Compositions and methods for depleting CD5 + cells

Abstract

The present invention relates to compositions and methods for depleting CD5 + cells. Provided are a conjugate represented by the formula Ab-Cy (wherein Ab is an antibody or an antigen-binding fragment thereof that binds to CD5, and Cy is a cytotoxin) and a pharmaceutical composition comprising the same for use in the treatment of e.g. Stem cell disorders, cancer, or autoimmune diseases, as well as other hematological and proliferative diseases. Compositions and methods for depleting populations of CD5 + cells, such as CD5 + cancer cells and CD5 + immune cells, can be used as a separate therapy for the direct treatment of cancer and autoimmune diseases by eradicating CD5-expressing cancer cells and autoreactive immune cells, and / or for preparing a patient for hematopoietic stem cell transplantation, for example by depleting a population of CD5 + immune cells that cross-react with non-autohematopoietic stem cells and produce an immune response against the non-autohematopoietic stem cells.

Description

[0001] This application is a divisional application of an application filed on November 29, 2018, with application number 201880088141.X, and invention name “Compositions and methods for depleting CD5+ cells”.

[0002] Related applications

[0003] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 592,214, filed on November 29, 2017, the contents of which are incorporated herein by reference. Background of the Invention

[0005] Despite progress in the field of medicine, there is still a need for treating the disease of the hematopoietic system such as diseases of specific blood cells, metabolic disorders, cancer and autoimmune conditions. Although hematopoietic stem cells have significant therapeutic potential, a limiting factor that has hindered their use clinically is the difficulty associated with ensuring that hematopoietic stem cell transplants are implanted into the host. The patient's own immune system often attacks the transplanted cells and mediates the rejection of the transplanted hematopoietic stem cells. In order to avoid rejection, before hematopoietic stem cell transplantation, the patient is treated with an immune system destroying agent (for example, chemotherapeutic agents or radiation). Unfortunately, the efforts to induce the tolerance of hematopoietic stem cell transplantation in the patient often lead to serious complications. Therefore, there is a need for new compositions and methods to improve hematopoietic stem cell transplantation. SUMMARY OF THE INVENTION

[0007] There is currently a need for compositions and methods for treating hematopoietic disorders such as autoimmune disorders, and for promoting the implantation of exogenous hematopoietic stem cell transplants so that the multipotency and hematopoietic function of these cells are maintained after transplantation. In one aspect, the present invention provides compositions and methods for directly treating a variety of hematopoietic disorders, metabolic disorders, cancers, and autoimmune diseases. The compositions and methods disclosed herein target immune cells for conditioning human patients for hematopoietic stem cell transplantation to treat diseases such as, but not limited to, blood cancers or autoimmune diseases.

[0008] On the other hand, the present invention is also characterized in that before a patient (such as a human patient) receives hematopoietic stem cell transplantation therapy, the patient is adjusted to promote the compositions and methods of the implantation of a hematopoietic stem cell transplant. The patient can be a patient suffering from an autoimmune disease or one or more blood disorders such as cancer, hemoglobinopathies or other hematopoietic disorders and therefore needs a hematopoietic stem cell transplant. As described herein, hematopoietic stem cells can differentiate into the various cell types in the hematopoietic lineage and can be administered to the patient to fill (populate) or refill (re-populate) the defective cell type in the patient. In certain aspects, the invention features antibodies and antibody-drug conjugates capable of binding to CD5, and methods of administering the same to patients to (i) directly treat blood disorders such as autoimmune diseases by selectively depleting populations of immune cells expressing CD5 (such as self-reactive T cells, B cells, or natural killer (NK) cells), and / or (ii) depleting populations of T cells, B cells, or NK cells before administering a hematopoietic stem cell transplant to a patient, thereby reducing the likelihood of hematopoietic stem cell graft rejection. The former activity can directly treat many autoimmune disorders because CD5 can be expressed by T cells, B cells, or NK cells that cross-react with self-antigens and produce an inappropriate immune response to self-antigens. In this case, administering an anti-CD5 antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate to a patient can cause depletion of populations of CD5+ autoimmune cells (such as T cells, B cells, or NK cells that cross-react with one or more self-antigens), thereby treating autoimmune disorders. The latter activity promotes the generation of an environment that is favorable for the engraftment of hematopoietic stem cells because T cells, B cells and / or NK cells that cross-react with one or more non-self antigens expressed by hematopoietic stem cells (such as one or more non-self MHC antigens expressed by hematopoietic stem cells) can generate an immune response against the transplanted hematopoietic stem cells and thus promote graft rejection. In the latter case, patients suffering from conditions such as cancer, autoimmune diseases or other hematopoietic system conditions can subsequently be administered a hematopoietic stem cell transplant to, for example, repopulate one or more blood cell lineages that are missing or depleted in the patient.In one aspect, the present invention thus provides methods for treating various hematopoietic conditions, such as sickle cell anemia, thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosine deaminase deficiency-severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia and Schwachman-Diamond syndrome, human immunodeficiency virus infection and acquired immunodeficiency syndrome, as well as cancer and autoimmune diseases.

[0009] In one aspect, the invention provides a method of depleting a population of CD5+ cells, e.g., in a human patient (such as a population of CD5+ T cells, CD5+ B cells, and / or CD5+ NK cells in a human patient) by administering to the patient an effective amount of an antibody, antigen-binding fragment thereof, or antibody-drug conjugate that binds to CD5.

[0010] In another aspect, the invention provides a method for depleting a population of CD5+ cells in a human patient in need of a hematopoietic stem cell transplant (such as a population of CD5+ T cells, CD5+ B cells and / or CD5+ NK cells in a human patient in need of a hematopoietic stem cell transplant) by administering to the patient an effective amount of an antibody, antigen-binding fragment thereof, or antibody-drug conjugate that binds to CD5, e.g., before the patient receives a transplant comprising hematopoietic stem cells.

[0011] In another aspect, the present invention provides a method for preventing or reducing the likelihood of rejection of a hematopoietic stem cell transplant in a human patient in need of hematopoietic stem cell transplantation therapy by administering an effective amount of an antibody, antigen-binding fragment thereof, or antibody-drug conjugate that binds to CD5 before the patient receives a transplant comprising hematopoietic stem cells.

[0012] In another aspect, provided herein is a method of depleting the endogenous T cell population in a human patient in need of hematopoietic stem cell transplantation therapy by administering an effective amount of an antibody, antigen-binding fragment thereof, or antibody-drug conjugate that binds to CD5 before the patient receives a transplant comprising hematopoietic stem cells.

[0013] In another aspect, the invention features a method, e.g., a method of treating a human patient in need of a hematopoietic stem cell transplant, the method comprising administering to the human patient a transplant comprising hematopoietic stem cells, wherein the patient has previously been administered an antibody or antigen-binding fragment thereof that binds to CD5. The antibody, antigen-binding fragment thereof, or antibody-drug conjugate can be administered to the patient in an amount sufficient to deplete a population of CD5+ cells in the patient (such as a population of CD5+T cells, CD5+B cells, and / or CD5+NK cells in the human patient).

[0014] In another aspect, the invention features a method, e.g., a method of treating a human patient in need of a hematopoietic stem cell transplant, comprising administering to the human patient an amount of an antibody, antigen-binding fragment thereof, or antibody-drug conjugate that binds to CD5 sufficient to deplete a population of CD5+ cells in the patient (such as a population of CD5+ T cells, CD5+ B cells, and / or CD5+ NK cells in the patient), and subsequently administering to the patient a transplant comprising hematopoietic stem cells.

[0015] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof is produced by hybridoma cell line ATCC CRL 8000. In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to the antibody or antigen-binding fragment thereof produced by hybridoma cell line ATCC CRL 8000.

[0016] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof comprises the following variable domains:

[0017] V having the following amino acid sequence L : and

[0018] V having the following amino acid sequence H :

[0019] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned variable domains.

[0020] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof comprises the following CDRs:

[0021] a CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 3);

[0022] CDR-H2 having the amino acid sequence NTHTGE (SEQ ID NO: 4);

[0023] CDR-H3 having the amino acid sequence RGYDWYFDV (SEQ ID NO: 5);

[0024] a CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 6);

[0025] a CDR-L2 having the amino acid sequence RANRLVD (SEQ ID NO: 7); and

[0026] CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 8).

[0027] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned CDRs.

[0028] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof comprises the following variable domains:

[0029] V having the following amino acid sequence L : and

[0030] V having the following amino acid sequence H :

[0031] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned variable domains.

[0032] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof comprises the following CDRs:

[0033] a CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 11);

[0034] CDR-H2 having the amino acid sequence NTHYGE (SEQ ID NO: 12);

[0035] CDR-H3 having the amino acid sequence RRGYDWYFDV (SEQ ID NO: 13);

[0036] a CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 14);

[0037] a CDR-L2 having the amino acid sequence RANRLES (SEQ ID NO: 15); and

[0038] CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 16).

[0039] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned CDRs.

[0040] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof comprises the following CDRs:

[0041] CDR-H1 having the amino acid sequence GYSITSGYY (SEQ ID NO: 17);

[0042] a CDR-H2 having the amino acid sequence ISYSGFT (SEQ ID NO: 18);

[0043] a CDR-H3 having the amino acid sequence AGDRTGSWFAY (SEQ ID NO: 19);

[0044] a CDR-L1 having the amino acid sequence QDISNY (SEQ ID NO: 20);

[0045] a CDR-L2 having the amino acid sequence ATS (SEQ ID NO: 21); and

[0046] CDR-L3 having the amino acid sequence LQYASYPFT (SEQ ID NO: 22).

[0047] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned CDRs.

[0048] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof comprises the following CDRs:

[0049] CDR-H1 having the amino acid sequence GYIFTNYG (SEQ ID NO: 23);

[0050] CDR-H2 having the amino acid sequence INTYNGEP (SEQ ID NO: 24);

[0051] a CDR-H3 having the amino acid sequence ARGDYYGYEDY (SEQ ID NO: 25);

[0052] a CDR-L1 having the amino acid sequence QGISNY (SEQ ID NO: 26);

[0053] a CDR-L2 having the amino acid sequence YTS (SEQ ID NO: 27); and

[0054] CDR-L3 having the amino acid sequence QQYSKLPWT (SEQ ID NO: 28).

[0055] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned CDRs.

[0056] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof comprises the following CDRs:

[0057] CDR-H1 having the amino acid sequence FSLSTSGMG (SEQ ID NO: 29);

[0058] CDR-H2 having the amino acid sequence WWDDD (SEQ ID NO: 30);

[0059] a CDR-H3 having the amino acid sequence RRATGTGFDY (SEQ ID NO: 31);

[0060] a CDR-L1 having the amino acid sequence QDVGTA (SEQ ID NO: 32);

[0061] a CDR-L2 having the amino acid sequence WTSTRHT (SEQ ID NO: 33); and

[0062] CDR-L3 having the amino acid sequence YNSYNT (SEQ ID NO: 34).

[0063] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned CDRs.

[0064] In some embodiments of any of the above aspects, the antibody or antigen-binding fragment thereof comprises a combination of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions listed below in Table 1. In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising a combination of the CDRs listed below in Table 1.

[0065] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of: a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a dual variable immunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment, a F(ab')2 molecule, and a tandem di-scFv. In some embodiments, the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.

[0066] In some embodiments, the antibody is conjugated to a cytotoxin.

[0067] In some embodiments, the cytotoxin is selected from the group consisting of amatoxin, Pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin, maytansine, maytansinoid, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine and indolinobenzodiazepine dimer, or a variant thereof.

[0068] In another aspect, the present invention provides a method for depleting a population of CD5+ cells in a human patient (such as a population of CD5+ T cells, CD5+ B cells and / or CD5+ NK cells in a human patient) by administering to the patient an effective amount of an antibody, fragment thereof, or antibody-drug conjugate capable of binding to CD5.

[0069] In another aspect, the present invention provides a method of depleting a population of CD5+ cells in a human patient in need of a hematopoietic stem cell transplant (such as a population of CD5+ T cells, CD5+ B cells and / or CD5+ NK cells in a human patient in need of a hematopoietic stem cell transplant) by administering an effective amount of an antibody, fragment thereof, or antibody-drug conjugate capable of binding to CD5 before the patient receives a transplant comprising hematopoietic stem cells.

[0070] In another aspect, the invention features a method, e.g., a method of treating a human patient in need of a hematopoietic stem cell transplant, the method comprising administering to the human patient a transplant comprising hematopoietic stem cells, wherein the patient has previously been administered an antibody, fragment thereof, or antibody-drug conjugate that binds to CD5 in an amount sufficient to deplete a population of CD5+ cells in the patient (such as a population of CD5+ T cells, CD5+ B cells, and / or CD5+ NK cells in the human patient).

[0071] In another aspect, the invention features a method, e.g., a method of treating a human patient in need of a hematopoietic stem cell transplant, comprising administering to the human patient an amount of an antibody, fragment thereof, or antibody-drug conjugate that binds to CD5 sufficient to deplete a population of CD5+ cells in the patient (such as a population of CD5+ T cells, CD5+ B cells, and / or CD5+ NK cells in the patient), and subsequently administering to the patient a transplant comprising the hematopoietic stem cells.

[0072] In some embodiments of any one of the aforementioned four aspects, an antibody or fragment thereof that binds to CD5 (e.g., CD5 on the surface of CD5+T cells, CD5+B cells, or CD5+NK cells) is covalently bound to an Fc domain, such as a dimeric Fc domain isolated from a human antibody (e.g., isolated from a human antibody of the IgG1, IgG2, IgG3, or IgG4 isotype). In some embodiments, the Fc domain is a monomeric Fc domain comprising a single polypeptide chain. In some embodiments, the N-terminus of the antibody or its fragment is bound to the Fc domain. In some embodiments, the C-terminus of the antibody or its fragment is bound to the Fc domain. The Fc domain can be conjugated to one or more copies of the antibody or its fragment. For example, a conjugate that can be used in conjunction with the methods described herein includes a dimeric Fc domain in which each polypeptide chain of the Fc domain is conjugated to an antibody or its fragment. The Fc domain, in turn, can be conjugated to a cytotoxin, such as a cytotoxin described herein (e.g., an amanita toxin such as α-amanitin, Pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin, maytansine, maytansine alkaloids, auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycins, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolobenzodiazepines, and indolobenzodiazepine dimers, or variants thereof).

[0073] In some embodiments, an anti-CD5 antibody or fragment thereof is covalently bound to a cytotoxin, such as a cytotoxin described herein (e.g., an amanita toxin such as α-amanitin, Pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin, maytansine, maytansine alkaloids, auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolebenzodiazepines, and indolebenzodiazepine dimers, or variants thereof). In some embodiments, the N-terminus of the antibody or fragment thereof is bound to the cytotoxin. In some embodiments, the C-terminus of the antibody or fragment thereof is bound to the cytotoxin. The cytotoxin, in turn, can be conjugated to an Fc domain.

[0074] In some embodiments, the anti-CD5 antibody or fragment thereof is covalently bound to a cytotoxin at one site on the antibody or fragment thereof (e.g., the N-terminus or C-terminus of the antibody or fragment thereof) and covalently bound to an Fc domain at another site on the antibody or fragment thereof (e.g., the opposite terminus of the antibody or fragment thereof).

[0075] 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 Fc domain.

[0076] In some embodiments of any of the above aspects, the cytotoxin is an amanitin toxin or a derivative thereof, such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin, amanullinic acid, and pro-amanitin. In one embodiment, the cytotoxin is amanitin. In some embodiments of any of the above aspects, the cytotoxin is an amanitin toxin, and the antibody or antigen-binding fragment thereof conjugated to the cytotoxin is represented by the formula Ab-ZL-Am, wherein Ab is an anti-CD5 antibody, an antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an amanitin toxin. In some embodiments, the amanitin toxin is conjugated to a linker. In some embodiments, the amanitin toxin-linker conjugate Am-LZ is represented by formula (I)

[0077]

[0078] Where R1 is H, OH, OR A OR C ;

[0079] R2 is H, OH, OR B OR C ;

[0080] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0081] R3 is H, R C or R D ;

[0082] R4 is H, OH, OR C , OR D 、R C or R D ;

[0083] R5 is H, OH, OR C , OR D 、R C or R D ;

[0084] R6 is H, OH, OR C , OR D 、R C or R D ;

[0085] R7 is H, OH, OR C , OR D 、R C or R D ;

[0086] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0087] R9 is H, OH, OR C OR D ;

[0088] X is -S-, -S(O)- or -SO2-;

[0089] R C Yes -LZ;

[0090] 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;

[0091] L is a linker such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (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 heteroarylene; a dipeptide, -C(=O)-, a peptide, or a combination thereof; and

[0092] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present within an antibody or antigen-binding fragment thereof that binds to CD5 on the surface of, for example, a CD5+ T cell, a CD5+ B cell, or a CD5+ NK cell.

[0093] In some embodiments, Am comprises exactly one R C substituent.

[0094] In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0095]

[0096] wherein S is a sulfur atom and represents a reactive substituent (e.g., a -SH group from a cysteine ​​residue) present in the antibody or antigen-binding fragment thereof that binds to CD5.

[0097] In some embodiments, LZ is

[0098]

[0099] In some embodiments, Am-LZ-Ab is:

[0100]

[0101] In some embodiments, Am-LZ is represented by formula (IA)

[0102]

[0103] Where R1 is H, OH, OR A OR C ;

[0104] R2 is H, OH, OR B OR C ;

[0105] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0106] R3 is H, R C or R D ;

[0107] R4 is H, OH, OR C , OR D 、R C or R D ;

[0108] R5 is H, OH, OR C , OR D 、R C or R D ;

[0109] R6 is H, OH, OR C , OR D 、R C or R D ;

[0110] R7 is H, OH, OR C , OR D 、R C or R D ;

[0111] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0112] R9 is H, OH, OR C OR D ;

[0113] X is -S-, -S(O)- or -SO2-;

[0114] R C Yes -LZ;

[0115] 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;

[0116] L is a linker such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (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 heteroarylene; a dipeptide, -C(=O)-, a peptide, or a combination thereof;

[0117] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present within an antibody or antigen-binding fragment thereof that binds to CD5, such as CD5 on the surface of a CD5+ T cell, a CD5+ B cell, or a CD5+ NK cell; and

[0118] where Am contains exactly one R C substituent.

[0119] In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0120]

[0121] In some embodiments, LZ is

[0122]

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

[0124]

[0125] In some embodiments, Am-LZ-Ab is

[0126] In some embodiments, Am-LZ is represented by formula (IB)

[0127]

[0128] Where R1 is H, OH, OR A OR C ;

[0129] R2 is H, OH, OR B OR C ;

[0130] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0131] R3 is H, R C or R D ;

[0132] R4 is H, OH, OR C , OR D 、R C or R D ;

[0133] R5 is H, OH, OR C , OR D 、R C or R D ;

[0134] R6 is H, OH, OR C , OR D 、R C or R D ;

[0135] R7 is H, OH, OR C , OR D 、R C or R D ;

[0136] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0137] R9 is H, OH, OR C OR D ;

[0138] X is -S-, -S(O)- or -SO2-;

[0139] R C Yes -LZ;

[0140] 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)-, a peptide, or a combination thereof;

[0141] L is a linker such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (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 heteroarylene;

[0142] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present within an antibody or antigen-binding fragment thereof that binds to CD5 on the surface of, for example, a CD5+ T cell, a CD5+ B cell, or a CD5+ NK cell; and

[0143] where Am contains exactly one R C substituent.

[0144] In some embodiments, R A and R B Together with the oxygen atom to which they are attached, they combine to form a 5-membered heterocycloalkyl group of the formula:

[0145]

[0146] wherein Y is -C(=O)-, -C(=S)-, -C(=NR E )-or-C(R E R E’ )-; and R E and R E’ Each independently is an optionally substituted C1-C6 alkylene-R C , optionally substituted C1-C6 heteroalkylene-R C , optionally substituted C2-C6 alkenylene-R C , optionally substituted C2-C6 heteroalkenylene-R C , optionally substituted C2-C6 alkynylene-R C , optionally substituted C2-C6 heteroalkynylene-R C , optionally substituted cycloalkylene-R C , optionally substituted heterocycloalkylene-R C , optionally substituted arylene-R C or optionally substituted heteroarylene-R C .

[0147] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB), wherein R1 is H, OH, OR A OR C ;

[0148] R2 is H, OH, OR B OR C ;

[0149] R A and R B Together with the oxygen atoms to which they are bound, they form:

[0150]

[0151] R3 is H or R C ;

[0152] R4 is H, OH, OR C , OR D 、R C or R D ;

[0153] R5 is H, OH, OR C , OR D 、R C or R D ;

[0154] R6 is H, OH, OR C , OR D 、R C or R D ;

[0155] R7 is H, OH, OR C , OR D 、R C or R D ;

[0156] R8 is OH, NH2, OR C or NHR C ;

[0157] R9 is H or OH; and

[0158] where R C and R D Each is as defined above.

[0159] In some embodiments, Am is represented by Formula (IA) or Formula (IB),

[0160] Where R1 is H, OH, OR A OR C ;

[0161] R2 is H, OH, OR B OR C ;

[0162] R A and R B Together with the oxygen atoms to which they are bound, they form:

[0163]

[0164] R3 is H or R C ;

[0165] R4 and R5 are each independently H, OH, OR C 、R C OR D ;

[0166] R6 and R7 are each H;

[0167] R8 is OH, NH2, OR C or NHR C ;

[0168] R9 is H or OH; and

[0169] Among them, X, R C and R D Each is as defined above.

[0170] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0171] Where R1 is H, OH or OR A ;

[0172] R2 is H, OH or OR B ;

[0173] R A and R B Together with the oxygen atoms to which they are bound, they form:

[0174]

[0175] R3, R4, R6 and R7 are each H;

[0176] R5 is OR C ;

[0177] R8 is OH or NH2;

[0178] R9 is H or OH; and

[0179] Where X and R C As defined above.

[0180] In some embodiments, Am is represented by Formula (IA) or Formula (IB),

[0181] wherein R1 and R2 are each independently H or OH;

[0182] R3 is R C ;

[0183] R4, R6 and R7 are each H;

[0184] R5 is H, OH or OC1-C6 alkyl;

[0185] R8 is OH or NH2;

[0186] R9 is H or OH; and

[0187] Where X and R C As defined above.

[0188] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0189] wherein R1 and R2 are each independently H or OH;

[0190] R3, R6 and R7 are each H;

[0191] R4 and R5 are each independently H, OH, OR C or R C ;

[0192] R8 is OH or NH2;

[0193] R9 is H or OH; and

[0194] Where X or R C As defined above.

[0195] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0196] wherein R1 and R2 are each independently H or OH;

[0197] R3, R6 and R7 are each H;

[0198] R4 and R5 are each independently H or OH;

[0199] R8 is OH, NH2, OR C or NHR C ;

[0200] R9 is H or OH; and

[0201] Where X and R C As defined above.

[0202] In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0203]

[0204] In some embodiments, LZ is

[0205]

[0206] In some embodiments, Am-LZ-Ab is

[0207]

[0208] In some embodiments, Am-LZ-Ab is

[0209]

[0210] In some embodiments, Am-LZ is represented by Formula (II), Formula (IIA), or Formula (IIB)

[0211]

[0212]

[0213] wherein X is S, SO or SO2; R1 is H or a linker that is covalently bound to the antibody or antigen-binding fragment thereof through a chemical moiety Z formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the antibody or antigen-binding fragment thereof; and R2 is H or a linker that is covalently bound to the antibody or antigen-binding fragment thereof through a chemical moiety Z formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the antibody or antigen-binding fragment thereof; wherein when R1 is H, R2 is a linker, and when R2 is H, R1 is a linker.

[0214] In some embodiments, the linker comprises -(CH) 2n - unit, wherein n is an integer from 2-6.

[0215] In some embodiments, R1 is a linker and R2 is H, and the linker and chemical moiety (together LZ) are

[0216]

[0217] In some embodiments, Am-LZ-Ab is

[0218]

[0219] In some embodiments, Am-LZ-Ab is

[0220]

[0221] In some embodiments, Am-LZ-Ab is

[0222]

[0223] In some embodiments of any of the above aspects, the cytotoxin is a maytansinoid selected from the group consisting of DM1 and DM4. In some embodiments, the cytotoxin is an auristatin selected from the group consisting of monomethyl auristatin E and monomethyl auristatin F. In some embodiments, the cytotoxin is an anthracycline selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin.

[0224] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine dimer represented by formula (IV):

[0225]

[0226] In some embodiments, the cytotoxin is conjugated to the antibody or antigen-binding fragment thereof by means of a maleimidocaproyl linker.

[0227] In some embodiments, the cytotoxin is an auristatin selected from the group consisting of monomethyl auristatin E and monomethyl auristatin F.

[0228] In some embodiments, the cytotoxin is an anthracycline selected from the group consisting of daunomycin, doxorubicin, epirubicin, and idarubicin.

[0229] In some embodiments, anti-CD5 antibodies, their antigen-binding fragments or antibody-drug conjugates are internalized by immune cells such as T cells, B cells or NK cells (e.g., CD5+T cells, CD5+B cells or CD5+NK cells) after being administered to the patient. For example, anti-CD5 antibodies, their antigen-binding fragments or antibody-drug conjugates can be internalized by T cells (e.g., after binding to cell surface CD5) through receptor-mediated endocytosis. In some embodiments, the cytotoxin covalently bound to the antibody or its antigen-binding fragment can be released intracellularly by chemical cleavage (e.g., by enzymatic cleavage or non-specific cleavage of a linker described herein). The cytotoxin can then approach its intracellular target (e.g., RNA polymerase, mitotic spindle apparatus, nuclear DNA, ribosomal RNA or topoisomerase, etc.) to promote the death of endogenous immune cells (e.g., CD5+T cells, CD5+B cells or CD5+NK cells) before hematopoietic stem cell transplantation therapy.

[0230] In some embodiments, anti-CD5 antibodies, antigen-binding fragments thereof, or antibody-drug conjugates can promote necrosis of immune cells such as T cells, B cells, or NK cells (e.g., CD5+T cells, CD5+B cells, or CD5+NK cells). In some embodiments, antibodies or antigen-binding fragments thereof can promote the death of endogenous immune cells (e.g., CD5+T cells, CD5+B cells, or CD5+NK cells) prior to transplantation therapy by recruiting one or more complement proteins, NK cells, macrophages, neutrophils, and / or eosinophils to immune cells after administration to the patient.

[0231] In some embodiments, the autologous transplant comprising hematopoietic stem cells is administered to the patient.For example, autologous hematopoietic stem cells can be taken out from the patient (such as the patient of needs hematopoietic stem cell transplantation therapy), and the cell can be administered to (for example, be infused into) the patient subsequently, to refill one or more cell types of hematopoietic pedigree. The hematopoietic stem cell extracted can, for example, be freshly infused into the experimenter after maintaining one or more hours, one day or more days or one week or more weeks in vitro. For example, the hematopoietic stem cell extracted can be infused into the patient again in 1 hour to approximately 1 week, 1 hour to approximately 72 hours, approximately 1 hour to approximately 48 hours or approximately 1 hour to approximately 24 hours after extracting from the patient. In some embodiments, the hematopoietic stem cell extracted is frozen for long-term storage before being infused into the patient again. For example, the hematopoietic stem cell extracted can be frozen and frozen for 1 week to 1 year or longer before being infused into the patient again.

[0232] In some embodiments, the allograft comprising hematopoietic stem cells is administered to the patient.For example, allogeneic hematopoietic stem cells can be from a donor, such as being the donor of HLA matching for the patient, for example, close family members on the patient's blood relationship take out.In some embodiments, allogeneic hematopoietic stem cells are HLA mismatches for the patient.After extracting allogeneic hematopoietic stem cells from a donor, the cell can be administered to (for example, be infused into) the patient subsequently, so as to refill one or more cell types of hematopoietic lineage. The hematopoietic stem cells extracted can, for example, be infused into the experimenter freshly after maintaining one or more hours, one or more days or one week or more weeks in vitro.For example, the hematopoietic stem cells extracted can be infused into the patient in 1 hour to about 1 week, 1 hour to about 72 hours, about 1 hour to about 48 hours or about 1 hour to about 24 hours after extracting from the donor.In some embodiments, the hematopoietic stem cells extracted were frozen so as to store for a long time before being infused into the patient. For example, extracted hematopoietic stem cells can be frozen and cryopreserved for a period of 1 week to 1 year or longer prior to infusion into the patient.

[0233] In some embodiments, a transplant comprising hematopoietic stem cells is administered to a patient after the concentration of the anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug conjugate has been substantially cleared from the patient's blood.

[0234] In some embodiments, a transplant comprising hematopoietic stem cells is administered to a patient 1 hour to 7 days (e.g., 6 hours to 3 days, 12 hours to 36 hours, or about 24 hours) after the concentration of the anti-CD5 antibody, antigen-binding fragment, or antibody-drug conjugate has been substantially cleared from the patient's blood.

[0235] In some embodiments, the hematopoietic stem cells or their progeny maintain hematopoietic stem cell functional potential two or more days (e.g., about 2 days to about 5 days, about 2 days to about 7 days, about 2 days to about 20 days, about 2 days to about 30 days, such as 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days or longer) after hematopoietic stem cells are transplanted into the patient.

[0236] In some embodiments, the population of CD5+ cells comprises CD34+ cells. For example, the proportion of CD34+ cells in a population of CD5+ cells can be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% of the total number of cells in the population.

[0237] In some embodiments, the hematopoietic stem cells or their progeny are capable of localizing to hematopoietic tissue (such as bone marrow) and / or reconstitute hematopoiesis following hematopoietic stem cell transplantation into a patient.

[0238] In some embodiments, upon transplantation into a patient, the hematopoietic stem cells result in the recovery of a population of cells selected from the group consisting of 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 B lymphocytes.

[0239] In some embodiments, the patient has cancer. The cancer can be a blood cancer or a type of leukemia, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, or chronic lymphocytic leukemia.

[0240] In some embodiments, the CD5+ cells include cancer cells.

[0241] In some embodiments, the anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug conjugate depletes cancer cells in a patient. For example, the antibody or antigen-binding fragment thereof can deplete 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or substantially all of the cancer cells in a patient.

[0242] In some embodiments, anti-CD5 antibodies, antigen-binding fragments thereof, or antibody-drug conjugates deplete cells (e.g., leukemia cells) of blood cancers in patients. In some embodiments, the cells of blood cancers are acute myeloid leukemia cells, acute lymphocytic leukemia cells, chronic myeloid leukemia cells, or chronic lymphocytic leukemia cells. In some embodiments, the cells of blood cancers are megakaryocytes, thrombocytes, platelets, red blood cells, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, or B lymphocytes.

[0243] In some embodiments, the population of CD5+ cells comprises immune cells, such as CD5+ T cells, CD5+ B cells, and / or CD5+ NK cells.

[0244] In some embodiments of any one of the above aspects, the method is used to treat one or more disorders, such as by, for example, depleting the population of immune cells that cross-react with the hematopoietic stem cell transplant (e.g., non-self MHC antigens expressed by the hematopoietic stem cell transplant) in the patient before hematopoietic stem cell transplantation therapy to prevent or reduce the possibility of rejection of the hematopoietic stem cell transplant that might otherwise be caused by the population of immune cells. After transplantation, hematopoietic stem cells can establish effective hematopoiesis to supplement the defective cell types in the patient or the cell types that are actively killed or have been killed by, for example, chemotherapy. For example, the patient can be a patient with a stem cell disorder. In some embodiments, the patient suffers from a hemoglobinopathy disorder, such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome. The patient may suffer from an immunodeficiency disorder, such as a congenital immunodeficiency disorder or an acquired immunodeficiency disorder (e.g., human immunodeficiency virus or acquired immunodeficiency syndrome). In some embodiments, the patient suffers from a metabolic disorder, such as glycogen storage disease, mucopolysaccharidosis, Gaucher's disease, Hurler's disease, sphingolipid storage disease and metachromatic leukodystrophy. In some embodiments, the patient suffers from a disorder selected from the group consisting of: adenosine deaminase deficiency and severe combined immunodeficiency, hyperimmunoglobulin M syndrome, Chediak-Higashi Disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, major thalassemia, systemic sclerosis, systemic lupus erythematosus and juvenile rheumatoid arthritis. In some embodiments, the patient suffers from an autoimmune disease, such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease (Crohn'sdisease) and type 1 diabetes. In some embodiments, the patient suffers from cancer or myeloproliferative disease, such as hematological cancer. In some embodiments, the patient has acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma or non-Hodgkin's lymphoma.In some embodiments, the patient has a myelodysplastic disease, such as myelodysplastic syndrome.

[0245] In some embodiments of any of the above aspects, the method is used to treat cancer, such as a cancer characterized by CD5+ cells (e.g., a leukemia characterized by CD5+ cells), by administering an antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate that depletes a population of CD5+ cancer cells in the patient, and / or by administering an antibody or its antigen-binding fragment prior to hematopoietic stem cell transplantation therapy to prevent or reduce the likelihood of rejection of the hematopoietic stem cell transplant that might otherwise be caused by a population of immune cells that cross-react with non-self antigens expressed by the hematopoietic stem cell transplant. In the latter case, the transplant can then reconstitute the population of cells that were depleted, for example, during the process of eradicating the cancer cells. The cancer can be a hematological cancer, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.

[0246] In some embodiments of any of the above aspects, the method is used to treat an autoimmune disease, such as by administering an anti-CD5 antibody, an antigen-binding fragment thereof, or an antibody-drug conjugate to deplete a population of CD5+ autoimmune cells (e.g., a population of self-reactive CD5+ T cells, B cells, and / or NK cells), and / or by administering the antibody or antigen-binding fragment thereof prior to hematopoietic stem cell transplantation therapy to prevent or reduce the likelihood of rejection of a hematopoietic stem cell transplant that might otherwise be caused by a population of immune cells that cross-react with the hematopoietic stem cell transplant (e.g., with non-self MHC antigens expressed by the hematopoietic stem cell transplant). In the latter case, the transplant can then reconstitute the population of cells that were depleted, for example, during the process of eradicating the autoimmune cells.The autoimmune disease can be, for example, scleroderma, multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), psoriasis, Type 1 diabetes mellitus, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Balo disease, Behcet's disease, disease), bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac disease-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, malignant atrophic papulosis (Degos disease), discoid lupus, dysautonomia, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis thyroiditis), hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki's disease, lichen planus, Lyme disease, Meniere's disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus-myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndromes, polymyalgia rheumatica, essential agammaglobulinemia, Raynaud phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome. syndrome, stiff-person syndrome, Takayasu's arteritis, temporal arteritis (also called "giant cell arteritis"), ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia ("vulvar vestibulitis"), and Wegener's granulomatosis.

[0247] Thus, in some embodiments of any of the above aspects, the invention features a method of treating a hemoglobinopathy disorder such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome. In some embodiments, the invention features a method of treating an immunodeficiency disorder such as a congenital immunodeficiency disorder or an acquired immunodeficiency disorder (e.g., human immunodeficiency virus or acquired immunodeficiency syndrome). In some embodiments, the invention features a method of treating a metabolic disorder such as glycogen storage disease, mucopolysaccharidosis, Gaucher's disease, Hurler's disease, sphingolipidosis, and metachromatic leukodystrophy. In some embodiments, the present invention features a method of treating a disorder selected from the group consisting of adenosine deaminase deficiency and severe combined immunodeficiency, hyperimmunoglobulinemia M syndrome, Chidon disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, and juvenile rheumatoid arthritis. In some embodiments, the present invention features a method of treating an autoimmune disease such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, and type 1 diabetes. In some embodiments, the present invention features a method of treating cancer or a myeloproliferative disorder such as a hematological cancer. In some embodiments, the present invention features a method of treating acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the patient has a myelodysplastic disease such as myelodysplastic syndrome. In these embodiments, the method may comprise administering to the patient an antibody, antigen-binding fragment thereof, or conjugate thereof that binds to CD5, such as the antibody, antigen-binding fragment thereof, or conjugate thereof according to any one of the aspects or embodiments of the invention. The method may further comprise administering to the patient a hematopoietic stem cell transplant, for example according to any one of the aspects or embodiments of the invention.

[0248] Similarly, in some embodiments of any of the above aspects, the present invention provides a method of directly treating cancer such as a cancer characterized by CD5+ cells (e.g., a leukemia characterized by CD5+ cells). In these embodiments, the method may comprise administering to the patient an antibody, an antigen-binding fragment thereof, or a conjugate thereof that binds to CD5, such as an antibody, an antigen-binding fragment thereof, or a conjugate thereof of any one of the aspects or embodiments of the present invention. The cancer may be a hematological cancer, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma.

[0249] Additionally, in some embodiments of any of the above aspects, the invention provides a method of treating an autoimmune disease such as MS, SLE, RA, IBD, psoriasis, type 1 diabetes, ADEM, Addison's disease, alopecia universalis, ankylosing spondylitis, APS, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, AIED, ALPS, autoimmune oophoritis, Barlow's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, CFIDS, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac disease-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, malignant atrophic papulosis, discoid lupus, dysautonomia, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome Graves' disease, 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, MCTD, myasthenia gravis, neuromyotonia, OMS, optic neuritis, Odd's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis, and Dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndrome, polymyalgia rheumatica, essential agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-person syndrome, Takayasu arteritis, temporal arteritis (also known as "giant cell arteritis"), ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia ("vulvar vestibulitis"), and Wegener's granulomatosis. In these embodiments, the method can comprise administering to the patient an antibody, antigen-binding fragment thereof, or conjugate thereof that binds to CD5, such as an antibody, antigen-binding fragment thereof, or conjugate thereof of any aspect or embodiment of the invention.

[0250] In another aspect, the invention features an antibody or antigen-binding fragment thereof capable of binding to CD5, wherein the antibody or antigen-binding fragment thereof is conjugated to a toxin.

[0251] In some embodiments, the antibody or antigen-binding fragment thereof is produced by hybridoma cell line ATCC CRL 8000. In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to the antibody or antigen-binding fragment thereof produced by hybridoma cell line ATCC CRL8000.

[0252] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following variable domains:

[0253] V having the following amino acid sequence L : and

[0254] V having the following amino acid sequence H :

[0255] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned variable domains.

[0256] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDRs:

[0257] a CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 3);

[0258] CDR-H2 having the amino acid sequence NTHTGE (SEQ ID NO: 4);

[0259] CDR-H3 having the amino acid sequence RGYDWYFDV (SEQ ID NO: 5);

[0260] a CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 6);

[0261] a CDR-L2 having the amino acid sequence RANRLVD (SEQ ID NO: 7); and

[0262] CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 8).

[0263] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned CDRs.

[0264] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following variable domains:

[0265] V having the following amino acid sequenceL : and

[0266] V having the following amino acid sequence H :

[0267] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned variable domains.

[0268] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDRs:

[0269] a CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 11);

[0270] CDR-H2 having the amino acid sequence NTHYGE (SEQ ID NO: 12);

[0271] CDR-H3 having the amino acid sequence RRGYDWYFDV (SEQ ID NO: 13);

[0272] a CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 14);

[0273] a CDR-L2 having the amino acid sequence RANRLES (SEQ ID NO: 15); and

[0274] CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 16).

[0275] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned CDRs.

[0276] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDRs:

[0277] CDR-H1 having the amino acid sequence GYSITSGYY (SEQ ID NO: 17);

[0278] a CDR-H2 having the amino acid sequence ISYSGFT (SEQ ID NO: 18);

[0279] a CDR-H3 having the amino acid sequence AGDRTGSWFAY (SEQ ID NO: 19);

[0280] a CDR-L1 having the amino acid sequence QDISNY (SEQ ID NO: 20);

[0281] a CDR-L2 having the amino acid sequence ATS (SEQ ID NO: 21); and

[0282] CDR-L3 having the amino acid sequence LQYASYPFT (SEQ ID NO: 22).

[0283] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned CDRs.

[0284] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDRs:

[0285] CDR-H1 having the amino acid sequence GYIFTNYG (SEQ ID NO: 23);

[0286] CDR-H2 having the amino acid sequence INTYNGEP (SEQ ID NO: 24);

[0287] a CDR-H3 having the amino acid sequence ARGDYYGYEDY (SEQ ID NO: 25);

[0288] a CDR-L1 having the amino acid sequence QGISNY (SEQ ID NO: 26);

[0289] a CDR-L2 having the amino acid sequence YTS (SEQ ID NO: 27); and

[0290] CDR-L3 having the amino acid sequence QQYSKLPWT (SEQ ID NO: 28).

[0291] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned CDRs.

[0292] In some embodiments, the antibody or antigen-binding fragment thereof comprises the following CDRs:

[0293] CDR-H1 having the amino acid sequence FSLSTSGMG (SEQ ID NO: 29);

[0294] CDR-H2 having the amino acid sequence WWDDD (SEQ ID NO: 30);

[0295] a CDR-H3 having the amino acid sequence RRATGTGFDY (SEQ ID NO: 31);

[0296] a CDR-L1 having the amino acid sequence QDVGTA (SEQ ID NO: 32);

[0297] a CDR-L2 having the amino acid sequence WTSTRHT (SEQ ID NO: 33); and

[0298] CDR-L3 having the amino acid sequence YNSYNT (SEQ ID NO: 34).

[0299] In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising the aforementioned CDRs.

[0300] In some embodiments, the antibody or antigen-binding fragment thereof comprises a combination of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions listed below in Table 1. In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising a combination of the CDRs listed below in Table 1.

[0301] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO: 257 and a light chain variable region as set forth in SEQ ID NO: 258. In some embodiments, the antibody or antigen-binding fragment thereof competitively inhibits binding of CD5 to an antibody or antigen-binding fragment thereof comprising a heavy chain variable region as set forth in SEQ ID NO: 257 and a light chain variable region as set forth in SEQ ID NO: 258.

[0302] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen-binding fragment thereof, a polyclonal antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, a dual variable immunoglobulin domain, a scFv, a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment, a F(ab')2 molecule, and a tandem di-scFv.

[0303] In some embodiments, the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.

[0304] In some embodiments, the antibody or antigen-binding fragment thereof conjugated to the cytotoxin is represented by the formula Ab-Cy, wherein Ab is an antibody or antigen-binding fragment thereof, and Cy is a cytotoxin. In some embodiments, the cytotoxin is selected from the group consisting of amanita phalloidins, pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin, maytansine, maytansine alkaloids, auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolebenzodiazepines, and indolebenzodiazepine dimers, or variants thereof.

[0305] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin non-toxic cyclic peptide, amanitin non-toxic cyclic peptide acid, and amanitin non-toxic cyclic peptide pro-. In some embodiments, the cytotoxin is an amanita toxin, and the antibody or antigen-binding fragment thereof conjugated to the cytotoxin is represented by the formula Ab-ZL-Am, wherein Ab is an antibody or antigen-binding fragment thereof, Z is a chemical moiety, L is a linker, and Am is an amanita toxin. In some embodiments, Am-LZ is represented by formula (I)

[0306]

[0307] Where R1 is H, OH, OR A OR C ;

[0308] R2 is H, OH, OR B OR C ;

[0309] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0310] R3 is H, R C or R D ;

[0311] R4 is H, OH, OR C , OR D 、R C or R D ;

[0312] R5 is H, OH, OR C , OR D 、R C or R D ;

[0313] R6 is H, OH, ORC , OR D 、R C or R D ;

[0314] R7 is H, OH, OR C , OR D 、R C or R D ;

[0315] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0316] R9 is H, OH, OR C OR D ;

[0317] X is -S-, -S(O)- or -SO2-;

[0318] R C Yes -LZ;

[0319] 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;

[0320] L is a linker such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (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 heteroarylene; a dipeptide, -C(=O)-, a peptide, or a combination thereof; and

[0321] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present within an antibody or antigen-binding fragment thereof that binds to CD5 on the surface of, for example, a CD5+ T cell, a CD5+ B cell, or a CD5+ NK cell.

[0322] In some embodiments, Am comprises exactly one R C substituent.

[0323] In some embodiments, Am-LZ is represented by formula (IA)

[0324]

[0325] Where R1 is H, OH, OR A OR C ;

[0326] R2 is H, OH, OR B OR C ;

[0327] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0328] R3 is H, R C or R D ;

[0329] R4 is H, OH, OR C , OR D 、R C or R D ;

[0330] R5 is H, OH, OR C , OR D 、R C or R D ;

[0331] R6 is H, OH, OR C , OR D 、R C or R D ;

[0332] R7 is H, OH, OR C , OR D 、R C or R D ;

[0333] R8 is OH, NH2, OR C , OR D 、NHRC or NR C R D ;

[0334] R9 is H, OH, OR C OR D ;

[0335] X is -S-, -S(O)- or -SO2-;

[0336] R C Yes -LZ;

[0337] 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;

[0338] L is a linker such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (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 heteroarylene, dipeptide, -C(=O)-, a peptide, or a combination thereof;

[0339] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present within an antibody, or an antigen-binding fragment thereof, that binds to CD5 on the surface of, for example, a CD5+ T cell, a CD5+ B cell, or a CD5+ NK cell; and

[0340] where Am contains exactly one R C substituent.

[0341] In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0342]

[0343] In some embodiments, LZ is

[0344]

[0345] In some embodiments, Am-LZ-Ab is

[0346]

[0347] In some embodiments, Am-LZ-Ab is

[0348]

[0349] In some embodiments, Am-LZ is represented by formula (IB)

[0350]

[0351] Where R1 is H, OH, OR A OR C ;

[0352] R2 is H, OH, OR B OR C ;

[0353] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0354] R3 is H, R C or R D ;

[0355] R4 is H, OH, OR C , OR D 、R C or R D ;

[0356] R5 is H, OH, OR C , OR D 、R C or R D ;

[0357] R6 is H, OH, OR C , OR D 、R C or R D ;

[0358] R7 is H, OH, OR C , OR D 、R C or R D ;

[0359] R8 is OH, NH2, OR C , ORD 、NHR C or NR C R D ;

[0360] R9 is H, OH, OR C OR D ;

[0361] X is -S-, -S(O)- or -SO2-;

[0362] R C Yes -LZ;

[0363] 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;

[0364] L is a linker such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (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 heteroarylene, dipeptide, -C(=O)-, a peptide, or a combination thereof;

[0365] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present within an antibody or antigen-binding fragment thereof that binds to CD5 on the surface of, for example, a CD5+ T cell, a CD5+ B cell, or a CD5+ NK cell; and

[0366] where Am contains exactly one R C substituent.

[0367] In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0368]

[0369] In some embodiments, LZ is

[0370]

[0371] In some embodiments, Am-LZ-Ab is

[0372]

[0373] In some embodiments, Am-LZ-Ab is

[0374]

[0375] In some embodiments, R A and R B Together with the oxygen atom to which they are attached, they combine to form a 5-membered heterocycloalkyl group of the formula:

[0376]

[0377] wherein Y is -C(=O)-, -C(=S)-, -C(=NR E )-or-C(R E R E’ )-;and

[0378] R E and R E’ Each independently is an optionally substituted C1-C6 alkylene-R C , optionally substituted C1-C6 heteroalkylene-R C , optionally substituted C2-C6 alkenylene-R C , optionally substituted C2-C6 heteroalkenylene-R C , optionally substituted C2-C6 alkynylene-R C , optionally substituted C2-C6 heteroalkynylene-R C , optionally substituted cycloalkylene-R C , optionally substituted heterocycloalkylene-R C , optionally substituted arylene-R C or optionally substituted heteroarylene-R C .

[0379] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB), wherein R1 is H, OH, OR A OR C ;

[0380] R2 is H, OH, OR B OR C ;

[0381] R Aand R B Together with the oxygen atoms to which they are bound, they form:

[0382]

[0383] R3 is H or R C ;

[0384] R4 is H, OH, OR C , OR D 、R C or R D ;

[0385] R5 is H, OH, OR C , OR D 、R C or R D ;

[0386] R6 is H, OH, OR C , OR D 、R C or R D ;

[0387] R7 is H, OH, OR C , OR D 、R C or R D ;

[0388] R8 is OH, NH2, OR C or NHR C ;

[0389] R9 is H or OH; and

[0390] Among them, X, R C and R D Each is as defined above.

[0391] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0392] Where R1 is H, OH, OR A OR C ;

[0393] R2 is H, OH, OR B OR C ;

[0394] R A and R B Together with the oxygen atoms to which they are bound, they form:

[0395]

[0396] R3 is H or R C ;

[0397] R4 and R5 are each independently H, OH, OR C 、R C OR D ;

[0398] R6 and R7 are each H;

[0399] R8 is OH, NH2, OR C or NHR C ;

[0400] R9 is H or OH; and

[0401] where R C As defined above.

[0402] In some embodiments, Am is represented by Formula (IA) or Formula (IB),

[0403] Where R1 is H, OH or OR A ;

[0404] R2 is H, OH or OR B ;

[0405] R A and R B Together with the oxygen atoms to which they are bound, they form:

[0406]

[0407] R3, R4, R6 and R7 are each H;

[0408] R5 is OR C ;

[0409] R8 is OH or NH2;

[0410] R9 is H or OH; and

[0411] Where X and R C As defined above.

[0412] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0413] wherein R1 and R2 are each independently H or OH;

[0414] R3 is R C ;

[0415] R4, R6 and R7 are each H;

[0416] R5 is H, OH or OC1-C6 alkyl;

[0417] R8 is OH or NH2;

[0418] R9 is H or OH; and

[0419] Where X and R C As defined above.

[0420] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0421] wherein R1 and R2 are each independently H or OH;

[0422] R3, R6 and R7 are each H;

[0423] R4 and R5 are each independently H, OH, OR C or R C ;

[0424] R8 is OH or NH2;

[0425] R9 is H or OH; and

[0426] Where X and R C As defined above.

[0427] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0428] wherein R1 and R2 are each independently H or OH;

[0429] R3, R6 and R7 are each H;

[0430] R4 and R5 are each independently H or OH;

[0431] R8 is OH, NH2, OR C or NHR C ;

[0432] R9 is H or OH; and

[0433] Where X and R C As defined above.

[0434] In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0435]

[0436] In some embodiments, LZ is

[0437]

[0438] In some embodiments, the Am-LZ-Ab precursor is

[0439]

[0440] The maleimide reacts with thiol groups on cysteines found in antibodies.

[0441] In some embodiments, the Am-LZ-Ab precursor is

[0442]

[0443] The maleimide reacts with thiol groups on cysteines found in antibodies.

[0444] In some embodiments, Am-LZ is represented by Formula (II), Formula (IIA), or Formula (IIB)

[0445]

[0446]

[0447] wherein X is S, SO or SO2; R1 is H or a linker that is covalently bound to the antibody or antigen-binding fragment thereof through a chemical moiety Z formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the antibody or antigen-binding fragment thereof; and R2 is H or a linker that is covalently bound to the antibody or antigen-binding fragment thereof through a chemical moiety Z formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the antibody or antigen-binding fragment thereof; wherein when R1 is H, R2 is a linker, and when R2 is H, R1 is a linker.

[0448] In some embodiments, the linker comprises -(CH) 2n - unit, wherein n is an integer from 2-6.

[0449] In some embodiments, R1 is a linker and R2 is H, and the linker and chemical moiety (together LZ) are

[0450]

[0451] In some embodiments, Ab-ZL-Am is

[0452]

[0453] In some embodiments, Ab-ZL-Am is

[0454]

[0455] In some embodiments, the Am-LZ-Ab precursor is one of the following:

[0456]

[0457] The maleimide reacts with thiol groups on cysteines found in antibodies.

[0458] In some embodiments, the cytotoxin is a maytansinoid selected from the group consisting of DM1 and DM4. In some embodiments, the cytotoxin is an auristatin selected from the group consisting of monomethyl auristatin E and monomethyl auristatin F. In some embodiments, the cytotoxin is an anthracycline selected from the group consisting of daunomycin, doxorubicin, epirubicin, and idarubicin.

[0459] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine dimer represented by formula (IV):

[0460]

[0461] In some embodiments, the cytotoxin is conjugated to the antibody or antigen-binding fragment thereof by means of a maleimidocaproyl linker.

[0462] In some embodiments, the cytotoxin is an auristatin selected from the group consisting of monomethyl auristatin E and monomethyl auristatin F.

[0463] In some embodiments, the cytotoxin is an anthracycline selected from the group consisting of daunomycin, doxorubicin, epirubicin, and idarubicin.

[0464] In another aspect, the invention features a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any of the above aspects or embodiments of the invention and a pharmaceutically acceptable excipient.

[0465] In some embodiments, the pharmaceutical composition is formulated for transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular, intratumoral, parenteral, topical, intrathecal, or intracerebroventricular administration to a human patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0467] Figure 1 The diagram depicts the results of an in vitro cell line binding assay in which each of the indicated anti-CD5 antibodies or a negative control (i.e., mIgG1) was incubated with MOLT-4 cells (i.e., a human T lymphoblastoid cell line) followed by incubation with a fluorophore-conjugated anti-IgG antibody. Signals were detected by flow cytometry and expressed as geometric mean fluorescence intensity (y-axis) as a function of anti-CD5 antibody concentration (x-axis).

[0468] Figure 2The diagram depicts the results of an in vitro primary cell binding assay in which the indicated anti-CD5 antibodies or a negative control (i.e., hIgG1) were incubated with primary human T cells followed by incubation with a fluorophore-conjugated anti-IgG antibody. Signals were detected by flow cytometry and expressed as geometric mean fluorescence intensity (y-axis) as a function of anti-CD5 antibody concentration (x-axis).

[0469] Figure 3A and Figure 3B The graph depicts the results of an in vitro T cell killing assay involving interchain conjugated amanitin ( Figure 3A ) or site-specific conjugation of amanitin with a proposed DAR of 2 ( Figure 3B ) of the anti-CD5-amanitin ADC (i.e., 5D7-AM or "CD5 AM"). Figure 3A In Figure 2, an analysis of anti-CD5-ADC T cell killing compared to unconjugated anti-CD5 5D7 antibody (i.e., "naked CD5") is shown. Figure 3B Figure 2 shows the results of a comparison of an anti-CD5 antibody with an anti-CD5 5D7 antibody having an H435A mutation that reduces the half-life of the antibody (i.e., "CD5 Fast 1 / 2 Life AM"). The results show the number of viable T cells (y-axis) as assessed using flow cytometry as a function of the concentration of ADC (CD5 5D7 AM, CD5 5D7D265C.H435A AM) or unconjugated antibody (CD5 5D7) (x-axis).

[0470] Figures 4A-4B Graph depicts the results of an in vivo T cell depletion assay showing peripheral blood ( ) of humanized NSG mice 7 days after a single administration of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of an anti-CD5 5D7 amanitin ADC with an interchain DAR of 6 (i.e., CD5 5D7-AM). Figure 4A ) and bone marrow ( Figure 4B ) in the absolute level of T cells (CD3+ cells; y axis). For comparison, Figures 4A-4B Also shown are the levels of T cell depletion following treatment of humanized NSG mice with the indicated controls (ie, 25 mg / kg anti-CD52 antibody; 3 mg / kg hIgG1-amanitin ADC (ie, hIgG1-AM), 25 mg / kg hIgG1, or PBS).

[0471] Figures 5A-5CGraph depicts the results of an in vivo T cell depletion assay showing peripheral blood ( ) of humanized NSG mice 7 days after a single administration of 1 mg / kg or 3 mg / kg of an anti-CD5 5D7-amanitin ADC with a site-specific DAR of 2 (i.e., 5D7-AM). Figure 5A ),marrow( Figure 5B ) and thymus ( Figure 5C ) in the absolute level of T cells (CD3+ cells; y axis). For comparison, Figures 5A-5C Also shown are the levels of T cell exhaustion following treatment of humanized NSG mice with 3 mg / kg of unconjugated anti-CD5 antibody or with the indicated controls, ie, 3 mg / kg hIgG1-amanitin-ADC ("hIgG1-AM") or PBS.

[0472] Details

[0473] The present invention is based in part on the discovery that antibodies or antigen-binding fragments thereof that bind to CD5 (also known as lymphocyte antigen T1 / Leu-1) can be used as therapeutic agents to (i) directly treat cancers and autoimmune diseases characterized by CD5+ cells, and (ii) promote engraftment of transplanted hematopoietic stem cells in patients in need of transplantation therapy by depleting a population of immune cells that cross-react with and generate an immune response to a non-autologous hematopoietic stem cell transplant. These therapeutic activities can occur, for example, by binding of an anti-CD5 antibody or antigen-binding fragment thereof to CD5 expressed on the surface of a cell, such as a cancer cell, an autoimmune cell, or an immune cell that cross-reacts with a non-autologous hematopoietic stem cell antigen, thereby inducing the death of the bound cell. In the case of depleting a population of cancer cells or autoimmune cells, the anti-CD5 antibody or antigen-binding fragment thereof can be used to directly treat cancer or autoimmune diseases, such as the cancers or autoimmune diseases described herein. In the case of depletion of a population of immune cells that cross-react with non-self hematopoietic stem cell antigens (e.g., non-self MHC antigens expressed by hematopoietic stem cell transplants), anti-CD5 antibodies or their antigen-binding fragments can be used to prevent or reduce the likelihood of transplant rejection in patients with stem cell disorders, cancer or autoimmune diseases and undergoing hematopoietic stem cell transplantation therapy. In such cases, depletion of CD5+ immune cells that cross-react with one or more non-self hematopoietic stem cell antigens (e.g., one or more non-self MHC antigens) enables the transplanted hematopoietic stem cells to be successfully implanted in the transplant recipient. As the transplanted cells implant, they can home to the hematopoietic tissue, and then effective hematopoiesis can be generated in the hematopoietic tissue. Transplanted hematopoietic stem cells can subsequently generate populations of cells that are defective or missing in the transplant recipient, such as 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 B lymphocytes. In this way, anti-CD5 antibodies or fragments thereof can be used to promote the successful engraftment of hematopoietic stem cells in patients, such as human patients with stem cell disorders described herein.

[0474] definition

[0475] As used herein, the term "about" refers to a value within 10% above or below the value being described. For example, the term "about 5 nM" means a range from 4.5 nM to 5.5 nM.

[0476] As used herein, the term "amanitin" refers to a member of the amanita toxin peptide family produced by the fungus Amanita phalloides, a synthetic amanita toxin, an amanita toxin variant, or a derivative thereof, such as a variant or derivative thereof that is capable of inhibiting RNA polymerase II activity. Also included are synthetic amanita toxins (see, e.g., U.S. Patent No. 9,676,702, incorporated herein by reference). As described herein, amanita toxins can be conjugated to an antibody or antigen-binding fragment thereof (thereby forming a conjugate (also referred to as an antibody drug conjugate (ADC)), for example, by way of a linker portion (L). Exemplary methods of amanita toxin conjugation and linkers that can be used in such methods are described below. Also described herein are exemplary linker-containing amanita toxins that can be used for conjugation to an antibody or antigen-binding fragment according to the compositions and methods.

[0477] In certain embodiments, amanita toxins useful in conjunction with the compositions and methods described herein include compounds according to Formula (III) below, such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin, amanitin acid, and pro-amanitin. Formula (III) is as follows:

[0478]

[0479] Where R1 is H, OH or OR A ;

[0480] R2 is H, OH or OR B ;

[0481] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0482] R3 is H or R D ;

[0483] R4 is H, OH, OR D or R D ;

[0484] R5 is H, OH, OR D or R D ;

[0485] R6 is H, OH, OR D or R D ;

[0486] R7 is H, OH, OR D or R D ;

[0487] R8 is OH, NH2 or OR D ;

[0488] R9 is H, OH or OR D ;

[0489] X is -S-, -S(O)-, or -SO2-; and

[0490] 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.

[0491] For example, in one embodiment, amanita toxins useful in conjunction with the compositions and methods described herein include the following compounds according to formula (IIIA):

[0492]

[0493] Where R1 is H, OH or OR A ;

[0494] R2 is H, OH or OR B ;

[0495] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0496] R3 is H or R D ;

[0497] R4 is H, OH, OR D or R D ;

[0498] R5 is H, OH, OR D or R D ;

[0499] R6 is H, OH, OR D or R D ;

[0500] R7 is H, OH, OR D or R D ;

[0501] R8 is OH, NH2 or OR D ;

[0502] R9 is H, OH or OR D ;

[0503] X is -S-, -S(O)-, or -SO2-; and

[0504] 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.

[0505] In one embodiment, amanita toxins useful in conjunction with the compositions and methods described herein also include compounds according to formula (IIIB) below:

[0506]

[0507] Where R1 is H, OH or OR A ;

[0508] R2 is H, OH or OR B ;

[0509] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0510] R3 is H or R D ;

[0511] R4 is H, OH, OR D or R D ;

[0512] R5 is H, OH, OR D or R D ;

[0513] R6 is H, OH, OR D or R D ;

[0514] R7 is H, OH, OR D or R D ;

[0515] R8 is OH, NH2 or ORD ;

[0516] R9 is H, OH or OR D ;

[0517] X is -S-, -S(O)-, or -SO2-; and

[0518] 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.

[0519] As described herein, amanita toxins can be conjugated to antibodies or antigen-binding fragments thereof, for example, by means of a linker moiety. Exemplary methods for conjugating amanita toxins and linkers useful for such methods are described below in the section entitled "Linkers for Chemical Conjugation" and in Table 1. Exemplary linker-containing amanita toxins useful for conjugation to antibodies, antigen-binding fragments according to the compositions and methods described herein are shown in the structural formulas (I), (IA), (IB), (II), (IIA), and (IIB) listed herein.

[0520] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to a particular antigen or undergoes an immunological reaction. Examples of antibodies include polyclonal antibodies, monoclonal antibodies, genetically engineered antibodies, and antibodies of otherwise modified form, including but not limited to chimeric antibodies, humanized antibodies, heteroconjugated antibodies (e.g., bispecific antibodies, trispecific antibodies, and tetraspecific antibodies, biantibodies, triantibodies, and tetraantibodies), and antigen-binding fragments of antibodies, including, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments. As used herein, Fab and F(ab')2 fragments refer to antibody fragments that lack the Fc fragment of a complete antibody. Examples of these antibody fragments are described herein.

[0521] Typically, antibodies comprise heavy and light chains containing antigen-binding regions. Each heavy chain is composed of a heavy chain variable region (herein referred to as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (herein referred to as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

[0522] As used herein, the term "antigen-binding fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody and binds to the antigen to which the intact antibody binds. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. An antibody fragment can be, for example, Fv, Fab, Fab', F(ab')2, scFv, a diabody, a single-chain antibody molecule (e.g., scFv), a triabody, an affibody, a nanobody, an aptamer, or a domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, which is a fragment of a V-chain antibody; L 、V H 、C L and C H 1 domain; (ii) F(ab')2 fragment, which is a bivalent fragment consisting of two Fab fragments connected by a disulfide bridge at the hinge region; (iii) H and C H 1 domain; (iv) a V fragment consisting of a single arm of the antibody L and V H Fv fragment composed of structural domains, (v) including V H and V L domain dAb; (vi) by V H (vii) a dAb fragment consisting of a V H or V L(viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more (e.g., two, three, four, five, or six) isolated CDRs, which may be optionally connected by a synthetic linker. In addition, although the two domains of the Fv fragment, V L and V H , are encoded by separate genes, but they can be joined by linkers using recombinant methods to enable them to form a single protein chain, where V L Area and V H The fragments are then paired with the corresponding domains to form monovalent molecules (referred to as single-chain Fv (scFv)); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art and can be screened for useful fragments in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis procedures known in the art.

[0523] As used herein, the term "anti-CD5 antibody" or "antibody that binds to CD5" refers to an antibody that specifically binds to CD5. An antibody that "binds" to an antigen of interest (i.e., CD5) is one that is capable of binding to that antigen with sufficient affinity such that the antibody can be used to target cells expressing the antigen. In a preferred embodiment, the antibody specifically binds to human CD5 (hCD5), the amino acid sequence of which is set forth in SEQ ID NO: 261.

[0524] As used herein, the term "bispecific antibody" refers to a hybrid antibody with two different antigen binding sites. A bispecific antibody is a multispecific antibody and can be produced by a variety of methods, including but not limited to the fusion of hybridomas or the connection of Fab' fragments. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79: 315-321; Kostelny et al., 1992, J. Immunol. 148: 1547-1553. The two binding sites of a bispecific antibody will bind to two different epitopes that may be located on the same or different protein targets. For example, one binding specificity can be for a T cell surface antigen (such as CD5), and the other binding specificity can be for a different T cell surface antigen or another cell surface protein (such as a receptor or receptor subunit involved in a signal transduction pathway that promotes cell growth).

[0525] As used herein, the term "complementarity determining region" (CDR) refers to the hypervariable regions found in both the light chain variable domain and the heavy chain variable domain of an antibody. The more highly conserved portions of the variable domains are called framework regions (FRs). Depending on the context and various definitions known in the art, the amino acid positions that delineate the hypervariable regions of an antibody can vary. Some positions within the variable domains can be considered as mixed hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria, while these positions are considered to be outside the hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions. The antibodies described herein can include modifications in these mixed hypervariable positions. The variable domains of native heavy and light chains each comprise four framework regions that primarily adopt a β-sheet configuration, connected by three CDRs that form a loop connecting the β-sheet structure and, in some cases, forming a portion of the β-sheet structure. The CDRs in each chain are held together in close proximity by the framework regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and, together with the CDRs from the other antibody chains, contribute to the formation of the target binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD., 1987). As used herein, unless otherwise indicated, the numbering of immunoglobulin amino acid residues is according to the immunoglobulin amino acid residue numbering system of Kabat et al.

[0526] As used herein, the terms "condition" and "conditioning" refer to a process in which a patient is prepared to receive a transplant comprising hematopoietic stem cells. Such a program promotes the implantation of a hematopoietic stem cell transplant (e.g., as inferred by a sustained increase in the number of viable hematopoietic stem cells in a blood sample isolated from the patient after the conditioning program and subsequent hematopoietic stem cell transplant). According to the methods described herein, by administering to the patient an antibody or antigen-binding fragment thereof that can bind to an antigen expressed by T cells (such as CD5), the patient can be conditioned to undergo hematopoietic stem cell transplantation therapy. As described herein, anti-CD5 antibodies can be covalently conjugated to a cytotoxin to form an antibody-drug conjugate (ADC). The antibody, its antigen binding fragment or antibody-drug conjugate capable of binding to one or more of the aforementioned antigens is administered to a patient in need of hematopoietic stem cell transplantation therapy and can promote the implantation of hematopoietic stem cell transplants, for example, by selectively exhausting endogenous immune cells, such as CD5+T cells (for example, CD4+ and / or CD8+T cells), CD5+B cells and / or CD5+NK cells, which cross-react with one or more non-self antigens expressed by hematopoietic stem cells (such as, one or more non-self MHC antigens expressed by hematopoietic stem cell transplants). This selective exhaustion of immune cells then prevents or reduces the possibility of graft rejection after transplanting exogenous (for example, autologous, allogeneic or syngeneic) hematopoietic stem cell transplants.

[0527] As used herein, the term "conjugate" refers to a compound formed by chemical bonding of a reactive functional group of a molecule (such as an antibody or its antigen-binding fragment) to a suitable reactive functional group of another molecule (such as a cytotoxin described herein). A conjugate can include a linker between two molecules (e.g., an anti-CD5 antibody and a cytotoxin) that are bound to each other. Examples of linkers that can be used to form a conjugate include peptide-containing linkers, such as those containing naturally occurring or non-naturally occurring amino acids such as D-amino acids. Linkers can be prepared using various strategies described herein and known in the art. Depending on the reactive component therein, the linker can be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under alkaline conditions, oxidation, disulfide reduction, nucleophilic cleavage or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20: 571-582, 2012).

[0528] As used herein, the term "coupling reaction" refers to a chemical reaction in which two or more substituents suitable for reacting with each other react to form a chemical moiety that links together (e.g., covalently) the molecular fragments to which each substituent is bound. Coupling reactions include those in which a reactive substituent bound to a fragment of a cytotoxin (such as a cytotoxin known in the art or described herein) reacts with a suitable reactive substituent bound to a fragment of an antibody or antigen-binding fragment thereof (such as an antibody or antigen-binding fragment thereof specific for CD5 known in the art or described herein). Examples of suitably reactive substituents include nucleophile / electrophile pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, etc.), diene / dienophile pairs (e.g., azide / alkyne pairs, etc.), and the like. Coupling reactions include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine condensation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, etc.), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reaction paradigms known in the art or described herein.

[0529] As used herein, "CRU (competitive repopulating unit)" refers to a unit of measure of long-term engrafted stem cells that can be detected after in vivo transplantation.

[0530] As used herein, "drug-to-antibody ratio" or "DAR" refers to the amount of cytotoxin, such as amanita cytoxin, attached to an antibody of an ADC. The DAR of an ADC can range from 1 to 8, although higher loadings are possible depending on the number of attachment sites on the antibody. Thus, in certain embodiments, the ADCs described herein have a DAR of about 1, 2, 3, 4, 5, 6, 7, or 8.

[0531] As used herein, the term "donor" refers to a human or animal from which one or more cells are isolated and then administered to a recipient. The one or more cells may be, for example, a population of hematopoietic stem cells.

[0532] As used herein, the term "diabody" refers to a bivalent antibody comprising two polypeptide chains, each of which comprises a polypeptide chain that is too short to allow V to be carried out on the same polypeptide chain. H and V L The V domains are connected by a linker (e.g., a linker consisting of five amino acids) that is intramolecularly associated with the domains. H and V LThis configuration forces each domain to pair with a complementary domain on another polypeptide chain to form a homodimeric structure. Thus, the term "triabody" refers to a trivalent antibody comprising three peptide chains, each consisting of a peptide chain that is so short that it does not allow V to be bound within the same peptide chain. H and V L A V domain is connected by a linker (e.g., a linker consisting of 1-2 amino acids) that is intramolecularly associated with the domain. H domain and a V L To fold into its native structure, peptides configured in this way typically trimerize to bind the V domains of adjacent peptide chains. H and V L The domains are positioned in spatial proximity to one another (see, eg, Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993).

[0533] As used herein, a "dual variable domain immunoglobulin" ("DVD-Ig") refers to an antigen binding protein in which the target-binding variable domains of two antibodies are combined by means of a linker to produce a tetravalent, dual-targeting single agent (see, e.g., Gu et al., Meth. Enzymol., 502:25-41, 2012).

[0534] As used herein, the term "endogenous" describes a substance, such as a molecule, cell, tissue, or organ (e.g., a hematopoietic stem cell or a cell of the hematopoietic lineage, such as a megakaryocyte, a thrombocyte, a platelet, an erythrocyte, a mast cell, a myeloblast, a basophil, a neutrophil, an eosinophil, a microglia, a granulocyte, a monocyte, an osteoclast, an antigen-presenting cell, a macrophage, a dendritic cell, a natural killer cell, a T lymphocyte (e.g., a CD4+ or CD8+ T lymphocyte), or a B lymphocyte) that is naturally found in a particular organism, such as a human patient, e.g., a human patient undergoing hematopoietic stem cell transplantation therapy as described herein.

[0535] As used herein, the term "engraftment potential" is used to refer to the ability of hematopoietic stem and progenitor cells to repopulate tissues, whether such cells are naturally circulating or provided by transplantation. The term includes all events associated with or causing implantation, such as the tissue homing of cells and the colonization of cells in the tissue of interest. Implantation efficiency or implantation rate can be evaluated or quantified using any clinically acceptable parameter known to those skilled in the art and can include, for example, assessing competitive repopulation units (CRUs); the incorporation or expression of markers in tissues to which stem cells have homed, colonized, or become implanted; or by evaluating the progress of the subject by disease progression, the survival of hematopoietic stem and progenitor cells, or the survival of the recipient. Implantation can also be determined by measuring the white blood cell count in the peripheral blood during the post-transplantation period. Implantation can also be assessed by measuring the recovery of myeloid cells by donor cells in a bone marrow aspirate sample.

[0536] As used herein, the term "excipient" refers to substances with which the active ingredients of a pharmaceutical agent are formulated. They may be included, for example, for the purpose of long-term stabilization or to impart therapeutic enhancement to the active ingredient in the final dosage form.

[0537] As used herein, the term "exogenous" describes a substance, such as a molecule, cell, tissue, or organ (e.g., a T cell, hematopoietic stem cell, or a cell of the hematopoietic lineage, such as a megakaryocyte, a thrombocyte, a platelet, an erythrocyte, a mast cell, a myeloblast, a basophil, a neutrophil, an eosinophil, a microglia, a granulocyte, a monocyte, an osteoclast, an antigen presenting cell, a macrophage, a dendritic cell, a natural killer cell, a T lymphocyte, or a B lymphocyte) that is not naturally found in a particular organism, such as a human patient. Exogenous substances include those that are provided to an organism from an external source or to cultured matter extracted therefrom.

[0538] As used herein, the term "framework region" or "FW region" includes amino acid residues adjacent to the CDRs of an antibody or its antigen-binding fragment. The FW region residues can be present in, for example, human antibodies, humanized antibodies, monoclonal antibodies, antibody fragments, Fab fragments, single-chain antibody fragments, scFv fragments, antibody domains, and bispecific antibodies.

[0539] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that generally comprises at least two full-length heavy chains and two full-length light chains, but may in some cases include fewer chains, such as antibodies naturally occurring in camelids which may comprise only heavy chains.

[0540] As used herein, the term "hematopoietic stem cell" ("HSC") refers to immature blood cells with the ability to self-renew and differentiate into mature blood cells, including a variety of lineages, including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), coagulocytes (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). Such cells can include CD34+ cells. CD34+ cells are immature cells expressing the CD34 cell surface marker. In humans, CD34+ cells are considered to include a subpopulation of cells having the stem cell properties defined above, while in mice, HSC is CD34-. In addition, HSC also refers to long-term repopulation HSC (LT-HSC) and short-term repopulation HSC (ST-HSC). LT-HSCs and ST-HSCs are distinguished based on their functional potential and expression of cell surface markers. For example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for mature lineage markers, which include CD5, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, and CD235A). In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, CD48-, and lin- (negative for mature lineage markers, including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL7ra), while ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, and lin- (negative for mature lineage markers, including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL7ra). Furthermore, under steady-state conditions, ST-HSCs are less quiescent and more proliferative than LT-HSCs. In some embodiments, the present invention relates to a method for the treatment of HSCs that is characterized by the presence of a large number of different stem cells, for example, LT-HSCs, which are capable of producing differentiated offspring, and which are capable of producing differentiated offspring more rapidly. However, LT-HSCs have a larger self-renewal potential (i.e., they survive the entire adult phase and can be continuously transplanted in continuous recipients), whereas ST-HSCs have a limited self-renewal capacity (i.e., they only survive a limited period of time and do not have continuous transplantation potential). Any of these HSCs can be used for methods described herein. ST-HSCs are particularly useful because they have a high degree of proliferative potential and can therefore produce differentiated offspring faster.

[0541] As used herein, the term "hematopoietic stem cell functional potential" refers to the functional properties of hematopoietic stem cells, including 1) multipotency (referring to the ability to differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), coagulocytes (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 produce daughter cells with equivalent potential to that of the parent cell, and further, this ability can be repeated throughout the life of an individual without exhaustion), and 3) the ability of hematopoietic stem cells or their progeny to be reintroduced into a transplant recipient, where they home to the hematopoietic stem cell niche and re-establish effective and sustained hematopoiesis.

[0542] As used herein, the term "major histocompatibility complex antigen" ("MHC," also referred to as "human leukocyte antigen" ("HLA") in the case of humans) refers to proteins expressed on the surface of cells that confer a unique antigenic identity to a cell. MHC / HLA antigens are target molecules that are recognized by T cells and NK cells as being derived from the same hematopoietic stem cell source as the immune effector cells ("self") or as being derived from another hematopoietic reconstitution 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, C in humans) allow each cell to be recognized as "self," while HLA class II antigens (DR, DP, and DQ in humans) participate in the reaction between lymphocytes and antigen-presenting cells. Both have been implicated in the rejection of transplanted organs. An important aspect of the HLA gene system is its polymorphism. Different alleles exist for each gene, MHC class I (A, B, and C) and MHC class II (DP, DQ, and DR). HLA alleles are represented by numbers and subscripts. For example, two unrelated individuals may carry the class I HLA-B genes B5 and Bw41, respectively. The allele products differ in one or more amino acids in the α and / or β domains. A wide range of specific antibodies or nucleic acid reagents are used to type an individual's HLA haplotype using white blood cells expressing class I and class II molecules. The genes commonly used for HLA typing are the genes for the six MHC class I and class II proteins: HLA-A; HLA-B; and HLA-DR, each with two alleles. The HLA genes are clustered in a "superlocus" located at chromosomal location 6p21. This "superlocus" encodes the six classical HLA genes and at least 132 protein-coding genes that play important roles in the regulation of the immune system and several other fundamental molecular and cellular processes. The entire locus roughly measures 3.6 Mb and has at least 224 loci. One effect of this clustering is a "haplotype," a set of alleles present on a single chromosome, inherited from one parent, that tend to be inherited as a group. The set of alleles inherited from each parent forms a haplotype, and some alleles tend to associate together. Identifying a patient’s haplotype can help predict the probability of finding a matching donor and aid in developing search strategies, because some alleles and haplotypes are more common than others, and their frequencies vary among different racial and ethnic groups.

[0543] As used herein, the term "HLA-matched" refers to a donor-recipient pair in which there is no mismatch in HLA antigens between the donor and recipient, such as a donor who provides a hematopoietic stem cell transplant to a recipient in need of hematopoietic stem cell transplantation therapy. HLA-matched (i.e., where all six alleles are matched) donor-recipient pairs have a reduced risk of graft rejection because endogenous T cells and NK cells are less likely to recognize the incoming graft as foreign and therefore less likely to generate an immune response against the transplant.

[0544] As used herein, the term "HLA-mismatched" refers to a donor-recipient pair in which at least one HLA antigen (particularly for HLA-A, HLA-B and HLA-DR) is mismatched between the donor and the recipient, such as a donor who provides a hematopoietic stem cell transplant to a recipient in need of hematopoietic stem cell transplantation therapy. In some embodiments, one haplotype is matched and the other is mismatched. HLA-mismatched donor-recipient pairs may have an increased risk of graft rejection relative to HLA-matched donor-recipient pairs because, in the case of HLA-mismatched donor-recipient pairs, endogenous T cells and NK cells are more likely to recognize the incoming graft as foreign, and such T cells and NK cells are therefore more likely to generate an immune response against the graft.

[0545] As used herein, the term "human antibody" refers to a protein in which substantially every portion (e.g., all CDRs, framework regions, C L 、C H Domain (e.g., C H 1. C H 2. C H 3) Hinge and V L and V HThe invention relates to antibodies that are essentially non-immunogenic in humans and have only minor sequence changes or variations (domains). Human antibodies can be produced in human cells (e.g., by recombinant expression) or produced by non-human animals or prokaryotic or eukaryotic cells that can express functionally rearranged human immunoglobulin (such as heavy chain and / or light chain) genes. When the human antibody is a single-chain antibody, it may include a linker peptide that is not found in natural human antibodies. For example, Fv can include a linker peptide connecting the variable region of the heavy chain and the variable region of the light chain, such as 2 to about 8 glycine or other amino acid residues. Such a linker peptide is considered to be of human origin. Human antibodies can be prepared by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. Human antibodies can also be produced using transgenic mice that are incapable of expressing functional endogenous immunoglobulins, but that can express human immunoglobulin genes (see, e.g., PCT Publication Nos. WO 1998 / 24893; WO 1992 / 01047; WO 1996 / 34096; WO 1996 / 33735; U.S. Pat. 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 one embodiment, human antibodies are produced using recombinant methods so that the glycosylation pattern of the antibodies differs from that of an antibody with the same sequence, if it occurs in nature.

[0546] As used herein, the term "humanized" antibody refers to a chimeric antibody generally comprising an amino acid sequence from non-human CDR and human framework region. In one embodiment, a humanized antibody is a human antibody wherein the residue (receptor antibody) from the CDR of the mankind is replaced by the residue (donor antibody) from the CDR of a non-human species with desired specificity, affinity and / or ability, the non-human species being such as mouse, rat, rabbit or non-human primate. Generally, humanized antibodies contain at least one and generally substantially all of two variable domains, wherein all or substantially all of the CDR regions correspond to those CDR regions of non-human immunoglobulins. All or substantially all of the FW regions can also be those FW regions of human immunoglobulin sequences. Humanized antibodies can also comprise at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin consensus sequence. Methods of antibody humanization are known in the art and have been described in, for example, Riechmann et al., Nature 332:323-327, 1988; US Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370.

[0547] As used herein, the term "immune cell" refers to a cell in the immune system that is involved in the generation and maintenance of an innate immune response or an adaptive immune response. Immune cells include lymphocytes that contain receptors that specifically bind to an antigen of interest and generate an immune response against the antigen of interest, such as a self-antigen in the case of autoimmune cells. Exemplary immune cells include mast cells, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

[0548] As used herein, the patient of " need " hematopoietic stem cell transplant includes showing that in one or more blood cell types, there is missing or defective patient, and suffers from the patient of stem cell disorder.Hematopoietic stem cell shows 1) multipotency usually and therefore can be divided into including but not limited to following multiple different blood lineages: granulocyte (for example, promyelocyte, neutrophil granulocyte, eosinophil, basophil), erythrocyte (for example, reticulocyte, erythrocyte), thrombocyte (for example, megakaryocyte, the megakaryocyte that produces platelet, platelet), monocyte (for example, monocyte, macrophage), dendritic cell, microglia, osteoclast and lymphocyte (for example, NK cell, B cell and T cell), 2) self-renewal and therefore can produce the daughter cell with the potentiality equivalent to mother cell, and 3) ability being reintroduced in transplant recipient, in transplant recipient, they home to hematopoietic stem cell niche and rebuild effective and lasting hematopoiesis. Thus, hematopoietic stem cells can be administered to a patient with a deficiency or defect in one or more hematopoietic lineage cell types in order to reconstitute the deficient or defective cell population in vivo. For example, the patient may have cancer, and the deficiency may be caused by the administration of a chemotherapeutic agent or other drug that selectively or non-specifically depletes the cancer cell population. Additionally or alternatively, the patient may have a non-malignant hemoglobinopathy that may cause a deficiency or defect in one or more blood cell types, such as sickle cell anemia, thalassemia, Fanconi anemia, and Wiskott-Aldrich syndrome. The subject may be a subject with adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome. The subject may have or be affected by an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune disorder. Additionally or alternatively, the subject may be suffering from or otherwise affected by a malignancy, such as a malignancy selected from the group consisting of: a hematological cancer (e.g., leukemia, lymphoma, multiple myeloma, or myelodysplastic syndrome) and neuroblastoma. In some embodiments, the subject is suffering from or otherwise affected by a metabolic disorder.For example, the subject may be suffering from or otherwise affected by a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher's disease, Hurler's disease, sphingolipidosis, and metachromatic leukodystrophy, or any other disease or disorder that may benefit from the treatments and therapies disclosed herein and includes, but is not limited to, the following: severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chidon disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell anemia, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and the inflammatory bowel disease described in "Bone Marrow Transplantation for Non-Malignant Disease," ASH Education Book, 1: those diseases or disorders described in 319-338 (2000), the disclosure of which is incorporated herein by reference in its entirety when it relates to a disease that can be treated by administering hematopoietic stem cell transplantation therapy. Additionally or alternatively, the patient who "needs" a hematopoietic stem cell transplant can be one of the aforementioned diseases or not, but a patient who exhibits a reduction in the level of one or more endogenous cell types within the hematopoietic lineage (e.g., as compared with other healthy subjects), such as 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 B lymphocytes. Those skilled in the art can easily determine, for example, by flow cytometry and fluorescence activated cell sorting (FACS) methods and other programs known in the art, whether the level of one or more of the aforementioned cell types or other blood cell types of a person is reduced relative to other healthy subjects.

[0549] The term "isolated" when used in the context of a protein (e.g., an antibody) refers to a protein that, by virtue of its origin or source of derivation, is not associated with naturally associated components with which it is naturally associated; is substantially free of other proteins from the same species; is expressed by cells from a different species; or does not occur in nature. Thus, a protein that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates would be "isolated" from its naturally associated components. A protein may also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

[0550] The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies, wherein the population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population, are identical and / or bind to the same epitope except for possible variant antibodies, such as naturally occurring mutations or variants during the production of a monoclonal antibody preparation, wherein such variants may be present in small amounts. Compared to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each mAb is directed against a single determinant on the antigen. The modifier "monoclonal" should not be understood as requiring the production of antibodies by any particular method.

[0551] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are suitable for contact with the tissues of subjects such as mammals (e.g., humans) without excessive toxicity, irritation, allergic response, and other problematic complications, commensurate with a reasonable benefit / risk ratio.

[0552] As used herein, the term "pharmaceutical composition" means a mixture comprising therapeutic compounds that is administered to a subject, such as a mammal, e.g., a human, to prevent, treat, or manage a particular disease or condition affecting the mammal, such as an autoimmune disorder, cancer, or blood disorder, etc., e.g., as described herein.

[0553] As used herein, the term "recipient" refers to a patient who receives a transplant, such as a transplant comprising a population of hematopoietic stem cells. The transplanted cells administered to the recipient can be, for example, autologous cells, syngeneic cells, or allogeneic cells.

[0554] As used herein, in the case of transplant (such as hematopoietic stem cell transplant), term " rejection " refers to that the recipient produces the immune response for the transplant entering, thereby reduces the process of the ability of the material (such as hematopoietic stem cell) of transplanting to persist in the recipient.Rejection of the transplant such as hematopoietic stem cell transplant can be for example quantitatively by measuring the quantity or concentration of the cells transplanted in a plurality of samples separated from the patient at different time points after transplanting.The quantity or concentration of the cells found to be transplanted from the sample separated from the patient decrease over time, for example, reduce 20%, 25%, 30%, 35%, 40%, 56%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, show that the patient suffers from transplant rejection.By contrast, the quantity or concentration of the cells found to be transplanted from the sample separated from the patient remain stable over time, for example, reduce less than 20%, 15%, 10%, 5% or less, show that the patient does not suffer from transplant rejection. Alternatively, transplant rejection can be quantitatively measured by measuring the quantity or concentration of the immunocytes (such as T cells and / or NK cells) cross-reacted by the MHC antigens expressed by the cells of the transplantation in a plurality of samples separated from the patient at different time points after transplanting. Find that the quantity or concentration of the immunocytes (such as T cells and / or NK cells) cross-reacted by the MHC antigens expressed by the cells of the transplantation from the sample separated from the patient increase over time, for example, increase by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 56%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 200%, 300% or more, show that the patient suffers from transplant rejection. In contrast, a finding that the number or concentration of immune cells, such as T cells and / or NK cells, that cross-react with the MHC antigens expressed by the transplanted cells in a sample isolated from the patient decreases over time, for example, by 20%, 25%, 30%, 35%, 40%, 56%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, indicates that the patient is not suffering from graft rejection.

[0555] As used herein, the term "sample" refers to a specimen obtained from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or dermis), pancreatic juice, chorionic villus samples, and cells).

[0556] As used herein, the term "scFv" refers to a single-chain Fv antibody in which the variable domains from the heavy and light chains of an antibody have been linked to form a single chain. An scFv fragment comprises a single polypeptide chain comprising the variable region (V) of the 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 )(e.g., CDR-H1, CDR-H2 and / or CDR-H3). L and V H The linker of the region can be a peptide linker consisting of proteinogenic amino acids. Alternative linkers can be used to increase the tolerance of the scFv fragment to proteolytic degradation (e.g., linkers containing D-amino acids), to enhance the solubility of the scFv fragment (e.g., hydrophilic linkers, such as linkers containing polyethylene glycol or polypeptides containing repeated glycine and serine residues), to improve the biophysical stability of the molecule (e.g., linkers containing cysteine ​​residues that form intramolecular or intermolecular disulfide bonds), or to reduce the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites). It will also be understood by those of ordinary skill in the art that the variable regions of the scFv molecules described herein can be modified so that they are different from the antibody molecules from which they are derived in terms of amino acid sequence. For example, nucleotide substitutions or amino acid substitutions (e.g., in CDR and / or framework residues) that cause conservative substitutions or changes in amino acid residues can be performed to maintain or enhance the ability of scFv to bind to the antigen recognized by the corresponding antibody.

[0557] In reference to the interaction of an antibody or antibody fragment with a second chemical species, the term "specific binding" or "specifically binds" means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, the antibody recognizes and binds to a specific protein structure, rather than recognizing and binding to proteins generally. If the antibody is specific for epitope "A," the presence of molecules containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A that is bound to the antibody. In one embodiment, if the antibody has at least about 10 -4 M, about 10 -5 M, about 10 -6 M, about 10 -7 M, about 10 -8 M, about 10 -9 M, about 10 -10 M, about 10 -11 M, about 10 -12 M or smaller (smaller means less than 10 -12 Number, such as 10 -13), then the antibody specifically binds to the target (e.g., CD5). In one embodiment, as used herein, the term "specifically binds to CD5" or "specifically binds to CD5" means that the antibody or its antigen-binding fragment binds to CD5 and has a specific affinity for CD5 as determined by surface plasmon resonance of 1.0×10 -7 The dissociation constant (K D In one embodiment, K D The determination is based on standard biolayer interferometry (BLI). However, it will be appreciated that an antibody may be capable of specifically binding to two or more sequence-related antigens. For example, in one embodiment, an antibody may specifically bind to both the human and non-human (e.g., mouse or non-human primate) species homologs of CD5.

[0558] As used herein, the terms "subject" and "patient" refer to a mammal, such as a human, who is receiving treatment for a particular disease or condition as described herein. For example, a patient, such as a human patient, can be a patient with an autoimmune disease as described herein, and the patient can be administered an anti-CD5 antibody or antibody-drug conjugate as described herein to (i) deplete a population of autoimmune cells (e.g., a population of autoimmune CD5+ T cells, B cells, and / or NK cells) and / or (ii) deplete a population of CD5+ immune cells (e.g., CD5+ T cells, B cells, and / or NK cells that cross-react with non-self antigens expressed by hematopoietic stem cells (e.g., non-self MHC antigens expressed by a hematopoietic stem cell transplant)), thereby preventing or reducing the likelihood of graft rejection prior to hematopoietic stem cell transplantation therapy.

[0559] As used herein, the phrase "substantially cleared from the blood" refers to a time point after administration of a therapeutic agent (such as an anti-CD5 antibody or antigen-binding fragment thereof) to a patient at which the concentration of the therapeutic agent in a blood sample isolated from the patient is such that the therapeutic agent is undetectable by conventional means (e.g., such that the therapeutic agent is undetectable above the noise threshold of the device or assay used to detect the therapeutic agent). Various techniques known in the art can be used to detect antibodies or antibody fragments, such as ELISA-based detection assays known in the art or described herein. Additional assays that can be used to detect antibodies and antibody fragments include immunoprecipitation techniques known in the art and immunoblotting assays, among others.

[0560] As used herein, the phrase "stem cell disorder" broadly refers to any disease, disorder or condition that can be treated or cured by modulating a subject's target tissue (e.g., by ablating an endogenous T cell population in the target tissue) and / or by implanting or transplanting stem cells in the subject's target tissue. For example, patients with type 1 diabetes have been shown to be cured by hematopoietic stem cell transplants and may benefit from modulation according to the compositions and methods described herein. Additional disorders that may be treated using the compositions and methods described herein include, but are not limited to, sickle cell anemia, thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome. The subject may be suffering from or affected by an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune disorder. Additionally or alternatively, the subject may be suffering from or otherwise affected by a malignancy, such as a malignancy selected from the group consisting of: a hematological cancer (e.g., leukemia, lymphoma, multiple myeloma, or myelodysplastic syndrome) and neuroblastoma. In some embodiments, the subject is suffering from or otherwise affected by a metabolic disorder. For example, the subject may have or be otherwise affected by a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher's disease, Hurler's disease, sphingolipidosis, and metachromatic leukodystrophy, or any other disease or disorder that may benefit from the treatments and therapies disclosed herein and includes, but is not limited to, the following: severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chidon disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, sickle cell anemia, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and the inflammatory bowel disease described in "Bone Marrow Transplantation for Non-Malignant Disease," ASH Education Book, 1:319-338 (2000), the disclosure of which is incorporated herein by reference in its entirety as it relates to conditions that can be treated by administering hematopoietic stem cell transplantation therapy.

[0561] As used herein, the term "transfection" refers to any of a number of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, and the like.

[0562] As used herein, the term "treat" or "treatment" refers to therapeutic treatment, wherein the purpose is to prevent or slow (mitigate) an undesirable physiological change or disorder, or to promote a beneficial phenotype in the patient being treated. Beneficial or desired clinical results include, but are not limited to, a reduction in the number of autoimmune cells present in a sample isolated from the patient, such as a population of CD5+ T cells, B cells, and / or NK cells that cross-react with self-antigens in the case of direct treatment of autoimmune disorders, or with antigens expressed by hematopoietic stem cells prior to hematopoietic stem cell transplantation in the case of treatment of autoimmune diseases by administering anti-CD5 antibodies, antigen-binding fragments thereof, and hematopoietic stem cell transplants. Additional beneficial results include an increase in the cell count or relative concentration of hematopoietic stem cells in a patient in need of a hematopoietic stem cell transplant following conditioning therapy and subsequent administration of an exogenous hematopoietic stem cell transplant to the patient. Beneficial outcomes of the therapies described herein may also include an increase in the cell count or relative concentration of one or more cells of the hematopoietic lineage, such as 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, or B lymphocytes, following conditioning therapy and subsequent hematopoietic stem cell transplantation therapy.

[0563] As used herein, the terms "variant" and "derivative" are used interchangeably and refer to naturally occurring, synthetic, and semisynthetic analogs of the compounds, peptides, proteins, or other substances described herein. Variants or derivatives of the compounds, peptides, proteins, or other substances described herein may retain or improve the biological activity of the original substance.

[0564] As used herein, the term "vector" includes nucleic acid vectors, such as plasmids, DNA vectors, plasmids, RNA vectors, viruses or other suitable replicons. The expression vectors described herein can include polynucleotide sequences and, for example, other sequence elements for expressing proteins and / or integrating these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences (such as promoters and enhancer regions) that instruct gene transcription. Other useful vectors for expressing antibodies and antibody fragments include polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of the mRNA produced by gene transcription. These sequence elements can include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to instruct the efficient transcription of the genes carried on the expression vector. The expression vectors described herein can also contain polynucleotides encoding markers for selecting cells containing such vectors. Examples of suitable markers include genes encoding tolerance to antibiotics such as ampicillin, chloramphenicol, kanamycin and nourseothricin.

[0565] As used herein, the term "alkyl" refers to a straight or branched chain alkyl group having, for example, from 1 to 20 carbon atoms in the chain. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like.

[0566] As used herein, the term "alkylene" refers to a straight or branched divalent alkyl group. The divalent positions can be on the same or different atoms within the alkyl chain. Examples of alkylene groups include methylene, ethylene, propylene, isopropylene, and the like.

[0567] As used herein, the term "heteroalkyl" refers to a straight or branched chain alkyl group having, for example, from 1 to 20 carbon atoms in the chain and also containing one or more heteroatoms (such as oxygen, nitrogen, or sulfur, etc.) in the chain.

[0568] As used herein, the term "heteroalkylene" refers to a straight or branched divalent heteroalkyl group. The divalent positions can be on the same or different atoms within the heteroalkyl chain. The divalent positions can be one or more heteroatoms.

[0569] As used herein, the term "alkenyl" refers to a straight or branched alkenyl group having, for example, from 2 to 20 carbon atoms in the chain. Examples of alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, tert-butenyl, hexenyl, and the like.

[0570] As used herein, the term "alkenylene" refers to a straight or branched divalent alkenyl group. The divalent positions can be on the same or different atoms within the alkenylene chain. Examples of alkenylene groups include ethenylene, propenylene, isopropenylene, butenylene, and the like.

[0571] As used herein, the term "heteroalkenyl" refers to a straight or branched alkenyl group having, for example, from 2 to 20 carbon atoms in the chain and also containing one or more heteroatoms (such as oxygen, nitrogen, or sulfur, etc.) in the chain.

[0572] As used herein, the term "heteroalkenylene" refers to a straight or branched divalent heteroalkenyl group. The divalent positions can be on the same or different atoms within the heteroalkenyl chain. The divalent positions can be one or more heteroatoms.

[0573] As used herein, the term "alkynyl" refers to a straight or branched chain alkynyl group having, for example, from 2 to 20 carbon atoms in the chain. Examples of alkynyl groups include propargyl, butynyl, pentynyl, hexynyl, and the like.

[0574] As used herein, the term "alkynylene" refers to a straight or branched divalent alkynyl group. The divalent positions can be on the same or different atoms within the alkynyl chain.

[0575] As used herein, the term "heteroalkynyl" refers to a straight or branched chain alkynyl group having, for example, from 2 to 20 carbon atoms in the chain and also containing one or more heteroatoms (such as oxygen, nitrogen, or sulfur, etc.) in the chain.

[0576] As used herein, the term "heteroalkynylene" refers to a straight or branched divalent heteroalkynyl group. The divalent positions can be on the same or different atoms within the heteroalkynyl chain. The divalent positions can be one or more heteroatoms.

[0577] As used herein, the term "cycloalkyl" refers to a saturated monocyclic, or fused, bridged, or spiro polycyclic ring structure having, for example, from 3 to 12 carbon ring atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[3.1.0]hexane, and the like.

[0578] 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 of cycloalkylene include cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, etc.

[0579] As used herein, the term "heterocycloalkyl" refers to a saturated, monocyclic, or fused, bridged, or spiral polycyclic ring structure having, for example, from 3 to 12 ring atoms selected from carbon atoms and heteroatoms, wherein the heteroatoms are selected from, for example, nitrogen, oxygen, and sulfur. The ring structure can contain one or more oxo groups, for example, on carbon, nitrogen, or sulfur ring members. By way of example, examples of heterocycloalkyl include, but are not limited to, dihydropyridyl, tetrahydropyridyl (piperidyl), tetrahydrothienyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidinone, tetrahydrofuranyl, tetrahydropyranyl, ditetrahydropyranyl, tetrahydroquinolyl, tetrahydroisoquinolyl, decahydroquinolyl, octahydroisoquinolyl, piperazinyl, quinuclidinyl, and morpholinyl.

[0580] As used herein, the term "heterocycloalkylene" refers to a divalent heterocycloalkyl group. The divalent positions can be on the same or different atoms within the ring structure.

[0581] As used herein, the term "aryl" refers to a monocyclic or polycyclic aromatic ring system containing, for example, from 6 to 19 carbon atoms. Aryl groups include, but are not limited to, phenyl, fluorenyl, naphthyl, and the like. The divalent position may be one or more heteroatoms.

[0582] As used herein, the term "arylene" refers to a divalent aromatic group. The divalent positions can be on the same or different atoms.

[0583] As used herein, the term "heteroaryl" refers to a monocyclic heteroaromatic or a bicyclic or tricyclic fused-ring heteroaromatic group in which one or more ring atoms is a heteroatom, such as nitrogen, oxygen, or sulfur. Heteroaryl groups include pyridyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,3,4-triazinyl, 1,2,3-triazinyl, benzofuranyl, [2,3-dihydro]benzofuranyl, isobenzofuranyl, benzothienyl, benzotriazolyl, isobenzothienyl, indole, benzothiophene, benzotriazolyl, isobenzothiophene, indole, benzothiophene ... indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolizinyl, quinazolinyl, phthalazinyl, quinoxalinyl, cinnolinyl, naphthyridinyl, pyrido[3,4-b]pyridinyl, pyrido[3,2-b]pyridinyl, pyrido[4,3-b]pyridinyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl, benzoquinolyl, etc.

[0584] 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 one or more heteroatoms.

[0585] Unless otherwise limited by the definition of an individual substituent, the foregoing chemical moieties, such as "alkyl," "alkylene," "heteroalkyl," "heteroalkylene," "alkenyl," "alkenylene," "heteroalkenyl," "heteroalkenylene," "alkynyl," "alkynylene," "heteroalkynyl," "heteroalkynylene," "cycloalkyl," "cycloalkylene," "heterocycloalkyl," "heterocycloalkylene," "aryl," "arylene," "heteroaryl," and "heteroarylene" groups can be optionally substituted, for example, with from 1 to 5 substituents selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, alkylcycloalkyl, alkylheterocycloalkyl, amino, ammonium, acyl, acyloxy, amido, aminocarbonyl, alkoxycarbonyl, urea, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxyl, trihalomethyl, cyano, hydroxyl, thiol, nitro, and the like. Typical substituents include, but are not limited to, -X, -R, -OH, -OR, -SH, -SR, NH2, -NHR, -N(R)2, -N +(R)3, -CX3, -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO2, -N3, -NC(=O)H, -NC(=O)R, -C(=O)H, -C(=O)R, -C(=O)NH2, -C(=O)N(R)2, -SO3-, -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NH2, -S(=O)2N(R)2, -S(=O)R, -OP(=O)(OH)2, -OP(=O)(OR)2, -P(=O)(OR)2, -P wherein each X is independently selected at each occurrence from F, Cl, Br, and I; and each R is independently selected at each occurrence from alkyl, aryl, heterocycloalkyl, or heteroaryl, a protecting group, and a prodrug moiety. In any case where a group is described as "optionally substituted," that group may be substituted, independently at each occurrence, with one or more of the substituents above. Substitution can include situations where adjacent substituents have undergone ring closure, such as ring closure of ortho-functional substituents to form, for example, lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals, aminals, and hemiaminals formed by ring closure, for example, to provide protecting groups.

[0586] It will be understood that, depending on the context, certain radical naming conventions may include monoradicals or diradicals. For example, when a substituent requires two points of attachment to the remainder of the molecule, it is understood that the substituent is a diradical. For example, a substituent identified as an alkyl that requires two points of attachment includes a diradical, such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc. Other radical naming conventions clearly indicate that the radical is a diradical, such as "alkylene," "alkenylene," "arylene," "heterocycloalkylene," etc.

[0587] In any case where a substituent is depicted as a diradical (ie, having two points of attachment to the rest of the molecule), it is understood that the substituent may be attached in any orientation configuration unless otherwise indicated.

[0588] anti-CD5 antibodies

[0589] The present invention is based in part on the discovery that anti-CD5 antibodies or antigen-binding fragments thereof can be used to directly treat cancer and autoimmune diseases, for example, due to the ability of such agents to kill CD5+ cancer cells (e.g., CD5+ leukemia cells) and CD5+ autoimmune cells (e.g., CD5+ autoimmune T cells, B cells and / or NK cells). In particular, the anti-CD5 antibodies described herein are conjugated to cytotoxins via a linker. Therefore, when describing an anti-CD5 antibody, unless otherwise indicated, conjugates thereof are also contemplated.

[0590] The present invention is also based in part on the following discovery that antibodies or antigen-binding fragments thereof that can bind to CD5 can be used as therapeutic agents to promote the implantation of transplanted hematopoietic stem cells in patients in need of transplant therapy by preventing or reducing the possibility of immune cell-mediated graft rejection. For example, anti-CD5 antibodies and antigen-binding fragments can bind to cell surface CD5 expressed by immune cells (such as T cells, B cells, or NK cells), which cross-react with non-self hematopoietic stem cell antigens (such as non-self MHC antigens expressed by hematopoietic stem cell transplants) and produce an immune response against them. The binding of such antibodies and antigen-binding fragments to hematopoietic stem cell-specific CD5+ immune cells can, for example, induce the death of the bound immune cells through the action of antibody-dependent cell-mediated cytotoxicity or by a cytotoxic agent conjugated to the antibody or its antigen-binding fragment. Therefore, the depletion of a population of CD5+ immune cells that cross-react with non-self hematopoietic stem cells can promote the implantation of hematopoietic stem cell transplants in patients in need by weakening the ability of the recipient's immune system to produce an immune response against the incoming transplant. In this way, patients suffering from stem cell disorders, cancer, autoimmune diseases, or other blood disorders described herein can be treated because a hematopoietic stem cell transplant can be provided to the subject to repopulate the lineage of cells that are missing and / or defective in the subject. The subject may be deficient in the population of cells due to, for example, chemotherapy that has been administered to the subject, which is intended to eradicate cancer cells but has also depleted healthy hematopoietic cells in the process.

[0591] For example, the present invention therefore provides compositions and methods for promoting the implantation of hematopoietic stem cells of transplantation by administering antibodies or their antigen-binding fragments that can be combined with antigens expressed by T cells. This administration can cause the selective exhaustion of a colony of endogenous T cells (such as CD4+ and CD8+T cells). This selective exhaustion of T cells then prevents graft rejection after transplanting exogenous (e.g., autologous, allogeneic or syngeneic) hematopoietic stem cell transplants. For example, selective exhaustion of CD4+ and / or CD8+T cells using anti-CD5 antibodies, antigen-binding fragments, antibody-drug conjugates or antibody-drug conjugates as described herein can weaken the T cell-mediated immune response of the hematopoietic stem cell transplant that may occur. The present invention is based in part on the following discovery: antibodies and their antigen-binding fragments that can be combined with CD5 can be administered to patients in need of hematopoietic stem cell transplantation therapy to promote the survival and implantation potential of the hematopoietic stem cells of transplantation.

[0592] The implantation of the hematopoietic stem cell transplant caused by the administration of anti-CD5 antibodies or their antigen-binding fragments can be represented by a variety of empirical measurements. For example, the implantation of the hematopoietic stem cell of the transplant can be evaluated by assessing the number of competitive refill units (CRU) present in the patient's bone marrow after administering an antibody or its antigen-binding fragment that can be combined with CD5 and subsequently administering a hematopoietic stem cell transplant. Additionally, people can be incorporated into a carrier used to transfect donor hematopoietic stem cells by reporter genes (such as enzymes that catalyze the chemical reaction that produces fluorescence, color-emitting or luminescent products), and subsequently monitor the corresponding signals in the tissue (such as bone marrow) to which the hematopoietic stem cells have been homed to observe the implantation of hematopoietic stem cell transplantation. People can also observe the implantation of hematopoietic stem cells by evaluating the number and survival of hematopoietic stem cells and progenitor cells as determined by fluorescence activated cell sorting (FACS) analytical methods known in the art. People can also determine the implantation of hematopoietic stem cells by measuring the white blood cell count in the peripheral blood during the post-transplant period, and / or by measuring the recovery of donor cells to bone marrow cells in a bone marrow aspirate sample.

[0593] The following sections provide a description of antibodies or antigen-binding fragments thereof, which can be administered to a patient in need of hematopoietic stem cell transplantation therapy to promote engraftment of the hematopoietic stem cell transplant, and methods of administering such therapeutic agents to a patient prior to hematopoietic stem cell transplantation.

[0594] Exemplary antibodies

[0595] The compositions and methods described herein include antibodies or fragments thereof that specifically bind to human CD5. Human CD5 is also known as LEU1 or T1. Human CD5 is a type I transmembrane glycoprotein found on the surface of thymocytes, T lymphocytes, and B lymphocyte subsets. Two isoforms of human CD5 have been identified. Isoform 1 comprises 438 amino acids and is described in: Jones et al. (1988) Nature 323 (6086), 346-349 and below (NCBI Reference Sequence: NP_001333385.1):

[0596]

[0597] It has been shown that T cells express CD5, which is a cell adhesion molecule and has been implicated in both the proliferative response of activated T cells and the T cell helper function. It has also been shown that CD5 acts as a receptor, delivering costimulatory signals to T cells by interacting with CD72 (a cell surface protein unique to B cells). Antibodies and antigen-binding fragments thereof that bind to CD5 can, for example, inhibit T cell activation and T cell-mediated immune responses to hematopoietic stem cell transplants by inhibiting the interaction between CD5 and CD72. Antibodies and antigen-binding fragments thereof that bind to CD5 can also be used, for example, to directly kill CD5+ T cells by conjugating the antibody or its antigen-binding fragment to a cytotoxin (such as described herein or known in the art), or by using an unconjugated antibody or its antigen-binding fragment that can recruit complement proteins to T cells.

[0598] Additionally, it has been shown that activated B cell subsets express CD5, and this expression pattern is particularly common in autoreactive B cells (Werner-Favre et al., European Journal of Immunology 19: 1209-1231 (1989), the disclosure of which is incorporated herein by reference in its entirety). It has also been shown that CD5 is expressed by NK cell subsets; it has been shown that there is a population of low-density CD5+ (CD5LOW+) NK cells, particularly in patients with multiple myeloma, and that this surface antigen has been implicated in NK cell activation (Ishiyama et al., Anticancer Research 14: 725-730 (1994), the disclosure of which is incorporated herein by reference in its entirety). Antibodies or antigen-binding fragments thereof that specifically bind to CD5 can therefore be used to attenuate the activation of B cells and NK cells. Antibodies or antigen-binding fragments thereof that bind to CD5 can also be used to directly kill CD5+ B cells and NK cells, for example, by conjugating the antibody or antigen-binding fragment thereof to a cytotoxin (such as those described herein or known in the art), or by using an unconjugated antibody or antigen-binding fragment thereof that is capable of recruiting complement proteins to B cells or NK cells.

[0599] The present invention includes antibodies and antigen-binding fragments thereof that specifically bind to CD5 polypeptides (e.g., human CD5 polypeptides) and uses thereof. In an exemplary embodiment, the antibody or antigen-binding fragment thereof that specifically binds to a CD5 polypeptide comprises a heavy chain variable region and a light chain variable region.

[0600] 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 CDR3 comprising the amino acid sequence of SEQ ID NO: 19. In one embodiment, the heavy chain variable region comprises one or more VH CDRs 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 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 a VH CDR1 comprising SEQ ID NO: 17, a VH CDR2 comprising SEQ ID NO: 18, and a VH CDR3 comprising SEQ ID NO: 19.

[0601] In one embodiment, the light chain variable region comprises one or more complementarity determining regions (CDRs). In one embodiment, 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 CDR3 comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the light chain variable region comprises one or more VL CDRs 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 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 a VL CDR1 comprising SEQ ID NO: 20, a VL CDR2 comprising SEQ ID NO: 21, and a VL CDR3 comprising SEQ ID NO: 22.

[0602] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 comprising SEQ ID NO: 17, a VH CDR2 comprising SEQ ID NO: 18, and a VH CDR3 comprising SEQ ID NO: 19, and a light chain variable region comprising a VL CDR1 comprising SEQ ID NO: 20, a VL CDR2 comprising SEQ ID NO: 21, and a VL CDR3 comprising SEQ ID NO: 22.

[0603] In certain embodiments, one or more CDRs (i.e., one or more heavy chain CDRs having SEQ ID NOs: 17-19, and / or one or more light chain CDRs having SEQ ID NOs: 20-22) can comprise a conservative amino acid substitution (or 2, 3, 4, or 5 amino acid substitutions) while maintaining the CD5 specificity of the antibody (i.e., a specificity similar to that of an antibody or antigen-binding fragment thereof comprising the heavy chain CDRs of SEQ ID NOs: 17 to 19 and the light chain CDRs of SEQ ID NOs: 20 to 22).

[0604] In certain embodiments, the anti-CD5 antibody or antigen-binding fragment thereof is murine antibody 5D7 or a humanized form thereof. Murine antibody 5D7 binds to human CD5 and is described in U.S. Patent Publication No. 2008 / 0245027, the contents of which are incorporated herein by reference for the antibody sequences disclosed herein. SEQ ID Nos: 29 to 34 described in Table 4 correspond to the CDRs of murine anti-CD5 antibody 5D7. The humanized form of anti-CD5 antibody 5D7 is described in SEQ ID NO: 257 (humanized heavy chain variable region) and SEQ ID NO: 258 (humanized light chain variable region). In one embodiment, the ADCs described herein and their uses include antibodies comprising the CDR sets listed in SEQ ID Nos: 29 to 34. In one embodiment, the ADCs described herein and their uses include antibodies comprising the heavy chain variable region and light chain variable region as listed in SEQ ID Nos: 257 and 258, respectively.

[0605] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 257. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 257, e.g., at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 257. In certain embodiments, the antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO: 257 or a variant of SEQ ID NO: 257 that (i) differs from SEQ ID NO: 257 in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 257 in at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 257 in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions and / or (iv) comprises a variant of SEQ ID NO: 257 that differs from SEQ ID NO: 257 in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; NO:257 is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical amino acid sequence, wherein in any of (i)-(iv), the amino acid substitution can be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified heavy chain variable region can have enhanced biological activity relative to the heavy chain variable region of SEQ ID NO:257 while maintaining the CD5 binding specificity of the antibody, i.e., having a binding specificity similar to that of the antibody or antigen-binding fragment thereof comprising SEQ ID NO:257.

[0606] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 258. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 258, e.g., at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 258. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain comprising SEQ ID NO: 258 or a variant of SEQ ID NO: 258 that (i) differs from SEQ ID NO: 258 in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 258 in up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 258 in 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions and / or (iv) comprises a variant of SEQ ID NO: 258 that differs from SEQ ID NO: 258 in 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; NO:258 is at least about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98% or about 99% identical amino acid sequence, wherein in any of (i)-(iv), the amino acid substitution can be a conservative amino acid substitution or a non-conservative amino acid substitution; and wherein the modified light chain variable region can have enhanced biological activity relative to the light chain variable region of SEQ ID NO:258 while maintaining the CD5 binding specificity of the antibody, i.e., having a binding specificity similar to that of the antibody or antigen-binding fragment thereof comprising SEQ ID NO:258.

[0607] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 257, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 257, and a light chain variable region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 258, e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 258. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO: 257, and a light chain variable region comprising SEQ ID NO: 258.

[0608] In another embodiment, an anti-CD5 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 29. 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 one or more VH CDRs 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 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 a VH CDR1 comprising SEQ ID NO: 29, a VH CDR2 comprising SEQ ID NO: 30, and a VH CDR3 comprising SEQ ID NO: 31.

[0609] In one embodiment, the light chain variable region comprises one or more complementarity determining regions (CDRs). In one embodiment, 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 CDR3 comprising the amino acid sequence of SEQ ID NO: 34. In one embodiment, the light chain variable region comprises one or more VL CDRs 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 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 a VL CDR1 comprising SEQ ID NO: 32, a VL CDR2 comprising SEQ ID NO: 33, and a VL CDR3 comprising SEQ ID NO: 34.

[0610] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 comprising SEQ ID NO: 29, a VH CDR2 comprising SEQ ID NO: 30, and a VH CDR3 comprising SEQ ID NO: 31, and a light chain variable region comprising a VL CDR1 comprising SEQ ID NO: 32, a VL CDR2 comprising SEQ ID NO: 33, and a VL CDR3 comprising SEQ ID NO: 34.

[0611] In certain embodiments, one or more CDRs (i.e., one or more heavy chain CDRs having SEQ ID NOs: 29-31, and / or one or more light chain CDRs having SEQ ID NOs: 32-34) can comprise a conservative amino acid substitution (or 2, 3, 4, or 5 amino acid substitutions) while maintaining the CD5 specificity of the antibody (i.e., a specificity similar to that of an antibody or antigen-binding fragment thereof comprising the heavy chain CDRs of SEQ ID NOs: 29 to 31 and the light chain CDRs of SEQ ID NOs: 32 to 34).

[0612] Antibodies and antigen-binding fragments thereof capable of binding to the CD5 antigen can be identified using techniques known in the art and described herein, such as by immunization, computational modeling techniques, and in vitro selection methods, such as phage display and cell-based display platforms described below.

[0613] Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those having one or both of the following variable regions, or an amino acid sequence having at least 85% sequence identity thereto (e.g., an amino acid sequence having 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity thereto):

[0614] V having the following amino acid sequence L : and

[0615] V having the following amino acid sequence H :

[0616]

[0617] Contains the aforementioned V L and V HAntibodies and antigen-binding fragments thereof comprising sequences are described, for example, in U.S. Patent No. 5,869,619, the disclosure of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof (such as he1 antibodies). In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises V sequences of SEQ ID NO: 1 and SEQ ID NO: 2. L and V H In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises a V chain comprised between SEQ ID NO: 1 and SEQ ID NO: 2. L and V H In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises the V sequences contained in SEQ ID NO: 1 and SEQ ID NO: 2. L and V H chain CDR, and V L and V H The rest of the sequence is identical to that of SEQ ID NO: 1 and SEQ ID NO: 2. L and V H The sequences have at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99% or greater sequence identity).

[0618] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises the following CDRs:

[0619] a CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 3);

[0620] CDR-H2 having the amino acid sequence NTHTGE (SEQ ID NO: 4);

[0621] CDR-H3 having the amino acid sequence RGYDWYFDV (SEQ ID NO: 5);

[0622] a CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 6);

[0623] a CDR-L2 having the amino acid sequence RANRLVD (SEQ ID NO: 7); and

[0624] CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 8).

[0625] Additional anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those having one or both of the following variable regions, or an amino acid sequence having at least 85% sequence identity thereto (e.g., an amino acid sequence having 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity thereto):

[0626] V having the following amino acid sequence L :

[0627] and

[0628] V having the following amino acid sequence H :

[0629]

[0630] Contains the aforementioned V L and V H Antibodies and antigen-binding fragments thereof comprising sequences of V1 and V2 are described, for example, in U.S. Patent No. 5,869,619, the disclosure of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof (such as he3 antibodies). In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises a V1 and V2 sequence of the antibody. L and V H The antibody comprises the CDRs of SEQ ID NO: 3 and SEQ ID NO: 4. L and V H In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises a V chain comprised between SEQ ID NO: 3 and SEQ ID NO: 4. L and V H chain CDR, and V L and V H The rest of the sequence is identical to that of SEQ ID NO: 3 and SEQ ID NO: 4. L and V H The sequences have at least 85% sequence identity (e.g., 85%, 90%, 95%, 97%, 98%, 99% or greater sequence identity).

[0631] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises the following CDRs:

[0632] a CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 11);

[0633] CDR-H2 having the amino acid sequence NTHYGE (SEQ ID NO: 12);

[0634] CDR-H3 having the amino acid sequence RRGYDWYFDV (SEQ ID NO: 13);

[0635] a CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 14);

[0636] a CDR-L2 having the amino acid sequence RANRLES (SEQ ID NO: 15); and

[0637] CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 16).

[0638] Antibodies and antigen-binding fragments thereof comprising the aforementioned CDR sequences are described, for example, in U.S. Patent No. 5,869,619, the disclosure of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0639] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, those described in U.S. Patent Nos. 5,821,123; 5,766,886; 5,770,196; 7,153,932; 5,621,083; 6,649,742; 6,146,631; 5,756,699; 5,744,580; 6,376,217; 5,837,491; and 6,146,850, the disclosure of each of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0640] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, those produced by the hybridoma cell line deposited as ATCC CRL 8000 (anti-CD5 murine antibody OKT1). Such antibodies are described in U.S. Pat. Nos. 4,515,894; 4,657,760; and 4,363,799, the disclosure of each of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0641] Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those having one or more or all of the following CDRs:

[0642] CDR-H1 having the amino acid sequence GYSITSGYY (SEQ ID NO: 17);

[0643] a CDR-H2 having the amino acid sequence ISYSGFT (SEQ ID NO: 18);

[0644] a CDR-H3 having the amino acid sequence AGDRTGSWFAY (SEQ ID NO: 19);

[0645] a CDR-L1 having the amino acid sequence QDISNY (SEQ ID NO: 20);

[0646] a CDR-L2 having the amino acid sequence ATS (SEQ ID NO: 21); and

[0647] CDR-L3 having the amino acid sequence LQYASYPFT (SEQ ID NO: 22).

[0648] Antibodies and antigen-binding fragments thereof comprising the aforementioned CDR sequences are described, for example, in U.S. Patent No. 8,679,500, the disclosure of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0649] Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those having one or more or all of the following CDRs:

[0650] CDR-H1 having the amino acid sequence GYIFTNYG (SEQ ID NO: 23);

[0651] CDR-H2 having the amino acid sequence INTYNGEP (SEQ ID NO: 24);

[0652] a CDR-H3 having the amino acid sequence ARGDYYGYEDY (SEQ ID NO: 25);

[0653] a CDR-L1 having the amino acid sequence QGISNY (SEQ ID NO: 26);

[0654] a CDR-L2 having the amino acid sequence YTS (SEQ ID NO: 27); and

[0655] CDR-L3 having the amino acid sequence QQYSKLPWT (SEQ ID NO: 28).

[0656] Antibodies and antigen-binding fragments thereof comprising the aforementioned CDR sequences are described in, for example, US Pat. No. 8,679,500.

[0657] Anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those having one or more or all of the following CDRs:

[0658] CDR-H1 having the amino acid sequence FSLSTSGMG (SEQ ID NO: 29);

[0659] CDR-H2 having the amino acid sequence WWDDD (SEQ ID NO: 30);

[0660] a CDR-H3 having the amino acid sequence RRATGTGFDY (SEQ ID NO: 31);

[0661] a CDR-L1 having the amino acid sequence QDVGTA (SEQ ID NO: 32);

[0662] a CDR-L2 having the amino acid sequence WTSTRHT (SEQ ID NO: 33); and

[0663] CDR-L3 having the amino acid sequence YNSYNT (SEQ ID NO: 34).

[0664] Antibodies and antigen-binding fragments thereof comprising the aforementioned CDR sequences are described, for example, in U.S. Patent Application Publication No. 2008 / 0254027, the disclosure of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0665] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, anti-CD5 antibodies described in PCT Application Publication No. WO 1992 / 014491, such as the anti-CD5 antibodies produced by the hybridoma cell line deposited with the Institut Pasteur as No. 1-1025 on January 10, 1991. The disclosure of PCT Application Publication No. WO 1992 / 014491 is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0666] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, anti-CD5 antibodies described in U.S. Patent Nos. 6,010,902 and 7,192,736, U.S. Patent Application Publication Nos. 2011 / 0250203 and 2017 / 0129128, and PCT Application Publication Nos. WO2016 / 172606; WO1994 / 023747; and WO1996 / 041608, the disclosures of each of which are incorporated herein by reference as they relate to anti-CD5 antibodies and antigen-binding fragments thereof.

[0667] In some embodiments, anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those containing a combination of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 regions as listed in Table 1 below.

[0668]

[0669]

[0670] Antibodies and antigen-binding fragments thereof comprising the aforementioned CDR sequences of Table 1 are described, for example, in U.S. Patent Application Publication No. 2011 / 0250203, the disclosure of which is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0671] Antibodies and fragments thereof for use in conjunction with the compositions and methods described herein include variants of those antibodies described above, such as antibody fragments containing or lacking an Fc domain, as well as humanized variants of the non-human antibodies described herein and antibody-like protein scaffolds containing one or more or all of the CDRs of the antibodies or antibody fragments described herein, or their equivalent regions (e.g., 10 Fn3 domain). Exemplary antigen-binding fragments of the aforementioned antibodies include 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, etc.

[0672] The aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can be used in various aspects of the invention described herein, including, for example, methods for depleting CD5+ cells in human subjects. As described herein, the aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can also be conjugated to an agent, such as a cytotoxin, such as amanita phalloidins. Additional anti-CD5 antibodies that can be used in aspects of the present invention are described in U.S. Patent No. 8,679,500, U.S. Patent Application Publication No. US2011 / 0250203, and U.S. Patent Application Publication No. US2008 / 0254027, the entire contents of each of which are incorporated herein by reference. Additional anti-CD5 antibodies that can be used in aspects of the present invention include, for example, the monoclonal antibody T101 described by Dillman et al. J. Clin. Oncol. (1984), 2(8): 881-891, and the monoclonal antibody Leu-1 described by Miller et al., Blood (1983), 62(5): 988-95.

[0673] In one embodiment, the anti-CD5 antibody or its binding fragment comprises a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification relative to the wild-type Fc region such that the molecule has an altered affinity or binding to FcgammaR (FcγR). Certain amino acid positions within the Fc region are known to make direct contact with FcγRs by crystallographic studies. In particular, amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C' / E loop), and amino acids 327-332 (F / G loop). (See Sondermann et al., 2000 Nature, 406: 267-273). The antibodies described herein may comprise a variant Fc region comprising modification of at least one residue that makes direct contact with FcγRs based on structural and crystallographic analysis. In one embodiment, the Fc region of an anti-CD5 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991), which is expressly incorporated herein by reference. "EU index as in Kabat" refers to the numbering of human IgG1 EU antibodies. In one embodiment, the Fc region comprises a D265A mutation. In one embodiment, the Fc region comprises a D265C mutation. In some embodiments, the Fc region of an antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index as in Kabat. In one embodiment, the Fc region comprises an L234A mutation. In some embodiments, the Fc region of an anti-CD5 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 235 according to the EU index as in Kabat. In one embodiment, the Fc region comprises an L235A mutation. In yet another embodiment, the Fc region comprises both L234A and L235A mutations. In another embodiment, the Fc region comprises D265C, L234A, and L235A mutations. In yet another embodiment, the Fc region comprises D265C, L234A, L235A, and H435A mutations. In another embodiment, the Fc region comprises D265C and H435A mutations.

[0674] The antibodies of the invention can also be further engineered by introducing additional Fc mutations, such as those described in, for example, (Dall'Acqua 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 279:6213-6), (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 et al. (2007) Drug Metab Dispos 35:86-94), (Vaccaro et al. (2005) Nat Biotechnol 23:1283-8), (Yeung et al. (2010) Cancer Res 70:3269-77), and (Kim et al. (1999) Eur J Immunol 29:2819-25), and including those at positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434, and 435. Exemplary mutations that can be made alone or in combination are T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A, and H435R mutations.

[0675] In some embodiments, anti-CD5 antibodies or their antigen-binding fragments are conjugated to cytotoxins (e.g., amanita phalloides toxin) by cysteine ​​residues in the Fc domains of antibodies or their antigen-binding fragments. In some embodiments, cysteine ​​residues are introduced by mutations in the Fc domains of antibodies or their antigen-binding fragments. For example, cysteine ​​residues can be selected from the group consisting of Cys118, Cys239, and Cys265. In one embodiment, the Fc region of an anti-CD5 antibody (or its fragment) comprises an amino acid substitution at amino acid 265 according to the EU index as in Kabat. In one embodiment, the Fc region comprises a D265C mutation. In one embodiment, the Fc region comprises a D265C and H435A mutation.

[0676] In one embodiment, the Fc region comprises a mutation that causes a shortened half-life. Antibodies with a short half-life may be useful in certain cases where the antibody is expected to function as a short-lived therapeutic agent, such as the conditioning step described herein, in which HSC is administered after the antibody is administered. Ideally, the antibody will be substantially cleared before HSC is delivered, and HSC can also express CD5 generally, but unlike endogenous stem cells, HSC is not a target for anti-CD5 antibodies. In one embodiment, the Fc region comprises a mutation at position 435 (according to the EU index of Kabat). In one embodiment, the mutation is an H435A mutation.

[0677] The aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can be used in various aspects of the invention described herein, including, for example, methods for depleting CD5+ cells in human subjects. The aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can also be conjugated with an agent as described herein, such as a cytotoxin, such as amanita phalloidins.

[0678] Methods for identifying anti-CD5 antibodies

[0679] Methods for high-throughput screening of molecules that bind to CD5 from libraries of antibodies or antibody fragments can be used to identify agents and affinity mature agents that can be used to modulate patients (e.g., human patients) in need of hematopoietic stem cell therapy and / or for direct treatment of cancer or autoimmune diseases as described herein. Such methods include in vitro display technologies known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display. The use of phage display to isolate antibodies or antigen-binding fragments that bind to biologically relevant molecules has been reviewed in, for example, Felici et al., Biotechnol. Annual Rev. 1: 149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26: 27-45, 1997; and Hoogenboom et al., Immunotechnology 4: 1-20, 1998, the disclosure of each of which is incorporated herein by reference when it relates to in vitro display technologies. Randomized combinatorial peptide libraries have been constructed to select polypeptides that bind to cell surface antigens, as described in Kay, Perspect.Drug Discovery Des. 2: 251-268, 1995 and Kay et al., Mol. Divers. 1: 139-140, 1996, the disclosure of each of which is incorporated herein by reference when it relates to the discovery of antigen-binding molecules. Proteins, such as multimeric proteins, have been successfully displayed as functional molecules on phages (see, e.g., EP 0 349 578; EP 4 527 839; and EP 0 589 877, and Chiswell and McCafferty, Trends Biotechnol. 10: 80-84 1992, the disclosure of each of which is incorporated herein by reference when it relates to the use of in vitro display technology for the discovery of antigen-binding molecules). In addition, functional antibody fragments, such as Fab and scFv fragments, have been expressed in in vitro display formats (see, e.g., McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, the disclosures of each of which are incorporated herein by reference as they relate to in vitro display platforms for the discovery of antigen-binding molecules).These and other techniques can be used to identify and improve the affinity of antibodies or antibody fragments that bind to CD5, which in turn can be used to deplete CD5+ T cells, B cells and / or NK cells in patients (e.g., human patients) in need of hematopoietic stem cell transplantation therapy and / or suffering from a cancer or autoimmune disease described herein.

[0680] Other techniques can be used to identify antibodies and antigen-binding fragments thereof that bind to CD5 on the surface of cells (e.g., T cells, B cells, or NK cells) and are internalized by the cells, for example, through receptor-mediated endocytosis. For example, the in vitro display technology described above can be suitable for screening antibodies and antigen-binding fragments thereof that bind to and subsequently internalize CD5 on the surface of T cells, B cells, or NK cells. Phage display represents such a technology that can be used in conjunction with this screening paradigm. In order to identify anti-CD5 antibodies and fragments thereof that bind to CD5 and are subsequently internalized by T cells, B cells, and / or NK cells, those skilled in the art can use the phage display technology described in Williams et al., Leukemia 19: 1432-1438, 2005, the disclosure of which is incorporated herein by reference in its entirety. For example, using mutagenesis methods known in the art, recombinant phage libraries can be produced that encode antibodies, antibody fragments such as scFv fragments, Fab fragments, diabodies, triabodies, and 10 Fn3 domains, etc., or antibodies containing randomized amino acid boxes (e.g., in one or more or all CDRs or equivalent regions of antibodies or antibody fragments). The framework regions, hinge regions, Fc domains, and other regions of antibodies or antibody fragments can be designed to be non-immunogenic in humans, for example, by having human germline antibody sequences or sequences that exhibit only minor changes relative to human germline antibodies.

[0681] Using phage display technology described herein or known in the art, a phage library comprising randomized antibodies or antibody fragments covalently bound to phage particles can be incubated with the CD5 antigen, for example, by first incubating the phage library with a blocking agent (such as, for example, milk protein, bovine serum albumin and / or IgG) to remove phage encoding antibodies or fragments thereof that exhibit non-specific protein binding and phage encoding antibodies or fragments thereof that bind to the Fc domain, and then incubating the phage library with a population of CD5+ T cells, B cells, or NK cells. The phage library can be incubated with T cells, B cells, or NK cells for a sufficient time (e.g., 30 minutes to 6 hours at 4°C, such as 1 hour at 4°C) to allow CD5-specific antibodies or antigen-binding fragments thereof to bind to cell surface CD5 and subsequently be internalized by the T cells, B cells, or NK cells. Phage containing antibodies or fragments thereof that do not show enough affinity for CD5 to such an extent that do not allow to be combined with T cells, B cells or NK cells and internalized by T cells, B cells or NK cells can be removed by washing cells at pH 2.8, for example, with cold (4 ° C) 0.1M glycine buffer. The phage bound to the antibody or fragment internalized by T cells, B cells and / or NK cells can be identified, for example, by lysing cells and recovering the internalized phage from cell culture medium. The phage can then be amplified in bacterial cells by, for example, incubating bacterial cells with the recovered phage in 2 × YT culture medium using methods known in the art. The phage recovered from the culture medium can then be characterized, for example, by determining the nucleic acid sequence of the gene (one or more than one) encoding the antibody or fragment thereof inserted into the phage genome. The encoded antibody or fragment can then be prepared from the beginning by chemical synthesis (for example, antibody fragment, such as scFv fragment) or by recombinant expression (for example, full-length antibody).

[0682] An exemplary method for in vitro evolution of anti-CD5 antibodies for use with the compositions and methods described herein is phage display. Phage display libraries can be generated by targeting similar regions of an antibody's CDRs or antibody-like scaffolds (e.g., 10 The antibodies are generated by performing a series of designed mutations or variations within the coding sequence of the BC, CD and DE loops of the Fn3 domain. The template sequence encoding the antibody into which these mutations are introduced can be, for example, a natural human germline sequence. These mutations can be performed using standard mutagenesis techniques known in the art. Thus, in addition to one or more amino acid variations, each mutant sequence encodes an antibody corresponding to the template. Retroviral and phage display vectors can be engineered using standard vector construction techniques known in the art. The P3 phage display vector, together with a compatible protein expression vector, can be used to generate phage display vectors for antibody diversification.

[0683] Mutagenized DNA provides sequence diversity, and each transformant phage displays a variant of the initial template amino acid sequence encoded by the DNA, resulting in a phage population (library) displaying a large number of different but structurally related amino acid sequences. Due to the well-defined structure of the antibody hypervariable region, it is expected that the amino acid variations introduced in phage display screening will change the binding properties of the binding peptide or domain without significantly changing its overall molecular structure.

[0684] In classical screening, a phage library can be contacted with CD5 or an epitope thereof and allowed to bind to CD5 or an epitope thereof. To facilitate separation of binders and non-binders, it is convenient to immobilize the target on a solid support. Phage with a CD5 binding moiety can form a complex with the target on the solid support, while non-binding phage remain in solution and can be washed away with excess buffer. The bound phage can then be released from the target by changing the buffer to an extreme pH (pH 2 or pH 10), changing the ionic strength of the buffer, adding a denaturant, or other known methods.

[0685] The recovered phage can then be amplified by infection of bacterial cells, and the screening process can be repeated with a new pool that is now depleted of non-binding antibodies and enriched for antibodies that bind to CD5. Even recovering a small amount of binding phage is sufficient to amplify the phage for subsequent iterative screening. After several rounds of selection, the gene sequence encoding the antibody or its antigen-binding fragment obtained from the selected phage clones in the binding pool is determined by conventional methods, thereby revealing the peptide sequence that gives the phage its binding affinity to the target. During the panning process, the sequence diversity of the population decreases with each round of selection until the desired peptide-binding antibody is retained. The sequences can converge to a few related antibodies or their antigen-binding fragments. The increase in the number of phage recovered in each round of selection indicates that the library has converged in the screening.

[0686] Another method for identifying anti-CD5 antibodies includes humanizing a non-human antibody that binds to CD5, for example, according to the following procedure. Non-human antibodies that bind to CD5 can be humanized, for example, according to the following procedure. The consensus human antibody heavy chain sequence and light chain sequence are known in the art (see, for example, the "VBASE" human germline sequence database; Kabat et al. Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, 1991; Tomlinson et al., J. Mol. Biol. 227: 776-798, 1992; and Cox et al., Eur. J. Immunol. 24: 827-836, 1994, the disclosure of each of which is incorporated herein by reference when it relates to consensus human antibody heavy chain sequence and light chain sequence). Using established procedures, one skilled in the art can identify the variable domain framework residues and CDRs of the consensus antibody sequence (e.g., by sequence alignment). One can replace one or more CDRs of the heavy and / or light chain variable domains of a common human antibody with one or more corresponding CDRs of a non-human antibody that binds to CD5 to generate a humanized antibody. Such CDR swapping can be performed using gene editing techniques described herein or known in the art.

[0687] An example of a consensus human antibody variable domain comprises the heavy chain variable domain identified in U.S. Pat. No. 6,054,297. (SEQ ID NO: 259) and the light chain variable domain The disclosure of this US patent is incorporated herein by reference as it relates to human antibody consensus sequences. The CDRs in the sequences above are shown in bold.

[0688] To generate a humanized antibody, one can recombinantly express a polynucleotide encoding the consensus sequence described above, wherein one or more variable region CDRs have been replaced with one or more variable region CDR sequences from a non-human antibody that binds to CD5. Because the affinity of an antibody for CD5 is primarily determined by its CDR sequence, the resulting humanized antibody is expected to have an affinity for CD5 that is approximately the same as the affinity of the non-human antibody from which the humanized antibody was derived. Methods for determining the affinity of an antibody for a target antigen include, for example, ELISA-based techniques described herein and known in the art, as well as surface plasmon resonance, fluorescence anisotropy, and isothermal titration calorimetry.

[0689] The internalization ability of the prepared antibodies or fragments thereof can be assessed, for example, using radionuclide internalization assays known in the art. For example, anti-CD5 antibodies or fragments thereof identified using in vitro display techniques described herein or known in the art can be functionalized by incorporating a radioisotope, such as 18 F. 75 Br, 77 Br, 122 I. 123 I. 124 I. 125 I. 129 I. 131 I. 211 At 67 Ga, 111 In, 99 Tc, 169 Yb, 186 Re、 64 Cu, 67 Cu, 177 Lu, 77 As、 72 As、 86 Y. 90 Y. 89 Zr, 212 Bi, 213 Bior 225 Ac. For example, radioactive halogens such as 18 F. 75 Br, 77 Br, 122 I. 123 I. 124 I. 125 I. 129 I. 131 I. 211 In some embodiments, the antibody or its fragment can be incorporated into an antibody or its fragment using a bead containing an electrophilic halogen reagent, such as a polystyrene bead (e.g., iodinated beads, ThermoFisherScientific, Inc., Cambridge, MA). Radiolabeled antibody or its fragment can be incubated with T cells, B cells and / or NK cells for a sufficient time (e.g., 4°C for 30 minutes to 6 hours, such as 4°C for 1 hour) to allow internalization. Cells can then be washed to remove uninternalized antibody or its fragment (e.g., using cold (4°C) 0.1M glycine buffer at pH 2.8). Internalized antibody or its fragment can be identified by comparing the radiation (e.g., gamma radiation) emitted by detecting the resulting T cells, B cells and / or NK cells with the radiation (e.g., gamma radiation) emitted by the washing buffer recovered.

[0690] In order to recombinantly produce an anti-CD5 antibody, a nucleic acid encoding an antibody, such as described above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody).

[0691] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. Regarding the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in Escherichia coli (E. coli). After expression, the antibody can be separated from the bacterial cell paste as a soluble fraction and can be further purified.

[0692] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension can be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells, as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); Buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, for example, in Mather et al., Annals of NY Acad. Sci. 383: 44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., Y0, NS0, Sp20 cell).

[0693] Antibody-drug conjugates (ADCs)

[0694] Cytotoxins

[0695] The antibodies and antigen-binding fragments thereof described herein (e.g., antibodies, antigen-binding fragments that recognize and bind to CD5) can be conjugated to a cytotoxin, such as Pseudomonas exotoxin A, deBouganin, diphtheria toxin, an amanita toxin (such as α-amanitin), saporin, maytansine, maytansine alkaloid, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolebenzodiazepines and indolebenzodiazepine dimers, or variants thereof, or another cytotoxic compound described herein or known in the art to (i) directly treat a cancer or autoimmune disease described herein, or (ii) deplete endogenous immune cells to prevent or reduce the likelihood of rejection of hematopoietic stem cells after transplantation into a patient (e.g., a human patient) in need of hematopoietic stem cell transplantation therapy. In some embodiments, the cytotoxic molecule is conjugated to an internalizing antibody or its antigen-binding fragment so that after the cell takes up the antibody or antigen-binding fragment, the cytotoxin can approach its intracellular target and kill endogenous T cells, B cells and / or NK cells. Suitable cytotoxins suitable for use with the compositions and methods described herein include DNA intercalators (e.g., anthracyclines), agents capable of destroying the mitotic spindle apparatus (e.g., vinca alkaloids, maytansine, maytansinoids and derivatives thereof), RNA polymerase inhibitors (e.g., amanita toxins such as α-amanitin and derivatives thereof), agents capable of destroying protein biosynthesis (e.g., agents exhibiting rRNA N-glycosidase activity, such as saporin and ricin A chain), and other cytotoxins known in the art.

[0696] In some embodiments, the cytotoxin of the antibody-drug conjugate is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is amanita phalloidin or a derivative thereof.

[0697] In some embodiments, the cytotoxin is an amanita toxin or a derivative thereof, such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanitin, amanitin amide, amanitin, amanitin acid, and pro-amanitin. The structures of various naturally occurring amanitin toxins are represented by Formula III and are disclosed, for example, in Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459.

[0698] In one embodiment, the cytotoxin is amanitin. For example, the antibodies or antigen-binding fragments described herein can be combined with amanitin toxin to form a conjugate represented by the formula Ab-ZL-Am, wherein Ab is an antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is amanitin toxin. Many positions on amanitin toxin or a derivative thereof can serve as positions for covalent bonding to the linking moiety L and thereby to the antibody or antigen-binding fragment thereof. For example, the antibodies and antigen-binding fragments described herein can be combined with amanitin toxin to form a conjugate represented by the formula Ab-ZL-Am, wherein Ab is an antibody or antigen-binding fragment thereof, Z is a chemical moiety, L is a linker, and Am is amanitin toxin. In some embodiments, Am-LZ is represented by formula (I)

[0699]

[0700] Where R1 is H, OH, OR A OR C ;

[0701] R2 is H, OH, OR B OR C ;

[0702] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0703] R3 is H, R C or R D ;

[0704] R4 is H, OH, OR C , OR D 、R C or R D ;

[0705] R5 is H, OH, OR C , OR D 、R C or R D ;

[0706] R6 is H, OH, OR C , OR D 、R C or R D ;

[0707] R7 is H, OH, OR C , OR D 、R C or R D ;

[0708] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0709] R9 is H, OH, OR C OR D ;

[0710] X is -S-, -S(O)- or -SO2-;

[0711] R C Yes -LZ;

[0712] 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;

[0713] L is a linker such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (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 heteroarylene, dipeptide, -C(=O)-, a peptide, or a combination thereof; and

[0714] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present within the antibody or antigen-binding fragment thereof that binds to CD5.

[0715] In some embodiments, Am comprises exactly one R C substituent.

[0716] In some embodiments, the linker comprises -(CH) 2n -unit, wherein n is an integer from 2 to 6. In some embodiments, the linker comprises -((CH2) n , wherein n is 6. In some embodiments, LZ is

[0717]

[0718] wherein S is a sulfur atom and represents a reactive substituent (e.g., a -SH group from a cysteine ​​residue) present in the antibody or antigen-binding fragment thereof that binds to CD5.

[0719] In some embodiments, LZ is

[0720]

[0721] In some embodiments, Am-LZ-Ab is

[0722]

[0723] In some embodiments, Am-LZ-Ab is

[0724]

[0725] In some embodiments, Am-LZ is represented by formula (IA)

[0726]

[0727] Where R1 is H, OH, OR A OR C ;

[0728] R2 is H, OH, OR B OR C ;

[0729] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0730] R3 is H, R C or R D ;

[0731] R4 is H, OH, OR C , OR D 、R C or R D ;

[0732] R5 is H, OH, OR C , OR D 、R C or R D ;

[0733] R6 is H, OH, OR C , OR D 、R Cor R D ;

[0734] R7 is H, OH, OR C , OR D 、R C or R D ;

[0735] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0736] R9 is H, OH, OR C OR D ;

[0737] X is -S-, -S(O)- or -SO2-;

[0738] R C Yes -LZ;

[0739] 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;

[0740] L is a linker such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (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 heteroarylene, dipeptide, -C(=O)-, a peptide, or a combination thereof;

[0741] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present within the antibody or antigen-binding fragment thereof that binds to CD5; and

[0742] where Am contains exactly one R C substituent.

[0743] In some embodiments, the linker comprises -((CH2) n , wherein n is 6. In some embodiments, LZ is

[0744]

[0745] In some embodiments, LZ is

[0746]

[0747] In some embodiments, Am-LZ-Ab is

[0748]

[0749] In some embodiments, Am-LZ-Ab is

[0750]

[0751] In some embodiments, Am-LZ is represented by formula (IB)

[0752]

[0753] Where R1 is H, OH, OR A OR C ;

[0754] R2 is H, OH, OR B OR C ;

[0755] R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group;

[0756] R3 is H, R C or R D ;

[0757] R4 is H, OH, OR C , OR D 、R C or R D ;

[0758] R5 is H, OH, OR C , OR D 、R C or R D ;

[0759] R6 is H, OH, OR C , OR D 、R C or RD ;

[0760] R7 is H, OH, OR C , OR D 、R C or R D ;

[0761] R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ;

[0762] R9 is H, OH, OR C OR D ;

[0763] X is -S-, -S(O)- or -SO2-;

[0764] R C Yes -LZ;

[0765] 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;

[0766] L is a linker such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (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 heteroarylene, dipeptide, -C(=O)-, a peptide, or a combination thereof;

[0767] Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present within the antibody or antigen-binding fragment thereof that binds to CD5; and

[0768] where Am contains exactly one R C substituent.

[0769] In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0770]

[0771] In some embodiments, LZ is

[0772]

[0773] In some embodiments, Am-LZ-Ab is

[0774]

[0775] In some embodiments, Am-LZ-Ab is

[0776]

[0777] In some embodiments, R A and R B Together with the oxygen atom to which they are attached, they combine to form a 5-membered heterocycloalkyl group of the formula:

[0778]

[0779] wherein Y is -C(=O)-, -C(=S)-, -C(=NR E )-or-C(R E R E’ )-;and

[0780] R E and R E’ Each independently is an optionally substituted C1-C6 alkylene-R C , optionally substituted C1-C6 heteroalkylene-R C , optionally substituted C2-C6 alkenylene-R C , optionally substituted C2-C6 heteroalkenylene-R C , optionally substituted C2-C6 alkynylene-R C , optionally substituted C2-C6 heteroalkynylene-R C , optionally substituted cycloalkylene-R C , optionally substituted heterocycloalkylene-R C , optionally substituted arylene-R C or optionally substituted heteroarylene-R C .

[0781] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0782] Where R1 is H, OH, OR A OR C ;

[0783] R2 is H, OH, OR B OR C ;

[0784] R A and R B Together with the oxygen atoms to which they are bound, they form:

[0785]

[0786] R3 is H or R C ;

[0787] R4 is H, OH, OR C , OR D 、R C or R D ;

[0788] R5 is H, OH, OR C , OR D 、R C or R D ;

[0789] R6 is H, OH, OR C , OR D 、R C or R D ;

[0790] R7 is H, OH, OR C , OR D 、R C or R D ;

[0791] R8 is OH, NH2, OR C or NHR C ;

[0792] R9 is H or OH; and

[0793] where R C and R D Each is as defined above.

[0794] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0795] Where R1 is H, OH, OR A OR C ;

[0796] R2 is H, OH, OR B ORC ;

[0797] R A and R B Together with the oxygen atoms to which they are bound, they form:

[0798]

[0799] R3 is H or R C ;

[0800] R4 and R5 are each independently H, OH, OR C 、R C OR D ;

[0801] R6 and R7 are each H;

[0802] R8 is OH, NH2, OR C or NHR C ;

[0803] R9 is H or OH; and

[0804] Where X and R C As defined above.

[0805] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0806] Where R1 is H, OH or OR A ;

[0807] R2 is H, OH or OR B ;

[0808] R A and R B Together with the oxygen atoms to which they are bound, they form:

[0809]

[0810] R3, R4, R6 and R7 are each H;

[0811] R5 is OR C ;

[0812] R8 is OH or NH2;

[0813] R9 is H or OH; and

[0814] Where X and R C As defined above. Such Amanita toxin conjugates are described, for example, in U.S. Patent Application Publication No. 2016 / 0002298, the disclosure of which is incorporated herein by reference in its entirety.

[0815] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0816] wherein R1 and R2 are each independently H or OH;

[0817] R3 is R C ;

[0818] R4, R6 and R7 are each H;

[0819] R5 is H, OH or OC1-C6 alkyl;

[0820] R8 is OH or NH2;

[0821] R9 is H or OH; and

[0822] where R C As defined above. Such Amanita toxin conjugates are described, for example, in US Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated herein by reference in its entirety.

[0823] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0824] wherein R1 and R2 are each independently H or OH;

[0825] R3, R6 and R7 are each H;

[0826] R4 and R5 are each independently H, OH, OR C or R C ;

[0827] R8 is OH or NH2;

[0828] R9 is H or OH; and

[0829] where R C As defined above. Such Amanita toxin conjugates are described, for example, in US Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0830] In some embodiments, Am-LZ is represented by Formula (IA) or Formula (IB),

[0831] wherein R1 and R2 are each independently H or OH;

[0832] R3, R6 and R7 are each H;

[0833] R4 and R5 are each independently H or OH;

[0834] R8 is OH, NH2, OR C or NHR C ;

[0835] R9 is H or OH; and

[0836] Where X and R C As defined above. Such amanita anitoxin conjugates are described, for example, in U.S. Patent Nos. 9,233,173 and 9,399,681 and US 2016 / 0089450, the disclosures of each of which are incorporated herein by reference in their entirety.

[0837] Additional Amanita toxins that can be used for conjugation to antibodies or antigen-binding fragments thereof according to the compositions and methods described herein are described, for example, in WO 2016 / 142049; WO 2016 / 071856; and WO 2017 / 046658, the disclosures of each of which are incorporated herein by reference in their entireties.

[0838] In some embodiments, Am-LZ is represented by Formula (II), Formula (IIA), or Formula (IIB),

[0839]

[0840] wherein X is S, SO or SO2; R1 is H or a linker that is covalently bound to the antibody or antigen-binding fragment thereof through a chemical moiety Z formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the antibody or antigen-binding fragment thereof; and R2 is H or a linker that is covalently bound to the antibody or antigen-binding fragment thereof through a chemical moiety Z formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the antibody or antigen-binding fragment thereof; wherein when R1 is H, R2 is a linker, and when R2 is H, R1 is a linker.

[0841] In some embodiments, the linker comprises -(CH2) n - unit, wherein n is an integer from 2 to 6. In some embodiments, R1 is a linker and R2 is H, and the linker and chemical moiety (together LZ) are

[0842]

[0843] In some embodiments, Am-LZ-Ab is one of the following

[0844]

[0845] In some embodiments, the amanita toxin is α-amanitin. In some embodiments, α-amanitin is a compound of formula III. In some embodiments, α-amanitin of formula III is attached to an antibody or antigen-binding fragment thereof that binds to CD5 via a linker L. Linker L can be in any of several possible positions (e.g., R 1 -R 9 ) is attached to an α-amanitin of Formula III to provide an α-amanitin-linker conjugate of Formula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker is attached at position R 1 In some embodiments, the linker is attached at position R 2 In some embodiments, the linker is attached at position R 3 In some embodiments, the linker is attached at position R 4 In some embodiments, the linker is attached at position R 5 In some embodiments, the linker is attached at position R 6 In some embodiments, the linker is attached at position R 7 In some embodiments, the linker is attached at position R 8 In some embodiments, the linker is attached at position R 9 In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the linker comprises a PAB-Cit-Val moiety. In some embodiments, the linker comprises a PAB-Ala-Val moiety. In some embodiments, the linker comprises -((C=O)(CH2) n - unit, wherein n is an integer from 1-6.

[0846] In some embodiments, the linker comprises -(CH2) n -unit, wherein n is an integer from 2 to 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 the chemical moiety Z (together referred to as LZ) are

[0847]

[0848] Antibodies and antigen-binding fragments for use with the compositions and methods described herein can be conjugated to amanita toxins such as α-amanitin or variants thereof using conjugation techniques known in the art or described herein. For example, antibodies and antigen-binding fragments thereof that recognize and bind to CD5 can be conjugated to amanita toxins such as α-amanitin or variants thereof, as described in US2015 / 0218220, the disclosure of which is incorporated herein by reference in its relation to, for example, amanita toxins such as α-amanitin and variants thereof and covalent linkers that can be used for covalent conjugation. Synthetic methods for preparing amanita toxins are described, for example, in U.S. Patent No. 9,676,702, which is incorporated herein by reference in its relation to the synthetic methods disclosed therein.

[0849] Antibodies or antigen-binding fragments for use with the compositions and methods described herein can be conjugated to an amanita toxin, such as α-amanitin, or a variant thereof, using conjugation techniques known in the art or described herein. For example, an antibody or antigen-binding fragment thereof that recognizes and binds to CD5 can be conjugated to an amanita toxin, such as α-amanitin, or a variant thereof, as described in US 2015 / 0218220, the disclosure of which is incorporated herein by reference with respect to, for example, an amanita toxin, such as α-amanitin, and a variant thereof, and a covalent linker that can be used for covalent conjugation.

[0850] Exemplary antibody-drug conjugates useful in conjunction with the methods described herein can be formed by reacting an antibody, or antigen-binding fragment thereof, with an amanita toxin conjugated to a linker containing a substituent suitable for reacting with a reactive residue on the antibody, or antigen-binding fragment thereof. Amanita toxins conjugated to linkers containing substituents suitable for reaction with reactive residues on the antibodies or antigen-binding fragments thereof described herein include, but are not limited to, the following: 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amanitin toxin; 7'C-(4-(6-(maleimido)hexanoylamido)piperidin-1-yl)-amanitin toxin; 7'C-(4-(6-(maleimido)hexanoylamido)hexanoyl)piperazin-1-yl)-amanitin toxin; 7'C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperazin-1-yl)-amanitin toxin; 7'C-(4-( 6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)-amanitin toxin; 7'C-(4-(2-(6-(maleimido)hexanoyl)ethyl)piperidin-1-yl)-amanitin toxin; 7'C-(4-(2-(6-(6-(maleimido)hexanoyl)hexanoyl)ethyl)piperidin-1-yl)-amanitin toxin; 7'C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin; 7'C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amanitin toxin; 7'C-(4-(2-(3-carboxypropionamido)ethyl)piperidin-1-yl)-amanitin toxin; 7'C-(4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)-amanitin toxin; 7'C-(4-(2-(3-(pyridin-2-yldisulfanyl)propionamido)ethyl)piperidin-1-yl)-amanitin toxin; 7'C-(4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)-amanitin toxin; 7'C-(4-(2-(maleimido)acetyl)piperazin-1-yl)-amanitin toxin; 7'C-(4-(3-(maleimido)propionyl)piperazin-1-yl)-amanitin toxin; 7'C-(4-(4-(maleimido)butyryl)piperazin-1-yl)-amanitin toxin; 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amanitin toxin; 7'C-(3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)-amanitin toxin; 7'C-(3-((6-(6-(maleimido)hexanamido)hexanamido)methyl)pyrrolidin-1-yl)-amanitin toxin;7'C-(3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrolidin-1-yl)-amanitin toxin; 7'C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-amanitin toxin; 7'C-(4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amanitin toxin; 7'C-(4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)-amanitin toxin; 7'C-(4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)- Amanita toxin; 7'C-(4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazin-1-yl)-amanitin toxin; 7'C-((4-(6-(maleimido)hexanoyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(6-(maleimido)hexanoyl)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(6-(maleimido)hexanoyl)piperazin-1-yl)methyl)-amanitin toxin; (R)-7'C-((3-((6-(maleimido)hexanoyl)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin; (S)-7'C-((3-((6 7'C-((4-(2-(6-(6-(maleimido)hexanoylamino)hexanoylamino)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-amanitin toxin; -yl)methyl)-amanitin toxin; 7'C-((4-(2-(6-(6-(maleimido)hexanoyl)hexanoyl)ethyl)piperazin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)ethyl)piperazin-1-yl)methyl)-amanitin toxin; 7'C-((3-((6-(6-(maleimido)hexanoyl)hexanoyl)-5-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin;7'C-((3-((6-(6-(maleimido)hexanoyl)hexanoyl)-R-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin; 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin; 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-R-methyl)pyrrolidin-1-yl)methyl)-amanitin toxin; 7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)methyl)pyrrolidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(3 7'C-((4-(6-(6-(maleimido)hexanoyl)piperazin-1-yl)methyl)-amanitin toxin; 7'C-((4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(maleimido)acetyl)piperazin-1-yl)methyl)-amanitin toxin; 7'C-((4-(3-(maleimido)propionyl)piperazin-1-yl)methyl)-amanitin toxin; 7'C-((4-(4-(maleimido)butanoyl)piperazin-1-yl)methyl) -Amanita toxin; 7'C-((4-(2-(2-(maleimido)acetamido)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((3-((6-(maleimido)hexanamido)methyl)azetidin-1-yl)methyl)-amanitin toxin; 7'C-((3-(2-(6-(maleimido)hexanamido)ethyl)azetidin-1-yl)methyl)-amanitin toxin methyl)-amanitin toxin; 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amanitin toxin; 7'C-((3-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)azetidin-1-yl)methyl)-amanitin toxin; 7'C-((3-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)azetidin-1-yl)methyl)-amanitin toxin; 7'C-((3-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)azetidin-1-yl)methyl)-amanitin toxin; 7'C-(((2-(6-(maleimido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amanitin toxin;7'C-(((4-(6-(maleimido)-N-methylhexanamido)butyl(methyl)amino)methyl)-amanitin; 7'C-((2-(2-(6-(maleimido)hexanamido)ethyl)aziridin-1-yl)methyl)-amanitin; 7'C-((2-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)aziridin-1-yl)methyl)-amanitin; 7'C-((4-(6-(6-(2-(aminooxy)acetamido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(1-(aminooxy)-2-oxo-6,9,12 ,15-tetraoxa-3-azaheptadecan-17-yl)piperazin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(2-(aminooxy)acetamido)acetyl)piperazin-1-yl)methyl)-amanitin toxin; 7'C-((4-(3-(2-(aminooxy)acetamido)propionyl)piperazin-1-yl)methyl)-amanitin toxin; 7'C-((4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(6-(2-(aminooxy)acetamido)hexanoyl)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(2-(2-(aminooxy)acetamido)hexanoyl)ethyl)piperidin-1-yl)methyl)-amanitin toxin; (aminooxy)acetamido)acetamido)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(2-(aminooxy)acetamido)butyramido)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-((4-(20-(aminooxy)-4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaicosyl)piperidin-1-yl)methyl)-amanitin toxin; 7'C-(((2-(6-(2-(aminooxy)acetamido)-N-methylhexamethyleneamino)ethyl)(methyl)amino)methyl)-amanitin toxin; 7'C-(((4-(6-(2-(aminooxy)acetamido)butyramido)ethyl)piperidin-1-yl)methyl)-amanitin toxin 7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-S-methyl)-amanitin; 7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amanitin; 7'C-((4-(2-(2-bromoacetamido)ethyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)methyl)-amanitin;7'C-((4-(2-(3-(pyridin-2-yldisulfanyl)propionamido)ethyl)piperidin-1-yl)methyl)-amanitin toxin; 6'O-(6-(6-(maleimido)hexanamido)hexyl)-amanitin toxin; 6'O-(5-(4-((maleimido)methyl)cyclohexanecarboxamido)pentyl)-amanitin toxin; 6'O-(2-((6-(maleimido)hexyl)oxy)-2-oxy 6'O-((6-(maleimido)hexyl)carbamoyl)-amanitin toxin; 6'O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexyl)carbamoyl)-amanitin toxin; 6'O-(6-(2-bromoacetamido)hexyl)-amanitin toxin; 7'C-(4-(6-(azido)hexanoyl)piperidin-1-yl)-amanitin toxin; 7'C -(4-(hex-5-ynylamino)piperidin-1-yl)-amanitin toxin; 7'C-(4-(2-(6-(maleimido)hexanoylamino)ethyl)piperazin-1-yl)-amanitin toxin; 7'C-(4-(2-(6-(6-(maleimido)hexanoylamino)hexanoylamino)ethyl)piperazin-1-yl)-amanitin toxin; 6'O-(6-(6-(11,12-didehydro-5,6-dihydro-diphenyl)piperidin-1-yl)-amanitin toxin [b,f] azocin-5-yl)-6-oxohexanamido)hexyl)-amanitin toxin; 6'O-(6-(hex-5-ynylamino)hexyl)-amanitin toxin; 6'O-(6-(2-(aminooxy)acetylamido)hexyl)-amanitin toxin; 6'O-((6-aminooxy)hexyl)-amanitin toxin; and 6'O-(6-(2-iodoacetamido)hexyl)-amanitin toxin. The foregoing linkers, as well as other linkers that can be used in conjunction with the compositions and methods described herein, are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0851] Additional cytotoxins that can be conjugated to antibodies and antigen-binding fragments thereof that recognize and bind to CD5 for use in the direct treatment of cancer or autoimmune conditions or for the regulation of patients undergoing hematopoietic stem cell transplantation therapy include, but are not limited to, the following: 5-ethynyluracil, abiraterone, acylfulvene, adecypenol, adozelesin, aldesleukin, hexamethylmelamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antarelix, anti-dorsal morphogenetic protein-1, anti-androgen, prostate cancer, anti-estrogen, anti-neoplaston, antisense oligonucleotides, glycine aphidicolin glycinate), apoptosis gene regulators, apoptosis regulators, apurinic nucleic acids, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, diazotyrosine, baccatin III derivative, balanol, batimastat, BCR / ABL antagonists, benzochlorins, benzoylstaurosporine, beta-lactam derivatives, beta-alethine, beta-betamycin B B), betulinic acid, bFGF inhibitors, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistratene A, bizelesin, breflate, bleomycin A2, bleomycin B2, bropirimine, budotitane, buthionine sulfoximine, calcipotriol, calphostin CC), camptothecin derivatives (e.g., 10-hydroxy-camptothecin), capecitabine, carboxamide-amino-triazole, carboxyamidotriazole, carzelesin, casein kinase inhibitors, castanospermine, cecropin B, cetrorelix, chlorin, chloroquinoxaline sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene and its analogs, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogs, conagenin, crambescidin 816, crisnatol, scutellaria cyclopentyl 8, scutellaria cyclopentyl A derivatives, curacin A, A), cyclopentanethrone, cycloplatam, cypemycin, cytarabine ocfosfate, cytolytic factor, estradiol phosphate (cytostatin), dacliximab, decitabine, dehydroepiphyseal B, 2'deoxycoformycin (DCF), deslorelin, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, epiphyseal B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin, diphenylspiromustine spiromustine), spondiacinol, docosanol, dolasetron, doxifene, droloxidene, dronabinol, duocarmycin SASA), ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, ethiprozin, epothilones, epithilones, epristeride, estramustine and its analogs, etoposide, etoposide 4'-phosphate (also known as etopofos), exemestane, dadrozole, dazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezellastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride hydrochloride), forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, homoharringtonine (HHT), hypericin, ibandronic acid, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridones, imiquimod, immunostimulatory peptides, iodobenzylguanidine, iodoxorubicin, sweet potato starch, irinotecan, iroplact, irsogladine, isobengazole, jasplakinolide, kahalalide F, lamellarin-N triacetate triacetate), lanreotide, leinamycin, lenograstim, lentinan sulfatesulfate), leptolstatin, letrozole, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol, lonidamine, losoxantrone, loxoribine, lurtotecan, lutetiumtexaphyrin, lysofylline, masorofol, maspin, matrix metalloproteinase inhibitors, menogaril, rnerbarone, meterelin , methioninase, metoclopramide, MIF inhibitors, mifepristone, miltefosine, mirimostim, mithracin, mitoguazone, dibromodulcitol, mitomycin and its analogs, mitonafide, mitoxantrone, mofarotene, molgramostim, mycaperoxideB. Myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin, nagrestip, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, nilutamide, nisamycin, nitrullyn, octreotide, okicenone, onapristone, ondansetron, oracin, ormaplatin, oxaliplatin, oxaunomycin, paclitaxel and its analogs, palauamin e), palmitoylrhizoxin, pamidronate, panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase, peldesine, pentosan polysulfate sodium, pentostatin, pentrozole, perflubron, perphosphamide, phenazinomycin, picibanil, pirarubicin, piritrexim, podophyllotoxin, porfiromycin, purine nucleoside phosphorylase inhibitors, raltitrexed, rhizotomycin, rogletimide, roxithromycin, rubiginone B1 B1), ruboxyl, safingol, saintopin, sarcophytol A, sargramostim, sobuzoxane, sonermin, sparphosphonate, spicamycin DD), spiromustine, stipiamide, sulfinosine, tallimustine, tegafur, temozolomide, teniposide, thaliblastine, thiocoraline, tirapazamine, topotecan, topsentin, triciribine, trimetrexate, varamine, vinorelbine, vinxaltine, vorozole, zeniplatin, and zilascorb.

[0852] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine dimer represented by formula (IV):

[0853]

[0854] A variety of linkers can be used to conjugate the antibodies and antigen-binding fragments described herein (eg, antibodies, antigen-binding fragments thereof that recognize and bind to CD5) to cytotoxic molecules.

[0855] As used herein, the term "linker" means a bivalent chemical moiety comprising a covalent bond or a chain of atoms that covalently attaches an antibody or fragment thereof (Ab) to a drug moiety (D) to form an antibody-drug conjugate (ADC; Ab-ZLD, wherein D is a cytotoxin) of the present disclosure. Suitable linkers have two reactive ends, one reactive end for conjugation to the antibody and the other reactive end for conjugation to the cytotoxin. The antibody-conjugating reactive end of the linker (reactive moiety, Z) is typically a site capable of conjugation to the antibody through a cysteine ​​thiol or lysine amine group on the antibody, and is therefore typically a thiol-reactive group, such as a double bond (as in maleimide) or a leaving group such as a chloro, bromo, iodo or R-sulfonyl group, or an amine-reactive group such as a carboxyl group; while the cytotoxin-conjugating reactive end of the linker is typically a site capable of conjugating to a cytotoxin by forming an amide bond with a basic amine or carboxyl group on the cytotoxin, and is therefore typically a carboxyl or basic amine group. When the term "linker" is used to describe a conjugated form of a linker, one or both reactive ends will be absent (such as reactive moiety Z, which has been converted to chemical moiety Z) or incomplete (such as merely the carbonyl group of a carboxylic acid) due to the formation of the bond between the linker and / or cytotoxin and between the linker and / or antibody or antigen-binding fragment thereof. Such conjugation reactions are further described below.

[0856] In some embodiments, the linker is cleavable under intracellular conditions so that the cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In yet other embodiments, the linker unit is non-cleavable and the drug is released by, for example, antibody degradation. The linker that can be used for the ADC of the present invention is preferably stable outside the cell, preventing the ADC molecules from aggregating, and keeping the ADC freely soluble in aqueous media and in a monomeric state. Before being transported or delivered into the cell, the ADC is preferably stable and remains intact, i.e., the antibody remains connected to the drug portion. The linker is stable outside the target cell and can be cleaved at a certain effective rate inside the cell. An effective linker will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug portion; (iii) remain stable and intact, i.e., not cleaved, until the conjugate has been delivered or transported to its targeted site; and (iv) maintain the cytotoxic, cell-killing, or cell-inhibitory effects of the cytotoxic portion. The stability of the ADC can be measured by standard analytical techniques such as mass spectrometry, HPLC, and separation / analysis techniques LC / MS. Covalent attachment of the antibody and drug moieties requires that the linker possess two reactive functional groups, i.e., divalency in the reactive sense. Bivalent linker reagents useful for attaching two or more functional or biologically active moieties (such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups) are known, and methods for their production of conjugates have been described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, p. 234-242).

[0857] Linkers include those that can be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference as it relates to linkers suitable for covalent conjugation).

[0858] Linkers that can be hydrolyzed under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, and the like. (See, for example, U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661, the disclosures of each of which are incorporated herein by reference in their entirety as they relate to linkers suitable for covalent conjugation. Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but are unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes).

[0859] Linkers that can be cleaved under reducing conditions include, for example, disulfides. A variety of disulfide linkers are known in the art, including, for example, those formed by the following: SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate) and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio) toluene), SPDB and SMPT (see, for example, Thorpe et al., 1987, Cancer Res. 47: 5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogeled., Oxford U. Press, 1987). See also U.S. Patent No. 4,880,935, the disclosure of each of which is incorporated herein by reference in its entirety as it relates to linkers suitable for covalent conjugation).

[0860] Examples of linkers useful in the synthesis of drug-antibody conjugates include those containing electrophiles suitable for reacting with nucleophilic substituents (such as amine and thiol moieties) present in the antibody or antigen-binding fragment, such as Michael acceptors (e.g., maleimide), activated esters, electron-deficient carbonyl compounds, and aldehydes. For example, linkers suitable for synthesizing drug-antibody conjugates include, but are not limited to, succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, etc., which are described, for example, in Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation. Additional linkers include non-cleavable maleimidocaproyl linkers, which are particularly useful for conjugation of microtubule disrupting agents (such as auristatins) and are described by Doronina et al., Bioconjugate Chem. 17: 14-24, 2006, the disclosure of which is incorporated herein by reference in its entirety with respect to linkers for chemical conjugation. Additional linkers suitable for the synthesis of drug-antibody conjugates as described herein include those capable of releasing cytotoxins by a 1,6-elimination process ("self-immolative" groups), such as p-aminobenzyl alcohol (PABC), 6-maleimidocaproic acid, pH-sensitive carbonates, and other reagents described in Jain et al., Pharm. Res. 32: 3526-3540, 2015, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the linker includes a self-degrading group, such as PAB or PABC (p-aminobenzyloxycarbonyl) mentioned above, which is described in, for example, Carl et al., J. Med. Chem. (1981) 24: 479-480; Chakravarty et al. (1983) J. Med. Chem. 26: 638-644; US 6214345; US20030130189; US20030096743; US6759509; US20040052793; US6218519; US6835807; US6268488; US20040018194; W098 / 13059; US20040052793; US6677435; US5621002; US20040121940; disclosed in W02004 / 032828.Other such chemical moieties capable of undergoing this process ("self-degrading linkers") include methylene carbamates and heteroaryl groups such as aminothiazole, aminoimidazole, aminopyrimidine, etc. Linkers containing such heterocyclic self-degrading groups are disclosed in, for example, U.S. Patent Publication Nos. 20160303254 and 20150079114 and U.S. Patent No. 7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237; US 2005 / 0256030; de Groot et al. (2001) J. Org. Chem. 66:8815-8830; and US 7223837.

[0861] A linker susceptible to enzymatic hydrolysis can be, for example, a peptide-containing linker that is cleaved by intracellular peptidases or proteases (including but not limited to lysosomal proteases or endosomal proteases). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically weakened during conjugation, and the serum stability of the conjugate is typically high. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Examples of suitable peptides include those containing amino acids such as valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine. The amino acid residues that constitute the amino acid linker component include naturally occurring amino acid residues, as well as minor amino acids and non-naturally occurring amino acid analogs such as citrulline. Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). In some embodiments, the linker comprises a dipeptide such as Val-Cit, Ala-Val or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg or Trp-Cit. Linkers containing dipeptides such as Val-Cit or Phe-Lys are disclosed in, for example, U.S. Patent No. 6,214,345, the disclosure of which is incorporated herein by reference in its entirety when it relates to linkers suitable for covalent conjugation. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the dipeptide is used in combination with a self-degrading linker.

[0862] Suitable linkers for use herein may also include one or more groups selected from the group consisting of C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which may be optionally substituted. Non-limiting examples of such groups include (CH2) n 、(CH2CH2O) n and -(C=O)(CH2) n - unit, wherein n is an integer from 1-6 selected independently at each occurrence.

[0863] In some embodiments, the linker may include one or more of the following: a hydrazine, a disulfide, a thioether, a dipeptide, a p-aminobenzyl (PAB) group, a heterocyclic self-immolative group, an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted C3-C6 cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, an acyl group, -C(=O)-, or -(CH2C2O) n - group, wherein n is an integer from 1 to 6. Those skilled in the art will recognize that one or more of the groups listed may exist in the form of a divalent (diradical) species, such as C1-C6 alkylene, etc.

[0864] In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In one embodiment, the p-aminobenzyl group is positioned between the cytotoxic drug and the protease cleavage site in the linker. In one embodiment, the p-aminobenzyl group is part of a p-aminobenzyloxycarbonyl unit. In one embodiment, the p-aminobenzyl group is part of a p-aminobenzylamido unit.

[0865] In some embodiments, the linker comprises PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0866] In some embodiments, the linker comprises a combination of one or more of the following: a peptide, an oligosaccharide, -(CH2) n -、-(CH2CH2O) n-, PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB or Ala-PAB.

[0867] In some embodiments, the linker comprises -(C=O)(CH2) n - unit, wherein n is an integer from 1-6.

[0868] In some embodiments, the linker comprises -(CH2) n - unit, wherein n is an integer from 2 to 6.

[0869] In certain embodiments, the linker of the ADC is N-β-maleimidopropyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB).

[0870] Linkers that can be used to conjugate an antibody or its antigen-binding fragment to a cytotoxic agent include those that are covalently bound to the cytotoxic agent at one end of the linker and contain a chemical moiety at the other end of the linker that is formed by a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the antibody or its antigen-binding fragment that binds to CD5. Reactive substituents that can be present in the antibody or its antigen-binding fragment that binds to CD5 include, but are not limited to, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine ​​residues, as well as propargyl, azido, halogenated aryl (e.g., fluoroaryl), halogenated heteroaryl (e.g., fluoroheteroaryl), halogenated alkyl, and halogenated heteroalkyl moieties of non-naturally occurring amino acids.

[0871] Examples of linkers useful in the synthesis of drug-antibody conjugates include those containing electrophiles suitable for reacting with nucleophilic substituents (such as amine and thiol moieties) present in the antibody or antigen-binding fragment, such as Michael acceptors (e.g., maleimide), activated esters, electron-deficient carbonyl compounds, and aldehydes. For example, linkers suitable for the synthesis of drug-antibody conjugates include, but are not limited to, succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, etc., which are described, for example, in Liu et al., 18: 690-697, 1979, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation. Additional linkers include the non-cleavable maleimidocaproyl linker, which is particularly useful for conjugation of microtubule disrupting agents such as auristatins and is described by Doronina et al., Bioconjugate Chem. 17:14-24, 2006, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation.

[0872] Those skilled in the art will recognize that any one or more of the chemical groups, moieties, and features disclosed herein can be combined in a variety of ways to form linkers useful for conjugation of antibodies and cytotoxins as disclosed herein. Other linkers that can be used in conjunction with the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0873] Linkers that can be used in conjunction with the antibody-drug conjugates described herein include, but are not limited to, linkers containing chemical moieties formed by coupling reactions as depicted below in Table 2. The curved lines represent the points of attachment to the antibody or antigen-binding fragment and the cytotoxic molecule, respectively.

[0874] Table 2. Exemplary chemical moieties formed by coupling reactions in the formation of antibody-drug conjugates

[0875]

[0876]

[0878]

[0879]

[0880] One skilled in the art will recognize that a reactive substituent Z attached to a linker and a reactive substituent on the antibody or antigen-binding fragment thereof participate in a covalent coupling reaction to produce the chemical moiety Z, and will recognize the reactive substituent Z. Thus, an antibody-drug conjugate useful in conjunction with the methods described herein can be formed by reacting the antibody or antigen-binding fragment thereof with a linker or cytotoxin-linker conjugate as described herein, the linker or cytotoxin-linker conjugate comprising the reactive substituent Z, which is suitable for reacting with a reactive substituent on the antibody or antigen-binding fragment thereof to form the chemical moiety Z.

[0881] As depicted in Table 2, examples of suitable reactive substituents on the linker and the antibody or antigen-binding fragment thereof include nucleophile / electrophile pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, etc.), diene / dienophile pairs (e.g., azide / alkyne pairs, or diene / α,β-unsaturated carbonyl pairs, etc.), etc. Coupling reactions between reactive substituents to form the chemical moiety Z include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine or hydroxylamine condensation, hydrazine formation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, etc.), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reaction paradigms known in the art or described herein. Preferably, the linker comprises an electrophilic functional group for reacting with the nucleophilic functional group on the antibody or antigen-binding fragment thereof.

[0882] Reactive substituents that may be present in an antibody or antigen-binding fragment thereof as disclosed herein include, but are not limited to, nucleophilic groups such as (i) an N-terminal amine group, (ii) a side chain amine group, e.g., lysine, (iii) a side chain thiol group, e.g., cysteine, and (iv) a sugar hydroxyl or amino group, wherein the antibody is glycosylated. Reactive substituents that may be present in an antibody or antigen-binding fragment thereof as disclosed herein include, but are not limited to, hydroxyl moieties of serine, threonine, and tyrosine residues; amino moieties of lysine residues; carboxyl moieties of aspartic acid and glutamic acid residues; and thiol moieties of cysteine ​​residues, as well as propargyl, azido, halogenated aryl (e.g., fluoroaryl), halogenated heteroaryl (e.g., fluoroheteroaryl), halogenated alkyl, and halogenated heteroalkyl moieties of non-naturally occurring amino acids. In some embodiments, reactive substituents present in an antibody or antigen-binding fragment thereof as disclosed herein include amine or thiol moieties. Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. By treating with a reducing agent such as DTT (dithiothreitol), antibodies can be made reactive for being put together with a linker reagent. Therefore, in theory, each cysteine ​​bridge will form two reactive thiol nucleophiles. Cause amine to be converted into thiol by the reaction of lysine and 2-iminothiolane (Traut reagent), other nucleophilic groups can be introduced into antibodies. By introducing one, two, three, four or more cysteine ​​residues (for example, preparing a mutant antibody comprising one or more non-natural cysteine ​​residues), reactive thiol groups can be introduced into antibodies (or their fragments). U.S. Patent No. 7,521,541 teaches engineering antibodies by introducing reactive cysteine ​​amino acids.

[0883] In some embodiments, the reactive moiety Z attached to the linker is a nucleophilic group that reacts with the electrophilic group present on the antibody. Useful electrophilic groups on the antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of the nucleophilic group can react with the electrophilic group on the antibody and form a covalent bond with the antibody. Useful nucleophilic groups include, but are not limited to, hydrazides, oximes, amino groups, hydroxyls, hydrazine, thiosemicarbazones, carboxylic acid hydrazines, and aryl hydrazides.

[0884] In some embodiments, Z is the reaction product between a reactive nucleophilic substituent present in the antibody or antigen-binding fragment thereof, such as an amine and thiol moiety, and a reactive electrophilic substituent Z. For example, Z can be a Michael acceptor (e.g., maleimide), an activated ester, an electron-deficient carbonyl compound, or an aldehyde, among others.

[0885] In some embodiments, the ADC comprises an anti-CD5 antibody conjugated to an amanita toxin of any of Formulas I, IA, IB, II, IIA, or IIB as disclosed herein via a linker and a chemical moiety Z. In some embodiments, the linker comprises a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises a PAB-Cit-Val moiety. In some embodiments, the linker comprises a PAB-Ala-Val moiety. In some embodiments, the linker comprises -((C=O)(CH2) n -unit, wherein n is an integer from 1 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH2) n -.

[0886] In some embodiments, the linker comprises -(CH2) n -unit, wherein n is an integer from 2 to 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 -((CH2) n -, where n is 6.

[0887] In some embodiments, the chemical moiety Z is selected from Table 1. In some embodiments, the chemical moiety Z is

[0888]

[0889] wherein S is a sulfur atom and represents a reactive substituent (e.g., a -SH group from a cysteine ​​residue) present in the antibody or antigen-binding fragment thereof that binds to CD5.

[0890] In some embodiments, the linker L and the chemical moiety Z (collectively referred to as LZ) are

[0891]

[0892] Those skilled in the art will recognize that, prior to conjugation to the antibody or antigen-binding fragment thereof, the linker-reactive substituent group structure includes a maleimide as group Z. The foregoing linker moieties and amanita toxin-linker conjugates, etc., that can be used in conjunction with the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220 and Patent Application Publication No. WO2017 / 149077, the disclosures of each of which are incorporated herein by reference in their entirety.

[0893] In some embodiments, prior to conjugation to the antibody or antigen-binding fragment thereof, the structure of the linker reactive substituent group is:

[0894]

[0895] Preparation of antibody-drug conjugates

[0896] In the ADC of Formula I as disclosed herein, the antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxic drug moieties (D) via a linker L and a chemical moiety Z as disclosed herein, for example, about 1 to about 20 drug moieties per antibody. The ADCs of the present disclosure can be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reacting a reactive substituent of the antibody or antigen-binding fragment thereof with a divalent linker reagent to form Ab-ZL as described above, followed by reaction with the drug moiety D; or (2) reacting a reactive substituent of the drug moiety with a divalent linker reagent to form DLZ, followed by reaction with a reactive substituent of the antibody or antigen-binding fragment thereof as described above to form an ADC of the formula DLZ-Ab, such as Am-ZL-Ab. Additional methods for preparing ADCs are described herein.

[0897] In another aspect, the antibody or antigen-binding fragment thereof has one or more lysine residues that can be chemically modified to introduce one or more sulfhydryl groups. ADCs are then formed by conjugation via the sulfur atom of the sulfhydryl group, as described above. Reagents that can be used to modify lysine include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).

[0898] In another aspect, the antibody or antigen-binding fragment thereof can have one or more carbohydrate groups that can be chemically modified to have one or more sulfhydryl groups. The ADC is then formed by conjugation via the sulfur atom of the sulfhydryl group as described above.

[0899] In yet another aspect, the antibody may have one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) group (see, e.g., Laguzza et al., J. Med. Chem. 1989, 32(3), 548-55). ADCs are then formed by conjugation with the corresponding aldehyde, as described above. Other protocols for modifying proteins for attachment or association of cytotoxins are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), incorporated herein by reference.

[0900] Methods for conjugating linker-drug moieties to cell-targeting proteins (such as antibodies, immunoglobulins, or fragments thereof) are found in, for example, U.S. Pat. No. 5,208,020; U.S. Pat. No. 6,441,163; WO2005037992; WO2005081711; and WO2006 / 034488, all of which are expressly incorporated herein by reference in their entirety.

[0901] Alternatively, a fusion protein comprising the antibody and the cytotoxic agent can be prepared, for example, by recombinant techniques or peptide synthesis. The length of DNA can include the corresponding regions encoding the two parts of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0902] Treatment

[0903] As described herein, hematopoietic stem cell transplantation therapy can be applied to the experimenter for needing treatment, to fill or refill one or more blood cell types.Hematopoietic stem cell usually shows multipotency and therefore can be divided into multiple different blood lineages, including but not limited to granulocyte (for example, promyelocyte, neutrophil, eosinophil, basophil), erythrocyte (for example, reticulocyte, erythrocyte), thrombocyte (for example, megakaryocyte, the megakaryocyte that produces platelet, platelet), monocyte (for example, monocyte, macrophage), dendritic cell, microglia, osteoclast and lymphocyte (for example NK cell, B cell and T cell).Hematopoietic stem cell can also self-renew and therefore can produce the daughter cell with the potentiality equal to mother cell, and feature is also the ability that is reintroduced into transplant recipient, in transplant recipient they home to hematopoietic stem cell niche and rebuild effective and lasting hematopoiesis.

[0904] Therefore, hematopoietic stem cells can be administered to one or more hematopoietic lineage cell types with missing or defective patients so that the colony of missing or defective cells is reconstructed in vivo, thereby treating the disease associated with the absence or depletion of endogenous blood cell colonies. Therefore, compositions and methods described herein can be used to treat non-malignant hemoglobinopathies (e.g., hemoglobinopathies selected from the group consisting of sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia and Wiskott-Aldrich syndrome). Additionally or alternatively, compositions and methods described herein can be used to treat immunodeficiency, such as congenital immunodeficiency. Additionally or alternatively, compositions and methods described herein can be used to treat acquired immunodeficiency (e.g., acquired immunodeficiency selected from the group consisting of HIV and AIDS). Compositions and methods described herein can be used to treat metabolic disorders (e.g., metabolic disorders selected from the group consisting of glycogen storage disease, mucopolysaccharidosis, Gaucher disease, Hurler disease, sphingolipid storage disease and metachromatic leukodystrophy).

[0905] Additionally or alternatively, the compositions and methods described herein can be used to treat malignant or proliferative disorders, such as hematological cancers, myeloproliferative diseases. In the case of cancer treatment, the compositions and methods described herein can be applied to the patient before hematopoietic stem cell transplantation therapy to exhaust the colony of immune cells that cross-react with non-self hematopoietic stem cells and produce an immune response against non-self hematopoietic stem cells, such as those expressing one or more non-self MHC antigens. This is used to prevent or reduce the possibility of rejection of the hematopoietic stem cell transplanted, allowing the transplanted hematopoietic stem cells to home to the stem cell niche and establish effective hematopoiesis. This can then allow the colony of cells exhausted during cancer cell eradication (such as during systemic chemotherapy) to be reconstructed. Exemplary hematological cancers that can be treated using the compositions and methods described herein include, but are not limited to, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B cell lymphoma, and non-Hodgkin's lymphoma, as well as other cancer conditions, including neuroblastoma.

[0906] Additional diseases that can be treated with the compositions and methods described herein include, but are not limited to, adenosine deaminase deficiency and severe combined immunodeficiency, hyperimmunoglobulinemia M syndrome, Chidon disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.

[0907] Antibodies or their antigen-binding fragments and conjugates described herein can be used to induce solid organ transplant tolerance (solid organ transplant tolerance). For example, compositions and methods described herein can be used to deplete or ablate a colony of immune cells before hematopoietic stem cell transplantation. After exhausting cells from target tissues like this, a colony of stem cells or progenitor cells (for example, hematopoietic stem cells from organ donors) from an organ donor can be administered to a transplant recipient, and after implanting such stem cells or progenitor cells, a temporary or stable mixed chimera can be achieved, thereby enabling long-term transplant organ tolerance to be produced without the need for further immunosuppressants. By administering anti-CD5 antibodies or their antigen-binding fragments, the possibility of transplanted graft rejection can be reduced, or rejection can be completely prevented. In this way, compositions and methods described herein can be used to induce transplant tolerance in solid organ transplant recipients (for example, kidney transplants, lung transplants, liver transplants, and heart transplants, etc.). For example, because a low percentage of temporary or stable donor implants is sufficient to induce the long-term tolerance of the transplanted organ, compositions and methods described herein are well suited for use in combination with inducing solid organ transplant tolerance.

[0908] In addition, the compositions and methods described herein can be used to directly treat cancers, such as cancers characterized by CD5+ cells. For example, the compositions and methods described herein can be used to treat leukemias, particularly in patients who exhibit CD5+ leukemic cells. By depleting CD5+ cancer cells, such as leukemic 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 hematological cancers, such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma.

[0909] In addition, the compositions and methods described herein can be used to treat autoimmune disorders. For example, antibodies or their antigen-binding fragments can be administered to subjects, such as human patients with autoimmune disorders in order to kill CD5+ immune cells. CD5+ immune cells can be autoreactive lymphocytes, such as T cells that express T cell receptors that specifically bind to autoantigens and produce immune responses against autoantigens. The compositions and methods described herein can be used to treat autoimmune disorders, such as those described below, by exhausting autoreactive CD5+ cells. Additionally or alternatively, the compositions and methods described herein can be used to treat autoimmune diseases by exhausting the colony of endogenous hematopoietic stem cells before hematopoietic stem cell transplantation therapy, in which case the transplanted cells can home to the niche produced by the endogenous cell exhaustion step and establish effective hematopoiesis. This in turn can reconstruct the colony of cells exhausted during autoimmune cell eradication.

[0910] Autoimmune diseases that can be treated using the compositions and methods described herein include, but are not limited to, psoriasis, psoriatic arthritis, Type 1 diabetes mellitus, Type 1 diabetes, rheumatoid arthritis (RA), 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 antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), and inflammatory bowel disease (IBD). S), autoimmune oophoritis, Barlow disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac disease-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, malignant atrophic papulosis, discoid lupus, dysautonomia, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's kidney 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, Ord's thyroiditis, pemphigus vulgaris , pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndromes, polymyalgia rheumatica, essential agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-person syndrome, Takayasu's arteritis, temporal arteritis (also called "giant cell arteritis"), ulcerative colitis, collagenous colitis, uveitis, vasculitis, vitiligo, vulvodynia ("vulvar vestibulitis"), and Wegener's granulomatosis.

[0911] For example, using the compositions and methods described herein, one skilled in the art can administer an anti-CD5 antibody or its antigen-binding fragment in an amount sufficient to treat an autoimmune disorder to a subject suffering from an autoimmune disorder. For example, the subject may suffer from scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease and / or type 1 diabetes. In order to improve one or more of these conditions, a physician skilled in the art can prescribe and administer an anti-CD5 antibody or its fragment, such as an antibody or its fragment that is bound to a cytotoxic agent, to the subject. The antibody or its fragment can be conjugated to a cytotoxic agent using the conjugation techniques and linkers described in detail above. A variety of cytotoxic agents can be conjugated to an anti-CD5 antibody or its antigen-binding fragment to deplete the population of endogenous, self-reactive CD5+T cells, B cells, or NK cells in the subject. For example, an antibody or its antigen-binding fragment can be conjugated to an Amanita toxin or another cytotoxic portion described herein.

[0912] When preparing for therapy, the physician can assess the number or concentration of autoreactive T cells, B cells and / or NK cells in the sample separated from the subject. This can, for example, be accomplished using FACS analysis techniques known in the art. Then, one skilled in the art can administer an antibody or fragment thereof, alone or in combination with a cytotoxin, to the subject in order to deplete the colony of autoreactive T cells, B cells and / or NK cells. In order to evaluate the effectiveness of therapy, the physician can determine the number or concentration of autoreactive T cells, B cells and / or NK cells in the sample separated from the patient at the time after administering an anti-CD5 antibody or fragment thereof. Determining the number or concentration of autoreactive T cells, B cells and / or NK cells in the sample separated from the subject after therapy relative to the number or concentration of T cells, B cells or NK cells before therapy provides an indication that the patient has a response to anti-CD5 antibodies or fragments thereof.

[0913] Antibody drug conjugates comprising anti-CD5 antibodies or antigen-binding fragments thereof can also be used in combination with CAR T therapy. Specifically, prior to CAR T therapy, an effective amount of an anti-CD5 antibody drug conjugate can be administered to a patient in need thereof to deplete naive T cells. Depleting naive T cells expressing CD5 using the methods and compositions described herein can provide more efficient transfer of engineered T cells used in CAR T therapy.

[0914] Administration and route of administration

[0915] The antibodies described herein or their antigen-binding fragments can be administered to patients (e.g., human patients in need of hematopoietic stem cell transplantation) in a variety of dosage forms. For example, the antibodies described herein or their antigen-binding fragments can be administered to patients in need of hematopoietic stem cell transplantation and / or suffering from cancer or autoimmune diseases in the form of an aqueous solution, such as an aqueous solution containing one or more pharmaceutically acceptable excipients. An exemplary pharmaceutically acceptable excipient for use with the compositions and methods described herein is a viscosity modifier. The aqueous solution can be sterilized using techniques known in the art.

[0916] The antibodies and antigen-binding fragments described herein can be administered by a variety of routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intraocularly, or parenterally. In any given case, the most appropriate route of administration will depend on the specific antibody or antigen-binding fragment being administered, the patient, the pharmaceutical formulation method, the method of administration (e.g., time of administration and route of administration), the patient's age, weight, sex, severity of the disease being treated, the patient's diet, and the patient's excretion rate.

[0917] An effective dosage of an antibody or antigen-binding fragment thereof described herein can range from about 0.001 mg / kg body weight to about 100 mg / kg body weight (e.g., about 0.001 mg / kg body weight to about 0.01 mg / kg body weight, about 0.01 mg / kg body weight to about 0.1 mg / kg body weight, about 0.1 mg / kg body weight to about 1 mg / kg body weight, about 1 mg / kg body weight to about 10 mg / kg body weight, about 10 mg / kg body weight to about 100 mg / kg body weight), for example, per single (bolus) administration, multiple administrations, or continuous administration, or to achieve an optimal serum concentration of the antibody or antigen-binding fragment thereof (e.g., about 0.0001 μg / kg body weight). g / mL to about 5000 μg / mL (e.g., a serum concentration of about 0.0001 μg / mL to about 0.001 μg / mL, about 0.001 μg / mL to about 0.01 μg / mL, about 0.01 μg / mL to about 0.1 μg / mL, about 0.1 μg / mL to about 1 μg / mL, about 1 μg / mL to about 10 μg / mL, about 10 μg / mL to about 100 μg / mL, about 100 μg / mL to about 1000 μg / mL, about 1000 μg / mL to about 2000 μg / mL, about 2000 μg / mL to about 3000 μg / mL, or about 3000 μg / mL to about 5000 μg / mL)). The dosage can be administered daily, weekly or monthly once or more times (e.g., 2-10 times) to a subject (e.g., a human) undergoing conditioning therapy in preparation for receiving a hematopoietic stem cell transplant. The antibody or its antigen-binding fragment can be administered to the patient at a time that best promotes the implantation of exogenous hematopoietic stem cells, e.g., prior to hematopoietic stem cell transplantation, at a time that best depletes CD5+T cells, B cells or NK cells that cross-react with non-self hematopoietic stem cell antigens (such as non-self MHC antigens). For example, anti-CD5 antibodies and antigen-binding fragments thereof can be administered to a patient undergoing hematopoietic stem cell transplantation therapy from about 1 hour to about 1 week (e.g., 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, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 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, about 6 days, or about 7 days) or earlier before administration of an exogenous hematopoietic stem cell transplant.The half-life of the antibody can be between about 1 hour and about 24 hours (e.g., 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, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours).

[0918] In one embodiment, the anti-CD5 antibody (or Fc-containing fragment thereof) has reduced half-life (compared to a wild-type Fc region), wherein the Fc region of the antibody comprises an H435A mutation (numbering according to the EU index).

[0919] According to the methods disclosed herein, physicians skilled in the art can adjust patients (such as human patients) before hematopoietic stem cell transplantation therapy to promote the implantation of exogenous hematopoietic stem cell transplants. To this end, physicians skilled in the art can administer antibodies or antigen-binding fragments thereof that can be combined with CD5 to human patients, such as anti-CD5 antibodies described herein. Antibodies or their fragments can be covalently conjugated to toxins (such as cytotoxic molecules described herein or known in the art) or Fc domains. For example, anti-CD5 antibodies or their antigen-binding fragments can be covalently conjugated to cytotoxins, such as Pseudomonas exotoxin A, deBouganin, diphtheria toxin, amanitin toxins (such as α-amanitin), saporin, maytansine, maytansinoids, auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycins, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolebenzodiazepines and indolebenzodiazepine dimers, or variants thereof. Such conjugation can be performed using covalent bond forming techniques described herein or known in the art. The antibody, antigen-binding fragment thereof, or antibody-drug conjugate can then be administered to the patient, for example, by intravenous administration, and then exogenous hematopoietic stem cells (such as autologous, syngeneic, or allogeneic hematopoietic stem cells) are transplanted into the patient.

[0920] The anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug conjugate can be administered in an amount sufficient to reduce the number of endogenous T cells, B cells, and / or NK cells (such as bone marrow resident T cells) by, for example, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more prior to hematopoietic stem cell transplantation therapy. For example, an anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug conjugate can be administered in an amount sufficient to reduce the number of endogenous T cells, B cells, and / or NK cells (such as bone marrow-resident T cells) by, e.g., about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 95%, or more, prior to hematopoietic stem cell transplantation therapy. For example, anti-CD5 antibodies, antigen-binding fragments thereof, or antibody-drug conjugates can be administered in an amount sufficient to reduce the number of endogenous T cells, B cells, and / or NK cells (such as bone marrow-resident T cells) prior to hematopoietic stem cell transplantation therapy by, for example, 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%, at least about 90%, at least about 95% or more. The reduction in T cell counts can be monitored using conventional techniques known in the art, such as by monitoring FACS analysis of cells expressing characteristic T cell surface antigens in blood samples extracted from the patient at different time intervals during the conditioning therapy. For example, a physician skilled in the art can extract bone marrow samples from the patient at multiple time points during the conditioning therapy and determine the extent of endogenous T cell reduction by performing FACS analysis, which uses antibodies bound to T cell marker antigens to illustrate the relative concentration of T cells in the sample. According to some embodiments, when the concentration of T cells has reached a minimum in response to conditioning therapy with an anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug conjugate, the physician can end conditioning therapy and can begin preparing the patient for hematopoietic stem cell transplantation therapy.

[0921] Anti-CD5 antibodies, their Fabs or antibody-drug conjugates can be administered to patients with aqueous solutions containing one or more pharmaceutically acceptable excipients (such as viscosity modifiers). Aqueous solutions can be sterilized using techniques described herein or known in the art. Before hematopoietic stem cell transplants are administered to patients, antibodies, their Fabs or antibody-drug conjugates can be administered to patients in a dosage of, for example, from about 0.001 mg / kg to about 100 mg / kg (e.g., about 0.001 mg / kg to about 0.01 mg / kg, about 0.01 mg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 1 mg / kg, about 1 mg / kg to about 10 mg / kg, about 10 mg / kg to about 100 mg / kg). The antibody, antigen-binding fragment thereof, or antibody-drug conjugate can be administered to the patient at a time that optimally promotes engraftment of the exogenous hematopoietic stem cells, for example, from about 1 hour to about 1 week (e.g., 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, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 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, about 6 days, or about 7 days) or earlier before administration of the exogenous hematopoietic stem cell transplant.

[0922] After the conditioning therapy is completed, the patient may then receive an infusion (e.g., intravenous infusion) of exogenous hematopoietic stem cells, such as by the same physician who performed the conditioning therapy or by a different physician. The physician may administer an infusion of autologous, syngeneic, or allogeneic hematopoietic stem cells to the patient, for example, at a dose of from about 1×10 3 Hematopoietic stem cells / kg is about 1×10 9 Hematopoietic stem cells / kg (about 1×10 3 Hematopoietic stem cells are approximately 1×10 4 Hematopoietic stem cells, about 1×10 4 Hematopoietic stem cells are approximately 1×10 5 Hematopoietic stem cells, about 1×10 5 Hematopoietic stem cells are approximately 1×10 6 Hematopoietic stem cells, about 1×10 6 Hematopoietic stem cells are approximately 1×10 7 hematopoietic stem cells, or about 1×10 8 Hematopoietic stem cells are approximately 1×10 9A physician can monitor the engraftment of a hematopoietic stem cell transplant, for example, by drawing a blood sample from the patient after administration of the transplant and determining an increase in the concentration of hematopoietic stem cells or cells of the hematopoietic lineage, such as 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 B lymphocytes. The analysis can be performed, for example, from 1 hour to 6 months or more (e.g., 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, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 49 hours, about 50 hours, about 51 hours, about 52 hours, about 53 hours, about 54 hours, about 55 hours, about 56 hours, about 57 hours, about 58 hours, about 59 hours, about 60 hours, about 61 hours, about 62 hours, about 63 hours, about 64 hours, about 65 hours, about 66 hours, about 67 hours, about 68 hours, about 69 hours, about 70 hours, about 71 hours, about 72 hours, about 73 hours, about 74 hours about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, or more). A finding that the concentration of hematopoietic stem cells or cells of the hematopoietic lineage has increased after transplantation therapy relative to the concentration of the corresponding cell type before transplantation therapy (e.g., an increase of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 500% or more) provides an indication that treatment with the anti-CD5 antibody, antigen-binding fragment thereof, or antibody-drug conjugate has successfully promoted the engraftment of the transplanted hematopoietic stem cell graft. Example

[0923] The following examples are presented so as to provide one of ordinary skill in the art with a description of how the compositions and methods described herein might be used, prepared, and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0924] Example 1: In vitro binding analysis of anti-CD5 antibodies.

[0925] To determine the binding characteristics of the anti-CD5 antibody 5D7 hIgG1, antibody binding studies were performed using biolayer interferometry (BLI) with a Pall ForteBio Octet Red 96 at 25 degrees Celsius in 1× PBS supplemented with 0.1% w / v bovine serum albumin. Purified human anti-CD5 antibody (5D7) was immobilized on an anti-human Fc biosensor (AHC; Pall ForteBio 18-5063) and incubated with 50 nM of purified human CD5 extracellular domain. The binding characteristics of the anti-CD5 antibody 5D7 are shown in Table 3. The anti-human CD5 antibody 5D7 used in Examples 1 to 5 is a humanized version of the murine antibody 5D7 (see US2008 / 0254027). The sequence of the antibody 5D7 used herein is described in SEQ ID Nos: 257 and 258 (heavy chain variable region and light chain variable region amino acid sequences) and SEQ ID Nos: 29 to 34 (heavy chain CDRs and light chain CDRs).

[0926] Table 3: Binding kinetics of 5D7 to the extracellular domain of human CD5

[0927] Antibody Concentration (nM) Response (nm) <![CDATA[K D (M)]]> <![CDATA[K ON (1 / Ms)]]> <![CDATA[K DIS (1 / s)]]> <![CDATA[Completely R 2 > 5D7 50 0.6696 1.41E-10 2.39E+05 3.36E-05 0.9996

[0928] Example 2: In vitro cell line binding analysis of anti-CD5 antibodies

[0929] MOLT-4 cells (i.e., immortalized human T lymphoblastoid cell line) were plated at 20,000 cells / well and stained with titrations of the indicated mouse anti-CD5 antibodies (i.e., L17F12, UCHT2, 205919, and CRIS-1) for 2 hours at 4°C. Secondary anti-mouse AF488 dye was added in a constant amount and placed at 4°C for 30 minutes. After washing, the plate was run on a flow cytometer and binding of the indicated antibodies (and negative control, i.e., mIgG1) was determined based on the geometric mean fluorescence intensity in the AF488 channel. Figure 1 Results from these assays are provided in .

[0930] like Figure 1 As shown in Figure 2, murine anti-CD5 antibodies L17F12 (Thermo Fisher), UCHT2 (BioLegend), 205919 (Novus Biologicals), and CRIS-1 (Novus Biologicals) bind to human T lymphoblastoid cells (i.e., MOLT-4 cells). 50 =207pM (L17), 354pM (UCH), 1350pM (205) and 43pM (CRIS).

[0931] Example 3: In vitro primary cell binding analysis of anti-CD5 antibodies

[0932] Primary human T cells were cultured at 8×10 4 Cells were plated at 500 cells / well and stained with titrations of the mouse anti-CD5 antibody 5D7 for 2 hours at 37°C. A secondary anti-mouse AF488 dye was added at a constant volume and incubated at 4°C for 30 minutes. After washing, the plate was run on a flow cytometer, and binding of the anti-CD5 5D7 antibody (and negative control, hIgG1) was determined based on the geometric mean fluorescence intensity in the AF488 channel. Figure 2 Results from these assays are provided in .

[0933] like Figure 2 As shown in Figure 2, the anti-CD5 antibody 5D7 binds to primary human T cells and has EC 50 =3.0pM.

[0934] Example 4. In vitro analysis of anti-CD5-amanitin antibody drug conjugates (ADCs) using an in vitro T cell killing assay

[0935] The anti-CD5 antibody 5D7 was conjugated to amanita toxin (amanitin) using a cleavable linker to form the anti-CD5 5D7 ADC. Anti-CD5 5D7-ADCs with a drug-to-antibody ratio (DAR) of approximately 6 (interchain DAR6) and anti-CD5 5D7-ADCs with a DAR of approximately 2 (prepared using site-specific conjugation via a D265C mutation) were tested. In addition, a short half-life variant of the anti-CD5 5D7-ADC was generated by introducing the H435A mutation in the Fc region.

[0936] Each anti-CD5 5D7-ADC was evaluated using an in vitro human T cell killing assay. Cryopreserved negatively selected primary human T cells were thawed and stimulated with anti-CD3 antibody and IL-2. At the start of the assay, 2×10 4 T cells were seeded into each well of a 384-well plate, and the indicated ADC or unconjugated anti-CD5 antibody was added to the wells at various concentrations between 0.003 nM and 30 nM, then placed in an incubator at 37°C and 5% CO2. The cells were analyzed by flow cytometry after five days of culture. The cells were stained with the viability marker 7-AAD and run on a volume flow cytometer.

[0937] The number of active T cells ( Figure 3A and Figure 3B ) were determined by FSC versus SSC and 7-AAD. Unconjugated anti-CD5 5D7 antibody was used as a control ( Figure 3A ).

[0938] like Figure 3AAs shown in Figure 2, anti-CD5 5D7-ADC with a DAR of about 6 exhibited potent and specific killing of human T cells (IC50 = 3.7 pM), while T cells remained viable in the presence of unconjugated ("naked") anti-CD5 5D7 antibody. Figure 3B As shown in Figure 3, an ADC with a site-specific (D265C) DAR of approximately 2 maintained a similarly potent level of T cell killing as the DAR 6 ADC (IC50 = 5.0 pM). A short half-life variant of the anti-CD5 5D7-ADC (H435A) exhibited similar levels of T cell killing (IC50 = 4.9 pM; Figure 3B ).

[0939] Example 5. Analysis of T cell exhaustion using the hNSG mouse model

[0940] In vivo T cell depletion assays were performed using humanized NSG mice (Jackson Laboratories). The anti-CD5 antibody 5D7 was conjugated to amanita toxin (amanitin) using a cleavable linker to form anti-CD5 5D7-ADC. As described above, anti-CD5 5D7-ADCs were prepared with a DAR of about 6 or a DAR of about 2. Each anti-CD5 5D7 ADC (DAR6 or DAR2) was administered to humanized mice as a single intravenous injection (0.3 mg / kg, 1 mg / kg, or 3 mg / kg for DAR6 ADC, or 1 mg / kg or 3 mg / kg for DAR2 ADC). Peripheral blood cells, bone marrow, or thymus samples were collected on day 7, and the absolute number of CD3+ T cells was determined by flow cytometry (for DAR6 ADC, see Figures 4A-4B , and for DAR2 ADC see Figures 5A-5C ).

[0941] like Figures 4A-4B As shown in Figure 2, humanized NSG mice treated with 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of DAR6 anti-CD5 5D7-ADC exhibited efficient T cell depletion in peripheral blood or bone marrow, while thymic T cells were depleted after treatment with 1 mg / kg or 3 mg / kg of DAR6 anti-CD5 5D7-ADC. Negative controls used in this in vivo experiment included human IgG1 not specific for CD5 as naked antibody (hIgG1) and human IgG1 conjugated to Amanita phalloidin (hIgG1-AM). Figures 4A to 4B As described in, naked hIgG1 and conjugated hIgG1 were tested against peripheral blood ( Figure 4A ) and bone marrow ( Figure 4B) had no effect, as these controls were comparable to the PBS control. Anti-CD52 antibody (antibody YTH34.5) was also used as a control and was also able to deplete peripheral and bone marrow T cells at a dose of 25 mg / kg.

[0942] like Figures 5A-5C As shown in Figure 3, humanized NSG mice treated with 1 mg / kg or 3 mg / kg of the site-specific DAR2 anti-CD5 5D7-ADC exhibited efficient T cell depletion in peripheral blood T cells, bone marrow T cells, and thymic T cells. Figures 5A to 5C In each figure, naked antibody 5D7 was also used as a control. Figure 5A As described in , antibody 5D7 was able to deplete peripheral T cells (relative to a nonspecific human IgG1 control or PBS), but not bone marrow T cells or thymic T cells, whereas Figure 5B and Figure 5C As described in , 5D7-AM ADC effectively depletes both myeloid and thymic T cells.

[0943] Table 4 Sequence summary

[0944]

[0945]

[0946]

[0947] Other implementation plans

[0948] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0949] Although the invention has been described with reference to specific embodiments thereof, it will be understood that the invention is capable of further modifications, and this application is intended to cover any variations, uses, or modifications of the invention that follow generally from the principles of the invention and include such departures from the invention as come within known or customary practice in the art to which the invention pertains and which may be applied to the essential features hereinbefore set forth and which fall within the scope of the appended claims.

[0950] This application also provides the following items:

[0951] 1. A method of depleting a population of CD5+ cells in a human patient, the method comprising administering to the patient an effective amount of an anti-CD5 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin.

[0952] 2. A method of depleting a population of CD5+ cells in a human patient in need of a hematopoietic stem cell transplant, the method comprising administering to the patient an effective amount of an anti-CD5 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin.

[0953] 3. A method of preventing rejection of a hematopoietic stem cell transplant in a human patient in need of a hematopoietic stem cell transplant, the method comprising administering to the human patient an effective amount of an anti-CD5 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin, prior to the human patient receiving the transplant comprising hematopoietic stem cells.

[0954] 4. A method of depleting a population of CD5+ cells in a human patient in need of a hematopoietic stem cell transplant, the method comprising administering to the patient an effective amount of an anti-CD5 antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin, prior to the patient receiving the transplant comprising hematopoietic stem cells.

[0955] 5. A method comprising administering to a human patient a transplant comprising hematopoietic stem cells, wherein the patient has previously been administered an anti-CD5 antibody or antigen-binding fragment thereof in an amount sufficient to deplete the population of CD5+ cells in the patient, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin.

[0956] 6. A method comprising:

[0957] a. administering to a human patient an anti-CD5 antibody or antigen-binding fragment thereof that binds to CD5 in an amount sufficient to deplete the population of CD5+ cells in the patient, wherein the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin; and

[0958] b. Subsequently administering to the patient a transplant comprising hematopoietic stem cells.

[0959] 7. The method of any one of items 1 to 6, wherein the antibody or antigen-binding fragment thereof is produced by the hybridoma cell line ATCC CRL 8000.

[0960] 8. The method of any one of items 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises the heavy chain variable region CDR set (CDR1, CDR2, and CDR3) and the light chain variable region CDR set (CDR1, CDR2, and CDR3) of antibody OKT1, wherein the antibody OKT1 is produced by a hybridoma cell line having ATCC accession number CRL8000.

[0961] 9. The method of any one of items 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises the following variable domains:

[0962] a. V having the following amino acid sequence L : and

[0963] b. V having the following amino acid sequence H : or

[0964] a. V having the following amino acid sequence L : and

[0965] b. V having the following amino acid sequence H :

[0966] 10. The method of any one of items 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs):

[0967] a. CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 3);

[0968] b. CDR-H2 having the amino acid sequence NTHTGE (SEQ ID NO: 4);

[0969] c. CDR-H3 having the amino acid sequence RGYDWYFDV (SEQ ID NO: 5);

[0970] d. CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 6);

[0971] e. CDR-L2 having the amino acid sequence RANRLVD (SEQ ID NO: 7); and

[0972] f. a CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 8); or

[0973] a. CDR-H1 having the amino acid sequence FSLSTSGMG (SEQ ID NO: 29);

[0974] b. CDR-H2 having the amino acid sequence WWDDD (SEQ ID NO: 30);

[0975] c. CDR-H3 having the amino acid sequence RRATGTGFDY (SEQ ID NO: 31);

[0976] d. CDR-L1 having the amino acid sequence QDVGTA (SEQ ID NO: 32);

[0977] e. CDR-L2 having the amino acid sequence WTSTRHT (SEQ ID NO: 33);

[0978] f. CDR-L3 having the amino acid sequence YNSYNT (SEQ ID NO: 34).

[0979] 11. The method of any one of items 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises the following variable domains:

[0980] a. V having the following amino acid sequence L : and

[0981] b. V having the following amino acid sequence H :

[0982] 12. The method of any one of items 1 to 6, wherein the antibody or antigen-binding fragment thereof comprises the following CDRs:

[0983] a....

Claims

1. A conjugate represented by the formula Ab-Cy, wherein Ab is an antibody or antigen-binding fragment thereof that binds to CD5, and Cy is a cytotoxin, wherein Cy is an amanita phalloidin toxin (Am) represented by the formula (IB): Where R1 is H, OH, OR A OR C ; R2 is H, OH, OR B OR C ; R A and R B When present, together with the oxygen atom to which they are bound, they combine to form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C or R D ; R4, R5, R6 and R7 are each independently H, OH, OR C , OR D , R C or R D ; R8 is OH, NH2, OR C , OR D 、NHR C or NR C R D ; R9 is H, OH, OR C OR D ; X is -S-, -S(O)- or -SO2-; R C Yes - LZ; R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is a linker which is optionally substituted C1-C6 alkylene, optionally substituted C1-C6 heteroalkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene, a dipeptide, -C(=O)-, a peptide, or a combination thereof; and Z is a chemical moiety formed by a coupling reaction between a reactive substituent present on L and a reactive substituent present in the antibody or antigen-binding fragment thereof, where Am contains exactly one R C substituents, and The antibody or antigen-binding fragment thereof comprises the following complementarity determining regions (CDRs): a. CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 3); b. CDR-H2 having the amino acid sequence NTHTGE (SEQ ID NO: 4); c. CDR-H3 having the amino acid sequence RGYDWYFDV (SEQ ID NO: 5); d. CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 6); e. CDR-L2 having the amino acid sequence RANRLVD (SEQ ID NO: 7); and f. CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 8); or a. CDR-H1 having the amino acid sequence FSLSTSGMG (SEQ ID NO: 29); b. CDR-H2 having the amino acid sequence WWWDDD (SEQ ID NO: 30); c. CDR-H3 having the amino acid sequence RRATGTGFDY (SEQ ID NO: 31); d. CDR-L1 having the amino acid sequence QDVGTA (SEQ ID NO: 32); e. 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).

2. The conjugate of claim 1, wherein the antibody or antigen binding fragment thereof is selected from the group consisting of a monoclonal antibody or antigen binding fragment thereof, a polyclonal antibody or antigen binding fragment thereof, a humanized antibody or antigen binding fragment thereof, a bispecific antibody or antigen binding fragment thereof, a dual variable immunoglobulin domain, a single-chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment, a F(ab')2 molecule, and a tandem di-scFv.

3. The conjugate of claim 2, wherein the antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. The conjugate of claim 3 , wherein the antibody has an IgG1 or IgG4 isotype.

5. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient.

6. The conjugate of claim 1, wherein the linker comprises PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB or Ala-PAB.

7. The conjugate of claim 1, wherein the linker comprises -(CH2) n - unit, wherein n is an integer from 2 to 6.

8. The conjugate of claim 1, wherein the linker L and the chemical moiety Z together are LZ, 9. The conjugate of claim 1, wherein Ab-ZL-Am is one of:

10. The conjugate of claim 1, wherein Ab-ZL-Am is one of:

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