Use of anti-CD 5 Antibody Drug Conjugates (ADCs) in allogeneic cell therapy
阅读说明：本技术 抗cd5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 5 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CD5+细胞以便促进对表达CAR的免疫细胞的接受的方法。将抗CD5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 CD5+ 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 5 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 the 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 5 Antibody Drug Conjugate (ADC) to a human subject having cancer or an autoimmune disease, wherein the anti-CD 5 ADC comprises an anti-CD 5 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 or 2, 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 5ADC 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 allogeneic cells expressing the CAR are therapeutically effectiveThe amount is about 1X 104Individual cell/kg to about 1.0X 108Individual cells/kg.
10. A method of treating a patient having a tumor, the method comprising (i) administering an anti-CD 5 ADC to a subject in need thereof, wherein the anti-CD 5 ADC comprises an anti-CD 5 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.
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 5 ADC 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 5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, respectively, and the anti-CD 5 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively.
14. The method of claim 13, wherein the anti-CD 5 antibody or antigen-binding fragment thereof is chimeric or humanized.
15. The method of any one of claims 1-14, wherein the anti-CD 5 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 5 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, ORAOR ORC；
R2Is H, OH, ORBOR ORC；
RAAnd RBWhen present, combine together with the oxygen atom to which they are bound to form an optionally substituted 5-membered heterocycloalkyl group;
R3is H, RCOr RD；
R4、R5、R6And R7Each independently is H, OH, ORC、ORD、RCOr RD；
R8Is OH, NH2、ORC、ORD、NHRCOr NRCRD；
R9Is H, OH, ORCOR ORD；
Q is-S-, -S (O) -or-SO2-；
RCis-L-Z;
RDis 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 5 antibody or antigen-binding fragment thereof, wherein Am contains exactly one RcAnd (4) a substituent.
21. The method of claim 20, wherein the linker (L) is optionally substituted C1-C6Alkyl, optionally substituted C1-C6Heteroalkyl, optionally substituted C2-C6Alkenyl, optionally substituted C 2-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 16, 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 claim 1 or 10, wherein the ADC is represented by any one of the following structures:
27. the method of claim 1 or 10, 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 1-30, 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 5 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.
The invention includes 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, the method comprising administering an anti-CD 5 Antibody Drug Conjugate (ADC) to the human subject having cancer or an autoimmune disease, wherein the anti-CD 5 ADC comprises an anti-CD 5 antibody or antigen-binding fragment thereof conjugated to a cytotoxin via a linker; and 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 (alemtuzumab) prior to, simultaneously with, or after administration of the immune cells expressing the CAR. In another embodiment, the human subject is not administered a lymphodepleting chemotherapeutic agent prior to, simultaneously with, or after administration of the immune cells expressing the CAR. In one embodiment, the lymphodepleting chemotherapeutic agent is fludarabine, cyclophosphamide, bendamustine and/or pentostatin.
In one embodiment, the method further comprises administering an anti-CD 5 ADC to the human subject prior to administration of the immune cells expressing the CAR.
In one embodiment, the method further comprises administering the anti-CD 5 ADC to the human subject about 12 hours to about 21 days prior to administration of the CAR-expressing immune cells.
In one embodiment, the immune cell is an allogeneic cell or an autologous cell. In one 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 10 4Individual cell/kg to about 1.0X 108Individual cells/kg.
The invention also features a method of treating a patient having a tumor, the method comprising administering an anti-CD 5 ADC to a patient in need thereof, wherein the anti-CD 5 ADC comprises an anti-CD 5 antibody or antigen-binding fragment thereof conjugated to a cytotoxin via a linker, and administering a therapeutically effective amount of about 1 x 106(ii) engineered CAR T cells/kg to about 1X 108Each engineered CAR T cell/kg is administered to the patient. 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 certain embodiments of the invention, the anti-CD 5 ADC is administered to the patient as a single dose or as multiple doses.
In one embodiment, the anti-CD 5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5, respectively, and the anti-CD 5 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising CDR1, CDR2, and CDR3 having the amino acid sequences set forth in SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No. 8, respectively.
In one embodiment, the anti-CD 5 antibody or antigen-binding fragment thereof is chimeric or humanized.
In another embodiment, the anti-CD 5 antibody or antigen-binding fragment thereof is an IgG1 isotype or an IgG4 isotype.
In yet another embodiment, 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 one embodiment, the RNA polymerase inhibitor is amatoxin. In another embodiment, the RNA polymerase inhibitor is amanitin (e.g., alpha-amanitin, beta-amanitin, gamma-amanitin, epsilon-amanitin, amanitin (amanin), amanitin amide (amaninamide), amanitin nontoxic cyclic peptide (amacullin), amanitin carboxylic acid (amaculinic acid), or amanitin nontoxic cyclic peptide (proamullin)).
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 CD5, 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, ORAOR ORC；
R2Is H, OH, ORBOR ORC；
RAAnd RBWhen present, combine together with the oxygen atom to which they are bound to form an optionally substituted 5-membered heterocycloalkyl group;
R3is H, RCOr RD；
R4、R5、R6And R7Each independently is H, OH, ORC、ORD、RCOr RD；
R8Is OH, NH2、ORC、ORD、NHRCOr NRCRD；
R9Is H, OH, ORCOR ORD；
Q is-S-, -S (O) -or-SO2-；
RCis-L-Z;
RDis 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 C 2-C6Heteroalkenyl, optionally substituted C2-C6Alkynyl, optionally substituted C2-C6Heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted 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 5 antibody or antigen-binding fragment thereof.
In this embodiment, linker-amanitin conjugate Am-L-Z is represented by formula (III)
Wherein R is1Is H, OH, ORAOR ORC；
R2Is H, OH, ORBOR ORC；
RAAnd RBWhen present, combine together with the oxygen atom to which they are bound to form an optionally substituted 5-membered heterocycloalkyl group;
R3is H, RCOr RD；
R4、R5、R6And R7Each independently is H, OH, ORC、ORD、RCOr RD；
R8Is OH, NH2、ORC、ORD、NHRCOr NRCRD；
R9Is H, OH, ORCOR ORD；
Q is-S-, -S (O) -or-SO2-；
RCis-L-Z;
RDis optionally substituted C1-C6Alkyl, optionally substituted C1-C6Heteroalkyl, optionally substituted C2-C6Alkenyl, optionally substituted C2-C6Heteroalkenyl, optionally substituted C2-C6Alkynyl, optionally substituted C 2-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 5 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 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.
In some embodiments, Am comprises exactly one RCAnd (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 5.
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 one embodiment, the ADC has a serum half-life of 3 days or less.
In one embodiment, the extracellular domain of the CAR is an scFv antibody.
In one embodiment, the extracellular domain of the CAR is a single chain T cell receptor (scTCR). In one embodiment, the extracellular domain of the CAR comprises a non-immunoglobulin scaffold protein.
In one embodiment, the extracellular domain of the CAR binds to a tumor antigen that is: CD19, CD22, CD30, CD7, BCMA, CD137, CD22, CD20, AFP, GPC3, MUC1, mesothelin, CD38, PD1, EGFR (e.g., EGFRvIII), MG7, BCMA, TACI, CEA, PSCA, CEA, HER2, MUC1, CD33, ROR2, NKR-2, PSCA, CD28, TAA, NKG2D, or 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 one embodiment, the cytoplasmic domain of the CAR comprises a CD3 delta cytoplasmic signaling domain.
The methods and compositions disclosed herein may be used to treat a human subject having a cancer including, but not limited to, 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, recurrent or refractory B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large cell lymphoma, recurrent or refractory diffuse large cell lymphoma, anaplastic large cell lymphoma, primary mediastinal B cell lymphoma, recurrent mediastinal large B cell lymphoma, refractory mediastinal large B cell lymphoma, hodgkin lymphoma, non-hodgkin lymphoma, recurrent or refractory non-hodgkin lymphoma, refractory large cell lymphoma, and combinations thereof, Refractory invasive non-hodgkin's lymphoma, B cell non-hodgkin's lymphoma, 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 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, recurrent plasma cell myeloma, refractory plasma cell myeloma, multiple myeloma, human myeloma, Relapsed or refractory multiple myeloma, bone multiple myeloma, brain malignant glioma, myelodysplastic syndrome, EGFR-positive colorectal cancer, glioblastoma multiforme, neoplasms, blast 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 5 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 1A and table 1B below. In certain embodiments, the anti-CD 5 antibody or antigen-binding fragment thereof comprises a combination of heavy chain variable regions and light chain variable regions as set forth in table 1A and table 1B.
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 5 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 5 antibody concentration (x-axis).
Fig. 2 graphically depicts the results of an in vitro primary cell binding assay in which an indicated anti-CD 5 antibody (i.e., "CD 55D 7") or negative control (i.e., hIgG1) was incubated with primary human T cells followed by incubation with fluorophore-conjugated anti-IgG antibody. The signal was detected by flow cytometry and expressed as the geometric mean fluorescence intensity (y-axis) as a function of anti-CD 5 antibody concentration (x-axis).
Fig. 3A and 3B graphically depict the results of an in vitro T cell killing assay comprising an anti-CD 5 amanitin ADC (i.e., 5D7-AM or "CD 55D 7 AM") with either interchain conjugated amanitin with an average drug-to-antibody ratio (DAR) of 6 (fig. 3A) or site-specifically conjugated amanitin with a DAR of 2 (fig. 3B). In fig. 3A, an anti-CD 5-ADC T cell killing assay is shown compared to a non-conjugated anti-CD 55D 7 antibody (i.e., "naked CD 55D 7"). In fig. 3B, an anti-CD 5-ADC T cell killing assay is shown compared to an H435A mutated anti-CD 55D 7 antibody (i.e., CD 55D 7D 265c.h435a AM) with a reduced half-life of the antibody (i.e., "CD 5 fast half-life AM"). The results show the number of surviving T cells (y-axis) as a function of ADC (CD 55D 7 AM, CD 55D 7D 265c.h435a AM) or unconjugated antibody (naked CD 55D 7) concentration (x-axis) as assessed using flow cytometry.
FIGS. 4A-4B 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. 4A) and bone marrow (FIG. 4B) of humanized NSG mice 7 days after a single administration of 0.3mg/kg, 1mg/kg, or 3mg/kg of anti-CD 55D 7 amanitine ADC with an interchain DAR of 6 (i.e., CD 55D 7-AM). For comparison, FIGS. 4A-4B also show the level of T cell depletion after treatment of humanized NSG mice with the indicated controls (i.e., 25mg/kg anti-CD 5 antibody, 3mg/kg hIgG 1-amanitine ADC (i.e., hIgG1-AM), 25mg/kg hIgG1, or PBS).
FIGS. 5A-5C 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), bone marrow (FIG. 5B), and thymus (FIG. 5C) of humanized NSG mice 7 days after a single administration of 1mg/kg or 3mg/kg of an anti-CD 55D 7-amanitine ADC with a site-specific DAR of 2 (i.e., CD 55D 7-AM). For comparison, figures 5A-5C also show the level of T cell depletion after treatment of humanized NSG mice with 3mg/kg of unconjugated anti-CD 5 antibody (i.e., CD 55D 7) 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 5 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 5 antibody" or "antibody that binds to CD 5" or "anti-CD 5 ADC" or "ADC that binds to CD 5" refers to an antibody or ADC that specifically binds to human CD5 when CD5 is present on the cell surface of a cell, such as a T cell. The amino acid sequence of human CD5 to which the anti-CD 5 antibody (or anti-CD 5 ADC) will bind is described below in SEQ ID NO: 20.
As used herein, the term "specifically binds" refers to an antibody (or ADC) that recognizes and binds a particular eggWhite structure (epitope) rather than the ability of general proteins. 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 ofDThe antibody then specifically binds to a target such as CD 5. In one embodiment, as used herein, the term "specifically binds to CD 5" or "specifically binds to CD 5" refers to binding to CD5 and having 1.0 x 10-7Dissociation constant (K) of M or lessD) As determined by surface plasmon resonance. In one embodiment, KDDetermined 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 5.
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 CD5, 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 5 ADC 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 5 antibody or ADC on CD 5-expressing cells, the term "depletion" refers to a reduction or elimination in the number of CD 5-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 CD5, antigen-binding fragment thereof, or specific anti-CD 5 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. Amanitins useful for use in conjunction with the compositions and methods described herein include compounds such as, but not limited to, compounds of formula (II), e.g., alpha-amanitin, beta-amanitin, gamma-amanitin, epsilon-amanitin, amanamide, amanitin nontoxic cyclic peptide, amanitin carboxylic acid, and amanitin nontoxic cyclic peptide. 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.
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, benzoyl 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. C 1-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. Arylalkyl groups contain from 6 to 20 carbon atoms, e.g., the alkyl portion of an arylalkyl group (including alkyl, alkenyl, or alkynyl groups) is from 1 to 6 carbon atoms, and the aryl portion is from 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. Unless otherwise limited by the definition of an individual substituent, the aforementioned chemical moieties, such as "alkyl", "alkylene", "heteroalkyl", "heteroalkylene", "alkenyl", "alkenylene", "heteroalkenyl", "heteroalkenylene", "alkynyl", "alkynylene", "heteroalkynyl", "heteroalkynylene", "cycloalkyl", "cycloalkylene", "heterocycloalkyl", "heterocycloalkylene", "aryl", "arylene", "heteroaryl", and "heteroarylene" groups may be optionally substituted.
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 radical naming conventions clearly indicate that the radical is a divalent radical, such as "alkyleneThe group "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 combining one or more water molecules with a molecule of substance, in which the water retains its molecular state as H2And O. Hydrates refer to, for example, 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 5 ADC to a human patient to facilitate the acceptance of CAR-expressing immune cells in CAR therapy.
anti-CD 5 ADC 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 CD5+ cells, e.g., CD5+ T cells, and eliminate CD5+ 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 5 Antibody Drug Conjugate (ADC) to deplete a population of CD 5-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 CD5 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 5 ADCs can be used to selectively target activated T cells in patients who will receive CAR cell therapy. anti-CD 5 ADCs as described herein may also be used to reduce the risk of CAR cell rejection by targeting and depleting CD5 positive cells in human patients who have received CAR therapy.
The compositions and methods described herein can be used to deplete CD5+ 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 5 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 5 ADC 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 5 ADC 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 5 ADC 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 5 ADC is administered to a human patient in need thereof about 3 days prior to administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 2 days prior to administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 1 day prior to administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 20 hours prior to administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 18 hours prior to administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered 15 hours prior to administration of the CAR cell therapy to a human patient in need thereof. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 12 hours prior to administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 6 hours prior to administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 4 hours prior to administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 2 hours prior to administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof concurrently with administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 2 hours after administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 4 hours after administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 6 hours after administration of the CAR cell therapy. In one embodiment, the anti-CD 5 ADC is administered to a human patient in need thereof about 12 hours after administration of the CAR cell therapy.
In some embodiments, the anti-CD 5 ADC 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 5 ADC 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 5 ADC 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 5 ADC is administered prior to administration of the CAR-expressing immune cells to a human patient in need thereof. In one embodiment, the anti-CD 5 ADC is administered to a human patient in combination with CAR therapy, wherein the anti-CD 5 ADC 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 5 ADC is administered to a human patient in combination with CAR therapy, wherein the anti-CD 5 ADC 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 5 ADC is administered to a human patient in combination with CAR therapy, wherein the anti-CD 5 ADC 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 5 ADC is administered to a human patient in combination with CAR therapy, wherein the anti-CD 5 ADC 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 5 ADC is administered to a human patient in combination with CAR therapy, wherein the anti-CD 5 ADC 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 5 ADC is administered to a human patient in combination with CAR therapy, wherein the anti-CD 5 ADC 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 5 ADC is administered to a human patient in combination with CAR therapy, wherein the anti-CD 5 ADC 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 5 ADC, wherein a decrease in the total number of T cells in the human patient after administration of the anti-CD 5 ADC relative to the level prior to administration indicates the efficacy of the anti-CD 5 ADC 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 5 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 5 ADC. In one embodiment, the level of endogenous T cells is determined one day or less prior to administration of the anti-CD 5 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 5 ADC 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 5 ADC 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 5 ADC 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 5 ADC is used in combination with another therapy in order to promote tolerance of the CAR-expressing immune cells. For example, an anti-CD 2 ADC may also be administered to a human patient prior to the human patient receiving CAR therapy. The anti-CD 2 ADC may be administered prior to the anti-CD 5 ADC, simultaneously with the anti-CD 5 ADC, or after the anti-CD 5 ADC, wherein both the anti-CD 2 ADC and the anti-CD 5 ADC 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 5 ADC 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 5 antibody drug conjugate is used to deplete CD5 expressing donor cells, e.g., activated CD5 expressing T cells, by administering the anti-CD 5 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 5 ADC 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 5 ADC 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 5 ADC is administered to a human patient in combination with CAR-T cell therapy. In one embodiment, the anti-CD 5 ADC 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 5 ADC 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 5 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 5 ADCs can be used as modulators of CAR therapy by promoting acceptance of CAR-expressing cells by eliminating endogenous CD5+ 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-3 CART (Shanghai GeneChem Co.); 302 GPC3-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-B38M CAR-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 5 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: 9).
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: 10).
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, CD5, 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 in a CAR for use herein is the CD8 transmembrane domain or a portion thereof. 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:11) or a functional portion thereof, such as LCYLLDGILF IYGVILTALF L (SEQ ID NO: 12).
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: 13). 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:16) or a functional fragment thereof, such as SEQ ID NO: 14.
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. 15 or a signaling portion thereof.
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"), fcsri, 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:
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:
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:18) or (GGGGS). times.3 (SEQ ID NO:19) 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 I C 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 may be incorporated between the extracellular domain (including the antigen binding domain) and the transmembrane domain (as described above) 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 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.
Upon identification of portions of the CAR construct, immune cells expressing the CAR are generated, whereby the immune cells express 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). Notably, the invention includes nucleic acids encoding the amino acid sequences disclosed 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 5 Antibody Drug Conjugates (ADC)
As described herein, anti-CD 5 ADCs can be used in combination with CAR therapy to treat cancer or autoimmune disease in a human patient. More specifically, anti-CD 5 ADCs can be used to deplete CD5+ cells (e.g., CD5+ T cells) in a human subject who also receives CAR therapy. The anti-CD 5ADC 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 5 ADCs are used as a regulatory step in combination with CAR therapy to facilitate the acceptance of engineered immune cells expressing CARs by recipient patients. One advantage of using anti-CD 5 ADCs as a modulation scheme is that endogenous T cells expressing CD5 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.
anti-CD 5 antibodies
ADCs that are capable of binding CD5 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 5 ADCs described herein include an anti-CD 5 antibody or antigen-binding portion thereof linked to a cytotoxin.
Human CD5 is also known as lymphocyte antigens T1, T1, Leu-1 and LEU 1. CD5 is expressed on human T cells. Two isoforms of human CD5 have been identified. Isoform 1 comprises 495 amino acids and is described in Gladkikh et al (2017) Cancer Med.6(12):2984 and Jones et al (1986) Nature 323(6086): 346). The amino acid sequence of CD5 (isoform 1) is provided below (NCBI reference sequence: NP-055022.2):
the second isoform of human CD5 is 438 amino acids and is identified below as the NCBI reference sequence: NP _ 001333385.1. Unlike isoform 1, CD5 isoform 2 is an intracellular protein. Isoform 2 contains a different 5'UTR compared to isoform 1 and lacks the in-frame portion of the 5' coding region. The resulting isoform 2 has a shorter N-terminus compared to isoform 1. CD5 isoform 2 lacks the leader peptide compared to isoform 1, and represents the intracellular isoform present in a subset of B lymphocytes. The ADCs described herein are specific for isoform 1 human CD5, which represents the extracellular form of human CD 5.
In one embodiment, the anti-CD 5 antibody that may be used in the methods and compositions described herein is antibody 5D7v (Ab5D7 v). The heavy chain variable region (VH) amino acid sequence of Ab5D7v is provided below as SEQ ID NO: 1.
The VH CDR amino acid sequence of Ab5D7v above is underlined and is as follows: FSLSTSGMG (VH CDR 1; SEQ ID NO: 3); WWDDD (VH CDR 2; SEQ ID NO: 4); and RRATGTGFDY (VH CDR 3; SEQ ID NO: 5).
The light chain variable region (VL) amino acid sequence of Ab5D7v is provided as SEQ ID NO:2 below.
The VL CDR amino acid sequence of Ab5D7v above is underlined and is as follows: QDVGTA (VL CDR 1; SEQ ID NO: 6); WTSTRHT (VL CDR 2; SEQ ID NO: 7); and YNSYNSYNT (VL CDR 3; SEQ ID NO: 8).
In one embodiment, the anti-CD 5 ADC comprises an anti-CD 5 antibody, the anti-CD 5 antibody comprises a heavy chain comprising a CDR1 domain comprising the amino acid sequence set forth in SEQ ID No. 3, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID No. 4, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID No. 5, and the anti-CD 5 antibody comprises a light chain comprising a CDR1 domain comprising the amino acid sequence set forth in SEQ ID No. 6, a CDR2 domain comprising the amino acid sequence set forth in SEQ ID No. 7, and a CDR3 domain comprising the amino acid sequence set forth in SEQ ID No. 8, wherein the antibody is conjugated to a cytotoxin via a linker.
In one embodiment, the anti-CD 5 ADC comprises an anti-CD 5 antibody, the anti-CD 5 antibody comprises a heavy chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID No. 1 and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID No. 2, wherein the antibody is conjugated to a cytotoxin via a linker.
In another embodiment, the anti-CD 5 antibody for the ADC described herein is a 5D7 antibody (see, e.g., US 20080254027, the disclosure of which is incorporated herein by reference). In another embodiment, the anti-CD 5 antibodies that can be used in the methods and compositions described herein (including ADCs) are variants of the 5D7 antibody (see, e.g., US 20080254027, the disclosure of which is incorporated herein by reference).
Furthermore, in certain embodiments, the anti-CD 5 ADC has a serum half-life in the human subject of 3 days or less.
Additional anti-CD 5 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. Optionally, anti-C D5 antibody can be usedOr XenoMouseTMAnd (4) generating. In making additional anti-CD 5 antibodies, the CD5 antigen is isolated and/or purified. The CD5 antigen may be a CD5 fragment from the extracellular domain of CD 5. Immunization of animals can be carried out by any method known in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990. Methods 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 CD5 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 CD5 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 5 antibodies for use in the anti-CD 5 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 5. 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. USA 88:7978-7982, 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 5 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 5 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 CD5, such as the extracellular epitope of CD 5.
In one embodiment, the anti-CD 5 antibodies used in the ADCs described herein are capable of internalizing into a cell. In identifying anti-CD 5 antibodies (or fragments thereof), additional techniques can be used to identify antibodies or pro-binding fragments that bind to CD5 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 CD5 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 5 antibodies or fragments thereof that bind CD5 and are subsequently internalized by CD5+ 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 5 antibody or fragment thereof can be assessed, for example, using radionuclide internalization assays known in the art. For example, an anti-CD 5 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. The radiolabeled antibody or fragment thereof may be incubated with 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 5 antibody (or fragment thereof) has a defined serum half-life. For example, in a human patient, an anti-CD 5 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 5 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 5 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. USA 77:4216(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).
In some embodiments, anti-CD 5 antibodies that can be used in conjunction with the compositions and methods described herein include those antibodies that comprise a combination of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions listed in table 1A and table 1B below.
Table 1A.
Table 1B.
Cytotoxins
Various cytotoxins may be conjugated to anti-CD 5 antibodies via linkers for use in the combination therapies described herein. In particular, the anti-CD 5 ADC comprises an antibody (or antigen-binding fragment thereof) conjugated (i.e., covalently attached by a linker) to a cytotoxic moiety (or cytotoxin). As used herein, the terms "cytotoxin," "cytotoxic moiety," and "drug" are used interchangeably. In various embodiments, the cytotoxic moiety 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 5 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 intercalating agents (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 α -amanitine and derivatives thereof), and agents capable of disrupting protein biosynthesis (e.g., agents exhibiting rRNA N-glycosidase activity, such as saporin and ricin a chains).
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 CD5 ADC 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 2, and are disclosed, for example, in Zantotti et al, int.J. peptide Protein Res.30,1987, 450-459.
TABLE 2 structural table of amanitin.
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 CD5, 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, ORAOR ORC；
R2Is H, OH, ORBOR ORC；
RAAnd RBWhen present, combine together with the oxygen atom to which they are bound to form a 5-membered heterocycloalkyl group;
R3is H, RCOr RD；
R4、R5、R6And R7Each of which is independently H, OH, ORC、ORD、RCOr RD；
R8Is OH, NH2、ORC、ORD、NHRCOr NRCRD；
R9Is H, OH, ORCOR ORD；
Q is-S-, -S (O) -or-SO2-；
RCis-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
RDIs 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, C 2-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, cycloalkylHeterocycloalkyl, 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.
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, ORAOR ORC；
R2Is H, OH, ORBOR ORC；
RAAnd RBWhen present, combine together with the oxygen atom to which they are bound to form a 5-membered heterocycloalkyl group;
R3is H, RCOr RD；
R4、R5、R6And R7Each of which is independently H, OH, ORC、ORD、RCOr R D；
R8Is OH, NH2、ORC、ORD、NHRCOr NRCRD；
R9Is H, OH, ORCOR ORD；
Q is-S-, -S (O) -or-SO2-；
RCis-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
RDIs 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, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, and nitro.
In some embodiments, the amanitin comprises one RCAnd (4) a substituent.
In some embodiments, RAAnd RBTaken 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 ═ NRE)-or- (CR)ERE’) -; and is
Wherein R isEAnd RE’Each independently is H, C1-C6alkylene-RC、C1-C6Heteroalkylidene-RC、C2-C6alkenylene-RC、C2-C6Heteroalkenylene-RC、C2-C6alkynylene-RC、C2-C6Heteroalkynylene-RCOr cycloalkylene-RCheterocycloalkylene-RCarylene-RCOr heteroarylene-RCOr a combination thereof; wherein each C1-C6alkylene-RC、C1-C6Heteroalkylidene-RC、C2-C6alkenylene-RC、C2-C6Heteroalkenylene-RC、C2-C6alkynylene-RC、C2-C6Heteroalkynylene-RCOr cycloalkylene-RCheterocycloalkylene-RCarylene-RCOr heteroarylene-RCOptionally 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, ORAOR ORC；
R2Is H, OH, ORBOR ORC；
RAAnd RBWhen present, combine with the oxygen atom to which they are bound to form:
wherein R is3Is H or RC。
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, ORAOR ORC；
R2Is H, OH, ORBOR ORC；
RAAnd RBWhen present, combine with the oxygen atom to which they are bound to form:
wherein
R3Is H or RC；
R4And R5Each independently is H, OH, ORC、RCOR ORD；
R6And R7Each is H;
R8is OH, NH2、ORCOr NHRC；
R9Is H or OH; and is
Wherein R isCAnd RDAs 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 ORA；
R2Is H, OH OR ORB；
RAAnd RBWhen present, combine with the oxygen atom to which they are bound to form:
wherein
R3、R4、R6And R7Each is H;
R5is ORC；
R8Is OH or NH2；
R9Is H or OH;
q is-S-, -S (O) -or-SO2-; and is
Wherein R isCAnd RDAs 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 RC；
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 isCAnd RDAs defined above. Such amatoxin-linker conjugates are described, for example, in U.S. patent applicationPublication 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, ORCOr RC；
R8Is OH or NH2；
R9Is H or OH;
q is-S-, -S (O) -or-SO2-; and is
Wherein R isCAnd RDAs 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 IIIA, wherein:
R1And R2Each independently is H or OH;
R3、R6and R7Each is H;
R4and R5Each independently is H or OH;
R8is OH, NH2、ORCOr NHRC；
R9Is H or OH;
q is-S-, -S (O) -or-SO2-; and is
Wherein R isCAnd RDAs 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.
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, ORAOR ORC；
R2Is H, OH, ORBOR ORC；
RAAnd RBWhen present, combine together with the oxygen atom to which they are bound to form a 5-membered heterocycloalkyl group;
R3is H, RCOr RD；
R4、R5、R6And R7Each of which is independently H, OH, ORC、ORD、RCOr RD；
R8Is OH, NH2、ORC、ORD、NHRCOr NRCRD；
R9Is H, OH, ORCOR ORD；
Q is-S-, -S (O) -or-SO2-；
RCis-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- ((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(ii) a 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
RDIs 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, 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, RAAnd RBTaken 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 ═ NRE)-or- (CR)ERE’) -; and is
Wherein R isEAnd RE’Each independently is H, C1-C6alkylene-RC、C1-C6Heteroalkylidene-RC、C2-C6alkenylene-RC、C2-C6Heteroalkenylene-RC、C2-C6alkynylene-RC、C2-C6Heteroalkynylene-RCOr cycloalkylene-RCheterocycloalkylene-RCarylene-RCOr heteroarylene-RCOr a combination thereof; wherein each C1-C6alkylene-RC、C1-C6Heteroalkylidene-RC、C2-C6alkenylene-RC、C2-C6Heteroalkenylene-RC、C2-C6alkynylene-RC、C2-C6Heteroalkynylene-RCOr cycloalkylene-RCheterocycloalkylene-RCarylene-RCOr heteroarylene-RCOptionally 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, an antibody or antigen-binding fragment thereof as described herein is conjugated to an amanitin-linker conjugate represented by formula IIIB or a derivative thereof, wherein
R1Is H, OH, ORAOR ORC；
R2Is H, OH, ORBOR ORC；
RAAnd RBWhen 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 RC。
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, ORAOR ORC；
R2Is H, OH, ORBOR ORC；
RAAnd RBWhen 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 RC；
R4And R5Each independently is H, OH, ORC、RCOR ORD；
R6And R7Each is H;
R8is OH, NH2、ORCOr NHRC；
R9Is H or OH; and is
Wherein R isCAnd RDAs 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 ORA；
R2Is H, OH OR ORB；
RAAnd RBWhen 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 ORC；
R8Is OH or NH2；
R9Is H or OH;
q is-S-, -S (O) -or-SO2-; and is
Wherein R isCAnd RDAs 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 RC；
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 isCAnd RDAs 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, ORCOr RC；
R8Is OH or NH2；
R9Is H or OH;
q is-S-, -S (O) -or-SO2-; and is
Wherein R isCAnd RDAs 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 independently is H or OH;
R3、R6and R7Each is H;
R4and R5Each independently is H or OH;
R8is OH, NH2、ORCOr NHRC；
R9Is H or OH;
q is-S-, -S (O) -or-SO2-; and is
Wherein R isCAnd RDAs 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 5 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-. United states patent
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, which then interact with cellular macromoleculesReact to cause damage to the cell, or 3) interaction 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"), a highly potent major metabolite of nemorubicin (nemorubicin). 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 137 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.
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 5 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 pyrazoles [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.
Examples of calicheamicins suitable for use in the present invention are disclosed in, for example, U.S. patent No. 4,671,958, U.S. patent No. 4,970,198, U.S. patent No. 5,053,394, U.S. patent No. 5,037,651, and U.S. patent No. 5,079,233, which are incorporated herein in their entirety.
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 5 antibody or antigen-binding fragment thereof as described herein via a linker.
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, Napthenanthrolin, narcotine (nartogastin), nedaplatin (nedaplatin), nemorubicin, neridronic acid, nilutamide (nilutamide), lissaxin (nisamycin), riluzin (nitsullyn), octreotide (octreotide), oxcarbazone (okinone), onapristone (onapristone), ondansetron (ondansetron), olaparin (oracin), ormaplatin (ormaplatin), oxaliplatin (oxaliplatin), enomycin (oxaaurocycin), taxol and its analogs, panomine (palauaminine), hexadecanorhizomycin (lmitoryloxin), pamidronic acid, panaxytriol (panomine), paminomycin (parazeocin), bepiastin (parazeocin), pezicin (parazeocin), parapenfluzine (peimine), paradoxin (penfluzine), paradoxine (penfluvastatin), pemphitin (penetretin (penfluvastatin), pemoline (penfluvastatin), fluvastatin (penfluvastatin), pexin (penfluvastatin), pexin (penflubenoxanthine, penfluvastatin), pexin (penflubenoxapexin), penflurbenoxapexin, penflubenoxapexin (penflurbenoxapexin, penflur, 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 5 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 a 1, 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 groups 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, in each caseThe substituents are independently selected 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. Joints comprising such moietiesDisclosed, 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-C24Cycloalkyl radical, 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)2)n-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 5.
In some embodiments, the linker comprises ((CH)2)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 3 below. The curves represent the attachment points to the antibody or antigen-binding fragment and the cytotoxic molecule, respectively.
TABLE 3 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 3, 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 4.
Table 4.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, a 1, 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 CD5 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 CD5 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 5 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 5 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 5 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 5 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 5 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, e.g. in order to reduce the risk of CAR-T cell rejection.
The anti-CD 5 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 5 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 5 ADC (e.g., serum concentrations of 0.0001 μ g/mL to 5000 μ g/mL). The dose of anti-CD 5 ADC 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 5 ADC. The anti-CD 5 ADC may be administered to a human patient in one or more doses. In one embodiment, the anti-CD 5 ADC 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.
Example 1: in vitro binding assay for anti-CD 5 antibody.
To determine the binding characteristics of the anti-CD 5 antibody 5D7 hIgG1, antibody binding studies were performed using biolayer interferometry (BLI) with Pall ForteBio Octet Red96 at 25 degrees celsius in 1 x PBS supplemented with 0.1% w/v bovine serum albumin. Purified human anti-CD 5 antibody (5D7) was immobilized on an anti-human Fc biosensor (AHC; Pall ForteBio 18-5063) and incubated with 50nM of purified human CD5 ectodomain. The binding characteristics of anti-CD 5 antibody 5D7 are shown in table 4. The anti-human CD5 antibody 5D7 used in examples 1 to 5 is a humanized form of the murine antibody 5D7 (see US 2008/0254027). The sequence of antibody 5D7 as used herein is described in SEQ ID Nos 53 and 54 (heavy and light chain variable region amino acid sequences) and 47 through 52 (heavy and light chain CDRs).
Table 4: binding kinetics of 5D7 to the extracellular domain of human CD5
Example 2: in vitro cell-binding assays for anti-CD 5 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 5 antibody (i.e., L17F12, UCHT2, 205919, and CRIS-1) 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 5 antibodies L17F12(Thermo Fisher), UCHT2(BioLegend), 205919(Novus Biologicals), and CRIS-1(Novus Biologicals) bind to human T lymphoblasts (i.e., MOLT-4 cells) with EC50207pM (L17), 354pM (uch), 1350pM (205), and 43pM (cris).
Example 3: in vitro primary cell binding assay for anti-CD 5 antibodies
Primary human T cells were plated at 8 × 104Individual cells/well were plated and stained with a titer of human anti-CD 5 antibody 5D7 at 37 ℃ for 2 hours. A constant amount of secondary anti-mouse AF488 dye was added for 30 minutes at 4 ℃. After washing, the plates were run on a flow cytometer and binding of the anti-CD 55D 7 antibody (and the negative control, i.e., 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, anti-CD 5 antibody 5D7 bound to primary human T cells with EC50＝3.0pM。
Example 4 in vitro analysis using an in vitro T cell killing assay anti-CD 5-amanitin Antibody Drug Conjugate (ADC)
anti-CD 5 antibody 5D7 was conjugated to amatoxin (amanitin) with a cleavable linker to form an anti-CD 55D 7 ADC. anti-CD 55D 7-ADCs with a drug-to-antibody ratio (DAR) of about 6 (inter-chain DAR6) and anti-CD 55D 7-ADCs with a DAR of about 2 (prepared using site-specific conjugation via D265C mutation) were tested. In addition, the anti-CD 55D 7-ADC rapid half-life variant was generated by introducing the H435A mutation in the Fc region.
Each anti-CD 55D 7-ADC was evaluated using an in vitro human T cell killing assay.
Cryopreserved negatively selected primary human T cells were thawed and stimulated with anti-CD 3 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 5 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 and 7-AAD. Unconjugated anti-CD 55D 7 antibody was used as a comparative object (fig. 3A).
As shown in figure 3A, anti-CD 55D 7-ADC with a DAR of about 6 exhibited efficient and specific killing of human T cells (IC50 ═ 3.7pM), while T cells remained viable in the presence of unconjugated ("naked") anti-CD 55D 7 antibody. As shown in figure 3B, ADCs with site-specific (D265C) DAR of about 2 maintained effective levels of T cell killing similar to those of DAR 6 ADCs (IC50 ═ 5.0 pM). The fast half-life variant of anti-CD 55D 7-ADC (H435A) showed similar levels of T cell killing (IC50 ═ 4.9 pM; fig. 3B).
Example 5 analysis of T cell depletion Using hNSG mouse model
In vivo T cell depletion assays were performed using humanized NSG mice (Jackson Laboratories). anti-CD 5 antibody 5D7 was conjugated to amatoxin (amanitin) with a cleavable linker to form an anti-CD 55D 7-ADC. As described above, the anti-CD 55D 7-ADC was prepared to have a DAR of about 6 or a DAR of about 2. Each anti-CD 55D 7ADC (DAR6 or DAR2) was administered to humanized mice as a single intravenous injection (0.3 mg/kg, 1mg/kg or 3mg/kg for DAR6 ADC, or 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 4A-4B for DAR2 ADC and figures 5A-5C for DAR6 ADC).
As shown in figures 4A-4B, humanized NSG mice treated with 0.3mg/kg, 1mg/kg, or 3mg/kg DAR6 anti-CD 55D 7-ADC exhibited efficient T cell depletion in peripheral blood or bone marrow, while thymic T cells were depleted after treatment with 1mg/kg or 3mg/kg DAR6 anti-CD 55D 7-ADC. The negative controls used in this in vivo experiment included human IgG1 (as naked antibody (huIgG1), and conjugated with amanitin (huIgG1-AM)) that was non-specific for CD 5. As depicted in fig. 4A-4B, the naked huIgG1 control and the conjugated control had no effect on T cell depletion in peripheral blood (fig. 4A) and bone marrow (fig. 4B), as these controls were comparable to the PBS control. anti-CD 5 antibody (antibody YTH34.5) was also used as a control, and peripheral and bone marrow T cells could also be depleted at a dose of 25 mg/kg.
As shown in fig. 5A-5C, humanized NSG mice treated with 1mg/kg or 3mg/kg site-specific DAR2 anti-CD 55D 7-ADC exhibited efficient T cell depletion in peripheral blood, bone marrow, and thymus T cells. In each of fig. 5A to 5C, naked antibody 5D7 was also used as a control. As depicted in fig. 5A, antibody 5D7 was able to deplete peripheral T cells (relative to non-specific human IgG1 control or PBS), but was unable to deplete bone marrow T cells or thymus T cells, whereas 5D7-AM ADC was effective in depleting both bone marrow and thymus, as depicted in fig. 5B and 5C.
Example 6 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 cells/kg allogeneic T cells were treated with a priming (priming) dose of allogeneic T cells. On day 3, mice were administered anti-CD 5- 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 5 antibody administered on day 3 instead of anti-CD 5 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 5ADC 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 CD5+ activated T cells in the spleen and peripheral blood of mice from each treatment group was determined on day 9. Throughout the study, mice were monitored for rejection symptoms.
Example 7 administration of an anti-CD 5 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 5 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 CD5+ T cells.
Subsequently, an anti-CD 5 ADC comprising an anti-CD 5 antibody conjugated to a cytotoxin via a linker is administered to the patient. The anti-CD 5 ADC is administered in an amount effective to deplete endogenous CD5+ activated T cells. Following administration of anti-CD 5 ADC, the level of CD5+ 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 5 ADC.
TABLE 5 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 5 Antibody Drug Conjugates (ADCs) in allogeneic cell therapy
<130> M103034 2060WO
<140>
<141>
<150> 62/773,047
<151> 2018-11-29
<150> 62/702,296
<151> 2018-07-23
<160> 282
<170> PatentIn version 3.5
<210> 1
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 1
Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30
Gly Met Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu
35 40 45
Trp Val Ala His Ile Trp Trp Asp Asp Asp Val Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Ala Ser Lys Asp Gln Val
65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Val Arg Arg Arg Ala Thr Gly Thr Gly Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 2
<211> 106
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 2
Asn Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Val Gly Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys His Gln Tyr Asn Ser Tyr Asn Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 3
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 3
Phe Ser Leu Ser Thr Ser Gly Met Gly
1 5
<210> 4
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 4
Trp Trp Asp Asp Asp
1 5
<210> 5
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 5
Arg Arg Ala Thr Gly Thr Gly Phe Asp Tyr
1 5 10
<210> 6
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 6
Gln Asp Val Gly Thr Ala
1 5
<210> 7
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 7
Trp Thr Ser Thr Arg His Thr
1 5
<210> 8
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 8
Tyr Asn Ser Tyr Asn Thr
1 5
<210> 9
<211> 46
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 9
Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
1 5 10 15
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
20 25 30
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
35 40 45
<210> 10
<211> 88
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 10
Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
1 5 10 15
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
20 25 30
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Pro Arg
35 40 45
Lys Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser
50 55 60
Asn Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro
65 70 75 80
Leu Phe Pro Gly Pro Ser Lys Pro
85
<210> 11
<211> 28
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 11
Leu Asp Pro Lys Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr
1 5 10 15
Gly Val Ile Leu Thr Ala Leu Phe Leu Arg Val Lys
20 25
<210> 12
<211> 21
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 12
Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu
1 5 10 15
Thr Ala Leu Phe Leu
20
<210> 13
<211> 27
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 13
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 14
<400> 14
000
<210> 15
<211> 112
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 15
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 16
<211> 66
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 16
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
1 5 10 15
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
20 25 30
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly
35 40 45
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
50 55 60
Trp Val
65
<210> 17
<211> 41
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 17
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 18
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 18
Gly Gly Gly Gly Ser
1 5
<210> 19
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 19
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 20
<211> 495
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 20
Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly
1 5 10 15
Met Leu Val Ala Ser Cys Leu Gly Arg Leu Ser Trp Tyr Asp Pro Asp
20 25 30
Phe Gln Ala Arg Leu Thr Arg Ser Asn Ser Lys Cys Gln Gly Gln Leu
35 40 45
Glu Val Tyr Leu Lys Asp Gly Trp His Met Val Cys Ser Gln Ser Trp
50 55 60
Gly Arg Ser Ser Lys Gln Trp Glu Asp Pro Ser Gln Ala Ser Lys Val
65 70 75 80
Cys Gln Arg Leu Asn Cys Gly Val Pro Leu Ser Leu Gly Pro Phe Leu
85 90 95
Val Thr Tyr Thr Pro Gln Ser Ser Ile Ile Cys Tyr Gly Gln Leu Gly
100 105 110
Ser Phe Ser Asn Cys Ser His Ser Arg Asn Asp Met Cys His Ser Leu
115 120 125
Gly Leu Thr Cys Leu Glu Pro Gln Lys Thr Thr Pro Pro Thr Thr Arg
130 135 140
Pro Pro Pro Thr Thr Thr Pro Glu Pro Thr Ala Pro Pro Arg Leu Gln
145 150 155 160
Leu Val Ala Gln Ser Gly Gly Gln His Cys Ala Gly Val Val Glu Phe
165 170 175
Tyr Ser Gly Ser Leu Gly Gly Thr Ile Ser Tyr Glu Ala Gln Asp Lys
180 185 190
Thr Gln Asp Leu Glu Asn Phe Leu Cys Asn Asn Leu Gln Cys Gly Ser
195 200 205
Phe Leu Lys His Leu Pro Glu Thr Glu Ala Gly Arg Ala Gln Asp Pro
210 215 220
Gly Glu Pro Arg Glu His Gln Pro Leu Pro Ile Gln Trp Lys Ile Gln
225 230 235 240
Asn Ser Ser Cys Thr Ser Leu Glu His Cys Phe Arg Lys Ile Lys Pro
245 250 255
Gln Lys Ser Gly Arg Val Leu Ala Leu Leu Cys Ser Gly Phe Gln Pro
260 265 270
Lys Val Gln Ser Arg Leu Val Gly Gly Ser Ser Ile Cys Glu Gly Thr
275 280 285
Val Glu Val Arg Gln Gly Ala Gln Trp Ala Ala Leu Cys Asp Ser Ser
290 295 300
Ser Ala Arg Ser Ser Leu Arg Trp Glu Glu Val Cys Arg Glu Gln Gln
305 310 315 320
Cys Gly Ser Val Asn Ser Tyr Arg Val Leu Asp Ala Gly Asp Pro Thr
325 330 335
Ser Arg Gly Leu Phe Cys Pro His Gln Lys Leu Ser Gln Cys His Glu
340 345 350
Leu Trp Glu Arg Asn Ser Tyr Cys Lys Lys Val Phe Val Thr Cys Gln
355 360 365
Asp Pro Asn Pro Ala Gly Leu Ala Ala Gly Thr Val Ala Ser Ile Ile
370 375 380
Leu Ala Leu Val Leu Leu Val Val Leu Leu Val Val Cys Gly Pro Leu
385 390 395 400
Ala Tyr Lys Lys Leu Val Lys Lys Phe Arg Gln Lys Lys Gln Arg Gln
405 410 415
Trp Ile Gly Pro Thr Gly Met Asn Gln Asn Met Ser Phe His Arg Asn
420 425 430
His Thr Ala Thr Val Arg Ser His Ala Glu Asn Pro Thr Ala Ser His
435 440 445
Val Asp Asn Glu Tyr Ser Gln Pro Pro Arg Asn Ser His Leu Ser Ala
450 455 460
Tyr Pro Ala Leu Glu Gly Ala Leu His Arg Ser Ser Met Gln Pro Asp
465 470 475 480
Asn Ser Ser Asp Ser Asp Tyr Asp Leu His Gly Ala Gln Arg Leu
485 490 495
<210> 21
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 21
Gly Tyr Thr Phe Thr Asn Tyr
1 5
<210> 22
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 22
Asn Thr His Thr Gly Glu
1 5
<210> 23
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 23
Arg Gly Tyr Asp Trp Tyr Phe Asp Val
1 5
<210> 24
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 24
Arg Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser
1 5 10
<210> 25
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 25
Arg Ala Asn Arg Leu Val Asp
1 5
<210> 26
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 26
Gln Gln Tyr Asp Glu Ser Pro Trp Thr
1 5
<210> 27
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 27
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Thr Leu Ile
35 40 45
Tyr Arg Ala Asn Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr
65 70 75 80
Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 28
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 28
Glu Ile Gln Leu Val Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Thr His Tyr Gly Glu Pro Thr Tyr Ala Asp Ser Phe
50 55 60
Lys Gly Thr Arg Thr Phe Ser Leu Asp Asp Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Gly
100 105 110
Thr Thr Val Thr Val Ser Ser
115
<210> 29
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 29
Gly Tyr Thr Phe Thr Asn Tyr
1 5
<210> 30
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 30
Asn Thr His Tyr Gly Glu
1 5
<210> 31
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 31
Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val
1 5 10
<210> 32
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 32
Arg Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser
1 5 10
<210> 33
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 33
Arg Ala Asn Arg Leu Glu Ser
1 5
<210> 34
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 34
Gln Gln Tyr Asp Glu Ser Pro Trp Thr
1 5
<210> 35
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 35
Gly Tyr Ser Ile Thr Ser Gly Tyr Tyr
1 5
<210> 36
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 36
Ile Ser Tyr Ser Gly Phe Thr
1 5
<210> 37
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 37
Ala Gly Asp Arg Thr Gly Ser Trp Phe Ala Tyr
1 5 10
<210> 38
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 38
Gln Asp Ile Ser Asn Tyr
1 5
<210> 39
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 39
Ala Thr Ser
1
<210> 40
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 40
Leu Gln Tyr Ala Ser Tyr Pro Phe Thr
1 5
<210> 41
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 41
Gly Tyr Ile Phe Thr Asn Tyr Gly
1 5
<210> 42
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 42
Ile Asn Thr Tyr Asn Gly Glu Pro
1 5
<210> 43
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 43
Ala Arg Gly Asp Tyr Tyr Gly Tyr Glu Asp Tyr
1 5 10
<210> 44
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 44
Gln Gly Ile Ser Asn Tyr
1 5
<210> 45
<211> 3
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 45
Tyr Thr Ser
1
<210> 46
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 46
Gln Gln Tyr Ser Lys Leu Pro Trp Thr
1 5
<210> 47
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 47
Phe Ser Leu Ser Thr Ser Gly Met Gly
1 5
<210> 48
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 48
Trp Trp Asp Asp Asp
1 5
<210> 49
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 49
Arg Arg Ala Thr Gly Thr Gly Phe Asp Tyr
1 5 10
<210> 50
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 50
Gln Asp Val Gly Thr Ala
1 5
<210> 51
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 51
Trp Thr Ser Thr Arg His Thr
1 5
<210> 52
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 52
Tyr Asn Ser Tyr Asn Thr
1 5
<210> 53
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 53
Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser
20 25 30
Gly Met Gly Val Gly Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu
35 40 45
Trp Val Ala His Ile Trp Trp Asp Asp Asp Val Tyr Tyr Asn Pro Ser
50 55 60
Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Ala Ser Lys Asp Gln Val
65 70 75 80
Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Val Arg Arg Arg Ala Thr Gly Thr Gly Phe Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 54
<211> 106
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 54
Asn Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Val Gly Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Trp Thr Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys His Gln Tyr Asn Ser Tyr Asn Thr
85 90 95
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 55
<211> 120
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 55
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Glu Asn Gly Ser Asp Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Arg Gly Gly Ala Val Ser Tyr Phe Asp Val Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 56
<211> 109
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 56
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
100 105
<210> 57
<211> 438
<212> PRT
<213> Intelligent (Homo sapiens)
<400> 57
Met Val Cys Ser Gln Ser Trp Gly Arg Ser Ser Lys Gln Trp Glu Asp
1 5 10 15
Pro Ser Gln Ala Ser Lys Val Cys Gln Arg Leu Asn Cys Gly Val Pro
20 25 30
Leu Ser Leu Gly Pro Phe Leu Val Thr Tyr Thr Pro Gln Ser Ser Ile
35 40 45
Ile Cys Tyr Gly Gln Leu Gly Ser Phe Ser Asn Cys Ser His Ser Arg
50 55 60
Asn Asp Met Cys His Ser Leu Gly Leu Thr Cys Leu Glu Pro Gln Lys
65 70 75 80
Thr Thr Pro Pro Thr Thr Arg Pro Pro Pro Thr Thr Thr Pro Glu Pro
85 90 95
Thr Ala Pro Pro Arg Leu Gln Leu Val Ala Gln Ser Gly Gly Gln His
100 105 110
Cys Ala Gly Val Val Glu Phe Tyr Ser Gly Ser Leu Gly Gly Thr Ile
115 120 125
Ser Tyr Glu Ala Gln Asp Lys Thr Gln Asp Leu Glu Asn Phe Leu Cys
130 135 140
Asn Asn Leu Gln Cys Gly Ser Phe Leu Lys His Leu Pro Glu Thr Glu
145 150 155 160
Ala Gly Arg Ala Gln Asp Pro Gly Glu Pro Arg Glu His Gln Pro Leu
165 170 175
Pro Ile Gln Trp Lys Ile Gln Asn Ser Ser Cys Thr Ser Leu Glu His
180 185 190
Cys Phe Arg Lys Ile Lys Pro Gln Lys Ser Gly Arg Val Leu Ala Leu
195 200 205
Leu Cys Ser Gly Phe Gln Pro Lys Val Gln Ser Arg Leu Val Gly Gly
210 215 220
Ser Ser Ile Cys Glu Gly Thr Val Glu Val Arg Gln Gly Ala Gln Trp
225 230 235 240
Ala Ala Leu Cys Asp Ser Ser Ser Ala Arg Ser Ser Leu Arg Trp Glu
245 250 255
Glu Val Cys Arg Glu Gln Gln Cys Gly Ser Val Asn Ser Tyr Arg Val
260 265 270
Leu Asp Ala Gly Asp Pro Thr Ser Arg Gly Leu Phe Cys Pro His Gln
275 280 285
Lys Leu Ser Gln Cys His Glu Leu Trp Glu Arg Asn Ser Tyr Cys Lys
290 295 300
Lys Val Phe Val Thr Cys Gln Asp Pro Asn Pro Ala Gly Leu Ala Ala
305 310 315 320
Gly Thr Val Ala Ser Ile Ile Leu Ala Leu Val Leu Leu Val Val Leu
325 330 335
Leu Val Val Cys Gly Pro Leu Ala Tyr Lys Lys Leu Val Lys Lys Phe
340 345 350
Arg Gln Lys Lys Gln Arg Gln Trp Ile Gly Pro Thr Gly Met Asn Gln
355 360 365
Asn Met Ser Phe His Arg Asn His Thr Ala Thr Val Arg Ser His Ala
370 375 380
Glu Asn Pro Thr Ala Ser His Val Asp Asn Glu Tyr Ser Gln Pro Pro
385 390 395 400
Arg Asn Ser His Leu Ser Ala Tyr Pro Ala Leu Glu Gly Ala Leu His
405 410 415
Arg Ser Ser Met Gln Pro Asp Asn Ser Ser Asp Ser Asp Tyr Asp Leu
420 425 430
His Gly Ala Gln Arg Leu
435
<210> 58
<211> 107
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 58
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Asn Ser Tyr
20 25 30
Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile
35 40 45
Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Tyr
65 70 75 80
Glu Asp Phe Gly Ile Tyr Tyr Cys Gln Gln Tyr Asp Glu Ser Pro Trp
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 59
<211> 118
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 59
Gln Ile Gln Leu Val Gln Ser Gly Pro Gly Leu Lys Lys Pro Gly Gly
1 5 10 15
Ser Val Arg Ile Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr
20 25 30
Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Arg Trp Met
35 40 45
Gly Trp Ile Asn Thr His Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe
50 55 60
Lys Gly Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr
65 70 75 80
Leu Gln Ile Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Phe Cys
85 90 95
Thr Arg Arg Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr
100 105 110
Thr Val Thr Val Ser Ser
115
<210> 60
<211> 42
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of the synthetic Polypeptides
<400> 60
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 61
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 61
Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met
1 5 10
<210> 62
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 62
Ser Gly Tyr Ser Phe Thr Asp Tyr Thr Met
1 5 10
<210> 63
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 63
Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met
1 5 10
<210> 64
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 64
Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met
1 5 10
<210> 65
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 65
Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met
1 5 10
<210> 66
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 66
Ser Gly Phe Thr Phe Ser Asn Tyr Ala Met
1 5 10
<210> 67
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 67
Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met
1 5 10
<210> 68
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 68
Ser Gly Tyr Ser Phe Thr Ala Tyr Asn Ile
1 5 10
<210> 69
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 69
Ser Gly Tyr Ser Phe Thr Ala Tyr Ser Met
1 5 10
<210> 70
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 70
Ser Gly Tyr Thr Phe Thr Asn Phe Ala Ile
1 5 10
<210> 71
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 71
Ser Gly Tyr Thr Phe Thr Asn Phe Ala Ile
1 5 10
<210> 72
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 72
Ser Gly Tyr Thr Phe Thr Asn Phe Ala Ile
1 5 10
<210> 73
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 73
Ser Gly Tyr Thr Phe Thr Asn Phe Ala Ile
1 5 10
<210> 74
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 74
Ser Gly Phe Asn Ile Lys Asp Thr Tyr Met
1 5 10
<210> 75
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 75
Ser Gly Tyr Ser Phe Thr Ser Tyr Trp Met
1 5 10
<210> 76
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 76
Ser Gly Phe Ser Leu Thr Asn Tyr Asp Val
1 5 10
<210> 77
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 77
Ser Gly Phe Ser Leu Thr Asn Tyr Asp Val
1 5 10
<210> 78
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 78
Ser Gly Phe Thr Phe Ser Asn Tyr Gly Met
1 5 10
<210> 79
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 79
Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met
1 5 10
<210> 80
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 80
Ser Gly Tyr Ile Phe Ala Asn Tyr Gly Met
1 5 10
<210> 81
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 81
Ser Gly Tyr Asn Phe Thr Asn Tyr Gly Met
1 5 10
<210> 82
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 82
Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met
1 5 10
<210> 83
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 83
Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Ile
1 5 10
<210> 84
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 84
Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr Ile
1 5 10
<210> 85
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 85
Ser Gly Asn Thr Phe Thr Asn Phe Tyr Leu
1 5 10
<210> 86
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 86
Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met
1 5 10
<210> 87
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 87
Ser Glu Phe Thr Phe Ser Asn Tyr Ala Met
1 5 10
<210> 88
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 88
Ser Gly Tyr Thr Phe Thr Ser Tyr Arg Met
1 5 10
<210> 89
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 89
Ser Gly Phe Asn Ile Lys Asp Thr Tyr Met
1 5 10
<210> 90
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 90
Ser Gly Tyr Ser Phe Thr Asp Tyr Thr Met
1 5 10
<210> 91
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 91
Ser Gly Tyr Met Phe Thr Asn His Gly Met
1 5 10
<210> 92
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 92
Ser Gly Tyr Met Phe Thr Asn Tyr Gly Met
1 5 10
<210> 93
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 93
Ser Gly Tyr Ile Phe Thr Asn Tyr Gly Met
1 5 10
<210> 94
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 94
Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Ile
1 5 10
<210> 95
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 95
Ser Gly Tyr Thr Phe Ile Asn Tyr Gly Met
1 5 10
<210> 96
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 96
Ser Gly Tyr Thr Phe Thr Asp Tyr Phe Ile
1 5 10
<210> 97
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 97
Ser Gly Tyr Ile Phe Thr Gly Tyr Asn Ile
1 5 10
<210> 98
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 98
Leu Ile Asn Pro Tyr Asn Gly Gly Thr Thr
1 5 10
<210> 99
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 99
Leu Ile Asn Pro Tyr Asn Gly Gly Thr Met
1 5 10
<210> 100
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 100
Leu Ile Asn Pro Tyr Asn Gly Gly Thr Met
1 5 10
<210> 101
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 101
Leu Ile Asn Pro Tyr Asn Gly Gly Thr Met
1 5 10
<210> 102
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 102
Leu Ile Asn Pro Tyr Asn Gly Gly Thr Thr
1 5 10
<210> 103
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 103
Ser Ile Ser Ser Gly Gly Asn Thr Phe
1 5
<210> 104
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 104
Ser Ile Ser Ser Gly Gly Ser Thr Tyr
1 5
<210> 105
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 105
Ser Ile Asp Pro Tyr Tyr Gly Asp Thr Lys
1 5 10
<210> 106
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 106
Ser Ile Asp Pro Tyr Tyr Gly Asp Thr Lys
1 5 10
<210> 107
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 107
Leu Ile Ser Ser Asn Ser Gly Asp Val Ser
1 5 10
<210> 108
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 108
Leu Ile Ser Thr Ser Ser Gly Asp Val Ser
1 5 10
<210> 109
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 109
Leu Ile Ser Ser Asn Ser Gly Asp Val Ser
1 5 10
<210> 110
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 110
Leu Ile Ser Ser Asn Ser Gly Asp Val Ser
1 5 10
<210> 111
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 111
Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys
1 5 10
<210> 112
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 112
Met Ile His Pro Ser Asp Ser Glu Thr Arg
1 5 10
<210> 113
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 113
Val Ile Trp Ser Gly Gly Asn Thr Asp
1 5
<210> 114
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 114
Val Ile Trp Ser Gly Gly Asn Thr Asp
1 5
<210> 115
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 115
Ala Ile Asn Ser Asn Gly Asp Ile Thr Tyr
1 5 10
<210> 116
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 116
Leu Ile Asn Pro Tyr Asn Gly Gly Thr Arg
1 5 10
<210> 117
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 117
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr
1 5 10
<210> 118
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 118
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr
1 5 10
<210> 119
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 119
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr
1 5 10
<210> 120
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 120
Trp Ile Tyr Pro Gly Gly Gly Asn Thr Arg
1 5 10
<210> 121
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 121
Trp Ile Tyr Pro Gly Gly Gly Asn Thr Arg
1 5 10
<210> 122
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 122
Cys Ile Tyr Pro Gly Asn Val Lys Thr Lys
1 5 10
<210> 123
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 123
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr
1 5 10
<210> 124
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 124
Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr
1 5 10
<210> 125
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 125
Arg Ile Asp Pro Tyr Asp Ser Gly Thr His
1 5 10
<210> 126
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 126
Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys
1 5 10
<210> 127
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 127
Leu Ile Asn Pro Tyr Asn Gly Gly Thr Arg
1 5 10
<210> 128
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 128
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr
1 5 10
<210> 129
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 129
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr
1 5 10
<210> 130
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 130
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr
1 5 10
<210> 131
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 131
Trp Ile Asp Pro Glu Asn Gly Arg Thr Glu
1 5 10
<210> 132
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 132
Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr
1 5 10
<210> 133
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 133
Glu Ile Tyr Pro Gly Ser Ser Asn Thr Tyr
1 5 10
<210> 134
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 134
Ala Val Tyr Pro Gly Asn Gly Asp Thr Ser
1 5 10
<210> 135
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 135
Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Asp Phe Asp Tyr Trp
1 5 10 15
<210> 136
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 136
Cys Ala Arg Asp Asn Tyr Gly Ser Ser Pro Asp Phe Asp Tyr Trp
1 5 10 15
<210> 137
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 137
Cys Ala Arg Asp Asn Tyr Gly Ser Ser Pro Tyr Phe Asp Tyr Trp
1 5 10 15
<210> 138
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 138
Cys Ala Arg Asp Asn Tyr Gly Ser Ser Pro Tyr Phe Asp Tyr Trp
1 5 10 15
<210> 139
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 139
Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Pro Asp Phe Asp Tyr Trp
1 5 10 15
<210> 140
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 140
Cys Val Arg Tyr Tyr Tyr Gly Val Thr Tyr Trp Tyr Phe Asp Val Trp
1 5 10 15
<210> 141
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 141
Cys Val Arg Tyr Tyr Tyr Gly Ile Arg Tyr Trp Tyr Phe Asp Val Trp
1 5 10 15
<210> 142
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 142
Cys Ala Arg Arg Met Ile Thr Met Gly Asp Trp Tyr Phe Asp Val Trp
1 5 10 15
<210> 143
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 143
Cys Ala Arg Arg Met Ile Thr Thr Gly Asp Trp Tyr Phe Asp Val Trp
1 5 10 15
<210> 144
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 144
Cys Ala Arg His Tyr Gly Ala His Asn Tyr Phe Asp Tyr Trp
1 5 10
<210> 145
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 145
Cys Ala Arg His Tyr Gly Ala Asn Asn Tyr Phe Asp Tyr Trp
1 5 10
<210> 146
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 146
Cys Ala Arg His Tyr Gly Ala His Asn Tyr Phe Asp Tyr Trp
1 5 10
<210> 147
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 147
Cys Ala Arg His Tyr Gly Ala His Asn Tyr Phe Asp Tyr Trp
1 5 10
<210> 148
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 148
Cys Ala Arg Glu Glu Asn Tyr Tyr Gly Thr Tyr Tyr Phe Asp Tyr Trp
1 5 10 15
<210> 149
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 149
Cys Ala Arg Trp Gly Asp His Asp Asp Ala Met Asp Phe Trp
1 5 10
<210> 150
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 150
Cys Ala Arg Asn His Gly Asp Gly Tyr Phe Asn Trp Tyr Phe Asp Val
1 5 10 15
Trp
<210> 151
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 151
Cys Ala Arg Asn His Gly Asp Gly Tyr Tyr Asn Trp Tyr Phe Asp Val
1 5 10 15
Trp
<210> 152
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 152
Cys Ala Arg Gly Thr Ala Trp Phe Thr Tyr Trp
1 5 10
<210> 153
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 153
Cys Ala Arg Asp Gly Asp Asp Gly Trp Asp Ile Asp Val Trp
1 5 10
<210> 154
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 154
Cys Ala Arg Arg Gly Thr Tyr Trp His Phe Asp Val Trp
1 5 10
<210> 155
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 155
Cys Ala Arg Arg Gly Ser Tyr Trp His Phe Asp Val Trp
1 5 10
<210> 156
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 156
Cys Ala Arg Arg Ser Thr Leu Val Phe Asp Tyr Trp
1 5 10
<210> 157
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 157
Cys Ala Arg Asn Gly Tyr Trp Tyr Phe Asp Val Trp
1 5 10
<210> 158
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 158
Cys Ala Arg Asn Gly Tyr Trp Tyr Phe Asp Val Trp
1 5 10
<210> 159
<211> 16
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 159
Cys Ala Lys Glu Gly Asp Tyr Asp Gly Thr Ala Tyr Phe Asp Tyr Trp
1 5 10 15
<210> 160
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 160
Cys Ala Arg Arg Arg Asp Gly Asn Phe Asp Tyr Trp
1 5 10
<210> 161
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 161
Cys Val Arg His Gly Tyr Phe Asp Val Trp
1 5 10
<210> 162
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 162
Cys Ala Phe Tyr Asp Gly Ala Tyr Trp
1 5
<210> 163
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 163
Cys Ala Ser Tyr Asp Pro Asp Tyr Trp
1 5
<210> 164
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 164
Cys Ala Arg Asp Thr Thr Ala Thr Tyr Tyr Phe Asp Tyr Trp
1 5 10
<210> 165
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 165
Cys Ala Arg Arg Val Ala Thr Tyr Phe Asp Val Trp
1 5 10
<210> 166
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 166
Cys Thr Arg Arg Ser His Ile Thr Leu Asp Tyr Trp
1 5 10
<210> 167
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 167
Cys Ala Arg Arg Arg Thr Thr Ala Phe Asp Tyr Trp
1 5 10
<210> 168
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 168
Cys Asn Asn Gly Asn Tyr Val Arg His Tyr Tyr Phe Asp Tyr Trp
1 5 10 15
<210> 169
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 169
Cys Thr Arg Arg Arg Glu Ile Thr Phe Asp Tyr Trp
1 5 10
<210> 170
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 170
Cys Ala Arg Ser Gly Ile Ser Pro Phe Thr Tyr Trp
1 5 10
<210> 171
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 171
Cys Ala Lys Tyr Asp Arg Phe Phe Ala Ser Trp
1 5 10
<210> 172
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 172
Ser Gln Gly Ile Ser Asn His Leu
1 5
<210> 173
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 173
Ser Gln Gly Ile Arg Asn Tyr Leu
1 5
<210> 174
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 174
Ser Gln Gly Ile Ser Asn His Leu
1 5
<210> 175
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 175
Ser Gln Gly Ile Asn Asn Tyr Leu
1 5
<210> 176
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 176
Ser Gln Gly Ile Ser Asn His Leu
1 5
<210> 177
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 177
Ser Gln Ser Val Asp His Asp Gly Asp Ser Tyr Met
1 5 10
<210> 178
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 178
Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met
1 5 10
<210> 179
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 179
Ser Gln Asp Ile Ser Asn Tyr Leu
1 5
<210> 180
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 180
Ser Gln Asp Ile Ser Thr Tyr Leu
1 5
<210> 181
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 181
Thr Ser Ser Ile Ser Ser Ser Tyr Leu
1 5
<210> 182
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 182
Asn Ser Ser Val Ser Ser Ser Tyr Leu
1 5
<210> 183
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 183
Thr Ser Ser Ile Ser Ser Ser Tyr Leu
1 5
<210> 184
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 184
Thr Ser Ser Ile Ser Ser Ser Tyr Leu
1 5
<210> 185
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 185
Ser Glu Asn Ile Tyr Tyr Asn Leu
1 5
<210> 186
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 186
Ser Glu Asn Ile Tyr Gly Tyr Phe
1 5
<210> 187
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 187
Ser Gln Asp Ile Asn Asn Tyr Ile
1 5
<210> 188
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 188
Ser Gln Asp Ile Asn Lys Tyr Ile
1 5
<210> 189
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 189
Ser Glu Asn Ile Tyr Ser Tyr Leu
1 5
<210> 190
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 190
Ser Gln Gly Ile Arg Asn Tyr Leu
1 5
<210> 191
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 191
Ser Gln Asp Val Arg Thr Asp Val
1 5
<210> 192
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 192
Ser Gln Asp Val Ile Thr Ala Val
1 5
<210> 193
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 193
Ser Gln Ser Ile Gly Thr Ser Ile
1 5
<210> 194
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 194
Ser Ser Gln Ser Leu Leu Asn Gln Lys Asn Tyr Leu
1 5 10
<210> 195
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 195
Ser Ser Ser Val Ser Ser Ser Tyr Leu
1 5
<210> 196
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 196
Ser Glu Asn Ile Tyr Tyr Asn Leu
1 5
<210> 197
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 197
Ser Gln Thr Ile Gly Thr Ser Ile
1 5
<210> 198
<211> 14
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 198
Ser Gln Ser Leu Leu Tyr Ser Ser Asp Gln Lys Asn Tyr Leu
1 5 10
<210> 199
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 199
Asn Ser Ser Val Ser Tyr Met
1 5
<210> 200
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 200
Ser Glu Asn Ile Tyr Tyr Asn Leu
1 5
<210> 201
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 201
Ser Ser Ser Leu Ser Tyr Met
1 5
<210> 202
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 202
Ser Gln Arg Ile Gly Thr Ser Met
1 5
<210> 203
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 203
Ser Gln Ser Ile Gly Thr Ser Ile
1 5
<210> 204
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 204
Ser Gln Asn Ile Gly Thr Ser Ile
1 5
<210> 205
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 205
Ile Ser Ser Val Ser Tyr Met
1 5
<210> 206
<211> 8
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 206
Ser Gln Thr Ile Ala Thr Ser Ile
1 5
<210> 207
<211> 13
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 207
Ser Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr Leu
1 5 10
<210> 208
<211> 12
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 208
Asn Glu Ser Val Glu Tyr Ser Gly Thr Ser Leu Met
1 5 10
<210> 209
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 209
Tyr Phe Thr Ser Ser
1 5
<210> 210
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 210
Tyr Phe Thr Ser Ser
1 5
<210> 211
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 211
Tyr Phe Thr Ser Ser
1 5
<210> 212
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 212
Tyr Tyr Thr Ser Ser
1 5
<210> 213
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 213
Tyr Phe Thr Ser Ser
1 5
<210> 214
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 214
Tyr Ala Ala Ser Asn
1 5
<210> 215
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 215
Tyr Ala Ala Ser Asn
1 5
<210> 216
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 216
Tyr Tyr Thr Ser Arg
1 5
<210> 217
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 217
Phe Tyr Thr Ser Arg
1 5
<210> 218
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 218
Tyr Gly Thr Ser Asn
1 5
<210> 219
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 219
Tyr Gly Thr Ser Asn
1 5
<210> 220
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 220
Tyr Gly Thr Ser Asn
1 5
<210> 221
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 221
Tyr Gly Thr Ser Asn
1 5
<210> 222
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 222
Tyr Asn Ala Asn Ser
1 5
<210> 223
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 223
Tyr Asn Ala Lys Thr
1 5
<210> 224
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 224
His Tyr Thr Ser Thr
1 5
<210> 225
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 225
His Tyr Thr Ser Thr
1 5
<210> 226
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 226
Tyr Asn Ala Lys Thr
1 5
<210> 227
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 227
Tyr His Thr Ser Thr
1 5
<210> 228
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 228
Tyr Ser Ala Ser Phe
1 5
<210> 229
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 229
Tyr Ser Ala Ser Tyr
1 5
<210> 230
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 230
Lys Ser Ala Ser Glu
1 5
<210> 231
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 231
Tyr Trp Ala Ser Thr
1 5
<210> 232
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 232
Tyr Ser Thr Ser Asn
1 5
<210> 233
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 233
Tyr Asn Ala Asn Ser
1 5
<210> 234
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 234
Lys Asn Ala Ser Glu
1 5
<210> 235
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 235
Tyr Trp Ala Ser Thr
1 5
<210> 236
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 236
Tyr Asp Thr Ser Lys
1 5
<210> 237
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 237
Tyr Asn Ala Asn Ser
1 5
<210> 238
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 238
Tyr Asp Thr Ser Asn
1 5
<210> 239
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 239
Lys Ser Ala Ser Glu
1 5
<210> 240
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 240
Lys Ser Ala Ser Glu
1 5
<210> 241
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 241
Lys Asp Ala Ser Glu
1 5
<210> 242
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 242
Tyr Ala Thr Ser Asn
1 5
<210> 243
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 243
Lys Asn Ala Ser Glu
1 5
<210> 244
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 244
Tyr Lys Val Ser Asn
1 5
<210> 245
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 245
Ser Ala Ala Ser Asn
1 5
<210> 246
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 246
Cys Gln Gln Tyr Ser Asn Leu Pro Tyr Thr Phe
1 5 10
<210> 247
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 247
Cys Gln Gln Tyr Ser Asn Leu Pro Tyr Thr Phe
1 5 10
<210> 248
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 248
Cys Gln Gln Tyr Ser Asn Leu Pro Tyr Thr Phe
1 5 10
<210> 249
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 249
Cys Gln Gln Tyr Ser Lys Ile Pro Tyr Thr Cys
1 5 10
<210> 250
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 250
Cys Gln Gln Tyr Ser Asn Leu Pro Tyr Thr Phe
1 5 10
<210> 251
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 251
Cys Gln Gln Asn Tyr Glu Asp Pro Thr Phe
1 5 10
<210> 252
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 252
Cys Gln Gln Ser Asn Glu Asp Pro Thr Phe
1 5 10
<210> 253
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 253
Cys Gln Gln Gly Asp Ala Leu Pro Trp Thr Phe
1 5 10
<210> 254
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 254
Cys Gln Gln Gly Asn Ser Leu Pro Phe Thr Phe
1 5 10
<210> 255
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 255
Cys Gln Gln Trp Ser Ser Arg Pro Pro Thr Phe
1 5 10
<210> 256
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 256
Cys Gln Gln Tyr Ser Gly Tyr Pro Leu Thr Phe
1 5 10
<210> 257
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 257
Cys Gln Gln Tyr Ser Asp Tyr Pro Leu Thr Phe
1 5 10
<210> 258
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 258
Cys Gln Gln Arg Ser Tyr Phe Pro Phe Thr Phe
1 5 10
<210> 259
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 259
Cys Lys Gln Val Tyr Asp Val Pro Phe Thr Phe
1 5 10
<210> 260
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 260
Cys Gln His His Tyr Gly Thr Pro Phe Thr Phe
1 5 10
<210> 261
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 261
Cys Leu Gln Tyr Asp Asn Leu Trp Thr Phe
1 5 10
<210> 262
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 262
Cys Leu Gln Tyr Asp Asn Leu Trp Thr Phe
1 5 10
<210> 263
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 263
Cys Gln His His Tyr Gly Tyr Pro Tyr Thr Phe
1 5 10
<210> 264
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 264
Cys Gln Gln Tyr Ser Asn Leu Pro Leu Thr Phe
1 5 10
<210> 265
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 265
Cys Gln Gln His Tyr Thr Ser Pro Trp Thr Phe
1 5 10
<210> 266
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 266
Cys Gln Gln His Tyr Ser Thr Pro Trp Thr Phe
1 5 10
<210> 267
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 267
Cys Gln Gln Ser Asn Arg Trp Pro Leu Thr Phe
1 5 10
<210> 268
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 268
Cys Gln Asn Asp Tyr Asp Tyr Pro Tyr Thr Phe
1 5 10
<210> 269
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 269
Cys His Gln Tyr His Arg Ser Pro Leu Thr Phe
1 5 10
<210> 270
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 270
Cys Gln Gln Thr Phe Asp Val Pro Trp Thr Phe
1 5 10
<210> 271
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 271
Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Tyr
1 5 10
<210> 272
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 272
Cys Gln Gln Tyr Tyr Asn Tyr Pro Leu Thr Phe
1 5 10
<210> 273
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 273
Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe
1 5 10
<210> 274
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 274
Cys Lys Gln Ala Tyr Asp Val Pro Trp Thr Phe
1 5 10
<210> 275
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 275
Cys Gln Gln Trp Ser Ser Phe Pro Pro Thr Phe
1 5 10
<210> 276
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 276
Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe
1 5 10
<210> 277
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 277
Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe
1 5 10
<210> 278
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 278
Cys Gln Gln Ser Asp Ser Trp Pro Leu Thr Phe
1 5 10
<210> 279
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 279
Cys Gln Gln Trp Ser Ser Asn Pro Arg Thr Phe
1 5 10
<210> 280
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 280
Cys Gln Gln Ser Asn Ser Trp Pro Leu Thr Phe
1 5 10
<210> 281
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 281
Cys Trp Gln Asn Thr His Phe Pro Gln Thr Phe
1 5 10
<210> 282
<211> 11
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Artificial sequence: description of synthetic peptides
<400> 282
Cys Gln Gln Ser Arg Gln Val Pro Leu Thr Phe
1 5 10