T-cell necrosis therapy

Abstract

T cell-depleting therapy [Solution] Provided herein are methods for depleting T cells for therapeutic use, including administering therapeutic anti-CD2 or anti-CD5 antibody-drug conjugates (ADCs). Provided are anti-CD2 ADCs or anti-CD5 ADCs for use as drugs to treat stem cell disorders, cancer, or autoimmune diseases, among other hematological and proliferative disorders. The described compositions and methods can be used to deplete populations of CD2+ or CD5+ cells, such as CD2+ or CD5+ cancer cells or CD2+ or CD5+ immune cells, and can also be used to prepare patients for hematopoietic stem cell transplantation or solid organ transplantation.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 857,744, filed June 5, 2019, the contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a sequence listing submitted electronically via EFS-Web in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on June 3, 2020, is named M103034_2170WO_SL.txt and is 91,817 bytes in size. [Background technology]

[0003] T cells are a type of lymphocyte that develop in the thymus gland and play a key role in immune responses. Although T cells are an important part of the immune system, abnormal T cell activity can cause disease in patients. For example, graft-versus-host disease (GVHD) is primarily caused by donor T cells within the graft, which trigger an immune response that damages host tissues. Other examples of disease-causing abnormal T cell activity include T cell-related cancers such as T cell lymphoma. Targeting T cell-based therapies remains a challenge in the art. Summary of the Invention [Problem to be solved by the invention]

[0004] Currently, there is a need for T cell-targeting compositions and methods for treating disorders of the hematopoietic system, such as autoimmune diseases, as well as compositions and methods that promote engraftment of xenogeneic hematopoietic stem cell grafts so that the pluripotency and hematopoietic function of these cells is maintained after transplantation.

[0005] Provided herein are T cell targeting compositions and methods for directly treating, inter alia, various disorders of the hematopoietic system, metabolic disorders, cancer, 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 cancer or autoimmune disease. [Means for solving the problem]

[0006] In one aspect, provided herein is a method of depleting T cells in a subject suffering from an autoimmune disease, the method comprising administering to the subject suffering from the autoimmune disease an anti-CD5 antibody drug conjugate (ADC) or an anti-CD2 ADC, wherein the ADC comprises an anti-CD5 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody or antigen-binding fragment thereof conjugated to a cytotoxin via a linker.

[0007] In one embodiment, the effective amount is an amount sufficient to substantially deplete endogenous CD5+ or CD2+ T cells in the subject's thymus.

[0008] In one embodiment, the subject has multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus (SLE), or systemic sclerosis (SSc).

[0009] In another aspect, provided herein is a method of treating a subject suffering from or at risk of developing steroid-resistant graft-versus-host disease (GVHD), the method comprising administering an anti-CD2 ADC or an anti-CD5 ADC to a subject suffering from steroid-resistant GVHD, such that the steroid-resistant GVHD is treated, wherein the ADC comprises an anti-CD5 antibody, or an antigen-binding fragment thereof, or an anti-CD2 antibody, or an antigen-binding fragment thereof, conjugated to a cytotoxin via a linker.

[0010] In one embodiment, the steroid-resistant GVHD is steroid-resistant acute GVHD.

[0011] In one embodiment, the subject has previously undergone an allogeneic HSC transplant.

[0012] In one embodiment, the subject has steroid-resistant acute GVHD grade 2 to grade 4 (Mount Sinai Acute GVHD International Consortium (MAGIC) criteria). In one embodiment, after administration of the anti-CD2 ADC or anti-CD5 ADC, the GVHD grade is reduced by one grade according to the MAGIC criteria.

[0013] In other aspects, provided herein are methods of treating a subject suffering from a T-cell malignancy, the method comprising administering to the subject an effective amount of an anti-CD2 ADC or an anti-CD5 ADC, wherein the ADC comprises an anti-CD5 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody, or antigen-binding fragment thereof, conjugated to a cytotoxin via a linker.

[0014] In one embodiment, the T cell malignancy is lymphoma. In a specific embodiment, the T cell malignancy is T cell acute lymphoblastic lymphoma (T-ALL), T cell large granular lymphocytic (LGL) leukemia, human T cell leukemia virus type 1 positive (HTLV-1+), adult T cell leukemia / lymphoma (ATL), T cell prolymphocytic leukemia (T-PLL), or peripheral T cell lymphoma (PTCL).

[0015] In one embodiment, the T cell malignancy is a relapsed and refractory T cell malignancy.

[0016] In one embodiment, the ADC comprises a humanized or human antibody. In one embodiment, the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE. In a specific embodiment, the IgG isotype is IgG1 or IgG4.

[0017] In some embodiments, the ADC is an anti-CD5 ADC (eg, 5D7 conjugated to an amatoxin).

[0018] In some embodiments, the ADC is an anti-CD2 ADC.

[0019] In one aspect, described herein are T cell targeting compositions and methods for conditioning a patient, such as a human patient, prior to undergoing hematopoietic stem cell transplantation therapy to promote engraftment of the hematopoietic stem cell graft. The patient may be suffering from one or more blood disorders, such as an autoimmune disease, cancer, hemoglobinopathy, or other hematopoietic condition, and therefore in need of a hematopoietic stem cell transplant.

[0020] As described herein, hematopoietic stem cells can be differentiated into multiple cell types in the hematopoietic lineage and can be administered to a patient to expand or repopulate cell types that are deficient in the patient.

[0021] In certain embodiments, the compositions described herein are antibody-drug conjugates (ADCs) that bind to T cells, specifically CD2 or CD5, and feature methods in which the compositions are administered to a patient to (i) directly treat hematologic disorders, such as autoimmune diseases, by selectively depleting immune cell populations that express CD2 or CD5, such as autoreactive T cells, B cells, and natural killer (NK) cells, and / or (ii) deplete populations of T cells, B cells, or NK cells prior to administering a hematopoietic stem cell transplant to the patient, thereby reducing the likelihood of hematopoietic stem cell graft rejection. The activity of CD2 or CD5 allows for the direct treatment of a wide range of autoimmune diseases, as these antigens can be expressed by T cells, B cells, or NK cells that cross-react with and mount inappropriate immune responses against self-antigens. In this case, administration of an anti-CD2 antibody-drug conjugate or an anti-CD5 antibody-drug conjugate to a patient causes depletion of a population of CD2+ (or CD5+) autoimmune cells, such as T cells, B cells, or NK cells, that cross-react with one or more self-antigens, thereby treating the autoimmune disease. This latter activity promotes the creation of an environment conducive to hematopoietic stem cell engraftment, because T cells, B cells, and / or NK cells that cross-react with one or more non-self antigens (e.g., non-self MHC antigens) expressed by hematopoietic stem cells can mount an immune response against the transplanted hematopoietic stem cells, thereby promoting graft rejection. In this latter case, a patient suffering from a disorder such as cancer, an autoimmune disease, or another condition of the hematopoietic system can subsequently receive a hematopoietic stem cell transplant, for example, to repopulate one or more populations of blood cells that are defective or depleted in the patient. Also provided herein are methods for treating various hematopoietic disorders, such as sickle cell anemia, thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, adenosine deaminase deficiency-severe combined immunodeficiency, metachromatic leukodystrophy, Diamond-Blackfan anemia and Shwachman-Diamond syndrome, human immunodeficiency virus infection, and acquired immunodeficiency syndrome, as well as cancer and autoimmune diseases.

[0022] In certain embodiments, described herein are methods of depleting T cells in a subject in need thereof, the method comprising administering an effective amount of an anti-CD5 or anti-CD2 antibody drug conjugate (ADC) to a subject in need thereof, wherein the subject is undergoing or prior to undergoing a hematopoietic stem cell (HSC) transplant or a solid organ transplant, and the ADC comprises an anti-CD5 or anti-CD2 antibody conjugated to a cytotoxin via a linker.

[0023] In one embodiment, when an effective amount of an anti-CD5 or anti-CD2 antibody drug conjugate (ADC) is administered to a subject in need thereof prior to receiving a hematopoietic stem cell (HSC) transplant or solid organ transplant, the method further comprises administering the HSC graft or solid organ transplant to the subject.

[0024] In one embodiment, the HSC transplant is an autologous HSC transplant.

[0025] In one embodiment, the HSC graft is administered to the subject after the level of ADC is substantially evident in the subject's blood.

[0026] In one embodiment, the HSCs or solid organ graft are administered to the subject between 1 hour and 7 days after the level of ADC becomes substantially evident in the subject's blood. In another embodiment, the HSCs or solid organ graft are administered to the subject between 6 hours and 3 days after the level of ADC becomes substantially evident in the subject's blood. In yet another embodiment, the HSCs or solid organ graft are administered to the subject between 12 hours and 36 days after the level of ADC becomes substantially evident in the subject's blood.

[0027] In one embodiment, the HSCs or solid organ transplant are administered to the subject about 24 hours after levels of the ADC are substantially evident in the subject's blood, and the ADC comprises an anti-CD5 or anti-CD2 antibody conjugated to a cytotoxin via a linker.

[0028] In a particular aspect, provided herein is a method for depleting T cells in a subject suffering from an autoimmune disease, the method comprising administering to the subject suffering from the autoimmune disease an effective amount of an anti-CD5 or anti-CD2 antibody-drug conjugate (ADC), wherein the ADC comprises an anti-CD5 or anti-CD2 antibody conjugated to a cytotoxin via a linker. In one embodiment, the subject suffers from multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus (SLE), or systemic sclerosis (SSc).

[0029] In one embodiment, the effective amount is an amount sufficient to substantially deplete endogenous CD5+ or CD2+ T cells in the subject's thymus.

[0030] In another aspect, provided herein is a method for treating a subject suffering from steroid-resistant graft-versus-host disease (GVHD), the method comprising administering an anti-CD2 or anti-CD5 ADC to a subject suffering from steroid-resistant GVHD to treat the steroid-resistant GVHD, wherein the ADC comprises an anti-CD5 or anti-CD2 antibody conjugated to a cytotoxin via a linker. In one embodiment, the steroid-resistant GVHD is steroid-resistant acute GVHD. In one embodiment, the subject has previously undergone allogeneic HSC transplantation. In one embodiment, the subject has steroid-resistant acute GVHD grade 2 to 4 (Mount Sinai Acute GVHD International Consortium (MAGIC) criteria). In one embodiment, after administration of the anti-CD2 or anti-CD5 ADC, the GVHD grade is reduced by one grade according to the MAGIC criteria.

[0031] In a further aspect, provided herein is a method of treating a subject suffering from a T-cell malignancy, the method comprising administering to the subject an effective amount of an anti-CD2 or anti-CD5 ADC, wherein the ADC comprises an anti-CD5 or anti-CD2 antibody conjugated to a cytotoxin via a linker. In one embodiment, the T-cell malignancy is lymphoma. In one embodiment, the T-cell malignancy is T-cell acute lymphoblastic lymphoma (T-ALL), T-cell large granular lymphocytic (LGL) leukemia, human T-cell leukemia virus type 1 positive (HTLV-1+), adult T-cell leukemia-lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), or peripheral T-cell lymphoma (PTCL). In one embodiment, the T-cell malignancy is a relapsed or refractory T-cell malignancy.

[0032] In one embodiment, the ADC comprises a humanized or human antibody.

[0033] In another embodiment, the antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgD and IgE, hi one embodiment, the IgG isotype is IgG1 or IgG4.

[0034] In one embodiment, the ADC is an anti-CD5 ADC.

[0035] In one embodiment, the ADC is an anti-CD2 ADC.

[0036] In one embodiment, the cytotoxin is selected from the group consisting of Pseudomonas exotoxin A, deboganin, diphtheria toxin, amatoxin, saporin, maytansine, maytansinoid, auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an indolinobenzodiazepine, or an indolinobenzodiazepine dimer.

[0037] In one embodiment, the cytotoxin is an RNA polymerase inhibitor. In one embodiment, the RNA polymerase inhibitor is an RNA polymerase II inhibitor. In one embodiment, the RNA polymerase inhibitor is an amatoxin.

[0038] In one embodiment, the ADC is represented by the formula Ab-ZL-Am, where Ab is An anti-CD5 or anti-CD2 antibody or antigen-binding fragment thereof, wherein L is a linker, Z is a chemical moiety, and Am is an amatoxin, and is represented by formula (I): [ka] (I) In the formula, R1 is H, OH, OR A , or OR C and R2 is H, OH, OR B , or OR C and R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C , or R D and R4, R5, R 6、 R7 is independently H, OH, or OR C , OR D , R C , R D and R8 is OH, NH2, OR C , OR D , NHR C or NR C R D can be, R9 is H, OH, OR C OR D and X is -S-, -S(O)-, or -SO2-; R C is -LZ, R Dis 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 an optionally substituted C1-C6 alkylene, an optionally substituted C1-C6 heteroalkylene, an optionally substituted C2-C6 alkenylene, an optionally substituted C2-C6 heteroalkenylene, an optionally substituted C2-C6 alkynylene, an optionally substituted C2-C6 heteroalkynylene, an optionally substituted cycloalkylene, an optionally substituted heterocycloalkylene, an optionally substituted arylene, an optionally substituted heteroarylene, a dipeptide, -C(=O)-, a peptide, or a combination thereof; Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an antibody or antigen-binding fragment thereof; Am contains exactly one RC substituent.

[0039] In one embodiment, Am-LZ is represented by formula (IA). [ka] In the formula, R1 is H, OH, OR A , or OR C and R2 is H, OH, OR B OR C and R A and R B when taken together with the oxygen atom to which they are attached, combine to form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C or R D and R4, R5, R6 and R7 are each independently H, OH, OR C, OR D , R C , or R D and R8 is OH, NH2, OR C , OR D , NHR C , or NR C R D and R9 is H, OH, OR C OR D and X is -S-, -S(O)- or -SO2-; R C is -LZ, R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroaromatic alkyl, 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 an optionally substituted C1-C6 alkylene, an optionally substituted C1-C6 heteroalkylene, an optionally substituted C2-C6 alkenylene, an optionally substituted C2-C6 heteroalkenylene, an optionally substituted C2-C6 alkynylene, an optionally substituted C2-C6 heteroalkynylene, an optionally substituted cycloalkylene, an optionally substituted heterocycloalkylene, an optionally substituted arylene, an optionally substituted heteroarylene, a dipeptide, -C(=O)-, a peptide, or a combination thereof; Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an antibody or antigen-binding fragment thereof; Am contains exactly one RC substituent.

[0040] In one embodiment, Am-LZ is represented by formula (IB). [ka] In the formula, R1 is H, OH, OR A , or OR C and R2 is H, OH, OR B OR C and R A and R B when taken together with the oxygen atom to which they are attached, combine to form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C or R D and R4, R5, R 6、 R7 is independently H, OH, or OR C , OR D , R C , R D and R8 is OH, NH2, OR C , OR D , NHR C or NR C R D and R9 is H, OH, OR C OR D and X is -S-, -S(O)- or -SO2-; R C is -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 an optionally substituted C1-C6 alkylene, an optionally substituted C1-C6 heteroalkylene, an optionally substituted C2-C6 alkenylene, an optionally substituted C2-C6 heteroalkenylene, an optionally substituted C2-C6 alkynylene, an optionally substituted C2-C6 heteroalkynylene, an optionally substituted cycloalkylene, an optionally substituted heterocycloalkylene, an optionally substituted arylene, an optionally substituted heteroarylene, a dipeptide, -C(=O)-, a peptide, or a combination thereof; Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an antibody or antigen-binding fragment thereof; Am contains exactly one Rc substituent.

[0041] In one embodiment, the ADC is represented by Ab-ZL-Am, where Ab is an antibody or antigenic fragment thereof, Z is a chemical moiety, L is a linker, and Am is an amatoxin, and the amatoxin-linker conjugate Am-LZ is represented by Formula (II), Formula (IIA), or Formula (IIB). [ka] wherein X is S, SO, or SO; R1 is H or a linker covalently attached to the antibody or antigen-binding fragment thereof via the chemical moiety Z formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the antibody or antigen-binding fragment thereof; R2 is H or a linker covalently attached to the antibody or antigen-binding fragment thereof via the chemical moiety Z formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the antibody or antigen-binding fragment thereof; When R1 is H, R2 is a linker, and when R2 is H, R1 is a linker.

[0042] In one embodiment, the cytotoxin of the ADC is a maytansinoid, such as DM1 or DM4.

[0043] In one embodiment, the cytotoxin of the ADC is an auristatin, such as monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF).

[0044] In one embodiment, the cytotoxin of the ADC is an anthracycline, such as daunorubicin, doxorubicin, epirubicin, or idarubicin.

[0045] In one embodiment, the cytotoxin of the ADC is a pyrrolobenzodiazepine dimeric derivative represented by formula (IV). [ka]

[0046] In one embodiment, the ADC is internalized by CD5+ or CD2+ immune cells after administration to a patient.

[0047] In one embodiment, the subject is a human.

[0048] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:288 and a light chain variable region comprising SEQ ID NO:289.

[0049] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:291 and a light chain variable region comprising SEQ ID NO:290. [Brief explanation of the drawings]

[0050] [Figure 1]1 is a graphical representation of the results of an in vitro cell line binding assay in which each of the indicated anti-CD2 antibodies or a negative control (i.e., mIgG1) was cultured with MOLT-4 cells (i.e., a human T lymphoblastoid cell line) followed by culture with a fluorochrome-conjugated anti-IgG antibody; the signal was detected by flow cytometry and is shown as geometric mean fluorescence intensity (y-axis) as a function of anti-CD2 antibody concentration (x-axis). [Figure 2] Graphical representation of the results of an in vitro primary cell line binding assay in which the indicated anti-CD2 antibody (RPA-2.10) or negative control (i.e., mIgG1) was incubated with primary human T cell lines followed by incubation with a fluorochrome-conjugated anti-IgG antibody. Signal was detected by flow cytometry and is shown as geometric mean fluorescence intensity (y-axis) as a function of anti-CD2 antibody concentration (x-axis). [Figure 3] Figures 3A and 3B graphically depict the results of in vitro T cell killing assays involving anti-CD2 amanitin ADCs (i.e., RPA-2.10-AM or "CD2AM") bearing interchain-conjugated amanitin at an average drug-to-antibody ratio of 6 (Figure 3A) or a site-specifically conjugated amanitin drug-to-antibody ratio of 2 (Figure 3B). In Figure 3A, anti-CD2 ADC cell killing analysis is shown compared to an unconjugated anti-CD2 antibody (i.e., "CD2 naked"). In Figure 3B, anti-CD2 antibody results are shown compared to an anti-CD2 antibody with the H435A mutation, which shortens the antibody's half-life. Results are shown as the number of viable T cells (y-axis) as a function of ADC (CD2 RPA-2.10 AM, CD2 D265C.H435A AM) and unconjugated antibody (CD2RPA-2.10) concentration (x-axis), assessed using flow cytometry. [Figure 4]1 is a graphical representation of the results of an in vitro natural killer (NK) cell killing assay involving an anti-CD2 amanitin ADC (i.e., RPA-2.10-AM or "CD2AM") having an interchain conjugated amanitin at a drug-to-antibody ratio of 6. Results are shown as viable NK cell levels (y-axis) as a function of ADC (CD2-AM) or control antibody (i.e., hIgG1, hIgG1-amanitin ("hIgG1-AM")) concentration (x-axis) assessed using the CellTiter Glo assay. [Figure 5] Figures 5A and 5B graphically depict the results of an in vivo T cell depletion assay showing the absolute amount of T cells (CD3+ cells, y-axis) in peripheral blood (Figure 5A) and bone marrow (Figure 5B) 7 days after a single dose of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of anti-CD2-amanitin ADC (RPA-2.10-AM) with an interchain drug-to-antibody ratio of 6. For comparison, Figures 5A and 5B also show the level of T cell depletion after treatment of humanized NSG mice with 25 mg / kg Ab1 (unconjugated anti-CD2 antibody) or the indicated controls (i.e., 25 mg / kg anti-CD52 antibody (clone YTH34.5), 3 mg / kg hIgG1-amanitin ADC ("hIgG1-AM"), 25 mg / kg hIgG1, or PBS). [Figure 6] Figures 6A-6C graphically depict the results of an in vivo T cell depletion assay showing the absolute levels of T cells (CD3+ cells, y-axis) in the peripheral blood (Figure 6A), bone marrow (Figure 6B), and thymus (Figure 6C) of humanized NSG mice 7 days after a single dose of an anti-CD2-amanitin ADC (i.e., RPA-2.10-AM) with a site-specific drug-to-antibody ratio of approximately 2. For comparison, Figures 6A-6C also show the levels of T cell depletion after treatment of humanized NSG mice with 3 mg / kg of unconjugated anti-CD2 antibody or the indicated controls (i.e., 3 mg / kg hIgG1-amanitin ADC ("hIgG1-AM") or PBS). [Figure 7]1 is a graphical representation of 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 cultured with MOLT-4 cells (i.e., a human T lymphoblast cell line) followed by culture with a fluorochrome-conjugated anti-IgG antibody; the signal was detected by flow cytometry and is shown as geometric mean fluorescence intensity (y-axis) as a function of anti-CD5 antibody concentration (x-axis). [Figure 8] 1 is a graphical representation of the results of an in vitro primary cell binding assay in which each of the indicated anti-CD5 antibodies or a negative control (i.e., hIgG1) was cultured with primary human T cells followed by incubation with a fluorochrome-conjugated anti-IgG antibody; the signal was detected by flow cytometry and is shown as geometric mean fluorescence intensity (y-axis) as a function of anti-CD5 antibody concentration (x-axis). [Figure 9] 9A-9B are graphical representations of the results of in vitro T cell killing assays involving anti-CD5 amanitin ADCs (i.e., 5D7-AM or "CD5 AM") with interchain-conjugated amanitin at an average drug-to-antibody ratio (DAR) of 6 (FIG. 9A) or site-specifically conjugated amanitin at a DAR of 2. In FIG. 9A, anti-CD5-ADC cell killing analysis is shown compared to unconjugated anti-CD5 5D7 antibody (i.e., "CD5 Naked"). In FIG. 9B, anti-CD5 antibody results are shown compared to an anti-CD5 5D7 antibody (i.e., "CD5 Fast ½ Life AM") with the H435A mutation, which reduces the half-life of the antibody (i.e., results show the number of viable T cells (y-axis) as a function of ADC (CD5 5D7 AM, CD5 5D7 D265C.H435A AM) or unconjugated antibody (CD5 5D7) concentration (x-axis), as assessed using flow cytometry. [Figure 10]Figure 10A shows graphical representations of the results of an in vivo T cell depletion assay demonstrating the absolute levels of T cells (CD3+ cells, y-axis) in the peripheral blood (Figure 10A) and bone marrow (Figure 10B) of humanized NSG mice 7 days after administration of a single dose of 0.3 mg / kg, 1 mg / kg, or 3 mg / kg of the interchain DAR6 anti-CD5 5D7-amanitin ADC (i.e., CD5 5D7-AM). For comparison, Figures 10A and 10B also show the levels of T cell depletion after treatment of humanized NSG mice with the indicated controls (i.e., 25 mg / kg anti-CD52 antibody, 3 mg / kg hIgG1-amanitin ADC (i.e., hIgG1-AM), 25 mg / kg hIgG1, or PBS). [Figure 11] Figure 11A-C graphically depicts the results of an in vivo T cell depletion assay showing the absolute levels of T cells (CD3+ cells, y-axis) in the peripheral blood (Figure 11A) and bone marrow (Figure 11B) of humanized NSG mice 7 days after a single dose of 1 mg / kg or 3 mg / kg of site-specific DAR2 anti-CD5 5D7 amanitin ADC (i.e., 5D7-AM). For comparison, Figures 11A-C also show the levels of T cell depletion after treatment of humanized NSG mice with 3 mg / kg of unconjugated anti-CD5 antibody or the indicated controls (i.e., 3 mg / kg hIgG1-amanitin ADC (hIgG1-AM or PBS)). [Figure 12]Figures 12A and 12B graphically depict the results of the depletion assays, demonstrating that both anti-CD5 ADC (CD5-AM) and anti-CD2 ADC (CD2-AM) can deplete Th1 and Th17 cell subsets in a polarized state. Figure 12A shows that both anti-CD5 ADC (CD5-AM) and anti-CD2 ADC (CD2-AM) were able to deplete Th1 cells with an IC50 of 2.73 pM, indicating that IFNγ signaling was downregulated, whereas the isotype control antibody was unable to deplete Th1 cells. Similarly, Figure 12B shows that both anti-CD5 ADC (CD5-AM) and anti-CD2 ADC (CD2-AM) were able to deplete Th1 cells with an IC50 of 2.73 pM, indicating that IL-17 signaling was downregulated, whereas the isotype control antibody was unable to deplete Th17 cells. [Figure 13] Figures 13A-13D show the results of in vivo survival studies, demonstrating that anti-CD5 ADC (CD5-AM) extends survival in patients with T-cell acute lymphoblastic leukemia. As shown in Figures 13A and 13C, anti-CD5 ADC (CD5-AM) or anti-CD2 ADC (CD2-AM) each extended survival by more than 20 days compared to the control group. Furthermore, the survival rate following a single administration of anti-CD5 ADC (CD5-AM) or anti-CD2 ADC (CD2-AM) appears to be comparable to that of a commercially available chemotherapy agent (Ara-C). As shown in Figures 13B and 13D, CD5 ADC (CD5-AM) or anti-CD2 ADC (CD2-AM) each reduced tumor burden in mice compared to the isotype and control groups. [Figure 14] Figures 14A and 14B graphically depict the results of in vivo studies, demonstrating that anti-CD5 ADCs prevent acute GvHD in a xenoantigen model. As shown in Figure 14A, mice treated with anti-CD5 ADCs (CD5-AM) experienced slight weight loss, but recovered by day 13 post-transplant. As shown in Figure 14B, anti-CD5 ADCs (CD5-AM) resulted in 80% sustained survival in this model. DETAILED DESCRIPTION OF THE INVENTION

[0051] The compositions and methods described herein are based, in part, on the discovery that antibody-drug conjugates (ADCs) that bind to CD2 (also known as T-cell surface antigens, LFA-2 and LFA-3 receptors) or CD5 (lymphocyte antigen T1 / Leu-1) can be used as therapeutic agents to (i) directly treat cancers and autoimmune diseases characterized by CD2+ or CD5+ cells, and (ii) promote engraftment of transplanted hematopoietic stem cells in patients requiring transplantation therapy by depleting populations of immune cells that cross-react with and initiate immune responses against the hematopoietic stem cell graft (e.g., by cross-reacting with non-self MHC antigens expressed by the hematopoietic stem cell graft). These therapeutic activities can occur, for example, by the binding of anti-CD2 or anti-CD5 antibody-drug conjugates to cancer cells, autoimmune cells, or immune cells that cross-react with non-self hematopoietic stem cell antigens (e.g., non-self MHC antigens), thereby inducing the death of the bound cells. When depleting populations of cancer cells or autoimmune cells, anti-CD2 ADCs or anti-CD5 ADCs can be used to directly treat autoimmune diseases, such as cancer or cancer-associated autoimmune diseases, as described herein. When depleting populations of immune cells that cross-react with non-self hematopoietic stem cell antigens, anti-CD2 ADCs or anti-CD5 ADCs can be used to prevent or reduce the likelihood of graft rejection in patients suffering from stem cell disorders, cancer, or autoimmune diseases and undergoing hematopoietic stem cell transplantation therapy. In such cases, depletion of CD2+ or 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) can enable successful engraftment of transplanted hematopoietic stem cells within the transplant recipient. As the transplanted cells engraft, they home to hematopoietic tissues, where they undergo productive hematopoiesis. The transplanted hematopoietic stem cells may subsequently give rise to populations of cells that are deficient in the transplant recipient, such as megakaryocytes, platelets, thrombocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.In this manner, anti-CD2 or anti-CD5 ADCs can be used to promote successful engraftment of hematopoietic stem cells in patients, such as human patients suffering from the stem cell disorders described herein.

[0052] definition As used herein, the term "about" refers to a value 5% higher or lower than the stated value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

[0053] As used herein, the term "amatoxin" refers to a member of the amatoxin family of peptides produced by mushrooms, a synthetic amatoxin, a mutant amatoxin, or a derivative thereof, such as a mutant or derivative thereof capable of inhibiting RNA polymerase II activity. Also included are synthetic amatoxins (see U.S. Patent No. 9,676,702, incorporated herein by reference). As described herein, amatoxins can be conjugated to antibodies or antigen-binding fragments thereof, for example, via a linker moiety (L) (thus forming a conjugate (also called an antibody-drug conjugate (ADC))). Exemplary methods of amatoxin conjugation and linkers useful in such processes are provided below and are known in the art. Exemplary linker-containing amatoxins useful for conjugation to antibodies or antigen-binding fragments according to compositions and methods are also described herein.

[0054] In certain embodiments, amatoxins useful with the compositions and methods described herein include compounds according to formula (III): α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, or proamanitin. Formula (III): [ka] and In the formula, R1 is H, OH, or OR A and R2 is H, OH, or ORB and R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D and R4 is H, OH, OR D or R D and R5 is H, OH, OR D or R D and R6 is H, OH, OR D or R D and R7 is H, OH, OR D or R D and R8 is OH, NH2 or OR D and R9 is H, OH or OR D and X is -S-, -S(O)-, or -SO2-; R D is an 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, or peptide.

[0055] For example, in one embodiment, amatoxins useful in conjunction with the compositions and methods described herein include compounds according to formula (IIIA): [ka] wherein R1 is H, OH, or OR Aand R2 is H, OH, or OR B and R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D and R4 is H, OH, OR D or R D and R5 is H, OH, OR D or R D and R6 is H, OH, OR D or R D and R7 is H, OH, OR D or R D and R8 is OH, NH2, or OR D and R9 is H, OH or OR D and X is -S-, -S(O)-, or -SO2-; R D is an optionally substituted alkyl (e.g., C-C alkyl), optionally substituted heteroalkyl (e.g., C-C heteroalkyl), optionally substituted alkenyl (e.g., C-C alkenyl), optionally substituted heteroalkenyl (e.g., C-C heteroalkenyl), optionally substituted alkynyl (e.g., C-C alkynyl), optionally substituted heteroalkynyl (e.g., C-C heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, or peptide.

[0056] In one embodiment, amatoxins useful in conjunction with the compositions and methods described herein include compounds according to formula (IIIB): [ka] wherein R1 is H, OH, or OR A and R2 is H, OH, or OR B and R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R3 is H or R D and R4 is H, OH, OR D or R D and R5 is H, OH, OR D or R D and R6 is H, OH, OR D or R D and R7 is H, OH, OR D or R D and R8 is OH, NH2 or OR D and R9 is H, OH or OR D and X is -S-, -S(O)-, or -SO2-; R D is an 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, or peptide.

[0057] As described herein, amatoxins can be attached to antibodies or antigen-binding fragments thereof, for example, via a linker moiety. Exemplary methods of amatoxin attachment and linkers useful for such processes are described in the section entitled "Linkers for Chemical Conjugation" and in Table 2 below. Exemplary linker-containing amatoxins useful for anti-CD2 antibodies, or antigen-binding fragments, or anti-CD5 antibodies, or antigen-binding fragments thereof, according to the compositions and methods described herein are shown in structural formulas (I), (IA), (IB), (II), (IIA), and (IIB) shown herein.

[0058] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen. Examples of antibodies include monoclonal and modified forms of antibodies, including, but not limited to, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi-, tri-, and tetra-specific antibodies, diabodies, triabodies), and antigen-binding fragments of antibodies (e.g., including Fab', F(ab')2, Fab, Fv, rlgG, and scFv fragments). The term "antibody" (Ab) is meant to include both intact molecules capable of specifically binding to a target protein, as well as antibody fragments thereof (e.g., including Fab and F(ab')2 fragments). As used herein, Fab and F(ab')2 fragments refer to antibody fragments that lack the Fc fragment of an intact antibody. Examples of these antibody fragments are described herein.

[0059] Generally, antibodies comprise heavy and light chains containing antigen-binding regions. Each heavy chain is composed of a heavy chain variable region (abbreviated herein 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 (abbreviated herein 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 divided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0060] As used herein, antibodies are generally isolated or recombinant. As used herein, "isolated" refers to a polypeptide, e.g., an antibody, that has been separated and / or recovered from the cell or cell culture in which it is expressed. Thus, an "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities. For example, an isolated antibody that specifically binds to CD2 or CD5 is substantially free of antibodies that specifically bind to antigens other than CD2 or CD5, respectively.

[0061] The term "antigen-binding fragment," as used herein, 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. The antibody fragment can be, for example, an Fv, Fab, Fab', F(ab')2, scFv, diabody, triabody, single-chain antibody molecule (such as scFv), affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb comprising the VH and VL domains; (vi) a dAb fragment consisting of the VH domain (see, e.g., Ward et al., Nature 341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) an isolated complementarity-determining region (CDR); and (ix) a combination of two or more (e.g., 2, 3, 4, 5, or 6) isolated CDRs, optionally linked by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a linker that allows them to be produced recombinantly as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv), see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988).These antibody fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments can be screened for utility in the same manner as are intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, by enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis procedures known in the art.

[0062] As used herein, the term "anti-CD2 antibody" or "antibody that binds to CD2" refers to an antibody that specifically binds to CD2. An antibody that "binds" an antigen of interest, i.e., CD2, is one that can bind to that antigen with sufficient affinity so that the antibody is useful for targeting cells that express the antigen. In a preferred embodiment, the antibody specifically binds to human CD2 (hCD2). CD2 is found on the cell surface of immune cells, such as T cells. The amino acid sequence of human CD2 to which an anti-CD2 antibody (or anti-CD2 conjugate) binds is set forth below in SEQ ID NO: 13. An "anti-CD2 antibody-drug conjugate" or "anti-CD2 ADC" refers to an ADC comprising an anti-CD2 antibody.

[0063] As used herein, the terms "anti-CD5 antibody" or "antibody that binds to CD5" refer to an antibody that specifically binds to CD5. An antibody that "binds" an antigen of interest, i.e., CD5, is one that can bind to that antigen with sufficient affinity so that the antibody is useful for targeting cells that express the antigen. In a preferred embodiment, the antibody specifically binds to human CD5 (hCD5), the amino acid sequence of which is set forth below in SEQ ID NO: 286. An "anti-CD5 antibody drug conjugate" or "anti-CD5 ADC" refers to an ADC comprising an anti-CD5 antibody.

[0064] As used herein, the term "bispecific antibody" refers to a hybrid antibody having two different antigen-binding sites. Bispecific antibodies are a type of multispecific antibody and can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking 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 bind to two different epitopes, which may reside on the same or different protein targets. For example, one binding specificity can be directed against a T cell surface antigen such as CD2, and the other can be directed against 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 enhances cell proliferation, among others.

[0065] As used herein, the term "complementarity-determining region" (CDR) refers to the hypervariable regions found in both the light and heavy chain variable domains of an antibody. The more highly conserved portions of the variable domains are called framework regions (FRs). The amino acid positions delineating the hypervariable regions of an antibody can vary depending on the context and the various definitions known in the art. Some positions within a variable domain can be considered hybrid hypervariable positions, in that these positions can be considered within a hypervariable region under some criteria, but outside of a hypervariable region under other criteria. One or more of these positions can also be found in an extended hypervariable region. The antibodies described herein can contain modifications at these hybrid hypervariable positions. Native heavy and light chain variable domains each contain four framework regions that primarily adopt a β-sheet structure, connected by three CDRs, which form loops that span, and in some cases, form part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by framework regions, in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and, together with the CDRs from other antibody chains, contribute to the formation of the antibody target binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987). As used herein, immunoglobulin amino acid residue numbering is done according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified.

[0066] As used herein, the terms "conditioning" and "conditioning" refer to the process of preparing a patient to receive a graft containing hematopoietic stem cells. Such a procedure promotes hematopoietic stem cell transplant engraftment (e.g., as inferred from a sustained increase in the amount of viable hematopoietic stem cells in a blood sample isolated from the patient after the conditioning procedure and subsequent hematopoietic stem cell transplant). In the methods described herein, a patient can be conditioned for hematopoietic stem cell transplantation therapy by administering to the patient an antibody or antigen-binding fragment thereof capable of binding to an antigen expressed by T cells, such as CD2 or CD5. As described herein, an anti-CD2 or anti-CD5 antibody can be covalently conjugated to a cytotoxin to form an antibody-drug conjugate (ADC). Administration of an antibody, antigen-binding fragment thereof, or antibody-drug conjugate capable of binding to one or more of the above-mentioned antigens to a patient in need of hematopoietic stem cell transplantation therapy can promote hematopoietic stem cell graft engraftment by selectively depleting endogenous immune cells, such as CD2+ T cells (such as CD4+ and / or CD8+ T cells) and / or CD2+ NK cells or CD5+ NK cells (such as CD4+ and / or CD8+ T cells), that cross-react with one or more non-self antigens (e.g., one or more non-self MHC antigens) expressed by hematopoietic stem cells. This selective depletion of immune cells prevents or reduces the likelihood of graft rejection after transplantation of an exogenous (e.g., autologous, allogeneic, or syngeneic) hematopoietic stem cell graft.

[0067] As used herein, the term "conjugate" refers to a compound formed by chemical bonding of a reactive functional group of one molecule, such as an antibody or antigen-binding fragment thereof, with a suitable reactive functional group of another molecule, such as a cytotoxin described herein. A conjugate may include a linker between the two molecules (e.g., an anti-CD2 antibody and a cytotoxin) attached to each other. Examples of linkers that can be used to form conjugates include peptide-containing linkers, such as those containing naturally occurring amino acids 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 moiety therein, the linker can be cleaved by, for example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012). It should be noted that the term "conjugate" (when referring to a compound) is also referred to interchangeably herein as "drug conjugate," "antibody drug conjugate," or "ADC."

[0068] 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 (e.g., covalently) the molecular fragments attached to each substituent. Coupling reactions include those in which a reactive substituent attached to a fragment that is a cytotoxin, such as a cytotoxin known in the art or described herein, reacts with an appropriately reactive substituent attached to an antibody, an antigen-binding fragment thereof, or a fragment that is an antibody, such as an antibody, an antigen-binding fragment thereof, or an antibody specific for CD2 or CD5 known in the art or described herein. Examples of appropriately reactive substituents include nucleophile / electrophile pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, among others), diene / dienophile pairs (e.g., azide / alkyne pairs, among others), 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, among others), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reaction modes known in the art or described herein.

[0069] As used herein, "CRU (competitive reconstituting unit)" refers to a measure of long-term engrafting stem cells that can be detected after in vivo transplantation.

[0070] As used herein, "drug-to-antibody ratio" or "DAR" refers to the number of cytotoxins (e.g., amatoxins) conjugated to the 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 binding sites on the antibody. Thus, in certain embodiments, the ADCs described herein have a DAR of 1, 2, 3, 4, 5, 6, 7, or 8.

[0071] As used herein, the term "donor" refers to a human or animal from which one or more cells are isolated prior to administration of the cells or their progeny to a recipient. The one or more cells may be, for example, a population of hematopoietic stem cells.

[0072] As used herein, the term "diabody" refers to a bivalent antibody containing two polypeptide chains, each of which contains a VH and a VL domain connected by a linker (e.g., a linker consisting of five amino acids) that is too short to allow intramolecular association of the VH and VL domains on the same peptide chain. This configuration allows each domain to pair with a complementary domain on another polypeptide chain to form a homodimeric structure. Accordingly, the term "triabody" refers to a trivalent antibody containing three peptide chains, each of which contains one VH and one VL domain connected by a linker (e.g., a linker consisting of one to two amino acids) that is too short to allow intramolecular association of the VH and VL domains on the same peptide chain. To fold into their native structure, peptides constructed in this manner typically trimerize to position the VH and VL domains of adjacent peptide chains in spatial proximity to one another (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993).

[0073] As used herein, a "dual variable domain immunoglobulin" ("DVD-Ig") refers to an antigen-binding protein that combines the target-binding variable domains of two antibodies via a linker to create a tetravalent, dual-targeting single agent (see, e.g., Gu et al., Meth. Enzymol., 502:25-41, 2012).

[0074] The term "effective amount" refers to the amount of a therapeutic agent, e.g., an anti-CD5 ADC or anti-CD2 ADC, needed to prevent or alleviate at least one sign or symptom of pain, and relates to an amount of a composition sufficient to provide the desired effect, e.g., for treating a subject with cancer. An effective amount also includes an amount sufficient to prevent or delay the onset of a disease symptom, alter the course of a disease symptom (e.g., slow the progression of a disease symptom), or reverse a disease symptom.

[0075] As used herein, the term "endogenous" refers to a substance, such as a molecule, cell, tissue, or organ, that is naturally found in a particular organism, such as a human patient, e.g., a human patient undergoing hematopoietic stem cell transplantation therapy described herein (e.g., a hematopoietic stem cell or cell of the hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, erythrocyte, mast cell, myeloblast, basophil, neutrophil, eosinophil, microglia, granulocyte, monocyte, osteoclast, antigen-presenting cell, macrophage, dendritic cell, natural killer cell, T lymphocyte (e.g., CD4+ or CD8+ T lymphocyte), or B lymphocyte).

[0076] As used herein, the term "engraftment potential" refers to the ability of hematopoietic stem and progenitor cells to reconstitute tissues, regardless of whether such cells are naturally circulating or provided by transplantation. This term encompasses all events surrounding or leading up to engraftment, such as tissue homing and colonization of the cells within the tissue of interest. Engraftment efficiency or rate can be assessed or quantified using any clinically acceptable parameter known to those skilled in the art, including, for example, assessment of competitive reconstituting units (CRUs), incorporation or expression of markers in the tissue(s) to which the stem cells have homed, colonized, or engrafted, or by assessing the progression of the subject by disease progression, hematopoietic stem and progenitor cell survival, or recipient survival. Engraftment can also be determined by measuring the number of white blood cells in peripheral blood during the post-transplant period. Engraftment can also be assessed by measuring bone marrow cell recovery by donor cells in a bone marrow aspirate sample.

[0077] As used herein, the term "excipient" refers to a substance that is formulated together with an active ingredient of a pharmaceutical product. They may be included, for example, for long-term stabilization or to provide a therapeutic enhancement to the active ingredient in the final dosage form.

[0078] As used herein, the term "exogenous" refers to a substance, such as a molecule, cell, tissue, or organ, that is not naturally found in a particular organism, such as a human patient (e.g., T cells, 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, or B lymphocytes, etc.). Exogenous materials include those provided to an organism from an external source or extracted therefrom for culture.

[0079] As used herein, the term "framework region" or "FW region" includes the amino acid residues that flank the CDRs of an antibody or antigen-binding fragment thereof. FW region residues may be present, for example, in human antibodies, humanized antibodies, monoclonal antibodies, antibody fragments, Fab fragments, single-chain antibody fragments, scFV fragments, antibody domains, and bispecific antibodies, among others.

[0080] The terms "full-length antibody" and "intact antibody" are used interchangeably to refer to an antibody in a substantially intact form, and do not refer to antibody fragments as defined herein. In one embodiment, an ADC described herein comprises an intact antibody, e.g., an anti-CD5 or anti-CD2 intact antibody. Thus, for an IgG antibody, an intact antibody comprises two heavy chains, each comprising a variable region, a constant region, and an Fc region, and two light chains, each comprising a variable region and a constant region. More specifically, an intact IgG comprises two light chains, each comprising a light chain variable region (VL) and a light chain ground region (CL), and two heavy chains, each comprising a heavy chain variable region (VH) and three heavy chain constant regions (CH1, CH2, and CH3). CH2 and CH3 represent the Fc region of the heavy chain.

[0081] As used herein, the term "hematopoietic stem cells" ("HSCs") refers to immature blood cells that have the ability to self-renew and differentiate into mature blood cells, including diverse lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Such cells may include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are believed to comprise a subpopulation of cells with the stem cell properties defined above, while in mice, HSCs are CD34-. HSCs are also referred to as long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). LT-HSCs and ST-HSCs are distinguished based on functional capacity and cell surface marker expression. For example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, and CD235A). In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamf1 / CD150+, CD48-, and lin- (negative for mature lineage markers, including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL7ra), whereas ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamf1 / CD150+, and lin- (negative for mature lineage markers, including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL7ra). Furthermore, ST-HSCs are less inactive and more proliferative than LT-HSCs under homeostatic conditions.However, LT-HSCs have greater self-renewal capacity (i.e., they survive through adulthood and can be serially transplanted through a series of recipients), while ST-HSCs have limited self-renewal (i.e., they survive for only a limited period of time and do not have serial transplantation capacity). Either of these HSCs can be used in the methods described herein. ST-HSCs are particularly useful because they are highly proliferative and therefore can give rise to differentiated progeny more quickly.

[0082] As used herein, the term "functional potential as a hematopoietic stem cell" refers to the functional properties of hematopoietic stem cells, including: 1) pluripotency (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, red blood cells), thrombocytes (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells)); 2) self-renewal (referring to the ability of hematopoietic stem cells to give rise to daughter cells with capabilities equivalent to those of the parent cell, which can occur repeatedly throughout an individual's lifetime without being depleted); 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 restore productive and sustained hematopoiesis.

[0083] As used herein, the term "major histocompatibility complex antigens" ("MHC," also referred to in the human context as "human leukocyte antigens" ("HLA")) refers to proteins expressed on the cell surface that confer a unique antigenic identity to the cell. MHC / HLA antigens are target molecules recognized by T cells and NK cells as originating from the same hematopoietic stem cell source as immune effector cells ("self") or from a different source of hematopoietic reconstituting cells ("non-self"). Two major classes of HLA antigens are recognized: HLA class I and HLA class II. HLA class I antigens (A, B, and C in humans) allow individual cells to be recognized as "self," while HLA class II antigens (DR, DP, and DQ in humans) are involved in reactions between lymphocytes and antigen-presenting cells. Both are involved in the rejection of transplanted organs. An important aspect of the HLA gene system is its polymorphism. Each gene, MHC class I (A, B, and C) and MHC class II (DP, DQ, and DR), exists in different alleles. HLA alleles are designated by a number and a subscript. For example, two unrelated individuals may each possess class I HLA-B, gene B5, and gene Bw41. The allele products differ in one or more amino acids in the α and / or β domains. Using a large panel of specific antibodies or nucleic acid reagents, leukocytes expressing class I and class II molecules are used to type an individual's HLA haplotype. Genes commonly used for HLA typing are the six MHC class I and class II proteins, with two alleles for each of HLA-A, HLA-B, and HLA-DR. HLA genes are clustered in a "superlocus" located at chromosome 6p21, which encodes six classical HLA genes and at least 132 protein-encoding genes that play important roles in regulating the immune system and several other fundamental molecular and cellular processes. The complete locus is approximately 3.6 Mb and contains at least 224 loci. One effect of this clustering is that "haplotypes", sets of alleles present on a single chromosome inherited from one parent, tend to be inherited as a group.The set of alleles inherited from each parent forms a haplotype, in which some alleles tend to be associated together. Because some alleles and haplotypes are more common than others and are distributed at different frequencies in different racial and ethnic groups, identifying a patient's haplotype can help predict the probability of finding a matched donor and aid in the development of search strategies.

[0084] As used herein, the term "HLA-matched" refers to a donor-recipient pair in which none of the HLA antigens are mismatched between the donor and recipient, such as a donor providing a hematopoietic stem cell graft to a recipient in need of hematopoietic stem cell transplantation therapy. HLA-matched (i.e., all six alleles matched) donor-recipient pairs have a reduced risk of graft rejection because endogenous T cells and NK cells are less likely to recognize the foreign graft as foreign and therefore less likely to mount an immune response against the graft.

[0085] As used herein, the term "HLA-mismatched" refers to a donor-recipient pair in which at least one HLA antigen, particularly with respect to HLA-A, HLA-B, HLA-C, and HLA-DR, is mismatched between the donor and recipient, such as a donor providing a hematopoietic stem cell graft 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 be at a higher risk of graft rejection than HLA-matched donor-recipient pairs because endogenous T cells and NK cells are more likely to recognize the foreign graft as foreign and therefore more likely to mount an immune response against the graft.

[0086] As used herein, the term "human antibody" refers to an antibody having antibody regions, such as variable and constant regions or domains, that substantially correspond to human germline immunoglobulin sequences. Human antibodies can be produced in human cell lines (e.g., by recombinant expression), non-human animals, or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. When a human antibody is a single-chain antibody, it can contain a linker peptide not found in native human antibodies. For example, an Fv can contain a linker peptide, such as two to about eight glycine or other amino acid residues, connecting the variable region of the heavy chain and the variable region of the light chain. Such a linker peptide is considered to be of human origin. Human antibodies can be produced by a variety of 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 which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes (see, e.g., WO 1998 / 24893, WO 1992 / 01047, 1996 / 34096, and 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, a human antibody is produced using recombinant methods such that the glycosylation pattern of the antibody differs from that of an antibody with the same sequence as occurs in nature.

[0087] As used herein, the term "humanized" antibody refers to a chimeric antibody, generally comprising amino acid sequences from non-human CDRs and human framework regions. In one embodiment, a humanized antibody is a human antibody in which residues from the recipient CDR are replaced by residues from a CDR of a non-human species, such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capacity. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. All or substantially all of the FW regions may also be human immunoglobulin sequences. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art and are described, for example, in Riechmann et al., Nature 332:323-7, 1988; U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370.

[0088] As used herein, the term "immune cell" refers to a cell of the immune system that is involved in the initiation and maintenance of innate or adaptive immune responses. Immune cells include lymphocytes that contain receptors that specifically bind to an antigen of interest, such as an autoantigen in the case of autoimmune cells, and initiate an immune response against it. 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.

[0089] As used herein, patients "in need of" hematopoietic stem cell transplantation include those with defects or deficiencies in one or more blood cell types, as well as those suffering from stem cell disorders. Hematopoietic stem cells generally: 1) exhibit pluripotency and can differentiate into multiple different blood lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells); 2) exhibit self-renewal and can generate daughter cells with capabilities equivalent to those of the parent cells; and 3) exhibit the ability to be reintroduced into the transplant recipient, where they home to the hematopoietic stem cell niche and restore productive and sustained hematopoiesis. Thus, hematopoietic stem cells can be administered to patients with defects or deficiencies in one or more cell types of the hematopoietic lineage to reconstitute defective or deficient cell populations in vivo. For example, the patient may have cancer, and the deficiency may be caused by the administration of chemotherapeutic or other agents that selectively or nonspecifically deplete cancerous cell populations. Additionally or alternatively, the patient may have a nonmalignant hemoglobinopathy that can cause defects or deficiencies in one or more blood cell types, such as sickle cell anemia, thalassemia, Fanconi anemia, and Wiskott-Aldrich syndrome. The subject may have adenosine deaminase severe combined immunodeficiency (ADA-SCID), HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwaman-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 have or be affected by a malignancy, e.g., a malignancy selected from the group consisting of a hematological cancer (such as leukemia, lymphoma, multiple myeloma, or myelodysplastic syndrome) and neuroblastoma. In some embodiments, the subject has or is otherwise affected by a metabolic disorder.For example, the subject may suffer from or be otherwise affected by: a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidosis, Gaucher disease, Hurler disease, sphingolipidosis, metachromatic leukodystrophy, or another disease or disorder that may benefit from the treatments and therapies disclosed herein, including, but not limited to, severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and "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 the administration of hematopoietic stem cell transplantation therapy. Additionally or alternatively, a patient "in need" of hematopoietic stem cell transplantation may be a patient who may or may not suffer from one of the aforementioned conditions, but who nevertheless exhibits reduced levels (e.g., compared to levels in otherwise healthy subjects) of one or more endogenous cell types within 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. One skilled in the art can readily determine whether an otherwise healthy subject has reduced levels of one or more of the aforementioned cell types or other blood cell types, for example, using flow cytometry and fluorescence-activated cell sorting (FACS) methods, among other procedures known in the art.

[0090] When used in the context of a protein such as an antibody, the term "isolated" refers to a protein that, by reason of its origin or source, is not associated with naturally associated components that accompany it in nature, is substantially free of other proteins of the same species, is expressed by cells of 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 is naturally derived would be "isolated" from its naturally associated components. A protein can also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art.

[0091] The term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical and / or bind the same epitope, except for variant antibodies, e.g., naturally occurring mutations or variants that arise during production of the monoclonal antibody preparation, and such variants may be present in minor amounts. In contrast to polyclonal antibody preparations, which 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 construed as requiring production of the antibody by any particular method.

[0092] As used herein, the term "patient at risk for GVHD" refers to a patient with one or more factors that contribute to the development of GVHD. Risk factors include, but are not limited to, allogeneic donor transplants (e.g., transplants of hematopoietic stem cells from bone marrow transplants), including HLA-mismatched donors and gender-mismatched donors, T cell-replete stem cell transplants, T cell-replete stem cell transplants, donor and recipient age, the presence of cytomegalovirus (CMV) or CMV antibodies in the transplant donor or host, escalation of total body irradiation (TBI), intensity of conditioning regimen, prophylaxis against acute GVHD, lack of a protective environment, splenectomy, immunoglobulin use, underlying diseases, ABO compatibility, history of herpes virus exposure, donor transfusion, performance score, antibiotic bowel irrigation, and post-allogeneic transplant transfusion.

[0093] As used herein, the term "patient at risk for autoimmune disease" refers to a patient who has one or more risk factors for developing an autoimmune disease, including, but not limited to, age (young to middle-aged), sex (female), ethnicity (African-American, American Indian, or Latino), family history of autoimmune disease, exposure to environmental factors, previous infection, chronic inflammation, and donor transplant (e.g., transplantation of hematopoietic stem cells from bone marrow transplant).

[0094] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human), without undue toxicity, irritation, allergic response, and other complications commensurate with a reasonable benefit / risk ratio.

[0095] As used herein, the term "pharmaceutical composition" refers to a mixture containing therapeutic compounds that is administered to a mammalian subject, e.g., a human, to prevent, treat, or control a particular disease or condition affecting the mammal, such as an autoimmune disorder, cancer, or blood disorder, among others.

[0096] As used herein, the term "recipient" refers to a patient receiving a transplant, such as a transplant containing a population of hematopoietic stem cells. The transplanted cells administered to the recipient can be, for example, autologous, syngeneic, or allogeneic cells.

[0097] As used herein, the term "rejection" in the context of a transplant, such as a hematopoietic stem cell transplant, refers to a process in which a recipient mounts an immune response against the incoming transplant, thereby reducing the ability of the transplanted material (e.g., hematopoietic stem cells) to remain in the recipient. Rejection of a transplant, such as a hematopoietic stem cell transplant, can be quantified, for example, by measuring the amount or concentration of transplanted cells in various samples isolated from the patient at different time points after transplant. A finding that the amount or concentration of transplanted cells in samples isolated from the patient decreases over time by, for example, about 20%, about 25%, about 30%, about 35%, about 40%, about 56%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or more indicates that the patient is suffering from graft rejection. Conversely, a finding that the amount or concentration of transplanted cells in samples isolated from the patient remains stable over time, e.g., by decreasing by less than about 20%, less than about 15%, less than about 10%, less than about 5%, or more, indicates that the patient is not suffering from graft rejection. Alternatively, graft rejection can be quantified by measuring the amount or concentration of immune cells (such as T cells and / or NK cells) that cross-react with MHC antigens expressed by the transplanted cells in various samples isolated from the patient at different time points after transplantation. A finding that the amount 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 increases over time by, for example, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 56%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 200%, about 300%, or more indicates that the patient is suffering from graft rejection.Conversely, a finding that the amount 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 by, for example, about 20%, about 25%, about 30%, about 35%, about 40%, about 56%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more indicates that the patient is not suffering from graft rejection.

[0098] As used herein, the term "sample" refers to a specimen (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or dermis), pancreatic juice, chorionic villus samples, and cells) obtained from a subject.

[0099] As used herein, the term "scFV" refers to a single-chain Fv antibody in which the variable domains of the heavy and light chains from an antibody are linked to form a single chain. The scFV fragment contains a single polypeptide chain comprising the variable region of the antibody light chain (VL) (e.g., CDR-L1, CDR-L2, and / or CDR-L3) and the variable region of the antibody heavy chain (VH) (e.g., CDR-H1, CDR-H2, and / or CDR-H3), separated by a linker. The linker connecting the VL and VH regions of the scFV fragment can be a peptide linker composed of proteogenic amino acids. Alternative linkers can be used to increase the resistance of the scFV fragment to proteolysis (e.g., linkers containing D-amino acids), to increase the solubility of the scFV fragment (e.g., hydrophilic linkers such as polyethylene glycol-containing linkers 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). Those skilled in the art will also understand that the variable regions of the scFV molecules described herein can be modified to differ in amino acid sequence from the antibody molecule from which they are derived. For example, nucleotide or amino acid substitutions resulting in conservative substitutions or amino acid residue changes (e.g., in CDR and / or framework residues) can be made to retain or enhance the ability of the scFV to bind to the antigen recognized by the corresponding antibody.

[0100] With respect to the interaction of an antibody or antigen-binding fragment with a second chemical species, the terms "specific binding" or "specifically binds" mean 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, antibodies recognize and bind to specific protein structures rather than proteins in general. If an antibody is specific for epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction involving labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody. In one embodiment, the KD of the antibody for its target is 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 10 -12 Less than M (10 -12 Less than 10 -12 A number less than, e.g., 10 -13 (meaning that the antibody specifically binds to a target, e.g., CD2). In one embodiment, the term "specific binding to CD2" or "specifically binds to CD2" as used herein refers to an antibody that binds to CD2 and has a dissociation constant (KD) of 1.0×10 as determined by surface plasmon resonance. -7 In another embodiment, the term "specific binding to CD5" or "specifically binds to CD5" refers to an antibody that binds to CD5 and has a dissociation constant (KD) of 1.0 x 10 M or less, as determined by surface plasmon resonance. -7KD refers to an antibody that has a KD of M or less. In one embodiment, the KD is determined by standard biolayer interferometry (BLI). However, it should be understood that an antibody may be capable of specifically binding to two or more antigens that are related in sequence. For example, in one embodiment, the antibody can specifically bind to both human and non-human (e.g., mouse or non-human primate) orthologs of CD2. In another embodiment, the antibody can specifically bind to both human and non-human (e.g., mouse or non-human primate) orthologs of CD5. Specific binding may also refer to an ADC comprising the antibody.

[0101] As used herein, the terms "subject" and "patient" refer to a mammal, such as a human, receiving treatment for a particular disease or condition as described herein. For example, a patient, such as a human patient, may be suffering from an autoimmune disease as described herein and may be administered an anti-CD2 antibody or antibody-drug conjugate or a CD5 antibody or antibody-drug conjugate described herein to (i) deplete a population of autoimmune cells (e.g., a population of autoimmune CD2+ T cells and / or NK cells, or a population of autoimmune CD5+ T cells, B cells, and / or NK cells), and / or (ii) deplete a population of CD2+ immune cells (e.g., CD2+ T cells and / or NK cells) or a population of CD5+ immune cells (e.g., CD5+ T cells, B cells, and / or NK cells) that cross-react with a non-self antigen expressed by hematopoietic stem cells (e.g., a non-self MHC antigen expressed by hematopoietic stem cells), thereby preventing or reducing the likelihood of graft rejection before hematopoietic stem cell transplantation therapy.

[0102] As used herein, the phrase "substantially removed from the blood" refers to a time point after administration of a therapeutic agent (anti-CD2 antibody or anti-CD5 antibody) to a patient when the concentration of the therapeutic agent in a blood sample isolated from the patient is such that the therapeutic agent cannot be detected by conventional means (e.g., when the therapeutic agent is not detectable 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 and immunoblot assays, among others known in the art.

[0103] As used herein, the phrase "stem cell disorder" broadly refers to any disease, disorder, or condition that can be treated or cured by conditioning a target tissue in a subject (e.g., by ablating endogenous T cell populations in the target tissue) and / or by engrafting or transplanting stem cells into the target tissue in a subject. For example, patients with type 1 diabetes have been shown to be cured by hematopoietic stem cell transplantation and may benefit from conditioning in accordance with the compositions and methods described herein. Additional disorders that can 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 Schwaman-Diamond syndrome. The subject may have or be otherwise affected by a genetic blood disorder (e.g., sickle cell anemia) or an autoimmune disorder. Additionally or alternatively, the subject may have or be 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 has or is otherwise affected by a metabolic disorder.For example, the subject may suffer from or be otherwise affected by: a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidosis, Gaucher disease, Hurler disease, sphingolipidosis, metachromatic leukodystrophy, or another disease or disorder that may benefit from the disclosed treatments and therapies, including, but not limited to: severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and "Bone Marrow Transplantation for Non-Malignant Disease" ASH Education Book, 1:319-338 (2000), the disclosure of which is incorporated by reference in its entirety as it relates to conditions that can be treated by the administration of hematopoietic stem cell transplantation therapy.

[0104] As used herein, the term "transfection" refers to any of a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, and DEAE-dextran transfection.

[0105] As used herein, the term "treat" or "treatment" refers to a therapeutic treatment whose purpose is to prevent or slow (alleviate) an undesirable physiological change or disorder or to promote a beneficial phenotype in the patient receiving treatment. Beneficial or desirable clinical outcomes depend on the disorder being treated and include, but are not limited to, a reduction in tumor burden; in the case of directly treating an autoimmune disease, a reduction in the amount of autoimmune cells present in a sample isolated from the patient, such as a population of CD2+ T cells and / or NK cells (or a population of CD5+ T cells, B cells, and / or NK cells) that cross-react with autologous antigens; or, in the case of treating an autoimmune disease by administering an anti-CD2 or anti-CD5 antibody and hematopoietic stem cell transplantation, a reduction in the amount of non-self antigens (e.g., non-self MHC antigens) expressed by hematopoietic stem cells prior to hematopoietic stem cell transplantation. Further beneficial outcomes include an increase in the cell number or relative concentration of hematopoietic stem cells in patients requiring hematopoietic stem cell transplantation after conditioning therapy and subsequent administration of an exogenous hematopoietic stem cell graft to the patient. Beneficial results of the treatments described herein may also include an increase in the cell number 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, after conditioning therapy and subsequent hematopoietic stem cell transplantation therapy. In certain embodiments, a patient is diagnosed with a disorder and then treated with a therapeutic agent, e.g., an anti-CD5 ADC. In other embodiments, a patient is at risk of developing a disorder, such as GVHD, and is treated as a preventative measure to reduce the risk of developing the disorder or to alleviate the symptoms of the disorder.

[0106] As used herein, the terms "variant" and "derivative" are used interchangeably and refer to natural, synthetic, and semi-synthetic 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 material.

[0107] As used herein, the term "vector" includes nucleic acid vectors such as plasmids, DNA vectors, plasmids, RNA vectors, viruses, or other suitable replicons. The expression vectors described herein may contain polynucleotide sequences and additional sequence elements used, for example, for protein expression and / or integration of these polynucleotide sequences into the genome of mammalian cells. Particular vectors that can be used to express the antibodies and antibody fragments of the present invention include plasmids containing regulatory sequences such as promoter and enhancer regions that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of the mRNA resulting from gene transcription. These sequence elements may include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of genes carried in the expression vector. The expression vectors described herein may contain a polynucleotide encoding a marker for selecting cells containing such a vector. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.

[0108] As used herein, the term "alkyl" refers to a straight- or branched-chain alkyl group having, for example, 1 to 20 carbon atoms in the chain. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and the like.

[0109] As used herein, the term "alkylene" refers to a straight or branched chain divalent alkyl group. The divalent positions can be on the same or different atoms within the alkyl chain. Examples of alkylene include methylene, ethylene, propylene, isopropylene, etc.

[0110] As used herein, the term "heteroalkyl" refers to a straight- or branched-chain alkyl group having, for example, 1 to 20 carbon atoms in the chain and further containing one or more heteroatoms in the chain (such as oxygen, nitrogen, or sulfur, among others).

[0111] As used herein, the term "heteroalkylene" refers to a straight-chain or branched-chain 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.

[0112] As used herein, the term "alkenyl" refers to a straight- or branched-chain alkenyl group having, for example, 2 to 20 carbon atoms in the chain. Examples of alkenyl groups include vinyl, propenyl, isopropenyl, butenyl, tert-butylenyl, hexenyl, and the like.

[0113] As used herein, the term "alkenylene" refers to a straight or branched chain divalent alkenyl group. The divalent positions can be on the same or different atoms within the alkenyl chain. Examples of alkenylene include ethenylene, propenylene, isopropenylene, butenylene, etc.

[0114] As used herein, the term "heteroalkenyl" refers to a straight- or branched-chain alkenyl group having, for example, 2 to 20 carbon atoms in the chain and further containing one or more heteroatoms in the chain (such as oxygen, nitrogen, or sulfur, among others).

[0115] As used herein, the term "heteroalkenylene" refers to a straight-chain or branched-chain 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.

[0116] As used herein, the term "alkynyl" refers to a straight- or branched-chain alkynyl group having, for example, 2 to 20 carbon atoms in the chain. Examples of alkynyl groups include propargyl, butynyl, pentynyl, hexynyl, and the like.

[0117] As used herein, the term "alkynylene" refers to a straight or branched chain divalent alkynyl group. The divalent positions can be on the same or different atoms within the alkynyl chain.

[0118] As used herein, the term "heteroalkynyl" refers to a straight- or branched-chain alkynyl group having, for example, 2 to 20 carbon atoms in the chain and further containing one or more heteroatoms in the chain (such as oxygen, nitrogen, or sulfur, among others).

[0119] As used herein, the term "heteroalkynylene" refers to a straight-chain or branched-chain 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.

[0120] As used herein, the term "cycloalkyl" refers to a saturated, monocyclic, or fused, bridged, or spiro polycyclic ring structure having, for example, 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.

[0121] 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, and the like.

[0122] As used herein, the term "heterocycloalkyl" refers to a monocyclic or fused, bridged, or spiropolycyclic ring structure that is saturated and has, e.g., 3 to 12 ring atoms per ring structure selected from carbon atoms and heteroatoms selected from, e.g., nitrogen, oxygen, and sulfur, among others. The ring structure may contain one or more oxo groups, e.g., on a carbon, nitrogen, or sulfur ring member. Examples of heterocycloalkyl include, by way of example and not limitation, dihydropyridyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydropyranyl, bistetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinuclidinyl, and morpholinyl.

[0123] 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. As used herein, the term "aryl" refers to a monocyclic or polycyclic aromatic ring system containing, for example, 6 to 19 carbon atoms. Aryl groups include, but are not limited to, phenyl, fluorenyl, naphthyl, and the like. The divalent positions can be one or more heteroatoms.

[0124] As used herein, the term "arylene" refers to a divalent aryl group. The divalent positions can be on the same or different atoms.

[0125] 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, benzofuryl, [2,3-dihydro]benzofuryl, isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, Indolyl, indolyl, isoindolyl, 3H-indolyl, benzimidazolyl, imidazo[1,2-a]pyridyl, benzothiazolyl, benzoxazolyl, quinolidinyl, quinazolinyl, phthalazinyl, quinoxalinyl, cinnolinyl, naphthyridinyl, pyrido[3,4-b]pyridyl, pyrido[3,2-b]pyridyl, pyrido[4,3-b]pyridyl, quinolyl, isoquinolyl, tetrazolyl, 5,6,7,8-tetrahydroquinolyl, 5,6,7,8-tetrahydroisoquinolyl, purinyl, pteridinyl, carbazolyl, xanthenyl, benzoquinolyl, and the like.

[0126] 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.

[0127] Unless otherwise limited by the definition of the individual substituents, the foregoing chemical moieties, e.g., "alkyl," "alkylene," "heteroalkyl," "heteroalkylene," "alkenyl," "alkenylene," "heteroalkenyl," "heteroalkenylene," "alkynyl," "alkynylene," "heteroalkynyl," "heteroalkynylene," "cycloalkyl," "cycloalkylene," "heterocycloalkyl," "heterocycloalkylene," "aryl," "arylene," "heteroaryl," and "heteroarylene" groups, may be optionally substituted with from 1 to 5 substituents selected from the group consisting of, for example, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, alkylaryl, alkylheteroaryl, alkylcycloalkyl, alkylheterocycloalkyl, amino, ammonium, acyl, acyloxy, acylamino, aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaryl, sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy, trihalomethyl, cyano, hydroxy, mercapto, nitro, and the like. Typical substituents include -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)2NH 2、 -S(=O)2N(R)2, -S(=O)R, -OP(=O)(OH) 2、 -OP(=O)(OR)2, -P(=O)(OR)2, -PO3, -PO3H2, -C(=O)X, -C(=S)R, -CO2H, -CO2R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NH 2、 -C(=O)N(R)2, -C(=S)NH 2、Examples include, but are not limited to, -C(=S)N(R), -C(=NH)NH, and -C(=NR)N(R). Each X, at each occurrence, is independently selected from F, Cl, Br, and I, and each R, at each occurrence, is independently selected from alkyl, aryl, heterocycloalkyl or heteroaryl, protecting groups, and prodrug moieties. Whenever a group is described as "optionally substituted," that group, at each occurrence, can be substituted with one or more of the above substituents. Substitution can include situations where adjacent substituents undergo ring closure, such as ring closure of adjacent functional substituents, to form, for example, lactams, lactones, cyclic anhydrides, acetals, hemiacetals, thioacetals, aminals, and heminaminals formed by ring closure, for example, to provide a protecting group.

[0128] It should be understood that certain radical naming conventions can include either monoradicals or diradicals depending on the context. For example, if a substituent requires two points of attachment to the rest of the molecule, the substituent is understood to be a diradical. For example, a substituent identified as alkyl, which requires two points of attachment, includes diradicals 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.

[0129] When a substituent is depicted as a diradical (i.e., having two points of attachment to the rest of the molecule), it is understood that the substituent may be attached in any directional orientation unless otherwise specified.

[0130] Anti-CD2 antibody drug conjugate The compositions and methods described herein are based in part on the discovery that anti-CD2 ADCs can be used to directly treat cancer and autoimmune diseases, for example, due to the ability of such agents to kill CD2+ cancer cells (e.g., CD2+ leukemia cells) and CD2+ autoimmune cells (e.g., CD2+ autoimmune T cells and / or NK cells). In particular, anti-CD2 antibodies are conjugated to cytotoxins via linkers. Thus, when anti-CD2 antibodies are described, their conjugates are also contemplated unless otherwise specified.

[0131] The compositions and methods described herein are also based in part on the discovery that ADCs capable of binding to CD2 can be used as therapeutic agents to promote the engraftment of transplanted hematopoietic stem cells in patients requiring transplantation therapy by preventing or reducing the likelihood of immune cell-mediated graft rejection. For example, anti-CD2 antibodies and antigen-binding fragments can cross-react with one or more non-self hematopoietic stem cell antigens, such as one or more non-self MHC antigens expressed by hematopoietic stem cells, and bind to cell-surface CD2 expressed by immune cells, such as T cells or NK cells, that initiate an immune response thereto. Binding of such antibodies and antigen-binding fragments to hematopoietic stem cell-specific CD2+ immune cells can induce the death of the bound immune cells, for example, by antibody-dependent cell-mediated cytotoxicity or by the action of a cytotoxic agent conjugated to the antibody or its antigen-binding fragment. Thus, depletion of the population of CD2+ immune cells that cross-react with non-self hematopoietic stem cells can promote the engraftment of hematopoietic stem cell transplants in patients requiring hematopoietic stem cell transplantation by weakening the ability of the recipient's immune system to initiate an immune response against the incoming graft. In this manner, a hematopoietic stem cell transplant can be provided to a subject to repopulate defective or deficient cell lineages in the subject, thereby treating patients suffering from stem cell disorders, cancer, autoimmune diseases, or other blood disorders described herein. For example, a subject may be deficient in a cell population due to chemotherapy administered to the subject with the intent of eradicating cancerous cells, but which also depletes healthy hematopoietic cells in the process.

[0132] For example, in certain embodiments, compositions and methods are provided that promote the engraftment of transplanted hematopoietic stem cells by administering an antibody or antigen-binding fragment thereof capable of binding to an antigen expressed by T cells. This administration can cause selective depletion of endogenous T cell populations, such as CD4+ and CD8+ T cells. This selective depletion of T cells can prevent graft rejection after transplantation of an exogenous (e.g., autologous, allogeneic, or syngeneic) hematopoietic stem cell transplant. For example, selective depletion of CD4+ and / or CD8+ T cells using the anti-CD2 antibodies, antigen-binding fragments, antibody-drug conjugates, or antibody-drug conjugates described herein can attenuate T cell-mediated immune responses that may occur against the transplanted hematopoietic stem cell graft. The compositions and methods described herein are based, in part, on the discovery that antibodies capable of binding to CD2 and antigen-binding fragments thereof can be administered to patients in need of hematopoietic stem cell transplantation therapy to promote the survival and engraftment potential of transplanted hematopoietic stem cells.

[0133] The engraftment of hematopoietic stem cell grafts following administration of an anti-CD2 antibody or its antigen-binding fragment can be determined by various empirical measurements. For example, engraftment of transplanted hematopoietic stem cells can be assessed by measuring the amount of competitive remodeling units (CRUs) present in the patient's bone marrow after administration of an antibody or its antigen-binding fragment capable of binding to CD2, followed by administration of a hematopoietic stem cell graft. Furthermore, hematopoietic stem cell graft engraftment can be observed by incorporating a reporter gene, such as an enzyme that catalyzes a chemical reaction that produces a fluorescent, chromogenic, or luminescent product, into a vector transfected with donor hematopoietic stem cells, followed by monitoring the corresponding signal in the tissue to which the hematopoietic stem cells home, such as the bone marrow. Hematopoietic stem cell graft engraftment can also be observed by assessing the quantity and survival of hematopoietic stem cells and progenitor cells, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis methods known in the art. Engraftment can also be determined by measuring peripheral blood white blood cell counts during the post-transplant period and / or by measuring bone marrow cell recovery with donor cells in bone marrow aspirate samples.

[0134] The following sections describe antibodies or antigen-binding fragments thereof that can be administered to patients in need of hematopoietic stem cell transplantation therapy to promote hematopoietic stem cell transplant engraftment, as well as methods of administering such therapeutics to patients prior to hematopoietic stem cell transplantation.

[0135] Anti-CD2 antibody The compositions and methods described herein include antibodies or fragments thereof that specifically bind to human CD2. Human CD2 is also known as T cell surface antigen T11 / Leu-5, T11, CD2 antigen (p50), and sheep erythrocyte receptor (SRBC). CD2 is expressed on T cells. Two isoforms of human CD2 have been identified. Isoform 1 contains 351 amino acids and is described in Seed, B. et al. (1987) 84:3365-69 (see also Sewell et al. (1986) 83:8718-22) and as follows (NCBI Reference Sequence: NP_001758.2): msfpckfvas flllifnvssk gavskeitna letwgalgqd inldipsfqm sddiddikwe ktsdkkkiaq frkeketfke kdtyklfkng tlkikhlktd dqdiykvsiy dtkgknvlek ifdlkiqerv skpkiswtci nttltcevmn gtdpelnlyq dgkhlklsqr vithkwttsl sakfkctagn kvskessvep vscpekgldi yliigicggg sllmvfvall vfyitkrkkq rsrrndeele trahrvatee rgrkphqipa stpqnpatsq hpppppghrs qapshrpppp ghrvqhqpqk rppapsgtqv hqqkgpplpr prvqpkpphg aaenslspss n (SEQ ID NO: 13) The second isoform of CD2 is 377 amino acids and is identified herein as NCBI reference sequence: NP_001315538.1.

[0136] T cells and NK cells have been shown to express CD2, a cell adhesion molecule and a specific marker for such lymphocytes. For example, CD2 interacts with other adhesion molecules, such as lymphocyte function-associated antigen-3 (LFA-3 / CD58), to enhance T cell activation. Antibodies and antigen-binding fragments thereof capable of binding to CD2 can suppress T cell activation and T cell-mediated immune responses to hematopoietic stem cell grafts, for example, by inhibiting the interaction between CD2 and LFA-3. Antibodies and antigen-binding fragments thereof that bind to this cell surface antigen can be identified using techniques known in the art and described herein, including immunization, computational modeling techniques, and in vitro selection methods such as the phage display and cell-based display platforms described below.

[0137] Described herein are antibodies and antigen-binding fragments thereof that specifically bind to CD2 polypeptides, e.g., human CD2 polypeptides, and uses thereof. In exemplary embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to CD2 polypeptides comprise a heavy chain variable region and a light chain variable region.

[0138] 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 having the amino acid sequence of SEQ ID NO: 1. In one embodiment, the heavy chain variable region comprises a VH CDR2 having the amino acid sequence of SEQ ID NO: 2. In one embodiment, the heavy chain variable region comprises a VH CDR3 having the amino acid sequence of SEQ ID NO: 3. In one embodiment, the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In one embodiment, the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In one embodiment, the heavy chain variable region comprises a VH CDR1 having SEQ ID NO: 1, a VH CDR2 having SEQ ID NO: 2, and a VH CDR3 having SEQ ID NO: 3.

[0139] 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 having the amino acid sequence of SEQ ID NO:4. In one embodiment, the light chain variable region comprises a VL CDR2 having the amino acid sequence of SEQ ID NO:5. In one embodiment, the light chain variable region comprises a VL CDR3 having the amino acid sequence of SEQ ID NO:6. In one embodiment, the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In one embodiment, the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In one embodiment, the light chain variable region comprises a VL CDR1 having SEQ ID NO:4, a VL CDR2 having SEQ ID NO:5, and a VL CDR3 having SEQ ID NO:6.

[0140] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 having SEQ ID NO:1, a VH CDR2 having SEQ ID NO:2, and a VH CDR3 having SEQ ID NO:3, and a light chain variable region comprising a VL CDR1 having SEQ ID NO:4, a VL CDR2 having SEQ ID NO:5, and a VL CDR3 having SEQ ID NO:6.

[0141] In certain embodiments, one or more of the CDRs (i.e., one or more heavy chain CDRs having SEQ ID NOs: 1-3, and / or one or more light chain CDRs having SEQ ID NOs: 4-6) may contain conservative amino acid substitutions (or two, three, four, or five amino acid substitutions) while retaining the CD2 specificity of the antibody (i.e., specificity similar to that of an antibody or antigen-binding fragment thereof comprising the heavy chain CDRs of SEQ ID NOs: 1-3 and the light chain CDRs of SEQ ID NOs: 4-6).

[0142] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 7. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence having at least 95% identity to SEQ ID NO: 7, e.g., at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 7. In a specific embodiment, the antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain having SEQ ID NO: 7 or a variant of SEQ ID NO: 7, wherein the variant (i) differs from SEQ ID NO: 7 in 1, 2, 3, 4 or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 7 in up to 5, 4, 3, 2 or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 7 in 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or deletions; and and / or (iv) an amino acid sequence at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 7, wherein in any of (i)-(iv), the amino acid substitutions may be conservative or non-conservative, and the modified heavy chain variable region may retain the CD2 binding specificity of the antibody, i.e., have similar binding specificity to an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 7, while having enhanced biological activity compared to the heavy chain variable region of SEQ ID NO: 7. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region that differs by one, two, three, or four amino acids from the amino acid sequence set forth in SEQ ID NO: 7. For example, the antibody or antigen-binding fragment thereof may comprise a heavy chain variable region that differs from the amino acid sequence set forth in SEQ ID NO: 7 at one, two, three, or four of positions 12, 13, 28, and / or 48. In one embodiment, the heavy chain variable region differs from the amino acid sequence set forth in SEQ ID NO: 7 at positions 12, 13, 28, and 48. In one embodiment, the heavy chain variable region comprises one, two, three, or four of the following substitutions relative to the sequence set forth in SEQ ID NO:7: K12Q, K13R, T28I, and M48V.In one embodiment, the heavy chain variable region comprises the substitutions K12Q, K13R, T28I, and M48V relative to SEQ ID NO:7.

[0143] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having an amino acid sequence having at least 95% identity to SEQ ID NO: 8, e.g., at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 8. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain having SEQ ID NO: 8 or a variant of SEQ ID NO: 8, wherein the variant (i) differs from SEQ ID NO: 8 in 1, 2, 3, 4 or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 8 in up to 5, 4, 3, 2 or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 8 in 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or deletions; and and / or (iv) an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8, wherein in any of (i) to (iv), the amino acid substitutions may be conservative or non-conservative amino acid substitutions, and the modified light chain variable region may retain the CD2 binding specificity of the antibody, i.e., have similar binding specificity as an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 8, while having enhanced biological activity compared to the light chain variable region of SEQ ID NO: 8.

[0144] In exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing an amino acid sequence having at least 95% identity to SEQ ID NO:7, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to SEQ ID NO:7, and a light chain variable region containing an amino acid sequence having at least about 95% identity to SEQ ID NO:8, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to SEQ ID NO:8. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having SEQ ID NO:7 and a light chain variable region having SEQ ID NO:8. In one embodiment, the antibody is an Ab1 antibody comprising a heavy chain variable region containing SEQ ID NO:7 and a light chain variable region containing SEQ ID NO:8.

[0145] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 9. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing an amino acid sequence having at least 95% identity to SEQ ID NO: 9, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to SEQ ID NO: 9. In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing an amino acid sequence having at least 95% identity to SEQ ID NO: 9, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to SEQ ID NO: 9, and a light chain variable region containing an amino acid sequence having at least about 95% identity to SEQ ID NO: 10, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to SEQ ID NO: 10. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing SEQ ID NO: 9 and a light chain variable region containing SEQ ID NO: 10. In one embodiment, the antibody is an Ab1 antibody comprising a heavy chain variable region containing SEQ ID NO: 9 and a light chain variable region containing SEQ ID NO: 10.

[0146] 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 containing the amino acid sequence of SEQ ID NO: 14. In one embodiment, the heavy chain variable region comprises a VH CDR2 containing the amino acid sequence of SEQ ID NO: 15. In one embodiment, the heavy chain variable region comprises a VH CDR3 containing the amino acid sequence of SEQ ID NO: 16. In one embodiment, the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In one embodiment, the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. In one embodiment, the heavy chain variable region comprises a VH CDR1 containing SEQ ID NO: 14, a VH CDR2 containing SEQ ID NO: 15, and a VH CDR3 containing SEQ ID NO: 16.

[0147] 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 containing the amino acid sequence of SEQ ID NO: 14. In one embodiment, the heavy chain variable region comprises a VH CDR2 containing the amino acid sequence of SEQ ID NO: 15. In one embodiment, the heavy chain variable region comprises a VH CDR3 containing the amino acid sequence of SEQ ID NO: 17. In one embodiment, the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17. In one embodiment, the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17. In one embodiment, the heavy chain variable region comprises a VH CDR1 containing SEQ ID NO: 14, a VH CDR2 containing SEQ ID NO: 15, and a VH CDR3 containing SEQ ID NO: 17.

[0148] 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 containing the amino acid sequence of SEQ ID NO: 18. In one embodiment, the light chain variable region comprises a VL CDR2 containing the amino acid sequence of SEQ ID NO: 19. In one embodiment, the light chain variable region comprises a VL CDR3 containing the amino acid sequence of SEQ ID NO: 20. In one embodiment, the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In one embodiment, the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In one embodiment, the light chain variable region comprises a VL CDR1 containing SEQ ID NO: 18, a VL CDR2 containing SEQ ID NO: 19, and a VL CDR3 containing SEQ ID NO: 20.

[0149] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 containing SEQ ID NO: 14, a VH CDR2 containing SEQ ID NO: 15, and a VH CDR3 containing SEQ ID NO: 16, and a light chain variable region comprising a VL CDR1 containing SEQ ID NO: 18, a VL CDR2 containing SEQ ID NO: 19, and a VL CDR3 containing SEQ ID NO: 20.

[0150] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 containing SEQ ID NO: 14, a VH CDR2 containing SEQ ID NO: 15, and a VH CDR3 containing SEQ ID NO: 17, and a light chain variable region comprising a VL CDR1 containing SEQ ID NO: 18, a VL CDR2 containing SEQ ID NO: 19, and a VL CDR3 containing SEQ ID NO: 20.

[0151] In certain embodiments, one or more of the CDRs (i.e., one or more heavy chain CDRs having SEQ ID NOs: 14-17 and / or one or more light chain CDRs having SEQ ID NOs: 18-19) may contain conservative amino acid substitutions (or two, three, four, or five amino acid substitutions) while retaining the CD2 specificity of the antibody (i.e., similar specificity to that of an antibody or antigen-binding fragment thereof comprising a heavy chain CDR of SEQ ID NO: 14-16 and a light chain CDR of SEQ ID NO: 18-20).

[0152] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 21. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing an amino acid sequence having at least about 95% identity to SEQ ID NO: 21, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to SEQ ID NO: 21. In a specific embodiment, the antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain containing SEQ ID NO: 21 or a variant of SEQ ID NO: 21, wherein the variant (i) differs from SEQ ID NO: 21 in 1, 2, 3, 4 or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 21 in up to 5, 4, 3, 2 or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 21 in 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or deletions; and / or (iv) an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 21, wherein in any of (i) to (iv), the amino acid substitutions may be conservative or non-conservative amino acid substitutions, and the modified heavy chain variable region may retain the CD2 binding specificity of the antibody, i.e., have similar binding specificity as an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 21, while having enhanced biological activity compared to the heavy chain variable region of SEQ ID NO: 21.

[0153] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 22. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing an amino acid sequence having at least about 95% identity to SEQ ID NO: 22, e.g., at least about 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 8. In a specific embodiment, the antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain containing SEQ ID NO: 21 or a variant of SEQ ID NO: 22, wherein the variant (i) differs from SEQ ID NO: 22 in 1, 2, 3, 4 or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 22 in up to 5, 4, 3, 2 or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 22 in 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or deletions; and / or (iv) an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 22, wherein in any of (i) to (iv), the amino acid substitutions may be conservative or non-conservative amino acid substitutions, and the modified heavy chain variable region may retain the CD2 binding specificity of the antibody, i.e., have similar binding specificity as an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 22, while having enhanced biological activity compared to the heavy chain variable region of SEQ ID NO: 22.

[0154] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region containing the amino acid sequence set forth in SEQ ID NO: 23. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region containing an amino acid sequence having at least about 95% identity to SEQ ID NO: 23, e.g., at least about 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 23. In a specific embodiment, the antibody comprises a modified heavy chain (LC) variable region comprising an LC variable domain containing SEQ ID NO: 23 or a variant of SEQ ID NO: 23, wherein the variant (i) differs from SEQ ID NO: 23 in 1, 2, 3, 4 or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 23 in up to 5, 4, 3, 2 or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 23 in 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or deletions; and / or (iv) an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 23, wherein in any of (i) to (iv), the amino acid substitutions may be conservative or non-conservative amino acid substitutions, and the modified heavy chain variable region may retain the CD2 binding specificity of the antibody, i.e., have similar binding specificity as an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 23, while having enhanced biological activity compared to the heavy chain variable region of SEQ ID NO: 23.

[0155] In exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing an amino acid sequence having at least about 95% identity to SEQ ID NO: 21, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to SEQ ID NO: 7, and a light chain variable region containing an amino acid sequence having at least about 95% identity to SEQ ID NO: 23, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to SEQ ID NO: 23. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing SEQ ID NO: 21, and a light chain variable region containing SEQ ID NO: 23.

[0156] In exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing an amino acid sequence having at least about 95% identity to SEQ ID NO: 22, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identity to SEQ ID NO: 22, and a light chain variable region containing an amino acid sequence having at least about 95% identity to SEQ ID NO: 23, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to SEQ ID NO: 23. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing SEQ ID NO: 22 and a light chain variable region containing SEQ ID NO:23.

[0157] Anti-CD2 antibodies that can be used in combination with the compositions and methods described herein include those with one, more than one, or all of the following CDRs: a. CDR-H1 having the amino acid sequence EYYMY (SEQ ID NO: 1); b. CDR-H2 having the amino acid sequence RIDPEDGSIDYVEKFKK (SEQ ID NO: 2); c. CDR-H3 having the amino acid sequence GKFNYRFAY (SEQ ID NO: 3); d. CDR-L1 having the amino acid sequence RSSQSLLHSSGNTYLN (SEQ ID NO: 4); e. CDR-L2 having the amino acid sequence LVSKLES (SEQ ID NO: 5), and f. CDR-L3 having the amino acid sequence MQFTHYPYT (SEQ ID NO: 6).

[0158] Antibodies and antigen-binding fragments thereof comprising the aforementioned CDR sequences are described, for example, in U.S. Pat. No. 6,849,258, the disclosure of which is incorporated herein by reference as it relates to anti-CD2 antibodies and antigen-binding fragments thereof.

[0159] Antibodies and fragments thereof disclosed in U.S. Patent Nos. 5,730,979, 5,817,311, 5,951,983, and 7,592,006, such as LO-CD2a, BTI-322, and the antibody produced by the hybridoma cell line deposited under ATCC Accession No. HB 11423 (e.g., an antibody or antigen-binding fragment thereof comprising one or more or all of the CDR sequences of antibody LO-CD2a isolated from the hybridoma cell line deposited under ATCC Accession No. HB 11423), can be used in conjunction with the compositions and methods disclosed herein. Exemplary antibodies that can be used in conjunction with the compositions and methods described herein include a humanized antibody, e.g., MEDI-507, containing one or more or all of the CDR sequences of the antibody isolated from the hybridoma cell line deposited under ATCC Accession No. HB 11423. MEDI-507 is a humanized anti-CD2 monoclonal antibody containing the CDR-H and CDR-L sequences (a) to (f) above, and is described in Branco et al., Transplantation 68:1588-1596 (1999). MEDI-507 is further described in International Publication Nos. 99 / 03502A1 and 1994 / 020619A1, U.S. Patent Nos. 7,592,006, 6,849,258, 5,951,983, 5,817,311, and 5,730,979, and U.S. Patent Application Publication Nos. 2011 / 0280868, 2004 / 0265315, and 2011 / 0091453, the disclosures of each of which are incorporated herein by reference as they relate to anti-CD2 antibodies and antigen-binding fragments thereof, such as the anti-CD2 antibody MEDI-507. In one embodiment, the anti-CD2 antibody is siplizumab or an antigen-binding fragment thereof.

[0160] The disclosures of the aforementioned scientific journal articles and US patents as they relate to anti-CD2 antibodies and antigen-binding fragments thereof are incorporated herein by reference.

[0161] Other anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, the anti-CD2 antibodies described in U.S. Patent Nos. 6,541,611 and 7,250,167, the disclosures of each of which are incorporated herein by reference as they relate to anti-CD2 antibodies and antigen-binding fragments thereof, e.g., anti-CD2 antibody LO-CD2b and the antibody produced by the hybridoma cell line deposited under ATCC Accession No. PTA-802. Exemplary antibodies that can be used in conjunction with the compositions and methods described herein include humanized antibodies containing one or more or all of the CDR sequences of the antibody isolated from the hybridoma cell line deposited under ATCC Accession No. PTA-802.

[0162] Other anti-CD2 antibodies that may be used in conjunction with the compositions and methods described herein include, for example, the anti-CD2 antibodies described in U.S. Patent Nos. 5,795,572 and 5,807,734, the disclosures of each of which are incorporated herein by reference as they relate to anti-CD2 antibodies and antigen-binding fragments thereof, e.g., the anti-CD2 antibody produced by the hybridoma cell line deposited under ATCC Accession No. HB 69277. For example, anti-CD2 antibodies and antigen-binding fragments thereof that may be used in conjunction with the compositions and methods described herein include those that include a hinge region having the amino acid sequence EPKSSDKTHTSPPSP (SEQ ID NO: 287), e.g., an scFv fragment that includes a hinge region having the amino acid sequence EPKSSDKTHTSPPSP (SEQ ID NO: 287). Incorporation of a hinge region having the amino acid sequence of SEQ ID NO: 287 can be advantageous because this hinge motif is mutated compared to the wild-type hinge region sequence to potentially eliminate reactive cysteine ​​residues that may promote undesired oxidative dimerization of single-chain antibody fragments, such as scFv fragments.

[0163] Other anti-CD2 antibodies that may be used in conjunction with the compositions and methods described herein include those described in U.S. Patent No. 6,764,688, such as, for example, anti-CD2 antibody TS2 / 18 and the antibody produced by the hybridoma cell line deposited under ATCC Accession No. HB-195. The disclosure of U.S. Patent No. 6,764,688 as it relates to anti-CD2 antibodies and antigen-binding fragments thereof is incorporated herein by reference.

[0164] Other anti-CD2 antibodies that may be used in conjunction with the compositions and methods described herein include, for example, the anti-CD2 antibodies described in U.S. Pat. Nos. 6,162,432, 6,558,662, 7,408,039, 7,332,157, 7,638,121, 7,939,062, and 7,115,259, U.S. Patent Application Publication Nos. 2006 / 0084107, 2014 / 0369974, 2002 / 0051784, and 2013 / 0183322, and PCT Publication No. WO 1992 / 016563, the disclosures of each of which are incorporated herein by reference as they relate to anti-CD2 antibodies and antigen-binding fragments thereof.

[0165] Antibodies and fragments thereof for use in conjunction with the methods described herein include variants of the above antibodies, such as antibody fragments that contain or lack an Fc domain, as well as humanized variants of the non-human antibodies and antibody-like protein scaffolds described herein (e.g., humanized variants of the non-human antibodies and antibody fragments described herein) that contain one or more or all of the CDRs or equivalent regions of the antibodies or antibody fragments described herein. 10 Exemplary antigen-binding fragments of the foregoing 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, among others.

[0166] In one embodiment, an anti-CD2 antibody or binding fragment thereof comprises an altered Fc region, the altered Fc region comprising at least one amino acid modification compared to a wild-type Fc region, such that the molecule has altered affinity or binding to FcgammaR (FcγR). Certain amino acid positions within the Fc region have been shown by crystallographic studies to make direct contact with FcγR, specifically amino acids 234-239 (hinge region), 265-269 (B / C loop), 297-299 (C' / E loop), and 327-332 (F / G loop) (see Sondermann et al., 2000, Nature, 406:267-273). The antibodies described herein comprise variant Fc regions, which comprise a modification of at least one residue that makes direct contact with FcγR based on structural and crystallographic analysis. In one embodiment, the Fc region of the anti-CD2 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NH1, MD (1991), which is expressly incorporated herein by reference. "Kabat's EU index" 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 the antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index of Kabat. In one embodiment, the Fc region comprises a L234A mutation. In some embodiments, the Fc region of the anti-CD2 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 235 according to the EU index of Kabat. In one embodiment, the Fc region comprises a L235A mutation. In yet another embodiment, the Fc region comprises both the L234A and L235A mutations. In a further embodiment, the Fc region comprises D265C, L234A, and L235A mutations.In a further embodiment, the Fc region comprises D265C, L234A, L235A and H435A mutations.In a further embodiment, the Fc region comprises D265C and H435A mutations.

[0167] The antibodies used herein are, 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 may include 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, 1253A, S254T, T256E, P2571, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations.

[0168] In some embodiments, the anti-CD2 antibody or antigen-binding fragment thereof is conjugated to a cytotoxin (e.g., an amatoxin) via a cysteine ​​residue in the Fc domain of the antibody or antigen-binding fragment thereof. In some embodiments, the cysteine ​​residue is introduced by mutation in the Fc domain of the antibody or antigen-binding fragment thereof. For example, the cysteine ​​residue may be selected from the group consisting of Cys118, Cys239, and Cys265. In one embodiment, the Fc region of the anti-CD2 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index of Kabat. In one embodiment, the Fc region comprises a D265C mutation. In one embodiment, the Fc region comprises a D265C and H435A mutations.

[0169] Thus, in one embodiment, the Fc region contains a mutation that results in a reduced half-life.An antibody with a short half-life may be advantageous in certain cases where the antibody is expected to function as a short-lived therapeutic agent, for example, in the conditioning step described herein in which the antibody is administered followed by HSCs.Ideally, the antibody is substantially removed before delivery of HSCs, which, unlike endogenous stem cells, generally also express CD2 but are not targeted by anti-CD2 antibodies.In one embodiment, the Fc region contains a mutation at position 435 (EU index according to Kabat).In one embodiment, the mutation is an H435A mutation.

[0170] The aforementioned anti-CD2 antibodies or antigen-binding fragments thereof can be used in various embodiments of the invention described herein, including, for example, methods for depleting CD2+ cells in a human subject. The aforementioned anti-CD2 antibodies or antigen-binding fragments thereof can also be conjugated to an agent, e.g., a cytotoxin such as an amatoxin, as described herein.

[0171] Anti-CD5 antibody drug conjugate The present disclosure is based in part on the discovery that anti-CD5 antibodies or antigen-binding fragments thereof, or anti-CD5 ADCs, can be used directly to treat cancers, such as T-cell malignancies and autoimmune diseases, 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). Anti-CD5 ADCs can also be used to treat patients at risk for graft-versus-host disease (GVHD).

[0172] In particular, the anti-CD5 antibodies described herein are conjugated to a cytotoxin via a linker. Thus, where an anti-CD5 antibody is described, the conjugate is also contemplated unless otherwise specified.

[0173] The disclosed embodiments described herein are further based in part on the discovery that antibodies capable of binding to CD5, or antibody-binding fragments thereof, can be used as therapeutic agents to promote engraftment of transplanted hematopoietic stem cells in patients requiring transplantation therapy by preventing or reducing the likelihood 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, that cross-react with one or more non-self hematopoietic stem cell antigens, such as one or more non-self MHC antigens expressed by hematopoietic stem cells, and initiate an immune response thereto. Binding of such antibodies and antigen-binding fragments to hematopoietic stem cell-specific CD5+ immune cells can induce the death of the bound immune cells, for example, by antibody-dependent cell-mediated cytotoxicity or by the action of a cytotoxic agent conjugated to the antibody or its antigen-binding fragment. Thus, depletion of the population of CD5+ immune cells that cross-react with non-self hematopoietic stem cells can promote hematopoietic stem cell engraftment in patients requiring hematopoietic stem cell transplantation by weakening the ability of the recipient's immune system to initiate an immune response against the incoming graft. In this manner, a hematopoietic stem cell transplant can be provided to a subject to repopulate defective or deficient cell lineages in the subject, thereby treating patients suffering from stem cell disorders, cancer, autoimmune diseases, or other blood disorders described herein. For example, a subject may be deficient in a cell population due to chemotherapy administered to the subject with the intent of eradicating cancerous cells, but which also depletes healthy hematopoietic cells in the process.

[0174] For example, provided herein are compositions and methods for promoting the engraftment of transplanted hematopoietic stem cells by administering an antibody or antigen-binding fragment thereof, or an ADC, capable of binding to an antigen expressed by T cells. This administration can cause selective depletion of endogenous T cell populations, such as CD4+ and CD8+ T cells. This selective depletion of T cells can prevent graft rejection after transplantation of an exogenous (e.g., autologous, allogeneic, or syngeneic) hematopoietic stem cell transplant. For example, selective depletion of CD4+ and / or CD8+ T cells using the anti-CD5 antibodies, antigen-binding fragments, antibody-drug conjugates, or antibody-drug conjugates described herein can attenuate T cell-mediated immune responses that may occur against the transplanted hematopoietic stem cell graft. The compositions and methods described herein are based in part on the discovery that antibodies and antigen-binding fragments thereof capable of binding to CD5 can be administered to patients in need of hematopoietic stem cell transplantation therapy to promote the survival and engraftment potential of transplanted hematopoietic stem cells.

[0175] The engraftment of hematopoietic stem cell grafts following administration of an anti-CD5 antibody or its antigen-binding fragment can be determined by various empirical measurements. For example, engraftment of transplanted hematopoietic stem cells can be assessed by measuring the amount of competitive remodeling units (CRUs) present in the patient's bone marrow after administration of an antibody or its antigen-binding fragment capable of binding to CD5, followed by administration of a hematopoietic stem cell graft. Furthermore, hematopoietic stem cell graft engraftment can be observed by incorporating a reporter gene, such as an enzyme that catalyzes a chemical reaction that produces a fluorescent, colored, or luminescent product, into a vector transfected with donor hematopoietic stem cells, followed by monitoring the corresponding signal in the tissue to which the hematopoietic stem cells home, such as the bone marrow. Hematopoietic stem cell graft engraftment can also be observed by assessing the quantity and survival of hematopoietic stem cells and progenitor cells, as determined, for example, by fluorescence-activated cell sorting (FACS) analysis methods known in the art. Engraftment can also be determined by measuring peripheral blood white blood cell counts during the post-transplant period and / or by measuring bone marrow cell recovery with donor cells in bone marrow aspirate samples.

[0176] The following sections describe antibodies or antigen-binding fragments thereof that can be administered to patients in need of hematopoietic stem cell transplantation therapy to treat autoimmune diseases, cancer, to treat or prevent graft-versus-host disease (GVHD), or to promote engraftment of a hematopoietic stem cell transplant, as well as methods of administering such therapeutics to patients prior to hematopoietic stem cell transplantation.

[0177] Anti-CD5 antibody The compositions and methods described herein include antibodies or fragments thereof that specifically bind to human CD5. Human CD5 is also called LEU1 or T1. Human CD5 is a type I transmembrane glycoprotein found on the surface of thymocytes, T lymphocytes, and a subset of B lymphocytes. Two isoforms of human CD5 have been identified. Isoform 1 contains 438 amino acids and is described in Jones et al. (1988) Nature 323 (6086), pp. 346-349 and below (NCBI Reference Sequence: NP_001333385.1): MVCSQSWGRS SKQWEDPSQASKVCQRLNCG VPLSLGPFLV TYTPQSSIICYGQL GSFSNCSHSRNDMCHS LGLTCLEPQKTTPPTTRPPPTTTPEPTAPP RLQLVAQSGG QHCAGVVEFYSGSLGGTISY EAQDKTQDLE NFLCNNLQCG SFLKHLPETE AGRAQDPGEP REHQPLPIQWKIQNSSCTSL EHCFRKIKPQ KSGRVLALLC SGFQPKVQSR LVGGSSICEG TVEVRQGAQWAALCDSSSAR SSLRWEEVCR EQQCGSVNSY RVLDAGDPTS RGLFCPHQKL SQCHELWERNSYCKKVFVTCQDPNPAGLAAGTVASIILAL VLLVVLLVVC GPLAYKKLVK KFRQKKQRQWIGPTGMNQNM SFHRNHTATV RSHAENPTAS HVDNEYSQPP RNSHLSAYPA LEGALHRSSMQPDNSSDSDY DLHGAQRL (SEQ ID NO: 286)

[0178] T cells have been shown to express CD5, a cell adhesion molecule involved in both the proliferation response of activated T cells and T cell helper function. It has also been shown to function as a receptor and deliver costimulatory signals to T cells by interacting with CD72, a cell surface protein exclusive to B cells. Antibodies and antigen-binding fragments thereof that bind to CD5 can suppress T cell activation and T cell-mediated immune responses to hematopoietic stem cell grafts, for example, by inhibiting the interaction between CD5 and CD72. Antibodies and antigen-binding fragments thereof that bind to CD5 can also be used to directly kill CD5+ T cells by conjugating the antibody or antigen-binding fragment 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 can recruit complement proteins to T cells.

[0179] Furthermore, a subset of activated B cells has been shown to express CD5, and this expression pattern is particularly common among autoreactive B cells (Werner-Favre et al., European Journal of Immunology 19:1209-1231 (1989), the entire disclosure of which is incorporated herein by reference). CD5 has also been shown to be expressed by a subset of NK cells; in particular, in patients with multiple myeloma, a population of low-density CD5+ (CD5LOW+) NK cells has been shown to exist, and this surface antigen has been implicated in NK cell activation (Ishiyama et al., Anticancer Research 14:725-730 (1994), the entire disclosure of which is incorporated herein by reference). Therefore, antibodies that specifically bind to CD5, or antigen-binding fragments thereof, can be used to inhibit the activation of B cells and NK cells. Antibodies and antigen-binding fragments thereof that bind CD5 can also be used to directly kill CD5+ B cells and NK cells by conjugating the antibody or antigen-binding fragment thereof to a cytotoxin (as described herein or known in the art) or by using an unconjugated antibody or antigen-binding fragment thereof that can recruit complement proteins to the B cell or NK cell.

[0180] Described herein are antibodies and antigen-binding fragments thereof that specifically bind to CD5 polypeptides, e.g., human CD5 polypeptides, and uses thereof. In exemplary embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to CD5 polypeptides comprise a heavy chain variable region and a light chain variable region.

[0181] 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 having the amino acid sequence of SEQ ID NO: 42. In one embodiment, the heavy chain variable region comprises a VH CDR2 having the amino acid sequence of SEQ ID NO: 43. In one embodiment, the heavy chain variable region comprises a VH CDR3 having the amino acid sequence of SEQ ID NO: 44. In one embodiment, the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44. In one embodiment, the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44. In one embodiment, the heavy chain variable region comprises a VH CDR1 having SEQ ID NO: 42, a VH CDR2 having SEQ ID NO: 43, and a VH CDR3 having SEQ ID NO: 44.

[0182] 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 having the amino acid sequence of SEQ ID NO: 45. In one embodiment, the light chain variable region comprises a VL CDR2 having the amino acid sequence of SEQ ID NO: 46. In one embodiment, the light chain variable region comprises a VL CDR3 having the amino acid sequence of SEQ ID NO: 47. In one embodiment, the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47. In one embodiment, the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47. In one embodiment, the light chain variable region comprises a VL CDR1 having SEQ ID NO: 45, a VL CDR2 having SEQ ID NO: 46, and a VL CDR3 having SEQ ID NO: 46.

[0183] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 having SEQ ID NO: 42, a VH CDR2 having SEQ ID NO: 43, and a VH CDR3 having SEQ ID NO: 44, and a light chain variable region comprising a VL CDR1 having SEQ ID NO: 45, a VL CDR2 having SEQ ID NO: 46, and a VL CDR3 having SEQ ID NO: 47.

[0184] In certain embodiments, one or more of the CDRs (i.e., one or more heavy chain CDRs having SEQ ID NOs: 42-44, and / or one or more light chain CDRs having SEQ ID NOs: 45-47) may contain conservative amino acid substitutions (or two, three, four, or five amino acid substitutions) while retaining the CD5 specificity of the antibody (i.e., similar specificity to that of an antibody or antigen-binding fragment thereof comprising the heavy chain CDRs of SEQ ID NOs: 42-44 and the light chain CDRs of SEQ ID NOs: 45-47).

[0185] In certain embodiments, the anti-CD5 antibody or antigen-binding fragment thereof is murine antibody 5D7, or a humanized version thereof. Murine antibody 5D7 binds to human CD5 and is described in U.S. Patent Publication No. 20008 / 0245027, the disclosure of which pertains to the antibody sequence herein is incorporated by reference. SEQ ID NOS: 54-59 in Table 5 correspond to the CDRs of murine anti-CD5 antibody 5D7. Humanized versions of anti-CD5 antibody 5D7 are set forth in SEQ ID NOS: 282 (humanized heavy chain variable region) and 283 (humanized light chain variable region). In one embodiment, the ADCs and uses thereof described herein include antibodies comprising the CDRs set forth in SEQ ID NOS: 54-59. In one embodiment, the ADCs and uses thereof described herein include antibodies comprising the heavy and light chain variable regions set forth in SEQ ID NOS: 282 and 283, respectively.

[0186] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing the amino acid sequence set forth in SEQ ID NO: 282. In another embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having an amino acid sequence having at least 95% identity to SEQ ID NO: 282, e.g., at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 282. In a specific embodiment, the antibody comprises a modified heavy chain (HC) variable region comprising an HC variable domain having SEQ ID NO: 282 or a variant of SEQ ID NO: 282, wherein the variant (i) differs from SEQ ID NO: 282 in 1, 2, 3, 4 or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 282 in up to 5, 4, 3, 2 or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 282 in 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or deletions. and / or (iv) an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 282, wherein in any of (i)-(iv), the amino acid substitutions may be conservative or non-conservative amino acid substitutions, and the modified heavy chain variable region may have enhanced biological activity compared to the heavy chain variable region of SEQ ID NO: 282 while retaining the CD5 binding specificity of the antibody, i.e., has similar binding specificity as an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 282.

[0187] In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 283. In another embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region having an amino acid sequence having at least 95% identity to SEQ ID NO: 283, e.g., at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 283. In a specific embodiment, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain having SEQ ID NO: 283 or a variant of SEQ ID NO: 283, wherein the variant (i) differs from SEQ ID NO: 283 in 1, 2, 3, 4 or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 283 in up to 5, 4, 3, 2 or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 283 in 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or deletions. and / or (iv) an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 283, wherein in any of (i) to (iv), the amino acid substitutions may be conservative or non-conservative amino acid substitutions, and the modified light chain variable region may retain the CD5 binding specificity of the antibody, i.e., have similar binding specificity as an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 283, while having enhanced biological activity compared to the light chain variable region of SEQ ID NO: 283.

[0188] In exemplary embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region containing an amino acid sequence having at least 95% identity to SEQ ID NO: 282, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to SEQ ID NO: 282, and a light chain variable region containing an amino acid sequence having at least about 95% identity to SEQ ID NO: 283, e.g., at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity to SEQ ID NO: 283. In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having SEQ ID NO: 282 and a light chain variable region having SEQ ID NO: 283.

[0189] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:288 and a light chain variable region comprising SEQ ID NO:289.

[0190] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising SEQ ID NO:291 and a light chain variable region comprising SEQ ID NO:290.

[0191] In another embodiment, the anti-CD5 antibody or antigen-binding fragment thereof may comprise a heavy chain variable region comprising a VH CDR1 containing the amino acid sequence of SEQ ID NO: 54. In one embodiment, the heavy chain variable region comprises a VH CDR2 containing the amino acid sequence of SEQ ID NO: 55. In one embodiment, the heavy chain variable region comprises a VH CDR3 containing the amino acid sequence of SEQ ID NO: 56. In one embodiment, the heavy chain variable region comprises one or more VH CDRs selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56. In one embodiment, the heavy chain variable region comprises two or more VH CDRs selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56. In one embodiment, the heavy chain variable region comprises a VH CDR1 containing SEQ ID NO: 54, a VH CDR2 containing SEQ ID NO: 55, and a VH CDR3 containing SEQ ID NO: 56.

[0192] 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 containing the amino acid sequence of SEQ ID NO:57. In one embodiment, the light chain variable region comprises a VL CDR2 containing the amino acid sequence of SEQ ID NO:58. In one embodiment, the light chain variable region comprises a VL CDR3 containing the amino acid sequence of SEQ ID NO:596. In one embodiment, the light chain variable region comprises one or more VL CDRs selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59. In one embodiment, the light chain variable region comprises two or more VL CDRs selected from the group consisting of SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:59. In one embodiment, the light chain variable region comprises a VL CDR1 containing SEQ ID NO:57, a VL CDR2 containing SEQ ID NO:58, and a VL CDR3 containing SEQ ID NO:59.

[0193] In an exemplary embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 containing SEQ ID NO: 54, a VH CDR2 containing SEQ ID NO: 55, and a VH CDR3 containing SEQ ID NO: 56, and a light chain variable region comprising a VL CDR1 containing SEQ ID NO: 57, a VL CDR2 containing SEQ ID NO: 58, and a VL CDR3 containing SEQ ID NO: 59.

[0194] In certain embodiments, one or more of the CDRs (i.e., one or more heavy chain CDRs having SEQ ID NOs: 54-56 and / or one or more light chain CDRs having SEQ ID NOs: 57-59) may contain conservative amino acid substitutions (or two, three, four, or five amino acid substitutions) while retaining the CD5 specificity of the antibody (i.e., similar specificity to that of an antibody or antigen-binding fragment thereof comprising the heavy chain CDRs of SEQ ID NOs: 54-56 and the light chain CDRs of SEQ ID NOs: 57-59).

[0195] 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 immunization, computational modeling techniques, and in vitro selection methods such as phage display and cell-based display platforms described below.

[0196] Anti-CD5 antibodies that can be used in combination with the compositions and methods described herein include those with one or both of the following variable regions, or an amino acid sequence with at least 85% sequence identity (e.g., an amino acid sequence with 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity): V having the following amino acid sequence: L DIQMTQSPSSMSASLGDRVTITCRASQDINSYLSWFQQKPGKSPKTLIYRANRL VDGVPSRFSGSGSGTDYTLTISSLQYEDFGIYYCQQYDESPWTFGGGTKLEIK (SEQ ID NO: 26), and V having the following amino acid sequence: H QIQLVQSGPGLKKPGGSVRISCAASGYTFTNYGMNWVKQAPGKGLRWMGWI NTHTGEPTYADDFKGRFTFSLDTSKSTAYLQINSLRAEDTATYFCTRRGYDWY FDVWGQGTTVTVSS (SEQ ID NO: 27).

[0197] The aforementioned V L and V H Antibodies and antigen-binding fragments thereof comprising the sequences of SEQ ID NO:26 and SEQ ID NO:27 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. In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises the sequences of SEQ ID NO:26 and SEQ ID NO:27. L and V HIn some embodiments, the anti-CD5 antibodies and antigen-binding fragments thereof comprise the V chain of SEQ ID NO:26 and SEQ ID NO:27. L and V H In some embodiments, the anti-CD5 antibodies and antigen-binding fragments thereof comprise CDRs containing the V chains of SEQ ID NO:26 and SEQ ID NO:27. L and V H CDRs contained in the V L and V H The remainder of the sequence is SEQ ID NO: 26 and SEQ ID NO: 27. L and V H having at least 85% (e.g., 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity to the sequence.

[0198] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof includes the following CDRs: CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 28), CDR-H2 having the amino acid sequence NTHTGE (SEQ ID NO: 29), CDR-H3 having the amino acid sequence RGYDWYFDV (SEQ ID NO: 30); CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 31), CDR-L2 having the amino acid sequence RANRLVD (SEQ ID NO: 32), and CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 33).

[0199] Additional anti-CD5 antibodies that can be used in combination with the compositions and methods described herein include those with one or both of the following variable regions, or an amino acid sequence with at least 85% sequence identity (e.g., an amino acid sequence with 85%, 90%, 95%, 97%, 98%, 99% or more sequence identity): V having the following amino acid sequence: L DIQMTQSPSSLSASVGDRVTITCRASQDINSYLSWFQQKPGKAPKTLIYRANRL ESGVPSRFSGSGSGTDYTLTIS SLQYEDFGIYYCQQYDESPWTFGGGTKLEIK (SEQ ID NO: 34), and V having the following amino acid sequence: H EIQLVQSGGGLVKPGGSVRISCAASGYTFTNYGMNWVRQAPGKGLEWMGWI NTHYGEPTYADSFKGTRTFSLDDSKNTAYLQINSLRAEDTAVYFCTRRGYDW YFDVWGQGGTTVTVSS (SEQ ID NO: 35)

[0200] In some embodiments, the aforementioned V L and V H Antibodies and antigen-binding fragments thereof comprising the 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, e.g., he3 antibodies. In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises the sequences of SEQ ID NO:28 and SEQ ID NO:29. L and V H CDRs contained in the V L and V H The remainder of the sequence is SEQ ID NO: 28 and SEQ ID NO: 29. L and V H having at least 85% (eg, 85%, 90%, 95%, 97%, 98%, 99% or more) sequence identity to the sequence.

[0201] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises the following CDRs: CDR-H1 having the amino acid sequence GYTFTNY (SEQ ID NO: 36), CDR-H2 having the amino acid sequence NTHYGE (SEQ ID NO: 37), CDR-H3 having the amino acid sequence RRGYDWYFDV (SEQ ID NO: 38); CDR-L1 having the amino acid sequence RASQDINSYLS (SEQ ID NO: 39), CDR-L2 having the amino acid sequence RANRLES (SEQ ID NO: 40), and CDR-L3 having the amino acid sequence QQYDESPWT (SEQ ID NO: 41).

[0202] Antibodies and antigen-binding fragments thereof comprising the foregoing CDR sequences are described, for example, in U.S. Pat. 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.

[0203] Other anti-CD5 antibodies that can be used in combination with the compositions and methods described herein are described, for example, in U.S. Pat. Nos. 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 disclosures of which are incorporated herein by reference as they relate to anti-CD5 antibodies and antigen-binding fragments thereof.

[0204] Other anti-CD5 antibodies that can be used in combination 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. Patent Nos. 4,515,894, 4,657,760, and 4,363,799, the disclosures of which are incorporated herein by reference as they relate to anti-CD5 antibodies and antigen-binding fragments thereof.

[0205] Still other anti-CD5 antibodies that can be used in combination with the compositions and methods described herein include those described in U.S. Pat. No. 8,679,500 and WO 2010 / 145895, such as the anti-CD5 antibody MAT304, the disclosures of which are incorporated herein by reference as they relate to anti-CD5 antibodies and antigen-binding fragments thereof.

[0206] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, anti-CD5 antibodies such as the anti-CD5 antibody T-101 described in U.S. Patent No. 4,675,386 (produced by the hybridoma deposited under ATCC Accession No. CRL-8023), Manske et al. (J Immunol 136:4721-4728(1986)), Shawler et al. (Cancer Res 44:5921-5927(1984)), Royston et al. (Blood 54 Suppl. 1:106a-106a(1979)), and Royston et al. (J Immunol 125:725-731(1980)). The disclosure of U.S. Patent No. 4,675,386 is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0207] 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 cells deposited as ATCC HB9285 (anti-CD5 conjugate zolimomab aritox-ricin protein A chain-linked antibody). Such antibodies are described in WO 1989006968; Henslee-Downey et al., Transplantation 61:738-45, 1996; Henslee et al., Transplant. Proc. 21:3004-3007, 1989; and Przepiorka et al., Ther. Immunol. 1:77-82, 1994, the disclosures of each of which are incorporated herein by reference as they relate to anti-CD5 antibodies and antigen-binding fragments thereof.

[0208] Additional anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include those described in U.S. Application No. 20110250203 and WO 2010 / 022737. See also the teachings of Koefoed et al., Br.J.Haematol, 2013 ("Koefoed et al."). The disclosures of U.S. Application No. 20110250203 and WO 2010 / 022737 and Koefoed et al. are incorporated herein by reference as they relate to anti-CD5 antibodies and antigen-binding fragments thereof.

[0209] Anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include one or more, or all, of the following CDRs: CDR-H1 having the amino acid sequence GYSITSGYY (SEQ ID NO: 42), CDR-H2 having the amino acid sequence ISYSGFT (SEQ ID NO: 43), CDR-H3 having the amino acid sequence AGDRTGSWFAY (SEQ ID NO: 44), CDR-L1 having the amino acid sequence QDISNY (SEQ ID NO: 45), CDR-L2 having the amino acid sequence ATS (SEQ ID NO: 46), and CDR-L3 having the amino acid sequence LQYASYPFT (SEQ ID NO: 47).

[0210] Antibodies and antigen-binding fragments thereof comprising the aforementioned CDR sequences are described, for example, in U.S. Pat. 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.

[0211] Anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include one or more, or all, of the following CDRs: CDR-H1 having the amino acid sequence GYIFTNYG (SEQ ID NO: 48), CDR-H2 having the amino acid sequence INTYNGEP (SEQ ID NO: 49), CDR-H3 having the amino acid sequence ARGDYYGYEDY (SEQ ID NO: 50); CDR-L1 having the amino acid sequence QGISNY (SEQ ID NO: 51), CDR-L2 having the amino acid sequence YTS (SEQ ID NO: 52), and CDR-L3 having the amino acid sequence QQYSKLPWT (SEQ ID NO: 53).

[0212] Antibodies and antigen-binding fragments thereof comprising the aforementioned CDR sequences are described, for example, in US Pat. No. 8,679,500.

[0213] Anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include one or more, or all, of the following CDRs: CDR-H1 having the amino acid sequence FSLSTSGMG (SEQ ID NO: 54), CDR-H2 having the amino acid sequence WWDDD (SEQ ID NO: 55), CDR-H3 having the amino acid sequence RRATGTGFDY (SEQ ID NO: 56), CDR-L1 having the amino acid sequence QDVGTA (SEQ ID NO: 57), CDR-L2 having the amino acid sequence WTSTRHT (SEQ ID NO: 58), and CDR-L3 having the amino acid sequence YNSYNT (SEQ ID NO: 59).

[0214] Antibodies and antigen-binding fragments thereof comprising the foregoing 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.

[0215] Other anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include those described in WO 1992 / 014491, such as, for example, the anti-CD5 antibodies produced by the hybridoma cell line deposited at the Pasteur Institute on January 10, 1991 under numbers 1-1025. The disclosure of WO 1992 / 014491 is incorporated herein by reference as it relates to anti-CD5 antibodies and antigen-binding fragments thereof.

[0216] Other anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include, for example, the anti-CD5 antibodies described in U.S. Pat. Nos. 6,010,902 and 7,192,736, U.S. Patent Application Publication Nos. 2011 / 0250203 and 2017 / 0129128, and WO 2016 / 172606, 1994 / 023747, and 1996 / 041608, the disclosures of which are incorporated herein by reference as they relate to anti-CD5 antibodies and antigen-binding fragments thereof.

[0217] In some embodiments, anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include those having a combination of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 regions as shown in Table 1 below.

[0218] [Table 1-1] [Table 1-2]

[0219] 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.

[0220] Antibodies and fragments thereof for use in conjunction with the compositions and methods described herein include variants of the above antibodies, such as antibody fragments that contain or lack an Fc domain, as well as humanized variants of the non-human antibodies and antibody-like protein scaffolds described herein (e.g., humanized variants of the non-human antibodies and antibody fragments described herein) that contain one or more or all of the CDRs or equivalent regions of the antibodies or antibody fragments described herein. 10 Exemplary antigen-binding fragments of the foregoing 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, among others.

[0221] The aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can be used in various embodiments described herein, including, for example, methods for depleting CD5+ cells in a human subject.The aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can also be conjugated to a drug, such as a cytotoxin, as described herein.Additional anti-CD5 antibodies that can be used in the compositions and methods described herein are described in U.S. Patent No. 8,679,500, U.S. Patent Application Publication No. 2011 / 0250203, and U.S. Patent Application Publication No. 2008 / 0254027, the entire contents of each of which are incorporated herein by reference. Additional anti-CD5 antibodies that may be used in embodiments of the compositions and methods described herein include, for example, the monoclonal antibody T101 described in Dillman et al., J. Clin. Oncol. (1984), 2(8):881-891, and the monoclonal antibody Leu-1 described in Miller et al., Blood (1983), 62(5)988-95.

[0222] In one embodiment, an anti-CD5 antibody or binding fragment thereof comprises an altered Fc region, the altered Fc region comprising at least one amino acid modification compared to a wild-type Fc region, such that the molecule has altered affinity or binding to FcgammaR (FcγR). Certain amino acid positions within the Fc region have been shown by crystallographic studies to make direct contact with FcγR, specifically amino acids 234-239 (hinge region), 265-269 (B / C loop), 297-299 (C' / E loop), and 327-332 (F / G loop) (see Sondermann et al., 2000, Nature, 406:267-273). The antibodies described herein comprise variant Fc regions, which comprise modifications of at least one residue that makes direct contact with FcγR based on structural and crystallographic analysis. In one embodiment, the Fc region of an anti-CD2 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NH1, MD (1991), which is expressly incorporated herein by reference. "Kabat's EU index" 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 the antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index of Kabat. In one embodiment, the Fc region comprises a 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 of Kabat. In one embodiment, the Fc region comprises a L235A mutation. In yet another embodiment, the Fc region comprises both the L234A and L235A mutations. In a further embodiment, the Fc region comprises D265C, L234A, and L235A mutations.In a further embodiment, the Fc region comprises D265C, L234A, L235A, and H435A mutations. In a further embodiment, the Fc region comprises D265C and H435A mutations.

[0223] The antibodies used herein are, 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 may include 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, 1253A, S254T, T256E, P2571, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations.

[0224] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof is conjugated to a cytotoxin (e.g., an amatoxin) via a cysteine ​​residue in the Fc domain of the antibody or antigen-binding fragment thereof. In some embodiments, the cysteine ​​residue is introduced by mutation in the Fc domain of the antibody or antigen-binding fragment thereof. For example, the cysteine ​​residue may be selected from the group consisting of Cys118, Cys239, and Cys265. In one embodiment, the Fc region of the anti-CD5 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index of Kabat. In one embodiment, the Fc region comprises a D265C mutation. In one embodiment, the Fc region comprises D265C and H435A mutations. In one embodiment, the Fc region comprises D265C and H435A mutations.

[0225] Thus, in one embodiment, the Fc region contains a mutation that results in a reduced half-life.An antibody with a short half-life may be advantageous in certain cases where the antibody is expected to function as a short-lived therapeutic agent, for example, in the conditioning step described herein, in which the antibody is administered followed by HSCs.Ideally, the antibody is substantially removed before delivery of HSCs, which, unlike endogenous stem cells, generally also express CD2 but are not targeted by anti-CD5 antibodies.In one embodiment, the Fc region contains a mutation at position 435 (EU index according to Kabat).In one embodiment, the mutation is an H435A mutation.

[0226] The aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can be used in various embodiments of the invention described herein, including, for example, methods for depleting CD5+ cells in a human subject. The aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can also be conjugated to an agent, e.g., a cytotoxin such as an amatoxin, as described herein.

[0227] Methods for identifying anti-CD2 and anti-CD5 antibodies Methods for high-throughput screening of libraries of antibodies or antibody fragments that bind to CD2 or CD5 can be used to identify and affinity mature agents useful for conditioning patients (e.g., human patients) in need of hematopoietic stem cell therapy and / or for directly treating cancer or autoimmune diseases as described herein. Such methods include in vitro display techniques known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display, among others. The use of phage display to isolate antibodies or antigen-binding fragments that bind to biologically relevant molecules has been reviewed, for example, in 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 disclosures of each of which are incorporated herein by reference as they relate to in vitro display techniques. 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 disclosures of each of which are incorporated herein by reference as they relate to the discovery of antigen-binding molecules. Proteins, such as multimeric proteins, have been successfully phage-displayed as functional molecules (see, e.g., EP0349578, EP4527839, and EP0589877, and Chiswell and McCafferty, Trends Biotechnol. 10:80-84, 1992, the disclosures of each of which are incorporated herein by reference as they relate to the use of in vitro display techniques for the discovery of antigen-binding molecules).Additionally, functional antibody fragments, such as Fab fragments 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 the use of in vitro display techniques for the discovery of antigen-binding molecules). These techniques can be used, inter alia, to identify and improve the affinity of antibodies and antigen-binding fragments thereof that bind to CD2 or CD5, which can then be used to deplete CD2+ T cells and / or NK cells (or CD5+ T cells and / or NK cells) in patients (e.g., human patients) in need of hematopoietic stem cell transplantation therapy and / or suffering from cancer or an autoimmune disease as described herein.

[0228] Additional techniques can be used to identify antibodies or antigen-binding fragments thereof that bind to CD2 or CD5 on the surface of cells (e.g., T cells, B cells, or NK cells) and are subsequently internalized by the cells, for example, by receptor-mediated endocytosis. For example, the in vitro display techniques described above can be adapted to screen for antibodies and antigen-binding fragments thereof that bind to CD2 on the surface of T cells or NK cells and are subsequently internalized. For example, the in vitro display techniques described above can be adapted to screen for antibodies and antigen-binding fragments thereof that bind to CD2 or CD5 on the surface of T cells, B cells, or NK cells and are subsequently internalized. Phage display represents one such technique that can be used in conjunction with this screening paradigm. To identify anti-CD2 antibodies and fragments thereof that bind to CD2 or CD5 and are subsequently internalized by T cells, B cells, and / or NK cells, those skilled in the art can use the phage display technique described in Williams et al., Leukemia 19:1432-1438, 2005, the entire disclosure of which is incorporated herein by reference. For example, mutagenesis techniques known in the art can be used to generate antibodies, antibody fragments, such as scFv fragments, Fab fragments, diabodies, triabodies, and 10 Recombinant phage libraries can be generated that encode antibodies containing randomized amino acid cassettes (e.g., one or more, or all, of the CDRs or equivalent regions, or antibodies or antibody fragments). The framework regions, hinges, 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 variations relative to human germline antibody sequences.

[0229] Using phage display techniques described herein or known in the art, a phage library containing randomized antibodies or antibody fragments covalently linked to phage particles can be incubated with CD2 or CD5 antigens, for example, by first incubating the phage library with a blocking agent (e.g., milk protein, bovine serum albumin, and / or IgG) to remove phage encoding antibodies or fragments thereof that exhibit nonspecific 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 CD2+ (or CD5+) T cells, B cells, or NK cells. The phage library can be incubated with T cells, B cells, or NK cells for a period of time sufficient to allow CD2-specific antibodies or antigen-binding fragments thereof (CD5-specific antibodies or antigen-binding fragments thereof) to bind to cell-surface CD2 or CD5 and subsequently be internalized by the T cells, B cells, or NK cells (e.g., for 30 minutes to 6 hours at 4°C, e.g., 1 hour at 4°C). Phage containing antibodies or fragments thereof that do not exhibit sufficient affinity for CD2 or CD5 to allow binding to and uptake by T cells or NK cells can then be removed by washing the cells, for example, with cold (4°C) 0.1 M glycine buffer at pH 2.8. Phage bound to antibodies or fragments thereof internalized by T cells, B cells, and / or NK cells can be identified, for example, by lysing the cells and recovering the internalized phage from the cell culture medium. The phage can then be amplified in bacterial cells, for example, by incubating the bacterial cells with the recovered phage in 2xYT medium using methods known in the art. The phage recovered from this medium can then be characterized, for example, by determining the nucleic acid sequence of the gene(s) encoding the antibody or fragment(s) inserted into the phage genome.The encoded antibodies, fragments thereof, can then be prepared de novo by chemical synthesis (eg, of antibody fragments such as scFV fragments) or by recombinant expression (eg, of full-length antibodies).

[0230] An exemplary method for the in vitro evolution of anti-CD2 or anti-CD5 antibodies for use in conjunction with the compositions and methods described herein is phage display. Phage display libraries are designed to detect CDRs of antibodies or similar regions of antibody-like scaffolds (e.g., 10 These antibodies can be generated by creating a series of designed mutations or alterations in the coding sequence of the BC, CD, and DE loops of the Fn3 domain. The template antibody-encoding sequence into which these mutations are introduced can be, for example, a naive human germline sequence. These mutations can be made using standard mutagenesis techniques known in the art. Thus, each mutant sequence encodes an antibody corresponding to the template except for one or more amino acid mutations. Retrovirus and phage display vectors can be engineered using standard vector construction techniques known in the art. The P3 phage display vector can be used in conjunction with a compatible protein expression vector to generate a phage display vector for antibody diversification.

[0231] The mutant DNA provides sequence diversity, with each transformant phage displaying one mutation of the initial template amino acid sequence encoded by its DNA, resulting in a phage population (library) displaying a vast number of different but structurally related amino acid sequences. Due to the well-defined structure of antibody hypervariable regions, amino acid mutations introduced in phage display screens are expected to alter the binding properties of a binding peptide or domain without significantly altering its overall molecular structure.

[0232] In a typical screening procedure, a phage library is contacted with and allowed to bind to CD2 or CD5 or an epitope thereof. To facilitate separation of bound and unbound phage, it is convenient to immobilize the target on a solid support. Phage bearing CD2- or CD5-binding moieties can form complexes with the target on the solid support, while unbound phage remain in solution and can be washed away with excess buffer. 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 means.

[0233] The recovered phage can then be amplified through infection of bacterial cells, and the screening process can be repeated using a new pool depleted of unconjugated antibodies and enriched for antibodies that bind to CD2 or CD5. The recovery of even a few binding phage is sufficient to amplify the phage for subsequent screening iterations. After several rounds of selection, the gene sequences encoding antibodies or antigen-binding fragments from selected phage clones in the binding pool are determined by conventional methods, thereby revealing the peptide sequences that confer the phage binding affinity for the target. During the panning process, the sequence diversity of the population decreases with each round of selection until only the desired peptide-binding antibodies remain. The sequences may converge to a small number of related antibodies or antigen-binding fragments thereof. An increase in the number of phage recovered with each round of selection is an indication that library convergence has occurred within the screen.

[0234] Another method for identifying anti-CD2 or anti-CD5 antibodies involves using a humanized non-human antibody that binds to CD2 or an anti-CD2 antibody, for example, according to the following procedure. A non-human antibody that binds to CD2 or an anti-CD5 antibody can be humanized, for example, according to the following procedure. Consensus human antibody heavy and light chain sequences are known in the art (see, e.g., the "VBASE" human germline sequence database; Kabat et al. Sequences of Proteins of Immunological Interest, 5th ed., USDapartment 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 disclosures of each of which are incorporated herein by reference as they relate to consensus human antibody heavy and light chain sequences). Using established procedures, those skilled in the art can identify the variable domain framework residues and CDRs of consensus antibody sequences (for example, by sequence alignment).To produce humanized antibodies, one or more CDRs of the heavy and / or light chain variable domains of consensus human antibodies can be replaced with one or more corresponding CDRs of non-human antibodies that bind to CD2 or CD5.This CDR exchange can be carried out using gene editing techniques described herein or known in the art.

[0235] An example of a consensus human antibody variable domain is the following heavy chain variable domain identified in U.S. Pat. No. 6,054,297: [ka] (SEQ ID NO: 11) and a light chain variable domain: [ka] (SEQ ID NO: 12), the disclosure of which is incorporated herein by reference as it relates to human antibody consensus sequences. The CDRs in the above sequence are shown in bold.

[0236] To produce a humanized antibody, a polynucleotide encoding the above consensus sequence can be recombinantly expressed, in which one or more variable region CDRs are replaced with one or more variable region CDR sequences of a non-human antibody that binds to CD2 or CD5. Because the affinity of an antibody for CD2 or CD5 is primarily determined by the CDR sequence, the resulting humanized antibody is expected to exhibit approximately the same affinity for CD2 or CD5 as the non-human antibody from which it 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, among others.

[0237] The uptake capacity of the prepared antibody or fragment thereof can be assessed, for example, using radionuclide uptake assays known in the art. For example, an anti-CD2 antibody or fragment thereof (or an anti-CD5 antibody or fragment thereof) identified using in vitro display techniques described herein or known in the art can be used to assess the uptake capacity of the prepared antibody or fragment thereof. 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 Bi or 225 It can be functionalized by incorporation of a radioisotope such as Ac. For example, 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 Radioactive halogens such as At can be incorporated into antibodies or fragments thereof using beads, such as polystyrene beads containing an electrophilic halogen reagent (e.g., iodinated beads, Thermo Fisher Scientific, Cambridge, MA). The radiolabeled antibody or fragment can be incubated with T cells, B cells, and / or NK cells for a time sufficient to allow uptake (e.g., 30 minutes to 6 hours at 4°C, e.g., 1 hour at 4°C). The cells can then be washed to remove unincorporated antibody or fragment (e.g., using cold (4°C) 0.1 M glycine buffer, pH 2.8). Internalized antibody or fragment can be identified by detecting radiation (e.g., gamma radiation) emitted from the resulting T cells, B cells, and / or NK cells compared to radiation (e.g., gamma radiation) emitted from the recovered wash buffer.

[0238] For recombinant production of an anti-CD2 or anti-CD5 antibody, for example, nucleic acid encoding the antibody as described above is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0239] Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly if glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0240] Vertebrate cells can also be used as hosts, for example, mammalian cell lines adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines include SV40 (COS-7) transformed monkey kidney CV1, human embryonic kidney (e.g., 293 or 293 cells described in Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney (BHK) cells, mouse Sertoli cells (e.g., TM4 cells described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney (CV1), African green monkey kidney (VERO-76), human cervical carcinoma (HELA), canine kidney (MDCK), buffalo rat hepatocytes (BRL 3A), human lung (W138), human hepatocytes (Hep G2), and mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals of NY Acad. Sci. 383:44-68 (1982). 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 specific mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Humana Press, Totowa, NJ), pp. 255-268 (2003). In one embodiment, the host cell is eukaryotic, e.g., Chinese hamster ovary (CHO) cells or lymphoid cells (such as Y0, NS0, or Sp20 cells).

[0241] Antibody-drug conjugates (ADCs) The anti-CD5 or CD2 antibody drug conjugate that can be used in the methods described herein comprises an anti-CD5 or anti-CD2 antibody conjugated to a cytotoxin via a linker.The anti-CD5 or anti-CD2 antibody that can be used in the methods described herein is known in the art and is described above.The cytotoxin, linker and conjugation method are described below.

[0242] cytotoxin The antibodies and antigen-binding fragments thereof described herein (e.g., antibodies, antigen-binding fragments that recognize and bind to CD2 or CD5) can be conjugated to a cytotoxin such as Pseudomonas exotoxin A, deBouganin, diphtheria toxin, amatoxins such as α-amanitin, saporin, maytansine, maytansinoids, auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolinobenzodiazepines, and indolinobenzodiazepine dimers, or variants thereof, or another cytotoxic compound described herein or known in the art, for (i) direct treatment of cancer or autoimmune diseases described herein, or (ii) depletion of endogenous immune cells to prevent or reduce the likelihood of rejection of hematopoietic stem cells upon transplantation into a patient (e.g., a human patient) in need of hematopoietic stem cell transplantation therapy. In some embodiments, a cytotoxic molecule is conjugated to the internalized antibody or antigen-binding fragment thereof so that, after cellular uptake of the antibody or antigen-binding fragment, the cytotoxin can access its intracellular target and kill endogenous T cells, B cells, and / or NK cells. Cytotoxins suitable for use with the compositions and methods described herein include, among others known in the art, DNA intercalators (e.g., anthracyclines), agents capable of disrupting the mitotic spindle apparatus (e.g., vinca alkaloids, maytansine, maytansinoids, and their derivatives), RNA polymerase inhibitors (e.g., amatoxins such as α-amanitin and their derivatives), and agents capable of disrupting protein biosynthesis (e.g., agents exhibiting rRNA N-glycosidase activity, such as saporin and ricin A chain).

[0243] In some embodiments, the cytotoxin of the antibody-drug conjugate is an RNA polymerase inhibitor, hi some embodiments, the RNA polymerase inhibitor is an amatoxin or a derivative thereof.

[0244] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, such as α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, and proamanulin. The structure of various naturally occurring amatoxins is represented by Formula III and is disclosed, for example, in Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, pp. 450-459.

[0245] In one embodiment, the cytotoxin is amanitin. For example, the antibodies or antigen-binding fragments described herein can be conjugated to an amatoxin to form a conjugate represented by the formula Ab-ZL-Am, where Ab is an antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an amatoxin. Many positions on an amatoxin or its derivatives can serve as a linking moiety L, and thus a position for covalently binding an antibody or its antigen-binding fragment. For example, the antibodies and antigen-binding fragments described herein can be conjugated to an amatoxin to form a conjugate represented by the formula Ab-ZL-Am, where Ab is an antibody or antigen-binding fragment thereof, Z is a chemical moiety, L is a linker, and Am is an amatoxin. In some embodiments, Am-LZ is represented by the formula (I): [ka] is expressed as In the formula, R1 is H, OH, OR A , or OR C and R2 is H, OH, OR B , or OR C and R A and R B when present, together with the oxygen atom to which they are attached, combine to form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C or R Dand R4 is H, OH, OR C , OR D , R C or R D and R5 is H, OH, OR C , OR D , R C or R D and R6 is H, OH, OR C , OR D , R C or R D and R7 is H, OH, OR C , OR D , R C or R D and R8 is OH, NH2, OR C , OR D , NHR C or NR C R D and R9 is H, OH, OR C OR D and X is -S-, -S(O)- or -SO2-; R C is -LZ, R D is an optionally substituted alkyl (e.g., C1-C6 alkyl), an optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), an optionally substituted alkenyl (e.g., C2-C6 alkenyl), an optionally substituted heteroalkenyl (e.g., C2-C6 heteroalkenyl), an optionally substituted alkynyl (e.g., C2-C6 alkynyl), an optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, or an optionally substituted heteroaryl; L is a linker that is an optionally substituted alkylene (e.g., C-C alkylene), an optionally substituted heteroalkylene (C-C heteroalkylene), an optionally substituted alkenylene (e.g., C-C alkenylene), an optionally substituted heteroalkenylene (e.g., C-C heteroalkenylene), an optionally substituted alkynylene (e.g., C-C alkynylene), an optionally substituted heteroalkynylene (e.g., C-C heteroalkynylene), an optionally substituted cycloalkylene, an optionally substituted heterocycloalkylene, an optionally substituted arylene, an optionally substituted heteroarylene, a dipeptide, -C(=O)-, a peptide, or a combination thereof; Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an antibody or antigen-binding fragment that binds to CD2 or CD5.

[0246] In some embodiments, Am contains exactly one R C substituent. In some embodiments, the linker contains -(CH)2n- units, where n is an integer from 2 to 6. In some embodiments, the linker contains -((CH2)n, where n is 6. In some embodiments, LZ is: [ka] In the above formula, S is a sulfur atom (eg, from the -SH group of a cysteine ​​residue) that represents a reactive substituent present in an antibody or antigen-binding fragment thereof that binds to CD117.

[0247] In some embodiments, LZ is: [ka]

[0248] In some embodiments, Am-LZ-Ab is as follows: [ka]

[0249] In some embodiments, Am-LZ-Ab is as follows: [ka]

[0250] In some embodiments, Am-LZ has the following formula (IA): [ka] is expressed as In the formula, R1 is H, OH, OR A OR C and R2 is H, OH, OR B OR C and R A and R B, if present, together with the oxygen atom to which they are attached, join to form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C or R D and R4 is H, OH, OR C , OR D , R C or R D and R5 is H, OH, OR C , OR D , R C or R D and R6 is H, OH, OR C , OR D , R C or R D and R7 is H, OH, OR C , OR D , R C or R D and R8 is OH, NH2, OR C , OR D , NHR C or NRC R D and R9 is H, OH, OR C OR D and X is -S-, -S(O)- or -SO2-; R C is -LZ, R D is an optionally substituted alkyl (e.g., C-C alkyl), an optionally substituted heteroalkyl (e.g., C-C heteroalkyl), an optionally substituted alkenyl (e.g., C-C alkenyl), an optionally substituted heteroalkenyl (e.g., C-C heteroalkenyl), an optionally substituted alkynyl (e.g., C-C alkynyl), an optionally substituted heteroalkynyl (e.g., C-C heteroalkynyl), an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, or an optionally substituted heteroaryl; L is a linker such as an optionally substituted alkylene (e.g., C-C alkylene), optionally substituted heteroalkylene (e.g., C-C heteroalkylene), optionally substituted alkenylene (e.g., C-C alkenylene), optionally substituted heteroalkenylene (e.g., C-C heteroalkenylene), optionally substituted alkynylene (e.g., C-C alkynylene), optionally substituted heteroalkynylene (e.g., C-C heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -C(=O)-, peptide, disulfide, hydrazone, or combinations thereof, and is a-(CHCHO) p - group, p is an integer of 1 to 6, ((CH2) m O) n (CH2) m - group, where n and m are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or combinations thereof. Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an antibody or antigen-binding fragment thereof that binds to CD2 or CD5, and Am contains exactly one RC substituent.

[0251] In some embodiments, the linker is -((CH) n and n is 6. In some embodiments, LZ is: [ka]

[0252] In some embodiments, LZ is as follows: [ka]

[0253] In some embodiments, the Am-LZ-Ab is as follows: [ka]

[0254] In some embodiments, the Am-LZ-Ab is as follows: [ka]

[0255] In some embodiments, Am-LZ has formula (IB): [ka] is expressed as In the formula, R1 is H, OH, OR A OR C and R2 is H, OH, OR B OR C and RA and R B when present, together with the oxygen atom to which they are attached, combine to form an optionally substituted 5-membered heterocycloalkyl group; R3 is H, R C or R D and R4 is H, OH, OR C , OR D , R C or R D and R5 is H, OH, OR C , OR D , R C or R D and R6 is H, OH, OR C , OR D , R C or R D and R7 is H, OH, OR C , OR D , R C or R D and R8 is OH, NH2, OR C , OR D , NHR C or NR C R D and R9 is H, OH, OR C OR D and X is -S-, -S(O)- or -SO2-; R C is -LZ, R Dis an optionally substituted alkyl (e.g., C-C alkyl), an optionally substituted heteroalkyl (e.g., C-C heteroalkyl), an optionally substituted alkenyl (e.g., C-C alkenyl), an optionally substituted heteroalkenyl (e.g., C-C heteroalkenyl), an optionally substituted alkynyl (e.g., C-C alkynyl), an optionally substituted heteroalkynyl (e.g., C-C heteroalkynyl), an optionally substituted cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, or an optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C-C alkylene), an optionally substituted heteroalkylene (C-C heteroalkylene), an optionally substituted alkenylene (e.g., C-C alkenylene), an optionally substituted heteroalkenylene (e.g., C-C heteroalkenylene), an optionally substituted alkynylene (e.g., C-C alkynylene), an optionally substituted heteroalkynylene (e.g., C-C heteroalkynylene), an optionally substituted cycloalkylene, an optionally substituted heterocycloalkylene, an optionally substituted arylene, an optionally substituted heteroarylene, a dipeptide, —C(═O)—, a peptide, a disulfide, a hydrazone, —(CHCHO) p - group (p is an integer of 1 to 6), ((CH2) m O) n (CH2) m - group (n and each m are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), or a combination thereof; Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an antibody or antigen-binding fragment thereof that binds to CD2 or CD5; Am just one R C Contains substituents.

[0256] In some embodiments, the linker L and the chemical moiety Z are both LZ. [ka]

[0257] In some embodiments, LZ is as follows: [ka]

[0258] In some embodiments, the Am-LZ-Ab is as follows: [ka]

[0259] In some embodiments, the Am-LZ-Ab is as follows: [ka]

[0260] In some embodiments, R A and R B together with the oxygen atom to which they are attached, combine to form a 5-membered heterocycloalkyl group of the formula: [ka] In the formula, Y is -C(=O)-, -C(=S)-, -C(=NR E )-, or -C(R E R E’ )- and R E and R E’ each independently represents an optionally substituted C-C alkylene-R C、 optionally substituted C1-C6 heteroalkylene-R C、 optionally substituted C2-C6 alkenylene-R C , optionally substituted C2-C6 heteroalkenylene-R C , 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 is.

[0261] In some embodiments, Am-LZ is represented by formula (IA) or (IB): R1 is H, OH, OR A OR C and R2 is H, OH, OR B OR C and R A and R B together with the oxygen atoms to which they are attached combine to form: [ka] R3 is H or R C and R4 is H, OH, OR C , OR D , R C or R D and R5 is H, OH, OR C , OR D , R C or R D and R6 is H, OH, OR C , OR D , R C or R D and R7 is H, OH, OR C , OR D , R C or R D and R8 is OH, NH2, OR C or NHR C and R9 is H or OH; X, R C and R Dare each as defined above.

[0262] In some embodiments, Am-LZ is represented by formula (IA) or (IB): R1 is H, OH, OR A OR C and R2 is H, OH, OR B OR C and R A and R B together with the oxygen atoms to which they are attached combine to form: [ka] R3 is H or R C and R4 and R5 are each independently H, OH, or OR C , R C OR D and R6 and R7 are each H; R8 is OH, NH2, OR C or NHR C and R9 is H or OH; X and R C are each as defined above.

[0263] In some embodiments, Am-LZ is represented by formula (IA) or (IB): In the formula, R1 is H, OH, or OR A and R2 is H, OH, or OR B and R A and R B together with the oxygen atoms to which they are attached combine to form: [ka] R3, R4, R6 and R7 are each H; R5 is OR Cand R8 is OH or NH2, R9 is H or OH; R C is as defined above. Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2016 / 0002298, the entire disclosure of which is incorporated herein by reference.

[0264] In some embodiments, Am-LZ is represented by formula (IA) or (IB), wherein R and R are each independently H or OH; R is R C and R4, R6 and R7 are each H; R5 is H, OH or OC1-C6 alkyl; R8 is OH or NH2, R9 is H or OH; X and R C is as defined above. Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2014 / 0294865, the entire disclosure of which is incorporated herein by reference.

[0265] In some embodiments, Am-LZ is represented by formula (IA) or (IB): wherein R1 and R2 are each independently H or OH; R3, R6 and R7 are each H; R4 and R5 are each independently H, OH, or OR C or R C and R8 is OH or NH2, R9 is H or OH; X and R C is as defined above. Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0266] In some embodiments, Am-LZ is represented by formula (IA) or (IB), wherein R and R are each independently H or OH; R3, R6 and R7 are each H; R4 and R5 are each independently H or OH; R8 is OH, NH2, OR C or NHR C and R9 is H or OH; R C is as defined above. Such amatoxin conjugates are described, for example, in U.S. Pat. Nos. 9,233,173 and 9,399,681, and U.S. Patent Application Publication No. 2016 / 0089450, the entire disclosures of which are incorporated herein by reference.

[0267] Additional amatoxins that can be used for conjugation to antibodies or antigen-binding fragments thereof in accordance with 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.

[0268] In some embodiments, Am-LZ has formula (II), (IIA), or (IIB): [ka] is expressed as wherein X is S, SO, or SO; R is H or a linker covalently attached to the antibody or antigen-binding fragment thereof via the chemical moiety Z, which is formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the antibody or antigen-binding fragment thereof; R is H or a linker covalently attached to the antibody or antigen-binding fragment thereof via the chemical moiety Z, which is formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the antibody or antigen-binding fragment thereof; when R is H, R is a linker; and when R is H, R is a linker.

[0269] In some embodiments, the linker is -(CH2) n― wherein n is an integer from 2 to 6. In some embodiments, R1 is a linker, R2 is H, and the linker and chemical moiety together are as follows: LZ. [ka]

[0270] In some embodiments, the Am-LZ-Ab is one of the following: [ka]

[0271] In some embodiments, the cytotoxin is α-amanitin. In some embodiments, the α-amanitin is a compound of Formula III. In some embodiments, the α-amanitin of Formula III is attached to an antibody or antigen-binding fragment thereof that binds to CD2 or CD5 via a linker L. The linker L can be attached to any of several possible positions (e.g., R 1 ~R 9 or any one of R to provide an α-amanitin-linker conjugate of formula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker is R 1In some embodiments, the linker is attached at the R 2 In some embodiments, the linker is attached at the R 3 In some embodiments, the linker is attached at the R 4 In some embodiments, the linker is attached at the R 5 In some embodiments, the linker is attached at the R 6 In some embodiments, the linker is attached at the R 7 In some embodiments, the linker is attached at the R 8 In some embodiments, the linker is attached at the R 9 In some embodiments, the linker comprises a hydrazine, disulfide, thioether, or 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 the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker is -((C=O)(CH2) n -unit, where n is an integer from 1 to 6.

[0272] In some embodiments, the linker is -(CH2) n -unit, where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH) n In some embodiments, the linker L and chemical moiety Z, taken together as LZ, are as follows: [ka]

[0273] Antibodies and antigen-binding fragments for use in conjunction with the compositions and methods described herein can be conjugated to amatoxins, such as α-amanitin, or variants thereof, using conjugation techniques known in the art or described herein. For example, as described in U.S. Patent Application Publication No. 2015 / 0218220, antibodies and antigen-binding fragments thereof that recognize and bind to CD2 or CD5 can be conjugated to amatoxins, such as α-amanitin, or variants thereof, the disclosure of which is incorporated herein by reference, for example, with respect to amatoxins, such as α-amanitin, and variants thereof, as well as covalent linkers that can be used for covalent conjugation. Synthetic methods for making amatoxins are described, for example, in U.S. Patent No. 9,676,702, which is incorporated herein by reference for the synthetic methods disclosed therein.

[0274] Antibodies or antigen-binding fragments for use in conjunction with the compositions and methods described herein can be conjugated to an amatoxin, such as α-amanitin, or a variant thereof, using conjugation techniques known in the art or described herein. For example, as described in U.S. Patent Application Publication No. 2015 / 0218220, an antibody or antigen-binding fragment thereof that recognizes and binds to CD2 or CD5 can be conjugated to an amatoxin, such as α-amanitin, or a variant thereof, the disclosure of which is incorporated herein by reference, for example, as it relates to amatoxins, such as α-amanitin, and variants thereof, as well as covalent linkers that can be used for covalent conjugation.

[0275] Exemplary antibody-drug conjugates useful in conjunction with the methods described herein can be formed by reaction of an antibody or antigen-binding fragment thereof with an amatoxin conjugated to a linker containing a substituent suitable for reaction with a reactive residue on the antibody or antigen-binding fragment thereof. Amatoxins conjugated to linkers containing a substituent suitable for reaction with a reactive residue on the antibody or antigen-binding fragment thereof described herein include, but are not limited to: 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin, 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin, 7'C-(4-(6-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin, 7'C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperazin-1-yl)-amatoxin, amatoxin, 7'C-(4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)-amatoxin, 7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)-amatoxin, 7'C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin, 7'C-( 4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amatoxine, 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine, 7'C-(4-(2-(3-carboxypropanamido)ethyl)piperidin-1-yl)-amatoxine, 7'C-(4-(2 -(2-bromoacetamido)ethyl)piperidin-1-yl)-amatoxine, 7'C-(4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)-amatoxine, 7'C-(4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)-amatoxine, 7'C-(4-(2-(maleimido)acetyl)piperazin-1-yl)-amatoxine,7'C-(4-(3-(maleimido)propanoyl)piperazin-1-yl)-amatoxine, 7'C-(4-(4-(maleimido)butanoyl)piperazin-1-yl)-amatoxine, 7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine, 7'C-(3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxine, 7'C-(3-((6-(6-(maleimido)hexanamido)hexane 7'C-(3-((4-(maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrolidin-1-yl)-amatoxine, 7'C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxine, 7'C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxine, 7'C-(4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine, 7'C-(4-(2-(4-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine 7'C-(4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)-amatoxine, 7'C-(4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazin-1-yl)-amatoxine, 7'C-((4-(6-(maleimido)hexanamido)piperidin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxine (R)-7'C-((3-((6-(maleimido)hexanoyl)piperazin-1-yl)methyl)-amatoxin, (R)-7'C-((3-((6-(maleimido)hexanoyl)methyl)pyrrolidin-1-yl)methyl)-amatoxin, (S)-7'C-((3-((6-(maleimido)hexanoyl)methyl)pyrrolidin-1-yl)methyl)-amatoxin, 7'C-((4-(2-(6-(6-(maleimido)hexanoyl)hexanoamido)ethyl)piperidin-1-yl)methyl)-amatoxin,7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine 7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine, 7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxine, 7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxine, -(maleimido)hexanamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin, 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxin, 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin, 7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidine 7'C-((4-(2-(3-carboxypropanamido)ethyl)piperazin-1-yl)methyl)-amatoxine, 7'C-((4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxine, 7'C-((4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(maleimido)acetyl)piperazin-1-yl)methyl)-amatoxine,7'C-((4-(3-(maleimido)propanoyl)piperazin-1-yl)methyl)-amatoxine, 7'C-((4-(4-(maleimido)butanoyl)piperazin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(2-(maleimido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexane 7'C-((3-((6-(maleimido)hexanamido)methyl)azetidin-1-yl)methyl)-amatoxine, 7'C-((3-(2-(6-(maleimido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxine, 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amatoxine, 7'C-((3-((2-(4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amatoxine, 7'C-((3-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amatoxine 7'C-((3-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)azetidin-1-yl)methyl)-amatoxine, 7'C-((3-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxine, 7'C-(((2-(6-(maleimido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxine, 7'C-(((4-(6-(maleimido)N-methylhexanamido)butyl(methyl)amino)methyl)-amatoxine hexanoyl)piperazin-1-yl)methyl)-amatoxine, 7'C-((2-(2-(6-(maleimido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxine, 7'C-((2-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxine, 7'C-((4-(6-(6-(2-(aminooxy)acetamido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxine, 7'C-((4-(1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-oyl)piperazin-1-yl)methyl)-amatoxin, 7'C-((4-(2-(2-(aminooxy)acetamido)acetyl)piperazin-1-yl)methyl)-amatoxin, 7'C-((4-(3-(2-(aminooxy)acetamido)propanoyl)piperazin-1-yl)methyl)-amatoxin, 7'C-((4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)methyl)-amatoxin, 7'C-((4-(2-(6-(2-( 7'C-((4-(2-(2-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin, 7'C-((4-(2-(2-(2-(aminooxy)acetamido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxin, 7'C-((4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxin, 7'C-((4-(20-(aminooxy)-4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaicosyl)piperidine lysin-1-yl)methyl)-amatoxin, 7'C-(((2-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxin, 7'C-(((4-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)butyl)(methyl)amino)methyl)-amatoxin, 7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-S-methyl)-amatoxin, 7'C-( (3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(2-bromoacetamido)ethyl)piperazin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)methyl)-amatoxine,6'O-(6-(6-(maleimido)hexanamido)hexyl)-amatoxine, 6'O-(5-(4-((maleimido)methyl)cyclohexanecarboxamido)pentyl)-amatoxine, 6'O-(2-((6-(maleimido)hexyl)oxy)-2-oxoethyl)-amatoxine, 6'O-((6-(maleimido)hexyl)carbamoyl)-amatoxine, 6'O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexyl)carbamoyl)-amatoxine, 6'O-(6-(2-bromoacetamido)hexyl)-amatoxin, 7'C-(4-(6-(azido)hexanamido)piperidin-1-yl)-amatoxin, 7'C-(4-(hex-5-ynoylamino)piperidin-1-yl)-amatoxin, 7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)-amatoxin, 7'C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)- amatoxin, 6'O-(6-(6-(11,12-didehydro-5,6-dihydro-dibenz[b,f]azocin-5-yl)-6-oxohexanamido)hexyl)-amatoxin, 6'O-(6-(hex-5-ynoylamino)hexyl)-amatoxin, 6'O-(6-(2-(aminooxy)acetylamido)hexyl)-amatoxin, 6'O-((6-aminooxy)hexyl)-amatoxin, and 6'O-(6-(2-iodoacetamido)hexyl)-amatoxin. The foregoing linkers are useful, inter alia, in conjunction with the compositions and methods described herein and 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.

[0276] Anti-CD5 or CD2 antibodies and antigen-binding fragments thereof, including those described herein, can be conjugated to a cytotoxin, which is an auristatin (U.S. Patent Nos. 5,635,483 and 5,780,588). Auristatins are antimitotic agents that interfere with microtubule dynamics, GTP hydrolysis, nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anticancer (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965) (U.S. Patent Nos. 5,635,483 and 5,780,588). The auristatin drug moiety can be attached to the antibody through the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172).

[0277] Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF (MMAE and MMAF, respectively), and are disclosed in Proceedings of the American Association for Cancer Research, Vol. 45, Abstract No. 623, submitted May 28, 2004, the entire disclosure of which is incorporated herein by reference.

[0278] An exemplary auristatin embodiment is MMAE, with the wavy line indicating the point of covalent attachment to the linker of the antibody-linker conjugate (-LZ-Ab or -L-Z' as described herein). [ka] where the wavy line indicates the point of covalent attachment to the linker of the antibody-drug or drug-linker conjugate (-LZ-Ab or -L-Z' as described herein).

[0279] Another exemplary auristatin embodiment is MMAF: [ka] where the wavy line indicates the point of covalent attachment to the linker of the antibody-linker conjugate (-LZ-Ab or -L-Z' as described herein) as disclosed in U.S. Patent Application Publication No. 2005 / 0238649.

[0280] Auristatins can be prepared according to the methods of U.S. Pat. Nos. 5,635,483, 5,780,588, Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465, Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277, Pettit, GR, et al. Synthesis, 1996, 719-725, Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 15:859-863, and Doronina (2003) Nat. Biotechnol. 21(7):778-784.

[0281] Maytansinoids The anti-CD5 or CD2 antibodies and antigen-binding fragments thereof described herein can be conjugated to a cytotoxin that is a microtubule-binding agent. In some embodiments, the microtubule-binding agent is maytansine, a maytansinoid, or a maytansinoid analog. Maytansinoids are mitotic inhibitors that act by binding to microtubules and inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Nathus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Pat. Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, and 4,331,598. and Nos. 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533. Maytansinoid drug moieties are attractive drug moieties for antibody-drug conjugates because (i) they are relatively easy to prepare by fermentation or chemical modification or derivatization of fermentation products, (ii) they can be derivatized with functional groups suitable for attachment to antibodies via non-disulfide linkers, (iii) they are stable in plasma, and (iv) they are effective against a variety of tumor cell lines.

[0282] Examples of suitable maytansinoids include esters of maytansinol, synthetic maytansinol, and maytansinol analogs and derivatives. Included herein is any cytotoxin that inhibits microtubule formation and is highly toxic to mammalian cells, such as maytansinoids, maytansinol, maytansinol analogs and derivatives.

[0283] Examples of suitable maytansinol esters include those with modified aromatic rings and those with modifications at other positions. Such suitable maytansinoids are described in U.S. Patent Nos. 4,137,230, 4,151,042, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, 4,309,428, 4,313,946, 4,315,929, and 4,316,949. No. 4, 317, 821, No. 4, 322, 348, No. 4, 331, 598, No. 4, 361, 650, No. 4, 362, 663, No. No. 4, 364, 866, No. 4, 424, 219, No. 4, 450, 254, No. 4, 322, 348, No. 4, 362, 663, No. Nos. 4,371,533, 5,208,020, 5,416,064, 5,475,092, 5,585,499, 5,846,545, 6,333,410, 7,276,497, and 7,473,796, the disclosures of each of which are incorporated herein by reference as they relate to maytansinoids and derivatives thereof.

[0284] In some embodiments, the antibody-drug conjugates (ADCs) of the present disclosure may be administered as cytotoxic agents, formally known as N 2 '-Deacetyl-N 2 It utilizes a thiol-containing maytansinoid called '-(3-mercapto-1-oxopropyl)-maytansine (DM1), which is represented by the following structural formula VI: [ka]

[0285] In another embodiment, the conjugate of the invention comprises a thiaminol-containing maytansinoid N 2 '-Deacetyl-N 2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (e.g., DM4) is utilized as the cytotoxic agent. DM4 is represented by the following structural formula VII: [ka]

[0286] Another maytansinoid comprising a side chain containing a sterically hindered thiol bond is N, represented by structural formula VIII below: 2 '-Deacetyl-N 2 '(4-mercapto-1-oxopentyl) maytansine (referred to as D3). [ka]

[0287] Each of the maytansinoids taught in U.S. Patent Nos. 5,208,020 and 7,276,497 can also be used in the conjugates of the present disclosure, and in this regard, the entire disclosures of U.S. Patent Nos. 5,208,020 and 7,276,497 are incorporated herein by reference.

[0288] Many positions on the maytansinoid can serve as the position for covalently attaching a linking moiety, and thus an antibody or antigen-binding fragment thereof (-L-Z-Ab or -L-Z' as described herein). For example, the C-3 position bearing a hydroxyl group, the hydroxymethyl-modified C-14 position, the hydroxy-modified C-15 position, and the hydroxyl group-modified C-20 position are all expected to be useful. In some embodiments, the C-3 position serves as the position for covalently attaching a linker moiety, and in some particular embodiments, the C-3 position of maytansinol serves as the position for covalently attaching a linking moiety. There are many linking groups known in the art for making antibody-maytansinoid conjugates, such as those disclosed in U.S. Patent Nos. 5,208,020 and 6,441,163, European Patent No. 0425235 B1, Chari et al., Cancer Research 52:127-131 (1992), and U.S. Patent No. US2005 / 0169933 A1, which are expressly incorporated herein by reference. Additional linking groups are described and exemplified herein.

[0289] The present invention also encompasses various isomers and mixtures of maytansinoids and conjugates.Specific compounds and conjugates of the present invention may exist in various stereoisomeric, enantiomeric, and diastereomeric forms.Descriptions for producing such antibody-maytansinoid conjugates are described in U.S. Patent Nos. 5,208,020, 5,416,064, 6,333,410, 6,441,163, 6,716,821, and 7,368,565, each of which is incorporated herein by reference in its entirety.

[0290] Anthracyclines In other embodiments, the anti-CD5 or CD2 antibodies and antigen-binding fragments thereof described herein can be conjugated to a cytotoxin that is an anthracycline molecule. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Research has shown that anthracyclines can act to kill cancer cells through a number of different mechanisms, including: 1) intercalation of the drug molecule into cellular DNA, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) drug-induced production of free radicals that then react with cellular macromolecules and cause cell damage; or 3) interaction of the drug molecule with cell membranes (see, e.g., C. Peterson et al., "Transport and Storage of Anthracyclines in Experimental Systems and Human Leukemia," Anthracycline Antibodies in Cancer Therapy; N. R. Bachur, "Free Radical Damage," pp. 97-102). Potentially cytotoxic anthracyclines are used to treat many cancers, including leukemia, breast carcinoma, lung cancer, ovarian adenocarcinoma, and sarcoma (see, e.g., P. H. Wiernik, Anthracyclines, in Experimental Systems and Human Leukemia: Current Status and New Developments, p11). Commonly used anthracyclines include doxorubicin, epirubicin, idarubicin, and daunomycin. In some embodiments, the cytotoxin is an anthracycline selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin. Representative anthracyclines include, but are not limited to, daunorubicin (Cerubicin, Bedford Laboratories), doxorubicin (ADRIAMYCIN®, Bedford Laboratories, also known as doxorubicin hydrochloride, hydroxydaunorubicin, and RUBEX®), epirubicin (ELLENCE®, Pfizer), and idarubicin (IDAMAYCIN®, Pfizer).

[0291] The anthracycline analog doxorubicin (ADRIAMYCIN®) is thought to interact with DNA by intercalating and inhibiting the progression of topoisomerase II, the enzyme that unwinds DNA for transcription. Doxorubicin stabilizes the topoisomerase II complex after DNA strand breaks for replication, preventing the DNA double helix from rejoining, thereby halting the replication process. Doxorubicin and daunorubicin are prototypic cytotoxic natural products that are anthracycline chemotherapeutic agents (Sessa et al., (2007) Cardiovasc. Toxicol. 7:75-79).

[0292] A non-limiting example of an anthracycline suitable for use herein is PNU-159682 ("PNU"). PNU exhibits over 3000 times the cytotoxicity of its parent nemorubicin (Quintieri et al., Clinical Cancer Research 2005, 11, 1608-1617). PNU has the structural formula: [ka] is expressed by

[0293] Many positions on anthracyclines, such as PNU, can serve as a linking moiety and thus a position for covalently attaching an anti-CD45 antibody or antigen-binding fragment thereof as described herein. For example, a linker can be introduced via modification to a hydroxymethyl ketone side chain.

[0294] In some embodiments, the cytotoxin has the structural formula: [ka] It is a PNU derivative represented by the formula: wherein the wavy line indicates the point of covalent attachment of the ADC to a linker as described herein.

[0295] In some embodiments, the cytotoxin has the structural formula: [ka] It is a PNU derivative represented by the formula: wherein the wavy line indicates the point of covalent attachment of the ADC to a linker as described herein.

[0296] Benzodiazepine cytotoxins The anti-CD45 antibodies and antigen-binding fragments thereof described herein (including, for example, bispecific and biparatopic antibodies) can be conjugated to a cytotoxin containing a benzodiazepine moiety, such as the PBDs and IGNs described herein. Pyrrolobenzodiazepines (PBDs) A PBD has the general structure: [ka]

[0297] They differ in the number, type, and position of substituents in both their aromatic ("A") and pyrrolo ("C") rings, as well as in the degree of saturation in the C ring. Diazepine B rings contain imine (N=C), carbinolamine (NH-CH(OH)), or carbinolamine metal ether (NH-CH(OMe)) groups at the N10-C11 position. This position is the ionizable group responsible for DNA alkylation. All known natural product PBDs have the (S) configuration at the chiral C11a position, which gives them a right-handed twist when viewed from the C ring toward the A ring. This provides the appropriate three-dimensional geometry for isohelicity with the minor groove of B-form DNA, providing a tight fit to the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc). Chem. Res., 19, 230-237 (1986)) The ability of PBDs to form adducts in the minor groove allows them to interfere with DNA processing, resulting in anticancer activity.

[0298] The biological activity of these molecules has previously been demonstrated to be due to the connection of two PBD units via a flexible alkylene phosphorus linker (Bose, DS, et al., J. Am. Chem. Soc., 114, 4939-4941 (1992); Thurston, DE, et al., J. Org. Chem., 61, 8141-8147 (1996)). PBD dimers are thought to form sequence-selective DNA lesions, such as the palindromic 5'-Pu-GATC-Py-3' interstrand crosslink (Smellie, M., et al., Biochemistry, 42, 8232-8239 (2003); Martin, C., et al., Biochemistry, 44, 4135-4147), which are thought to be primarily responsible for their biological activity. Gregson et al. (Chem. Commun. 1999, 797-798, "Complex 1" and Gregson et al. (J. Med. Chem. 2001, 44, 1161-1174, "Complex 4a") describe an advantageous dimeric pyrrolobenzodiazepine compound. This complex, also known as SG2000, has the following structural formula: [ka]

[0299] Generally, modifications to pyrrolidine alkenes provide a handle for homovalently attaching conjugates, thus providing their anti-substance or antigen-binding fragments (-L-Z' and -LZ-Ab, as described herein). Alternatively, a linker can be attached at the N10 position.

[0300] In some embodiments, the cytotoxin has the following structural formula: [ka] It is a pyrrolobenzodiazepine dimer represented by the formula: wherein n is an integer from 2 to 5. The compound of this formula where n is 3 is known as DSB-120 (Bose et. Al., J. Am. Chem Soc. 1992, 114, 4939-4941).

[0301] In some embodiments, the cytotoxin has the following structural formula: [ka] It is a pyrrolobenzodiazepine dimer represented by the formula: where n is an integer from 2 to 5. When n is 3, the compound of this formula is known as SJG-136 (Gregson et. Al., J. Med. Chem. 2001, 44, 737-748). When n is 5, the compound of this formula is known as DRG-16 (Gregson et. Al., Med. Chem. 2004, 47:1161-1174).

[0302] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine dimer having the formula: [ka] where the wavy line indicates the point of covalent attachment to the linker of the ADC described herein. This PBD-based ADC is disclosed, for example, in Sutherland et al., Blood 2013 122:1455-1463, the entire disclosure of which is incorporated herein by reference.

[0303] In some embodiments, the cytotoxin is a PBD having the formula: [ka] wherein n is 3 or 5, and the wavy line indicates the point of covalent attachment of an ADC described herein to a linker.

[0304] In some embodiments, the cytotoxin is a PBD dimer represented by structural formula (I): [ka] wherein the wavy line indicates the point of covalent attachment of the ADC described herein to the linker.

[0305] Indolinobenzodiazepines (IGN) In some embodiments, an antibody or antigen-binding fragment thereof that binds CD45 described herein can be conjugated to an indolinobenzodiazepine ("IGN") or an IGN-containing cytotoxin. In some embodiments, the IGN cytotoxin is an indolinobenzodiazepine dimer or an indolinobenzodiazepine pseudodimer.

[0306] Indolinobenzodiazepine dimers have high in vitro potency against cancer cells (low pM range IC 50 IGN dimers are a relatively new chemical class of cytotoxins. Similar to the PBD dimer SJG-136, IGN dimers bind to the minor groove of DNA and covalently bond to guanine residues via two imine functional groups within the dimer, crosslinking DNA. An IGN dimer (IGN6, in which the methylene group in the PBD moiety is replaced by a phenyl ring) exhibited approximately 10-fold greater potency in vivo than SJG-136, likely due to a faster rate of DNA IGN adduct formation (see, e.g., Miller et al., "A New Class of Antibody-Drug Conjugates with Potent DNA Alkylating Activity," Mol. Cancer Ther. 2016, 15(8), 1870-1878). On the other hand, IGN pseudodimers consist of a single reactive indolinobenzodiazepine imine, and the second indolinobenzodiazepine in the dimeric cytotoxin exists in its reduced (amine) form. Thus, IGN pseudodimers alkylate DNA through the single imine site contained in the dimer, but do not crosslink DNA.

[0307] In some embodiments, the cytotoxin is an IGN pseudodimer having the following structural formula: [ka] In the formula, the wavy line indicates the point of attachment of the linker.

[0308] In some embodiments, the cytotoxin-linker conjugate, including the reactive substituent Z′, prior to conjugation to the antibody is summarized as Cy-L-Z′ and has the following structure: [ka]

[0309] This cytotoxin-linker conjugate is referred to herein as DGN549 and is present in the ADC IMGN632, both of which are disclosed, for example, in WO2017004026, which is incorporated herein by reference.

[0310] In some embodiments, the cytotoxin is an indolinobenzodiazepine pseudodimer having the structure: [ka] where the wavy line indicates the point of attachment of the linker. This IGN pseudo-dimeric cytotoxin is referred to herein as DGN462 and is disclosed, for example, in U.S. Patent Application Publication No. 20170080102, which is incorporated herein by reference.

[0311] In some embodiments, the cytotoxin-linker conjugate, including chemical moiety Z, prior to conjugation to the antibody is summarized as Cy-LZ and has the following structure: [ka] where the wavy line indicates the point of attachment of the antibody (e.g., an anti-CD45 antibody or fragment thereof). This cytotoxin-linker conjugate is present in ADC IMGN779 and is disclosed, for example, in U.S. Patent Application Publication No. 20170080102, previously incorporated by reference.

[0312] Kalicare sewing machine In other embodiments, the antibodies and antigen-binding fragments thereof described herein can be conjugated to cytotoxins that are enediyne antitumor compounds (e.g., calicheamicin, ozogamicin). The calicheamicin family of antibiotics can cause cleavage of unstranded DNA at sub-picomolar concentrations. For preparation of calicheamicin family members, see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all to American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, those disclosed in, for example, Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the aforementioned U.S. patents to American Cyanamid.

[0313] An exemplary calicheamicin is designated γ1 and has the following structural formula: [ka]

[0314] In some embodiments, the calicheamicin is a gamma-calicheamicin derivative or an N-acetyl gamma-calicheamicin derivative. Structural analogs of calicheamicin that can be used include, but are not limited to, those disclosed in, for example, Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the aforementioned U.S. patents. Calicheamicin contains a methyl trisulfide moiety that can react with an appropriate thiol to form a disulfide, while simultaneously introducing a functional group useful for attaching the calicheamicin derivative, via a linker, to a bispecific binding agent as described herein. For the preparation of conjugates of the calicheamicin family, see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all to American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, those disclosed in, for example, Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the aforementioned U.S. patents to American Cyanamid.

[0315] In some embodiments, the cytotoxin of the ADCs disclosed herein is a calicheamicin disulfide derivative having the formula: [ka] In the formula, the wavy line indicates the point of attachment of the linker.

[0316] Additional cytotoxins that can be conjugated to antibodies, and antigen-binding fragments thereof, that recognize and utilize CD2 or CD5 for use in the direct treatment of cancer, autoimmune conditions, or in the preparation of patients (e.g., human patients) for hematopoietic stem cell transplantation therapy include, but are not limited to, 5-ethynyluracil, abiraterone, acylfulvene, adecipenol, adzelesin, aldesleukin, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anaphylactoid ... Grelide, anastrozole, andrographolide, angiogenesis antagonist, antarelix, antithoracic vertebrate morphogenetic protein-1, antiandrogen, prostate cancer, antiestrogen, antineoplaston, antisense oligonucleotide, aphidicolin glycinate, apoptotic gene regulator, apoptosis inhibitor, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase, asulaculin, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, bacactin I II derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorins, benzoylstaurosporine, beta-lactam derivatives, beta-arretin, beta-clamycin B, betolic acid, bFGF inhibitors, bicalutamide, bisantrene, bisatyridine, bisnafide, bisstraten A, bizelesin, bleflate, bleomycin A2, bleomycin B2, bropiromim, budotitanium, buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives (e.g., 10-hydroxycamptothecin) , capecitabine, carboxamido-amino-triazole, carboxyamidotriazole, carzelesin, casein kinase inhibitor, castanospermine, cecropin B, cetrorelix, chlorin, chloroquinoxaline sulphonamide, cicaprost, cis-porphyrin, cladribine, clomiphene analogues, clotrimazole, collismycin A, collismycin B, combrestatin A4, combrestatin analogues, conagenin, clambecidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A,Cyclopentaanthraquinone, cycloplatam, sipemycin, cytarabine ocfosfate, cytolytic factors, cytostatin, dacliximab, decitabine, dehydrodidemnin B, 2'deoxycoformycin (DCF), deslorelin, dexphosphamide, dexrazoxane, dexverapamil, diaticuone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin, diphenylspiromustine, discodermolide, docosanol, dolasterone, doxifluridine, Droloxifene, dronabinol, doucarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epothilone, epithilone, epristeride, estramustine analogues, etoposide, etoposide 4'-phosphate (also known as etopofos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, flezelastine, fluasterone, fludarabine, fluorodaunornithine hydrochloride, forfenimex, forfenilone Mestan, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, gallocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepzulfam, homoharringtonine (HHT), hypericin, ibandronic acid, idarubicin, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridone, imiquimod, immunostimulating peptides, iobenguane, iododoxorubicin, ipomeanol, irinotecan, ilopract, irsogladine, isobengazole, jasplakia Nolide, Kahalalide F, Lamellarin N Triacetate, Lanreotide, Leinamycin, Lenoglastin, Lentinan Sulfate, Leptolstatin, Letrozole, Lipophilic Platinum Compounds, Lisoclinamide 7, Lobaplatin, Lometrexol, Lonidamine, Losoxantrone, Loxoribine, Lurtotecan, Lutetium Texaphyrin, Lisofylline, Natansin, Masoprocol, Maspin, Matrix Metalloproteinase Inhibitors, Menogaril, Melbarone, Meterelin, Methioninase, Metoclopramide, Mifepristone, Miltefosine,Millimostin, mithracin, mitoguazone, mitolactol, mitomycin analogues, mitonafide, mitoxantrone, mofalotene, mofgramostim, micaperoxide B, miriaporone, N-acetyldynalin, N-substituted benzamides, nafarelin, nagressip, navavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase, nilutamiud, octreotide, oxenon, onapristone, ondansetron, oracin, ormaplatin, oxaliplatin, oxaunomycin, paclitaxel analogues, palaumin, palmitoylrhizoxin, pamidronate, panaxytriol, panomyphen, parabactin, pazelliptin, pegaspargase, perdecin, sodium Pentosan polysulfate, pentostatin, pentrozole, perflubron, periosfamide, phenazinomycin, picibanil, pirarubicin, piriterexim, podophyllotoxin, porfiromycin, purine nucleoside phosphorylase inhibitors, raltitrexed, rhizoxin, logretimide, rohitukin, rubiginone B1, ruboxil, safingol, saintpin, sarcophyt A, sargramostim, sobuzoxane, sonermin, spahisinic acid, spicamycin D, spiromustine, stipiamide, sulfinosine, tallimustine, tegafur, temozolomide, teniposide, saliblastine, thiocoraline, tirapazamine, topotecan, topsentin, triciribine, trimetrexate, veramine, vinorelbine, vinxartin, vorozole, zeniplatin, and zilascorub.

[0317] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine dimer represented by formula (IV). [ka]

[0318] A variety of linkers can be used to conjugate the antibodies and antigen-binding fragments described herein that recognize and bind to CD2 or CD5.

[0319] As used herein, the term "linker" refers to a bivalent chemical moiety comprising a covalent bond or chain of atoms that covalently attaches an anti-CD5 or CD2 antibody or fragment thereof (Ab) to a drug moiety (D) to form an antibody-drug conjugate (ADC, Ab-ZLD, where D is a cytotoxin) of the present disclosure. Suitable linkers have two reactive ends, one for conjugation to the antibody and the other for conjugation to the cytotoxin. The reactive end of the linker for antibody conjugation (reactive moiety, Z) is typically a site available for conjugation to an antibody via a cysteine ​​thiol or lysine amine group on the antibody, and is typically a thiol-reactive group (e.g., maleimide) such as a double bond (e.g., maleimide) or a leaving group (e.g., chloro, bromo, iodo, R-sulfanyl group), or an amine-reactive group such as a carboxyl group. On the other hand, the reactive end of the linker for antibody conjugation is a site that can be conjugated to a cytotoxin via the formation of an amide bond with a basic amine or carboxyl group on the cytotoxin, typically a carboxyl or basic amine group. When the term "linker" is used to describe a conjugated form of a linker, one or both of the reactive ends are absent (e.g., reactive moiety Z converted to chemical moiety Z) or incomplete (e.g., only the carbonyl of a carboxylic acid) for the formation of bonds between the linker and / or the cytotoxin, and between the linker and / or the antibody or antigen-binding fragment thereof. Such conjugation reactions are further described herein below.

[0320] Various linkers can be used to conjugate the antibodies or antibody fragments described herein to cytotoxic molecules. In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In yet other embodiments, the linker unit is not cleavable, and the drug is released, for example, by antibody degradation. Linkers useful in the present ADCs are preferably stable extracellularly, prevent aggregation of the ADC molecules, and keep the ADC freely soluble in aqueous media and in a monomeric state. Prior to transport or delivery to cells, the ADC is preferably stable and remains intact, i.e., the antibody remains linked to the drug moiety. The linker is stable outside the target cells and can be cleaved at an effective rate within the cells. An effective linker (i) maintains the specific binding properties of the antibody, (ii) enables intracellular delivery of the conjugate or drug moiety, (iii) remains stable and intact (i.e., not cleaved) until the conjugate is delivered or transported to its target site, and (iv) maintains the cytotoxic, cell-killing, or cytostatic effect of the cytotoxic moiety. The stability of ADCs can be measured by standard analytical techniques such as mass spectrometry, HPLC, and the separation / analysis technique LC / MS. Covalent attachment of an antibody to a drug moiety requires that the linker have two reactive functional groups, i.e., be bivalent in the sense of reactivity. Bivalent linker reagents useful for joining two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, reporter groups, etc., are known, and methods have been described for the resulting conjugates (Hermanson, GT (1996) Bioconjugate Techniques, Academic Press: New York, pp. 234-242).

[0321] Linkers include, for example, those that can be cleaved 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).

[0322] Linkers hydrolyzable under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, and the like. See, e.g., U.S. Pat. Nos. 5,122,368, 5,824,805, 5,622,929, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123, and Neville et al., 1989, Biol. Chem. 264:14653-14661. The disclosures of each of these 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.

[0323] Linkers that can be cleaved under reducing conditions include, for example, disulfides. For example, various disulfide linkers are known in the art, including those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB and SMPT. See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel, ed., Oxford University Press, 1987); see also U.S. Pat. No. 4,880,935, the disclosures of each of which relate to linkers suitable for covalent conjugation and are incorporated herein by reference in their entireties.

[0324] Examples of linkers useful in the synthesis of drug-antibody conjugates include, among others, those containing electrophiles suitable for reaction with nucleophilic substituents, such as amine or thiol moieties, present in antibodies or antigen-binding fragments, such as Michael acceptors (e.g., maleimides), 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-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among others, as described, for example, in Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation. Additional linkers include non-cleavable maleimidocaproyl linkers, which are particularly useful for conjugating microtubule-disrupting agents such as auristatins, and are described in 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. Additional linkers suitable for the synthesis of the drug-antibody conjugates described herein include those capable of releasing cytotoxins by a 1,6-elimination process ("self-immolative" groups), such as p-aminobenzyl alcohol (PABC), 6-maleimidohexanoic 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 comprises a self-immolative group such as PAB or PABC (paraaminobenzyloxycarbonyl) as described above, e.g., in Carl et al., J. Med. Chem. (1981) 24:479-480; Chakravarty et al (1983) J. Med. Chem. 26:638-644, U.S. Patent No. 6,214,345, U.S. Patent Application Publication No. 20030130189, U.S. Patent Application Publication No. 20030096743, U.S. Patent No. 6,759,509, U.S. Patent Application Publication No. 20040052793, U.S. Patent No. 6,218,519, U.S. Patent No. 6,835,807, U.S. Patent No. 6,268,488, U.S. Patent Application Publication No. 20040018194, WO 98 / 13059, U.S. Patent Application Publication No. 20040052793, U.S. Patent No. 6,677,435, U.S. Patent No. 5,621,002, U.S. Patent Application Publication No. 20040121940, and WO 2004 / 032828. Other such chemical moieties ("self-immolative linkers") that are capable of this process include methylene carbamates and heteroaryl groups such as aminothiazoles, aminoimidazoles, aminopyrimidines, etc. Linkers containing such heterocyclic self-immolative groups are disclosed, for example, in U.S. Patent Publication Nos. 20160303254 and 20150079114, as well as U.S. Patent No. 7,754,681, Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237, US2005 / 0256030, de Groot et al. (2001) J. Org. Chem. 66:8815-8830, and US7223837.

[0325] A linker susceptible to enzymatic hydrolysis can be, for example, a peptide-containing linker that is cleaved by intracellular peptidases or protease enzymes, including, but not limited to, lysosomal or endosomal proteases. One advantage of using intracellular proteolytic release of a therapeutic agent is that the drug is generally attenuated when conjugated, and the serum stability of the conjugate is generally 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. Amino acid residues that comprise amino acid linker components include naturally occurring ones as well as minor amino acids and unnatural 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, Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit. Linkers containing dipeptides such as Val-Cit or Phe-Lys are disclosed, for example, in U.S. Pat. No. 6,214,345, the disclosure of which is incorporated herein by reference in its entirety as it relates to linkers suitable for covalent conjugation. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, a dipeptide is used in combination with a self-immolative linker.

[0326] Linkers suitable for conjugating the antibodies or antibody fragments described herein with cytotoxic molecules include those capable of releasing the cytotoxin via a 1,6-elimination process. Chemical moieties capable of this release process include p-aminobenzyl (PAB) groups, 6-maleimidohexanoic 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, particularly as it relates to linkers suitable for covalent attachment.

[0327] In some embodiments, the linker comprises a self-immolative group such as the aforementioned PAB or PABC (paraaminobenzyloxycarbonyl), which are described, for example, in Carl et al., J. Med. Chem. (1981) 24:479-480; Chakravarty et al. al (1983) J. Med. Chem. 26:638-644, U.S. Patent No. 6,214,345, U.S. Patent Application Publication No. 20030130189, U.S. Patent Application Publication No. 20030096743, U.S. Patent No. 6,759,509, U.S. Patent Application Publication No. 20040052793, U.S. Patent No. 6,218,519, U.S. Patent No. 6,835,807, U.S. Patent No. 6,268,488, U.S. Patent Application Publication No. 20040018194, WO 98 / 13059, U.S. Patent Application Publication No. 20040052793, U.S. Patent No. 6,677,435, U.S. Patent No. 5,621,002, U.S. Patent Application Publication No. 20040121940, and WO 2004 / 032828. Other such chemical moieties ("self-immolative linkers") that are capable of this process include methylene carbamates and heteroaryl groups such as aminothiazoles, aminoimidazoles, and aminopyrimidines. Linkers containing such heterocyclic self-immolative groups are disclosed, for example, in U.S. Patent Publication Nos. 20160303254 and 20150079114, as well as 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 U.S. Patent No. 7,223,837. In some embodiments, dipeptides are used in combination with self-immolative linkers.

[0328] Suitable linkers may contain groups with solubility enhancing properties, for example (CH2CH2O) pLinkers containing units (polyethylene glycol, PEG) can enhance solubility, as can alkyl chains substituted with amino, sulfonic acid, phosphonic acid, or phosphate residues. Linkers containing such moieties are disclosed, for example, in U.S. Patent Nos. 8,236,319 and 9,504,756, which are incorporated herein by reference in their entirety for their relatedness to linkers suitable for covalent conjugation. Additional solubility-enhancing groups include, for example, acyl and carbamoyl sulfamido groups, having the following structures: [ka] In the formula, a is 0 or 1; R 10 is hydrogen, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C1-C 24 (Hetero)aryl groups, C1-C 24 Alkyl(hetero)aryl groups, C1-C 24 (Hetero)arylalkyl groups, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and C3-C 24 (hetero)arylalkyl groups, each of which is selected from the group consisting of O, S, NR 11 R 12 and R may be substituted and / or optionally interrupted by one or more heteroatoms selected from 11 and R 12 are independently selected from the group consisting of hydrogen and C1-C4 alkyl groups, or R 10 is a cytotoxin, which is optionally connected to N via a spacer moiety. Linkers containing such groups are described, for example, in U.S. Pat. No. 9,636,421 and U.S. Patent Application Publication No. 2017 / 0298145, the disclosures of which are incorporated by reference in their entireties as they relate to linkers suitable for covalent attachment to cytotoxins and antibodies or antigen-binding fragments thereof.

[0329] In some embodiments, the linker is a hydrazine, a disulfide, a thioether, a dipeptide, a p-aminobenzyl (PAB) group, a heterocyclic autoimmune group, an optionally substituted C1-C6 alkyl, an optionally substituted C1-C6 heteroalkyl, an optionally substituted C2-C6 alkenyl, an optionally substituted C2-C6 heteroalkenyl, an optionally substituted C2-C6 alkynyl, an optionally substituted C2-C6 heteroalkynyl, an optionally substituted C3-C6 cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, a solubility-enhancing group, acyl, -(C=O)-, or -(CH2CHO) p - groups, where p is an integer from 1 to 6. One of ordinary skill in the art will recognize that one or more of the listed groups may exist in the form of a divalent (diradical) species (e.g., C1-C6 alkylene, etc.).

[0330] In some embodiments, the linker comprises the moiety *-L1L2-**: L1 is absent or -(CH2) m NR 13 C(=O)-, -(CH2) m NR 13 -, -(CH2) m X3(CH2) m -, [ka] and L2 is absent or -(CH2) m -, -NR 13 (CH2) m -, -(CH2) m NR 13 C(=O)(CH2) m -, -X4, -(CH2) m NR 13 C(=O)X4, - (CH2) m NR 13 C(=O)-, -((CH2) m O) n (CH2)m -、-((CH2) m O) n (CH2) m X3(CH2) m -、-NR 13 ((CH2) m O) n X3(CH2) m -、-NR 13 ((CH2) m O) n (CH2) m X3(CH2) m -、-X1X2C(=O)(CH2) m -、-(CH2) m (O(CH2) m ) n -、-(CH2) m ARN 13 (CH2) m -、-(CH2) m ARN 13 C(=O)(CH2) m X3(CH2) m -、-(CH2) m C(=O)NR 13 (CH2) m ARN 13 C(=O)(CH2) m -、-(CH2) m C(=O)-、-(CH2) m ARN 13 (CH2) m C(=O)X2X1C(=O)-、-(CH2) m X3(CH2) m C(=O)X2X1C(=O)-、-(CH2) m C(=O)NR 13 (CH2) m -、-(CH2) m C(=O)NR 13 (CH2) m X3(CH2) m -、-(CH2) m X3(CH2) m ARN 13 C(=O)(CH2) m -、-(CH2) m X3(CH2) m C(=O)NR 13 (CH2)m -、-(CH2) m O) n (CH2) m ARN 13 C(=O)(CH2) m -、-(CH2) m C(=O)NR 13 (CH2) m (O(CH2) m ) n -、-(CH2) m (O(CH2) m ) n C(=O)-、-(CH2) m ARN 13 (CH2) m C(=O)-、-(CH2) m C(=O)NR 13 (CH2) m ARN 13 C(=O)-、-(CH2) m (O(CH2) m ) n X3(CH2) m -、-(CH2) m X3((CH2) m O) n (CH2) m -、-(CH2) m X3(CH2) m C(=O)-、-(CH2) m C(=O)NR 13 (CH2) m O) n (CH2) m X3(CH2) m -、-(CH2) m X3(CH2) m (O(CH2) m ) n ARN 13 C(=O)(CH2) m -、-(CH2) m X3(CH2) m (O(CH2) m ) n C(=O)-、-(CH2) m X3(CH2) m (O(CH2) m ) n -、-(CH2) m C(=O)NR13 (CH2) m C(=O)-、-(CH2) m C(=O)NR 13 (CH2) m (O(CH2) m ) n C(=O)-、-((CH2) m O) n (CH2) m ARN 13 C(=O)(CH2) m -、-(CH2) m C(=O)NR 13 (CH2) m C(=O)NR 13 (CH2) m -、-(CH2) m ARN 13 C(=O)(CH2) m ARN 13 C(=O)(CH2)-(CH2) m X3(CH2) m C(=O)NR 13 -、-(CH2) m C(=O)NR 13 -、-(CH2) m X3-、-C(R 13 )2(CH2) m -、-(CH2) m C(R 13 )2NR 13 -、-(CH2) m C(=O)NR 13 (CH2) m ARN 13 -、-(CH2) m C(=O)NR 13 (CH2) m ARN 13 C(=O)NR 13 -、-(CH2) m C(=O)X2X1C(=O)-、-C(R 13 )2(CH2) m ARN 13 C(=O)(CH2) m -、-(CH2) m C(=O)NR 13 (CH2) m C(R 13 )2NR 13 -、-C(R13 )2(CH2) m X3(CH2) m -、-(CH2) m X3(CH2) m C(R 13 )2NR 13 -、-C(R 13 )2(CH2) m OC(=O)NR 13 (CH2) m -、-(CH2) m NR 13 C(=O)O(CH2) m C(R 13 )2NR 13 -、-(CH2) m X3(CH2) m NR 13 -、-(CH2) m X3(CH2) m (O(CH2) m ) n NR 13 -、-(CH2) m NR 13 -、-(CH2) m C(=O)NR 13 (CH2) m (O(CH2) m ) n NR 13 -、-(CH2) m (O(CH2) m ) n NR 13 -、-(CH2CH2O) n (CH2) m -、-(CH2) m (OCH2CH2) n、 -(CH2) m O(CH2) m -、-(CH2) m S(=O)2-、-(CH2) m C(=O)NR 13 (CH2) m S(=O)2-、-(CH2) m X3(CH2) m S(=O)2-、-(CH2) m X2X1C(=O)-、-(CH2) m (O(CH2) m )n C(=O)X2X1C(=O)-,-(CH2) m (O(CH2) m ) n X2X1C(=O)-、-(CH2) m X3(CH2) m X2X1C(=O)-、-(CH2) m X3(CH2) m (O(CH2) m ) n X2X1C(=O)-、-(CH2) m X3(CH2) m C(=O)NR 13 (CH2) m NR 13 C(=O)-,-(CH2) m X3(CH2) m C(=O)NR 13 (CH2) m C(=O)-,-(CH2) m X3(CH2) m C(=O)NR 13 (CH2) m (O(CH2) m ) n C(=O)-,-(CH2) m C(=O)X2X1C(=O)NR 13 (CH2) m -、-(CH2) m X3(O(CH2) m ) n C(=O)-,-(CH2) m NR 13 C(=O)((CH2) m Or) n (CH2) m -、-(CH2) m (O(CH2) m ) n C(=O)NR 13 (CH2) m -、-(CH2) m NR 13 C(=O)NR 13 (CH2) m -or-(CH2) m X3(CH2) m NR 13 C(=O)- X1 is expressed by the following formula: [ka] X2 is expressed by the following formula: [ka] X3 is expressed by the following formula: [ka] X4 is expressed by the following formula: [ka] , In the formula, R 13 are each independently selected at each occurrence from H and C1-C6 alkyl; m is independently selected in each occurrence from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; n is independently selected in each occurrence from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14; where a single asterisk (*) indicates the point of attachment to a cytotoxin (e.g., an amatoxin), a double asterisk (**) indicates the point of attachment to a reactive substituent Z' or chemical moiety Z, and both L1 and L2 are absent.

[0331] In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, 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 some embodiments, the p-aminobenzyl group is part of a p-aminobenzylamide unit.

[0332] 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.

[0333] In some embodiments, the linker comprises one or more combinations of a peptide, an oligosaccharide, -(CH2)p-, -(CH2CHO)p-, PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0334] In some embodiments, the linker comprises -(C=O)(CH2)p- units, where p is an integer from 1 to 6.

[0335] In certain embodiments, the linker comprises the following structure: [ka] where the wavy line indicates the point of attachment of the cytotoxin and the reactive moiety Z'. In another embodiment, the linker comprises the structure: [ka] where the wavy lines indicate the attachment points of the cytotoxin and reactive moiety Z'. The peptide-propionyl linker is, for example, as disclosed in WO 2017 / 149077. and WO 2017 / 023144, which is incorporated herein by reference in its entirety. The cytotoxins disclosed in US Pat. No. 149077 are also incorporated herein by reference.

[0336] In certain embodiments, the linker of the ADC is maleimidocaproyl-Val-Ala-para-aminobenzyl (mc-Val-Ala-PAB).

[0337] In certain embodiments, the linker of the ADC is maleimidocaproyl-Val-Cit-para-aminobenzyl (mc-vc-PAB).

[0338] In some embodiments, the linker comprises the following structure: [ka]

[0339] In some embodiments, the linker comprises MCC (4-[N-maleimidomethyl]cyclohexane-1-carboxylate).

[0340] 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 conjugating antibodies and cytotoxins as disclosed herein. Additional linkers useful in conjunction with the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the entire disclosure of which is incorporated herein by reference.

[0341] Linkers suitable for use herein may further include one or more groups selected from C-C alkylene, C-C heteroalkylene, C-C alkenylene, C-C heteroalkenylene, C-C alkynylene, C-C heteroalkynylene, C-C cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which may be optionally substituted. Non-limiting examples of such groups include (CH) n , (CH2CH2O) n , and -(C=O)(CH2) n- units, where n is an integer from 1 to 6 and is independently selected in each occurrence.

[0342] In some embodiments, the linker is selected from the group consisting of hydrazine, disulfide, thioether, dipeptide, p-aminobenzyl (PAB) group, heterocyclic self-immolative group, optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, acyl, -(C=O)-, or -(CH2CHO) n - groups, where n is an integer from 1 to 6. One of ordinary skill 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 a C1-C6 alkylene.

[0343] 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-aminobenzylamide unit.

[0344] 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.

[0345] In some embodiments, the linker is 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 in combination.

[0346] In some embodiments, the linker is -(C=O)(CH2) n -unit, where n is an integer from 1 to 6.

[0347] In some embodiments, the linker is -(CH) n -unit, where n is an integer from 2 to 6.

[0348] In certain embodiments, the linker of the ADC is N-β-maleimidopropyl-Val-Ala-paraaminobenzyl (BMP-Val-Ala-PAB).

[0349] Linkers that can be used to conjugate an antibody or antigen-binding fragment thereof to a cytotoxic agent include those that are covalently attached to the cytotoxic agent at one end of the linker and contain a chemical moiety at the other end of the linker formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in an antibody or antigen-binding fragment thereof that binds to CD2 or CD5. Reactive substituents that can be present in an antibody or antigen-binding fragment thereof that binds to CD2 or 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, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-naturally occurring amino acids.

[0350] Examples of linkers useful in the synthesis of drug-antibody conjugates include, among others, those containing an electrophile suitable for reaction with a nucleophilic substituent, such as an amine or thiol moiety, present in an antibody or antigen-binding fragment, such as a Michael acceptor (e.g., maleimide), activated ester, electron-deficient carbonyl compound, and aldehyde. 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-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among others, as described, for example, in Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation. Additional linkers include non-cleavable maleimidocaproyl linkers, which are particularly useful for conjugating microtubule-disrupting agents such as auristatins, and are described in 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.

[0351] 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 multiple ways to form linkers useful for conjugating the antibodies and cytotoxins disclosed herein. Additional linkers useful in conjunction with the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the entire disclosure of which is incorporated herein by reference.

[0352] Linkers useful in combination with the antibody-drug conjugates described herein include, but are not limited to, linkers that contain chemical moieties formed by coupling reactions such as those shown below in Table 2. The wavy lines indicate the points of attachment to the antibody or antigen-binding fragment and to the cytotoxic molecule.

[0353] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0354] Those skilled in the art will recognize that a reactive substituent Z attached to a linker and a reactive substituent on an antibody or antigen-binding fragment thereof participate in a covalent coupling reaction to generate the chemical moiety Z, and will recognize the reactive substituent Z. Thus, antibody-drug conjugates useful in conjunction with the methods described herein can be formed by reacting an antibody or antigen-binding fragment thereof with a linker or cytotoxin-linker conjugate, as described herein, wherein the linker or cytotoxin-linker conjugate comprises a reactive substituent Z suitable for reaction with a reactive substituent on an antibody or antigen-binding fragment thereof to form the chemical moiety Z.

[0355] In some embodiments, Z' is -NR 13 C(=O)CH=CH2, -N3, -SH, -S(=O)2(CH=CH2), -(CH2)2S(=O)2(CH=CH2), -NR 13 S(=O)2(CH=CH2), -NR 13 C(=O)CHR 14 , -NR 13 C(=O)CH2Br, -NR 13C(=O)CH2I, -NHC(=O)CH2Br, -NHC(=O)CH2I, -ONH2, -C(O)NHNH2, -CO2H, -NH2, -NH(C=O), -NC(=S), [ka] [ka] and During the ceremony, R 13 is independently selected in each occurrence from H and C1-C6 alkyl; R 14 is -S(CH2) n CHR 15 NHC(=O)R 13 and R 15 is R 13 or -C(=O)OR 13 and R 16 is independently selected in each occurrence from H, C1-C6 alkyl, F, Cl, and —OH; R 17 is independently selected in each occurrence from H, C1-C6 alkyl, F, Cl, —NH2, —OCH3, —OCH2CH3, —N(CH3)2, —CN, —NO2, and —OH; R 18 is independently selected in each occurrence from H, C1-C6 alkyl, F, benzyloxy substituted with —C(═O)OH, benzyl substituted with —C(═O)OH, C1-C4 alkoxy substituted with —C(═O)OH, and C1-C4 alkyl substituted with —C(═O)OH; m is independently selected in each occurrence from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10; n is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14, respectively.

[0356] As shown in Table 2, examples of suitable reactive substituents on linkers and antibodies or antigen-binding fragments thereof include nucleophile / electrophile pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, among others), diene / dienophile pairs (e.g., azide / alkyne pairs or diene / α,β-unsaturated carbonyl pairs, among others). 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 condensation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, among others), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reaction modes known in the art or described herein. Preferably, the linker contains an electrophilic functional group for reacting with a nucleophilic functional group on an antibody or antigen-binding fragment thereof.

[0357] As disclosed herein, the reactive substituents present in an antibody or antigen-binding fragment thereof 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 (where the antibody is glycosylated). These include, but are not limited to, the hydroxyl moieties of serine, threonine, and tyrosine residues, the amino moiety of lysine residues, the carboxyl moiety of aspartic acid and glutamic acid residues, and the thiol moieties of propargyl, azido, haloaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of unnatural amino acids. In some embodiments, the reactive substituents present in an antibody or antigen-binding fragment thereof disclosed herein comprise an amine or thiol moiety. Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Thus, each cysteine ​​bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by reacting lysines with 2-iminothiolane (Traut's reagent), resulting in the conversion of amines to thiols. Reactive thiol groups can be introduced into antibodies (or fragments thereof) by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing mutant antibodies containing one or more non-natural cysteine ​​amino acid residues). U.S. Patent No. 7,521,541 teaches engineering antibodies by the introduction of reactive cysteine ​​amino acids.

[0358] In some embodiments, the reactive moiety Z attached to the linker is a nucleophilic group reactive with an electrophile present on the antibody. Useful electrophiles on antibodies include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of the nucleophilic group can react with an electrophile on the antibody to form a covalent bond with the antibody. Useful nucleophilic groups include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In some embodiments, Z is the product of the reaction between a reactive nucleophilic substituent present in an antibody or an antigen-binding fragment thereof (e.g., an amine moiety and a thiol moiety) and the reactive electrophilic substituent Z. For example, Z can be, among others, a Michael acceptor (e.g., maleimide), an activated ester, an electron-deficient carbonyl compound, and an aldehyde.

[0359] In some embodiments, an ADC comprises an anti-CD5 antibody or an anti-CD5 antibody conjugated via a linker and a chemical moiety Z to an amatoxin of any of Formulas I, IA, IB, II, IIA, or IIB disclosed herein. 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 the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker is -((C=O)(CH2) n -unit, where n is an integer from 1 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n -It is.

[0360] In some embodiments, the linker is -(CH2) n It contains a - unit, and n is an integer of 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH) n In some embodiments, the linker is -(CH2). n In some embodiments, the linker is -((CH) n - and n is 6.

[0361] In some embodiments, the chemical moiety Z is selected from Table 2. In some embodiments, the chemical moiety Z is represented by: [ka] , wherein S is a sulfur atom representing a reactive substituent present in an antibody or antigen-binding fragment thereof that binds to CD2 or CD5 (e.g., from the —SH group of a cysteine ​​residue).

[0362] In some embodiments, the linker L and chemical moiety Z, collectively LZ, are represented by: [ka]

[0363] Those skilled in the art will recognize that the structure of the linker reactive substituent prior to conjugation to an antibody or antigen-binding fragment thereof includes a maleimide group as the Z group. The foregoing linker moieties and amatoxin-linker conjugates useful in conjunction with, among other things, the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220 and WO 2017 / 149077, the entire disclosures of each of which are incorporated herein by reference.

[0364] In some embodiments, the structure of the linker reactive substituent prior to conjugation to an antibody or antigen-binding fragment thereof is as follows: [ka]

[0365] Preparation of antibody-drug conjugates In the ADCs of Formula I disclosed herein, an antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxic drug moieties (D) (e.g., about 1 to about 20 drug moieties per antibody) via a linker, L, and a chemical moiety, Z, as disclosed herein. The ADCs of the present disclosure can be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those of skill in the art, including: (1) reacting a reactive substituent on an antibody or antigen-binding fragment thereof with a bivalent linker reagent to form Ab-ZL, as described above, followed by reaction with a drug moiety, D; or (2) reacting a reactive substituent on a drug moiety with a bivalent linker reagent to form DLZ, followed by reaction with a reactive substituent on an antibody or antigen-binding fragment thereof, as described above, to form an ADC of formula DLZ-Ab, such as Am-ZL-Ab. Additional methods for preparing ADCs are described herein.

[0366] As described herein, in another embodiment, 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. The sulfhydryl groups are then conjugated through the sulfur atom to form an ADC, as described herein above. Reagents that can be used for lysine modification include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent). In another embodiment, 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 sulfhydryl groups are then conjugated through the sulfur atom to form an ADC, as described herein above.

[0367] In yet another embodiment, the antibody can 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 via the corresponding aldehyde, as described herein above. Other protocols for modifying proteins for the 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. Methods for attaching linker drug moieties to cell-targeting proteins such as antibodies, immunoglobulins, or fragments thereof can be found, for example, in U.S. Pat. No. 5,208,020, U.S. Pat. No. 6,441,163, WO 2005037992, WO 2005081711, and WO 2006034488, all of which are expressly incorporated herein by reference in their entirety.

[0368] Alternatively, a fusion protein comprising an antibody and a cytotoxic agent can be produced, for example, by recombinant techniques or peptide synthesis. The length of DNA can include regions encoding the two portions of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not disrupt the desired properties of the conjugate.

[0369] Treatment method Anti-CD2 or anti-CD5 ADCs can be used to target T cells in the patient's thymus, and T cell-specific ADCs can be used to deplete endogenous T cells and "reboot" the subject's immune system.

[0370] In vivo T cell depletion is typically achieved using chemotherapy, such as antithymocyte globulin (ATG). Current methods achieve T cell depletion while leaving the subject's immune system intact. T cell depletion can be used to treat subjects who have undergone or will undergo hematopoietic stem cell (HSC) transplantation, such as autologous HSC transplantation.

[0371] As described herein, hematopoietic stem cell transplantation therapy can be administered to a subject in need of treatment to expand or repopulate one or more blood cell types. Hematopoietic stem cells typically exhibit pluripotency and can therefore differentiate into multiple different blood lineages, including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Hematopoietic stem cells can also self-renew, thus giving rise to daughter cells with potential equivalent to that of the parent cells, and are characterized by their ability to be reintroduced into the transplant recipient, where they home to the hematopoietic stem cell niche and restore productive and sustained hematopoiesis.

[0372] Thus, hematopoietic stem cells can be administered to patients with defects or deficiencies in one or more cell types of the hematopoietic lineage to reconstitute defective or deficient cell populations in vivo, thereby treating pathologies associated with defects or deficiencies in endogenous blood cell populations. Thus, the 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, the compositions and methods described herein can be used to treat immunodeficiencies, such as congenital immunodeficiencies. Additionally or alternatively, the compositions and methods described herein can be used to treat acquired immunodeficiencies (e.g., acquired immunodeficiencies selected from the group consisting of HIV and AIDS). The compositions and methods described herein can be used to treat metabolic disorders (e.g., a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidoses, and metachromatic leukodystrophy).

[0373] Additionally or alternatively, the compositions and methods described herein can be used to treat malignant tumors or proliferative disorders, such as hematological cancers and myeloproliferative disorders. For cancer treatment, the compositions and methods described herein can be administered to patients prior to hematopoietic stem cell transplantation therapy to deplete immune cell populations that cross-react with non-autologous hematopoietic stem cells and initiate an immune response against them. This helps prevent or reduce the likelihood of rejection of the transplanted hematopoietic stem cell graft and allows the transplanted hematopoietic stem cells to home to the stem cell niche and establish productive hematopoiesis. This can then repopulate the population of cells that are depleted during cancer cell eradication, such as during systemic chemotherapy. 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 cancerous conditions, including neuroblastoma.

[0374] Anti-CD2 or anti-CD5 antibody-drug conjugates (ADCs) can be used to treat disorders associated with aberrant T cell activity. For example, anti-CD2 ADCs or anti-CD5 ADCs can be used to treat T cell malignancies (e.g., lymphomas affecting T cells), where an effective amount of an anti-CD2 or anti-CD5 ADC is administered to a subject suffering from a T cell malignancy to reduce the proliferation or proliferation of malignant T cells. A "T cell malignancy" is a cancer formed within T cells. In particular, the methods disclosed herein can be used to treat patients suffering from T cell malignancies associated with CD5+ expression. In certain embodiments, the malignant T cells express CD5+, such that treatment with an anti-CD5 ADC targets and depletes the malignant T cells, resulting in a therapeutic effect. In certain embodiments, the T cell malignancy is a relapsed, refractory T cell malignancy. Examples of T-cell malignancies that can be treated using the methods disclosed herein include T-cell acute lymphoblastic lymphoma (T-ALL; also called precursor T-cell leukemia, T-cell acute lymphoblastic leukemia), T-cell large granular lymphocyte (LGL) leukemia, human T-cell leukemia virus type 1 positive (HTLV-1+), adult T-cell leukemia / lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), and peripheral T-cell lymphoma (PTCL).

[0375] In certain embodiments, anti-CD2 ADCs or anti-CD5 ADCs can be used to treat human patients with T-cell lymphoma. Examples of T-cell lymphomas that can be treated using the methods and compositions disclosed herein include T-cell childhood systemic EBV+ T-cell lymphoma, extranodal NK- / T-cell lymphoma, nasal type, enteropathy-associated T-cell lymphoma, monomorphic epithelial intestinal T-cell lymphoma, indolent T-cell, gastrointestinal lymphoproliferative disease, hepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, mycosis fungoides, Sézary syndrome, primary cutaneous CD30-positive T-cell, lymphoproliferative disorders (e.g., lymphomatoid papulosis, primary cutaneous anaplastic large cell lymphoma), primary cutaneous gamma delta T-cell lymphoma, primary cutaneous CD8-positive aggressive epidermotropic cytotoxic T-cell lymphoma, primary cutaneous acral CD8-positive T-cell lymphoma, primary cutaneous CD4 These include, but are not limited to, TFH-positive small- and medium-cell T-cell lymphoma, angioimmunoblastic T-cell lymphoma, follicular T-cell lymphoma, nodal peripheral T-cell lymphoma with a TFH phenotype, anaplastic large cell lymphoma (ALK+), anaplastic large cell lymphoma (ALK-), and breast implant-associated anaplastic large cell lymphoma.

[0376] In some embodiments, a human patient suffering from a T-cell malignancy is treated by administering an anti-CD5 ADC, e.g., an anti-CD5 antibody conjugated via a linker to an amatoxin described herein.

[0377] In some embodiments, a human patient suffering from a T-cell malignancy is treated by administering an anti-CD2 ADC, e.g., an anti-CD2 antibody conjugated via a linker to an amatoxin described herein.

[0378] Further conditions that may be treated using the methods and compositions disclosed herein include, but are not limited to, adenosine deaminase deficiency and severe combined immunodeficiency, hyperimmunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, and juvenile rheumatoid arthritis.

[0379] The anti-CD5 or CD2 ADCs described herein can be used to induce solid organ transplant tolerance. For example, the compositions and methods described herein can be used to deplete or remove populations of immune cells prior to hematopoietic stem cell transplantation. Following cell depletion from such target tissues, a population of stem or progenitor cells (e.g., hematopoietic stem cells from an organ donor) can be administered to the transplant recipient, and subsequent engraftment of such stem or progenitor cells can achieve temporary or stable mixed chimerism, thereby enabling long-term transplant tolerance without the need for additional immunosuppressants. Administration of anti-CD2 or anti-CD5 ADCs can reduce the likelihood of rejection of the transplanted graft or may even prevent rejection entirely. Thus, the compositions and methods described herein can be used to induce transplant tolerance in solid organ transplant recipients (e.g., kidney, lung, liver, and heart transplants, among others). The compositions and methods described herein are well suited for use in inducing solid organ transplant tolerance, for example, because a low percentage of temporary or stable donor grafts is sufficient to induce long-term tolerance of the transplanted organ.

[0380] Furthermore, the compositions and methods described herein can be used to directly treat cancers, such as cancers characterized by CD2+ or CD5+ cells. For example, the compositions and methods described herein can be used to treat leukemia, particularly in patients exhibiting CD2+ or CD5+ leukemia cells. By depleting CD2+ or CD5+ cancerous cells, such as leukemia cells, the compositions and methods described herein can be used to directly treat various cancers. Examples of cancers that can be treated in this way include blood 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.

[0381] Furthermore, the compositions and methods described herein can be used to treat autoimmune diseases. The methods and compositions disclosed herein can also be used to substantially deplete endogenous CD5+ or CD2+ T cells in the thymus of subjects suffering from autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus (SLE), or systemic sclerosis (SSc). For example, an antibody or antigen-binding fragment thereof can be administered to a subject, such as a human patient, suffering from an autoimmune disease to kill CD2+ or CD5+ immune cells. The CD2+ or CD5+ immune cells can be autoreactive lymphocytes, such as T cells, that express T cell receptors that specifically bind to and mediate an immune response against an autoantigen. By depleting autoreactive CD2+ or CD5+ cells, the compositions and methods described herein can be used to treat autoimmune conditions, such as those described below. Additionally or alternatively, the compositions and methods described herein can be used to treat autoimmune diseases by depleting endogenous hematopoietic stem cell populations prior to hematopoietic stem cell transplantation therapy, where the transplanted cells can home into the niche created by the endogenous cell depletion process and establish productive hematopoiesis, which can then reconstitute the population of cells depleted during autoimmune cell eradication.

[0382] Autoimmune diseases that may 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), human systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), lymphocytic colitis, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, Autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Barrow's disease, Behçet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, chronic fatigue and immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac sprue dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythematosus, autonomic dysfunction, endometriosis, essential mixed cryoglobulinemia, fibromyalgia Myalgia / fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, Lyme disease, Meniere's disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus-myoclonus ataxia (OMS), optic neuritis, Ord's thy...

Claims

1. 1. A composition comprising an anti-CD5 antibody-drug conjugate (ADC) for use in a method of treating a human subject suffering from or at risk of developing steroid-resistant graft-versus-host disease (GVHD), the method comprising administering the composition to the subject, wherein the ADC comprises an anti-CD5 antibody, or an antigen-binding fragment thereof, conjugated to a cytotoxin via a linker; the cytotoxin is an amatoxin; The ADC is represented by the formula Ab-Z-L-Am, where Ab is the anti-CD5 antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an amatoxin, and has the formula (IB): 【Chemistry 1】 is represented by In the formula, R １ is H, OH, OR Ａ or OR Ｃ and R ２ is H, OH, OR Ｂ or OR Ｃ and R Ａ and R Ｂ when present, together with the oxygen atom to which they are attached, combine to form an optionally substituted 5-membered heterocycloalkyl group; R ３ is H, R Ｃ or R Ｄ and R ４ , R ５ , R ６ , R ７ are each independently H, OH, or OR Ｃ , OR Ｄ , R Ｃ or R Ｄ and R ８ OH, NH ２ , OR Ｃ , OR Ｄ , N.H.R. Ｃ or NR Ｃ R Ｄ can be, R ９ is H, OH, OR Ｃ or OR Ｄ and X is -S-, -S(O)- or -SO ２ - and R Ｃ is -L-Z, R Ｄ is an optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ Heteroalkyl, optionally substituted C ２ -C ６ Alkenyl, optionally substituted C ２ -C ６ Heteroalkenyl, optionally substituted C ２ -C ６ Alkynyl, optionally substituted C ２ -C ６ heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl or optionally substituted heteroaryl; L is an optionally substituted C １ -C ６ Alkylene, optionally substituted C １ -C ６ Heteroalkylene, optionally substituted C ２ -C ６ Alkenylene, optionally substituted C ２ -C ６ Heteroalkenylene, optionally substituted C ２ -C ６ Alkynylene, optionally substituted C ２ -C ６ heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, —C(═O)—, peptide, or combinations thereof; Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in the antibody or antigen-binding fragment thereof; Am is exactly one R Ｃ containing a substituent, and The anti-CD5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 54, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 55, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 56, and a light chain variable region comprising a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 58, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 59; A composition for preventing or treating GVHD, wherein the anti-CD5 ADC depletes T cells.

2. (a) the anti-CD5 antibody, or antigen-binding fragment thereof, comprises a heavy chain comprising a variable region set forth in the amino acid sequence of SEQ ID NO: 282 and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO: 283; or (b) The composition of claim 1, wherein the anti-CD5 antibody or antigen-binding fragment thereof comprises a heavy chain comprising a variable region set forth in the amino acid sequence of SEQ ID NO: 288 and a light chain comprising a variable region comprising the amino acid sequence set forth in SEQ ID NO:

289.

3. The composition according to claim 1 or 2, wherein the steroid-resistant GVHD is steroid-resistant acute GVHD.

4. The composition according to any one of claims 1 to 3, wherein the subject has previously undergone allogeneic HSC transplantation.

5. The composition of claim 3 or 4, wherein the subject has steroid-resistant acute GVHD grade 2 to grade 4 (Mount Sinai Acute GVHD International Consortium (MAGIC) criteria).

6. The composition according to any one of claims 3 to 5, wherein after administration of the anti-CD5 ADC, the GVHD grade according to the MAGIC criteria is reduced by one grade.

7. The composition according to any one of claims 1 to 6, wherein the ADC is internalized by CD5+ immune cells after administration to a patient.

8. The ADC is represented by the formula Ab-ZL-Am, where Ab-ZL-Am is 【Chemistry 2】 The composition according to any one of claims 1 to 7, characterized in that

9. The ADC is represented by the formula Ab-ZL-Am, where Ab-ZL-Am is 【Transformation 3】 The composition according to any one of claims 1 to 7, wherein the composition is any one of the following:

10. The ADC is represented by the formula Ab-ZL-Am, where Ab-ZL-Am is 【Chemistry 4】 The composition according to any one of claims 1 to 7, characterized in that

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