Compositions and methods for depletion of CD5+ cells

Abstract

To provide novel compositions and methods for improving hematopoietic stem cell transplantation.SOLUTION: The invention provides anti-CD5 antibodies, antigen-binding fragments thereof, and antibody drug conjugates thereof, for use in treating, for example, a stem cell disorder, cancer, or autoimmune disease, among other hematological and proliferative diseases. Compositions and methods for depleting populations of CD5+ cells, such as CD5+ cancer cells and CD5+ immune cells are described, and can be used to treat cancers and autoimmune diseases directly as stand-alone therapies by eradicating cancerous cells and autoreactive immune cells that express CD5 and / or to prepare a patient for hematopoietic stem cell transplantation, for instance, by depleting populations of CD5+ immune cells that cross-react with, and mount an immune response against, non-self hematopoietic stem cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Provisional Patent Application No. 62 / 592,21, filed November 29, 2017. This application claims the benefit of priority to US Provisional Patent Application No. 2004 / 0109994, the contents of which are incorporated herein by reference. [Background technology]

[0002] Despite advances in medicine, certain blood cell disorders, metabolic disorders, cancer, autoimmune diseases, etc. There remains a need for treatments for hematopoietic pathologies. Hematopoietic stem cells have great therapeutic potential. Although they have potential benefits, limitations that have hindered their clinical use include the inability to transfer hematopoietic stem cell grafts to the host. The difficulty is that the patient's own immune system often rejects the transplanted cells. attack and mediate the rejection of transplanted hematopoietic stem cells. Before hematopoietic stem cell transplantation, patients are given substances that destroy the immune system, such as chemotherapy or radiation. Unfortunately, attempts to induce tolerance to hematopoietic stem cell transplantation in patients have been unsuccessful. This often leads to serious complications. There is a need for new compositions and methods for this purpose. Summary of the Invention

[0003] Currently, compositions and methods for treating diseases of the hematopoietic system, such as autoimmune diseases, and promotes the engraftment of exogenous hematopoietic stem cell grafts and maintains the pluripotency and hematopoietic functions of these cells after transplantation. There is a need for compositions and methods for ensuring that the functionality of the cells is maintained. The invention is directed to, inter alia, the direct treatment of various diseases of the hematopoietic system, metabolic disorders, cancer, and autoimmune diseases. The compositions and methods disclosed herein provide compositions and methods for the treatment of cancer. Hematopoietic stem cell transplantation to treat diseases such as, but not limited to, blood cancer or autoimmune disorders To condition human patients for HIV-1, immune cells are targeted.

[0004] In another aspect, the present invention further provides a method for enhancing the engraftment of hematopoietic stem cells. Compositions for conditioning a patient, such as a human patient, prior to undergoing cell transplantation therapy The subject may have an autoimmune disease, or cancer, a hemoglobinopathy, or suffer from one or more blood disorders, such as other hematopoietic conditions, and therefore require hematopoietic stem cell transplantation As used herein, hematopoietic stem cells are cells that are involved in the production of hematopoietic stem cells. can differentiate into multiple cell types and can be used to transfer or retransfer deficient cell types in patients. In certain embodiments, the present invention provides a method for administering to a patient an antibody capable of binding to CD5. and administering the antibodies and antibody-drug conjugates to a patient to (i) induce the proliferation of autoreactive T cells. CD5-expressing immune cell populations, such as lymphocytes, B cells, or natural killer (NK) cells By selectively depleting the ATPases, we are able to directly treat blood disorders such as autoimmune diseases. and / or (ii) administering to the patient a hematopoietic stem cell graft, Depleting B-cell or NK-cell populations, thereby potentially reducing hematopoietic stem cell graft rejection The present invention is characterized by a method for reducing the sensitivity of the antibody to cross-react with autoantigens and to reduce the sensitivity of the antibody to autoantigens. CD5 can be expressed by T cells, B cells, or NK cells that initiate an appropriate immune response. Therefore, the former activity allows for the direct treatment of a wide range of autoimmune diseases. 5. Administering the Antibody, Antigen-Binding Fragment thereof, or Antibody-Drug Conjugate to a Patient and CD5+ T cells, B cells, or NK cells that cross-react with one or more self-antigens. causing depletion of populations of autoimmune cells, thereby treating autoimmune conditions One or more non-self antigens expressed by hematopoietic stem cells, e.g. T cells, B cells, and / or IgG4 that cross-react with one or more non-self MHC antigens expressed by the or NK cells mount an immune response against the transplanted hematopoietic stem cells, thereby preventing graft rejection. The latter activity may promote the generation of an environment conducive to hematopoietic stem cell engraftment. In the latter case, subsequent diseases such as cancer, autoimmune diseases, or other pathologies of the hematopoietic system may occur. Affected patients may, for example, have one or more genes that are missing or depleted in the patient. To repopulate blood lineages, hematopoietic stem cell grafts can be administered. The present invention provides a method for treating various hematopoietic conditions, such as sickle cell anemia, thalassemia, and Fanconi anemia. , Wiskott-Aldrich syndrome, adenosine deaminase deficiency-severe combined immunodeficiency Diabetes, Metachromatic Leukodystrophy, Diamond-Blackfan Anemia and Schwack's Mann-Diamond syndrome, human immunodeficiency virus infection, and acquired immunodeficiency syndrome and methods for treating, inter alia, cancer and autoimmune diseases.

[0005] In one aspect, the present invention provides a method for detecting a CD5+ cell population, e.g., in a human patient, e.g., in a human patient. a population of CD5+ T cells, CD5+ B cells, and / or CD5+ NK cells in Antibodies, antigen-binding fragments thereof, or antibody-drug conjugates that bind to CD5 A method of depletion is provided by administering an effective amount to a patient.

[0006] In another aspect, the present invention provides a method for detecting CD5+ Cell populations, such as CD5+ T cells, C in human patients requiring hematopoietic stem cell transplantation A population of CD5+ B cells, and / or CD5+ NK cells can be obtained, for example, by transplanting hematopoietic stem cells from a patient. an antibody that binds to CD5, an antigen-binding fragment thereof, or The present invention provides a method for depleting an antibody-drug conjugate by administering an effective amount of the antibody-drug conjugate to a patient. Provide.

[0007] In a further aspect, the present invention provides a method for the treatment of hematopoietic stem cell transplantation in a human patient in need of hematopoietic stem cell transplantation therapy. Hematopoietic stem cell graft rejection can be prevented by screening patients for CD5 an effective amount of an antibody, antigen-binding fragment thereof, or antibody-drug conjugate that binds to The present invention provides a method for preventing or reducing the likelihood of such a condition by administering

[0008] In another aspect, the present invention provides a method for treating endogenous hematopoietic stem cell transplantation in a human patient in need of hematopoietic stem cell transplantation therapy. The patient's T cell population is isolated by an antibody that binds to CD5 before receiving a graft containing hematopoietic stem cells. administering an effective amount of the antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate. This provides a method for depleting the

[0009] In another aspect, the present invention provides a method for treating a human patient, e.g., in need of a hematopoietic stem cell transplant. The method comprises administering to a human patient a graft comprising hematopoietic stem cells, wherein the patient is and administering to the subject an antibody or antigen-binding fragment thereof that binds to D5. The antibody, antigen-binding fragment thereof, or antibody-drug conjugate may be administered to a patient. CD5+ cell populations in human patients, e.g., CD5+ T cells, CD5+ B cells, and The antibody may be administered to a patient in an amount sufficient to deplete the population of CD5+ NK cells and / or CD5+ NK cells.

[0010] In a further aspect, the present invention provides a method for treating, for example, a human patient in need of a hematopoietic stem cell transplant. a method of treating a human patient comprising administering to the human patient an antibody, antigen-binding fragment thereof, or Alternatively, the antibody-drug conjugate may be administered to a CD5+ cell population in a patient, e.g., Deplete populations of CD5+ T cells, CD5+ B cells, and / or CD5+ NK cells that are involved in the proliferation of CD5+ T cells. and subsequently administering to the patient a graft comprising hematopoietic stem cells. and

[0011] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof of the invention The compound is produced by the hybridoma cell line ATCC CRL 8000. In some embodiments, the antibodies or antigen-binding fragments thereof of the invention are isolated from hybridoma cells. Antibodies or antigen-binding fragments thereof produced by cell line ATCC CRL 8000 It competitively inhibits CD5 binding to IgG.

[0012] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof It contains the following variable domains: Amino acid sequence DIQMTQSPSSMSASLGDRVTITCRASQDINSYLSWFQQ KPGKSPKTLIYRANRLVDGVPSRFSGSGSGTDYTLTISSL QYEDFGIYYCQQYDESPWTFGGGTKLEIK (SEQ ID NO: 1) V L and; Amino acid sequence QIQLVQSGPGLKKPGGSVRISCAASGYTFTNYGMNWVK QAPGKGLRWMGWINTHTGEPTYADDFKGRFTFSLDTSKST AYLQINSLRAEDTATYFCTRRGYDWYFDVWGQGTTVTVSS (SEQ ID NO: 2) H .

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the aforementioned variable domains. The antibody or antigen-binding fragment thereof competitively inhibits CD5 binding to an antibody or antigen-binding fragment thereof containing the antibody.

[0014] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof Contains the following CDRs: and CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 3); and CDR-H2 having the amino acid sequence NTHTGE (SEQ ID NO: 4); and a CDR-H3 having the amino acid sequence RGYDWYFDV (SEQ ID NO: 5); and CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 6); and a CDR-L2 having the amino acid sequence RANRLVD (SEQ ID NO: 7); CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 8).

[0015] In some embodiments, the antibody or antigen-binding fragment thereof comprises the aforementioned CDRs. It competitively inhibits the binding of CD5 to an antibody or antigen-binding fragment thereof.

[0016] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof It contains the following variable domains: Amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQ KPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSL QYEDFGIYYCQQYDESPWTFGGGTKLEIK (SEQ ID NO: 9) V L and; Amino acid sequence EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVR QAPGKGLEWWMGWINTHYGEPTYADSFKGTRTFSLDDSKNT AYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGGTTTVVS V with S (SEQ ID NO: 10) H .

[0017] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the aforementioned variable domains. The antibody or antigen-binding fragment thereof competitively inhibits CD5 binding to an antibody or antigen-binding fragment thereof containing the antibody.

[0018] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof Contains the following CDRs: and CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 11); and CDR-H2 having the amino acid sequence NTHYGE (SEQ ID NO: 12); and a CDR-H3 having the amino acid sequence RRGYDWYFDV (SEQ ID NO: 13); and CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 14); and a CDR-L2 having the amino acid sequence RANRLES (SEQ ID NO: 15); CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 16).

[0019] In some embodiments, the antibody or antigen-binding fragment thereof comprises the aforementioned CDRs. It competitively inhibits the binding of CD5 to an antibody or antigen-binding fragment thereof.

[0020] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof Contains the following CDRs: and CDR-H1 having the amino acid sequence GYSITSGYY (SEQ ID NO: 17); and CDR-H2 having the amino acid sequence ISYSGFT (SEQ ID NO: 18); and a CDR-H3 having the amino acid sequence AGDRTGSWFAY (SEQ ID NO: 19); and CDR-L1 having the amino acid sequence QDISNY (SEQ ID NO: 20); and a CDR-L2 having the amino acid sequence ATS (SEQ ID NO: 21); CDR-L3 having the amino acid sequence LQYASYPFT (SEQ ID NO: 22).

[0021] In some embodiments, the antibody or antigen-binding fragment thereof comprises the aforementioned CDRs. It competitively inhibits the binding of CD5 to an antibody or antigen-binding fragment thereof.

[0022] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof Contains the following CDRs: and CDR-H1 having the amino acid sequence GYIFTNYG (SEQ ID NO: 23); and a CDR-H2 having the amino acid sequence INTYNGEP (SEQ ID NO: 24); and a CDR-H3 having the amino acid sequence ARGDYYGYEDY (SEQ ID NO: 25); and CDR-L1 having the amino acid sequence QGISNY (SEQ ID NO: 26); and a CDR-L2 having the amino acid sequence YTS (SEQ ID NO: 27); CDR-L3 having the amino acid sequence QQYSKLPWT (SEQ ID NO: 28).

[0023] In some embodiments, the antibody or antigen-binding fragment thereof comprises the aforementioned CDRs. It competitively inhibits the binding of CD5 to an antibody or antigen-binding fragment thereof.

[0024] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof Contains the following CDRs: CDR-H1 having the amino acid sequence FSLSTSGMG (SEQ ID NO: 29); and CDR-H2 having the amino acid sequence WWDDD (SEQ ID NO: 30); and a CDR-H3 having the amino acid sequence RRATGTGFDY (SEQ ID NO: 31); and CDR-L1 having the amino acid sequence QDVGTA (SEQ ID NO: 32); and a CDR-L2 having the amino acid sequence WTSTRHT (SEQ ID NO: 33); CDR-L3 having the amino acid sequence YNSYNT (SEQ ID NO: 34).

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises the aforementioned CDRs. It competitively inhibits the binding of CD5 to an antibody or antigen-binding fragment thereof.

[0026] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-H4 listed in Table 1 below In some embodiments, the antibody comprises a combination of CDR-L2, CDR-L3, and CDR-L4 regions. The antibody or antigen-binding fragment thereof may be an antibody comprising a combination of CDRs as set forth in Table 1 below. Competitively inhibits the binding of CD5 to the antibody or its antigen-binding fragment.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody. antibody or antigen-binding fragment thereof, polyclonal antibody or antigen-binding fragment thereof a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, fragments, dual variable immunoglobulin domains, single-chain Fv molecules (scFv), diabodies Dibodies, triabodies, nanobodies, antibody-like protein scaffolds, Fv fragments, Fab fragments fragment, F(ab')2 molecule, and tandem di-scFv. In some embodiments, the antibodies include IgG, IgA, IgM, IgD, and IgE. The isotype is selected from the group consisting of:

[0028] In some embodiments, the antibody is conjugated to a cytotoxin.

[0029] In some embodiments, the cytotoxin is amatoxin, Pseudomonas exotoxin A, de Bouganin, diphtheria toxin, saporin, maytansin, maytansinoid, au Ristatin, anthracycline, calicheamicin, irinotecan, SN-38, dutasteride Ocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolino Benzodiazepine and indolinobenzodiazepine dimers or their variants is selected from the group consisting of:

[0030] In another aspect, the present invention provides a CD5+ cell population in a human patient, e.g., The population of CD5+ T cells, CD5+ B cells, and / or CD5+ NK cells that are present in the nucleus are referred to as C. Antibodies, fragments thereof, or antibody-drug conjugates capable of binding to D5 The present invention provides a method for depleting a patient's immune system by administering to the patient an effective amount of

[0031] In a further aspect, the present invention provides a method for treating CD in a human patient in need of hematopoietic stem cell transplantation. 5+ cell populations, e.g., CD5+ T cells in human patients requiring hematopoietic stem cell transplantation , CD5+ B cells, and / or CD5+ NK cell populations, wherein the patient is Before receiving the graft, patients should be given an antibody, fragment thereof, or and administering an effective amount of an antibody-drug conjugate to the subject. .

[0032] In another aspect, the present invention provides a method for treating a human patient, e.g., in need of a hematopoietic stem cell transplant. The method comprises administering to a human patient a graft comprising hematopoietic stem cells, wherein the patient is The antibody, fragment thereof, or antibody-drug conjugate that binds to D5 is administered to a patient. CD5+ cell populations in human patients, e.g., CD5+ T cells, CD5+ B cells, and and / or previously administered in an amount sufficient to deplete the population of CD5+ NK cells. It is characterized by:

[0033] In a further aspect, the present invention provides a method for treating, for example, a human patient in need of a hematopoietic stem cell transplant. A method of treating a human patient comprising administering to the patient an antibody, fragment thereof, or antibody that binds to CD5. The body-drug conjugate is administered to a CD5+ cell population in the patient, e.g., a CD5 sufficient to deplete populations of CD5+ T cells, CD5+ B cells, and / or CD5+ NK cells. and subsequently administering to the patient a graft comprising hematopoietic stem cells. The present invention is characterized by a method comprising:

[0034] In some embodiments of any of the foregoing four aspects, the target cells are CD4+ T cells (e.g., CD5+ T cells, CD4+ T cells, an antibody or antibody that binds to CD5 (on the surface of CD5+ B cells, or CD5+ NK cells) Fragments have been isolated from human antibodies (e.g., IgG1, IgG2, IgG3, or or a dimeric Fc domain (isolated from a human antibody of the IgG4 isotype) In some embodiments, the Fc domain is covalently linked to the single-chain polypeptide. In some embodiments, the antibody or its Fc domain is a monomeric Fc domain containing a nucleotide. In some embodiments, the N-terminus of the fragment is linked to an Fc domain. The C-terminus of the antibody or its fragment is linked to the Fc domain. or one or more copies of a fragment thereof, for example: Conjugates that can be used in conjunction with the methods described herein include those comprising each polypeptide of the Fc domain. a dimeric Fc domain in which the peptide chains are conjugated to an antibody or fragment thereof The Fc domain then binds to a cytotoxin described herein (e.g., α-amanitin, etc. amatoxin, Pseudomonas exotoxin A, deBouganin, diphtheria toxin, Porins, maytansine, maytansinoids, auristatins, anthracyclines, potassium Caremicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepines pyrrolobenzodiazepine dimers, indolinobenzodiazepines, and indolinobenzidines conjugated to cytotoxins such as benzodiazepine dimers, or their variants good.

[0035] In some embodiments, the anti-CD5 antibody or fragment thereof is Cytotoxins (e.g., amatoxins such as α-amanitin, Pseudomonas exotoxin A, de Bouganin, diphtheria toxin, saporin, maytansin, maytansinoid, au Ristatin, anthracycline, calicheamicin, irinotecan, SN-38, dutasteride Ocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolino benzodiazepines, and indolinobenzodiazepine dimers, or their variants) In some embodiments, the antibody or fragment thereof is covalently attached to a cytotoxin such as In some embodiments, the N-terminus of the antibody or its fragment is conjugated to a cytotoxin. The C-terminus of the fragment is conjugated to a cytotoxin, which is then coupled to the Fc domain. It may be conjugated.

[0036] In some embodiments, the anti-CD5 antibody or fragment thereof is an antibody or fragment thereof. at one site of a fragment (e.g., the N-terminus or C-terminus of an antibody or fragment thereof) ) covalently attached to the cytotoxin at another site on the antibody or fragment thereof (e.g., The antibody or fragment thereof is covalently linked to the Fc domain at one end (opposite end of the antibody or fragment thereof).

[0037] In some embodiments, the Fc domain is a human IgG1 isotype Fc domain. In some embodiments, the Fc domain is a human IgG2 isotype Fc domain. In some embodiments, the Fc domain is a human IgG3 isotype Fc domain. In some embodiments, the Fc domain is a human IgG4 isotype F It is the c domain.

[0038] In some embodiments of any of the above aspects, the cytotoxin is α-amanitin, β-amanitin, Amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, flax amatoxins or derivatives thereof, such as amanuric acid, and proamanurin. In some embodiments of any of the above aspects, the cytotoxin is amanitin. The toxin is an amatoxin, and the toxin is an antibody conjugated to a cytotoxin, or its antigen-binding fragment. The fragment is represented by the formula Ab-ZL-Am, where Ab is the anti-CD5 antibody and its antigen-binding fragment. is a segment, L is a linker, Z is a chemical moiety, and Am is an amatoxin In some embodiments, the amatoxin is conjugated to a linker. In some embodiments, the amatoxin-linker conjugate Am-LZ is of formula (I): Represented: [ka] (wherein R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D and; R4 is H, OH, OR C , OR D , R C , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, OR C , ORD , R C , or R D and; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D and; R9 is H, OH, OR C , or OR D and; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a linker, for example, an optionally substituted alkylene (e.g., C1-C6 alkylene). cyclohexane), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic groups; Alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heptane Tetraarylene; dipeptide, -C(=O)-, peptide, or combination thereof can be; Z is a reactive group present on L and a CD5+ T cell, a CD5+ B cell, or Antibodies that bind to CD5, such as those on the surface of CD5+ NK cells, or their antigen-binding fragments It is a chemical moiety formed by a coupling reaction with a reactive substituent present in the (This is the case.)

[0039] In some embodiments, Am is exactly one R C Contains substituents.

[0040] In some embodiments, the linker L and chemical moiety Z together form LZ. considered, [ka] (wherein S is the antigen present in an antibody, or antigen-binding fragment thereof, that binds to CD5) is a sulfur atom representing a reactive substituent (e.g., from the -SH group of a cysteine ​​residue) be.

[0041] In some embodiments, LZ is [ka] is.

[0042] In some embodiments, Am-LZ-Ab is: [ka] is.

[0043] In some embodiments, Am-LZ is represented by formula (IA): [ka] (wherein R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D and; R4 is H, OH, OR C , OR D , R C , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, OR C , OR D , R C , or R D and; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D and; R9 is H, OH, OR C , or OR D and; X is -S-, -S(O)-, or -SO2-; RC is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a linker, for example, an optionally substituted alkylene (e.g., C1-C6 alkylene). cyclohexane), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic groups; Alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heptane Tetraarylene; dipeptide, -C(=O)-, peptide, or combinations thereof and; Z is a reactive group present on L and a CD5+ T cell, CD5+ B cell, or CD Antibodies that bind to CD5, such as those on the surface of 5+ NK cells, or their antigen-binding fragments a chemical moiety formed by a coupling reaction with a reactive substituent present in the where Am is exactly one R C (including substituents).

[0044] In some embodiments, the linker L and chemical moiety Z together form LZ. considered, [ka] is.

[0045] In some embodiments, the LZ is: [ka] is.

[0046] In some embodiments, Am-LZ-Ab is: [ka] is.

[0047] In some embodiments, Am-LZ-Ab is: [ka] is.

[0048] In some embodiments, Am-LZ is represented by formula (IB): [ka] (wherein R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or RD and; R4 is H, OH, OR C , OR D , R C , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, OR C , OR D , R C , or R D and; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D and; R9 is H, OH, OR C , or OR D and; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, dipeptide, -C(=O)-, peptide or a combination thereof; L is a linker, for example, an optionally substituted alkylene (e.g., C1-C6 alkylene). cyclohexane), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic groups; Alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heterocycloalkylene; Being Roariren; Z is a reactive group present on L and a CD5+ T cell, CD5+ B cell, or CD Antibodies that bind to CD5, such as those on the surface of 5+ NK cells, or their antigen-binding fragments a chemical moiety formed by a coupling reaction with a reactive substituent present in the where Am is exactly one R C (including substituents).

[0049] In some embodiments, R A and R B along with the oxygen atoms to which they are attached , combined to form the formula: [ka] (Wherein, Y is —C(═O)—, —C(═S)—, —C(═NR E )-, or -C(R E R E’ )-and;R E and R E’ are each independently an optionally substituted C- C6 alkylene-R C , optionally substituted C1-C6 heteroalkylene-R C , arbitrarily placed Substituted C2-C6 alkenylene-R C , optionally substituted C2-C6 heteroalkenyl N-R C , optionally substituted C2-C6 alkynylene-R C , optionally substituted C2-C 6 heteroalkynylene-R C , optionally substituted cycloalkylene-R C , optionally substituted Heterocycloalkylene-R C , optionally substituted arylene-R C , or optionally Substituted heteroarylene-R C ) to form a 5-membered heterocycloalkyl group.

[0050] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB), and R is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached. [ka] Forming; R3 is H or R C and; R4 is H, OH, OR C , OR D , R C , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, ORC , OR D , R C , or R D and; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , or NHR C and; R9 is H or OH; R C and R D are as defined above.

[0051] In some embodiments, Am is represented by formula (IA) or formula (IB): R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached. [ka] Forming; R3 is H or R C and; R4 and R5 each independently represent H, OH, OR C , R C , or OR D and ; R6 and R7 are each H; R8 is OH, NH2, OR C , or NHR C and; R9 is H or OH; X, R C , and R D are as defined above.

[0052] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 is H, OH, or OR A and; R2 is H, OH, or OR B and; R A and R B are bonded together with the oxygen atoms to which they are attached. [ka] Forming; R3, R4, R6, and R7 are each H; R5 is OR C and; R8 is OH or NH2; R9 is H or OH; X and R C is as defined above.

[0053] In some embodiments, Am is represented by formula (IA) or formula (IB): R1 and R2 are each independently H or OH; R3 is R C and; R4, R6, and R7 are each H; R5 is H, OH, or OC1-C6 alkyl; R8 is OH or NH2; R9 is H or OH; X and R C is as defined above.

[0054] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are each independently H, OH, or OR C , or R C and; R8 is OH or NH2; R9 is H or OH; X or R C is as defined above.

[0055] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are each independently H or OH; R8 is OH, NH2, OR C , or NHR C and; R9 is H or OH; X or R C is as defined above.

[0056] In some embodiments, the linker L and chemical moiety Z together form LZ. considered, [ka] is.

[0057] In some embodiments, the LZ is: [ka] is.

[0058] In some embodiments, Am-LZ-Ab is: [ka] is.

[0059] In some embodiments, Am-LZ-Ab is: [ka] is.

[0060] In some embodiments, Am-LZ is represented by Formula (II), Formula (IIA), or Formula (II B) is represented by: [ka] or [ka] wherein X is S, SO, or SO; R is H or a linker, The reactive substituents present on the linker and the the antibody or a linker covalently linking the antigen-binding fragment to the a reactive group present on the linker and an antibody or its antigen-binding fragment; via chemical moiety Z formed by coupling reaction with reactive substituents present in the methyl group. is a linker covalently attached to the antibody or antigen-binding fragment thereof via when R2 is H, R1 is a linker).

[0061] In some embodiments, the linker is -(CH) 2n -unit, n is an integer between 2 and 6 is.

[0062] In some embodiments, R1 is a linker, R2 is H, and the linker and The learning areas are grouped together as the LZ. [ka] is.

[0063] In some embodiments, Am-LZ-Ab is: [ka] is.

[0064] In some embodiments, Am-LZ-Ab is: [ka] is.

[0065] In some embodiments, Am-LZ-Ab is: [ka] is.

[0066] In some embodiments of any of the above aspects, the cytotoxin consists of DM1 and DM4. In some embodiments, the cytotoxin is a maytansinoid selected from the group an auristatin selected from the group consisting of methylauristatin E and monomethylauristatin F; In some embodiments, the cytotoxin is daunorubicin, doxorubicin, or urinary inactivation inhibitor. an anthracycline selected from the group consisting of bicin, epirubicin, and idarubicin It is.

[0067] In some embodiments, the cytotoxin has formula (IV): [ka] It is a pyrrolobenzodiazepine dimer represented by the formula:

[0068] In some embodiments, the cytotoxin is attached to the antibody via a maleimidocaproyl linker. or an antigen-binding fragment thereof.

[0069] In some embodiments, the cytotoxin is monomethyl auristatin E and monomethyl The auristatin is selected from the group consisting of auristatin F.

[0070] In some embodiments, the cytotoxin is daunorubicin, doxorubicin, epirubicin, The anthracycline is selected from the group consisting of idarubicin, idarubicin, and idarubicin.

[0071] In some embodiments, an anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug The conjugate may, after administration to a patient, induce T cells, B cells, or NK cells (e.g., C CD5+ T cells, CD5+ B cells, or CD5+ NK cells) For example, an anti-CD5 antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate The ATP is released by receptor-mediated endocytosis (e.g., upon binding to cell surface CD5). ), which can be taken up by T cells. In some embodiments, the antibody, or its antigen-binding fragment, The cytotoxin covalently attached to the fragment can be released by chemical cleavage (e.g., by using the ligands described herein). The cytotoxin can then be released into the cell (by enzymatic or nonspecific cleavage of the anchor). is the number of endogenous immune cells (e.g., CD5+ T cells, CD5+ B cells) before hematopoietic stem cell transplantation treatment. , or CD5+ NK cells) to promote the death of their intracellular targets (e.g., RNA polymerase, mitotic spindle, nuclear DNA, ribosomal RNA, or topoisomerase etc.)

[0072] In some embodiments, an anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug The conjugates may be used to target T cells, B cells, or NK cells (e.g., CD5+ T cells, CD It can promote necrosis of immune cells such as CD5+ B cells, or CD5+ NK cells. In some embodiments, the antibody, or antigen-binding fragment thereof, is administered to a patient and one or more complement proteins, NK cells, macrophages, neutrophils, and / or neutrophils. By recruiting immune cells with eosinophils, endogenous immune cells (e.g., CD4+) can be replenished prior to transplant treatment. These antibodies may promote the death of CD5+ T cells, CD5+ B cells, or CD5+ NK cells.

[0073] In some embodiments, an autologous transplant comprising hematopoietic stem cells is administered to the patient. Autologous hematopoietic stem cells are collected from a patient, e.g., a patient in need of hematopoietic stem cell transplantation therapy. and the cells are subsequently administered to the patient (e.g., The recovered hematopoietic stem cells can be cultured ex vivo for, for example, 1 hour, 1 day, Alternatively, the cells may be maintained for a week or more and then reinfused into the subject. After collection from the patient, blood stem cells are collected within 1 hour to approximately 1 week, 1 hour to approximately 72 hours, or approximately 1 hour to approximately The patient may be reinfused within 48 hours, or between about 1 hour and about 24 hours. In this form, the collected hematopoietic stem cells are frozen for long-term storage before being reinfused into the patient. For example, harvested hematopoietic stem cells may be stored for one week to one year, or longer, before being reinfused into the patient. It can be frozen and stored frozen for longer periods.

[0074] In some embodiments, an allograft comprising hematopoietic stem cells is administered to the patient. Allogeneic hematopoietic stem cells are obtained from a donor who is HLA-matched to the patient, such as a close relative of the patient. In some embodiments, the allogeneic hematopoietic stem cells may be obtained from a donor. HLA-incompatible. After collecting allogeneic hematopoietic stem cells from the donor, one of the hematopoietic To reintroduce these cells, the cells can be administered (e.g., infused) to the patient. The hematopoietic stem cells were maintained ex vivo for, e.g., 1 hour, 1 day, or 1 week or longer. For example, the recovered hematopoietic stem cells may be reinfused into the subject after recovery from a donor. After, 1 hour to about 1 week, 1 hour to about 72 hours, about 1 hour to about 48 hours, or about 1 hour In some embodiments, the recovered hematopoietic stem cells may be infused into the patient over a period of about 24 hours. The cells are frozen for long-term storage before being infused into the patient. are frozen and cryopreserved for a period of one week to one year or longer before being injected into the patient. It can be preserved.

[0075] In some embodiments, the hematopoietic stem cell-containing graft is administered with an anti-CD5 antibody, its antigen-binding fragment, or a combination thereof. fragment, or the concentration of the antibody-drug conjugate, is substantially removed from the patient's blood. It is then administered to the patient.

[0076] In some embodiments, the hematopoietic stem cell-containing graft is administered with an anti-CD5 antibody, its antigen-binding fragment, or a combination thereof. fragment, or the concentration of the antibody-drug conjugate, is substantially removed from the patient's blood. 1 hour to 7 days (e.g., 6 hours to 3 days, 12 hours to 36 hours, or approximately 24 hours) after It is administered to the patient during

[0077] In some embodiments, the hematopoietic stem cells or their progeny are transplanted into a patient. to 2 days or more (e.g., about 2 to about 5 days, about 2 to about 7 days, about 2 to about 20 days, about 2 to Approximately 30 days, for example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, 30th, or They maintain the functional capacity of hematopoietic stem cells even after a period of time (longer than 10 years).

[0078] In some embodiments, the CD5+ cell population comprises CD34+ cells. The percentage of CD34+ cells in the 5+ cell population was approximately 10% of the total number of cells in the population, 2 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or or 100%.

[0079] In some embodiments, the hematopoietic stem cells or their progeny are derived from hematopoietic stem cells transplanted into a patient. It can subsequently localize to hematopoietic tissues such as bone marrow and / or restore hematopoiesis.

[0080] In some embodiments, when transplanted into a patient, the hematopoietic stem cells produce megakaryocytes, thrombocytes, platelets, , erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, Osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes The method results in recovery of a cell population selected from the group consisting of lymphocytes, leukocytes, and B lymphocytes.

[0081] In some embodiments, the patient is suffering from cancer. The cancer may be a hematological cancer, i.e., acute Myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, or chronic lymphocytic leukemia It can be any type of leukemia.

[0082] In some embodiments, the CD5+ cells comprise cancer cells.

[0083] In some embodiments, an anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug The antibody conjugate depletes cancer cells in a patient. For example, the antibody or its antigen The binding fragments were expressed in 10%, 20%, 30%, 40%, and 50% of the cancer cells in the patient. , 60%, 70%, 80%, 90%, or substantially all cancer cells may be depleted.

[0084] In some embodiments, an anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug The monoconjugate depletes blood cancer cells (e.g., leukemia cells) in the patient. In some embodiments, the hematological cancer cells are selected from the group consisting of acute myeloid leukemia cells, acute lymphocytic leukemia cells, and leukemia cells. cells, chronic myeloid leukemia cells, or chronic lymphocytic leukemia cells. In this state, blood cancer cells include megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, and basophils. leukocytes, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages The antigen-binding protein (AGP) is a cytoplasmic antigen (CAG) that binds to a target cell, such as a dendritic cell, a natural killer cell, a T lymphocyte, or a B lymphocyte.

[0085] In some embodiments, the CD5+ cell population comprises CD5+ T cells, CD5+ B cells, and and / or immune cells such as CD5+ NK cells.

[0086]

[0013] In some embodiments of any of the above aspects, the method comprises administering to the patient a therapeutically effective amount of a compound that is capable of inhibiting or preventing the progression of a disease, including ... This is caused by immune cell populations that cross-react with the hematopoietic stem cell graft, such as non-self MHC antigens. To prevent or reduce the likelihood of hematopoietic stem cell graft rejection, which may occur in the future. For example, depleting immune cell populations in patients prior to hematopoietic stem cell transplantation therapy. After transplantation, hematopoietic stem cells are used to treat one or more diseases. These are cell types that are deficient in the patient or that are actively killed, for example, by chemotherapeutic methods. Productive hematopoiesis can be established to replenish lost or killed cell types. The patient may be a patient suffering from a stem cell disease. In some embodiments, the patient is a patient suffering from a sickle cell disease. cytic anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiscott- Patients with hemoglobinopathies such as Aldrich syndrome have a congenital immune disorder. deficiency or acquired immunodeficiency disease (e.g., human immunodeficiency virus or acquired immunodeficiency In some embodiments, the patient may be suffering from an immunodeficiency disease, such as HIV-1. Patients with glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidosis Some patients suffer from metabolic disorders such as leukodystrophy, leukodystrophy, and metachromatic leukodystrophy. In this form, patients have adenosine deaminase deficiency and severe combined immunodeficiency, hyperimmune group Roblin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta thyroid disease, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, and In some embodiments, the patient is suffering from a disease selected from the group consisting of juvenile rheumatoid arthritis. In this study, patients were diagnosed with a variety of diseases, including scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, and type 1 diabetes. In some embodiments, the patient is suffering from a cancer such as a hematological cancer. In some embodiments, the patient is suffering from acute myeloid leukemia or a myeloproliferative disorder. Leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, Have diffuse large B-cell lymphoma or non-Hodgkin's lymphoma. In an embodiment, the patient is suffering from a myelodysplastic disease, such as a myelodysplastic syndrome.

[0087] In some embodiments of any of the above aspects, the method comprises detecting CD5+ cancer cells in the patient. population-depleting antibodies, antigen-binding fragments thereof, or antibody-drug conjugates and / or non-self antibodies expressed by hematopoietic stem cell grafts. Rejection of hematopoietic stem cell grafts, which may otherwise be caused by immune cell populations that cross-react with the marker, is prevented. Antibodies or other treatments may be administered prior to hematopoietic stem cell transplantation to prevent or reduce the likelihood of failure. characterized in CD5+ cells, for example by administering its antigen-binding fragment. To directly treat cancers, such as those characterized by CD5+ cells (e.g., leukemias). In the latter case, transplantation is used to replace cell populations that have been depleted in the process of eradicating cancer cells, for example. Cancers include acute myeloid leukemia, acute lymphoblastic leukemia, and chronic myeloid leukemia. Hematologic malignancies, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non- It can be a blood cancer such as Hodgkin's lymphoma.

[0088] In some embodiments of any of the above aspects, the method further comprises: depletion of autoreactive CD5+ T cell, B cell, and / or NK cell populations The anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug conjugate Gates expressed by administering and / or hematopoietic stem cell grafts Immune cell populations that cross-react with hematopoietic stem cell grafts, such as non-self MHC antigens, can cause To prevent or reduce the likelihood of hematopoietic stem cell graft rejection, administration of the antibody or its antigen-binding fragment prior to hematopoietic stem cell transplantation therapy, etc. In the latter case, transplantation is used to treat autoimmune diseases, e.g. Cell populations that are depleted during the eradication of autoimmune cells can be reconstituted later. For example, it may be: scleroderma, multiple sclerosis (MS), human systemic lupus (SLE), related Treatment of rheumatoid arthritis (RA), inflammatory bowel disease (IBD), psoriasis, type 1 diabetes mellitus (type 1 diabetes) disease), acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis, anti- Phospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune liver inflammation, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune Immune oophoritis, Baro's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic Fatigue and immune deficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease, scar Scarlet pemphigoid, celiac disease-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Dogo Disease, discoid lupus, autonomic dysfunction, endometriosis, essential mixed cryoglobulinemia, fibromyalgia Fibromyalgia-fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome ( GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura , idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, Lyme disease, Meniere's disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia , Opsoclonus-myoclonus syndrome (OMS), optic neuritis, Odo thyroiditis, vulgaris Pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, Polyarteritis nodosa, polyglandular syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-person syndrome, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis") Also known as ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia (vulvar vestibulopathy) granulomatosis), as well as Wegener's granulomatosis.

[0089] Thus, in some embodiments of any of the above aspects, the present invention provides a method for treating sickle cell anemia, Thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome In some embodiments, the present invention features a method for treating a hemoglobinopathy disease, such as a steroid hormone, a steroid hormone, or a steroid hormone. The present invention relates to a method for treating congenital or acquired immunodeficiency diseases (e.g., human immunodeficiency virus The present invention features a method for treating an immunodeficiency disease, such as HIV-1 or acquired immunodeficiency syndrome (AIDS). In some embodiments, the present invention provides a method for treating glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Herpes simplex virus, and the like. Treat metabolic disorders such as Lehr disease, sphingolipidosis, and metachromatic leukodystrophy In some embodiments, the present invention features a method for treating adenosine deaminase Deficiency and severe combined immunodeficiency, hyperimmunoglobulin M syndrome, Chediak Higashi disease, inherited Inherited lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic selected from the group consisting of sclerosis, systemic lupus erythematosus, and juvenile rheumatoid arthritis In some embodiments, the present invention features methods for treating diseases such as scleroderma, multifocal sclerosis, and rheumatoid arthritis. Those treating autoimmune diseases such as sclerosis, ulcerative colitis, Crohn's disease, and type 1 diabetes In some embodiments, the present invention features a method for treating cancer, such as a hematological cancer, or bone marrow proliferation. In some embodiments, the present invention features a method for treating acute myeloid proliferation disorders. Leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, 1. A method for treating diffuse large B-cell lymphoma or non-Hodgkin's lymphoma. In some embodiments, the patient is afflicted with a myelodysplastic disease, such as myelodysplastic syndrome. In these embodiments, the method comprises administering to a subject an antibody that binds to CD5, its antigen-binding fragment, ment, or a conjugate thereof, such as an antibody of any aspect or embodiment of the invention; administering the antigen-binding fragment, or a conjugate thereof, to a patient The method may further comprise, for example, in accordance with the method of any aspect or embodiment of the present invention, This may include administering a stem cell graft to the patient.

[0090] Similarly, in some embodiments of any of the above aspects, the invention provides a method for detecting a cell type characterized by CD5+ cells. Directly treat cancers, including cancers characterized by CD5+ cells (e.g., leukemias characterized by CD5+ cells). In these embodiments, the methods provide methods for detecting CD5, including the use of antibodies that bind to CD5, its antigen, and Binding fragments, or conjugates thereof, e.g., any aspect or embodiment of the present invention administering the antibody, antigen-binding fragment thereof, or conjugate thereof to a patient. The cancer may include acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic myeloid leukemia, lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma It can be a blood cancer such as lymphoma.

[0091] Furthermore, in some embodiments of any of the above aspects, the present invention provides a method for treating autoimmune diseases such as: Provides methods for treating diseases including: MS, SLE, RA, IBD, psoriasis, type 1 diabetes, and ADE M, Addison's disease, alopecia universalis, ankylosing spondylitis, APS, aplastic anemia, autoimmune lysis. Blood anemia, autoimmune hepatitis, AIED, ALPS, autoimmune oophoritis, Barrow's disease, Bech Whetstone's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, CFIDS, chronic inflammatory demyelinating multiple myelopathy Hemorrhagic fever, Crohn's disease, cicatricial pemphigoid, celiac disease-dermatitis herpetiformis, cold agglutinin disease, CR EST syndrome, Dego's disease, discoid lupus, autonomic dysfunction, endometriosis, essential mixed clonus Oglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome, Graves' disease, G BS, Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, especially Acute pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, leprosy Meniere's disease, MCTD, myasthenia gravis, neuromyotonia, OMS, optic neuritis, thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, Primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndrome, polymyalgia rheumatica, primary non-cancer Maglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, sclerosing dermatosis, Sjögren's syndrome, stiff-person syndrome, Takayasu's arteritis, temporal arteritis ("giant arteritis"). (also known as "cytoplasmic arteritis"), ulcerative colitis, uveitis, vasculitis, vitiligo, Vulvar pain ("vulvar vestibulitis"), and Wegener's granulomatosis. The method comprises administering to a subject an antibody, antigen-binding fragment thereof, or conjugate thereof that binds to CD5; For example, an antibody, antigen-binding fragment thereof, or a The method may include administering to the patient a conjugate of

[0092] In another aspect, the present invention provides an antibody capable of binding to CD5, or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof is conjugated to a toxin. It is being done.

[0093] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are hybridized It is produced by the doma cell line ATCC CRL 8000. In some embodiments, The antibodies or antigen-binding fragments of the present invention are derived from the hybridoma cell line ATCC 61444. Binding of CD5 to antibodies or antigen-binding fragments thereof produced by RL 8000 It competitively inhibits the binding of

[0094] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following variable domains: Contains: Amino acid sequence DIQMTQSPSSMSASLGDRVTITCRASQDINSYLSWFQQ KPGKSPKTLIYRANRLVDGVPSRFSGSGSGTDYTLTISSL QYEDFGIYYCQQYDESPWTFGGGTKLEIK (SEQ ID NO: 1) V L and; Amino acid sequence QIQLVQSGPGLKKPGGSVRISCAASGYTFTNYGMNWVK QAPGKGLRWMGWINTHTGEPTYADDFKGRFTFSLDTSKST AYLQINSLRAEDTATYFCTRRGYDWYFDVWGQGTTVTVSS (SEQ ID NO: 2) H .

[0095] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the aforementioned variable domains. The antibody or antigen-binding fragment thereof competitively inhibits CD5 binding to an antibody or antigen-binding fragment thereof containing the antibody.

[0096] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDRs: nothing: and CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 3); and CDR-H2 having the amino acid sequence NTHTGE (SEQ ID NO: 4); and a CDR-H3 having the amino acid sequence RGYDWYFDV (SEQ ID NO: 5); and CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 6); and a CDR-L2 having the amino acid sequence RANRLVD (SEQ ID NO: 7); CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 8).

[0097] In some embodiments, the antibody or antigen-binding fragment thereof comprises the aforementioned CDRs. It competitively inhibits the binding of CD5 to an antibody or antigen-binding fragment thereof.

[0098] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following variable domains: Contains: Amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQ KPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSL QYEDFGIYYCQQYDESPWTFGGGTKLEIK (SEQ ID NO: 9) V L and; Amino acid sequence EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVR QAPGKGLEWWMGWINTHYGEPTYADSFKGTRTFSLDDSKNT AYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGGTTTVVS V with S (SEQ ID NO: 10) H .

[0099] In some embodiments, the antibody or antigen-binding fragment thereof comprises one or more of the aforementioned variable domains. The antibody or antigen-binding fragment thereof competitively inhibits CD5 binding to an antibody or antigen-binding fragment thereof containing the antibody.

[0100] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDRs: nothing: and CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 11); and CDR-H2 having the amino acid sequence NTHYGE (SEQ ID NO: 12); and a CDR-H3 having the amino acid sequence RRGYDWYFDV (SEQ ID NO: 13); and CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 14); and a CDR-L2 having the amino acid sequence RANRLES (SEQ ID NO: 15); CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 16).

[0101] In some embodiments, the antibody or antigen-binding fragment thereof comprises the aforementioned CDRs. It competitively inhibits the binding of CD5 to an antibody or antigen-binding fragment thereof.

[0102] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDRs: nothing: and CDR-H1 having the amino acid sequence GYSITSGYY (SEQ ID NO: 17); and CDR-H2 having the amino acid sequence ISYSGFT (SEQ ID NO: 18); and a CDR-H3 having the amino acid sequence AGDRTGSWFAY (SEQ ID NO: 19); and CDR-L1 having the amino acid sequence QDISNY (SEQ ID NO: 20); and a CDR-L2 having the amino acid sequence ATS (SEQ ID NO: 21); CDR-L3 having the amino acid sequence LQYASYPFT (SEQ ID NO: 22).

[0103] In some embodiments, the antibody or antigen-binding fragment thereof comprises the aforementioned CDRs. It competitively inhibits the binding of CD5 to an antibody or antigen-binding fragment thereof.

[0104] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDRs: nothing: and CDR-H1 having the amino acid sequence GYIFTNYG (SEQ ID NO: 23); and a CDR-H2 having the amino acid sequence INTYNGEP (SEQ ID NO: 24); and a CDR-H3 having the amino acid sequence ARGDYYGYEDY (SEQ ID NO: 25); and CDR-L1 having the amino acid sequence QGISNY (SEQ ID NO: 26); and a CDR-L2 having the amino acid sequence YTS (SEQ ID NO: 27); CDR-L3 having the amino acid sequence QQYSKLPWT (SEQ ID NO: 28).

[0105] In some embodiments, the antibody or antigen-binding fragment thereof comprises the aforementioned CDRs. It competitively inhibits the binding of CD5 to an antibody or antigen-binding fragment thereof.

[0106] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDRs: nothing: CDR-H1 having the amino acid sequence FSLSTSGMG (SEQ ID NO: 29); and CDR-H2 having the amino acid sequence WWDDD (SEQ ID NO: 30); and a CDR-H3 having the amino acid sequence RRATGTGFDY (SEQ ID NO: 31); and CDR-L1 having the amino acid sequence QDVGTA (SEQ ID NO: 32); and a CDR-L2 having the amino acid sequence WTSTRHT (SEQ ID NO: 33); CDR-L3 having the amino acid sequence YNSYNT (SEQ ID NO: 34).

[0107] In some embodiments, the antibody or antigen-binding fragment thereof comprises the aforementioned CDRs. It competitively inhibits the binding of CD5 to an antibody or antigen-binding fragment thereof.

[0108] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of antibodies, antibodies, and antigen-binding fragments thereof. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and C In some embodiments, the antibody or its antigen comprises a combination of the DR-L3 region. The binding fragments are antibodies or antigens thereof comprising the CDR combinations set forth in Table 1 below. Competitively inhibits CD5 binding to the binding fragment.

[0109] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 257 and a light chain variable region set forth in SEQ ID NO: 258. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of: and a light chain variable region shown in SEQ ID NO: 258, or an antibody or its antigen-binding domain. It competitively inhibits CD5 binding to the antibody fragment.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody. antibody or antigen-binding fragment thereof, polyclonal antibody or antigen-binding fragment thereof a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, fragments, dual variable immunoglobulin domains, scFv, diabodies, triabodies , nanobody, antibody-like protein scaffold, Fv fragment, Fab fragment, F(a b') two molecules, and tandem di-scFv.

[0111] In some embodiments, the antibody is selected from the group consisting of IgG, IgA, IgM, IgD, and IgE. The antibody has an isotype selected from the group consisting of:

[0112] In some embodiments, an antibody conjugated to a cytotoxin, or an antigen-binding fragment thereof, The fragment is represented by the formula Ab-Cy, where Ab is an antibody or an antigen-binding fragment thereof. and Cy is a cytotoxin. In some embodiments, the cytotoxin is an amatoxin, Cy Pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin, maytansin maytansinoids, auristatins, anthracyclines, calicheamicin, irinotecans, Notecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine Azepine dimers, indolinobenzodiazepines, and indolinobenzodiazepine dimers The polypeptide is selected from the group consisting of:

[0113] In some embodiments, the cytotoxin is α-amanitin, β-amanitin, γ-amanitin, tin, ε-amanitin, amanin, amaninamide, amanulin, amanuric acid, and In some embodiments, the compound is an amatoxin, such as proamanulin, or a derivative thereof. The cytotoxin is an amatoxin, and the antibody conjugated to the cytotoxin or its antigen-binding The synthetic fragments are represented by the formula Ab-ZL-Am, where Ab is an antibody or its antigen-binding fragment. fragment, Z is a chemical moiety, Li is a linker, and Am is an amatoxin. In some embodiments, Am-LZ is represented by formula (I): [ka] (wherein R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D and; R4 is H, OH, OR C , OR D , R C , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, OR C , OR D , R C , or R D and; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D and; R9 is H, OH, OR C , or OR D and; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a linker, for example, an optionally substituted alkylene (e.g., C1-C6 alkylene). cyclohexane), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic groups; Alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroaryl dipeptide, -C(=O)-, peptide, or a combination thereof; Z is a reactive group present on L and a CD5+ T cell, CD5+ B cell, or CD Antibodies that bind to CD5, such as those on the surface of 5+ NK cells, or their antigen-binding fragments (These are chemical moieties formed by coupling reactions with reactive substituents present in the hydroxyl group.)

[0114] In some embodiments, Am is exactly one R C Contains substituents.

[0115] In some embodiments, Am-LZ is represented by formula (IA): [ka] (wherein R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D and; R4 is H, OH, OR C , OR D , R C , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, OR C , OR D , R C , or R D and; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D and; R9 is H, OH, OR C , or OR D and; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a linker, for example, an optionally substituted alkylene (e.g., C1-C6 alkylene). cyclohexane), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic groups; Alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroaryl -C(=O)-, a peptide, or a combination thereof; Z is a reactive group present on L and a CD5+ T cell, CD5+ B cell, or CD Antibodies that bind to CD5, such as those on the surface of 5+ NK cells, and their antigen-binding fragments is a chemical moiety formed by a coupling reaction with an existing reactive substituent; where Am is exactly one R C (including substituents).

[0116] In some embodiments, the linker L and chemical moiety Z together form LZ. considered, [ka] is.

[0117] In some embodiments, the LZ is: [ka] is.

[0118] In some embodiments, Am-LZ-Ab is: [ka] is.

[0119] In some embodiments, Am-LZ-Ab is: [ka] is.

[0120] In some embodiments, Am-LZ is represented by formula (IB): [ka] (wherein R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D and; R4 is H, OH, OR C , OR D , RC , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, OR C , OR D , R C , or R D and; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D and; R9 is H, OH, OR C , or OR D and; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a linker, for example, an optionally substituted alkylene (e.g., C1-C6 alkylene). cyclohexane), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic groups; Alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroaryl -C(=O)-, a peptide, or a combination thereof; Z is a reactive group present on L and a CD5+ T cell, CD5+ B cell, or CD Antibodies that bind to CD5, such as those on the surface of 5+ NK cells, or their antigen-binding fragments a chemical moiety formed by a coupling reaction with a reactive substituent present in the where Am is exactly one R C (including substituents).

[0121] In some embodiments, the linker L and chemical moiety Z together form LZ. considered, [ka] is.

[0122] In some embodiments, the LZ is: [ka] is.

[0123] In some embodiments, Am-LZ-Ab is: [ka] is.

[0124] In some embodiments, Am-LZ-Ab is: [ka] is.

[0125] In some embodiments, R A and R B along with the oxygen atoms to which they are attached , combined to form the formula: [ka] (Wherein, Y is —C(═O)—, —C(═S)—, —C(═NR E )-, or -C( R E R E’ )-and; R E and R E’ are each independently an optionally substituted C-C alkylene-R C , optionally substituted C1-C6 heteroalkylene-R C , optionally substituted C2-C6 Alkenylene-R C , optionally substituted C2-C6 heteroalkenylene-R C , arbitrarily placed Substituted C2-C6 alkynylene-R C , optionally substituted C2-C6 heteroalkynyl N-R C , optionally substituted cycloalkylene-R C , optionally substituted heterocycloa Rukiren-R C , optionally substituted arylene-R C or optionally substituted heteroaryl Liren-R C ) to form a 5-membered heterocycloalkyl group.

[0126] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB), and R is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached. [ka] Forming; R3 is H or R C and; R4 is H, OH, OR C , OR D , R C , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, OR C , OR D , R C , or R D and; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , or NHR C and; R9 is H or OH; X, R C and R D are as defined above.

[0127] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached. [ka] Forming; R3 is H or R C and; R4 and R5 each independently represent H, OH, OR C , R C , or OR D and ; R6 and R7 are each H; R8 is OH, NH2, OR C , or NHR C and; R9 is H or OH; R C is as defined above.

[0128] In some embodiments, Am is represented by formula (IA) or formula (IB): R1 is H, OH, or OR A and; R2 is H, OH, or OR B and; R A and R B are bonded together with the oxygen atoms to which they are attached. [ka] Forming; R3, R4, R6, and R7 are each H; R5 is OR C and; R8 is OH or NH2; R9 is H or OH; X and R C is as defined above.

[0129] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 and R2 are each independently H or OH; R3 is R C and; R4, R6, and R7 are each H; R5 is H, OH, or OC1-C6 alkyl; R8 is OH or NH2; R9 is H or OH; X and R C is as defined above.

[0130] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are each independently H, OH, or OR C , or R C and; R8 is OH or NH2; R9 is H or OH; X or R C is as defined above.

[0131] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are each independently H or OH; R8 is OH, NH2, OR C , or NHR C and; R9 is H or OH; A and R C is as defined above.

[0132] In some embodiments, the linker L and chemical moiety Z together form LZ. considered, [ka] is.

[0133] In some embodiments, the LZ is: [ka] is.

[0134] In some embodiments, the Am-LZ precursor is: [ka] and Maleimides react with thiol groups found on cysteines in antibodies.

[0135] In some embodiments, the Am-LZ precursor is: [ka] and Maleimides react with thiol groups found on cysteines in antibodies.

[0136] In some embodiments, Am-LZ is represented by Formula (II), Formula (IIA), or Formula (II B) is represented by: [ka] or [ka] wherein X is S, SO, or SO; R is H or a linker, The reactive substituents present on the linker and the the antibody or a linker covalently linking the antigen-binding fragment to the a reactive group present on the linker and an antibody or its antigen-binding fragment; via chemical moiety Z formed by coupling reaction with reactive substituents present in the methyl group. is a linker covalently attached to the antibody or antigen-binding fragment thereof via when R2 is H, R1 is a linker).

[0137] In some embodiments, the linker is -(CH) 2n -unit, n is an integer between 2 and 6 is.

[0138] In some embodiments, R1 is a linker, R2 is H, and the linker and The learning areas are grouped together as the LZ. [ka] is.

[0139] In some embodiments, Ab-ZL-Am is: [ka] is.

[0140] In some embodiments, Ab-ZL-Am is: [ka] is.

[0141] In some embodiments, the Am-LZ precursor is: [ka] maleimides react with thiol groups found on cysteines in antibodies. do.

[0142] In some embodiments, the cytotoxin is selected from the group consisting of DM1 and DM4. In some embodiments, the cytotoxin is a monomethyl aurista. auristatin selected from the group consisting of monomethylauristatin E and monomethylauristatin F. In some embodiments, the cytotoxin is daunorubicin, doxorubicin, epirubicin, or the like. The anthracycline is selected from the group consisting of cyclosporine, cyclosporine, and idarubicin.

[0143] In some embodiments, the cytotoxin has formula (IV): [ka] It is a pyrrolobenzodiazepine dimer represented by the formula:

[0144] In some embodiments, the cytotoxin is attached to the antibody via a maleimidocaproyl linker. or an antigen-binding fragment thereof.

[0145] In some embodiments, the cytotoxin is monomethyl auristatin E and monomethyl The auristatin is selected from the group consisting of auristatin F.

[0146] In some embodiments, the cytotoxin is daunorubicin, doxorubicin, epirubicin, The anthracycline is selected from the group consisting of idarubicin, idarubicin, and idarubicin.

[0147] In another aspect, the invention provides an antibody of any above aspect or embodiment of the invention, or a The present invention features a pharmaceutical composition comprising an antigen-binding fragment and a pharmaceutically acceptable excipient.

[0148] In some embodiments, the pharmaceutical composition is administered transdermally, subcutaneously, intranasally, intravenously, or intravenously to a human patient. Formulated for intramuscular, intraocular, intratumoral, parenteral, topical, intrathecal or intracerebroventricular administration. [Brief explanation of the drawings]

[0149] [Figure 1]

[0023] Figure 1 graphically depicts the results of an in vitro cell line binding assay in which MOLT-4 cells (i.e., a human T lymphoblastoid cell line) were incubated with the indicated anti-CD5 antibodies or a negative control (i.e., mIgG1), followed by incubation with a fluorochrome-labeled anti-IgG antibody. Signal was detected by flow cytometry and is shown as geometric mean fluorescence intensity (y-axis) as a function of anti-CD5 antibody concentration (x-axis). [Figure 2] Figure 1 graphically depicts the results of an in vitro primary cell binding assay in which human primary T cells were incubated with the indicated anti-CD5 antibodies or a negative control (i.e., hIgG1), followed by incubation with a fluorochrome-labeled anti-IgG antibody. Signals were detected by flow cytometry and are shown as geometric mean fluorescence intensity (y-axis) as a function of anti-CD5 antibody concentration (x-axis). [Figure 3A] Figure 3A graphically depicts the results of an in vitro T cell killing assay involving an anti-CD5-amanitin ADC (i.e., 5D7-AM or "CD5 AM") with an interchain-conjugated amanitin having an average drug-antibody ratio (DAR) of 6. In Figure 3A, anti-CD5-ADC T cell killing analysis is shown compared to unconjugated anti-CD5 5D7 antibody (i.e., "CD5 Naked"). Results show the number of viable T cells (y-axis) as a function of ADC (CD5 5D7 AM) or unconjugated antibody (CD5 5D7) concentration (x-axis), as assessed using flow cytometry. [Figure 3B]Figure 3B graphically depicts the results of an in vitro T cell killing assay involving an anti-CD5-amanitin ADC (i.e., 5D7-AM or "CD5 AM") with a site-specifically conjugated amanitin having a DAR of 2. In Figure 3B, the results for the anti-CD5 antibody are shown in comparison to an anti-CD5 5D7 antibody (i.e., "CD5 Fast 1 / 2 Life AM") with an H435A mutation that shortens the half-life of the antibody. The results show the number of viable T cells (y-axis) as a function of ADC (CD5 5D7 AM, CD5 5D7 D265C.H435A AM) concentration (x-axis) assessed using flow cytometry. [Figure 4A] Figure 4A graphically depicts 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 of humanized NSG mice 7 days after a single dose of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of an anti-CD5 5D7-amanitin ADC (i.e., CD5 5D7-AM) with an interchain DAR of 6. For comparison, Figure 4A also shows the level of T cell depletion after treatment of humanized NSG mice with the indicated controls (i.e., 25 mg / kg anti-CD52 antibody; 3 mg / kg hIgG1-amanitin ADC (i.e., hIgG1-AM), 25 mg / kg hIgG1, or PBS). [Figure 4B] Figure 4B graphically depicts the results of an in vivo T cell depletion assay showing the absolute levels of T cells (CD3+ cells; y-axis) in the bone marrow of humanized NSG mice 7 days after a single dose of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of an anti-CD5 5D7-amanitin ADC (i.e., CD5 5D7-AM) with an interchain DAR of 6. For comparison, Figure 4B also shows the level of T cell depletion after treatment of humanized NSG mice with the indicated controls (i.e., 25 mg / kg anti-CD52 antibody; 3 mg / kg hIgG1-amanitin ADC (i.e., hIgG1-AM), 25 mg / kg hIgG1, or PBS). [Figure 5A]Figure 5A graphically depicts 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 of humanized NSG mice 7 days after a single dose of 1 mg / kg or 3 mg / kg of an anti-CD5 5D7 amanitin ADC (i.e., 5D7-AM) with a site-specific DAR of 2. For comparison, Figure 5A also shows the level of T cell depletion after treatment of humanized NSG mice with 3 mg / kg of unconjugated anti-CD5 antibody or the indicated control (i.e., 3 mg / kg of hIgG1-amanitin-ADC ("hIgG1-AM") or PBS). [Figure 5B] FIG. 5B graphically depicts the results of an in vivo T cell depletion assay showing the absolute levels of T cells (CD3+ cells; y-axis) in the bone marrow of humanized NSG mice 7 days after a single dose of 1 mg / kg or 3 mg / kg of an anti-CD5 5D7 amanitin ADC (i.e., 5D7-AM) with a site-specific DAR of 2. For comparison, FIG. 5B also shows the level of T cell depletion after treatment of humanized NSG mice with 3 mg / kg of unconjugated anti-CD5 antibody or the indicated control (i.e., 3 mg / kg of hIgG1-amanitin-ADC ("hIgG1-AM") or PBS). [Figure 5C] FIG. 5C graphically depicts the results of an in vivo T cell depletion assay showing the absolute levels of T cells (CD3+ cells; y-axis) in the thymus of humanized NSG mice 7 days after a single dose of 1 mg / kg or 3 mg / kg of an anti-CD5 5D7 amanitin ADC (i.e., 5D7-AM) with a site-specific DAR of 2. For comparison, FIG. 5C also shows the level of T cell depletion after treatment of humanized NSG mice with 3 mg / kg of unconjugated anti-CD5 antibody or the indicated control (i.e., 3 mg / kg of hIgG1-amanitin-ADC ("hIgG1-AM") or PBS). DETAILED DESCRIPTION OF THE INVENTION

[0150] The present invention relates to antibodies that bind to CD5 (also known as lymphocyte antigen T1 / Leu-1), or or antigen-binding fragments thereof as therapeutic agents, characterized in (i) CD5+ cells; (ii) non-autologous hematopoietic stem cell transplantation, which can directly treat cancer and autoimmune diseases Transplant treatment by depleting immune cell populations that cross-react with the transplant and initiate an immune response. It is believed that this method can promote the engraftment of transplanted hematopoietic stem cells in patients in need. These therapeutic activities are based in part on the discovery that, for example, anti-CD5 antibodies, or their anti-CD5 counterparts, The antigen-binding fragment cross-reacts with non-self hematopoietic stem cell antigens, including cancer cells, autoimmune cells, and or by binding to CD5 expressed on the surface of cells such as immune cells. This can occur by inducing death, such as by depleting populations of cancer cells or autoimmune cells. Anti-CD5 antibodies, or antigen-binding fragments thereof, can be used to treat cancer autologous tumors as described herein. Immunologic diseases such as cancer or autoimmune diseases can be directly treated. Cross-react with antigens (e.g., non-self MHC antigens expressed by hematopoietic stem cell grafts) To deplete immune cell populations, use anti-CD5 antibodies, or antigen-binding fragments thereof. patients with stem cell disease, cancer, or autoimmune disease who have undergone hematopoietic stem cell transplantation treatment. This can prevent or reduce the likelihood of graft rejection in patients receiving In such cases, one or more non-self hematopoietic stem cell antigens (e.g., one or more non-self MHC antigens) ) by depleting CD5+ immune cells that cross-react with the transplanted hematopoietic stem cells. Once the transplanted cells have engrafted, they can be used to create The transplanted hematopoietic stem cells can home to the blood tissue where they can undergo productive hematopoiesis. The cells then become megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, and neutrophils. leukocytes, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells cells, natural killer cells, T lymphocytes, and B lymphocytes in transplant recipients. Thus, anti-CD5 The antibodies, or fragments thereof, may be used to treat patients suffering from a stem cell disorder as described herein. Successful engraftment of hematopoietic stem cells in a patient, such as a human patient, can be promoted.

[0151] (definition) As used herein, the term "about" refers to a value within 10% above or below the stated value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

[0152] As used herein, the term "amatoxin" refers to a compound produced by Amanita phalloides. Members of the amatoxin family of peptides, synthetic amatoxins, mutant amatoxins or derivatives thereof, e.g., mutations capable of inhibiting RNA polymerase II activity It also includes synthetic amatoxins (e.g., see the references herein). (See U.S. Patent No. 9,676,702, incorporated herein by reference.) As used herein, The amatoxin can be linked to an antibody, or its antigen-binding fragment, for example, via a linker moiety (L). The antibody may be conjugated to a drug fragment (hence, a conjugate (antibody drug conjugate) Amatoxin compounds useful in such processes are also known as anti-cancer drugs (ADCs). Exemplary methods of conjugation and linkers are described below. Exemplary linkers useful for conjugating to antibodies or antigen-binding fragments according to Containing amatoxins are also described herein.

[0153] In certain embodiments, amatoxins useful in combination with the compositions and methods described herein are Amanitin includes α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, and amanitin. , amaninamide, amanulin, amanuric acid, and proamanulin, etc., The compound of formula (III) is as follows: [ka] (wherein R1 is H, OH, or OR A and; R2 is H, OH, or OR B and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D and; R4 is H, OH, OR D , or R D and; R5 is H, OH, OR D , or R D and; R6 is H, OH, OR D , or R D and; R7 is H, OH, OR D , or R D and; R8 is OH, NH2, or OR D and; R9 is H, OH, or OR D and; X is -S-, -S(O)-, or -SO2-; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0154] For example, in one embodiment, amatoxin useful in conjunction with the compositions and methods described herein Xines include compounds of formula (IIIA): [ka] (wherein R1 is H, OH, or OR A and; R2 is H, OH, or OR B and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D and; R4 is H, OH, OR D , or R D and; R5 is H, OH, OR D , or R D and; R6 is H, OH, OR D , or R D and; R7 is H, OH, OR D , or R D and; R8 is OH, NH2, or OR D and; R9 is H, OH, or OR D and; X is -S-, -S(O)-, or -SO2-; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0155] In one embodiment, amatoxins useful in combination with the compositions and methods described herein include Also included are compounds of formula (IIIB): [ka] (wherein R1 is H, OH, or OR A and; R2 is H, OH, or OR B and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D and; R4 is H, OH, OR D , or R D and; R5 is H, OH, OR D , or R D and; R6 is H, OH, OR D , or R D and; R7 is H, OH, OR D , or R D and; R8 is OH, NH2, or OR D and; R9 is H, OH, or OR D and; X is -S-, -S(O)-, or -SO2-; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0156] As described herein, amatoxins can be attached to antibodies, e.g., via a linker moiety, or can be conjugated to an antigen-binding fragment thereof. Examples of amatoxin conjugates Exemplary methods and linkers useful in such processes are described in "Linkers for Chemical Conjugation." The compositions and compositions described herein are described in the section entitled "Binders" and in Table 1 below. Exemplary linkers useful for conjugating to antibodies, antigen-binding fragments according to the method The amatoxins contained therein are those having the structural formulas (I), (IA), (IB), and (II) listed herein. , (IIA), and (IIB).

[0157] As used herein, the term "antibody" refers to an antibody that specifically binds to a particular antigen or binds to a particular An antibody is an immunoglobulin molecule that is immunologically reactive with an antigen. Examples of antibodies include chimeric antibodies. antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi-, tri-, and tetra-specific antibodies) , diabodies, triabodies, and tetrabodies) Polyclonal antibodies, monoclonal antibodies, genetically engineered antibodies, and other Modified forms of antibodies, as well as, for example, Fab', F(ab')2, Fab, Fv, rlg G, and antigen-binding fragments of antibodies, including scFv fragments. As used herein, Fab and F(ab')2 fragments refer to intact These antibody fragments lack the Fc fragment of the antibody. Examples are provided herein.

[0158] Generally, antibodies comprise a heavy chain containing an antigen-binding region and a light chain. Each heavy chain contains a heavy chain variable region. The heavy chain constant region is composed of a heavy chain constant region (herein abbreviated as HCVR or VH). Each light chain consists of three domains: CH1, CH2, and CH3. The light chain constant region (LCVR or VL) is composed of a light chain constant region. The VH and VL regions consist of one domain, CL. The VH and VL regions further consist of framework regions Complementarity-determining regions (CDRs) interspersed with more highly conserved regions called FRs Each VH and VL can be subdivided into three CDRs and four hypervariable regions called It consists of two FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Heavy chain and The variable region of the light chain contains a binding domain that interacts with an antigen. The constant region of the antibody is responsible for the immune system various cells (e.g., effector cells) and the first component of the classical complement system (Clq) It can mediate the binding of immunoglobulins to host tissues or factors, including

[0159] As used herein, the term "antigen-binding fragment" refers to a portion of an intact antibody. It refers to a molecule other than an intact antibody that contains and binds to the antigen to which the intact antibody binds. The body's antigen-binding function can be performed by fragments of a full-length antibody. Examples of the antibody include Fv, Fab, Fab', F(ab')2, scFv, diabody, monoclonal antibody, and the like. Single-chain antibody molecules (e.g., scFv), triabodies, affibodies, nanobodies, aptamers, The term "antigen-binding fragment" of an antibody may be a mer, or a domain antibody. Examples of binding fragments that may be included include, but are not limited to: (i) Fab fragment, V L , V H , C L and a CH1 domain (ii) F(ab')2 fragment, disulfide bridged in the hinge region (iii) a bivalent fragment comprising two Fab fragments linked by V H Domain and C H (iv) a single-arm Fd fragment of an antibody; V L and V H Fv fragment consisting of domains; (v) V H Domains and V L Domestic (vi) V H A dAb fragment consisting of the domain (e.g., Wa rd et al., Nature 341:544-546, 1989); (v ii) V H or V L (viii) isolated complementarity-determining regions; (CDR); and (ix) two or more (e.g., and combinations of isolated CDRs (e.g., two, three, four, five, or six). The two domains of the Fv fragment, V L and V H are encoded by separate genes However, they have been developed using recombinant methods. L and V H The regions are paired to form a monovalent molecule The linker allows them to be made as a single protein chain forming a (known as single-chain Fv (scFv); see, e.g., Bird et al. al., Science 242:423-426, 1988 and Huston e t al.,Proc.Natl.Acad.Sci.USA 85:5879-588 3, 1988). These antibody fragments can be produced using conventional techniques known to those skilled in the art. The fragments can be used in the same manner as intact antibodies for utility. Antigen-binding fragments can be screened using recombinant DNA techniques, by enzymatic or chemical cleavage of tactic immunoglobulins, or in certain cases, It can be produced by chemical peptide synthesis procedures known in the art.

[0160] As used herein, the term "anti-CD5 antibody" or "antibody that binds to CD5" refers to This refers to an antibody that specifically binds to CD5. An antibody that "binds" to the antigen of interest, i.e., CD5. The antibody binds to the antigen with sufficient affinity so that it is useful for targeting cells that express the antigen. In a preferred embodiment, the antibody is human CD5 (hCD5). CD5), and its amino acid sequence is set forth in SEQ ID NO:261.

[0161] As used herein, the term "bispecific antibody" refers to an antibody having two different antigen-binding sites. A bispecific antibody is a type of multispecific antibody, and refers to a hybrid antibody that Various methods, including but not limited to, fusion of plasmids or linking of Fab' fragments, are also possible. For example, Songsivilai and Lachma nn,1990,Clin.Exp.Immunol.79:315-321;Kost elny et al.,1992,J.Immunol.148:1547-1553 The two binding sites of a bispecific antibody may be on the same or different protein targets. For example, one of the binding specificities can be determined by the CD 5, and the other can be directed against another T cell surface antigen, or Receptors or receptor subunits involved in signal transduction pathways that enhance cell proliferation, among others. The antibody can be directed to another cell surface protein, such as a ribozyme.

[0162] As used herein, the term "complementarity determining region" (CDR) refers to the light and heavy chains of an antibody. The more conserved parts of the variable domains are referred to as hypervariable regions found in both the nucleotide and nucleotide chains. The amino acids that delineate the hypervariable regions of an antibody are called framework regions (FR). The position may vary depending on the context and various definitions known in the art. Some positions within the domain are within hypervariable regions under certain criteria. Although some sequences can be considered to be within the hypervariable region, others are considered to be outside the hypervariable region under a different set of criteria. These positions can be considered hybrid hypervariable positions in that they are both nucleotides. The above may also be present in the extended hypervariable region. The antibodies described herein may contain these hybrids. The variable domains of naturally occurring heavy and light chains, respectively, may contain modifications at hypervariable positions. It contains four framework regions that form a sheet, connected by three CDRs. These form loops that connect the β-sheet structure and in some cases The CDRs in each chain form part of the FR1-CDR1-FR2-CDR2-FR3 - in the order CDR3-FR4, held together in close proximity by framework regions, Together with the CDRs from other antibody chains, they contribute to the formation of the target binding site of the antibody (Kabat et al. t al.,Sequences of Proteins of Immunolog ical Interest,National Institute of Heal (See, e.g., Bethesda, MD., 1987). As used herein, immunoglobulin The numbering of immunoglobulin amino acid residues follows that of Kabat et al., unless otherwise indicated. This is done according to the phosphorus amino acid residue numbering system.

[0163] As used herein, the terms "conditioning" and "conditioning "To prepare" refers to the process of preparing a patient to receive a graft containing hematopoietic stem cells. Such procedures promote hematopoietic stem cell transplant engraftment (e.g., conditioning procedures and Persistence of viable hematopoietic stem cell abundance in blood samples isolated from patients following hematopoietic stem cell transplantation In the methods described herein, antigens expressed by T cells, For example, an antibody or antigen-binding fragment thereof capable of binding to CD5 or the like is administered to a patient. By administering this medicine, patients can be conditioned for hematopoietic stem cell transplantation. As described herein, the anti-CD5 antibody can be used as an antibody-drug conjugate (AD C). capable of binding to one or more of the aforementioned antigens to a patient in need of cell transplant therapy. Administration of the antibody, antigen-binding fragment thereof, or antibody-drug conjugate can be, for example, One or more non-self antigens expressed by hematopoietic stem cells, e.g., by hematopoietic stem cell grafts CD5+ T cells (e.g., CD4 + and / or CD8+ T cells), CD5+ B cells, and / or CD5+ NK cells Selective depletion of endogenous immune cells, such as blastocysts, promotes hematopoietic stem cell graft survival. This selective depletion of immune cells can then be promoted by the administration of exogenous (e.g., autologous, allogeneic) To prevent or reduce the likelihood of graft rejection after transplantation of hematopoietic stem cell grafts (i.e., allogeneic or syngeneic) Fall.

[0164] As used herein, the term "conjugate" refers to a compound comprising an antibody or its antigen-binding fragment. The reactive functional group of one molecule, such as a cytotoxin, can be linked to the reactive functional group of another molecule, such as a cytotoxin described herein. A conjugate refers to a compound formed by chemical bonding with an appropriate reactive functional group. A linker may be included between the two molecules (e.g., an anti-CD5 antibody and a cytotoxin) that are linked to each other. Examples of linkers that can be used to form conjugates include natural amino acids or Peptides containing phosphorus, including those containing non-naturally occurring amino acids such as D-amino acids, Linkers include various linkers described herein and known in the art. Depending on the reactive moiety therein, the linker can be prepared using various strategies, e.g. For example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, acid can be cleaved by oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (e.g., Leriche et al.,Bioorg.Med.Chem.,20:571-5 82, 2012).

[0165] As used herein, the term "coupling reaction" refers to the process of combining two suitable compounds to react with each other. Two or more substituents react to form molecular fragments (e.g., covalent bonds) attached to each substituent. Coupling reactions involve the formation of chemical moieties that are bonded together. fragments of cytotoxins, such as those known in the art or described herein; The reactive substituent attached to the antibody may be an antibody, an antigen-binding fragment thereof, or an antibody, e.g., an antibody an antibody, an antigen-binding fragment thereof, or a polypeptide known in the art or described herein. Reacts with appropriately reactive substituents attached to fragments of antibodies specific for CD5 Examples of suitably reactive substituents include nucleophilic / electrophilic pairs (e.g., Thiol / haloalkyl pair, amine / carbonyl pair, or thiol / α,β-unsaturated carbonyl pairs), diene / dienophile pairs (e.g., azide / alkyne pairs, among others). Coupling reactions include, but are not limited to, thiol alkylation, hydrolysis, etc. Alkyl alkylation, amine alkylation, amine condensation, amidation, esterification, disulfide amide formation, cycloadditions (e.g., [4+2] Diels-Alder cycloadditions, [3+ 2] Huisgen cycloaddition), nucleophilic aromatic substitution, electrophilic aromatic substitution, and Other reaction modes known or described herein are included.

[0166] As used herein, a "CRU (competitive retransfer unit)" refers to a cell that is transferred to a cell after in vivo transplantation. It refers to a measure of long-term engrafting stem cells that can be detected in vitro.

[0167] As used herein, "drug-to-antibody ratio" or "DAR" refers to the ratio of the antibody-to-antibody ratio of an ADC to the drug-to-antibody ratio. The DAR of an ADC ranges from 1 to 8. However, higher loadings are possible depending on the number of linking sites on the antibody. In some embodiments, the ADCs described herein have a nucleotide sequence of about 1, 2, 3, 4, 5, 6, 7, or 8 It has a DAR of

[0168] As used herein, the term "donor" refers to a donor or donors from which one or more cells are derived. refers to a human or animal from which the progeny of one or more cells are isolated prior to administration to a recipient. can be, for example, a hematopoietic stem cell population.

[0169] As used herein, the term "diabody" refers to a molecule containing two polypeptide chains. A bivalent antibody, in which each polypeptide chain is connected to a V on the same peptide chain. H Domains and V L domain A linker that is too short (e.g., consisting of five amino acids) to allow intramolecular association of V linked by a linker H and VL This configuration contains a homo domain. Each domain pairs with a complementary domain on another polypeptide chain to form a dimeric structure. Thus, the term "triabody" refers to a trivalent antibody containing three peptide chains. Each of these is a V within the same peptide chain. H Domains and V L Enables intramolecular association of domains Linkers that are too short to be used (e.g., linkers consisting of 1-2 amino acids) ) connected by one V H Domain and One V L domains and their natural To fold into a structure, peptides constructed in this way typically require adjacent peptides. Chain V H and V L Trimerize domains to place them in close spatial proximity to each other (e.g., Holliger et al.,Proc.Natl.Acad.Sci.USA 90:6444-48,1993).

[0170] As used herein, a "dual variable domain immunoglobulin" ("DVD-Ig") The target-binding variable domains of two antibodies are combined via a linker to form a tetravalent, dual-targeting single antibody. It refers to an antigen-binding protein that produces an agent (see, e.g., Gu et al., Meth. En zymol.,502:25-41,2012).

[0171] As used herein, the term "endogenous" refers to a gene that is present in a human patient, e.g., a cell line described herein. The molecules found naturally in certain organisms, such as human patients undergoing hematopoietic stem cell transplantation therapy, a substance such as a cell, tissue, or organ (e.g., a hematopoietic stem cell or cell of the hematopoietic system, e.g., megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, Gliocytes, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes (e.g., CD4+ or CD8+ T lymphocytes), or B lymphocytes).

[0172] As used herein, the term "engraftment potential" refers to the ability of hematopoietic stem and progenitor cells to repopulate tissues. It refers to the ability of such cells to circulate naturally or be supplied by transplantation. This term refers to the tissue homing of cells and their proliferation within the tissue of interest. It encompasses all events surrounding engraftment, including cell colonization. Efficiency or engraftment rate can be measured using any clinically accepted parameter known to those skilled in the art. can be assessed or quantified using, for example, assessment of competitive repopulation units (CRU); Markers of stem cell homing, colonization, or engraftment in one or more tissues or disease progression, hematopoietic stem and progenitor cell proliferation, Engraftment is assessed by assessing the progression of the subject based on cell survival and recipient survival. Engraftment can also be assessed by measuring the number of white blood cells in the peripheral blood during bone marrow transplantation. It can also be assessed by measuring bone marrow cell recovery with donor cells in aspirate samples. can.

[0173] As used herein, the term "excipient" refers to a substance that is formulated with the active ingredient of a drug. They are used, for example, for the purpose of long-term stabilization or to provide a therapeutic benefit to the active ingredient in the final dosage form. It may be included to provide enhancement.

[0174] As used herein, the term "exogenous" refers to a substance that is not naturally found in a particular organism, such as a human patient. molecules, cells, tissues, or organs (e.g., T cells, hematopoietic stem cells, or Hematopoietic cells, such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, and basophils , neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages , dendritic cells, natural killer cells, T lymphocytes, or B lymphocytes). Substances include those supplied to the organism from an external source or cultures extracted therefrom. Examples include:

[0175] As used herein, the term "framework region" or "FW region" refers to a region of an antibody or the amino acid residues adjacent to the CDRs of an antigen-binding fragment thereof. can be, for example, human antibodies, humanized antibodies, monoclonal antibodies, antibody fragments, among others. , Fab fragment, single-chain antibody fragment, scFv fragment, antibody domain , and bispecific antibodies.

[0176] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein. These antibodies are used for their anti-cancer activity and generally contain at least two full-length heavy chains and two full-length light chains. refers to antibodies naturally occurring in camelids, which in some cases may contain only heavy chains It may contain fewer chains, such as

[0177] As used herein, the term "hematopoietic stem cell" ("HSC") refers to a hematopoietic stem cell that has the capacity for self-renewal. , which have the ability to differentiate into mature blood cells comprising a variety of lineages, including, but not limited to: Immature blood cells: granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets) , monocytic cells (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and phospholipids. lymphocytes (e.g., NK cells, B cells, and T cells). Such cells contain CD34 + cell CD34 may be mentioned. + The cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells comprise a subpopulation of cells with the stem cell properties defined above. In mice, HSCs are CD34-. Furthermore, HSCs are thought to be long-term Also referred to as reimplanted HSCs (LT-HSCs) and short-term reimplanted HSCs (ST-HSCs). HSCs and ST-HSCs are distinguished based on functional capacity and cell surface marker expression. For example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (CD5, CD3, CD4, CD7, CD8, CD10, Mature lineage markers, including CD11B, CD19, CD20, CD56, and CD235A In mice, bone marrow LT-HSCs express CD34-, SCA-1 +, C-kit+, CD135-, Slamfl / CD150+, CD48-, and l in-(Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and Negative for mature lineage markers, including IL-7ra and IL-7ra, whereas ST-HSCs CD34+, SCA-1+, C-kit+, CD135-, Slamfl / CD150+ , and lin-(Ter119, CD11b, Gr1, CD3, CD4, CD8, B2 20, and IL7ra). -HSCs are less inactive and more proliferative than LT-HSCs under homeostatic conditions. However, LT-HSCs have a greater self-renewal capacity (i.e., they mature (survive through the first stage and can be serially transplanted through successive recipients), whereas ST- HSCs have limited self-renewal (i.e., they survive for only a limited period of time and require continuous Any of these HSCs may be used in the methods described herein. ST-HSCs are highly proliferative and therefore differentiate more rapidly. They are particularly useful because they can give rise to offspring.

[0178] As used herein, the term "hematopoietic stem cell functional capacity" refers to the functional capacity of hematopoietic stem cells, including: Functional characteristics include: 1) multipotency (granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils) ), red blood cells (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryocytes, platelet-producing megakaryocytes , platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells), 2) self-renewal (referring to the ability of hematopoietic stem cells to differentiate into multiple different blood lineages without the need for stem cells) This refers to the ability of a cell to produce daughter cells with the same capabilities as the original cell, and this ability is not depleted. can occur repeatedly throughout an individual's life without any disruption), and 3) hematopoietic stem cells or The progeny are then reintroduced into the transplant recipient, where they home to the hematopoietic stem cell niche. The ability to stimulate hematopoiesis and restore productive and sustained hematopoiesis.

[0179] As used herein, the term "major histocompatibility complex antigen" ("MHC", in humans) refers to Human leukocyte antigens (HLA), also known as human leukocyte antigens (HLA), give cells their unique antigenic identity. MHC / HLA antigens are proteins expressed on the cell surface that confer immune effect. They may be derived from the same hematopoietic stem cell source as the culprit cells ("autologous") or from another hematopoietic reconstitution Recognized by T cells and NK cells as derived from an adult cell source ("non-self"). Two major classes of HLA antigens are recognized: HLA class I and HLA Class II. HLA class I antigens (A, B, and C in humans) distinguish each cell from its “self.” HLA class II antigens (DR, DP, and D in humans) are recognized as "self" by the immune system. Q) is involved in the reaction between lymphocytes and antigen-presenting cells. Both are involved in the rejection of transplanted organs. An important aspect of the HLA gene system is its polymorphism. MHC class I (A, B, and C) and MHC class II (DP, DQ, and DR) , present in different alleles. HLA alleles are designated by a number and a subscript. For example, two unrelated individuals share class I HLA-B, genes B5, and Bw 41. Allelic products may possess the α and / or β domains (one or more ) differ by one or more amino acids. Using the HLA haplotype of an individual, the leukocytes expressing class I and class II molecules are identified. HLA typing is performed using a ball. Six genes are commonly used for HLA typing. MHC class I and class II proteins of HLA-A; HLA-B and H There are two alleles for each of the HLA and DR genes. They are clustered in a "super locus" present in p21, which is a subset of six classical Transplanted HLA genes and the regulation of the immune system and several other fundamental molecular and cellular processes At least 132 proteins encoding genes that play important roles in the process The complete locus is approximately 3.6 Mb and contains at least 224 genes. One effect of this clustering is to create "haplotypes," i.e., The set of alleles present on a single chromosome inherited from a single parent is collectively inherited. The set of alleles inherited from each parent is called the haplotype. form a prototype, in which several alleles tend to be associated together. Some alleles and haplotypes are more frequent than others and differ Because of the different frequencies distributed among racial and ethnic groups, patients' haplotypes Identifying donors helps predict the probability of finding a suitable donor and develops search strategies. tsu.

[0180] As used herein, the term "HLA-matched" refers to a patient who is HLA-matched and does not require hematopoietic stem cell transplantation therapy. between donors and recipients, such as donors who provide hematopoietic stem cell grafts to recipients who This refers to a donor-recipient pair in which none of the HLA antigens are incompatible. A donor-recipient pair (i.e., all six alleles are matched) Induced T cells and NK cells are less likely to recognize the incoming graft as foreign, Therefore, they are less likely to mount an immune response against the graft, reducing the risk of graft rejection. The price decreases.

[0181] As used herein, the term "HLA incompatible" refers to a patient who is in need of hematopoietic stem cell transplantation therapy. donors who provide hematopoietic stem cell grafts to recipients who are Among them, at least one HLA-A, HLA-B, and HLA-DR are specifically involved. Refers to a donor-recipient pair that is mismatched for LA antigens. One haplotype is matched and the other is mismatched. In this case, endogenous T cells and NK cells may recognize the incoming graft as foreign. and therefore the potential for T cells and NK cells to mount an immune response against the graft. Therefore, HLA-mismatched donor-recipient pairs are more likely to be mismatched than HLA-matched donor-recipient pairs. There is a higher risk of graft rejection compared to control patients.

[0182] As used herein, the term "human antibody" refers to an antibody in which substantially all portions of the protein (e.g., For example, all CDRs, framework regions, and C L , C H Domain (e.g., C H 1. C H 2. C H 3), hinges, and V L and V H domain) is substantially Human antibodies are non-immunogenic and contain only minor sequence changes or mutations. , in human cells (e.g., by recombinant expression) or functionally rearranged human A non-human immunoglobulin (e.g., heavy and / or light chain) gene expressing Human antibodies can be produced by human animals or by prokaryotic or eukaryotic cells. If it is an antibody, it may contain a linker peptide not found in naturally occurring human antibodies. For example, Fv has 2 to about 8 glycine residues linking the heavy chain variable region and the light chain variable region, or The linker peptide may include other amino acid residues, such as Human antibodies are considered to be of human origin. Human antibodies are antibody libraries derived from human immunoglobulin sequences. Various methods known in the art, including phage display methods using libraries, have been proposed. Human antibodies can be produced by methods that do not express functional endogenous immunoglobulins. Transgenic mice that cannot express human immunoglobulin genes These can also be produced using a variety of methods (e.g., PCT Publication No. WO1998 / 24893; WO1992 / 01047;WO1996 / 34096;WO1996 / 33735;rice National Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569, 825;5,661,016;5,545,806;5,814,318;5,885, 793; 5,916,771; and 5,939,598). In human antibodies, the glycosylation pattern of the antibody has the same sequence as when it occurs in nature. These antibodies are produced using recombinant methods that are different from the antibodies that have been developed.

[0183] As used herein, the term "humanized" antibody refers to an antibody that has non-human CDRs and a human framework. In one embodiment, a humanized antibody refers to a chimeric antibody, which typically contains amino acid sequences derived from the amino acid sequence of the target protein. The CDR-derived residues of the recipient may be selected to provide the desired specificity, affinity, and / or capacity. of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate A human antibody (recipient antibody) is a human antibody in which the residues derived from the DR have been substituted. The antibody contains substantially all of at least one, and typically two, variable domains, all or substantially all of the CDR regions of a non-human immunoglobulin correspond to those of a non-human immunoglobulin Even if all or substantially all of the FW regions are from human immunoglobulin sequences, Humanized antibodies contain at least a portion of an immunoglobulin constant region (Fc), typically a human The antibody may also include those of a human immunoglobulin consensus sequence. Methods for antibody humanization are known in the art. For example, Riechmann et al., Nature 332: 323-327,1988;U.S. Patent Nos. 5,530,101;5,585,089;5, 693,761; 5,693,762; and 6,180,370. There are.

[0184] As used herein, the term "immune cell" refers to a cell that is involved in the development of an innate or adaptive immune response. These refer to cells of the immune system involved in the initiation and maintenance of immune cells. They contain receptors that specifically bind to antigens of interest, such as autoantigens, and initiate an immune response against them. Exemplary immune cells include mast cells, basophils, neutrophils, eosinophils, Microglia, granulocytes, monocytes, antigen-presenting cells, macrophages, dendritic cells, natural killer cells These include lymphocytes, T lymphocytes, and B lymphocytes.

[0185] As used herein, a patient "in need" of a hematopoietic stem cell transplant includes one or more This includes patients who exhibit defects or deficiencies in certain blood cell types, as well as patients with stem cell disorders. Blood stem cells generally exhibit 1) multipotency and therefore can give rise to granulocytes (e.g., promyelocytes, neutrophils, etc.); erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryocytes, cytoplasmic cells (e.g., platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, Contains microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells) 2) can differentiate into multiple different blood lineages, including but not limited to: and thus can produce daughter cells with abilities equivalent to those of the mother cell; 3) demonstrate the ability to be reintroduced into the transplant recipient, where they host the hematopoietic stem cell niche; This restores productive and sustained hematopoiesis, thus reducing the risk of deficiency or To reconstitute cell populations in vivo, hematopoietic stem cells are induced to differentiate into one or more cell types of the hematopoietic system. The antibody can be administered to a patient with a defective or deficient cell type. For example, the patient may have cancer. The deficiency may be due to a chemical reaction that selectively or nonspecifically depletes the cancer cell population. This may be caused by the administration of chemotherapy or other drugs. cytic anemia, thalassemia, Fanconi anemia, and Wiskott-Aldrich syndrome Non-malignant abnormal hemoglobinuria, which can cause a defect or deficiency of one or more blood cell types, such as The subject may have adenosine deaminase severe combined immunodeficiency syndrome (ADNS). All diseases (ADA SCID), HIV / AIDS, Metachromatic Leukodystrophy, Diamond Patients with de Blackfan anemia and Shwachman-Diamond syndrome The subject may be a patient suffering from an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune Suffering from or susceptible to the effects of a disease. Subjects will have a blood cancer (e.g., leukemia, lymphoma, multiple myeloma, or myelodysplastic syndrome) ) and neuroblastoma, or In some embodiments, the subject is diagnosed with a metabolic disorder. For example, the subject may have or be otherwise affected by: or may be affected by: glycogen storage disease, mucositis Polysaccharidosis, Gaucher disease, Hurler disease, sphingolipidosis, metachromatic leukodystrophy or a metabolic disorder selected from the group consisting of, but not limited to, Any other illnesses or diseases that may benefit from the treatments and therapies disclosed herein: Severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM ) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, septicemia Thalassemia, thalassemia major, sickle cell anemia, systemic sclerosis, systemic lupus erythematosus, Multiple sclerosis, juvenile rheumatoid arthritis, and "Bone Marrow Transplant" antation for Non-Malignant Disease,” ASH Diseases listed in Education Book, 1:319-338(2000) The disclosure relates to diseases or conditions that can be treated by administering hematopoietic stem cell transplantation therapy. The disclosure of which is incorporated herein by reference in its entirety. Alternatively, a patient "in need" of a hematopoietic stem cell transplant may be suffering from one of the above conditions. or are not affected, but still contain megakaryocytes, thrombocytes, platelets, red blood cells, mast cells, bone marrow cells, and Medulloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells , macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes The levels of one or more endogenous cell types within the hematopoietic system (e.g., in otherwise healthy subjects) Those skilled in the art can also recognize that the level of ATP in an otherwise healthy subject may be decreased. Regarding the presence of decreased levels of one or more of the aforementioned cell types or other blood cell types, For example, flow cytometry, among other procedures known in the art, can be used to determine whether the This can be easily determined using the ELISA and fluorescence-activated cell sorting (FACS) methods. Cut.

[0186] When used in the context of a protein, e.g., an antibody, the term "isolated" refers to the origin or or the source of its origin, in association with components that are naturally associated with it or with which it is naturally associated. a protein that is substantially free from other proteins from the same species; proteins expressed by cells from a species; or proteins not occurring in nature Therefore, it refers to chemically synthesized proteins or naturally occurring cells. Proteins synthesized in different cell lines are "isolated" from components with which they are naturally associated. The protein can be purified using protein purification techniques well known in the art. By isolating the compound in a manner that makes it substantially free from components with which it is naturally associated, It can also be done as follows.

[0187] The term "monoclonal antibody" or "mAb" refers to a antibody obtained from a population of substantially homogeneous antibodies. The antibodies that make up the population are identical and / or have the same enzyme. It refers to antibodies that bind to a specific target, but does not include possible mutant antibodies, e.g., naturally occurring mutants. or mutations that occur during the manufacture of monoclonal antibody preparations, Except for those that may be present in trace amounts. Typically, different antibodies are directed against different determinants (epitopes). In contrast to polyclonal antibody preparations, each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" refers to the production of antibodies by any particular method. It should not be construed as requiring

[0188] As used herein, the term "pharmaceutically acceptable" means a compound that does not cause undue toxicity, irritation, allergies, or GI response, approximately 100% without other problematic complications, in mammals (e.g., humans), etc. compounds suitable for contact with the tissues of a subject and commensurate with a reasonable benefit / risk ratio; Refers to materials, compositions, and / or dosage forms.

[0189] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical composition administered to a subject, such as a mammal, e.g., a human. in particular in the treatment of autoimmune diseases, cancer, or Preventing, treating, or controlling certain diseases or conditions that affect mammals, such as blood disorders "A therapeutic compound" refers to a mixture containing a therapeutic compound administered to treat a disease.

[0190] As used herein, the term "recipient" refers to a recipient of a transplant, such as a graft, containing a hematopoietic stem cell population. The term "transplant" refers to a patient who receives a transplant of transplanted cells. The transplanted cells administered to the recipient may be, for example, autologous cells. The cells may be cells, syngeneic cells, or allogeneic cells.

[0191] As used herein, the term "rejection" in the context of transplantation, such as hematopoietic stem cell transplantation, is used to refer to a condition in which the patient is receiving a transplant. " is when the recipient mounts an immune response against the incoming graft, thereby killing the transplant. A process that reduces the ability of the transferred material (e.g., hematopoietic stem cells) to persist in the recipient. For example, the amount of transplanted cells in various samples isolated from a patient at different times after transplantation or quantifying graft rejection, such as hematopoietic stem cell transplants, by measuring the concentration A decrease in the amount or concentration of transplanted cells in a sample isolated from a patient over time can be For example, 20%, 25%, 30%, 35%, 40%, 56%, 50%, 55%, 60%, Findings of 65%, 70%, 75%, 80%, 85%, 90%, and 95% or greater indicate that the patient Conversely, the presence of IgG in samples isolated from patients indicates that the patient is experiencing graft rejection. The amount or concentration of transplanted cells remains stable over time, e.g., less than 20%, less than 15%, A finding of only a decrease of less than 10%, less than 5%, or less than that indicates that the patient is not receiving transplantation. This indicates that the patient did not experience any rejection or that the transplant was delayed at different times after transplantation. T cells isolated from various samples that cross-react with MHC antigens expressed by transplanted cells and / or by measuring the amount or concentration of immune cells such as NK cells. The graft rejection can be quantified. The amount of immune cells, such as T cells and / or NK cells, that cross-react with the identified MHC antigens may also be increased. or the concentration increases over time, for example, by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 56%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, Findings of 90%, 95%, 100%, 200%, and 300% or greater indicate that the patient is receiving the graft. Conversely, the transplanted cells in the samples isolated from the patient immune cells such as T cells and / or NK cells that cross-react with MHC antigens expressed by The decrease in cell amount or concentration over time is, for example, 20%, 25%, 30%, 35%, 40% , 56%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% A finding of 95% or greater indicates that the patient is not rejecting the graft. There are.

[0192] As used herein, the term "sample" refers to a specimen (e.g., blood, Blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissues (e.g., placenta or dermis), pancreatic juice, chorionic villus samples, and cells).

[0193] As used herein, the term "scFv" refers to a fragment of heavy and light chain variable domains derived from an antibody. A single-chain Fv antibody is one in which the fragments are linked to form a single chain. is the variable region (V) of an antibody light chain separated by a linker. L )(e.g. CDR-L1 , CDR-L2, and / or CDR-L3) and the variable region of the antibody heavy chain (V H )(for example , CDR-H1, CDR-H2, and / or CDR-H3) The V of the scFv fragment contains L Area and V H The linker that connects the regions is Alternatively, the linker may be a peptide linker composed of proteinogenic amino acids. , to increase the resistance of the scFv fragment to proteolysis (e.g., D-amino acids amino acid-containing linkers), to increase the solubility of the scFv fragment (e.g., Polyethylene glycol-containing linkers or linkers containing repeating glycine and serine residues hydrophilic linkers such as polypeptides), to improve the biophysical stability of the molecule (e.g. For example, phosphorus phosphates containing cysteine ​​residues that form intramolecular or intermolecular disulfide bonds. car), or to weaken the immunogenicity of the scFv fragment (e.g., glycosylation The variable regions of the scFv molecules described herein may be linked to the nucleotide sequences of the nucleotides of the nucleotide sequence ... sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the nucleotide sequence of the Regions can be modified so that they differ in amino acid sequence from the antibody molecule from which they are derived. For example, conservative substitutions or changes in amino acid residues may also be used. Nucleotide or amino acid substitutions that result in (at work residues), the ability of the scFv to bind to the antigen recognized by the corresponding antibody The method can be implemented to maintain or enhance the

[0194] The terms "specific binding" or "specifically bind" refer to binding to an antibody or antibody fragment. In relation to the interaction with the second chemical species, the interaction may involve a specific structure on the chemical species (e.g., an antigenic determinant). This means that the antibody is dependent on the presence of a specific target (epitope); for example, antibodies usually Antibodies recognize and bind to specific protein structures, not proteins. If the epitope is specific for "A", then the presence of a molecule containing epitope "A" (i.e., free, unlabeled A) The presence of the antibody reduces the amount of labeled "A" bound to the antibody in a reaction involving labeled "A" and the antibody. In one embodiment, when an antibody specifically binds to a target, e.g., CD5, the target of the antibody On the other hand, K D is at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 - 8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less (less than 1 0 -12 A number less than, e.g. 10 -13 In one embodiment, as used herein, When referring to a CD5-specific antibody, the term "CD5-specific binding" or "specifically binds to CD5" refers to an antibody or It binds to CD5 and has a dissociation constant (K) determined by surface plasmon resonance D ) is 1.0 × 1 0 -7 In one embodiment, K D is a standard biolayer interference However, antibodies may be derived from two or more related sequences. It should be understood that the antibody may be capable of specifically binding to an antigen of, for example, In one embodiment, the antibody is a human or non-human (e.g., mouse or non-human primate) antibody to CD5. It can specifically bind to both orthologs.

[0195] As used herein, the terms "subject" and "patient" refer to a human subject, as described herein. A mammal, such as a human, undergoing treatment for a particular disease or condition. For example, a human patient. The patient may be a patient suffering from an autoimmune disease as described herein, of an anti-CD5 antibody or antibody-drug conjugate to (i) an autoimmune cell population (e.g., Deplete autoimmune CD5+ T cell, B cell, and / or NK cell populations and / or (ii) by CD5+ immune cell populations (e.g., hematopoietic stem cells) and non-self antigens expressed, e.g., non-self MHC antigens expressed by hematopoietic stem cell grafts. depletion of cross-reactive CD5+ T cells, B cells, and / or NK cells, before hematopoietic stem cell transplantation treatment to prevent or reduce the likelihood of graft rejection It can be administered to

[0196] As used herein, the phrase "substantially removed from the blood" refers to the therapeutic agent (anti-CD5 At some point after administration of a therapeutic antibody (e.g., antibody, or antigen-binding fragment thereof) to a patient, The concentration of the therapeutic drug in a blood sample isolated from a subject is such that the therapeutic drug is not detectable by conventional means. (e.g., the therapeutic agent exceeds the noise threshold of the device or assay used to detect the therapeutic agent) refers to a point in time when the signal is not detectable by the signal (such that the signal is not detectable by the signal). Various techniques known in the art can be used to detect IgG, such as the ELISA-based detection assays described in It can be used to detect antibodies, or antibody fragments. Additional assays that can be used to detect ment include those known in the art. Among these are immunoprecipitation techniques and immunoblot assays.

[0197] As used herein, the phrase "stem cell disease" refers to a condition that conditions a target tissue in a subject. (e.g., by depleting the endogenous T cell population in the target tissue) and / or by engrafting or transplanting stem cells into a target tissue in a subject. It broadly refers to any disease, disorder, or condition that can be cured or eliminated. For example, type 1 diabetes has been shown to be cured by hematopoietic stem cell transplantation, and the compositions and methods described herein may benefit from conditioning in accordance with the methods and procedures described herein. Additional diseases that can be treated using the described compositions and methods include sickle cell anemia. Anemia, thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, ADA SC ID, HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan poverty blood, and Shwachman-Diamond syndrome The subject may have an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune disease. Additionally or alternatively, the subject may have blood Cancer (e.g., leukemia, lymphoma, multiple myeloma, or myelodysplastic syndrome) and neuroblastoma the presence or effects of a malignant tumor, such as a malignant tumor selected from the group consisting of carcinoma, In some embodiments, the subject may have or be susceptible to a metabolic disorder. For example, the subject may have or not have: If not, they may be affected by: glycogen storage diseases, mucopolysaccharidosis, gorse syndrome From the group consisting of leukemia, Hurler's disease, sphingolipidosis, and metachromatic leukodystrophy Selected metabolic disorders or therapeutic agents disclosed herein include, but are not limited to: Any other disease or condition that may benefit from treatment and therapy: severe combined immunodeficiency syndrome (SIS) Allergies, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Diak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia A major, sickle cell anemia, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, Juvenile rheumatoid arthritis and "Bone Marrow Transplantation" n for Non-Malignant Disease,” ASH Educat Illnesses or diseases listed in Ion Book, 1:319-338 (2000) The disclosure relates to a medical condition that can be treated by administering hematopoietic stem cell transplantation therapy. No. 60 / 699,999, filed on Oct. 23, 2003, which is incorporated herein by reference in its entirety.

[0198] As used herein, the term "transfection" refers to electroporation. , lipofection, calcium phosphate precipitation, DEAE-dextran transfection are commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as recombinant DNA technology. Refers to any of a wide variety of techniques used.

[0199] As used herein, the term "treat" or "treatment" refers to the treatment of a disease or condition where the purpose is to to prevent or slow down (alleviate) an undesired physiological change or disease, or to refers to a therapeutic treatment that aims to promote a beneficial phenotype in a patient. Desirable clinical outcomes include, but are not limited to: directly treating autoimmune diseases; In some cases, CD5+ T cells, B cells, and / or NK cells cross-react with autoantigens. a reduction in the amount of autoimmune cells present in a sample isolated from a patient, such as a population of cells; or Treatment of autoimmune diseases with anti-CD5 antibodies or their antigen-binding fragments and hematopoietic stem cell transplants When administered with , C that cross-reacts with antigens expressed by hematopoietic stem cells Reagents isolated from patients, such as populations of D5+ T cells, B cells, and / or NK cells Reduction of the amount of autoimmune cells present in the tissue prior to hematopoietic stem cell transplantation. The results showed that conditioning therapy and Hematopoietic stem cell counts or relative concentrations after subsequent administration of an exogenous hematopoietic stem cell graft to the patient Beneficial results of the treatments described herein include an increase in the degree of conditioning therapy. Megakaryocytes, thrombocytes, platelets, erythrocytes, and mast cells after hematopoietic stem cell transplantation , myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presentation cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, or B lymphocytes It may also include an increase in the cell number or relative concentration of one or more cells of the hematopoietic system, such as lymphocytes.

[0200] As used herein, the terms "variant" and "derivative" are used interchangeably and Natural, synthetic, and Refers to semi-synthetic analogs of the compounds, peptides, proteins, or other substances described herein. Variants or derivatives of a substance may retain or improve the biological activity of the original material.

[0201] As used herein, the term "vector" includes plasmids, DNA vectors, and nucleic acid vectors, such as nucleotides, RNA vectors, viruses, or other suitable replicons. The expression vectors described herein can be used to express polynucleotide sequences and, e.g., Expression of proteins and / or integration of these polynucleotide sequences into the genome of mammalian cells The antibodies and antibody fragments of the present invention may contain additional sequence elements used for integration. Particular vectors that can be used for expression of the polypeptides include promoter regions that direct gene transcription. The present invention also includes plasmids containing regulatory sequences such as promoter regions and enhancer regions. Other useful vectors for expressing fragments include those that increase the rate of translation of these genes. or polynucleotides that improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements may be used, for example, in the coding sequence of a gene carried by an expression vector. 5' and 3' untranslated regions and polyadenylation signals to direct efficient transcription of The expression vectors described herein may be used to transform cells containing such vectors. The vector may also contain a polynucleotide encoding a marker for selection. Examples include ampicillin, chloramphenicol, kanamycin, and nourceotrichin. Examples of genes that encode resistance to antibiotics such as cyclosporin.

[0202] As used herein, the term "alkyl" refers to an alkyl group having, for example, 1 to 20 carbon atoms in the chain. Examples of alkyl groups include methyl, ethyl, n -propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, Examples include pentyl, isopentyl, tert-pentyl, hexyl, and isohexyl. do.

[0203] As used herein, the term "alkylene" refers to a straight or branched chain divalent alkyl group. The divalent positions can be on the same or different atoms within the alkyl chain. Examples include methylene, ethylene, propylene, isopropylene, and the like.

[0204] As used herein, the term "heteroalkyl" refers to an alkyl group having, for example, 1 to 20 carbon atoms in the chain. and one or more heteroatoms (e.g., oxygen, nitrogen, or refers to a straight or branched chain alkyl group containing sulfur.

[0205] As used herein, the term "heteroalkylene" refers to a straight or branched chain divalent heteroalkylene. The divalent positions may be on the same or different atoms within the heteroalkyl chain. The divalent positions may be one or more heteroatoms.

[0206] As used herein, the term "alkenyl" refers to an alkyl group having, for example, 2 to 20 carbon atoms in the chain. Examples of alkenyl groups include vinyl, propyl, and propyl groups. Examples include butenyl, isopropenyl, butenyl, tert-butylenyl, and hexenyl. do.

[0207] As used herein, the term "alkenylene" refers to a straight or branched chain divalent alkenylene. The divalent positions can be on the same or different atoms within the alkenyl chain. Examples of phenylene include ethenylene, propenylene, isopropenylene, and butenylene. It can be obtained.

[0208] As used herein, the term "heteroalkenyl" refers to an alkyl group having, for example, 2 to 20 alkyl groups in the chain. carbon atoms and in addition one or more heteroatoms in the chain (e.g., oxygen, nitrogen, or or sulfur-containing straight or branched chain alkenyl groups.

[0209] As used herein, the term "heteroalkenylene" refers to a straight or branched chain divalent heteroalkenylene. The divalent positions may be the same or different atoms within the heteroalkenyl chain. The divalent position can be one or more heteroatoms.

[0210] As used herein, the term "alkynyl" refers to an alkynyl group having, for example, 2 to 20 carbon atoms in the chain. Examples of alkynyl groups include propargyl, alkynyl groups, and alkynyl groups having a straight or branched chain. , butynyl, pentynyl, hexynyl, and the like.

[0211] As used herein, the term "alkynylene" refers to a straight or branched chain divalent alkynylene. The divalent positions can be on the same or different atoms within the alkynyl chain.

[0212] As used herein, the term "heteroalkynyl" refers to a heteroalkynyl group having, for example, 2 to 20 alkyl groups in the chain. carbon atoms and in addition one or more heteroatoms in the chain (e.g., oxygen, nitrogen, or or sulfur-containing straight or branched chain alkynyl groups.

[0213] As used herein, the term "heteroalkynylene" refers to a straight or branched chain divalent heteroalkynylene. The divalent positions can be the same or different atoms within the heteroalkynyl chain. The divalent position can be one or more heteroatoms.

[0214] As used herein, the term "cycloalkyl" refers to a saturated group, e.g., a group having 3 to 12 carbon atoms. refers to a monocyclic, or fused, bridged, or spiro polycyclic ring structure having 10 carbon ring atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[3.1.0]hexane, etc. Examples include:

[0215] As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl group. The divalent positions can be on the same or different atoms within the ring structure. Examples include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, etc. Examples include:

[0216] As used herein, the term "heterocycloalkyl" refers to a saturated, e.g., carbon-, and heteroatoms selected, for example, from nitrogen, oxygen, and sulfur, among others. monocyclic, or fused, bridged, or otherwise, having 3 to 12 ring atoms per ring structure selected from the group consisting of The ring structure may have, for example, carbon, nitrogen, or sulfur ring members. It may contain one or more oxo groups. Examples of heterocycloalkyl include, by way of example and not limitation, Dihydropyridyl, tetrahydropyridyl (piperidyl), tetrahydrothiophene nyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydro furanyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinone quinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl Examples include aryl, piperazinyl, quinuclidinyl, and morpholinyl.

[0217] As used herein, the term "heterocycloalkylene" refers to a divalent heterocycloalkylene. The divalent positions can be on the same or different atoms within the ring structure.

[0218] As used herein, the term "aryl" refers to an aryl group containing, for example, 6 to 19 carbon atoms. Aryl groups include phenyl, fluorenyl, naphthalene, and phenyl. The divalent position may be one or more heteroatoms. It can be a child.

[0219] As used herein, the term "arylene" refers to a divalent aryl group. can be on the same or different atoms.

[0220] As used herein, the term "heteroaryl" refers to an aryl group in which one or more ring atoms is a heteroatom. monocyclic heteroaromatic rings, for example, nitrogen, oxygen, or sulfur, or bicyclic or tricyclic rings; Heteroaryl groups include: pyridinyl, phenyl ... pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thienyl Azolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl Azolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2, 5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1 ,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl, isoben Zofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, indolyl, isobenzothienyl Soindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyridin benzothiazolyl, benzoxazolyl, quinolizinyl, quinazolinyl, phthalazinyl quinoxalinyl, cinnolinyl, naphthyridinyl, pyrido[3,4-b]pyridyl, Pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroquinolyl Isoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl, benzoquinolyl Lil, etc.

[0221] As used herein, the term "heteroarylene" refers to a divalent heteroaryl group. The divalent positions can be on the same or different atoms. The divalent positions can be on one or more heteroatoms. It can be an atom.

[0222] Unless otherwise limited by the definition of an individual substituent, the aforementioned chemical moieties, e.g., "alkane" alkyl, alkylene, heteroalkyl, heteroalkylene, alkenyl "Alkenylene", "heteroalkenyl", "heteroalkenylene", "alkynyl" "alkynylene," "heteroalkynyl," "heteroalkynylene," "cycloalkynyl," "cycloalkylene," "heterocycloalkyl," "heterocycloalkylene" "aryl", "arylene", "heteroaryl", and "heteroarylene" groups may be optionally substituted with 1 to 5 substituents selected from the group consisting of: Good: alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, a alkylaryl, alkylheteroaryl, alkylcycloalkyl, alkylheteroaryl Chloroalkyl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl Carbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl , sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy, tri halomethyl, cyano, hydroxy, mercapto, nitro, etc. Typical substituents include: These include, but are not limited to: -X, -R, -OH, -OR, -SH, -SR , NH2, -NHR, -N(R)2, -N + (R)3, -CX3, -CN, -OCN, - SCN, -NCO, -NCS, -NO, -NO2, -N3, -NC(=O)H, -NC( =O)R, -C(=O)H, -C(=O)R, -C(=O)NH2, -C(=O)N(R )2, -SO3-, -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O )2NH2, -S(=O)2N(R)2, -S(=O)R, -OP(=O)(OH)2, -OP(=O)(OR)2, -P(=O)(OR)2, -PO3, -PO3H2, -C( =O)X, -C(=S)R, -CO2H, -CO2R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NH2, -C(=O)N(R)2, -C(=S)NH2, -C(=S)N(R)2, -C(=NH)NH2, and -C(= NR)N(R)2; each X is independently selected from F, Cl, Br, and I; each R is independently selected from alkyl, aryl, heterocycloalkane, The group "any" is selected from alkyl or heteroaryl, protecting groups and prodrug moieties. Whenever "substituted with" is described, that group represents, independently in each occurrence, , may be substituted with one or more of the above substituents. Substitution is by closure of adjacent functional substituents. The adjacent substituents, such as a ring, undergo ring closure to form, for example, lactams, lactams, etc. ton, cyclic anhydrides, acetals, hemiacetals, thioacetals, aminals, and This may include situations where a hemiaminal is formed, for example to provide a protecting group.

[0223] The naming convention for a particular radical can be either monoradical or diradical, depending on the context. For example, it is understood that a substituent may have two bonds to the rest of the molecule. Where a bond is required, the substituent is understood to be a diradical. For example, Substituents identified as alkyl requiring a bond include -CH2-, -CH2CH2- Other radical naming conventions include diradicals such as -CH2CH(CH3)CH2-. The rule is that the radical is "alkylene," "alkenylene," "arylene," "heterocycle," It clearly indicates that the radical is a diradical such as "trimethylsilyl alkylene."

[0224] The substituent is a diradical (i.e., has two points of attachment to the rest of the molecule) Wherever shown as a group, the substituents may be in any directional conformation unless otherwise stated. It will be understood that the bond may be at any position.

[0225] (anti-CD5 antibody) The present invention relates to an anti-CD5 antibody, or an antigen-binding fragment thereof, that can be used to treat, for example, such a drug. The agent targets CD5+ cancer cells (e.g., CD5+ leukemia cells) and CD5+ autoimmune cells ( For example, CD5+ autoimmune T cells, B cells, and / or NK cells) for their ability to kill This is based in part on the discovery that steroids can be used to directly treat cancer and autoimmune diseases. In particular, the anti-CD5 antibodies described herein can be conjugated to a cytotoxin via a linker. Therefore, wherever anti-CD5 antibodies are mentioned, unless otherwise indicated, , and conjugates thereof are also contemplated.

[0226] The present invention further provides antibodies, or antigen-binding fragments thereof, capable of binding to CD5. by preventing or reducing the likelihood of immune cell-mediated transplant rejection. Treatments to enhance engraftment of transplanted hematopoietic stem cells in patients requiring transplantation therapy The present invention is based in part on the discovery that anti-CD5 antibodies, and The antigen-binding fragment may be a non-self MHC antigen expressed by the hematopoietic stem cell graft. T cells, B cells, or NK cells that cross-react with non-self hematopoietic stem cell antigens and initiate an immune response It can bind to cell surface CD5 expressed by immune cells such as lymphocytes. Binding of novel antibodies and their antigen-binding fragments to hematopoietic stem cell-specific CD5+ immune cells In this case, for example, by antibody-dependent cell-mediated cytotoxicity or by the antibody or its antigen. The bound immune cells are activated by the action of a cytotoxic drug conjugated to the binding fragment. Therefore, CD5+ immune cells that cross-react with non-autologous hematopoietic stem cells can induce the death of depleting the immune cell population in the recipient, which mounts an immune response against the incoming graft By weakening the immune system's ability to deliver hematopoietic stem cell transplants to patients in need In this way, the subject can be free from defects and / or deficiencies. A hematopoietic stem cell graft can be provided to a subject to repopulate the deficient cell lineage. and therefore, those suffering from stem cell disorders, cancer, autoimmune diseases, or other blood disorders described herein. The subject can be treated with a drug, for example, for the purpose of eradicating cancer cells. The chemotherapy administered to the subjects depleted healthy blood-forming cells in the process. In some cases, the cell population is deficient.

[0227] For example, the present invention therefore provides a method for detecting a leukocyte antigen capable of binding to an antigen expressed by a T cell. The administration of antibodies or antigen-binding fragments thereof to the transplanted hematopoietic stem cells enhances the engraftment of the transplanted hematopoietic stem cells. The present invention provides compositions and methods for promoting the development of CD4+ and CD8+ It is possible to selectively deplete endogenous T cell populations, such as T cells. Depletion is then followed by transplantation of an exogenous (e.g., autologous, allogeneic, or syngeneic) hematopoietic stem cell graft. Transplant rejection can be prevented, for example, by administering an anti-C antibody as described herein. D5 antibody, antigen-binding fragment, antibody-drug conjugate, or antibody-drug conjugate Selective depletion of CD4+ and / or CD8+ T cells using duplicative immunoglobulins (DGI) enhances the survival of transplanted endothelial cells. This can attenuate the T cell-mediated immune response that may occur against the blood stem cell graft. binds to CD5 to promote the survival and engraftment potential of transplanted hematopoietic stem cells. and antigen-binding fragments thereof, which can be used to treat hematopoietic stem cell transplantation. This is based in part on the discovery that it can be administered to patients with

[0228] Hematopoietic stem cell graft survival after administration of anti-CD5 antibody or its antigen-binding fragment can be determined by various empirical measurements, such as the viability of transplanted hematopoietic stem cells. The method comprises administering an antibody or antigen-binding fragment thereof capable of binding to CD5, and Following this, the patient receives a hematopoietic stem cell graft, which is then transferred to the competitive repopulating units ( It can be evaluated by calculating the amount of CRU. The method involves transfecting donor hematopoietic stem cells with a reporter gene, such as an enzyme that catalyzes a chemical reaction that produces a luminescent product. The cells then integrate the corresponding signal into the transfected vector. By monitoring the tissues where hematopoietic stem cells home, such as bone marrow, The engraftment of the cell graft can be monitored. Hematopoietic stem cells and The engraftment of hematopoietic stem cells can also be monitored by assessing the quantity and viability of hematopoietic stem cells and progenitor cells. Engraftment can also be assessed by measuring peripheral blood white blood cell counts during the post-transplant period and and / or by measuring bone marrow cell recovery by donor cells in bone marrow aspirate samples. It can be determined.

[0229] The following sections provide information to patients in need of hematopoietic stem cell transplantation therapy regarding the survival of hematopoietic stem cell grafts. an antibody, or antigen-binding fragment thereof, that can be administered to promote Methods for administering such therapeutic agents to patients prior to hematopoietic stem cell transplantation are described.

[0230] Exemplary Antibodies The compositions and methods described herein include antibodies that specifically bind to human CD5, or antibodies thereto Human CD5 is also called LEU1 or T1. , a type I transmembrane glycoprotein found on the surface of thymocytes, T lymphocytes, and a subset of B lymphocytes Two isoforms of human CD5 have been identified. M1 contains 438 amino acids, as described by Jones et al. (1988) Nature 323 (6086), 346-349 and the following (NCBI reference sequence: NP_0 01333385.1) as described in: MVCSQSWGRS SKQWEDPSQASKVCQRLNCG VPLSLGPF LV TYTPQSSIICYGQL GSFSNCSHSRNDMCHS LGLTC LEPQKTTPPTTRPPPTTTPEPTAPP RLQLVAQSGG QHC AGVVEFYSGSLGGTISY EAQDKTQDLE NFLCNNLQCG SFLKHLPETE AGRAQDPGEP REHQPLPIQWKIQNSSCT SL EHCFRKIKPQ KSGRVLALLC SGFQPKVQSR LVGG SSICEG TVEVRQGAQWAALCDSSSSAR SSLRWEEVCR E QQCGSVNSY RVLDAGDPTS RGLFCPHQKL SQCHELWE RNSYCKKVFVTCQDPNPAGLAAGTVASIILAL VLLVVLL VVC GPLAYKKLVK KFRQKKQRQWIGPTGMNQNM SFHR NHTATV RSHAENPTAS HVDNEYSQPP RNSHLSAYPA LEGALHRSSMQPDNSSDSDY DLHGAQRL (SEQ ID NO: 261)

[0231] T cells are composed of cell adhesion molecules and are involved in the proliferation response and T cell helper function of activated T cells. It has been shown that CD5, which is involved in both It functions by interacting with CD72, a cell surface protein found only on B cells. It has also been shown that antibodies that bind to CD5 transmit costimulatory signals to T cells. or an antigen-binding fragment thereof, e.g., inhibits the interaction of CD5 with CD72. suppresses T cell activation and T cell-mediated immune responses to hematopoietic stem cell grafts. Antibodies that bind to CD5, and antigen-binding fragments thereof, can be used to, for example, , antibodies, or antigen-binding fragments thereof, can be coupled to cytotoxins (e.g., those described herein). or by conjugation to a cytotoxin (such as a cytotoxin known in the art). or unconjugated antibodies capable of recruiting complement proteins to T cells; It is also possible to directly kill CD5+ T cells by using antigen-binding fragments of do.

[0232] Furthermore, a subset of activated B cells has been shown to express CD5; This expression pattern is particularly common among autoreactive B cells (Werner-Favr e et al.,European Journal of Immunology 19:1209-1231 (1989), the disclosure of which is incorporated herein by reference in its entirety. CD5 has also been shown to be expressed by a subset of NK cells. particularly among patients with multiple myeloma, there is a high concentration of low-density CD5+ (CD5LOW+) NK cells. It has been shown that the surface antigen is involved in the activation of NK cells ( Ishiyama et al., Anticancer Research 14:7 25-730 (1994), the disclosure of which is incorporated herein by reference in its entirety. Therefore, antibodies that specifically bind to CD5, or antigen-binding fragments thereof, can be used. Antibodies that bind to CD5 can be used to attenuate the activation of B cells and NK cells. or antigen-binding fragments thereof can be used to, for example, The fragment may be combined with a cytotoxin (e.g., a compound described herein or known in the art). Complement proteins are targeted to B cells or NK cells by conjugating them to cytotoxins (which can be used to treat complement disorders). Unconjugated antibodies, or antigen-binding fragments thereof, capable of being recruited to cells can also be used to directly kill CD5+ B cells and NK cells.

[0233] The present invention relates to a method for producing a CD5 polypeptide, which specifically binds to a CD5 polypeptide, for example, a human CD5 polypeptide. Exemplary embodiments include antibodies, and antigen-binding fragments thereof, and uses thereof. In one embodiment, an antibody that specifically binds to a CD5 polypeptide, or an antigen-binding fragment thereof, is The component comprises a heavy chain variable region and a light chain variable region.

[0234] In one embodiment, the heavy chain variable region comprises one or more complementarity determining regions (CDRs). In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 17. In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the heavy chain variable region comprises a VH C comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 17, SEQ ID NO: 18, and DR3. In one embodiment, the VH CDRs comprise one or more VH CDRs selected from the group consisting of SEQ ID NO: 19. The heavy chain variable region is selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. In one embodiment, the heavy chain variable region comprises two or more VH CDRs selected from SEQ ID NO: 1. VH CDR1 comprising SEQ ID NO: 7, VH CDR2 comprising SEQ ID NO: 18, and VH CDR3 comprising SEQ ID NO: 19. VH CDR3.

[0235] In one embodiment, the light chain variable region comprises one or more complementarity determining regions (CDRs). In embodiments, the light chain variable region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 20. In one embodiment, the light chain variable region comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 21. In one embodiment, the light chain variable region comprises a VL C comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the light chain variable region comprises SEQ ID NO: 20, SEQ ID NO: 21, and DR3. In one embodiment, the VL CDRs comprise one or more VL CDRs selected from the group consisting of SEQ ID NO: 22. The light chain variable region is selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. In one embodiment, the light chain variable region comprises two or more VL CDRs selected from SEQ ID NO:2. 0, VL CDR1 comprising SEQ ID NO: 21, and VL CDR2 comprising SEQ ID NO: 22. VL CDR3.

[0236] In an exemplary embodiment, the antibody, or antigen-binding fragment thereof, comprises SEQ ID NO: 17. VH CDR1 comprising SEQ ID NO: 18, VH CDR2 comprising SEQ ID NO: 19 a heavy chain variable region comprising a VL CDR1 comprising SEQ ID NO: 20 and a VL CDR3 comprising SEQ ID NO: 21; a light chain variable region comprising a VL CDR2 comprising SEQ ID NO: 21 and a VL CDR3 comprising SEQ ID NO: 22 Includes.

[0237] In certain embodiments, one or more CDRs (i.e., one having SEQ ID NOs: 17-19) or more heavy chain CDRs, and / or one or more light chain CDRs having SEQ ID NOs: 20 to 22) The CD5 specificity of the antibody (i.e., the heavy chain CDRs of SEQ ID NOs: 17-19 and SEQ ID NO: 20) ~22 light chain CDRs and an antibody, or antigen-binding fragment thereof, with similar specificity Conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining ).

[0238] In certain embodiments, the anti-CD5 antibody, or antigen-binding fragment thereof, is a murine antibody 5D7, or a humanized version thereof. The murine antibody 5D7 binds to human CD5, The present invention is described in U.S. Patent Publication No. 20008 / 0245027 and disclosed therein. The contents relating to the antibody sequences listed in Table 4 are incorporated herein by reference. SEQ ID NOs: 29 to 34 correspond to the CDRs of the murine anti-CD5 antibody 5D7. The humanized version of D7 has SEQ ID NO: 257 (humanized heavy chain variable region) and SEQ ID NO: 25 8 (humanized light chain variable region). In one embodiment, the ADCs and and uses thereof include antibodies comprising the CDRs set forth in SEQ ID NOS: 29 to 34. In some embodiments, the ADCs and their uses described herein are directed to SEQ ID NOs: 257 and 258. Includes antibodies comprising the heavy and light chain variable regions, respectively, as shown.

[0239] In one embodiment, the antibody, or antigen-binding fragment thereof, is selected from the group consisting of: In another embodiment, the antibody, or its antigen-binding domain, comprises a heavy chain variable region comprising an amino acid sequence A hybrid fragment has at least 95% identity to SEQ ID NO: 257, e.g., SEQ ID NO: At least 95%, 96%, 97%, 98%, 99%, or 100% for No. 257 In certain embodiments, the antibody comprises a heavy chain variable region comprising an amino acid sequence having the identity of , SEQ ID NO: 257, or a variant of SEQ ID NO: 257. The variants of SEQ ID NO: 257 include: (i) a variant of SEQ ID NO: 1, 2, 3, 4 or differs by five amino acid substitutions, additions, or deletions; (ii) SEQ ID NO: 25 7 means that a maximum of 5, 4, 3, 2, or 1 amino acid substitution, addition, or deletion (iii) SEQ ID NO: 257 is 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 (iv) differs by substitution, addition, or deletion of up to 5 amino acids; and / or At least about 75%, 80%, 85%, 90%, 95%, 96%, 9% for number 257 7%, 98% or 99% identical amino acid sequence, wherein any of (i) to (i In v), the amino acid substitution is a conservative amino acid substitution or a non-conservative amino acid substitution. the modified heavy chain variable region retains the CD5 binding specificity of the antibody, i.e., 257, or an antigen-binding fragment thereof, having similar binding specificity to an antibody comprising the sequence It may have enhanced biological activity compared to the heavy chain variable region of number 257.

[0240] In one embodiment, the antibody, or antigen-binding fragment thereof, is selected from the group consisting of: In another embodiment, the antibody, or its antigen-binding domain, comprises a light chain variable region comprising an amino acid sequence A hybrid fragment has at least 95% identity to SEQ ID NO: 258, e.g., SEQ ID NO: At least 95%, 96%, 97%, 98%, 99%, or 100% for No. 258 In certain embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence having the identity of , SEQ ID NO: 258, or a modified light chain comprising an LC variable domain comprising a variant of SEQ ID NO: 258. The variants of SEQ ID NO: 258 include: (i) a variant of SEQ ID NO: 258 that is 1, 2, 3, 4, or 5; or differs by five amino acid substitutions, additions, or deletions; (ii) SEQ ID NO: 25 8 means that a maximum of 5, 4, 3, 2, or 1 amino acid substitution, addition, or deletion (iii) SEQ ID NO: 258 is 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 (iv) differs by substitution, addition, or deletion of up to 5 amino acids; and / or At least about 75%, 80%, 85%, 90%, 95%, 96%, 9% for number 258 7%, 98% or 99% identical amino acid sequence, wherein any of (i) to (i In v), the amino acid substitution is a conservative amino acid substitution or a non-conservative amino acid substitution. the modified light chain variable region retains the CD5 binding specificity of the antibody, i.e., 258, or an antigen-binding fragment thereof, having similar binding specificity to an antibody comprising the sequence It may have enhanced biological activity compared to the light chain variable region of number 258.

[0241] In an exemplary embodiment, the antibody, or antigen-binding fragment thereof, is selected from the group consisting of SEQ ID NO:257. at least 95% identity to, e.g., at least 95% to SEQ ID NO: 257; Contains an amino acid sequence with 96%, 97%, 98%, 99%, or 100% identity heavy chain variable region and at least 95% identity to SEQ ID NO: 258, e.g., SEQ ID NO: At least 95%, 96%, 97%, 98%, 99%, or 100% of the 258 and a light chain variable region comprising an amino acid sequence having identity to the antibody. The antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 257 and a heavy chain variable region comprising SEQ ID NO: 258 and a light chain variable region comprising:

[0242] In another embodiment, the anti-CD5 antibody, or antigen-binding fragment thereof, is SEQ ID NO: 29 The heavy chain variable region may comprise a VH CDR1 comprising the amino acid sequence In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 30. In one embodiment, the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 31. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: In one embodiment, the heavy chain comprises one or more VH CDRs selected from the group consisting of: The variable region is selected from the group consisting of SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In one embodiment, the heavy chain variable region comprises two or more VH CDRs comprising SEQ ID NO: 29. VH CDR1 comprising SEQ ID NO: 30, VH CDR2 comprising SEQ ID NO: 31 CDR3.

[0243] In one embodiment, the light chain variable region comprises one or more complementarity determining regions (CDRs). In embodiments, the light chain variable region comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 32. In one embodiment, the light chain variable region comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 33. In one embodiment, the light chain variable region comprises a VL C comprising the amino acid sequence of SEQ ID NO: 34. In one embodiment, the light chain variable region comprises SEQ ID NO: 32, SEQ ID NO: 33, and DR3. In one embodiment, the VL CDRs comprise one or more VL CDRs selected from the group consisting of SEQ ID NO: 34. The light chain variable region is selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34. In one embodiment, the light chain variable region comprises two or more VL CDRs selected from SEQ ID NO:3. 2, VL CDR1 comprising SEQ ID NO: 33, and VL CDR2 comprising SEQ ID NO: 34. VL CDR3.

[0244] In an exemplary embodiment, the antibody, or antigen-binding fragment thereof, comprises SEQ ID NO:29. VH CDR1 comprising SEQ ID NO: 30, VH CDR2 comprising SEQ ID NO: 31 a heavy chain variable region comprising a VL CDR1 comprising SEQ ID NO: 32 and a VL CDR3 comprising SEQ ID NO: 33; a light chain variable region comprising a VL CDR2 comprising SEQ ID NO: 33 and a VL CDR3 comprising SEQ ID NO: 34; Includes.

[0245] In certain embodiments, one or more CDRs (i.e., one having SEQ ID NOs: 29-31) or more heavy chain CDRs, and / or one or more light chain CDRs having SEQ ID NOs: 32 to 34) The CD5 specificity of the antibody (i.e., the heavy chain CDRs of SEQ ID NOs: 29-31 and SEQ ID NO: 32) ~34 light chain CDRs and an antibody, or antigen-binding fragment thereof, with similar specificity Conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining ).

[0246] Antibodies and antigen-binding fragments thereof capable of binding to the CD5 antigen are Using techniques known in the art and described herein, e.g., immunization, computational modeling, ring technology, as well as phage display and cell-based display technologies described below. These can be identified by in vitro selection methods such as the I-platform.

[0247] Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those with the following potential: one or both of the variable regions, or have at least 85% sequence identity thereto amino acid sequences (e.g., 85%, 90%, 95%, 97%, 98%, 99%, 9% or more sequence identity): Amino acid sequence DIQMTQSPSSMSASLGDRVTITCRASQDINSYLSWFQQ KPGKSPKTLIYRANRLVDGVPSRFSGSGSGTDYTLTISSL QYEDFGIYYCQQYDESPWTFGGGTKLEIK (SEQ ID NO: 1) V L and; Amino acid sequence QIQLVQSGPGLKKPGGSVRISCAASGYTFTNYGMNWVK QAPGKGLRWMGWINTHTGEPTYADDFKGRFTFSLDTSKST AYLQINSLRAEDTATYFCTRRGYDWYFDVWGQGTTVTVSS (SEQ ID NO: 2) H .

[0248] The aforementioned V L and V H Antibodies and antigen-binding fragments thereof containing the sequences are e.g. For example, see U.S. Pat. No. 5,869,619, the disclosure of which is directed to he1 anti- For information regarding anti-CD5 antibodies and antigen-binding fragments thereof, see references herein. In some embodiments, an anti-CD5 antibody or its antigen binding molecule is The combined fragment is V of SEQ ID NO: 1 and SEQ ID NO: 2. L and V H Contains chains. In embodiments, the anti-CD5 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2. Number 2 V L and V H In some embodiments, the CDRs comprise the CDRs contained in the anti-CD 5 antibodies or antigen-binding fragments thereof may be selected from the group consisting of SEQ ID NO: 1 and V of SEQ ID NO: 2 L and V H CDRs contained in the V chain L and V H The remainder of the sequence is SEQ ID NO: 1 and and V of sequence number 2 L and V H At least 85% sequence identity (e.g., 8 5%, 90%, 95%, 97%, 98%, 99% or more sequence identity).

[0249] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof has the following characteristics: DR includes: and CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 3); and CDR-H2 having the amino acid sequence NTHTGE (SEQ ID NO: 4); and a CDR-H3 having the amino acid sequence RGYDWYFDV (SEQ ID NO: 5); and CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 6); and a CDR-L2 having the amino acid sequence RANRLVD (SEQ ID NO: 7); CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 8).

[0250] Additional anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include: One or both of the variable regions below, or at least 85% sequence identity thereto (e.g., 85%, 90%, 95%, 97%, 98% %, 99% or more sequence identity) of the amino acid sequence: Amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQ KPGKAPKTLIYRANRLESGVPSRFSGSGSGTDYTLTISSL QYEDFGIYYCQQYDESPWTFGGGTKLEIK (SEQ ID NO: 9) V L and; Amino acid sequence EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVR QAPGKGLEWWMGWINTHYGEPTYADSFKGTRTFSLDDSKNT AYLQINSLRAEDTAVYFCTRRGYDWYFDVWGQGGTTTVVS V with S (SEQ ID NO: 10) H .

[0251] The aforementioned V L and V H Antibodies and antigen-binding fragments thereof containing the sequences are e.g. For example, see U.S. Pat. No. 5,869,619, the disclosure of which is directed to the he3 anti- For information regarding anti-CD5 antibodies and antigen-binding fragments thereof, see references herein. In some embodiments, an anti-CD5 antibody or its antigen binding molecule is The combined fragment is V of SEQ ID NO: 3 and SEQ ID NO: 4. Land V H V of an antibody containing chain L oh Yobi V H In some embodiments, the anti-CD5 antibody or its CDRs are The antigen-binding fragments of SEQ ID NO: 3 and SEQ ID NO: 4 are L and V H contained in the chain CDRs containing V L and V H The remainder of the sequence is V of SEQ ID NO:3 and SEQ ID NO:4 L and V H At least 85% sequence identity (e.g., 85%, 90%, 9 5%, 97%, 98%, 99% or more sequence identity).

[0252] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof has the following characteristics: DR includes: and CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 11); and CDR-H2 having the amino acid sequence NTHYGE (SEQ ID NO: 12); and a CDR-H3 having the amino acid sequence RRGYDWYFDV (SEQ ID NO: 13); and CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 14); and a CDR-L2 having the amino acid sequence RANRLES (SEQ ID NO: 15); CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 16).

[0253] Antibodies and antigen-binding fragments thereof containing the aforementioned CDR sequences are available from, for example, The disclosure of which is described in Japanese Patent No. 5,869,619, relates to anti-CD5 antibodies and and antigen-binding fragments thereof, which are incorporated herein by reference. do.

[0254] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, For example, U.S. Patents 5,821,123; 5,766,886; 5,770,196; 7,1 53,932;5,621,083;6,649,742;6,146,631;5,7 56,699; 5,744,580; 6,376,217; 5,837,491; and and anti-CD5 antibodies described in US Pat. No. 6,146,850, each of which disclosures is incorporated herein by reference. The present invention is incorporated by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof. It will be incorporated into the specification.

[0255] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, For example, the hybridoma deposited as ATCC CRL 8000 (anti-CD5 mouse antibody OKT1) Such antibodies include those produced by primate cell lines. 4,515,894; 4,657,760; and 4,363,799. Each of these disclosures relates to anti-CD5 antibodies and antigen-binding fragments thereof. and the like, which are hereby incorporated by reference.

[0256] Anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include those listed below. This includes having one or more or all of the following DRs: and CDR-H1 having the amino acid sequence GYSITSGYY (SEQ ID NO: 17); and CDR-H2 having the amino acid sequence ISYSGFT (SEQ ID NO: 18); and a CDR-H3 having the amino acid sequence AGDRTGSWFAY (SEQ ID NO: 19); and CDR-L1 having the amino acid sequence QDISNY (SEQ ID NO: 20); and a CDR-L2 having the amino acid sequence ATS (SEQ ID NO: 21); CDR-L3 having the amino acid sequence LQYASYPFT (SEQ ID NO: 22).

[0257] Antibodies and antigen-binding fragments thereof containing the aforementioned CDR sequences are available from, for example, The disclosure of which is described in the specification of Japanese Patent No. 8,679,500, is directed to anti-CD5 antibodies and and antigen-binding fragments thereof, which are incorporated herein by reference. do.

[0258] Anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include those listed below. This includes having one or more or all of the following DRs: and CDR-H1 having the amino acid sequence GYIFTNYG (SEQ ID NO: 23); and a CDR-H2 having the amino acid sequence INTYNGEP (SEQ ID NO: 24); and a CDR-H3 having the amino acid sequence ARGDYYGYEDY (SEQ ID NO: 25); and CDR-L1 having the amino acid sequence QGISNY (SEQ ID NO: 26); and a CDR-L2 having the amino acid sequence YTS (SEQ ID NO: 27); CDR-L3 having the amino acid sequence QQYSKLPWT (SEQ ID NO: 28).

[0259] Antibodies and antigen-binding fragments thereof containing the aforementioned CDR sequences are available from, for example, This is described in US Patent No. 8,679,500.

[0260] Anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include those listed below. This includes having one or more or all of the following DRs: CDR-H1 having the amino acid sequence FSLSTSGMG (SEQ ID NO: 29); and CDR-H2 having the amino acid sequence WWDDD (SEQ ID NO: 30); and a CDR-H3 having the amino acid sequence RRATGTGFDY (SEQ ID NO: 31); and CDR-L1 having the amino acid sequence QDVGTA (SEQ ID NO: 32); and a CDR-L2 having the amino acid sequence WTSTRHT (SEQ ID NO: 33); CDR-L3 having the amino acid sequence YNSYNT (SEQ ID NO: 34).

[0261] Antibodies and antigen-binding fragments thereof containing the aforementioned CDR sequences are available from, for example, The disclosure of which is described in the specification of the US Patent Application Publication No. 2008 / 0254027, The present specification is incorporated by reference as it relates to CD5 antibodies and antigen-binding fragments thereof. be incorporated into the book.

[0262] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, For example, anti-CD5 antibodies described in PCT Application Publication No. WO1992 / 014491, e.g. For example, the hive deposited at the Pasteur Institute on January 10, 1991 under number 1-1025 Examples of such antibodies include anti-CD5 antibodies produced by CD54 cells. The disclosure of US Pat. No. 992 / 014491 relates to anti-CD5 antibodies and antigen-binding fragments thereof. and the like, which are hereby incorporated by reference.

[0263] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, See, for example, U.S. Patent Nos. 6,010,902 and 7,192,736, U.S. Patent Application Publication No. Patent Publications Nos. 2011 / 0250203 and 2017 / 0129128, and PC Publication Nos. WO2016 / 172606; WO1994 / 023747; and WO1 and the anti-CD5 antibodies described in US Pat. No. 996 / 041608; each of these disclosures is directed to anti-C D5 antibodies and antigen-binding fragments thereof, will be incorporated into

[0264] In some embodiments, anti-cancer drugs that may be used in conjunction with the compositions and methods described herein The CD5 antibody contains CDR-H1, CDR-H2, CDR-H3, and CDR-H4 listed in Table 1 below. and those containing a combination of CDR-L1, CDR-L2, and CDR-L3 regions. can be. [Table 1-1] [Table 1-2]

[0265] Antibodies and antigen-binding fragments thereof containing the CDR sequences of Table 1 above can be used, for example, For example, see U.S. Patent Application Publication No. 2011 / 0250203, the disclosure of which are incorporated by reference as they relate to anti-CD5 antibodies and antigen-binding fragments thereof. incorporated herein.

[0266] Antibodies and fragments thereof for use in conjunction with the compositions and methods described herein The present invention also includes variants of the above antibodies, such as antibody fragments that contain or lack an Fc domain, as well as antibody fragments that contain or lack an Fc domain. , one or more or all of the CDRs of an antibody or antibody fragment described herein or equivalent regions thereof. Protein scaffolds (e.g., 10 Fn3 domain). The original binding fragments include, inter alia, dual variable immunoglobulin domains, single chain Fv molecules, (scFv), diabodies, triabodies, nanobodies, antibody-like protein scaffolds, Fv fragments, Fab fragments, F(ab')2 molecules, and tandem di-scFv Examples include:

[0267] The aforementioned anti-CD5 antibodies, or antigen-binding fragments thereof, can be administered to, for example, a human subject. The present invention can be used in various aspects of the invention described herein, including methods for depleting CD5+ cells in vivo. The aforementioned anti-CD5 antibodies, or antigen-binding fragments thereof, can be used in As described, conjugation to a drug, e.g., a cytotoxin, e.g., an amatoxin, Additional anti-CD5 antibodies that may be used in embodiments of the present invention are described in U.S. Pat. ,500, U.S. Patent Application Publication No. 2011 / 0250203, and U.S. The invention is described in the specification of the patent application 2008 / 0254027, and each of these The entire contents of which are incorporated herein by reference. Additional anti-CD20 antibodies that may be used in embodiments of the present invention 5 Antibodies include those described, for example, in Dillman et al., J. Clin. Oncol. (1 984), 2(8):881-891, and the monoclonal antibody T101 described in As described in Iller et al., Blood (1983), 62(5):988-95 An example is the monoclonal antibody Leu-1.

[0268] In one embodiment, the anti-CD5 antibody or binding fragment thereof comprises a modified Fc region. The modified Fc region alters the affinity of the molecule for Fc gamma R (FcγR). or at least a portion thereof, as compared to the wild-type Fc region, such that the binding thereto is altered. The specific amino acid positions within the Fc region are identified by crystallographic studies. It is known that the amino acids 234 to 239 ( hinge region), amino acids 265–269 (B / C loop), amino acids 297–299 (C ' / E loop), and amino acid 327–332 (F / G) loop. (Sonderma (See nn et al., 2000 Nature, 406:267-273). The antibodies described herein have at least one FcγR that makes direct contact with FcγR based on structural crystallography. In one embodiment, the anti-CD5 antibody (or The Fc region of a polypeptide (or a fragment thereof) is defined as a polypeptide of the invention, which is expressly incorporated herein by reference. bat et al., Sequences of Proteins of Immu nological interest,5th Ed.Public Health Amino Acids 2 according to the EU Index in Service, NH1, MD (1991) Contains an amino acid substitution at position 65. The "EU index in Kabat" is 1 refers to EU antibody numbering. In one embodiment, the Fc region comprises a D265A mutation. In one embodiment, the Fc region comprises a D265C mutation. or fragment thereof) is an Fc region of the In one embodiment, the Fc region comprises an amino acid substitution at amino acid 234. In some embodiments, the Fc region of an anti-CD5 antibody (or fragment thereof) The region contains an amino acid substitution at amino acid 235 according to the EU index in Kabat. In one embodiment, the Fc region comprises a L235A mutation. In a further embodiment, the Fc region comprises L234A and L235A mutations. The c region contains the D265C, L234A, and L235A mutations. In this state, the Fc region contains the D265C, L234A, L235A, and H435A mutations. In a further embodiment, the Fc region comprises D265C and H435A mutations. nothing.

[0269] The antibodies of the invention can be further engineered to introduce additional Fc mutations, such as those described below. The antibody half-life can be further modulated by adding et al. (2006) J Biol Chem 281:23514-24), ( Zalevsky et al. (2010) Nat Biotechnol 28: 157-9), (Hinton et al. (2004) J Biol Chem 2 79:6213-6), (Hinton et al. (2006) J Immunol 176:346-56), (Shields et al. (2001) J Biol Chem 276:6591-604), (Petkova et al. (2006 )Int Immunol 18:1759-69), (Datta-Mannan e (2007) Drug Metab Dispos 35:86-94), ( Vaccaro et al. (2005) Nat Biotechnol 23:1 283-8), (Yeung et al. (2010) Cancer Res 70: 3269-77) and (Kim et al. (1999) Eur J Immuno l 29:2819-25), positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434 and 435, alone or in combination. Exemplary mutations that can be combined are T250Q, M252Y, 1253A, S254 T, T256E, P2571, T307A, D376V, E380A, M428L, H4 33K, N434S, N434A, N434H, N434F, H435A and H435 R mutation.

[0270] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof is an antibody or The antigen-binding fragment binds to a cytotoxin (e.g., a cysteine ​​residue in the Fc domain) In some embodiments, the cysteine ​​residue is conjugated to a cysteine-containing nucleotide. The group is introduced by mutation in the Fc domain of the antibody or antigen-binding fragment thereof. For example, cysteine ​​residues Cys118, Cys239, and Cys26 5. In one embodiment, the anti-CD5 antibody (or a fragment thereof) The Fc region of the IgG1 gene is the amino acid sequence at amino acid 265 according to the EU index in Kabat. In one embodiment, the Fc region comprises a D265C mutation. In , the Fc region contains D265C and H435A mutations.

[0271] Thus, in one embodiment, the Fc region comprises a mutation that results in a decreased half-life. Antibodies with short half-lives are useful in certain instances where the antibody is expected to function as a short-lived therapeutic agent, e.g. For example, in the conditioning step described herein, the antibody is administered followed by the HSCs. Unlike endogenous stem cells, they may generally express CD5. Ideally, the antibody would be substantially removed before delivery of HSCs that are not targeted by anti-CD5 antibodies. In one embodiment, the Fc region comprises an Fc region at position 435 (EU index according to Kabat). In one embodiment, the mutation is a H435A mutation. do.

[0272] The aforementioned anti-CD5 antibodies, or antigen-binding fragments thereof, can be administered to, for example, a human subject. The present invention can be used in various aspects of the invention described herein, including methods for depleting CD5+ cells in vivo. The aforementioned anti-CD5 antibodies, or antigen-binding fragments thereof, can be used in As described, conjugation to a drug, e.g., a cytotoxin, e.g., an amatoxin, It can also be done as follows.

[0273] (Method for identifying anti-CD5 antibodies) High-throughput screening of antibodies or antibody fragment libraries for molecules that bind to CD5. Using a method for high-throughput screening, for conditioning a subject (e.g., a human patient) and / or for use in the methods described herein. The aim of this study is to develop affinity matured drugs that are useful for directly treating cancer or autoimmune diseases, such as Such methods include, among others, phage display, bacterial display, and display, yeast display, mammalian cell display, ribosome display, Display of mRNA and display of cDNA are well known in the art. In vitro display techniques include binding to biologically relevant molecules. The use of phage display to isolate antibodies, or antigen-binding fragments, has been described, e.g. For example, Felici et al.,Biotechnol.Annual Rev.1 :149-183,1995;Katz,Annual Rev.Biophys.Bi omol.Struct. 26:27-45, 1997; and Hoogenboo Reviewed in m et al., Immunotechnology 4:1-20, 1998. The disclosures of each of these relate to in vitro display technology. The contents of which are incorporated herein by reference. Discovery Des.2:251-268,1995 and Kay et al. As described in I., Mol. Divers. 1:139-140, 1996 , randomized combinatorial sequencing to select polypeptides that bind to cell surface antigens. Peptide libraries have been constructed, and each of these disclosures contributes to the discovery of antigen-binding molecules. The present invention is incorporated herein by reference in its entirety. Proteins have been successfully phage-displayed as functional molecules (e.g., EP03 49578; EP4527839; and EP0589877, and Chiswel l and McCafferty, Trends Biotechnol.10:80 1992, each of which discloses in The present invention is incorporated by reference as it relates to the use of in vitro display technology. In addition, functional antibody fragments such as Fab and scFv fragments can be synthesized. The fragment has been expressed in an in vitro display format (e.g., M cCafferty et al.,Nature 348:552- 554,199 0;Barbas et al.,Proc.Natl.Acad.Sci. USA 88:7978-7982, 1991; and Clackson et al., Na 352:624-628, 1991, each of which discloses antigen binding. In vitro display platform for the discovery of synthetic molecules (These techniques involve, among other things, the detection of antibodies that bind to CD5. can be used to identify and improve the affinity of antibodies or antibody fragments that and then requiring hematopoietic stem cell transplantation therapy and / or CD5+ T cells in a patient (e.g., a human patient) suffering from cancer or an autoimmune disease It can be used to deplete cells, B cells, and / or NK cells.

[0274] binds to CD5 on the surface of a cell (e.g., a T cell, a B cell, or a NK cell), e.g. Antibodies taken up by cells by receptor-mediated endocytosis and their antigen-binding Additional techniques can be used to identify the binding fragments. For example, T cell , B cells, or NK cells, and subsequently internalized antibodies that bind to CD5 on the surface of the cells; and To screen for these antigen-binding fragments, the in vitro method described above was used. Phage display technology can be applied to this screen. represents one such technique that can be used in conjunction with the CD5 immunization paradigm. anti-CD5 antibodies that bind to and are subsequently taken up by T cells, B cells, and / or NK cells; and fragments thereof, one skilled in the art can refer to Williams et al. Leukemia 19:1432-1438, 2005. technology, the disclosure of which is incorporated herein by reference in its entirety. For example, mutagenesis of antibodies, antibody flags, etc. may be performed using mutagenesis techniques known in the art. fragments, such as scFv fragments, Fab fragments, diabodies, among others Triabodies, and 10 Fn3 domain or a randomized amino acid cassette (e.g., C One or more or all of DR or its equivalent regions, or an antibody or antibody fragment and generating a recombinant phage library encoding antibodies containing the fragment. The framework region, hinge, Fc domain, and other regions, e.g., human germline antibody sequences or sequences corresponding to human germline antibodies. and non-immunogenic in humans by having sequences that show only minor variations compared to the It can be designed so that

[0275] Phage display techniques described herein or known in the art phage particles, containing randomized antibodies, or antibody fragments, covalently attached to the phage particles. The phage library can be incubated with the CD5 antigen, e.g. For example, first, the phage library is blocked with a blocking agent (e.g., milk protein, bovine blood Incubate with serum albumin and / or IgG to remove nonspecific proteins. Phages encoding antibodies or fragments thereof that exhibit protein binding and Fc domains and then removing phage encoding antibodies or fragments thereof that bind to the The phage library was incubated with a population of CD5+ T cells, B cells, or NK cells. The phage library is incubated with T cells, B cells, or NK cells. Incubate together for a sufficient time to allow the CD5-specific antibodies, or their antigen-binding fragments, to bind to the antigen. The agonist binds to cell surface CD5 and is then taken up by T cells, B cells, or NK cells. (e.g., 30 minutes to 6 hours at 4°C, e.g., 1 hour at 4°C) It binds to and allows for uptake by T cells, B cells, or NK cells. Phages containing antibodies or fragments thereof that do not exhibit sufficient CD5 affinity to The cells can then be washed with, for example, 0.1 M glycine buffer at pH 2.8 (4°C). It can be removed by T cells, B cells, and / or NK cells. Phages bound to the antibodies, or fragments thereof, can e.g., lyse cells. The internalized phage can be identified by recovering them from the cell culture medium. The phages are then transferred to, for example, bacterial cells in 2xYT medium with the recovered phages. in bacterial cells by incubation using methods known in the art. The phage recovered from the culture medium can then be amplified, e.g., The gene(s) encoding the antibody or fragment thereof inserted into the genome The encoded antibody or its These fragments are then synthesized (e.g., antibody fragments such as scFv fragments) De novo, by chemical synthesis (e.g., of antibodies) or recombinant expression (e.g., of full-length antibodies). It can be prepared.

[0276] In vitro synthesis of anti-CD5 antibodies for use in the compositions and methods described herein An exemplary method for evolution is phage display. The fragment may be a CDR of an antibody or a similar region of an antibody-like scaffold (e.g., 10 Fn3 domain A series of mutations or variants designed into the coding sequence of the BC, CD, and DE loops These mutations can be generated by creating a template antibody into which these mutations are introduced. The encoding sequence can be, for example, a native human germline sequence. Mutations can be made using standard mutagenesis techniques known in the art. Thus, each mutant sequence encodes an antibody identical to the template except for one or more amino acid mutations. Retroviral and phage display vectors are known in the art. Compatible protein expression vectors can be engineered using standard vector construction techniques. Phage display for antibody diversification using P3 phage display vectors You can create a play vector.

[0277] The mutated DNA provides sequence diversity, and each transformed phage is encoded by the DNA. We present a single variation of the initial template amino acid sequence and generate a vast number of different but structurally related The present invention provides a phage library that displays a large number of amino acid sequences. Due to the well-defined structure of the region, the amino acid mutations introduced in the phage display screening The difference is that the binding of the binding peptide or domain is altered without significantly changing its overall molecular structure. It is expected to change properties.

[0278] In a typical screen, a phage library is cloned into CD5 or its epitopes. The bound and unbound substances can be easily separated. To facilitate this, it is convenient to immobilize the target on a solid support. The phage can form a complex with the target on the solid support, but the unbound phage The ATP remains in solution and can be washed away with excess buffer. The buffer is then diluted to an extreme pH ( pH 2 or pH 10), changing the ionic strength of the buffer, or adding denaturants The bound phage can then be released from the target by adding phage to the target or by other known means. Cut.

[0279] The recovered phage can then be amplified through infection of bacterial cells, this time resulting in non-binding The antibody binding pool is then depleted and the new pool is enriched for antibodies that bind to CD5. The process can be repeated. Even the recovery of a small number of binding phages is sufficient for subsequent screening. After several rounds of selection, the selected phages in the binding pool are The phage clones selected encode antibodies or antigen-binding fragments thereof. The gene sequence is determined by conventional methods, and the binding affinity of the phage for the target is then determined. During the panning process, the sequence diversity of the population is determined by The number of sequences is reduced with each round of selection until the desired peptide-binding antibodies remain. The antibodies recovered at each round of selection may converge on related antibodies or antigen-binding fragments thereof. An increase in the number of pages is an indication that library convergence has occurred within the screen. .

[0280] Another method for identifying anti-CD5 antibodies is to identify humanized antibodies that bind to CD5, for example, by the following procedure. The non-human antibody that binds to CD5 can be prepared, for example, by the following procedure. Consensus human antibody heavy and light chain sequences can be humanized according to the techniques described in the art. The human germline sequence database (e.g., "VBASE" human germline sequence database; Ka bat et al.Sequences of Proteins of Immun ological interest, Fifth Edition, US De part of Health and Human Services,NI H Publication No. 91 -3242,1991;Tomlinso n et al., J. Mol. Biol. 227:776-798, 1992; and and Cox et al..Eur.J.Immunol.24:827-836,199 4, each of which discloses consensus human antibody heavy and light chain sequences. (The text is hereby incorporated by reference as if fully set forth.) Using established procedures, one skilled in the art can identifies the variable domain framework residues and CDRs of the consensus antibody sequence (e.g., Those skilled in the art can identify the sequence of the humanized antibody. To achieve this, one or more CDs of the heavy and / or light chain variable domains of a consensus human antibody are R can be replaced with one or more corresponding CDRs of a non-human antibody that binds to CD5. This CDR exchange can be performed using any of the methods described herein or known in the art. This can be done using editing techniques.

[0281] An example of a consensus human antibody variable domain is the heavy chain variable domain EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQA PGKGLEWVAVISENGSDTYYADSVKGRFTISRDDSKNTLY LQMNSLRAEDTAVYYCARDRGGAVSYFDVWGQGTLVTVSS (SEQ ID NO: 259) and a light chain variable domain DIQMTQSPSSLSASVGDRVTITCRASQDVSSYLAWYQQKP GKAPKLLIYAASSLESGVPSRFSGSGGSGTDFTLTISSLQP EDFATYYCQQYNSLPYTFGQGTKVEIKRT (SEQ ID NO: 260) and identified in U.S. Patent No. 6,054,297, the disclosure of which is incorporated herein by reference in its entirety. The CDRs in the above sequences are incorporated herein by reference. are shown in bold.

[0282] To produce a humanized antibody, one or more variable region CDRs are ligated to a non-human antibody that binds to CD5. and encoding the above consensus sequence substituted with one or more variable region CDR sequences of the corresponding The polynucleotide can be recombinantly expressed. Since it is primarily determined by the CDR sequences, the resulting humanized antibody will be It is expected that the CD5 affinity of the antibody will be similar to that of the non-human antibody. Methods for determining antibody affinity include, for example, those described herein and known in the art. ELISA-based techniques, as well as surface plasmon resonance, fluorescence anisotropy, and and isothermal titration calorimetry, among others.

[0283] The uptake capacity of the prepared antibodies, or fragments thereof, can be determined by methods known in the art, for example. Radionuclide uptake assays known in the art can be used to assess the In vitro displays as described herein or known in the art Anti-CD5 antibodies, or fragments thereof, identified using the techniques 18 F, 75 B r, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At, 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re, 64 Cu, 67 Cu , 177 Lu, 77 As, 72 As, 86 Y, 90 Y, 89 Zr, 212 Bi, 213 B i, or 225 It can be functionalized by incorporation of radioisotopes such as Ac For example, 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 Radioactive halogens such as At are reacted with electrophilic halogen reagents. Beads such as polystyrene beads (e.g., iodinated beads, ThermoFisher) Her Scientific, Cambridge, MA) using antibodies or fragments thereof. The fragments are capable of being uptaken by T cells, B cells, and / or NK cells. Incubate for a sufficient time (e.g., 30 minutes to 6 hours at 4°C, e.g., 1 hour at 4°C) to The cells can then be washed to remove any unincorporated antibodies or their fragments. Fragments can be removed (e.g., by adding cold (4°C) 0.1 M glycine at pH 2.8). The incorporated antibodies, or their fragments, are then transferred to T cells. Radiation (e.g., gamma rays) emitted from the cytoplasmic reticulum, B cells, and / or NK cells was collected. Identification by detecting radiation (e.g., gamma rays) emitted by the wash buffer It is possible.

[0284] For recombinant production of an anti-CD5 antibody, a nucleic acid encoding the antibody, e.g., as described above, can be used. and one for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures. The sequences can be determined (e.g., specifically for the genes encoding the heavy and light chains of an antibody). by using oligonucleotide probes that can bind to

[0285] Suitable host cells for cloning or expressing antibody-encoding vectors include those described herein. For example, antibodies may be produced from prokaryotic or eukaryotic cells, particularly those described in the literature. When Fc effector functions are not required, antibodies can be produced in bacteria. For bacterial expression of fragments and polypeptides, see, e.g., U.S. Pat. See US Pat. Nos. 648,237, 5,789,199, and 5,840,523 (L.I. Charlton, Methods Describing the Expression of Antibody Fragments in Bacteria in Molecular Biology,Vol.248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. After expression, the antibody is isolated from the bacterial cell paste in a soluble fraction and then purified. It can be further purified.

[0286] Vertebrate cells can also be used as hosts, for example, cells adapted to grow in suspension. Other examples of useful mammalian host cell lines are: : SV40 (COS-7) transformed monkey kidney CV1 strain; human embryonic kidney strain ( For example, Graham et al., J. Gen Virol. 36:59 (197 7) 293 or 293 cells described in); baby hamster kidney cells (BHK); Mouse Sertoli cells (e.g., Mather, Biol. Reprod. 23:243- TM4 cells described in 251 (1980); monkey kidney cells (CV1); African miliary Dorisal kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (M DCK; Buffalo rat hepatocytes (BRL 3A); Human lung cells (W138); Human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, e.g. , Mather et al.,Annals NYAcad.Sci.383:4 4-68 (1982); MRC 5 cells; and FS4 cells. Host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells. cells (Urlaub et al., Proc. Natl. Acad. Sci. USA77 :4216 (1980)); and myeloma cells such as Y0, NS0 and Sp2 / 0. For a review of specific mammalian host cell lines suitable for antibody production, see e.g. For example, Yazaki and Wu,Methods in Molecular Bi ology, Vol.248 (BKCLo,ed.,Humana Pres. s, Totowa, NJ), pp. 255-268 (2003). In the present case, the host cell is eukaryotic, e.g., Chinese hamster ovary (CHO) cells or Lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0287] (Antibody-drug conjugates (ADCs)) (cytotoxin) The antibodies described herein, and antigen-binding fragments thereof (e.g., antibodies that recognize CD5, Antibodies and antigen-binding fragments that bind to Pseudomonas exotoxin A, deBoug anin, diphtheria toxin, amatoxins such as α-amanitin, saporin, maytansin maytansinoids, auristatins, anthracyclines, calicheamicin, irinotecans, Notecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine Azepine dimers, indolinobenzodiazepines, and indolinobenzodiazepine dimers cytotoxins, such as cytotoxins, or variants thereof, or any of the cytotoxins described herein or in the art. Conjugation to other cytotoxic compounds known in the art for the purposes of: (i) directly treating a cancer or autoimmune disease as described herein; or (ii) transplanted into a patient (e.g., a human patient) in need of hematopoietic stem cell transplantation therapy. Depletion of endogenous immune cells to prevent or reduce the likelihood of hematopoietic stem cell rejection during transplantation In some embodiments, the antibody, or antigen-binding fragment thereof, is lysed. Following internalization, the cytotoxin accesses intracellular targets and is secreted by endogenous T cells, B cells, and Cytotoxic molecules are incorporated into antibodies, or other molecules, so that they can kill NK cells and / or and the antigen-binding fragment of the antibody is conjugated to the antibody. Suitable cytotoxins suitable for use with include, among others known in the art, D NA intercalators (e.g., anthracyclines), drugs that can disrupt the mitotic spindle agents (e.g., vinca alkaloids, maytansine, maytansinoids, and their derivatives) conductors), RNA polymerase inhibitors (e.g., amatoxins such as α-amanitin, and and its derivatives), drugs that can inhibit protein biosynthesis (e.g., saporin and and drugs that exhibit rRNA N-glycosidase activity, such as ricin A chain.

[0288] In some embodiments, the cytotoxin of the antibody-drug conjugate inhibits RNA polymerase activity. In some embodiments, the RNA polymerase inhibitor is an amatoxin. or a derivative thereof.

[0289] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, and Manitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamine These include amanulin, amanuric acid, and proamanuline. The structure of synthon is represented by formula III and is described, for example, in Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459 are.

[0290] In one embodiment, the cytotoxin is amanitin. These antigen-binding fragments are conjugates of the formula Ab-ZL-Am. Ab may be an antibody or its antigen-binding fragment. ment, L is a linker, Z is a chemical moiety, and Am is an amatoxin. Many positions on the amatoxins or their derivatives are compatible with the linking site L and thus with the antibody or can serve as a site for covalent attachment of such antigen-binding fragments. For example, the antibodies and antigen-binding fragments described herein can be represented by the formula Ab-ZL-Am. Ab may be an antibody or a toxin to form a conjugate that is or an antigen-binding fragment thereof, Z is a chemical moiety, L is a linker, and Am is an amatoxin. In some embodiments, Am-LZ is represented by formula (I): [ka] (wherein R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C, or R D and; R4 is H, OH, OR C , OR D , R C , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, OR C , OR D , R C , or R D and; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D and; R9 is H, OH, OR C , or OR D and; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a linker, for example, an optionally substituted alkylene (e.g., C1-C6 alkylene). cyclohexane), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic groups; Alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroaryl -C(=O)-, a peptide, or a combination thereof; Z is a compound that combines the reactive substituents present on L with an antibody that binds to CD5, or an antigen-binding fragment thereof. A chemical moiety formed by coupling with a reactive substituent present in the fragment. be).

[0291] In some embodiments, Am is exactly one R C Contains substituents.

[0292] In some embodiments, the linker is -(CH) 2n -unit, n is an integer between 2 and 6 In some embodiments, the linker is -((CH) n and n is 6. In some embodiments, LZ is [ka] where S is a nucleotide present in an antibody, or antigen-binding fragment thereof, that binds to CD117. is a sulfur atom representing a reactive substituent (e.g., from the -SH group of a cysteine ​​residue) )

[0293] In some embodiments, LZ is [ka] is.

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

[0295] In some embodiments, Am-LZ-Ab is: [ka] is.

[0296] In some embodiments, Am-LZ is represented by formula (IA): [ka] (wherein R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D and; R4 is H, OH, OR C , OR D , R C , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, OR C , OR D , R C , or R D and; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D and; R9 is H, OH, OR C , or OR D and; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a linker, for example, an optionally substituted alkylene (e.g., C1-C6 alkylene). cyclohexane), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic groups; Alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heptane Tetraarylene, dipeptide, -C(=O)-, peptide, or a combination thereof can be; Z is a compound that combines the reactive substituents present on L with an antibody that binds to CD5, or an antigen-binding fragment thereof. A chemical moiety formed by coupling with a reactive substituent present in the fragment. can be; where Am is exactly one R C (including substituents).

[0297] In some embodiments, the linker is -((CH) n where n is 6. In some embodiments, LZ is [ka] is.

[0298] In some embodiments, LZ is [ka] is.

[0299] In some embodiments, Am-LZ-Ab is: [ka] is.

[0300] In some embodiments, Am-LZ-Ab is: [ka] is.

[0301] In some embodiments, Am-LZ is represented by formula (IB): [ka] (wherein R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached, if present. forming an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D and; R4 is H, OH, OR C , OR D , R C , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, OR C , OR D , R C , or R D and; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D and; R9 is H, OH, OR C , or OR D and; X is -S-, -S(O)-, or -SO2-; R C is -LZ; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a linker, for example, an optionally substituted alkylene (e.g., C1-C6 alkylene). cyclohexane), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroaromatic groups; Alkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroaryl -C(=O)-, a peptide, or a combination thereof; Z is a compound that combines the reactive substituents present on L with an antibody that binds to CD5, or an antigen-binding fragment thereof. A chemical moiety formed by coupling with a reactive substituent present in the fragment. can be; where Am is exactly one R C (including substituents).

[0302] In some embodiments, the linker L and chemical moiety Z together form LZ. considered, [ka] is.

[0303] In some embodiments, the LZ is: [ka] is.

[0304] In some embodiments, Am-LZ-Ab is: [ka] is.

[0305] In some embodiments, Am-LZ-Ab is: [ka] is.

[0306] In some embodiments, R A and R B along with the oxygen atoms to which they are attached , combined to form the formula: [ka] (Wherein, Y is —C(═O)—, —C(═S)—, —C(═NR E )-, or -C( R E R E’ )-and; R E and R E’ are each independently an optionally substituted C-C alkylene-R C , optionally substituted C1-C6 heteroalkylene-R C , optionally substituted C2-C6 Alkenylene-R C , optionally substituted C2-C6 heteroalkenylene-R C , arbitrarily placed Substituted C2-C6 alkynylene-R C , optionally substituted C2-C6 heteroalkynyl N-R C , optionally substituted cycloalkylene-R C , optionally substituted heterocycloa Rukiren-R C , optionally substituted arylene-R C or optionally substituted heteroaryl Liren-R C ) to form a 5-membered heterocycloalkyl group.

[0307] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB), and R is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached. [ka] Forming; R3 is H or R C and; R4 is H, OH, OR C , OR D , R C , or R D and; R5 is H, OH, OR C , OR D , R C , or R D and; R6 is H, OH, OR C , OR D , R C , or R Dand; R7 is H, OH, OR C , OR D , R C , or R D and; R8 is OH, NH2, OR C , or NHR C and; R9 is H or OH; R C and R D are as defined above.

[0308] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 is H, OH, OR A , or OR C and; R2 is H, OH, OR B , or OR C and; R A and R B are bonded together with the oxygen atoms to which they are attached. [ka] Forming; R3 is H or R C and; R4 and R5 each independently represent H, OH, OR C , R C , or OR D and ; R6 and R7 are each H; R8 is OH, NH2, OR C , or NHR C and; R9 is H or OH; X and R C is as defined above.

[0309] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 is H, OH, or ORA and; R2 is H, OH, or OR B and; R A and R B are bonded together with the oxygen atoms to which they are attached. [ka] Forming; R3, R4, R6, and R7 are each H; R5 is OR C and; R8 is OH or NH2; R9 is H or OH; X and R C is as defined above. Such amatoxin conjugates is described, for example, in U.S. Patent Application Publication No. 2016 / 0002298, No. 6,229,699, the disclosure of which is incorporated herein by reference in its entirety.

[0310] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 and R2 are each independently H or OH; R3 is R C and; R4, R6, and R7 are each H; R5 is H, OH, or OC1-C6 alkyl; R8 is OH or NH2; R9 is H or OH; R C is as defined above. Such amatoxin conjugates may be, for example, For example, see U.S. Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated herein by reference in its entirety.

[0311] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are each independently H, OH, or OR C , or R C and; R8 is OH or NH2; R9 is H or OH; R C is as defined above. Such amatoxin conjugates may be, for example, For example, see U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0312] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): R1 and R2 are each independently H or OH; R3, R6, and R7 are each H; R4 and R5 are each independently H or OH; R8 is OH, NH2, OR C , or NHR C and; R9 is H or OH; X and R C is as defined above. Such amatoxin conjugates See, for example, U.S. Pat. Nos. 9,233,173 and 9,399,681, and and U.S. Patent Application Publication No. 2016 / 0089450, and each of these No. 6,299,499, the disclosures of which are incorporated herein by reference in their entireties.

[0313] In accordance with the compositions and methods described herein, antibodies, or antigen-binding fragments thereof, Additional amatoxins that can be used for conjugation to 42049; WO 2016 / 071856; and WO 2017 / 046658 No. 6,239,999, the disclosures of each of which are incorporated herein by reference in their entirety.

[0314] In some embodiments, Am-LZ is represented by Formula (II), Formula (IIA), or Formula (II B) is represented by: [ka] or [ka] wherein X is S, SO, or SO; R is H or a linker, The reactive substituents present on the linker and the the antibody or a linker covalently linking the antigen-binding fragment to the a reactive group present on the linker and an antibody or its antigen-binding fragment; via chemical moiety Z formed by coupling reaction with reactive substituents present in the methyl group. is a linker covalently attached to the antibody or antigen-binding fragment thereof via when R2 is H, R1 is a linker).

[0315] In some embodiments, the linker is -(CH) n -unit, n is an integer between 2 and 6 In some embodiments, R1 is a linker, R2 is H, and the linker and The and chemical moieties together form the LZ, [ka] is.

[0316] In some embodiments, Am-LZ-Ab is [ka] It is one of them.

[0317] In some embodiments, the cytotoxin is α-amanitin. α-amanitin is a compound of formula III. In some embodiments, the α-amanitin of formula III -Amanitin binds to CD5 via a linker L, or its antigen-binding flag The linker L may be attached to any one of several possible positions (e.g. For example, R 1 ~R 9 (any of the compounds of formula III) binds to α-amanitin of formula I, IA, IB α-amanitin-linker conjugates of II, IIA, or IIB may be provided. In some embodiments, the linker is located at position R 1 In some embodiments, , the linker is at position R 2 In some embodiments, the linker is attached at position R 3 in In some embodiments, the linker is located at position R 4 Some implementations In the embodiment, the linker is located at position R 5 In some embodiments, the linker is attached at position Place R 6 In some embodiments, the linker is attached at position R 7 Combine with. In some embodiments, the linker is located at position R 8 In some embodiments, the linker - is position R 9In some embodiments, the linker is a hydrazine, disulfide, or In some embodiments, the linker comprises a thioether, a dipeptide, a thioether, or a dipeptide. The dipeptide is selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the site PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. (C=O)(CH2) n -unit, and n is an integer of 1 to 6.

[0318] In some embodiments, the linker is -(CH) n -unit, n is an integer between 2 and 6 In some embodiments, the linker is -PAB-Cit-Val-((C= O)(CH2) n In some embodiments, the linker is -PAB-Ala- Val-((C=O)(CH2) n In some embodiments, the linker L and and chemical moiety Z are considered together as LZ, [ka] is.

[0319] Antibodies and antigen-binding fragments for use in the compositions and methods described herein The α-amino acids can be conjugated to α-amino acids using conjugation techniques known to those skilled in the art or described herein. It can be conjugated to an amatoxin such as manitin or a variant thereof. For example, antibodies that recognize and bind to CD5, and antigen-binding fragments thereof, are α-amyloids. and U.S. Pat. No. 6,229,693. As described in the specification of application publication no. 2015 / 0218220, the disclosure of which is , for example, amatoxins such as α-amanitin and its variants, as well as covalently bound conjugates. The present invention is incorporated by reference as it relates to covalent linkers that can be used in conjugates. Synthetic methods for producing amatoxins are described, for example, in U.S. Pat. 76,702, which is related to the synthetic methods disclosed therein. and is incorporated herein by reference.

[0320] Antibodies, or antigen-binding fragments, for use in the compositions and methods described herein The α-amino acids can be conjugated to α-amino acids using conjugation techniques known to those skilled in the art or described herein. It can be conjugated to an amatoxin such as manitin or a variant thereof. For example, antibodies, or antigen-binding fragments thereof, that recognize and bind to CD5 are α-ama The compound can be conjugated to an amatoxin such as nitine or a variant thereof, and is disclosed in U.S. Pat. As described in the specification of Patent Application Publication No. 2015 / 0218220, the disclosure of which The compounds include amatoxins, such as α-amanitin and its variants, as well as covalently bound covalent bonds. For covalent linkers that can be used in conjugates, see incorporated herein.

[0321] Exemplary antibody-drug conjugates useful in conjunction with the methods described herein are antibodies, or or an antigen-binding fragment thereof with the reactivity on said antibody or antigen-binding fragment thereof Reaction with amatoxins conjugated to linkers containing substituents suitable for reaction with the residue The antibody or antigen-binding fragment thereof described herein may be formed by Amatoxins conjugated to linkers containing substituents suitable for reaction with reactive residues include , including, but not limited to: 7'C-(4-(6-(maleimido)he (Xanoyl)piperazin-1-yl)-amatoxine; 7'C-(4-(6-(maleimidazole) 7'C-(4-(6-( 6-(Maleimido)hexanoyl)piperazin-1-yl)-Amatoki Syn;7'C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperidine 7'C-(4-(6-(4-((maleimido)methyl)-lazin-1-yl)-amatoxin; (I)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)-amatoki Syn;7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperidine -1-yl)-amatoxin;7'C-(4-(2-(6-(6-(maleimido)hexa 7'C-hexanamido)ethyl)piperidin-1-yl)-amatoxine; (4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl) Piperidin-1-yl)-amatoxin; 7'C-(4-(2-(6-(4-((Male Amido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidine- 1-yl)-amatoxin;7'C-(4-(2-(3-carboxypropanamido)e 7'C-(4-(2-(2-bromoacetate) 7'C-(4-(2-(3 -(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl 7'C-(4-(2-(4-(maleimido)butanamido)ethyl)-amatoxin; )piperidin-1-yl)-amatoxine;7'C-(4-(2-(maleimido)acetyl) 7'C-(4-(3-(maleimido)propionyl)piperazin-1-yl)-amatoxin; (4-(4-(maleimide)-(4-((aminoyl)piperazin-1-yl)-amatoxin; )butanoyl)piperazin-1-yl)-amatoxine;7'C-(4-(2-(6-( 4-((Maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl )piperidin-1-yl)-amatoxine; 7'C-(3-((6-(maleimido)hexyl) 7'C-(3-((6- (6-(maleimido)hexanamido)hexanamido)methyl)pyrrolidin-1-yl )-Amatoxin;7'C-(3-((4-((maleimido)methyl)cyclohexane 7'C-(3-((6 -((4-(maleimido)methyl)cyclohexanecarboxamido)hexanamide)methyl 7'C-(4-(2-(6-(2-(amatoxin) Aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amato 7'C-(4-(2-(4-(2-(aminooxy)acetamido)butanamido) 7'C-(4-(4-(2-(amatoxin) 7'-aminoxy)acetamido)butanoyl)piperazin-1-yl)-amatoxine C-(4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazine-1 -yl)-amatoxin;7'C-((4-(6-(maleimido)hexanamide)piperazine Lysin-1-yl)methyl)-amatoxin; 7'C-((4-(2-(6-(maleimine hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C -((4-(6-(maleimido)hexanoyl)piperazin-1-yl)methyl)-amannoyl Toxin; (R)-7'C-((3-((6-(maleimido)hexanamido)methyl) (S)-7'C-((3-((6-( Maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin ;7'C-((4-(2-(6-(6-(maleimido)hexanamide)hexanamide )ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-( 4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidine-1 -yl)methyl)-amatoxin;7'C-((4-(2-(6-(4-((maleimide )Methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidine-1- 7'C-((4-(2-(6-(maleimido)hexane) Amido)ethyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-( 2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazine -1-yl)methyl)-amatoxin;7'C-((4-(2-(4-((maleimide) Methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl)-a 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexane (Hexanamide) ethyl) piperazin-1-yl) methyl) - ama Toxin; 7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido 7'C-((3-amino)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxin; -((6-(6-(maleimido)hexanamido)hexanamido)-R-methyl)pyro Lysin-1-yl)methyl)-amatoxin;7'C-((3-((4-((maleimide )Methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-1-yl)methyl 7'C-((3-((4-((maleimido)methyl)cyclohexyl) (R-methyl)pyrrolidin-1-yl)methyl)-amatoxin;7 'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamide )Hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C- ((4-(2-(3-carboxypropanamido)ethyl)piperazin-1-yl)methyl 7'C-((4-(6-(6-(maleimido)hexanamide) Xanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(6- (4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)pipera 7'C-((4-(2-(maleimido)acetate)-4 ... 7'C-((4-(3-(maleimide)piperazin-1-yl)methyl)-amatoxine; Imido)propanoyl)piperazin-1-yl)methyl)-amatoxine; 7'C-(( 4-(4-(maleimido)butanoyl)piperazin-1-yl)methyl)-amatoxine 7'C-((4-(2-(2-(maleimido)acetamido)ethyl)piperidine-1 -yl)methyl)-amatoxin;7'C-((4-(2-(4-(maleimido)butane Amido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-( 2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanea 7'C-((3-(( 6-(Maleimido)hexanamido)methyl)azetidin-1-yl)methyl)-amato 7'C-((3-(2-(6-(maleimido)hexanamido)ethyl)azetidin 7'C-((3-((4-((maleimide) Methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-azetidin-1-yl 7'C-((3-(2-(4-((maleimido)methyl)cyclohexane 7'C-((3 -(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamide)hexa 7'C-(((2 -(6-(maleimido)-N-methylhexanamido)ethyl)(methyl)amino)methyl 7'C-(((4-(6-(maleimido)-N-methylhexane) 7'C-((2-(2-(6 -(Maleimido)hexanamido)ethyl)aziridin-1-yl)methyl)-Amatoki Syn;7'C-((2-(2-(6-(4-((maleimido)methyl)cyclohexane Ruboxamido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxyl 7'C-((4-(6-(6-(2-(aminooxy)acetamido)hexaneamine 7'C-((4-(hexanoyl)piperazin-1-yl)methyl)-amatoxine (1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azahene Butadecan-17-oyl)piperazin-1-yl)methyl)-amatoxin;7'C- ((4-(2-(2-(aminooxy)acetamido)acetyl)piperazin-1-yl )methyl)-amatoxin;7'C-((4-(3-(2-(aminooxy)acetamido 7'C-((4 ... (4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)methyl 7'C-((4-(2-(6-(2-(aminooxy)acetamino) hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C -((4-(2-(2-(2-(aminooxy)acetamido)acetamido)ethyl) Piperidin-1-yl)methyl)-amatoxin; 7'C-((4-(2-(4-(2- (Aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)methyl )-Amatoxin;7'C-((4-(20-(aminooxy)-4,19-dioxo- 6,9,12,15-tetraoxa-3,18-diazaicosyl)piperidin-1-yl )methyl)-amatoxin;7'C-(((2-(6-(2-(aminooxy)acetate Amido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxy 7'C-(((4-(6-(2-(aminooxy)acetamido)-N-methylhexyl 7'C-((3-( ... (6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamide )methyl)pyrrolidin-1-yl)-S-methyl)-amatoxine;7'C-((3-( (6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamide )-R-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((4-( 2-(2-Bromoacetamido)ethyl)piperazin-1-yl)methyl)amatoxy 7'C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl )methyl)-amatoxin;7'C-((4-(2-(3-(pyridin-2-yldisulfonyl) (phenyl)propanamido)ethyl)piperidin-1-yl)methyl)-amatoxine; 6'O-(6-(6-(maleimido)hexanamido)hexyl)-amatoxin;6' O-(5-(4-((maleimido)methyl)cyclohexanecarboxamido)pentyl) -Amatoxin; 6'O-(2-((6-(maleimido)hexyl)oxy)-2-ox 6'O-((6-(maleimido)hexyl)carbamoyl)-amatoxin -Amatoxin; 6'O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexa 6'O-(6-(2-bromoacetamido)hexyl)-amatoxin; 7'C-(4-(6-(azido)hexanamido)piperidine- 1-yl)-amatoxin;7'C-(4-(hex-5-ynoylamino)piperidine -1-yl)-amatoxin;7'C-(4-(2-(6-(maleimido)hexaneamino) 7'C-(4-(2-(6-(6 -(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)- Amatoxin; 6'O-(6-(6-(11,12-didehydro-5,6-dihydro-di Benzo[b,f]azocin-5-yl)-6-oxohexanamido)hexyl)-aza Toxin; 6'O-(6-(hex-5-ynoylamino)hexyl)-amatoxin; 6'O-(6-(2-(aminooxy)acetylamido)hexyl)-amatoxin;6 'O-((6-aminooxy)hexyl)-amatoxin; and 6'O-(6-(2- iodoacetamido)hexyl)-amatoxin. The aforementioned linkers are, inter alia, those described herein. and, for example, U.S. Patent Application Publication No. 2015 / 0159994. No. 0218220, the disclosure of which is incorporated herein by reference in its entirety. will be incorporated into

[0322] For use in the direct treatment of cancer, autoimmune conditions, or in preparation for hematopoietic stem cell transplantation treatments Recognize and bind CD5 for conditioning a patient (e.g., a human patient). Additional cells that can be conjugated to antibodies and antigen-binding fragments thereof Cytotoxins include, among others, but are not limited to: 5-ethynyluracil ru, abiraterone, acylfulvene, adecipenol, adzelesin, aldesleukin , altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, Amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, Angiogenesis inhibitors, antarelix, anti-dorsal morphogenetic protein-1, anti-androgen anti-cancer drugs, anti-estrogens, anti-neoplastic agents, antisense oligonucleotides, affinity Dicholine glycinate, apoptotic gene modulator, apoptosis regulator -, apurinic acid, asulaculin, atamestane, atlimustine, axinastatin 1, Axinastatin 2, Axinastatin 3, Azasetron, Azatoxin, Azatyrosine , baccatin III derivatives, balanol, batimastat, BCR / ABL antagonists beta-lactam derivatives, beta- Retin, betaclamycin B, betulinic acid, bFGF inhibitors, bicalutamide, bisantre bisaziridinyl spermine, bisnafide, bisstraten A, bizelesin, brev bleomycin A2, bleomycin B2, bropirimine, budotitanium, buthio thiamin sulfoximine, calcipotriol, calphostin C, camptothecin derivatives (e.g. For example, 10-hydroxy-camptothecin), capecitabine, carboxamido-amino- Triazoles, carboxyamidotriazoles, carzelesin, casein kinase inhibitors , castanospermine, cecropin B, cetrorelix, chlorin, chloroquinoxal sulfonamides, cicaprost, cis-porphyrins, cladribine, clomiphene and and its analogs, clotrimazole, colismycin A, colismycin B, combreta Statin A4, combretastatin analogues, conagenin, clambecidin 816, chris Nutol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentasiloxane Anthraquinone, cycloplatin, cypemycin, cytarabine octphosphate, cells Lytic factors, cytostatin, dacliximab, decitabine, dehydrodidemnin B, 2' Deoxycoformycin (DCF), deslorelin, dexphosphamide, dexlazo Xanthone, dexverapamil, diazicon, didemnin B, didox, diethylnorspe Lumin, dihydro-5-azacytidine, dihydrotaxol, dioxamycin, diphen Nirspiromustine, discodermolide, docosanol, dolasetron, doxiflurry Jin, droloxifene, dronabinol, duocarmycin SA, ebselen, eco Mustine, edelfosine, edrecolomab, eflornithine, elemene, emiteflu, Epothilone, epithilone, epristeride, estramustine and its analogues, etopo Etoposide, etoposide 4'-phosphate (also known as etopophos), exemestane, and fad Lozole, fazarabine, fenretinide, filgrastim, finasteride, flavonoids Piridol, flazelastine, fluasterone, fludarabine, fluorodaunorni hydrochloride Syn, Forfenimex, Formestane, Fostriecin, Fotemustine, Gado Gallium texaphyrin, gallium nitrate, gallocitabin, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, homoharringtonine (H HT), hypericin, ibandronic acid, idoxifene, idramantone, ilmofosine , Ilomastat, Imidazoacridone, Imiquimod, Immunostimulating peptides, Iobengua Iododoxorubicin, Ipomeanol, Irinotecan, Ilopract, Irsogura Zin, Isobengazole, Jasplakinolide, Kahalalide F, Lamellarin-N Triacetate Tate, lanreotide, leinamycin, lenograstim, lentinan sulfate, Leptolus tatin, letrozole, lipophilic platinum compounds, lysocrinamide 7, lobaplatin, romethon Rexol, lonidamine, loxoxantrone, loxoribine, lurtotecan, lutetium Mutexaphyrin, rhizophyllin, massoprocol, maspin, matrix metalloprotease enzyme inhibitors, menogaril, lunervalon, meterelin, methioninase, metoclopramide Plamid, MIF inhibitors, ifepristone, miltefosine, millimostim, mitralactone mitoguazone, mitolactol, mitomycin and its analogues, mitonafide, Mitoxantrone, mofarotene, molgramostin, mycaperoxide B, myriapo Ron, N-acetyldinaline, N-substituted benzamides, nafarelin, Nagresschip, Napavine, nafterpin, nartograstim, nedaplatin, nemorubicin, neridrone Acid, nilutamide, nisamycin, nitrulline, octreotide, oxenon, onapris Ondansetron, Oracin, Ormaplatin, Oxaliplatin, Oxauno Isin, paclitaxel and its analogues, palauamine, palmitoylrhizoxin, paclitaxel Midronic acid, panaxytriol, panomyphen, parabactin, pazelliptin, pegasus Pargase, Perdecin, Pentosan Polysulfate Sodium, Pentostatin, Pentrozolo perflubron, perfosfamide, phenazinomycin, picibanil, piral Bicin, piritrexim, podophyllotoxin, porfiromycin, purine nucleosides Doxorubicin inhibitors, raltitrexed, rhizoxin, rogletimide, rohitukin, Rubiginone B1, Ruboxyl, Safingol, Saintpin, Sarcophytol A, Rugramostim, sobuzoxane, sonermin, sparfosinic acid, spicamycin D, Lomustine, stipiramide, sulfinosine, tallimustine, tegafur, temozolomide , teniposide, thaliblastine, thiocoraline, tirapazamine, topotecan, topsenti vinorelbine, triciribine, trimetrexate, veramine, vinorelbine, vinxartine, bolus Lozole, Zeniplatin, and Zilascorub.

[0323] In some embodiments, the cytotoxin has formula (IV): [ka] (IV) It is a pyrrolobenzodiazepine dimer represented by the formula:

[0324] A variety of linkers can be used to link the antibodies and antigen-binding fragments (e.g., For example, antibodies that recognize and bind to CD5 or their antigen-binding fragments can be combined with cytotoxic molecules. It can be conjugated. As used herein, the term "linker" refers to a linker that is attached to an antibody or fragment thereof (Ab). is covalently attached to a drug moiety (D) to form an antibody-drug conjugate (ADC; Ab- Divalent chemicals involving a covalent bond or chain of atoms forming a ZLD, where D is a cytotoxin A suitable linker has two reactive ends, one for conjugation to the antibody. One for the gate and the other for the conjugate to the cytotoxin. The conjugate reactive end (reactive site, Z) typically corresponds to a cysteine ​​thiol on the antibody. is a moiety that can be conjugated to an antibody via a carboxyl group or a lysine amine group. , thus typically containing a thiol-reactive group such as a double bond (such as in a maleimide), or a leaving group such as chloro, bromo, iodo, or R-sulfanyl group, or a carboxyl group the amine-reactive group, such as a carboxyl group; whereas the antibody-conjugate reactive end of the linker The terminus is typically an amide bond with a basic amine or carboxyl group on the cytotoxin. A site that can be conjugated to a cytotoxin via the formation of a cytotoxin. Typically, it is a carboxyl group or a basic amine group. When used to describe a linker of the form, between the linker and / or the cytotoxin, if and linker and / or antibody or antigen-binding fragment thereof. For example, reactive moiety Z is a chemical moiety that lacks one or both reactive ends. converted to position Z) or incompletely (e.g., only the carbonyl of the carboxylic acid Such conjugation reactions are further described herein below. do.

[0325] In some embodiments, cleavage of the linker releases the drug unit from the antibody in an intracellular environment. In yet another embodiment, the linker is cleavable under intracellular conditions to release The linker unit is not cleaved and the drug is released, for example, by antibody degradation. Linkers useful in ADCs of the present invention are preferably stable extracellularly and prevent aggregation of the ADC molecules. This keeps the ADC freely soluble in aqueous media and in a monomeric state. Prior to delivery, the ADC preferably remains stable and intact, i.e., the antibody The linker remains linked to the drug moiety. The linker is stable outside the target cell and An effective linker is one that (i) binds specifically to the antibody and can be cleaved at a fairly efficient rate within the antibody. (ii) maintain the binding properties; (iii) enable intracellular delivery of the conjugate or drug moiety; ii) the conjugate remains stable and intact until delivered or transported to its target site; (iv) the cytotoxicity of the cytotoxic moiety, i.e., remains uncleaved; The stability of the ADCs was assessed by mass spectrometry, HPLC, and and separation / analysis techniques such as LC / MS. Covalent bonding to the functional moiety requires that the linker has two reactive functional groups, i.e., reactive It is necessary to have bivalency in the sense of peptides, nucleic acids, drugs, toxins, antibodies, haptens, etc. and reporter groups. Bivalent linker reagents useful for this purpose are known, and methods are known to those skilled in the art to The jugate has been described (Hermanson, GT (1996) Bio conjugate Techniques;Academic Press:New York, pp. 234-242).

[0326] The linker may be, for example, hydrolyzed by enzymes, photolysis, hydrolysis under acidic conditions, or hydrolysis under basic conditions. Cleavage by hydrolysis, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage under Examples include those that can be used for the synthesis of hydroxybenzoates (e.g., Leriche et al., Bioorg. M ed. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference. The present invention is incorporated by reference herein as it relates to suitable linkers for conjugates. (This is the case.)

[0327] Examples of linkers that can be hydrolyzed under acidic conditions include hydrazones and semicarbazones. , thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketters (See, for example, U.S. Patent Nos. 5,122,368; 5,824,805; No. 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123;Neville et al. al., 1989, Biol. Chem. 264:14653-14661; Each of these disclosures discloses, as related to linkers suitable for covalent conjugates: The entire contents of which are incorporated herein by reference. Such linkers may be used in a variety of media, such as blood. It is relatively stable under neutral pH conditions, but is stable at pH 5.5 or 5, which are the approximate pH of the lysosome. It is unstable below .0.

[0328] Examples of linkers that are cleavable under reducing conditions include disulfides. Various disulfide linkers are known in the art, for example, those formed using Examples of compounds that can be used include: SATA (N-succinimidyl-S-acetylthiol) acetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate nate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha -(2-pyridyl-dithio)toluene), SPDB and SMPT (e.g., Thorp e et al.,1987,Cancer Res. 47:5924-5931;W awrzynczak et al.,In Immunoconjugates:An tibody Conjugates in Radioimagery and Th erapy of cancer (CW Vogel ed.,Oxford U.S. Press, 1987. See also U.S. Pat. No. 4,880,935, which The disclosure of which is incorporated herein by reference in its entirety as it relates to linkers suitable for covalent conjugates. is incorporated herein by reference.

[0329] Examples of linkers useful in the synthesis of drug-antibody conjugates include, among others, Michael acceptors. derivatives (e.g., maleimides), activated esters, electron-deficient carbonyl compounds, and aldehydes. Amine or thiol sites present in an antibody or antigen-binding fragment, such as a hydroxyl group For example, drugs containing electrophilic species suitable for reaction with nucleophilic substituents such as Suitable linkers for the synthesis of the dimer conjugates include, but are not limited to, 4-( N-Maleimidomethyl)-cyclohexane-L-carboxylate succinimidyl (SMCC ), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleic acid Midobenzoyl-N-hydroxysuccinimidyl 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. The linkers are incorporated herein by reference. These include non-cleavable microtubules, which are particularly useful for conjugating microtubule-disrupting agents such as auristatins. and Doronina et al., Bioco injugate Chem. 17:14-24, 2006, the disclosure of which is , which are incorporated herein by reference as they relate to linkers for chemical conjugates. Additional linkers suitable for the synthesis of the drug-antibody conjugates described herein include: those capable of releasing cytotoxins by a 1,6-elimination process ("self-immolative" groups); For example, p-aminobenzyl alcohol (PABC), 6-maleimidohexanoic acid, pH Sensitive carbonate, and Jain et al., Pharm. Res. 32:35 26-3540, 2015, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the linker includes the aforementioned PAB or a self-immolative group such as PABC (para-aminobenzyloxycarbonyl), These are described, for example, in Carl et al., J. Med. Chem. (1981) 24 :479-480;Chakravarty et al(1983) J.Med.Ch em. 26:638-644; U.S. Patent No. 6,214,345; U.S. Patent Application Publication No. No. 030130189; U.S. Patent Application Publication No. 20030096743; U.S. Japanese Patent No. 6759509; U.S. Patent Application Publication No. 20040052793 ;U.S. Patent No. 6,218,519;U.S. Patent No. 6,835,807;U.S. Patent 6268488; U.S. Patent Application Publication No. 20040018194; WO9 8 / 13059; U.S. Patent Application Publication No. 20040052793; U.S. Patent No. 66 77435; U.S. Pat. No. 5,621,002; U.S. Pat. App. Pub. No. 2004 This process is disclosed in WO2004 / 032828. Other such chemical moieties capable of effecting linkage ("self-immolative linkers") include methyl carbamates and heteroaryl groups, e.g., aminothiazole, aminoimidazole , aminopyrimidine, etc. Linkers containing such heterocyclic self-immolative groups include, for example, For example, U.S. Patent Publication Nos. 20160303254 and 20150079114, and U.S. Pat. No. 7,754,681; Hay et al. (1999) Bi oorg.Med.Chem.Lett.9:2237;U.S. Patent Application Publication No. 2005 / Specification No. 0256030; de Groot et al (2001) J.Org.Ch em. 66:8815-8830; and U.S. Pat. No. 7,223,837. are.

[0330] Linkers that are susceptible to enzymatic hydrolysis may be used, for example, to transport proteins to the lysosome or endosome. Intracellular peptidase or protease enzymes, including but not limited to proteases The linker may be a peptide-containing linker that is cleaved by the One advantage of using an antibody is that the therapeutic agent is usually attenuated when in a conjugated state. In some embodiments, the serum stability of the conjugates is generally high. , the peptidyl linker is at least two amino acids long or at least three amino acids long. Exemplary amino acid linkers are dipeptides, tripeptides, tetrapeptides or Examples of suitable peptides include valine, alanine, citrulline, amino acids such as phenylalanine, lysine, leucine, and glycine The amino acid residues constituting the amino acid linker component include those containing natural amino acids. , as well as minor amino acids and unnatural amino acid analogs such as citrulline Exemplary dipeptides include valine-citrulline (vc or val-cit) and valine-citrulline (vc or val-cit). Alanine-phenylalanine (af or ala-phe) is an exemplary tripeptide. The peptides include glycine-valine-citrulline (gly-val-cit) and glycine In some embodiments, the amino acid sequence of ... The linker is Val-Cit, Ala-Val, or Phe-Lys, Val-L ys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-A rg, or containing dipeptides such as Trp-Cit, Val-Cit or Phe-L Linkers containing dipeptides such as ys are described, for example, in U.S. Pat. No. 6,214,345. The disclosure relates to linkers suitable for covalent conjugates. The entire contents of which are incorporated herein by reference. The anchor comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the dipeptide is used in combination with a self-immolative linker.

[0331] Linkers suitable for use herein further include C1-C6 alkylene, C1-C 6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2- C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, and a combination thereof. The group may include one or more groups selected from the group consisting of aryl, ... Non-limiting examples of such groups include (CH2) n , (CH2CH2O) n , and -(C=O )(CH2) n -unit, n is an integer from 1 to 6, and independently in each occurrence be selected.

[0332] In some embodiments, the linker is a hydrazine, disulfide, thioether, diamine, or Peptide, p-aminobenzyl (PAB) group, heterocyclic self-immolative group, optionally substituted C1- C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C 6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2- C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3 -C6 cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl aryl, optionally substituted heteroaryl, acyl, -C(=O)-, or -(CH2C H2O) n -group, where n is an integer from 1 to 6. One or more of the attached groups may be in the form of a divalent (diradical) species, e.g., C1-C6 alkylene, etc. You will realize that it can exist.

[0333] In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In embodiments, the p-aminobenzyl group is a protease cleavage site between the cytotoxic drug and the linker. In one embodiment, the p-aminobenzyl group is In one embodiment, the p-aminobenzyl group is part of a p-aminocarbonyl unit. It is part of the benzylamide unit.

[0334] In some embodiments, the linker is PAB, Val-Cit-PAB, Val-A la-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-L ys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala -Ala-Asn-PAB, or Ala-PAB.

[0335] In some embodiments, the linker is a peptide, an oligosaccharide, -(CH2) n -,-( CH2CH2O) n -, PAB, Val-Cit-PAB, Val-Ala-PAB, V al-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-P AB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn -PAB, or Ala-PAB in combination with one or more of:

[0336] In some embodiments, the linker is -(C=O)(CH) n -units, n is An integer between 1 and 6.

[0337] In some embodiments, the linker is -(CH) n -unit, n is an integer between 2 and 6 It's a number.

[0338] In certain embodiments, the linker of the ADC is N-beta-maleimidopropyl-Val -Ala-para-aminobenzyl (BMP-Val-Ala-PAB).

[0339] To conjugate an antibody, or an antigen-binding fragment thereof, to a cytotoxic agent Linkers that can be used include a linker that is covalently attached to the cytotoxic drug at one end of the linker and At the other end of the linker, a reactive substituent present on the linker is coupled to an antibody that binds to CD5, or The antigen-binding fragment of the present invention is formed from a coupling reaction between the reactive substituents present in the antigen-binding fragment of the present invention and the Antibodies that bind to CD5 or those that contain a chemical moiety that binds to the antigen. Reactive substituents that may be present in the fragments include, but are not limited to: hydroxyl groups of serine, threonine, and tyrosine residues, and amino groups of lysine residues , the carboxyl groups of aspartic acid and glutamic acid residues; and the thiol groups of cysteine ​​residues. ol groups, as well as propargyl, azido, haloaryl (e.g., fluoroaryl) , haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and non-natural The haloheteroalkyl moiety of natural amino acids.

[0340] Examples of linkers useful in the synthesis of drug-antibody conjugates include, inter alia: , Michael acceptors (e.g., maleimides), activated esters, electron-deficient carbonyl compounds amines or amines present in the antibody or antigen-binding fragment, such as aldehydes, aldehydes, and aldehydes. contains electrophilic species suitable for reaction with nucleophilic substituents such as thiol moieties. For example, suitable linkers for the synthesis of drug-antibody conjugates include, but are not limited to: , especially 4-(N-maleimidomethyl)-cyclohexane-L-carboxylic acid succinic Succinimidyl (SMCC), N-succinimidyl iodoacetate (SIA), Sulfo-S MCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS ), sulfo-MBS, and succinimidyl iodoacetate, such as L Iu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference. The present invention is incorporated by reference as it relates to linkers for chemical conjugates. Additional linkers include those that are particularly useful for conjugating microtubule-disrupting agents such as auristatins. Examples of suitable non-cleavable maleimidocaproyl linkers include those described by Doronina et al. al., Bioconjugate Chem. 17:14-24, 2006 No. 6,299,499, the disclosure of which is incorporated by reference as it relates to linkers for chemical conjugates. incorporated herein.

[0341] Any one or more of the chemical groups, moieties and features disclosed herein may be combined in multiple ways. In combination, these compounds are useful in antibody-cytotoxin conjugates as disclosed herein. It will be appreciated by those skilled in the art that linkers may be formed using the compositions described herein. Additional linkers useful in conjunction with the compositions and methods are described, for example, in U.S. Patent Application Publication No. 201 No. 5 / 0218220, the disclosure of which is incorporated herein by reference in its entirety. It will be incorporated into the specification.

[0342] Linkers useful in conjunction with the antibody-drug conjugates described herein include, but are not limited to, However, phosphorus-containing chemical moieties formed by coupling reactions as shown in Table 2 below are also available. The curves are for antibodies, or antigen-binding fragments thereof, and cytotoxic molecules. The attachment points to each are shown. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0343] Those skilled in the art will readily appreciate that the reactive substituent Z attached to the linker and the antibody or its antigen-binding fragment can be easily combined. Reactive substituents on the cations of ... and will recognize the reactive substituent Z. Antibody-drug conjugates useful in conjunction with the methods may be antibodies, as described herein, or and a linker or a cytotoxin-linker conjugate. The linker or cytotoxin-linker conjugate can be formed by reaction of an antibody, or an antigen-binding fragment thereof. forming chemical moiety Z. As shown in Table 3, the linker and antibody or Examples of suitable reactive substituents on an antigen-binding fragment of , thiol / haloalkyl pair, amine / carbonyl pair, or thiol / α,β-unsaturated pair carbonyl pairs, etc.), diene / dienophile pairs (e.g., azide / alkyne pairs, among others, or diene / α,β-unsaturated carbonyl pair). Forms chemical moiety Z Coupling reactions between reactive substituents for the synthesis of hydroxyl groups include, but are not limited to, thiol alkylation, Hydroxyl alkylation, amine alkylation, amine or hydroxylamine condensation, Hydrazine formation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition), nucleophilic aromatic rings aromatic substitution, electrophilic aromatic substitution, and others known in the art or described herein. Preferably, the linker is an antibody, or an antigen-binding fragment thereof. The compound contains an electrophilic functional group for reacting with a nucleophilic functional group on the compound.

[0344] Antibodies that may be present in an antibody, or antigen-binding fragment thereof, as disclosed herein. Reactive substituents include, but are not limited to, (i) N-terminal amine groups, (ii) side chain amine groups, e.g., (iii) side chain thiol groups, e.g., cysteine; and (iv) antibodies that are glycosylated. Nucleophilic groups such as sugar hydroxyl groups or amino groups that are sylated are included. Reactive substitutions that may be present in an antibody, or antigen-binding fragment thereof, as disclosed herein. Groups include, but are not limited to: serine, threonine, and tyrosine. the hydroxyl site of lysine residues; the amino site of lysine residues; aspartic acid and glutamic acid the carboxyl site of the carboxylic acid residue; and the thiol site of the cysteine ​​residue, as well as the propanol site. Gyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., For example, fluoroheteroaryl, haloalkyl, and haloheteroaryl of unnatural amino acids. In some embodiments, an antibody as disclosed herein, or an antigen thereof The reactive substituents present in the binding fragment are amine or thiol moieties. The antibody has reducible interchain disulfides, i.e., cysteine ​​bridges. Conjugation with linker reagents is achieved by treatment with a reducing agent such as dithiothreitol. Thus, each cysteine ​​bridge can theoretically forms two reactive thiol nucleophiles. The additional nucleophilic group converts the amine to a thiol. The antibody was prepared by reacting lysine with 2-iminothiolane (Traut's reagent), which is a lysine-containing compound. The reactive thiol groups can be 1, 2, 3, 4 or more. Introducing cysteine ​​residues (e.g., containing one or more non-naturally occurring cysteine ​​amino acid residues) (preparing a mutant antibody containing the nucleotide sequence) U.S. Patent No. 7,521,541 describes the use of reactive cysteine ​​amino acids. It teaches the engineering of antibodies by transfection.

[0345] In some embodiments, the reactive moiety Z attached to the linker is an electrophilic moiety present on the antibody. Useful electrophilic groups on antibodies include aldehyde groups and The heteroatom of the nucleophilic group may be an anti- Useful nucleophilic groups include: are hydrazides, oximes, aminos, hydroxyls, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides. It will not be done. In some embodiments, Z is present in an antibody, or antigen-binding fragment thereof. reactive nucleophilic substituents, such as amine and thiol moieties, and reactive electrophilic substituents Z is the product of a reaction between Z and, for example, Z is, among others, a Michael acceptor (e.g., imide), activated ester, electron-deficient carbonyl compound, or aldehyde .

[0346] In some embodiments, the ADC can be linked to any of the compounds described herein via a linker and chemical moiety Z. an amatoxyl group of any of formula I, IA, IB, II, IIA, or IIB as shown; In some embodiments, the linker comprises an anti-CD5 antibody conjugated to a diamine. In some embodiments, the linker comprises a Val-Ala and a Val- In some embodiments, the linker comprises a dipeptide selected from the group consisting of para- In some embodiments, the linker comprises an aminobenzyl group (PAB). In some embodiments, the linker comprises the site PAB-Ala- In some embodiments, the linker comprises -((C=O)(CH) n -single In some embodiments, the linker is -PAB- Cit-Val-((C=O)(CH2) n -It is.

[0347] In some embodiments, the linker is -(CH) n -unit, n is an integer between 2 and 6 In some embodiments, the linker is -PAB-Cit-Val-((C= O)(CH2) n In some embodiments, the linker is -PAB-Ala- Val-((C=O)(CH2) n In some embodiments, the linker is - (CH2) n In some embodiments, the linker is -((CH) n - and , where n is 6.

[0348] In some embodiments, the chemical moiety Z is selected from Table 1. is the chemical moiety Z [ka] (wherein S is the antigen present in an antibody, or antigen-binding fragment thereof, that binds to CD5) is a sulfur atom representing a reactive substituent (e.g., from the -SH group of a cysteine ​​residue) be.

[0349] In some embodiments, the linker L and chemical moiety Z together form LZ. considered, [ka] is.

[0350] Those skilled in the art will appreciate that the linker-reactive substituent structure is a suitable nucleotide sequence for an antibody or antigen-binding fragment thereof. It will be appreciated that the Z group may include maleimide prior to conjugation with the carboxylate. The aforementioned linker moieties and agonists are particularly useful in conjunction with the compositions and methods described herein. The toxin-linker conjugates are described, for example, in U.S. Patent Application Publication No. 2015 / 021 8220 and Patent Application Publication No. WO 2017 / 149077. No. 6,229,699, the disclosure of each of which is incorporated herein by reference in its entirety.

[0351] In some embodiments, the linker-reactive substituent structure is an antibody or antigen-binding fragment thereof. Before conjugation with Fragment, [ka] is.

[0352] (Preparation of antibody-drug conjugates) In the ADCs of Formula I as disclosed herein, the antibody or its antigen-binding fragment The fragment is connected to 1 via a linker L and a chemical moiety Z as disclosed herein. more than one cytotoxic drug moiety (D), e.g., about 1 to about 20 drug moieties per antibody The ADCs of the present disclosure are conjugated to organic compounds known to those of skill in the art, including: It can be prepared by several routes, employing chemical reactions, conditions, and reagents: ) reacting a reactive substituent of the antibody or antigen-binding fragment thereof with a bivalent linker reagent; forming Ab-ZL as described herein above, followed by reaction with a drug moiety, D; and (2) reacting a reactive substituent on the drug moiety with a bivalent linker reagent to form a DLZ; and subsequently reacting with a reactive substituent of an antibody or antigen-binding fragment thereof as described herein above. reacting to form an ADC of the formula DLZ-Ab, e.g., Am-ZL-Ab. AD Additional methods for preparing C are described herein.

[0353] In another embodiment, the antibody or antigen-binding fragment is chemically modified to include one or more sequences. The resulting product has one or more lysine residues to which a hydroxyl group can be introduced. C is a conjugate through the sulfhydryl group's sulfur atom, as described herein above. Reagents that can be used to modify lysines include N-sulfonyl esters, Chuccinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride Examples of suitable ion exchangers include, but are not limited to, ion exchangers containing ion exchange salts (Traut's reagent). In another embodiment, the antibody or antigen-binding fragment thereof is chemically modified to include one or more may have one or more carbohydrate groups, which may have sulfhydryl groups of The ADC may be conjugated via the sulfhydryl group's sulfur atom, as described herein above. formed by the gate.

[0354] In yet another embodiment, the antibody can be oxidized to provide aldehyde (-CHO) groups. and may have one or more carbohydrate groups (see, e.g., Laguzza, et al., J. (See Med. Chem. 1989, 32(3), 548-55). The ADC is then As described herein above, by conjugation via the corresponding aldehyde Other protocols for modifying proteins for cytotoxin binding or association include: Coligan et al.,Current Protocols in Prot. ein Science, vol. 2, John Wiley & Sons (2002) and is incorporated herein by reference.

[0355] Linker-drug moieties are attached to cells such as antibodies, immunoglobulins or fragments thereof. -Methods for conjugating to target proteins are described, for example, in U.S. Pat. No. 5,208 ,020; U.S. Pat. No. 6,441,163; WO2005037992 No. WO2005081711; and WO2006 / 034488. No. 6,299,133, all of which are expressly incorporated herein by reference in their entireties.

[0356] Alternatively, a fusion protein comprising an antibody and a cytotoxic agent can be produced, e.g., by recombinant techniques or can be produced by peptide synthesis. The length of the DNA can be adjusted to separate the two parts of the conjugate. The coding regions may be contiguous or conjugated. separated by a region encoding a linker peptide that does not destroy the desired properties of the It may be included in the form.

[0357] (Treatment method) As described herein, hematopoietic stem cell transplantation therapy is a method of administering hematopoietic stem cell transplantation therapy to a subject in need of therapy. The method can be applied to transfer or retransfer more than one blood cell type. They are normally multipotent and therefore granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils) , erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and and lymphocytes (e.g., NK cells, B cells, and T cells), Hematopoietic stem cells can further self-renew and differentiate into multiple different blood lineages. Therefore, daughter cells with the same ability as the mother cells can be produced, and the transplantation reintroduced into the spirient, where they home to the hematopoietic stem cell niche and become productive and It is also characterized by its ability to restore sustained hematopoiesis.

[0358] Therefore, to reconstitute defective or deficient cell populations in vivo, Hematopoietic stem cells are administered to patients with defects or deficiencies in one or more cell types of the hematopoietic system, This allows for the treatment of conditions associated with defects or depletion of endogenous blood cell populations. Therefore, the compositions and methods described herein are useful for treating non-malignant hemoglobinopathies (e.g., sickle cell disease). Red cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Al Hemoglobinopathy selected from the group consisting of Dritz syndrome Additionally or alternatively, the compositions and methods described herein can be used to treat It can be used to treat immune deficiencies, such as genetic immunodeficiency. Alternatively, the compositions and methods described herein can be used to treat acquired immune deficiencies (e.g., HIV and and AIDS) The compositions and methods described herein can be used to treat metabolic disorders (e.g., glycogen storage disorders). Mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidoses, and metachromatic albuginea and (b) a metabolic disorder selected from the group consisting of: can.

[0359] Additionally or alternatively, the compositions and methods described herein can be used to treat hematological cancers, myeloproliferative It can be used to treat malignancies or proliferative disorders such as cancer. In the case of hematopoietic stem cell transplantation, those expressing one or more non-self MHC antigens, etc. to deplete immune cell populations that cross-react with non-autologous hematopoietic stem cells and initiate immune responses. The compositions and methods described herein may be administered to a patient, such as a transplanted graft. Helps prevent or reduce the likelihood of rejection of blood stem cell grafts, and transplanted hematopoietic This allows stem cells to home to the stem cell niche and establish productive hematopoiesis. and subsequently reconstitute populations of cells depleted during cancer cell eradication, such as during systemic chemotherapy. Exemplary conditions that can be treated using the compositions and methods described herein include: Common blood cancers include acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia. Myeloid leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma tumors, as well as other cancerous conditions, including, but not limited to, neuroblastoma .

[0360] Additional diseases that can be treated with the compositions and methods described herein include adenosine triphosphate (ATP), thrombolytic therapy, and steroid therapy. Deaminase deficiency and severe combined immunodeficiency, hyperimmunoglobulin M syndrome, Cheddi Ack-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia Jar, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis These include, but are not limited to, gussets.

[0361] Antibodies, or antigen-binding fragments thereof, and conjugates described herein can be used to induce solid organ transplant tolerance. The compositions and methods are used to deplete or remove immune cell populations prior to hematopoietic stem cell transplantation. Following depletion of cells from such target tissues, stem cells from organ donors can be used to A population of cells or progenitor cells (e.g., hematopoietic stem cells from an organ donor) is transferred to the transplant recipient. and administering to the subject a transient or stable mixture of stem or progenitor cells followed by engraftment of such stem or progenitor cells. Chimerism can be achieved, thereby allowing long-term transfer without the need for additional immunosuppressive agents. Enables transplant tolerance by administering anti-CD5 antibodies or their antigen-binding fragments. , which can reduce the likelihood of graft rejection or prevent rejection altogether. In this manner, the compositions and methods described herein are useful in solid organ transplant applications. Transplantation in patients (e.g., kidney, lung, liver, and heart transplants, among others) The compositions and methods described herein can be used to induce tolerance in solid organ transplant patients. The invention is well suited for use in connection with the induction of transplant tolerance, for example, because of transient or stable Even low rates of donor engraftment are sufficient to induce long-term tolerance of the transplanted organ. be.

[0362] Additionally, the compositions and methods described herein are useful in treating cancers such as those characterized by CD5+ cells. For example, the compositions and methods described herein can be used to directly treat cancer. The method and method may be used to treat leukemia, particularly in patients exhibiting CD5+ leukemia cells. By depleting CD5+ cancer cells, such as leukemia cells, The compositions and methods described herein can be used to directly treat a variety of cancers. Exemplary cancers that can be treated in this manner include acute myeloid leukemia, acute lymphoblastic leukemia, and Leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma These include blood cancers such as follicular lymphoma, and non-Hodgkin's lymphoma.

[0363] Additionally, the compositions and methods described herein may be used to treat autoimmune diseases. For example, antibodies or their antigens can be used to kill CD5+ immune cells. administering the binding fragment to a subject, such as a human patient, suffering from an autoimmune disease. CD5+ immune cells are T cells that specifically bind to self-antigens and initiate immune responses. They may be autoreactive lymphocytes, such as T cells, that express the CD5+ receptor. By depleting the Additionally or alternatively, the present invention can treat autoimmune conditions such as those described herein. Using the described compositions and methods, a population of endogenous hematopoietic stem cells can be obtained prior to hematopoietic stem cell transplantation therapy. Autoimmune diseases can be treated by depleting the They are able to home into the niche created during the endogenous cell depletion step and establish productive hematopoiesis. This can then reconstitute the population of cells depleted during autoimmune cell eradication. Cut.

[0364] Autoimmune diseases that can be treated using the compositions and methods described herein include: Examples include, but are not limited to: psoriasis, psoriatic arthritis, type 1 diabetes mellitus Disease (type 1 diabetes), rheumatoid arthritis (RA), human systemic lupus erythematosus (SLE), multiple sclerosis ( MS), inflammatory bowel disease (IBD), lymphocytic colitis, acute disseminated encephalomyelitis (ADEM) , Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid syndrome (APS), regenerative disorders Benign anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune otitis interna (AIED), Autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Baro's disease, Behcet's disease Disease, Bullous Pemphigoid, Cardiomyopathy, Chagas' Disease, Chronic Fatigue and Immune Dysfunction Syndrome (CFIDS) , chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac disease-dermatosis herpetiformis Dermatitis, cold agglutinin disease, CREST syndrome, Dogaud's disease, discoid lupus, autonomic imbalance, uterus Endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa , idiopathic and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, Interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, Lyme disease, Meniere's disease, mixed connective tissue Myasthenia gravis, neuromyotonia, opsoclonus-myoclonus syndrome OMS, optic neuritis, thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyxitis Myositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndrome, rheumatoid arthritis Polymyalgia nervosa, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatoid arthritis fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-person syndrome, hypertension Arteritis of the gallbladder, temporal arteritis (also known as "giant cell arteritis"), ulcerative colitis, ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia ("vulvar vestibulitis"), and Wegener's granulomatosis.

[0365] For example, using the compositions and methods described herein, one of skill in the art can easily identify and treat autoimmune diseases. and administering to a subject suffering from the disease an anti-CD5 antibody in an amount sufficient to treat the autoimmune disease, and For example, a subject can be treated with scleroderma, multifocal sclerosis, or a combination of scleroderma and multifocal sclerosis. You may have idiopathic sclerosis, ulcerative colitis, Crohn's disease, and / or type 1 diabetes. To ameliorate one or more of these conditions, a physician of ordinary skill in the art may administer an anti-CD5 antibody, or A fragment thereof, e.g., an antibody conjugated to a cytotoxic drug, or a fragment thereof, is tested. The antibody, or fragment thereof, can be formulated and administered to the body. Conjugated to a cytotoxic drug using conjugation technology and linkers The population of endogenous autoreactive CD5+ T cells, B cells, or NK cells in a subject may be To deplete the CD5 receptor, various cytotoxic agents were administered to the anti-CD5 antibody, or its antigen-binding fragment. For example, an antibody or its antigen-binding fragment can be conjugated to the antibody. The cytotoxin is conjugated to an amatoxin or another cytotoxic moiety described herein. That's fine.

[0366] In preparation for treatment, the physician will measure the number of autoreactive T cells, B cells, and and / or the amount or concentration of NK cells can be assessed. This can be done using FACS analysis techniques known in the art. , anti-inflammatory drugs, such as anti-inflammatory drugs, to deplete populations of autoreactive T cells, B cells, and / or NK cells. The antibody, or a fragment thereof, alone or conjugated to a cytotoxin, is administered to the subject. To assess the effectiveness of treatment, a physician may administer an anti-CD5 antibody or or a fragment thereof in a sample isolated from the patient at a time point after administration of the antibody or a fragment thereof, The amount or concentration of B cells and / or NK cells can be determined. The amount or abundance of autoreactive T cells, B cells, and / or NK cells in a sample isolated from a subject The concentration of T cells, B cells, or NK cells was compared with the amount or concentration of T cells, B cells, or NK cells before treatment. This will reveal whether the patient has responded to anti-CD5 antibodies or fragments thereof.

[0367] Antibody-drug conjugates containing anti-CD5 antibodies, or antigen-binding fragments thereof, are It can also be used in combination with CAR T therapy, specifically to deplete natural T cells. and administering to a patient in need thereof an effective amount of an anti-CD5 antibody drug conjugate to The methods and compositions described herein can be administered to a subject in need thereof prior to CAR T therapy. It is used in CAR T therapy by depleting natural T cells expressing CD5 using This allows for more efficient transfer of engineered T cells.

[0368] (Route of Administration and Dosage) The antibodies described herein, or antigen-binding fragments thereof, can be administered to a patient (e.g., a patient with hematopoietic It can be administered to human patients (e.g., those in need of stem cell transplantation therapy) in a variety of dosage forms. For example, the antibodies described herein, or antigen-binding fragments thereof, can be administered to patients undergoing hematopoietic stem cell transplantation. Patients in need of medical treatment and / or suffering from cancer or autoimmune diseases administered to a subject in the form of an aqueous solution, such as an aqueous solution containing one or more pharmaceutically acceptable excipients. Exemplary pharmaceutical agents for use in the compositions and methods described herein include: An acceptable excipient for the aqueous solution is a viscosity adjusting agent. can be sterilized using

[0369] The antibodies and antigen-binding fragments described herein can be administered orally, transdermally, subcutaneously, intranasally, or intravenously. Administration can be by a variety of routes, such as intravenous, intramuscular, intraocular, or parenteral. The optimal route of administration in a given case will depend on the specific antibody or antigen-binding fragment thereof being administered. fragment, patient, how the drug is formulated, and how it is administered (e.g., time and route of administration). , the patient's age, weight, sex, the severity of the disease being treated, the patient's diet, and the patient's excretion rate. is determined by.

[0370] An effective amount of an antibody, or antigen-binding fragment thereof, described herein may be administered, for example, in a single dose ( For example, about 0.001 to about 10 per bolus, multiple, or continuous administration. 0 mg / kg (e.g., about 0.001 mg / kg to about 0.01 mg / kg, about 0.01 m g / kg ~ approx. 0.1 mg / kg, approx. 0.1 mg / kg ~ approx. 1 mg / kg, approx. 1 mg / kg ~ about 10 mg / kg, about 10 mg / kg to about 100 mg / kg of body weight , or the optimal serum concentration of the antibody, or antigen-binding fragment thereof (e.g., about 0.00 Serum concentrations of 0.1 to approximately 5000 μg / mL (e.g., approximately 0.0001 to 0.001 μg / mL) L, approx. 0.001~0.01μg / mL, approx. 0.01~0.1μg / mL, approx. 0.1~1 μg / mL, approx. 1~10μg / mL, approx. 10~100μg / mL, approx. 100~1000μ g / mL, approximately 1000–2000 μg / mL, approximately 2000–3000 μg / mL, or The dose can be in the range of approximately 3000-5000 μg / mL serum concentration. Subjects undergoing conditioning treatment in preparation for undergoing stem cell transplantation (e.g., administered once or more times (e.g., 2–10 times) daily, weekly, or monthly. The antibody or antigen-binding fragment thereof is administered to the patient by injecting exogenous hematopoietic stem cells prior to hematopoietic stem cell transplantation. Non-self hematopoietic stem cell antigens, such as non-self MHC antigens, are administered at a time that optimally promotes cell engraftment. At the time of optimal depletion of CD5+ T cells, B cells, or NK cells that cross-react with the antigen, For example, anti-CD5 antibodies and their antigen-binding fragments can be administered to patients. The patient was administered the exogenous hematopoietic stem cell graft approximately 1 hour before administration. ~ about 1 week (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, Approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours, approximately 12 hours, approximately 13 hours, approximately 14 hours, approx. 15 hours, approx. 16 hours, approx. 17 hours, approx. 18 hours, approx. 19 hours, approx. 20 hours , about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days The antibody can be administered about 1 day, about 6 days, or about 7 days) or more before the first dose. , about 1 hour to about 24 hours (for example, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours) , about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, approx. 14 hours, approx. 15 hours, approx. 16 hours, approx. 17 hours, approx. 18 hours, approx. 19 hours , about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours) do.

[0371] In one embodiment, the half-life of the anti-CD5 antibody (or Fc-containing fragment thereof) is If the Fc region of the antibody contains the H435A mutation (numbering according to the EU index), Short (compared to wild-type Fc region).

[0372] According to the methods disclosed herein, the exogenous hematopoietic stem cell graft is administered prior to hematopoietic stem cell transplantation therapy. To promote engraftment, practitioners of skill in the art may condition a patient, such as a human patient. For this purpose, the skilled practitioner may use antibodies or antibodies capable of binding to CD5. or antigen-binding fragments thereof, such as the anti-CD5 antibodies described herein. The antibodies, or fragments thereof, can be administered to a patient. or a toxin, such as a cytotoxic molecule conjugated to an Fc domain or known in the art. For example, an anti-CD5 antibody or its antigen-binding The combined fragments were used to identify Pseudomonas exotoxin A, deBouganin, diphtheria toxin, Amatoxins such as α-amanitin, saporin, maytansin, maytansinoids, and au Ristatin, anthracycline, calicheamicin, irinotecan, SN-38, dutasteride Ocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolino Benzodiazepines, indolinobenzodiazepine dimers, or their variants The conjugation can be carried out by covalent bonding to the cytotoxin. Covalent bonding techniques described herein or known in the art may be used. The antibody, antigen-binding fragment thereof, or antibody-drug conjugate can be The patient then receives, for example, by intravenous administration, exogenous hematopoietic stem cells (e.g., autologous, syngeneic, or allogeneic hematopoietic stem cells) can be administered prior to transplantation into the patient.

[0373] Prior to hematopoietic stem cell transplantation treatment, endogenous T cells, B cells, and / or NK cells, e.g. The amount of bone marrow resident T cells is, for example, about 10%, about 20%, about 30%, about 40%, about 50% , about 60%, about 70%, about 80%, about 90%, about 95% or more an amount of an anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug conjugate sufficient to For example, prior to hematopoietic stem cell transplantation therapy, endogenous T cells, B cells, , and / or NK cells, e.g., bone marrow resident T cells, e.g., about 10% to 20% , about 20% to 30%, about 30% to 40%, about 40% to 50%, about 50% to 60%, about 60 %~70%, about 70%~80%, about 80%~90%, about 90%~95%, or more an amount of an anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug sufficient to reduce For example, the conjugate can be administered to the patient prior to hematopoietic stem cell transplantation therapy. The amount of T cells, B cells, and / or NK cells, e.g., bone marrow resident T cells, can be reduced, e.g., by at least about 10%, at least about 20%, at least about 30%, at least about 40%, At least about 50%, at least about 60%, at least about 70%, at least about 80%, an amount of an antibody sufficient to reduce the administering a CD5 antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate The reduction of T cell numbers can be achieved using conventional techniques known in the art, e.g. For example, characteristic T This can be monitored by, for example, FACS analysis of cells expressing the cell surface antigen. For example, a physician skilled in the art may take bone marrow samples from a patient at various times during the conditioning treatment. and performing FACS analysis to determine the relative concentration of T cells in the sample that bind to T cell marker antigens. The extent of endogenous T cell depletion can be determined by antibody probing According to some embodiments, the concentration of T cells is determined by the administration of anti-CD5 antibodies, their antigen-binding fragments, or to a minimum in response to conditioning treatment with either the antibody or antibody-drug conjugate. When this occurs, the doctor will end the conditioning treatment and prepare the patient for hematopoietic stem cell transplantation. You may begin preparing for the event.

[0374] The anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug conjugate is administered by a viscosity It may be administered to a patient in an aqueous solution containing one or more pharmaceutically acceptable excipients, such as a modifier. The aqueous solution can be prepared by techniques described herein or known in the art. Before administering the hematopoietic stem cell graft to the patient, the antibody, its anti- The original binding fragment, or antibody-drug conjugate, is added to, for example, about 0.001 mg / kg to about 100 mg / kg (e.g., about 0.001 mg / kg to about 0.01 mg / kg, Approx. 0.01mg / kg ~ approx. 0.1mg / kg, approx. 0.1mg / kg ~ approx. 1mg / kg, approx. At a dose of 1 mg / kg to approximately 10 mg / kg, approximately 10 mg / kg to approximately 100 mg / kg The antibody, antigen-binding fragment thereof, or antibody-drug conjugate can be administered to a patient. The adjugate is administered to the patient at a time that optimally promotes engraftment of the exogenous hematopoietic stem cells, e.g., About 1 hour to about 1 week (e.g., about 1 hour, about 2 hours, about 3 hours) after administration of the hematopoietic stem cell graft 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 hour 1 hour, approximately 12 hours, approximately 13 hours, approximately 14 hours, approximately 15 hours, approximately 16 hours, approximately 17 hours, Approximately 18 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days) or more before It can be given.

[0375] After the conditioning treatment was completed, the patient was then placed in the same Infusion of exogenous hematopoietic stem cells (e.g., intravenous infusion) from the same physician or from a different physician ) can be administered. Physicians may administer autologous, syngeneic, or allogeneic hematopoietic stem cell infusions, e.g. , about 1×10 3 ~Approx. 1×10 9 Hematopoietic stem cells / kg (approximately 1×10 3 Hematopoietic stem cells ~ approx. 1×10 4 , about 1×10 4 Hematopoietic stem cells ~ approx. 1×10 5 , about 1×10 5 Hematopoietic stem cells ~ approx. 1×10 6 , Approximately 1×10 6 Hematopoietic stem cells ~ approx. 1×10 7 , or approximately 1 × 10 8 Hematopoietic stem cells ~ approx. 1×10 9 The physician may, for example, take a blood sample from the patient and administer the dose to the patient. After the administration of the graft, hematopoietic stem cells or cells of the hematopoietic system (megakaryocytes, thrombocytes, platelets, erythrocytes, Mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, Antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and The engraftment of hematopoietic stem cells can be monitored by measuring the increase in the concentration of erythrocytes (e.g., B lymphocytes). This analysis can be performed, for example, within about 1 hour to about 6 hours after hematopoietic stem cell transplantation. It can be performed for months or more (e.g., about 1 hour, about 2 hours, about 3 hours, Approximately 4 hours, approximately 5 hours, approximately 6 hours, approximately 7 hours, approximately 8 hours, approximately 9 hours, approximately 10 hours, approximately 11 hours 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 1 8 hours, approximately 19 hours, approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, About 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, approximately 11 weeks, approximately 12 weeks 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 1 9 weeks, approximately 20 weeks, approximately 21 weeks, approximately 22 weeks, approximately 23 weeks, approximately 24 weeks, or longer (Top) The concentration of hematopoietic stem cells or cells of the hematopoietic system is compared to the concentration of the corresponding cell type before transplant treatment. In comparison, after transplant treatment (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, approximately 8%, approximately 9%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60% , approximately 70%, approximately 80%, approximately 90%, approximately 100%, approximately 200%, approximately 500%, or more (Top) The finding that increased activity was due to anti-CD5 antibodies, their antigen-binding fragments, or antibody-drug Treatment with the antibody conjugate successfully promoted engraftment of transplanted hematopoietic stem cell grafts. This is one indicator of the above. [Example]

[0376] The following examples illustrate how the compositions and methods described herein can be used and prepared. are provided to illustrate to one skilled in the ...

Claims

1. A conjugate of the formula Ab-Cy, wherein Ab is an antibody or antigen-binding fragment thereof that binds to CD5 and Cy is a cytotoxin, the cytotoxin comprising: Formula (I): 【Chemistry 1】 (In the formula, R １ is H, OH, OR Ａ , or OR Ｃ and R ２ is H, OH, OR Ｂ , or OR Ｃ and R Ａ and R Ｂ when present, together with the oxygen atom to which they are attached, combine to form an optionally substituted 5-membered heterocycloalkyl group; R ３ is H, R Ｃ , or R Ｄ and R ４ , R ５ , R ６ , and R ７ are each independently H, OH, OR Ｃ , OR Ｄ , R Ｃ , or R Ｄ and R ８ OH, NH ２ , OR Ｃ , OR Ｄ , N.H.R. Ｃ , or NR Ｃ R Ｄ and R ９ is H, OH, OR Ｃ , or OR Ｄ and X is -S-, -S(O)-, or -SO ２ - and; R Ｃ is -L-Z; R Ｄ is an optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ Heteroalkyl, optionally substituted C ２ -C ６ Alkenyl, optionally substituted C ２ -C ６ Heteroalkenyl, optionally substituted C ２ -C ６ Alkynyl, optionally substituted C ２ -C ６ heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted C １ -C ６ Alkylene, optionally substituted C １ -C ６ Heteroalkylene, optionally substituted C ２ -C ６ Alkenylene, optionally substituted C ２ -C ６ Heteroalkenylene, optionally substituted C ２ -C ６ Alkynylene, optionally substituted C ２ -C ６ heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene; a dipeptide, —C(═O)—, a peptide, or a combination thereof; Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the antibody or antigen-binding fragment thereof; Am is exactly one R Ｃ (including substituents) The conjugate is an amatoxin (Am) represented by the formula:

2. 2. The conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): a. a CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 3); b. a CDR-H2 having the amino acid sequence NTHTGE (SEQ ID NO: 4); c. a CDR-H3 having the amino acid sequence RGYDWYFDV (SEQ ID NO: 5); d. a CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO:6); e. a CDR-L2 having the amino acid sequence RANRLVD (SEQ ID NO:7); f. CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO:8); or a. a CDR-H1 having the amino acid sequence FSLSTSGMG (SEQ ID NO:29); b. a CDR-H2 having the amino acid sequence WWDDD (SEQ ID NO: 30); c. a CDR-H3 having the amino acid sequence RRATGTGFDY (SEQ ID NO:31); d. a CDR-L1 having the amino acid sequence QDVGTA (SEQ ID NO:32); e. a CDR-L2 having the amino acid sequence WTSTRHT (SEQ ID NO:33); f. CDR-L3 having the amino acid sequence YNSYNT (SEQ ID NO:34).

3. The conjugate of claim 1 , wherein the antibody or antigen-binding fragment thereof comprises the following CDRs: a. CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 11); b. a CDR-H2 having the amino acid sequence NTHYGE (SEQ ID NO: 12); c. a CDR-H3 having the amino acid sequence RRGYDWYFDV (SEQ ID NO: 13); d. a CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 14); e. a CDR-L2 having the amino acid sequence RANRLES (SEQ ID NO: 15); f. CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 16).

4. R Ａ and R Ｂ together with the oxygen atom to which they are attached to form the formula: 【Chemistry 2】 (Wherein, Y is —C(═O)—, —C(═S)—, —C(═NR Ｅ ) - or -C(R Ｅ R Ｅ’ )- and; R Ｅ and R Ｅ’ each independently represents an optionally substituted C １ -C ６ Alkylene-R Ｃ , optionally substituted C １ -C ６ Heteroalkylene-R Ｃ , optionally substituted C ２ -C ６ Alkenylene-R Ｃ , optionally substituted C ２ -C ６ Heteroalkenylene-R Ｃ , optionally substituted C ２ -C ６ Alkynylene-R Ｃ , optionally substituted C ２ -C ６ Heteroalkynylene-R Ｃ , optionally substituted cycloalkylene-R Ｃ , optionally substituted heterocycloalkylene-R Ｃ , optionally substituted arylene-R Ｃ or optionally substituted heteroarylene-R Ｃ The conjugate of claim 1 , wherein the heterocycloalkyl group is a 5-membered heterocycloalkyl group of the formula:

5. R Ａ and R Ｂ are bonded together with the oxygen atoms to which they are attached. 【Transformation 3】 The conjugate of claim 4, which forms:

6. 2. The conjugate of claim 1, (a) R １ is H, OH, or OR Ａ and R ２ is H, OH, or OR Ｂ and R Ａ and R Ｂ are bonded together with the oxygen atoms to which they are attached. 【Chemistry 4】 Forming; R ３ , R ４ , R ６ , and R ７ are each H; R ５ is OR Ｃ and R ８ is OH or NH ２ and R ９ is H or OH; (b) R １ and R ２ are each independently H or OH; R ３ is R Ｃ and R ４ , R ６ , and R ７ are H, respectively; R ５ is H, OH, or OC １ -C ６ is alkyl; R ８ is OH or NH ２ and R ９ is H or OH; (c) R １ and R ２ are each independently H or OH; R ３ , R ６ , and R ７ are H, respectively; R ４ and R ５ are each independently H, OH, OR Ｃ , or R Ｃ and R ８ is OH or NH ２ and R ９ is H or OH; or (d) R １ and R ２ are each independently H or OH; R ３ , R ６ , and R ７ are H, respectively; R ４ and R ５ are each independently H or OH; R ８ is OR Ｃ Or NHR Ｃ and R ９ is H or OH.

7. The conjugate of claim 1, wherein Cy is an amatoxin (Am) represented by formula (II), formula (IIA), or formula (IIB): 【Transformation 5】 (Wherein X is S, SO, or SO ２ and R １ is H or a linker that is covalently attached to the antibody or antigen-binding fragment thereof via a chemical moiety Z formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the antibody or antigen-binding fragment thereof; R ２ is H or a linker that is covalently attached to the antibody or antigen-binding fragment thereof via a chemical moiety Z formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the antibody or antigen-binding fragment thereof; R １ When is H, R ２ is the linker, and R ２ When is H, R １ is the linker).

8. the antibody or antigen-binding fragment thereof comprises an Fc domain comprising a cysteine ​​residue introduced by mutation; 2. The conjugate of claim 1, wherein the cysteine ​​residue is selected from the group consisting of Cys118, Cys239, and Cys265 (numbering according to the EU index).

9. 9. The conjugate of claim 8, wherein the Fc region comprises a D265C mutation.

10. 10. The conjugate of claim 9, wherein the Fc region comprises D265C, L234A, and L235A mutations (numbering according to the EU index).

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