteine thiol groups to which drug moieties can be attached; cysteine thiol residues in antibodies are mainly present in the form of disulfide bridges. In certain embodiments, the antibody may be reduced under partially or fully reducing conditions with a reducing agent such as Dithiothreitol (DTT) or Tricarbonylethylphosphine (TCEP) to produce a reactive cysteine thiol group. In certain embodiments, higher drug loadings, e.g., n >5, may cause aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody drug conjugates.

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

The loading of the ADC (drug/antibody ratio) 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 reduction conditions for cysteine thiol group modification, (iv) engineering the amino acid sequence of the antibody by recombinant techniques such that the number and position of cysteine residues are modified to control the number and/or position of linker-drug attachments.

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 mitotic spindles (e.g., vinca alkaloids, maytansine alkaloids and derivatives thereof), RNA polymerase inhibitors (e.g., amatoxins, such as alpha-amanitine and derivatives thereof), and agents capable of disrupting protein biosynthesis (e.g., agents exhibiting rnan-glycosidase activity, such as saporin and ricin a chain).

In some embodiments, the cytotoxin is a microtubule binding agent (e.g., a maytansine or maytansine alkaloid), amatoxin, pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolopendrons, and indolopendrons dimers, or a variant thereof, or another cytotoxic compound described herein or known in the art.

In some embodiments, the cytotoxin of the antibody drug conjugate is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is amatoxin or a derivative thereof. In some embodiments, the cytotoxin of an antibody drug conjugate as disclosed herein is an amatoxin or a derivative thereof, such as α -amanitin, β -amanitin, γ -amanitin, epsilon-amanitin, amanamide, amanitin nontoxic cyclic peptide, amanitin monocarboxylic acid, pre-amanitin nontoxic cyclic peptide, or a derivative thereof.

Additional details regarding cytotoxins that can be used against CD2ADC useful in the methods of the invention are described below.

Amanitin shiitake venom

In some embodiments, the RNA polymerase inhibitor is amatoxin or a derivative thereof. In some embodiments, the cytotoxin of an antibody drug conjugate as disclosed herein is an amatoxin or a derivative thereof, such as α -amanitin, β -amanitin, γ -amanitin, epsilon-amanitin, amanamide, amanitin nontoxic cyclic peptide, amanitin monocarboxylic acid, pre-amanitin nontoxic cyclic peptide, or a derivative thereof. The structures of various naturally occurring amatoxins are represented by formula II and accompanying Table 1, and are disclosed, for example, in Zantotti et al, int.J. peptide Protein Res.30,1987, 450-459.

Table 1 structural table of naturally occurring amanitins.

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

A number of positions on amatoxin or a derivative thereof may be used as the position to which the linking moiety L is covalently bonded and thus covalently bonded to the antibody or antigen-binding fragment thereof. In some embodiments, the cytotoxin in the ADC of formula I is amatoxin according to formula (II) or a derivative thereof. In one embodiment, the ADC is represented by the formula Ab-Z-L-Am, wherein Ab is an antibody or antigen-binding fragment thereof that binds CD2, L is a linker, Z is a chemical moiety, and Am is amatoxin. In this embodiment, linker-amanitin conjugate Am-L-Z is represented by formula (III)

Wherein:

R₁is H, OH, OR_AOR OR_C；

R₂Is H, OH, OR_BOR OR_C；

R_AAnd R_BWhen present, combine together with the oxygen atom to which they are bound to form a 5-membered heterocycloalkyl group;

R₃is H, R_COr R_D；

R₄、R₅、R₆And R₇Each of which is independently H, OH, OR_C、OR_D、R_COr R_D；

R₈Is OH, NH₂、OR_C、OR_D、NHR_COr NR_CR_D；

R₉Is H, OH, OR_COR OR_D；

Q is-S-, -S (O) -or-SO₂-；

R_Cis-L-Z' or-L-Z-Ab, wherein L is a linker and is optionally substituted C₁-C₆Alkyl, optionally substituted C₁-C₆Heteroalkyl, optionally substituted C₂-C₆Alkenyl, optionally substituted C₂-C₆Heteroalkenyl, optionally substituted C₂-C₆Alkynyl radical, renOptionally substituted C₂-C₆Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl; comprises a dipeptide; or- ((CH₂)_mO)_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 the coupling reaction of Z' with a functional group on Ab; and is

R_DIs C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl group, C₂-C₆Alkenyl radical, C₂-C₆Heteroalkenyl, C₂-C₆Alkynyl, C₂-C₆Heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or combinations thereof, wherein each C is₁-C₆Alkyl radical, C₁-C₆Heteroalkyl group, C₂-C₆Alkenyl radical, C₂-C₆Heteroalkenyl, C₂-C₆Alkynyl, C₂-C₆(ii) heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with 1 to 5 substituents independently selected for each occurrence from the group consisting of: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl (sulfanyl), halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro. In some embodiments, the amanitin comprises one R_CAnd (4) a substituent.

Formula (III) includes amatoxin and a linker, and in some embodiments, includes linkers, chemical moieties, and antibodies.

In some embodiments, the cytotoxin is amatoxin, and the linker-amatoxin conjugate or the antibody-linker-amatoxin conjugate is represented by formula (IIIA):

wherein:

R₁is H, OH, OR_AOR OR_C；

R₂Is H, OH, OR_BOR OR_C；

R_AAnd R_BWhen present, combine together with the oxygen atom to which they are bound to form a 5-membered heterocycloalkyl group;

R₃is H, R_COr R_D；

R₄、R₅、R₆And R₇Each of which is independently H, OH, OR_C、OR_D、R_COr R_D；

R₈Is OH, NH₂、OR_C、OR_D、NHR_COr NR_CR_D；

R₉Is H, OH, OR_COR OR_D；

Q is-S-, -S (O) -or-SO₂-；

R_Cis-L-Z' or-L-Z-Ab, wherein L is a linker and is optionally substituted C₁-C₆Alkyl, optionally substituted C₁-C₆Heteroalkyl, optionally substituted C₂-C₆Alkenyl, optionally substituted C₂-C₆Heteroalkenyl, optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl; or comprises a dipeptide; or- ((CH₂)_mO)_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 the coupling reaction of Z' with a functional group on Ab; and is

R_DIs C₁-C₆Alkyl radical, C₁-C₆A heteroalkyl group,C₂-C₆Alkenyl radical, C₂-C₆Heteroalkenyl, C₂-C₆Alkynyl, C₂-C₆Heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or combinations thereof, wherein each C is₁-C₆Alkyl radical, C₁-C₆Heteroalkyl group, C₂-C₆Alkenyl radical, C₂-C₆Heteroalkenyl, C₂-C₆Alkynyl, C₂-C₆(ii) heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with 1 to 5 substituents independently selected for each occurrence from the group consisting of: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro. In some embodiments, the amanitin comprises one R_CAnd (4) a substituent.

In some embodiments, R_AAnd R_BTaken together with the oxygen atom to which they are bound, form a 5-membered heterocycloalkyl group of the formula:

wherein Y is- (C ═ O) -, - (C ═ S) -, - (C ═ NR_E)-or- (CR)_ER_E’) -; and is

Wherein R is_EAnd R_E’Each independently is H, C₁-C₆alkylene-R_C、C₁-C₆Heteroalkylidene-R_C、C₂-C₆alkenylene-R_C、C₂-C₆Heteroalkenylene-R_C、C₂-C₆alkynylene-R_C、C₂-C₆Heteroalkynylene-R_COr cycloalkylene-R_Cheterocycloalkylene-R_Carylene-R_COr heteroarylene-R_COr a combination thereof; wherein each C₁-C₆alkylene-R_C、C₁-C₆Heteroalkylidene-R_C、C₂-C₆alkenylene-R_C、C₂-C₆Heteroalkenylene-R_C、C₂-C₆alkynylene-R_C、C₂-C₆Heteroalkynylene-R_COr cycloalkylene-R_Cheterocycloalkylene-R_Carylene-R_COr heteroarylene-R_COptionally substituted with 1 to 5 substituents independently selected for each occurrence from the group consisting of: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

Formula (IIIA) includes amatoxin and a linker, and in some embodiments, includes a linker, a chemical moiety, and an antibody.

In some embodiments, the cytotoxin is amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIA, wherein

R₁Is H, OH, OR_AOR OR_C；

R₂Is H, OH, OR_BOR OR_C；

R_AAnd R_BWhen present, combine with the oxygen atom to which they are bound to form:

wherein R is₃Is H or R_C。

In some embodiments, the cytotoxin is amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIA, wherein

R₁Is H, OH, OR_AOR OR_C；

R₂Is H, OH, OR_BOR OR_C；

R_AAnd R_BWhen present, combine with the oxygen atom to which they are bound to form:

wherein

R₃Is H or R_C；

R₄And R₅Each independently is H, OH, OR_C、R_COR OR_D；

R₆And R₇Each is H;

R₈is OH, NH₂、OR_COr NHR_C；

R₉Is H or OH; and is

Wherein R is_CAnd R_DAs defined above.

In some embodiments, the cytotoxin is amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIA, wherein:

R₁is H, OH OR OR_A；

R₂Is H, OH OR OR_B；

R_AAnd R_BWhen present, combine with the oxygen atom to which they are bound to form:

wherein

R₃、R₄、R₆And R₇Each is H;

R₅is OR_C；

R₈Is OH or NH₂；

R₉Is H or OH;

q is-S-, -S (O) -or-SO₂-; and is

Wherein R is_CAnd R_DAs defined above. Such amatoxin-linker conjugates are described, for example, in U.S. patent application publication No. 2016/0002298, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the cytotoxin is amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIA, wherein:

R₁and R₂Each independently is H or OH;

R₃is R_C；

R₄、R₆And R₇Each is H;

R₅is H, OH or OC₁-C₆An alkyl group;

R₈is OH or NH₂；

R₉Is H or OH;

q is-S-, -S (O) -or-SO₂-; and is

Wherein R is_CAnd R_DAs defined above. Such amatoxin-linker conjugates are described, for example, in U.S. patent application publication No. 2014/0294865, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the cytotoxin is amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIA, wherein:

R₁and R₂Each independently is H or OH;

R₃、R₆and R₇Each is H;

R₄and R₅Each independently is H, OH, OR_COr R_C；

R₈Is OH or NH₂；

R₉Is H or OH;

q is-S-, -S (O) -or-SO₂-; and is

Wherein R is_CAnd R_DAs defined above. Such amatoxin-linker conjugates are described, for example, in U.S. patent application publication No. 2015/0218220, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the cytotoxin is amatoxin represented by formula IIIA or a derivative thereof, wherein:

R₁and R₂Each independently is H or OH;

R₃、R₆and R₇Each is H;

R₄and R₅Each independently is H or OH;

R₈is OH, NH₂、OR_COr NHR_C；

R₉Is H or OH;

q is-S-, -S (O) -or-SO₂-; and is

Wherein R is_CAnd R_DAs defined above. Such amanitin conjugates are described, for example, in U.S. patent nos. 9,233,173 and 9,399,681, the disclosure of each of which is incorporated herein by reference in its entirety.

In some embodiments, the cytotoxin is amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIB, wherein:

wherein:

R₁is H, OH, OR_AOR OR_C；

R₂Is H, OH, OR_BOR OR_C；

R_AAnd R_BWhen present, combine together with the oxygen atom to which they are bound to form a 5-membered heterocycloalkyl group;

R₃is H, R_COr R_D；

R₄、R₅、R₆And R₇Each of which is independently H, OH, OR_C、OR_D、R_COr R_D；

R₈Is OH, NH₂、OR_C、OR_D、NHR_COr NR_CR_D；

R₉Is H, OH, OR_COR OR_D；

Q is-S-, -S (O) -or-SO₂-；

R_Cis-L-Z' or-L-Z-Ab, wherein L is a linker and is optionally substituted C₁-C₆Alkyl, optionally substituted C₁-C₆Heteroalkyl, optionally substituted C₂-C₆Alkenyl, optionally substituted C₂-C₆Heteroalkenyl, optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl; or comprises a dipeptide; or- ((CH₂)_mO)_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 the coupling reaction of Z' with a functional group on Ab; and is

R_DIs C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl group, C₂-C₆Alkenyl radical, C₂-C₆Heteroalkenyl, C₂-C₆Alkynyl, C₂-C₆Heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, or combinations thereof, wherein each C is₁-C₆Alkyl radical, C₁-C₆Heteroalkyl group, C₂-C₆Alkenyl radical, C₂-C₆Heteroalkenyl, C₂-C₆Alkynyl, C₂-C₆The heteroalkynyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is optionally substituted with 1 to 5 substituents, independently for each occurrenceA group consisting of: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

Formula (IIIB) includes amatoxin and a linker, and in some embodiments, includes a linker, a chemical moiety, and an antibody.

In some embodiments, R_AAnd R_BTaken together with the oxygen atom to which they are bound, form a 5-membered heterocycloalkyl group of the formula:

wherein Y is- (C ═ O) -, - (C ═ S) -, - (C ═ NR_E)-or- (CR)_ER_E’) -; and is

Wherein R is_EAnd R_E’Each independently is H, C₁-C₆alkylene-R_C、C₁-C₆Heteroalkylidene-R_C、C₂-C₆alkenylene-R_C、C₂-C₆Heteroalkenylene-R_C、C₂-C₆alkynylene-R_C、C₂-C₆Heteroalkynylene-R_COr cycloalkylene-R_Cheterocycloalkylene-R_Carylene-R_COr heteroarylene-R_COr a combination thereof; wherein each C₁-C₆alkylene-R_C、C₁-C₆Heteroalkylidene-R_C、C₂-C₆alkenylene-R_C、C₂-C₆Heteroalkenylene-R_C、C₂-C₆alkynylene-R_C、C₂-C₆Heteroalkynylene-R_COr cycloalkylene-R_Cheterocycloalkylene-R_Carylene-R_COr heteroarylene-R_COptionally 1 to 5Substituents selected independently for each occurrence from the group consisting of: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

In some embodiments, the antibody or antigen-binding fragment thereof as described herein is conjugated to a amatoxin-linker conjugate or derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIB, wherein

R₁Is H, OH, OR_AOR OR_C；

R₂Is H, OH, OR_BOR OR_C；

R_AAnd R_BWhen present, combine with the oxygen atom to which they are bound to form a 5-membered heterocycloalkyl group of the formula:

wherein R is₃Is H or R_C。

In some embodiments, the cytotoxin is amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIB, wherein

R₁Is H, OH, OR_AOR OR_C；

R₂Is H, OH, OR_BOR OR_C；

R_AAnd R_BWhen present, combine with the oxygen atom to which they are bound to form a 5-membered heterocycloalkyl group of the formula:

wherein

R₃Is H or R_C；

R₄And R₅Each independently is H, OH, OR_C、R_COR OR_D；

R₆And R₇Each is H;

R₈is OH, NH₂、OR_COr NHR_C；

R₉Is H or OH; and is

Wherein R is_CAnd R_DAs defined above.

In some embodiments, the cytotoxin is amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIB, wherein:

R₁is H, OH OR OR_A；

R₂Is H, OH OR OR_B；

R_AAnd R_BWhen present, combine with the oxygen atom to which they are bound to form a 5-membered heterocycloalkyl group of the formula:

wherein

R₃、R₄、R₆And R₇Each is H;

R₅is OR_C；

R₈Is OH or NH₂；

R₉Is H or OH;

q is-S-, -S (O) -or-SO₂-; and is

Wherein R is_CAnd R_DAs defined above. Such amatoxin-linker conjugates are described, for example, in U.S. patent application publication No. 2016/0002298, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the cytotoxin is amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIB, wherein:

R₁and R₂Each independently is H or OH;

R₃is R_C；

R₄、R₆And R₇Each is H;

R₅is H, OH or OC₁-C₆An alkyl group;

R₈is OH or NH₂；

R₉Is H or OH;

q is-S-, -S (O) -or-SO₂-; and is

Wherein R is_CAnd R_DAs defined above. Such amatoxin-linker conjugates are described, for example, in U.S. patent application publication No. 2014/0294865, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the cytotoxin is amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIB, wherein:

R₁and R₂Each independently is H or OH;

R₃、R₆and R₇Each is H;

R₄and R₅Each independently is H, OH, OR_COr R_C；

R₈Is OH or NH₂；

R₉Is H or OH;

q is-S-, -S (O) -or-SO₂-; and is

Wherein R is_CAnd R_DAs defined above. Such amatoxin-linker conjugates are described, for example, in U.S. patent application publication No. 2015/0218220, the disclosure of which is incorporated herein by reference in its entirety.

In some embodiments, the cytotoxin is amatoxin or a derivative thereof, and the amatoxin-linker conjugate is represented by formula IIIB, wherein:

R₁and R₂Each of which isIndependently is H or OH;

R₃、R₆and R₇Each is H;

R₄and R₅Each independently is H or OH;

R₈is OH, NH₂、OR_COr NHR_C；

R₉Is H or OH;

q is-S-, -S (O) -or-SO₂-; and is

Wherein R is_CAnd R_DAs defined above. Such amatoxin-linker conjugates are described, for example, in U.S. patent nos. 9,233,173 and 9,399,681, the disclosure of each of which is incorporated herein by reference in its entirety.

Auristatin

The anti-CD 2 antibodies and antigen-binding fragments thereof described herein can be conjugated to a cytotoxin that is an auristatin (U.S. patent nos. 5,635,483, 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 both 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). An auristatin drug moiety may be attached to an antibody via the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02/088172).

Exemplary auristatin embodiments include N-terminally attached monomethylauristatin drug moieties DE and DF (MMAE and MMAF, respectively), which are disclosed in sensor et al, filed 3/28/2004, Proceedings of the American Association for Cancer Research, volume 45, abstract No. 623, the disclosure of which is expressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE:

wherein the wavy line indicates the point of covalent attachment of the linker of the antibody-drug or drug-linker conjugate (-L-Z-Ab or-L-Z', as described herein).

Another exemplary auristatin embodiment is MMAF,

wherein the wavy line indicates the point of covalent attachment of the linker of the antibody-linker conjugate (-L-Z-Ab or-L-Z', as described herein), as disclosed in US 2005/0238649.

Auristatins can be prepared according to the following method: U.S. Pat. nos. 5,635,483; U.S. Pat. nos. 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, G.R., 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-.

Maytansine alkaloids

The antibodies and antigen binding fragments thereof described herein may be conjugated to a cytotoxin that is a microtubule binding agent. In some embodiments, the microtubule binding agent is maytansine, a maytansine alkaloid, or an analog of a maytansine alkaloid. Maytansinoids are mitotic inhibitors that bind to microtubules and act by inhibiting tubulin polymerization. Maytansine was first isolated from the east African shrub Maytenus serrata (Maytenus serrata) (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogues are disclosed in the following: 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, 4,309,428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663 and 4,371,533. Maytansinoid drug moieties are attractive drug moieties in antibody drug conjugates because they: (i) is relatively easy to prepare by fermentation or chemical modification, derivatizes the fermentation product, (ii) is easy to derivatize with functional groups suitable for conjugation to antibodies via non-disulfide linkers, (iii) is stable in plasma, and (iv) is effective on a variety of tumor cell lines.

Examples of suitable maytansinoids 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.

Examples of suitable maytansinol esters include those having a modified aromatic ring and those having modifications at other positions. Such suitable maytansinoids are disclosed in: U.S. Pat. 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, 4,361,650, 4,362,663, 4,364,866, 4,424,219, 4,450,254, 4,322,348, 4,362,663, 4,371,533, 5,208,020, 5,416,064, 5,475,092, 5,585,499, 5,846,545, 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 maytansinoids and derivatives thereof.

In some embodiments, the Antibody Drug Conjugates (ADCs) of the present disclosure utilize a thiol-containing maytansine alkaloid (DM1), formally designated N, as the cytotoxic agent²' -Deacetyl-N²' - (3-mercapto-1-oxopropyl) -maytansine. DM1 is represented by the following structural formula IV:

in another embodiment, the conjugates of the invention utilize a thiol-containing maytansine alkaloid N²' -Deacetyl-N²' (4-methyl-4-mercapto-1-oxopentyl) -maytansine (e.g., DM4) as a cytotoxic agent. DM4 is represented by the following structural formula V:

another maytansinoid comprising a side chain containing a sterically hindered thiol bond is N²' -Deacetyl-N-²' (4-mercapto-1-oxopentyl) -maytansine (referred to as DM3), represented by the following structural formula VI:

each of the maytansinoids 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.

Many positions on maytansinoids can be used as the position for covalent bonding to a linking moiety, and thus to an antibody or antigen-binding fragment thereof (-L-Z-Ab or-L-Z', as described herein). For example, the C-3 position having 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 having a hydroxyl group are all expected to be useful. In some embodiments, the C-3 position serves as a position for covalent bonding to a linker moiety, and in some particular embodiments, the C-3 position of maytansinol serves as a position for covalent bonding to a linker moiety. There are many linker groups known in the art for use in preparing antibody-maytansine alkaloid conjugates, including, for example, those disclosed in: U.S. Pat. nos. 5,208,020, 6,441,163 and EP 0425235B 1; chari et al, Cancer Research 52: 127-; and U.S.2005/0169933a, the disclosures of which are expressly incorporated herein by reference. Additional linker groups are described and exemplified herein.

The invention also includes various isomers and mixtures of maytansine alkaloids and conjugates. Certain compounds and conjugates of the invention can exist in a variety of stereoisomeric, enantiomeric, and diastereomeric forms. Several descriptions for the production of such antibody-maytansine alkaloid conjugates are provided in the following: U.S. Pat. nos. 5,208,020, 5,416,064, 6,333,410, 6,441,163, 6,716,821, and 7,368,565, each of which is incorporated herein in its entirety.

Anthracyclines

In other embodiments, the antibodies and antigen binding fragments thereof described herein may be conjugated to a cytotoxin that is an anthracycline molecule. Anthracyclines are antibiotic compounds that exhibit cytotoxicity. Research has shown that anthracyclines can operate to kill cells by a number of different mechanisms, including: 1) intercalating a drug molecule into the DNA of a cell, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) free radicals are generated by the drug and then react with cellular macromolecules to cause damage to the cell, or 3) interactions of drug molecules with the cell membrane [ see, for example,Anthracycline Antibiotics In Cancer Therapypeterson et al, "Transport And Storage Of And acyclic In Experimental Systems And 35 Human Leukamia"; bachur, Free radial Damage, supra, at pages 97-102]. Due to their cytotoxic potential, anthracyclines have been used to treat a number of cancers, such as leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma. [ see for example,Anthracycline:Current Status And New DevelopmentsP.H-Wiernik, page 11]. Commonly used anthracyclines include doxorubicin, epirubicin, idarubicin and daunomycin.

Representative examples of anthracyclines include, but are not limited to, daunorubicin (Cerubidine; Bedford Laboratories), doxorubicin (doxorubicin; 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 unzips 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 prototypical cytotoxic natural product anthracycline chemotherapeutics (Sessa et al, (2007) cardiovasc. toxicol.7: 75-79).

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

more than one position on an anthracycline such as PNU may be used as a position to which the linking moiety L is covalently bonded and thus covalently bonded to the anti-CD 2 antibody or antigen-binding fragment thereof as described herein. For example, the linker may be introduced by modification of the hydroxymethyl ketone side chain.

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

wherein the wavy line indicates the point of covalent attachment of the linker of the ADC as described herein.

Pyrrolobenzodiazepines (PBD)

In other embodiments, the anti-CD 2 antibodies or antigen-binding fragments thereof described herein can be conjugated to a cytotoxin that is a Pyrrolobenzodiazepine (PBD) or to a cytotoxin comprising a PBD. 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, anthranilic (antrramycin), dimeric PBDs, and those disclosed in, for example: hartley, J.A, (2011) The level of pyrazolodiazepines as inhibitors, expert Opin Inv Drug,20(6), 733-containing 744 and Antonow D, Thurston DE (2011) Synthesis of DNA-interactive pyro [2,1-c ] [1,4] benzodiazepines (PBDs), Chem Rev 111: 2815-containing 2864.

In some embodiments, the cytotoxin can be a pyrrolobenzodiazepine dimer represented by the following structural formula:

wherein the wavy line indicates the point of covalent attachment of the linker of the ADC as described herein. Such PBD-based ADCs are disclosed, for example, in Sutherland et al, Blood 2013122: 1455-1463, which is incorporated herein by reference in its entirety.

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

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

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

wherein the wavy line indicates the point of covalent attachment of the linker of the ADC as described herein.

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

this particular cytotoxin-linker conjugate is referred to as tesirine (SG3249) and has been described, for example, in Howard et al, ACS med. chem. lett.2016,7(11), 983-.

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

this particular cytotoxic linker conjugate is referred to as 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.

Calicheamicin

In other embodiments, the antibodies and antigen binding fragments thereof described herein can be conjugated to a cytotoxin that is an enediyne antitumor antibiotic (e.g., calicheamicin, ozomicin).

Antibiotics of the calicheamicin family are capable of generating double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates, see U.S. Pat. nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all belonging to the 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-; lode et al, Cancer Research 58: 2925-; and the aforementioned American Cyanamid U.S. patent.

An exemplary calicheamicin is designated gamma₁Which is abbreviated herein as γ, and has the structural formula:

in some embodiments, the calicheamicin is a gamma calicheamicin derivative or an N-acetyl gamma calicheamicin derivative. Structural analogs of calicheamicin that may be used include, but are not limited to, those disclosed in, for example: hinman et al, Cancer Research 53: 3336-; lode et al, Cancer Research 58: 2925-; and the aforementioned U.S. patents. Calicheamicin comprises a methyl trisulfide moiety that can be reacted with an appropriate thiol to form a disulfide, while introducing a functional group that can be used to attach calicheamicin derivatives to an anti-CD 137 antibody or antigen-binding fragment thereof as described herein via a linker. For the preparation of calicheamicin family conjugates, see U.S. Pat. nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all belonging to the 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-; lode et al, Cancer Research 58: 2925-; and the aforementioned American Cyanamid U.S. patent.

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

wherein the wavy lines indicate the attachment points of the joint.

Additional cytotoxins

In other embodiments, the antibodies and antigen-binding fragments thereof described herein may be conjugated to cytotoxins other than or in addition to those disclosed herein above. Additional cytotoxins suitable for use with the compositions and methods described herein include, but are not limited to, 5-ethynyluracil, abiraterone, acylfulvene (acylfulvene), adenosine (adecylenol), adolesin (adozelesin), aldesleukin, altretamine, ammostemin, amamaduox, amifostine, aminolevulinic acid (aminolevulinic acid), amrubicin (amrubicin), amsacrine, anagrelide (anagrelide), anastrozole (anastrozole), andrographolide, angiogenesis inhibitors, enriches (antarelix), anti-dorsal morphogenetic protein-1 (anti-dorsalmonetic protein-1), antiandrogen, prostate cancer, anti-estrogen, anti-oncone (antineopton), antisense oligonucleotides, glycine-nonglutin, adenine dinucleotide gene, modulators, apoptosis modulators, nucleic acid apoptosis regulators, and apoptotroptosis, Oxanilin (asulamine), atamestane (atamestane), amoxastine (atrimustine), apremistatin 1(axinastatin 1), apremistatin 2(axinastatin 2), apremistatin 3(axinastatin 3), azasetron (azasetron), azatolysin (azatoxixin), diazotyrosine, baccatin III derivatives (baccatin III derivatives), banlangonol (balanol), batimastat (batimastat), BCR/ABL antagonists, benzodichlorin (benzochlororins), benzoylstaurosporin (benzoylstaurosporine), beta lactam derivatives, beta-alexin (betagliptin), betagliptin B (aclamycins B), zetimylvanic acid, betahistidinin inhibitors, betahistidinin (biaquinamide), betanidine (betanidine-apine), betagliptin B (biamycin B), betahistidinin B (bleridine), betanidine (biazosterine), betahistidinin (biaquinamide), betahistidinine (biamycin A), biaquinamide (biamine (biaquinamide), biamide (biaquinacrine (B), betamethacin B), bleomycin (biaquinamide), betahistidinine (biaquinamide), betanidine (biaquinacrine (B), betanidine (B), betahistidinine (biaquinacrine (B), betahistidinine (B), betanidine (biaquinacrine (B), betanidine (biaquinacrine (bia, Butortitanium (budotitane), sulfoximine, calcipotriol, calphostin C (calphostin C), camptothecin derivatives (e.g., 10-hydroxy-camptothecin), capecitabine (capecitabine), formamide-aminotriazole (carboxamide-amino-triazole), carboxyamidotriazole (carboxyyamidotrizole), capecitabine (carzelesin), casein kinase inhibitors, castanospermine, cecropin B (cecropin B), cetrorelix (cetrorelix), chlorin, chloroquinoxaline, cicaprost (ciprocast), cis-porphyrin, cladribine (claddibine), clomiphene and analogs thereof, clotrimazole, clarithromycin A (collismycin A), clarithromycin B (collymycin B), collymycin A (brestatin A4), cleistamycin A (4), bellatinib (816), bellatin A), bellatin (816), cryptococcus peptide C (816), cryptolitorine C (calcipotanin C), and (calcipotanin C(s), carboxim-amino-triazole (carboxim), carboxim-tris (cloristine), clofibrate A, clofibrate (clofibrate), clofibrate A, clofibrate (clofibrate), clofibrate B, clo, Nostoc cyclopeptide A derivatives, karacin A (CURACIN A), cyclopentaxanthone, cyclopalm (cycloplatam), cipomycin (cyclopycin), cytarabine sodium octadecylphosphate (cytarabine ocfosfate), cytolysin, hexestrol phosphate (cytostatin), daclizumab, decitabine, dehydromembrane-sphingosine B, 2'deoxycoformycin (2' Deoxycoronafcillin) (DCF), deslorelin (deslorelin), dexifosfamide (dexfosfamide), dexrazoxane (dexrazoxane), dexverapamil (dexverapamide), dizoquinone (diazepine), hymexamycin B, didox (didox), diethylnoramine (diethylnortryptamine), dihydro-5-azacytidine (dihydrazadin-5-diazacycline), dihydrodoxycycline (dihydrodoxycycline), doxycycline (doxycycline), doxycycline (doxycycline), doxycycline, Dronabinol, duocarmycin SA (duocanycin SA), ebselen, etokavastin, edelfosine, esomeprazole, efloroglucine, eflornithine (eflornithine), elemene, ethimidifluoride, epothilone, epithilones, epristeride (epristeride), estramustine and its analogs, etoposide 4' -phosphate (also known as etofosos), exemestane, fazole, fazarabine, veboxamide (fenretinide), filgrastim, finasteride, flavelipid (flavopiridol), flevelastine (flezetidine), fubastard (flustereonlone (flusterbine), fludarabine, fludaunorubicin hydrochloride (flurunavirenzinulinone), fosfamycin hydrochloride (forrmex), melphalan (flusterbinetinine), gefitinib (flusterbinetinine), gadetazolirtine (gadine), gadolinicine (gadolinitum), glutethimide (gadicine), glutethimide (e), glutethimide (gadicine), glutethimide (e), prussion (hepsulfam), homoharringtonine (homoharringtonine) (HHT), hypericin, ibandronic acid, idoxifene (idoxifene), idromantone (idramantone), imofovir (ilmofosine), ilomastat (ilomastat), imidazolacridones (imidazoacridones), imiquimod (imiquimod), immunostimulating peptides, iodobenzylguanidine, iododoxorubicin, Ipomol, irinotecan, ilolat (irolact), issoragladine (irsogladine), isobenzoguanazole (isobenozole), Jestrigiline (jalapinolide), carhalarolide F (kahalalilide F), trexolin-N (lamellarin-N), lanoline peptide (lanreotide), lipophilic polysaccharide (lipoloratadine), lentinolamide (7), lipolat-amide (7), ritol (amide), ritol (amide), and (amide), salts thereof, and salts thereof, Losoxantrone (losoxantrone), losoxantrone (loxorine), lurotecan (lurotecan), lutetium porphyrin (lutetium texaphyrin), lisofenac (lysofyline), maxol, masculin (maspin), matrix metalloproteinase inhibitors, methonuril (menogaril), rneberarone, metirelin (meterelin), methionine, metoclopramide, MIF inhibitors, mifepristone (ifeptone), miltefosine (miltefosine), mirimostimastim (mirimostimustim), mithramycin (mithracin), mitoguazone (mitoguazone), dibromodulcitol, mitomycin and its analogs, mitonaphthylamine (mitonafide), mitoxantrone (mitoxantrone), farotene (monatin), lamivudine (momatriovenin), milnaciprone (N), milnacipratropine (N), milnacipranone (N), milnacipranolide (N), milnacipranolide (vitamin B), milnacipranolide (e), milnacipranolide (milnacipranolide), milnacipranolide (vitamin B, Naproxen (naphalopin), naltostatin (nartogastin), nedaplatin (nedaplatin), nemorubicin (nemorubicin), neridronic acid, nilutamide (nilutamide), lissamycin (nisamycin), ritulin (nitrilyn), octreotide (octreotide), oxcarbazone (okinone), onapristone (onapristone), ondansetron (ondansetron), olaparin (oracin), ormaplatin (ormaplatin), oxaliplatin (oxaliplatin), enomycin (oxaauromycin), taxol and its analogs, pannaomine (palauamine), hexadecanoxymycin (palitoloxacin), pamidronic acid, panaxytriol, panomorphine (panoxacillin), parafibrate (paraflavin), penoxerucin), pentostatin (paraflavin), paraflavin (penoxpreneomycin), paraflavin (pezidine), pemoline (pervone (penoxsulosin), pemetrexedin (pentostatin), pemphitin (paradoxine (penoxsulosin), pemoline (pentostatin), pemoline (pervomycin), pemphibenoxazidine (pervomycin), pemoline (pervopeptaibenoxapezidine), pemoline (pervomycin), pemoline (pervomycin), pem, Podophyllotoxin (podophyllyloxin), podophyllotoxin (porfiromycin), purine nucleoside phosphorylase inhibitors, raltitrexed (raltitrexed), rhizomycin, roguinimine (rogletimine), rohituzone, lurbiglong B1(rubiginone B1), rupotexed (rubixyl), saflufenol (saflufenol), santopril (saintopin), myofol A (sarrophytol A), sargramostim (sargramostim), sobuzosin (sobuzoxazone), sondamine (sonermin), spepatinophosphate (sparfosic acid), spicamycin D (spicamycin D), spiromustine (spirastine), stevespertidine (stipidine), stipaminoside (stigmastatin), tazocine (tazarine), tipatin (tipatin), tipetrexed (tipetrexed), teniposide (tipipramine), thiepinine (tretin), thielavine (tretin), tezomib (tretin (picrin), teosine (tretin), tretin (tretinoine), tretinoine (tretinoine), tretinoine (tretinose (tretinoine), tre, Vilazone (vinxaline), vorozole (vorozole), zeniplatin (zeniplatin) and benzal vitamin c (zilascorb).

Joint

As used herein, the term "linker" means a divalent chemical moiety comprising a chain of covalent bonds or atoms that covalently attaches an anti-CD 2 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 terminus (reactive moiety, Z') of the linker is typically a site capable of conjugation to the antibody via a cysteine thiol or lysine amine group on the antibody, and is thus 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 cytotoxic conjugation reactive end of the linker is generally the site capable of conjugation to the cytotoxin. Non-limiting examples of linker-cytotoxin conjugation include, for example, formation of an amide bond via a carboxyl or basic amine group on the linker, respectively, with a basic amine or carboxyl group on the cytotoxin, or formation of an ether via alkylation of an OH group on the cytotoxin (e.g., via a leaving group on the linker), and the like. In some embodiments, the cytotoxin linker is conjugated by forming an amide bond with a basic amine or carboxyl group on the 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 conjugated form, one or both reactive termini will be absent (such as reactive moiety Z', already converted to chemical moiety Z) or incomplete (such as the carbonyl of a carboxylic acid only) due to the formation of bonds between the linker and/or cytotoxin and between the linker and/or antibody or antigen-binding fragment thereof. Such conjugation reactions are described further below.

A variety of linkers can be used to conjugate the described antibodies, antigen binding fragments, and ligands to cytotoxic molecules. In some embodiments, the linker is cleavable under intracellular conditions such that cleavage of the linker releases the drug unit from the antibody in an intracellular environment. In yet other embodiments, the linker unit is non-cleavable and the drug is released by, for example, antibody degradation. The linkers useful in the ADCs of the present invention are preferably stable extracellularly, preventing aggregation of the ADC molecules, and maintaining the ADC in a readily soluble and monomeric state in aqueous media. Prior to transport or delivery into a cell, preferably the ADC is stable and remains intact, i.e. the antibody remains linked to the drug moiety. The linker is stable outside the target cell and can be cleaved at an effective rate inside the cell. The effective joint will: (i) maintaining the specific binding characteristics of the antibody; (ii) allowing intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact, i.e., not cleaved, until the conjugate has been delivered or transported to the site it is targeted; and (iv) maintaining the cytotoxic, cell killing or cytostatic effect of the cytotoxic moiety. The stability of the ADC can be measured by standard analytical techniques such as mass spectrometry, HPLC and separation/analysis techniques LC/MS. Covalent attachment of the antibody and drug moiety requires that the linker have two reactive functional groups, i.e., a bivalent property in the reactive sense. Bivalent linker reagents useful for attaching two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens and reporter groups, are known and their methods of generating conjugates have been described (Hermanson, G.T. (1996) Bioconjugate Techniques; Academic Press: New York, p.234-242).

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., Lerich et al, bioorg.Med.chem.,20: 571-. Suitable cleavable linkers may include, for example, chemical moieties such as hydrazines, disulfides, thioethers, or dipeptides.

Linkers hydrolyzable under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, and the like. (see, e.g., 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 disclosure of each of which is incorporated herein by reference in its entirety as it relates to a linker suitable for covalent conjugation). Such linkers are relatively stable under neutral pH conditions, such as those in blood, but are unstable below pH5.5 or 5.0 (the approximate pH of lysosomes).

Linkers cleavable under reducing conditions include, for example, disulfides. A variety of disulfide linkers are known in the art, including, for example, disulfide linkers formed as follows can be used: 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, e.g., Thorpe et al, 1987, Cancer Res.47: 5924-.

The linker susceptible to enzymatic hydrolysis may be, for example, a peptide-containing linker that is cleaved by an intracellular peptidase or protease, including but not limited to lysosomal or endosomal proteases. One advantage of using intracellular proteolytic release of the therapeutic agent is that, when conjugated, the agent is generally impaired and the serum stability of the conjugate is generally higher. In some embodiments, the peptidyl linker is at least two amino acids in length or at least three amino acids in length. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Examples of suitable peptides include peptides comprising the following amino acids: such as valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine. Amino acid residues that constitute a component of an amino acid linker include naturally occurring amino acid residues, as well as small numbers 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 (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, for example, in U.S. patent No. 6,214,345, the disclosure of which is incorporated herein by reference in its entirety as it relates to linkers suitable for covalent conjugation. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit.

Linkers suitable for conjugating the antibodies, antigen-binding fragments, and ligands described herein to cytotoxic molecules include those that are capable of releasing a cytotoxin by a1, 6-elimination process. Chemical moieties capable of such elimination 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 as it relates to linkers suitable for covalent conjugation.

In some embodiments, the linker comprises 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-; chakravarty et al (1983) J.Med.chem.26: 638-; US 6214345; US 20030130189; US 20030096743; US 6759509; US 20040052793; US 6218519; US 6835807; US 6268488; US 20040018194; w098/13059; US 20040052793; US 6677435; US 5621002; US 20040121940; w02004/032828). Other such chemical moieties ("autocleavable linkers") capable of performing this process include methylene carbamates and heteroaryl groups such as aminothiazoles, aminoimidazoles, aminopyrimidines, and the like. Linkers containing such heterocyclic autodegradable groups are disclosed in the following: 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, the dipeptide is used in combination with an autolytic linker.

Linkers suitable for use herein may also include one or more optionsA group selected from: c₁-C₆Alkylene radical, C₁-C₆Heteroalkylidene radical, C₂-C₆Alkenylene radical, C₂-C₆Heteroalkenylene radical, C₂-C₆Alkynylene, C₂-C₆Heteroalkynylene, C₃-C₆Cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which may be optionally substituted. Non-limiting examples of such groups include (CH)₂)_p、(CH₂CH₂O)_pAnd- (C ═ O) (CH)₂)_p-A unit where p is an independently selected integer from 1 to 6 for each case.

In some embodiments, each C is₁-C₆Alkyl radical, C₁-C₆Heteroalkyl group, C₂-C₆Alkenyl radical, C₂-C₆Heteroalkenyl, C₂-C₆Alkynyl, C₂-C₆Heteroalkynyl, C₃-C₆The cycloalkyl, heterocycloalkyl, aryl, or heteroaryl groups may be optionally substituted with 1 to 5 substituents independently selected for each occurrence from the group consisting of: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

In some embodiments, each C is₁-C₆Alkyl radical, C₁-C₆Heteroalkyl group, C₂-C₆Alkenyl radical, C₂-C₆Heteroalkenyl, C₂-C₆Alkynyl, C₂-C₆Heteroalkynyl, C₃-C₆The cycloalkyl, heterocycloalkyl, aryl or heteroaryl group may be optionally interrupted by one or more heteroatoms selected from O, S and N.

In some embodiments, each C is₁-C₆Alkyl radical, C₁-C₆Heteroalkyl group, C₂-C₆Alkenyl radical, C₂-C₆Heteroalkenyl, C₂-C₆Alkynyl, C₂-C₆Heteroalkynyl, C₃-C₆The cycloalkyl, heterocycloalkyl, aryl or heteroaryl group may be optionally interrupted by one or more heteroatoms selected from O, S and N and may be optionally substituted with 1 to 5 substituents independently selected for each occurrence from the group consisting of: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, hydroxy, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.

Suitable linkers may comprise groups with enhanced solubility characteristics. For example, Comprising (CH)₂CH₂O)_pThe solubility may be enhanced by a linker for the unit (polyethylene glycol, PEG), as may an alkyl chain substituted with an amino, sulfonic, phosphonic or phosphoric acid residue. Linkers comprising such moieties are disclosed, for example, in U.S. patent nos. 8,236,319 and 9,504,756, the disclosure of each of which is incorporated herein by reference in its entirety as it relates to linkers suitable for covalent conjugation. Other solubility enhancing groups include, for example, acyl and carbamoyl sulfonamide groups having the following structure:

wherein a is 0 or 1; and is

R¹⁰Selected from the group consisting of: hydrogen, C₁-C₂₄Alkyl radical, C₃-C₂₄Cycloalkyl radical, C₁-C₂₄(hetero) aryl radical, C₁-C₂₄Alkyl (hetero) aryl group and C₁-C₂₄(hetero) arylalkyl radical, C₁-C₂₄Alkyl radical, C₃-C₂₄CycloalkanesRadical, C₂-C₂₄(hetero) aryl radical, C₃-C₂₄Alkyl (hetero) aryl group and C₃-C₂₄(hetero) arylalkyl groups, each of which may be optionally substituted and/or optionally interrupted by one or more heteroatoms selected from O, S and NR¹¹R¹²Wherein R is¹¹And R¹²Independently selected from the group consisting of: hydrogen and C₁-C₄An alkyl group; or R¹⁰Is a cytotoxin, wherein the cytotoxin is attached to the N, optionally via a spacer moiety. Linkers comprising such groups are described, for example, in U.S. patent No. 9,636,421 and U.S. patent application publication No. 2017/0298145, the disclosures of which are incorporated herein by reference in their entirety as they relate to linkers suitable for covalent conjugation to cytotoxins and antibodies or antigen-binding fragments thereof.

In some embodiments, the linker may comprise one or more of: hydrazine, disulfide, thioether, dipeptide, p-aminobenzyl (PAB) group, heterocyclic autodegradable group, optionally substituted C₁-C₆Alkyl, optionally substituted C₁-C₆Heteroalkyl, optionally substituted C₂-C₆Alkenyl, optionally substituted C₂-C₆Heteroalkenyl, optionally substituted C₂-C₆Alkynyl, optionally substituted C₂-C₆Heteroalkynyl, optionally substituted C₃-C₆Cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, solubility-enhancing group, acyl, - (C ═ O) -, or- (CH)₂CH₂O)_p-a group, wherein p is an integer from 1 to 6. One skilled in the art will recognize that one or more of the groups listed may be present as a divalent (divalent radical) species, such as C₁-C₆Alkylene groups, and the like.

In some embodiments, the linker comprises 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 para-aminobenzyl group is part of a para-aminobenzylamido unit.

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 (Ac) -PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

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

In some embodiments, the linker comprises a combination of one or more of: peptides, oligosaccharides, - (CH)₂)_p-、-(CH₂CH₂O)_p-, PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys (Ac) -PAB, Phe-Lys (Ac) -PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB or Ala-PAB.

In some embodiments, the linker comprises- (C ═ O) (CH)₂)_p-units, wherein p is an integer from 1 to 6.

In some embodiments, the linker comprises- (CH)_2n-units, wherein n is an integer from 2 to 6. In some embodiments, the linker comprises- ((CH)₂)_nWherein n is 6. In some embodiments, L-Z is

Wherein S is a sulfur atom, represents a reactive substituent (e.g., an-SH group from a cysteine residue) present within an antibody or antigen-binding fragment thereof that binds CD 2.

In some embodiments, the linker comprises ((C)H₂)_mO)_n(CH₂)_m-a group and a 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 a triazole. In some embodiments, ((CH)₂)_mO)_n(CH₂)_mThe radicals and triazoles together comprising

Wherein n is 1 to 10 and the wavy line indicates the point of attachment to another linker component, chemical moiety Z or amatoxin. Other linkers useful in the methods and compositions described herein are described in US 2019/0144504, which is incorporated herein by reference.

In a particular embodiment, the linker comprises a PAB-Ala-Val-propionyl group represented by the structure

Wherein the wavy line indicates the point of attachment of the cytotoxin and the reactive moiety Z'. In another particular embodiment, the linker comprises a PAB-Cit-Val-propionyl group represented by the structure

Wherein the wavy line indicates the point of attachment of the cytotoxin and the reactive moiety Z'. Such PAB-dipeptide-propionyl linkers are disclosed, for example, in patent application publication No. WO2017/149077, which is incorporated herein by reference in its entirety. Furthermore, the cytotoxins disclosed in WO2017/149077 are incorporated herein by reference.

One 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 the 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 by reference herein in its entirety.

In certain embodiments, the intermediate, which is a linker precursor, is reacted with the drug moiety under appropriate conditions. In certain embodiments, reactive groups are used on the drug and/or intermediate or linker. The reaction product between the drug and the intermediate or derivatized drug is then reacted with the antibody or antigen-binding fragment under appropriate conditions. Alternatively, the linker or intermediate may be reacted first with the antibody or derivatized antibody and then with the drug or derivatized drug. Such conjugation reactions will now be described more fully.

Many different reactions can be used to covalently attach a linker or drug-linker conjugate to an antibody or antigen-binding fragment thereof. Suitable attachment points on the antibody molecule include amine groups of lysine, free carboxylic acid groups of glutamic and aspartic acids, thiol groups of cysteine, and various portions of aromatic amino acids. For example, non-specific covalent attachment can be performed using a carbodiimide reaction to link a carboxyl (or amino) group on a compound to an amino (or carboxyl) group on an antibody moiety. In addition, bifunctional agents such as dialdehydes or imidates may also be used to link amino groups on compounds to amino groups on antibody moieties. Schiff base reactions can also be used for drug attachment to binding agents. This method involves periodic acid oxidation of a drug containing ethylene glycol or hydroxyl groups to form an aldehyde, which is then reacted with a binding agent. Attachment occurs via schiff base formation with the amino group 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 skilled artisan and are within the scope of the present disclosure.

Linkers useful for conjugation with 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 in table 2 below. The curves represent the attachment points to the antibody or antigen-binding fragment and the cytotoxic molecule, respectively.

TABLE 2 exemplary chemical moieties Z formed by coupling reactions in the formation of antibody drug conjugates

One 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 that can be used in conjunction with the methods described herein can be formed by reacting an antibody or antigen-binding fragment thereof with a linker or cytotoxin-linker conjugate as described herein, the linker or cytotoxin-linker conjugate including a reactive substituent Z ', the reactive substituent Z' being suitable for reacting with a reactive substituent on the antibody or antigen-binding fragment thereof to form a chemical moiety Z.

As depicted in table 2, examples of suitable reactive substituents on the linker and antibody or antigen-binding fragment thereof include nucleophile/electrophile pairs (e.g., thiol/haloalkane pairs, amine/carbonyl pairs, or thiol/α, β -unsaturated carbonyl pairs, etc.), diene/dienophile pairs (e.g., azide/alkyne pairs or diene/α, β -unsaturated carbonyl pairs, among others), and the like. Coupling reactions between reactive substituents forming chemical moiety Z include, but are not limited to, thiol alkylation, hydroxyalkylation, amine alkylation, amine or hydroxylamine condensation, hydrazine formation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] diels-alder cycloaddition, [3+2] Huisgen cycloaddition, among others), 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.

Reactive substituents that may be present within 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 pendant amine group, e.g., lysine, (iii) a pendant thiol group, e.g., cysteine, and (iv) a sugar hydroxyl or amino group, wherein the antibody is glycosylated. Reactive substituents that may be present within an antibody or antigen-binding fragment thereof as disclosed herein include, but are not limited to, hydroxyl moieties of serine, threonine, and tyrosine residues; the amino moiety of a lysine residue; the carboxyl portion of aspartic and glutamic acid residues; and the thiol moiety of a cysteine residue, as well as the propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of a non-naturally occurring amino acid. In some embodiments, the reactive substituent present within an antibody or antigen-binding fragment thereof as disclosed herein comprises an amine or thiol moiety, which is an amine or thiol moiety. Some antibodies have reducible interchain disulfides, i.e., cysteine bridges. The antibody can be made reactive for conjugation to a linker reagent by treatment with a reducing agent such as DTT (dithiothreitol). Thus, in theory, each cysteine bridge will form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into the antibody by reaction of lysine with 2-iminothiolane (2-iminothiolane) (Traut's reagent), resulting in conversion of the amine to a thiol. Reactive thiol groups can be introduced into an antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., making a mutant antibody comprising one or more non-natural cysteine amino acid residues). U.S. patent No. 7,521,541 teaches the engineering of antibodies by the introduction of reactive cysteine amino acids.

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 antibodies include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of the nucleophilic group can react with an electrophilic group on the antibody and form a covalent bond with the antibody. Useful nucleophilic groups include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazide carboxylate, and aryl hydrazide.

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

Table 3.Complementary reactive substituents and chemical moieties

For example, linkers suitable for the synthesis of drug antibody conjugates and drug ligand conjugates include, but are not limited to, reactive substituents Z', such as maleimide or haloalkyl groups. These may be attached to the linker by the following 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, as described, for example, in Liu et al, 18:690-697,1979, the disclosure of which is incorporated herein by reference as it relates to a linker for chemical conjugation.

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 that is particularly useful for conjugation of microtubule disrupting agents such as auristatins. Such linkers are described by Doronina et al Bioconjugate chem.17:14-24,2006, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation.

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

In some embodiments, the reactive substituent is an N-maleimido group, a halogenated 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, (hetero) cycloalkynyl group, 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, an oxynitride group, a nitrone group, a nitrilimine (nitrile imine) group, a diazonium group, a ketone group, an (O-alkyl) hydroxyamino group, a hydrazine group, a halogenated N-maleimido group, a1, 1-bis (sulfonylmethyl) methylcarbonyl group or an eliminated derivative thereof, 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, A carbonyl halide group or an allenamide group, each of which may be optionally substituted. In some embodiments, the reactive substituent comprises a cycloalkene group, a cycloalkyne group, or an optionally substituted (hetero) cycloalkyne group.

Non-limiting examples of amatoxin-linker conjugates comprising a reactive substituent Z 'suitable for reaction 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) -amatoxin, 7'C- (4- (6- (maleimido) hexanoylamino) piperidin-1-yl) -amatoxin, 7' C- (4- (6- (6- (maleimido) hexanoylamino) hexanoyl) piperazin-1-yl) -amatoxin, 7'C- (4- (4- ((maleimido) methyl) cyclohexanecarbonyl) piperazin-1-yl) -amatoxin, and a conjugate comprising a reactive substituent Z' suitable for reaction with a reactive residue on an antibody or antigen-binding fragment thereof, 7' C- (4- (6- (4- ((maleimido) methyl) cyclohexanecarboxamido) hexanoyl) piperazin-1-yl) -amatoxin, 7' C- (4- (2- (6- (maleimido) hexanoylamino) ethyl) piperidin-1-yl) -amatoxin, 7' C- (4- (2- (6- (6- (maleimido) hexanoylamino) ethyl) piperidin-1-yl) -amatoxin, 7' C- (4- (2- (4- ((maleimido) methyl) cyclohexanecarboxamido) ethyl) piperidin-1-yl) -amatoxin, 7' C- (4- (2- (6- (4- ((maleimido) formamido) ethyl) piperidin-1-yl) -amatoxin Yl) cyclohexanecarboxamido) hexanoylamino) ethyl) piperidin-1-yl) -amatoxin, 7'C- (4- (2- (3-carboxypropionylamino) ethyl) piperidin-1-yl) -amatoxin, 7' C- (4- (2- (2-bromoacetylamino) ethyl) piperidin-1-yl) -amatoxin, 7'C- (4- (2- (3- (pyridin-2-yldisulfanyl) propionylamino) ethyl) piperidin-1-yl) -amatoxin, 7' C- (4- (2- (4- (maleimido) butyrylamino) ethyl) piperidin-1-yl) -amatoxin, and salts thereof, 7' C- (4- (2- (maleimido) acetyl) piperazin-1-yl) -amatoxin, 7' C- (4- (3- (maleimido) propionyl) piperazin-1-yl) -amatoxin, 7' C- (4- (4- (maleimido) butyryl) piperazin-1-yl) -amatoxin, 7' C- (4- (2- (6- (4- ((maleimido) methyl) cyclohexanecarboxamido) caproamido) ethyl) piperidin-1-yl) -amatoxin, 7' C- (3- ((6- (maleimido) caproamido) methyl) pyrrolidin-1-yl) -amatoxin, and methods of making and using the same, 7'C- (3- ((6- (6- (maleimido) hexanoylamino) methyl) pyrrolidin-1-yl) -amatoxin, 7' C- (3- ((4- ((maleimido) methyl) cyclohexanecarboxamido) methyl) pyrrolidin-1-yl) -amatoxin, 7'C- (3- ((6- ((4- (maleimido) methyl) cyclohexanecarboxamido) methyl) pyrrolidin-1-yl) -amatoxin, 7' C- (4- (2- (6- (2- (aminooxy) acetamido) hexanoamido) ethyl) piperidin-1-yl) -amatoxin, and a pharmaceutical composition comprising the compound, 7' C- (4- (2- (4- (2- (aminooxy) acetylamino) butyrylamino) ethyl) piperidin-1-yl) -amatoxin, 7' C- (4- (4- (2- (aminooxy) acetylamino) butyryl) piperazin-1-yl) -amatoxin, 7' C- (4- (6- (2- (aminooxy) acetylamino) hexanoyl) piperazin-1-yl) -amatoxin, 7' C- ((4- (6- (maleimido) hexanoylamino) piperidin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (6- (maleimido) hexanoylamino) ethyl) piperidin-1-yl) methyl) -amatoxin A peptide, 7' C- ((4- (6- (maleimido) hexanoyl) piperazin-1-yl) methyl) -amatoxin, (R) -7' C- ((3- ((6- (maleimido) hexanoylamino) methyl) pyrrolidin-1-yl) methyl) -amatoxin, (S) -7' C- ((3- ((6- (maleimido) hexanoylamino) methyl) pyrrolidin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (6- (6- (maleimido) hexanoylamino) ethyl) piperidin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (4- ((maleimido) methyl) cyclohexaden-1-yl) methyl) -amatoxin Alkanecarboxamido) ethyl) piperidin-1-yl) methyl) -amatoxin, 7'C- ((4- (2- (6- (4- ((maleimido) methyl) cyclohexanecarboxamido) hexanoamido) ethyl) piperidin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (6- (maleimido) hexanoamido) ethyl) piperazin-1-yl) methyl) -amatoxin, 7'C- ((4- (2- (6- (6- (maleimido) hexanoamido) ethyl) piperazin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (4- ((maleimido) methyl) cyclohexanecarboxamido) ethyl) piperazine toxin -1-yl) methyl-amatoxin, 7'C- ((4- (2- (6- (4- ((maleimido) methyl) cyclohexanecarboxamido) hexanoamido) ethyl) piperazin-1-yl) methyl) -amatoxin, 7' C- ((3- ((6- (6- (maleimido) hexanoamido) -S-methyl) pyrrolidin-1-yl) methyl) -amatoxin, 7'C- ((3- ((6- (6- (maleimido) hexanoamido) -R-methyl) pyrrolidin-1-yl) methyl) -amatoxin, 7' C- ((3- ((4- ((maleimido) methyl) cyclohexanecarboxamido) S-methyl) pyrrolidin-1-yl) methyl-amatoxin, 7'C- ((3- ((4- ((maleimido) methyl) cyclohexanecarboxamido) -R-methyl) pyrrolidin-1-yl) methyl) -amatoxin, 7' C- ((3- ((6- (4- ((maleimido) methyl) cyclohexanecarboxamido) methyl) pyrrolidin-1-yl) methyl) -amatoxin, 7'C- ((4- (2- (3-carboxypropionamido) ethyl) piperazin-1-yl) methyl) -amatoxin, 7' C- ((4- (6- (6- (maleimido) hexanoamido) hexanoyl) piperazin-1-yl) methyl) -amatoxin Amatoxin, 7' C- ((4- (6- (4- ((maleimido) methyl) cyclohexanecarboxamido) hexanoyl) piperazin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (maleimido) acetyl) piperazin-1-yl) methyl) -amatoxin, 7' C- ((4- (3- (maleimido) propionyl) piperazin-1-yl) methyl) -amatoxin, 7' C- ((4- (4- (maleimido) butyryl) piperazin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (2- (maleimido) acetamido) ethyl) piperidin-1-yl) methyl) -amatoxin Muscarine, 7'C- ((4- (2- (4- (maleimido) butyrylamino) ethyl) piperidin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (6- (4- ((maleimido) methyl) cyclohexanecarboxamido) ethyl) piperidin-1-yl) methyl) -amatoxin, 7'C- ((3- ((6- (maleimido) hexanoylamino) methyl) azetidin-1-yl) methyl) -amatoxin, 7' C- ((3- (2- (6- (maleimido) hexanoylamino) ethyl) azetidin-1-yl) methyl) -amatoxin, and amatoxin, 7'C- ((3- ((4- ((maleimido) methyl) cyclohexanecarboxamido) methyl) azetidin-1-yl) methyl) -amatoxin, 7' C- ((3- (2- (4- ((maleimido) methyl) cyclohexanecarboxamido) ethyl) azetidin-1-yl) methyl) -amatoxin, 7'C- ((3- (2- (6- (4- ((maleimido) methyl) cyclohexanecarboxamido) hexanoamido) ethyl) azetidin-1-yl) methyl) -amatoxin, 7' C- (((2- (6- (maleimido) -N-methylhexanoamido) ethyl) (methyl) amino) methyl) -amatoxin, and salts thereof, 7' C- (((4- (6- (maleimido) -N-methylhexanamido) butyl (methyl) amino) methyl) -amatoxin, 7' C- ((2- (2- (6- (maleimido) hexanoylamino) ethyl) aziridin-1-yl) methyl) -amatoxin, 7' C- ((2- (2- (6- (4- ((maleimido) methyl) cyclohexanecarboxamido) hexanoylamino) ethyl) azetidin-1-yl) methyl) -amatoxin, 7' C- ((4- (6- (2- (aminooxy) acetamido) hexanoyl) methyl) -amatoxin, 7' C- ((4- (6- (2- (aminooxy) acetylamino) hexanoyl) piperazin-1-yl) methyl) -amatoxin, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof, 7'C- ((4- (1- (aminooxy) -2-oxo-6, 9,12, 15-tetraoxa-3-azaheptadecan-17-o-yl) piperazin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (2- (aminooxy) acetamido) acetyl) piperazin-1-yl) methyl) -amatoxin, 7'C- ((4- (3- (2- (aminooxy) acetamido) propionyl) piperazin-1-yl) methyl) -amatoxin, 7' C- ((4- (4- (2- (aminooxy) acetamido) butyryl) piperazin-1-yl) methyl) -amatoxin, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof, 7'C- ((4- (2- (6- (2- (aminooxy) acetylamino) hexanoylamino) ethyl) piperidin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (2- (2- (aminooxy) acetylamino) ethyl) piperidin-1-yl) methyl) -amatoxin, 7'C- ((4- (2- (4- (2- (aminooxy) acetylamino) butyrylamino) ethyl) piperidin-1-yl) methyl) -amatoxin, 7' C- ((4- (20- (aminooxy) -4, 19-dioxo-6, 9,12, 15-tetraoxa-3, 18-diazicosyl) piperidin-1-yl) methyl-amatoxin, 7' C- (((2- (6- (2- (aminooxy) acetamido) -N-methylhexanoamido) ethyl) (methyl) amino) methyl) -amatoxin, 7' C- (((4- (6- (2- (aminooxy) acetamido) -N-methylhexanoamido) butyl) (methyl) amino) methyl) -amatoxin, 7' C- ((3- ((6- (4- ((maleimido) methyl) cyclohexanecarboxamido) caproamido) methyl) pyrrolidin-1-yl) -S-methyl) -amatoxin, and pharmaceutically acceptable salts thereof, 7'C- ((3- ((6- (4- ((maleimido) methyl) cyclohexanecarboxamido) hexanoylamino) -R-methyl) pyrrolidin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (2-bromoacetamido) ethyl) piperazin-1-yl) methyl) -amatoxin, 7'C- ((4- (2- (2-bromoacetamido) ethyl) piperidin-1-yl) methyl) -amatoxin, 7' C- ((4- (2- (3- (pyridin-2-yldisulfonyl) propionylamino) ethyl) piperidin-1-yl) methyl) -amatoxin, 6'O- (6- (6- (maleimido) hexanoylamino) hexyl) -amatoxin, 6' O- (5- (4- ((maleimido) methyl) cyclohexanecarboxamido) pentyl) -amatoxin, 6'O- (2- ((6- (maleimido) hexyl) oxy) -2-oxoethyl) -amatoxin, 6' O- ((6- (maleimido) hexyl) carbamoyl) -amatoxin, 6'O- ((6- (4- ((maleimido) methyl) cyclohexanecarboxamido) hexyl) carbamoyl) -amatoxin, 6' O- (6- (2-bromoacetamido) hexyl) -amatoxin, amatoxin, 7' C- (4- (6- (azido) hexanoylamino) piperidin-1-yl) -amatoxin, 7' C- (4- (hex-5-ynoylamino) piperidin-1-yl) -amatoxin, 7' C- (4- (2- (6- (maleimido) hexanoylamino) ethyl) piperazin-1-yl) -amatoxin, 7' C- (4- (2- (6- (6- (maleimido) hexanoylamino) ethyl) piperazin-1-yl) -amatoxin, 6' O- (6- (6- (11, 12-didehydro-5), 6-dihydro-dibenzo [ b, f ] azacyclo-cin (azocin) -5-yl) -6-oxohexanoylamino) hexyl-amatoxin, 6'O- (6- (hex-5-ynoylamino) hexyl) -amatoxin, 6' O- (6- (2- (aminooxy) acetylamino) hexyl) -amatoxin, 6'O- ((6-aminooxy) hexyl) -amatoxin and 6' O- (6- (2-iodoacetamido) hexyl) -amatoxin.

In some embodiments, chemical moiety Z is selected from table 3 or table 4. In some embodiments, the chemical moiety Z is:

wherein S is a sulfur atom, represents a reactive substituent present within an antibody or antigen-binding fragment thereof (such as an anti-CD 5 antibody).

In some embodiments, amanitin as disclosed herein is conjugated with a linker-reactive moiety-L-Z' having the formula:

wherein the wavy line indicates the point of attachment to a substituent on the cytotoxin (e.g., amatoxin). The linker-reactive substituent group L-Z' may alternatively be referred to as N- β -maleimidopropanoyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB).

In some embodiments, amanitin as disclosed herein is conjugated with a linker-reactive moiety-L-Z' having the formula:

wherein the wavy line indicates the point of attachment to a substituent on the cytotoxin (e.g., amatoxin). The linker-reactive substituent group L-Z' may alternatively be referred to as N- β -maleimidopropanoyl-Val-Cit-p-aminobenzyl (BMP-Val-Cit-PAB).

In some embodiments, linker L and chemical moiety Z (collectively referred to as L-Z) are

Wherein S is a sulfur atom, represents a reactive substituent present within 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 amatoxin.

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

the foregoing linker moieties and amatoxin-linker conjugates, as well as other linker moieties and amatoxin-linker conjugates, which 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 disclosure of each of which is incorporated herein by reference in its entirety.

The foregoing linker moieties and amatoxin-linker conjugates, as well as other linker moieties and amatoxin-linker conjugates, which 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 disclosure of each of which is incorporated herein by reference in its entirety.

In one embodiment, the CD2 antibody or antigen-binding fragment described herein may bind to amatoxin so as to form a conjugate represented by the formula Ab-Z-L-Am, wherein Ab is CD2 antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is amatoxin, each as described herein.

In some embodiments, Am-L-Z-Ab is:

in some embodiments, Am-L-Z-Ab is:

in some embodiments, Am-L-Z-Ab is:

in some embodiments, Am-L-Z-Ab is:

in some embodiments, Am-L-Z-Ab is:

preparation of antibody drug conjugates

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

In another aspect, the anti-CD 2 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 of the sulphur 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).

In another aspect, the anti-CD 2 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 of the sulphur atom of the sulfhydryl group as described above.

In yet another aspect, the anti-CD 2 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 ADCs were then formed by conjugation of the corresponding aldehydes 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.

Methods for conjugating linker-drug moieties to cell-targeting proteins such as antibodies, immunoglobulins, or fragments thereof are found, for example, in U.S. Pat. nos. 5,208,020; U.S. Pat. nos. 6,441,163; WO 2005037992; WO 2005081711; and WO2006/034488, all of which are hereby expressly incorporated by reference in their entirety.

Route of administration and dosage

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

The ADCs described herein may 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 ADCs described herein may 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 may be sterilized using techniques known in the art.

Pharmaceutical formulations comprising anti-CD 2 ADCs as described herein are prepared by mixing such ADCs with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16 th edition, Osol, a. eds. (1980)), either as a lyophilized formulation or as an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other 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 a non-ionic surfactant such as polyethylene glycol (PEG).

The amount of ADC administered should be sufficient to deplete cells, e.g., activated T cells, that reject CAR cell therapy. Determination of a therapeutically effective dose is within the ability of a practitioner in the art, however, by way of example, in embodiments of the methods described herein for treating an immune disease or cancer using systemic administration of an ADC, an effective human dose will be in 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 may affect the recommended dosage. Depending on the mode of administration employed, repeated systemic doses are envisaged in order to maintain effective levels, for example, in order to reduce the risk of CAR-T cell rejection.

The anti-CD 2 ADCs described herein may be administered by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular, or parenteral administration. The most suitable route of administration in any given case will depend on the particular ADC, the patient, the method of pharmaceutical formulation, the method of administration (e.g. time of administration and route of administration), the age, weight, sex, severity of the disease being treated, the diet of the patient and the rate of excretion from the patient.

Effective doses of anti-CD 2 ADCs described herein may range, for example, from about 0.001mg/kg body weight to about 100mg/kg body weight per single (e.g., bolus) administration, multiple administrations, or continuous administration, or may range to achieve optimal serum concentrations of anti-CD 2ADC (e.g., serum concentrations of 0.0001 μ g/mL to 5000 μ g/mL). The dose of anti-CD 2ADC can be administered daily, weekly, or monthly or more times (e.g., 2-10 times) to a human subject that has received CAR therapy, is receiving CAR therapy concurrently, or will receive CAR therapy at a time point after delivery of the anti-CD 2 ADC. The anti-CD 2ADC may be administered to a human patient in one or more doses. In one embodiment, the anti-CD 2ADC may be administered prior to CAR therapy in an amount sufficient to reduce the amount of host-reactive T cells by, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

Examples

The following examples are provided to provide those of ordinary skill in the art with a description of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

The data in examples 1-6 below are described in international patent application No. WO 2019/108860, which is incorporated herein by reference.

Example 1: in vitro binding assay for anti-CD 2 antibody.

To determine the binding characteristics of the anti-CD 2 antibodies RPA-2.10mIgG1 and Ab1 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. The indicated purified human antibody (Ab1-hIgG1) or murine antibody (RPA-2.10mIgG1) was immobilized on an anti-human Fc biosensor (AHC; Pall ForteBio 18-5063) or anti-murine Fc biosensor (AMQ; Pall ForteBio18-5090) and incubated with 50nM purified human CD2 ectodomain (Sigma Aldrich and catalog # 5086). The apparent monovalent affinity (K) of each IgG for the purified extracellular domain of human CD2 is shown in Table 3_D) Apparent binding Rate (K)_ON) And apparent dissociation rate (K)_DIS) They were determined by ForteBio data analysis software version 10 calculation with a 1:1 binding model partial full fit.

Further characterization of the anti-CD 2 antibodies is provided in examples 2 to 6.

Table 3: kinetics of binding of indicated IgG to the extracellular domain of human CD2

Example 2: in vitro cell-binding assays for anti-CD 2 antibodies

MOLT-4 cells (i.e., immortalized human T lymphoblast cell line) were plated at 20,000 cells/well and stained with a titer of the indicated murine anti-CD 2 antibody (i.e., RPA-2.10, TS1/8, BH1, UMCD2, 1e7e8.g4, or LT2) for 2 hours at 4 ℃. A constant amount of secondary anti-mouse AF488 dye was added for 30 minutes at 4 ℃. After washing, the plate was run on a flow cytometer and binding of the indicated antibody (and the negative control, i.e., mIgG1) was determined based on the geometric mean fluorescence intensity in the AF488 channel. The results from these assays are provided in fig. 1.

As shown in FIG. 1, murine anti-CD 2 antibodies RPA-2.10, TS1/8, BH1, UMCD2, 1E7E8.G4, and LT2 bind to human T lymphoblastoid cells (i.e., MOLT-4 cells), with EC being incorporated in the cells₅₀160pM (RPA-2.10), 125pM (TS 1/8), 639pM (BH1), 151pM (UMCD2), 134pM (1E7E8) and 60pM (LT 2).

Example 3: in vitro primary cell binding assay for anti-CD 2 antibodies

Primary human T cells were plated at 8 × 10⁴Individual cells/well were plated and stained with a titer of the murine anti-CD 2 antibody RPA-2.10 for 2 hours at 37 ℃. A constant amount of secondary anti-mouse or anti-human AF488 dye was added relative to the primary antibody, lasting 30 minutes at 4 ℃. After washing, the plates were run on a flow cytometer and binding of the indicated antibodies (and negative controls, i.e., mIgG1 or hIgG1) was determined based on the geometric mean fluorescence intensity in the AF488 channel. The results from these assays are provided in fig. 2.

As shown in figure 2, the murine anti-CD 2 antibody RPA-2.10 binds to primary human T cells. Wherein EEC₅₀＝1.84pM(RPA-2.10)。

Example 4 in vitro analysis of anti-CD 2 amanitin Antibody Drug Conjugates (ADCs) Using an in vitro T cell killing assay

The anti-CD 2 antibody RPA 2.10 was conjugated to amanitin with a cleavable linker to form anti-CD 2-ADC. An anti-CD 2-ADC having a mean interchain drug-to-antibody ratio (DAR) of 6 was prepared from the murine anti-CD 2 antibody RPA-2.10. A second anti-CD 2-ADC with an average DAR of 2 was prepared using human chimeric variants of RPA-2.10 conjugated to amanitin using site-specific conjugation. Furthermore, a fast half-life variant of anti-CD 2-ADC was generated by introducing the H435A mutation. Each anti-CD 2-ADC was evaluated using an in vitro T cell killing assay.

Cryopreserved negatively selected primary human T cells were thawed and stimulated with anti-CD3 antibody and IL-2. At the start of the assay, each well of a 384 well plate was inoculated with 2X 10⁴T cells and the indicated ADC or unconjugated anti-CD 2 antibody was added to the wells at different concentrations between 0.003nM and 30nM and then placed at 37 ℃ and 5% CO₂In an incubator. After five days of culture, cells were analyzed by flow cytometry. Cells were stained with viability marker 7-AAD and run on a volume flow cytometer. The number of surviving T cells (FIGS. 3A and 3B) was determined by FSC vs SSC and7-AAD staining determination. Unconjugated anti-CD 2 antibody (RPA 2.10) was used as a comparative subject (fig. 3A).

As shown in figure 3A, anti-CD 2-ADC with an interchain drug-to-antibody ratio of 6 exhibited efficient and specific killing of T cells (IC50 ═ 5.0pM), while T cells remained viable in the presence of unconjugated ("naked") anti-CD 2 antibody. As shown in figure 3B, human chimeric anti-CD 2-ADC with site-specific drug-to-antibody ratio of 2 maintained an effective level of T cell killing similar to that of DAR6 ADC (IC50 ═ 1.0 pM). Furthermore, the short half-life variant of anti-CD 2-ADC (H435A) showed similar levels of T cell killing as anti-CD 2-ADC with WT half-life (IC50 ═ 6.3 pM; fig. 3B).

Example 5 in vitro analysis of anti-CD 2 amanitin Antibody Drug Conjugates (ADCs) Using an in vitro T cell killing assay

The anti-CD 2 antibody RPA 2.10 was conjugated to amanitin with a cleavable linker to form an interchain anti-CD 2-ADC with an average interchain drug-to-antibody ratio (DAR) of 6. In vitro Natural Killer (NK) cell killing assays were used to evaluate anti-CD 2-ADC.

Primary human CD56+ CD3-NK cells were cultured for 4 days with recombinant IL-2 and IL-15. At the start of the assay, 30,000 NK cells freshly isolated from healthy human donors were seeded in each well of 384-well plates, and the indicated ADCs or controls (i.e., IgG1 or IgG 1-amanitine ADC) were added to the wells at different concentrations between 0.003nM and 30nM, then placed at 37 ℃ and 5% CO₂In an incubator. After 4 days of culture, NK cell viability was analyzed by CellTiter-Glo assay (FIG. 4).

As shown in figure 4, anti-CD 2-ADC showed effective killing of NK cells with an IC50 of 5.2 pM. The lack of complete killing by anti-CD 2-ADC is consistent with the fact that CD2 is expressed only on about 75% of NK cells.

Example 6 analysis of T cell depletion Using hNSG mouse model

In vivo T cell depletion assays were performed using humanized NSG mice (Jackson Laboratories). The anti-CD 2 antibody RPA 2.10 was conjugated to amanitin with a cleavable linker to form anti-CD 2-ADC. One anti-CD 2-ADC with a mean interchain drug-antibody ratio (DAR) of 6 was prepared with murine RPA 2.10, while the other anti-CD 2-ADC with a mean site-specific DAR of 2 was prepared with human chimeric RPA 2.10. Each anti-CD 2-ADC (DAR6 and DAR2) was administered to the humanized mouse model in a single intravenous injection (0.3 mg/kg, 1mg/kg, or 3mg/kg for DAR6 ADC and 1mg/kg or 3mg/kg for DAR2 ADC). Peripheral blood, bone marrow, or thymus samples were collected on day 7 and the absolute number of CD3+ T cells was determined by flow cytometry (see figures 5A and 5B for DAR2ADC and figures 6A-6C for DAR6 ADC).

As shown in figures 5A-5B, humanized NSG mice treated with 0.3mg/kg, 1mg/kg, or 3mg/kg inter-chain DAR6 anti-CD 2-ADC exhibited efficient T cell depletion in peripheral blood or bone marrow, while thymic T cells were depleted after treatment with 3mg/kg DAR6 anti-CD 2-ADC. For comparison, fig. 5A and 5B also show the level of T cell depletion after treatment of humanized NSG mice with 25mg/kg Ab1 (unconjugated anti-CD 2 antibody) or with the indicated control (i.e., 25mg/kg anti-CD 52 antibody (YTH34.5 clone)), 3mg/kg hIgG 1-amanitine ADC ("hIgG 1-AM"), 25mg/kg hIgG1, or PBS).

As shown in figures 6A-6C, humanized NSG mice treated with 1mg/kg or 3mg/kg site-specific DAR2 anti-CD 2-ADC exhibited efficient T cell depletion in peripheral blood or bone marrow, while thymus T cells exhibited about 59% depletion after treatment with 3mg/kg DAR2 anti-CD 2-ADC. For comparison, FIGS. 6A-6C also show the level of T cell depletion after treatment of humanized NSG mice with 3mg/kg of unconjugated anti-CD 2 antibody or with the indicated control (i.e., 3mg/kg hIgG 1-amanitine ADC ("hIgG 1-AM") or PBS).

Example 7 administration of allogeneic CAR-T cells in a mouse model

The following study was conducted to assess the level of CAR-T cells present in allogeneic recipients under different conditions.

The study used the murine allogeneic CAR-T model.

On day 0, mice of the first treatment group were administered 1 × 10 by intravenous infusion⁷Cell/kg to 1X 10⁹Individual cell/kg allogeneic TCells, to be treated with a priming (priming) dose of allogeneic T cells. On day 3, mice were administered anti-CD 2- α -amanitin ADC at a dose of 3 mg/kg. On day 10, after the ADCs were substantially cleared from the mouse blood, the mice were administered allogeneic CAR-T cells. CAR-T cells were from the same donor as allogeneic T cells administered on day 0.

Mice in the second treatment group were treated using the same protocol as the first treatment group, but with unconjugated anti-CD 2 antibody administered on day 3 instead of anti-CD 2 ADC.

Mice of the third treatment group were treated using the same protocol as the first treatment group, but with an isotype control antibody conjugated to a-amanitin instead of anti-CD 2ADC on day 3.

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

Mice in the fifth treatment group were administered allogeneic CAR-T cells on day 10 without prior treatment.

Mice in the sixth treatment group were administered autologous CAR-T cells on day 10 without prior treatment.

The number of CAR-T cells present in the spleen and peripheral blood of mice from each treatment group was determined on day 14, day 17 and day 30. The number of CD2+ 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 to determine symptoms of CAR-T cell rejection and the presence of CAR-T cells.

Example 8 administration of an anti-CD 2 antibody drug conjugate to a human patient to prevent rejection of allogeneic cell therapy

A human patient is selected to receive an allogeneic cell therapy, such as an allogeneic CAR cell therapy. To inhibit or prevent rejection of allogeneic cells, an anti-CD 2 Antibody Drug Conjugate (ADC) is administered according to the methods disclosed herein. The doctor performs the following treatment steps.

First, an initial amount of allogeneic cells is administered intravenously to a patient in an amount sufficient to elicit a primed immune response to the allogeneic cells. In the priming step, allogeneic cells are administered to the patient in order to elicit an immune response, resulting in endogenous activated CD2+ T cells.

Subsequently, an anti-CD 2ADC comprising an anti-CD 137 antibody conjugated to a cytotoxin via a linker is administered to the patient. The anti-CD 2ADC is administered in an amount effective to deplete endogenous CD2+ activated T cells. Following administration of anti-CD 2ADC, the level of CD2+ activated T cells in the patient is assessed to confirm depletion.

Next, a therapeutically effective amount of the CAR-expressing allogeneic cells is administered to the patient. The allogeneic cells are derived from the same donor as the cells administered to the patient during the priming step. Recipient patient acceptance of allogeneic cells is facilitated and the risk of rejection is reduced relative to a patient receiving allogeneic cell therapy without sensitization and administration of anti-CD 2 ADC.

TABLE 4 sequence summary

Other embodiments

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

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Sequence listing

<110> Meizhenda therapeutic Co

<120> use of anti-CD 2 Antibody Drug Conjugates (ADCs) in allogeneic cell therapy

<130> M103034 2050WO

<140>

<141>

<150> 62/773,108

<151> 2018-11-29

<150> 62/702,301

<151> 2018-07-23

<160> 43

<170> PatentIn version 3.5

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<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<223> Artificial sequence: description of synthetic peptides

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Glu Tyr Tyr Met Tyr

1 5

<210> 2

<211> 17

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<213> Artificial Sequence (Artificial Sequence)

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Arg Ile Asp Pro Glu Asp Gly Ser Ile Asp Tyr Val Glu Lys Phe Lys

1 5 10 15

Lys

<210> 3

<211> 9

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<213> Artificial Sequence (Artificial Sequence)

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Gly Lys Phe Asn Tyr Arg Phe Ala Tyr

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<210> 4

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<213> Artificial Sequence (Artificial Sequence)

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Arg Ser Ser Gln Ser Leu Leu His Ser Ser Gly Asn Thr Tyr Leu Asn

1 5 10 15

<210> 5

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Leu Val Ser Lys Leu Glu Ser

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<210> 6

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Met Gln Phe Thr His Tyr Pro Tyr Thr

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<210> 7

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<213> Intelligent (Homo sapiens)

<400> 7

Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn

1 5 10 15

Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu

20 25 30

Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe

35 40 45

Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp

50 55 60

Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu

65 70 75 80

Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His

85 90 95

Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr

100 105 110

Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile Gln Glu

115 120 125

Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu

130 135 140

Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu Tyr Gln

145 150 155 160

Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr His Lys Trp

165 170 175

Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn Lys Val

180 185 190

Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys Pro Glu Lys Gly Leu

195 200 205

Asp Ile Tyr Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met

210 215 220

Val Phe Val Ala Leu Leu Val Phe Tyr Ile Thr Lys Arg Lys Lys Gln

225 230 235 240

Arg Ser Arg Arg Asn Asp Glu Glu Leu Glu Thr Arg Ala His Arg Val

245 250 255

Ala Thr Glu Glu Arg Gly Arg Lys Pro His Gln Ile Pro Ala Ser Thr

260 265 270

Pro Gln Asn Pro Ala Thr Ser Gln His Pro Pro Pro Pro Pro Gly His

275 280 285

Arg Ser Gln Ala Pro Ser His Arg Pro Pro Pro Pro Gly His Arg Val

290 295 300

Gln His Gln Pro Gln Lys Arg Pro Pro Ala Pro Ser Gly Thr Gln Val

305 310 315 320

His Gln Gln Lys Gly Pro Pro Leu Pro Arg Pro Arg Val Gln Pro Lys

325 330 335

Pro Pro His Gly Ala Ala Glu Asn Ser Leu Ser Pro Ser Ser Asn

340 345 350

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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

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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

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<211> 28

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<400> 10

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> 11

<211> 21

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<213> Artificial Sequence (Artificial Sequence)

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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> 12

<211> 27

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<223> Artificial sequence: description of synthetic peptides

<400> 12

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> 13

<211> 66

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<400> 13

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> 14

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 14

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> 15

<211> 42

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 15

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> 16

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<213> Artificial Sequence (Artificial Sequence)

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<400> 16

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> 17

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

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Gly Gly Gly Gly Ser

1 5

<210> 18

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 18

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 19

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 19

Glu Tyr Tyr Met Tyr

1 5

<210> 20

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 20

Arg Ile Asp Pro Glu Asp Gly Ser Ile Asp Tyr Val Glu Lys Phe Lys

1 5 10 15

Lys

<210> 21

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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<400> 21

Gly Lys Phe Asn Tyr Arg Phe Ala Tyr

1 5

<210> 22

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

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Arg Ser Ser Gln Ser Leu Leu His Ser Ser Gly Asn Thr Tyr Leu Asn

1 5 10 15

<210> 23

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 23

Leu Val Ser Lys Leu Glu Ser

1 5

<210> 24

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 24

Met Gln Phe Thr His Tyr Pro Tyr Thr

1 5

<210> 25

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 25

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Leu Met

35 40 45

Gly Arg Ile Asp Pro Glu Asp Gly Ser Ile Asp Tyr Val Glu Lys Phe

50 55 60

Lys Lys Lys Val Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Lys Phe Asn Tyr Arg Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 26

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 26

Asp Val Val Met Thr Gln Ser Pro Pro Ser Leu Leu Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Ser Gly Asn Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Pro Leu Ile Tyr Leu Val Ser Lys Leu Glu Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Gly Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Phe

85 90 95

Thr His Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 27

<211> 118

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 27

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Gln Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Glu Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Leu Val

35 40 45

Gly Arg Ile Asp Pro Glu Asp Gly Ser Ile Asp Tyr Val Glu Lys Phe

50 55 60

Lys Lys Lys Val Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Lys Phe Asn Tyr Arg Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 28

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 28

Asp Val Val Met Thr Gln Ser Pro Pro Ser Leu Leu Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Ser Gly Asn Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Pro Leu Ile Tyr Leu Val Ser Lys Leu Glu Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Gly Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Phe

85 90 95

Thr His Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 29

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 29

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> 30

<211> 109

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 30

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> 31

<211> 351

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 31

Met Ser Phe Pro Cys Lys Phe Val Ala Ser Phe Leu Leu Ile Phe Asn

1 5 10 15

Val Ser Ser Lys Gly Ala Val Ser Lys Glu Ile Thr Asn Ala Leu Glu

20 25 30

Thr Trp Gly Ala Leu Gly Gln Asp Ile Asn Leu Asp Ile Pro Ser Phe

35 40 45

Gln Met Ser Asp Asp Ile Asp Asp Ile Lys Trp Glu Lys Thr Ser Asp

50 55 60

Lys Lys Lys Ile Ala Gln Phe Arg Lys Glu Lys Glu Thr Phe Lys Glu

65 70 75 80

Lys Asp Thr Tyr Lys Leu Phe Lys Asn Gly Thr Leu Lys Ile Lys His

85 90 95

Leu Lys Thr Asp Asp Gln Asp Ile Tyr Lys Val Ser Ile Tyr Asp Thr

100 105 110

Lys Gly Lys Asn Val Leu Glu Lys Ile Phe Asp Leu Lys Ile Gln Glu

115 120 125

Arg Val Ser Lys Pro Lys Ile Ser Trp Thr Cys Ile Asn Thr Thr Leu

130 135 140

Thr Cys Glu Val Met Asn Gly Thr Asp Pro Glu Leu Asn Leu Tyr Gln

145 150 155 160

Asp Gly Lys His Leu Lys Leu Ser Gln Arg Val Ile Thr His Lys Trp

165 170 175

Thr Thr Ser Leu Ser Ala Lys Phe Lys Cys Thr Ala Gly Asn Lys Val

180 185 190

Ser Lys Glu Ser Ser Val Glu Pro Val Ser Cys Pro Glu Lys Gly Leu

195 200 205

Asp Ile Tyr Leu Ile Ile Gly Ile Cys Gly Gly Gly Ser Leu Leu Met

210 215 220

Val Phe Val Ala Leu Leu Val Phe Tyr Ile Thr Lys Arg Lys Lys Gln

225 230 235 240

Arg Ser Arg Arg Asn Asp Glu Glu Leu Glu Thr Arg Ala His Arg Val

245 250 255

Ala Thr Glu Glu Arg Gly Arg Lys Pro His Gln Ile Pro Ala Ser Thr

260 265 270

Pro Gln Asn Pro Ala Thr Ser Gln His Pro Pro Pro Pro Pro Gly His

275 280 285

Arg Ser Gln Ala Pro Ser His Arg Pro Pro Pro Pro Gly His Arg Val

290 295 300

Gln His Gln Pro Gln Lys Arg Pro Pro Ala Pro Ser Gly Thr Gln Val

305 310 315 320

His Gln Gln Lys Gly Pro Pro Leu Pro Arg Pro Arg Val Gln Pro Lys

325 330 335

Pro Pro His Gly Ala Ala Glu Asn Ser Leu Ser Pro Ser Ser Asn

340 345 350

<210> 32

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 32

Gly Phe Thr Phe Ser Ser Tyr

1 5

<210> 33

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 33

Ser Gly Gly Gly Phe

1 5

<210> 34

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 34

Ser Ser Tyr Gly Glu Ile Met Asp Tyr

1 5

<210> 35

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 35

Ser Ser Tyr Gly Glu Leu Met Asp Tyr

1 5

<210> 36

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 36

Arg Ala Ser Gln Arg Ile Gly Thr Ser Ile His

1 5 10

<210> 37

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 37

Tyr Ala Ser Glu Ser Ile Ser

1 5

<210> 38

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of synthetic peptides

<400> 38

Gln Gln Ser His Gly Trp Pro Phe Thr Phe

1 5 10

<210> 39

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 39

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Gly Gly Gly Phe Leu Tyr Tyr Leu Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu

65 70 75 80

His Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Ser Ser Tyr Gly Glu Ile Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 40

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 40

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Ser Gly Gly Gly Phe Leu Tyr Tyr Leu Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu

65 70 75 80

His Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Ser Ser Tyr Gly Glu Leu Met Asp Tyr Trp Gly Gln Gly Thr Ser

100 105 110

Val Thr Val Ser Ser

115

<210> 41

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 41

Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Arg Ile Gly Thr Ser

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Thr Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Val Ala Asp Tyr Tyr Cys Gln Gln Ser His Gly Trp Pro Phe

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Glu

100 105

<210> 42

<211> 324

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 42

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 43

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Artificial sequence: description of the synthetic Polypeptides

<400> 43

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105