T cell depleting therapy

Abstract

To provide T cell depleting therapies.SOLUTION: The disclosure provides a method for depleting T cells for therapeutic uses, comprising administering a therapeutic anti-CD2 or anti-CD5 antibody-drug conjugate (ADC) for treatment. Provided herein are anti-CD2 ADC or anti-CD5 ADC for use as an agent to treat hematological diseases and proliferative diseases, in particular stem cell disorders, cancer or autoimmune diseases. Disclosed compositions and methods can be used to deplete cell populations of CD2+ or CD5+, including CD2+ or CD5+ cancer cells or CD2+ or CD5+ immune cells to treat hematological diseases and proliferative diseases, in particular, stem cell disorders, cancer or autoimmune diseases, and for preparing a patient to hematopoietic stem cell transplantation or solid organ transplantation.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Provisional Patent Application No. 62 / 857,744, filed June 5, 2019. No. 60 / 699,999, filed on May 1, 2003, the contents of which are incorporated herein by reference.

[0002] Sequence Listing This application is a seeking application filed electronically in ASCII format via EFS-Web. June 2020, which includes the entirety of the enlisted materials. The ASCII copy, created on the 3rd, is M103034_2170WO_SL.t It is called xt 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. T cells are an important part of the immune system, but abnormal T cell activity can lead to disease in patients. For example, graft-versus-host disease (GVHD) can occur primarily in the graft. These are caused by immune T cells, which trigger an immune response that damages host tissues. Other examples of abnormal T cell activity that can lead to T cell-related cancers such as T cell lymphoma Therapeutics that target T cells remain a challenge in the art. Summary of the Invention [Problem to be solved by the invention]

[0004] Currently, T cell targeting compositions and methods for treating disorders of the hematopoietic system, such as autoimmune diseases, are available. , as well as exogenous hematopoietic stem cells to maintain the pluripotency and hematopoietic function of these cells after transplantation. There is a need for compositions and methods that promote the engraftment of cell grafts.

[0005] Provided herein are, inter alia, various disorders of the hematopoietic system, metabolic disorders, cancer, and autoimmune diseases. T cell targeting compositions and methods for directly treating disease. The products and methods may be used to treat diseases such as, but not limited to, blood cancer or autoimmune diseases. Targeting immune cells to modulate human patients for therapeutic hematopoietic stem cell transplantation . [Means for solving the problem]

[0006] In one aspect, the present invention provides a method for depleting T cells in a subject suffering from an autoimmune disease. The present invention provides a method for administering an anti-CD5 antibody drug conjugate ( the ADC is administered to the cell via a linker. an anti-CD5 antibody or antigen-binding fragment thereof conjugated to a cytotoxin; It includes an anti-CD2 antibody or an antigen-binding fragment thereof.

[0007] In one embodiment, the effective amount is a measure of the endogenous CD5+ or CD2+ T cells in the thymus of the subject. The amount is sufficient to substantially deplete the cells.

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

[0009] In another embodiment, the present invention relates to a method for treating a patient suffering from steroid-resistant graft-versus-host disease (GVHD). The present invention provides a method for treating a subject having or at risk of developing GVHD, the method comprising: The method involves administering an anti-CD2 ADC or an anti-CD5 ADC to treat steroid-resistant GVHD. administering an ADC to a subject suffering from steroid-resistant GVHD, wherein the ADC Anti-CD5 antibodies or their antigen-binding flags conjugated to cytotoxins via a carrier ment, or an anti-CD2 antibody or antigen-binding fragment thereof.

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

[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 Graft-versus-Human Disease International Consortium (MAGIC) criteria. In this condition, after administration of anti-CD2 ADC or anti-CD5 ADC, This reduces the GVHD grade by one.

[0013] In another aspect, provided herein is a method of treating a subject suffering from a T-cell malignancy. The method comprises administering to a subject an effective amount of an anti-CD2 ADC or an anti-CD5 ADC. The ADC comprises an anti-CD5 antibody or antibody conjugated to a cytotoxin via a linker. or an anti-CD2 antibody or an antigen-binding fragment thereof. include.

[0014] In one embodiment, the T cell malignancy is lymphoma. Cellular malignancies include T-cell acute lymphoblastic lymphoma (T-ALL), T-cell large granular lymphoma (T-LAM), and LGL leukemia, human T-cell leukemia virus type 1 positive (HTLV-1+), adult T-cell leukemia Acute myeloid leukemia / lymphoma (ATL), T-cell prolymphocytic leukemia (T-PLL), or peripheral It is a type of 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. The antibody is an antibody selected from the group consisting of IgG, IgA, IgM, IgD and IgE. In certain embodiments, the IgG isotype is IgG1 or I gG4.

[0017] In some embodiments, the ADC is an anti-CD5 ADC (e.g., amatoxin-coated It is a conjugated 5D7).

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

[0019] In one aspect, described herein are methods for enhancing hematopoietic stem cell graft survival. and T cell targeting compositions for conditioning patients, such as human patients, prior to undergoing hematopoietic stem cell transplantation therapy. The patient may have an autoimmune disease, cancer, hemoglobinopathy, or other inflammatory disease. suffer from one or more blood disorders, such as hematopoietic leukemia, that require a hematopoietic stem cell transplant It may be the same as the above.

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

[0021] In certain embodiments, the compositions described herein target T cells, specifically CD2 or an antibody-drug conjugate (ADC) that binds to CD5, and administering the composition to a patient; In response to these factors, (i) CD4+ cells, such as autoreactive T cells, B cells, and natural killer (NK) cells, By selectively depleting immune cell populations expressing CD2 or CD5, autoimmune diseases can be prevented. and / or (ii) administering hematopoietic stem cell transplants to patients. Before administration, the T cell, B cell, or NK cell populations are depleted, thereby increasing the number of hematopoietic stem cells. The present invention features a method for reducing the likelihood of transplant rejection. T cells, B cells, or N cells that cross-react with and initiate an inappropriate immune response against self-antigens It can be expressed by K cells, and the activity of the former may contribute to a wide range of autoimmune diseases. In this case, anti-CD2 antibody-drug conjugates or anti-C Administration of the D5 antibody-drug conjugate to a patient results in cross-reactivity with one or more autoantigens. A collection of CD2+ (or CD5+) autoimmune cells, such as T cells, B cells, or NK cells, This latter activity can cause the depletion of ATP, thereby treating autoimmune diseases. promotes the creation of an environment conducive to hematopoietic stem cell engraftment because cross-react with one or more non-self antigens (e.g., non-self MHC antigens) expressed on the T cells, B cells, and / or NK cells mount an immune response against the transplanted hematopoietic stem cells. In this latter case, the immune system can initiate immune responses, leading to graft rejection. Patients suffering from disorders such as diseases or other conditions of the hematopoietic system may be diagnosed with a defect in e.g. or hematopoietic stem cells to repopulate one or more populations of depleted blood cells. The transplant can be administered subsequently. Also, as used herein, the present invention relates to the treatment of sickle cell anemia, thalassemia, and the like. , 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 Treating various hematopoietic disorders, including immunodeficiency syndromes, cancer, and autoimmune diseases It also provides the law.

[0022] In certain embodiments, provided herein are methods for depleting T cells in a subject in need thereof. The present invention describes a method for administering an effective amount of an anti-CD5 or anti-CD2 antibody drug conjugate ( ADC) to a subject in need thereof, wherein the subject has hematopoietic stem cells (HSCs). ) undergoing or prior to a transplant or solid organ transplant, and the ADC These include anti-CD5 or anti-CD2 antibodies conjugated to a cytotoxin via a cytotoxin-containing antibody.

[0023] In one embodiment, an effective amount of an anti-CD5 or anti-CD2 antibody drug conjugate (AD C) require it before undergoing hematopoietic stem cell (HSC) transplantation or solid organ transplantation When administered to a subject, the method comprises administering to the subject an HSC graft or a solid organ graft. It further includes:

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

[0025] In one embodiment, the HSC graft comprises a graft in which the level of ADC is substantially clear from the subject's blood. Once this has been achieved, it is administered to the subject.

[0026] In one embodiment, the HSCs or solid organ transplant are administered by injecting the ADC into the blood of the subject. In another embodiment, the subject is administered between 1 hour and 7 days after the onset of the disease is substantially apparent. In this study, HSC or solid organ transplants were administered to subjects whose blood levels of ADC were substantially clear. In yet another embodiment, the compound is administered to a subject between 6 hours and 3 days after the compound has been administered. HSC or solid organ transplants are administered to subjects whose blood levels of ADC are substantially clear. The test is administered to the subject between 12 hours and 36 days after the test.

[0027] In one embodiment, the HSCs or solid organ transplant are administered by injecting the ADC into the blood of the subject. The ADC is administered to a subject approximately 24 hours after the ADC becomes substantially clear from the Examples include anti-CD5 or anti-CD2 antibodies conjugated to a cytotoxin.

[0028] In certain embodiments, the present invention provides methods for depleting T cells in subjects suffering from autoimmune diseases. The present invention provides a method for administering an effective amount of anti-CD5 or anti-CD6 antibody to a subject suffering from an autoimmune disease. 2. administering an antibody drug conjugate (ADC), wherein the ADC is linked via a linker In one embodiment, the antibody comprises an anti-CD5 or anti-CD2 antibody conjugated to a cytotoxin. The subjects are those with multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus (SLE), or has systemic sclerosis (SSc).

[0029] In one embodiment, the effective amount is a measure of the endogenous CD5+ or CD2+ T cells in the thymus of the subject. The amount is sufficient to substantially deplete the cells.

[0030] In another embodiment, the present invention relates to a method for treating a patient suffering from steroid-resistant graft-versus-host disease (GVHD). The present invention provides a method of treating a subject with steroid-resistant steroids, the method comprising administering an anti-CD2 or anti-CD5 ADC to a subject. and administering the compound to a subject suffering from steroid-resistant GVHD to treat the steroid-resistant GVHD. The ADCs consist of anti-CD5 or anti-CD20 antibodies 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 an allogeneic HSC transplant. In one embodiment, the subject has steroid-resistant acute GVHD grade 2 to 4 (Mount Syndrome). In one embodiment, the patient has the following criteria: After administration of anti-CD2 or anti-CD5 ADC, GVHD grade 1 according to the MAGIC criteria The grade will be lowered.

[0031] In a further aspect, provided herein is a method of treating a subject suffering from a T-cell malignancy. the method comprises administering to a subject an effective amount of an anti-CD2 or anti-CD5 ADC; ADCs are anti-CD5 or anti-CD2 antibodies conjugated to a cytotoxin via a linker. In one embodiment, the T cell malignancy is lymphoma. T-cell malignancies include T-cell acute lymphoblastic lymphoma (T-ALL), T-cell large granular lymphoma (T-LAM), and 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), or In one embodiment, the T-cell malignancy is refractory to relapse. It is a curable T-cell malignancy.

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

[0033] In another embodiment, the antibodies comprise IgG, IgA, IgM, IgD, and IgE. In one embodiment, the IgG isotype is It 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 Pseudomonas exotoxin A, deboganin, diphtheria toxins, amatoxins, saporins, maytansin, maytansinoids, auristatins, a Irinotecan, SN-38, duocarmycin, Pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolinobenzodiazepines 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 this case, 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, L is a linker, Z is a chemical moiety and Am is an amatoxin, 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 are optionally substituted together with the oxygen atoms to which they are attached, if present. forming a 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 in can be, 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 D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 heteroaryl alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroaryl alkynyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroaryl alkynyl, 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 heteroaromatic group, alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 heptylene alkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C 6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene chloroalkylene, optionally substituted arylene, optionally substituted heteroarylene, diaryl peptide, -C(=O)-, peptide, or a combination thereof; Z is a group consisting of a reactive substituent present on L and a group consisting of a reactive substituent present in the antibody or antigen-binding fragment thereof. is a chemical moiety formed from a coupling reaction between a reactive substituent and 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 present together with the oxygen atom to which they are attached, they are bonded and optionally substituted. forming a 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 ORD and X is -S-, -S(O)- or -SO2-; R C is -LZ, R D is optionally substituted C1-C6 alkyl, optionally substituted C1-C6 hetero Roa alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroaromatic alkyl, optionally substituted C2-C6 aryl, optionally substituted C2-C6 hetero ... alkenyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 hetero Alkynyl, 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 hetero C2-C6 alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 Heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2 -C6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted hetero cycloalkylene, optionally substituted arylene, optionally substituted heteroarylene , a dipeptide, -C(=O)-, a peptide, or a combination thereof; Z is a reactive substituent present on L and a substituent present in the antibody or antigen-binding fragment thereof. is a chemical moiety formed from a coupling reaction between reactive substituents present, 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 present together with the oxygen atom to which they are attached, they are bonded and optionally substituted. forming a 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 in can be, 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 heteroaryl alkyl, optionally substituted C2-C6 alkenyl, optionally substituted C2-C6 heteroaryl alkynyl, optionally substituted C2-C6 alkynyl, optionally substituted C2-C6 heteroaryl alkynyl, 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 heteroaryl, alkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 heteroaryl Alkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 Heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocyclo alkylene, optionally substituted arylene, optionally substituted heteroarylene, diphenyl peptide, -C(=O)-, peptide, or a combination thereof; Z is a group consisting of a reactive substituent present on L and a group consisting of a reactive substituent present in the antibody or antigen-binding fragment thereof. is a chemical moiety formed from a coupling reaction between a reactive substituent and Am contains exactly one Rc substituent.

[0041] In one embodiment, the ADC is represented by Ab-ZL-Am, where Ab is an antibody. or an antigenic fragment thereof, wherein Z is a chemical moiety, L is a linker, and Am is The amatoxin-linker conjugate Am-LZ is represented by the formula (II) , represented by formula (IIA) or formula (IIB). [ka] wherein X is S, SO, or SO; R1 is H or a reactive substituent present on the linker and a reactive substituent present in the antibody. or an antigen-binding fragment thereof, a linker covalently attached to the antibody or antigen-binding fragment thereof via R2 is H or a reactive substituent present on the linker and a reactive substituent present in the antibody. or an antigen-binding fragment thereof, a linker covalently attached to the antibody or antigen-binding fragment thereof via 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, e.g., DM1 or DM It is 4.

[0043] In one embodiment, the cytotoxin of the ADC is an auristatin, e.g., monomethylauristatin. These are monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF).

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

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

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

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

[0048] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof is SEQ ID NO: 28 8, and a light chain variable region comprising SEQ ID NO:289.

[0049] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof is SEQ ID NO: 29 1, 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 include (i) a population of cells characterized by CD2+ cells or CD5+ cells. (ii) for the direct treatment of cancer and autoimmune diseases, and (iii) hematopoietic stem cell transplants. by depleting populations of immune cells that cross-react with and mount an immune response against them. (e.g., cross-reacting with non-self MHC antigens expressed by hematopoietic stem cell grafts) to promote the engraftment of transplanted hematopoietic stem cells in patients requiring transplantation therapy. To do this, CD2 (also known as T-cell surface antigen, LFA-2, and LFA-3 receptors) or Antibody-drug conjugates (ADs) that bind to CD5 (lymphocyte antigen T1 / Leu-1) C) can be used as therapeutic agents. These therapeutic activities are based in part on the discovery that For example, anti-CD2 or anti-CD5 antibody drug conjugates may target non-autologous hematopoietic stem cell antigens (e.g., For example, they bind to cancer cells, autoimmune cells, or immune cells that cross-react with non-self MHC antigens. This can occur by binding to the ATP-dependent ATP-dependent ATP complex, thereby inducing the death of the bound cell. When depleting populations of autoimmune cells, anti-CD2 ADCs or anti-CD5 ADCs are useful. Use to directly treat cancer or autoimmune diseases, such as cancer autoimmune diseases, as described in the specification. Depleting populations of immune cells that cross-react with non-self hematopoietic stem cell antigens , anti-CD2 ADCs or anti-CD5 ADCs are used to treat stem cell disorders, cancer, or autoimmune diseases. To prevent or reduce the likelihood of graft rejection in patients with hematopoietic stem cell transplantation In such cases, one or more non-autologous hematopoietic stem cells can be used to reduce the CD2+ immune cells that cross-react with a cell antigen (e.g., one or more non-self MHC antigens). Depletion of CD5+ cells or CD5+ immune cells results in the depletion of transplanted hematopoietic stem cells within the transplant recipient. As the transplanted cells engraft, they become part of the hematopoietic tissue. The transplanted hematopoietic stem cells then , megakaryocytes, platelets, 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 are deficient in transplant recipients. In this way, anti-CD2 ADCs or Anti-CD5 ADCs can be used to treat human patients suffering from stem cell disorders, such as those described herein. This can promote successful engraftment of hematopoietic stem cells in patients with

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

[0053] As used herein, the term "amatoxin" refers to a compound produced by mushrooms. Members of the amatoxin family of peptides, synthetic amatoxins, and mutant amatoxins or a mutant or derivative thereof capable of inhibiting RNA polymerase II activity. Also refers to synthetic amatoxins (USA, incorporated herein by reference). As described herein, amatoxins are also included. The linker is attached to an antibody or antigen-binding fragment thereof, for example, via a linker moiety (L). In this way, the conjugate (also called antibody drug conjugate (ADC)) Exemplary methods of amatoxin binding and methods useful for such processes Suitable linkers are shown below and are known in the art. Exemplary linker-containing amatoxins useful for conjugation to antibodies or antigen-binding fragments is also described herein.

[0054] In certain embodiments, Amatoki spp. are useful with the compositions and methods described herein. Examples of amanitin include compounds according to the following formula (III), α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, amanulin acid, or proamanulin, of formula (III): [ka] and In the formula, R1 is H, OH, or OR A and R2 is H, OH, or OR B and R A and R B are optionally substituted together with the oxygen atoms to which they are attached, if present. forming a 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, ORD or R D and R8 is OH, NH2 or OR D and R9 is H, OH or OR D and X is -S-, -S(O)-, or -SO2-; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted hydroxyl heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C-C alkenyl), optionally substituted heteroalkenyl (e.g., C-C 6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl) , optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), optionally optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl or peptide.

[0055] For example, in one embodiment, useful in combination with the compositions and methods described herein Suitable amatoxins include compounds according to formula (IIIA): [ka] wherein R1 is H, OH, or OR A and R2 is H, OH, or OR B and R A and R B are optionally substituted together with the oxygen atoms to which they are attached, if present. forming a substituted 5-membered heterocycloalkyl group, R3 is H or R D and R4 is H, OH, OR D or R D and R5 is H, OH, OR D or R D and R6 is H, OH, OR D or R D and R7 is H, OH, OR D or R D and R8 is OH, NH2, or OR D and R9 is H, OH or OR D and X is -S-, -S(O)-, or -SO2-; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2- C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl ), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), optionally optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl or peptide.

[0056] In one embodiment, amatoxins useful in combination with the compositions and methods described herein Xines 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 Bare optionally substituted together with the oxygen atoms to which they are attached, if present. forming a substituted 5-membered heterocycloalkyl group, R3 is H or R D and R4 is H, OH, OR D or R D and R5 is H, OH, OR D or R D and R6 is H, OH, OR D or R D and R7 is H, OH, OR D or R D and R8 is OH, NH2 or OR D and R9 is H, OH or OR D and X is -S-, -S(O)-, or -SO2-; R D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted hydroxyl heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C-C alkenyl), optionally substituted heteroalkenyl (e.g., C-C 6 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 It is aryl, optionally substituted heteroaryl or peptide.

[0057] As described herein, amatoxins can be attached to antibodies or or antigen-binding fragments thereof. The methods and linkers useful in such processes are described in the "Linkers for Chemical Bonding" section. The compositions and methods described herein are described in the preceding section and in Table 2 below. anti-CD2 antibody, or antigen-binding fragment thereof, or anti-CD5 antibody, or Exemplary linker-containing amatoxins useful for antigen-binding fragments thereof are shown herein. These are shown in structural formulas (I), (IA), (IB), (II), (IIA) and (IIB).

[0058] As used herein, the term "antibody" refers to a molecule that specifically binds to a particular antigen or It also refers to an immunoglobulin molecule that is immunologically reactive with a specific antigen. Examples of antibodies include Antibodies include monoclonal and modified forms of antibodies, including chimeric antibodies, humanized antibodies, and the like. antibodies, heteroconjugate antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies, diabodies, antibodies (e.g., Fab', F (ab')2, including Fab, Fv, rgG and scFv fragments) The term "antibody" (Ab) refers to a molecule that specifically binds to a target protein. The complete molecule can be used to generate antibodies, as well as antibody fragments thereof (e.g., Fab and F( ab') and b') fragments. In this case, Fab and F(ab')2 fragments lack the Fc fragment of the intact antibody. Examples of these antibody fragments are described herein.

[0059] Generally, antibodies comprise a heavy chain and a light chain which contain an antigen-binding region. Each heavy chain contains a heavy chain variable region. The heavy chain constant region (abbreviated herein as HCVR or VH) and the heavy chain constant region The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. A light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VL and VL regions are separated by more conserved regions called framework regions (FR). They can also be further divided into hypervariable regions called complementarity-determining regions (CDRs). VH and VL consist of three CDRs and four FRs, and are arranged from the amino terminus to the carboxy terminus. The sequence is arranged at the end of the loop in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Constant regions of the body regulate the function of various cells of the immune system (e.g., effector cells) and the classical complement system. It mediates the binding of immunoglobulins to host tissues or factors, including the first component (Clq) of the It is possible.

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

[0061] The term "antigen-binding fragment" as used herein refers to an antigen-binding fragment of an intact antibody. A molecule other than an intact antibody that contains a portion of the antibody and binds to the antigen to which the intact antibody binds. The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The fragments include, for example, Fv, Fab, Fab', F(ab')2, scFv, diabodies, Dibodies, triabodies, single-chain antibody molecules (such as scFv), affibodies, nanobodies, and a The term "antigen-binding fragment" of an antibody may be a peptide, a domain antibody, or a domain antibody. Examples of binding fragments encompassed by include, but are not limited to: i) a monovalent flag consisting of a Fab fragment, VL, VH, CL, and CH1 domains (ii) F(ab')2 fragment, via disulfide bridges in the hinge region a bivalent fragment comprising two linked Fab fragments, (iii) a VH and a C (iv) an Fd fragment consisting of the H1 domain, and (iv) a VL and VH domain of a single arm of an antibody. (v) a dAb comprising a VH and VL domain; (vi) an Fv fragment comprising a VH and VL domain; ) dAb fragments consisting of the VH domain (see, e.g., Ward et al., Nat (See, e.g., J. Med. Soc. 341:544-546, 1989), (vii) VH or VL domains (viii) an isolated complementarity-determining region (CDR); (ix) any Two or more (e.g., 2, 3, 4, 5, or 6) amino acids optionally linked by a synthetic linker In addition, the two domains of the Fv fragment are The VL and VH are encoded by separate genes, but they are recombined. Using this method, the VL and VH regions are paired together to form a single protein chain that forms a monovalent molecule. These can be linked by a linker that allows them to be made into single chain Fvs ( scFv), see, e.g., Bird et al., Science 242: pp. 423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art. Fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic synthesis of intact immunoglobulins, by electrochemical cleavage, or in some cases by chemical methods known in the art. It can be produced by conventional peptide synthesis procedures.

[0062] As used herein, the terms "anti-CD2 antibody" or "antibody that binds to CD2" The term refers to an antibody that specifically binds to CD2. The antibody may be synthesized with sufficient affinity so that the antibody is useful for targeting cells expressing the antigen. In a preferred embodiment, the antibody is a human CD4+ antibody capable of binding to the antigen. CD2 specifically binds to human CD2 (hCD2), which is found on the surface of immune cells such as T cells. The amino acid sequence of human CD2 to which anti-CD2 antibodies (or anti-CD2 conjugates) bind is The sequence is set forth below in SEQ ID NO: 13. "Anti-CD2 antibody drug conjugate" or "anti-CD2 ADC" refers to an ADC that comprises an anti-CD2 antibody.

[0063] As used herein, the terms "anti-CD5 antibody" or "antibody that binds to CD5" The term refers to an antibody that specifically binds to CD5. The antibody may be synthesized with sufficient affinity so that the antibody is useful for targeting cells expressing the antigen. In a preferred embodiment, the antibody is a human CD4+ antibody capable of binding to the antigen. 5 (hCD5), the amino acid sequence of which is set forth in SEQ ID NO: 286 below. "Anti-CD5 antibody drug conjugate" or "anti-CD5 ADC" refers to an antibody drug conjugate that Refers to ADCs containing antibodies.

[0064] As used herein, the term "bispecific antibody" refers to a antibody that binds two different antigen-binding moieties. A bispecific antibody is a type of multispecific antibody, and refers to a hybrid antibody having two or more sites. including, but not limited to, hybridoma fusion or linking of Fab' fragments. It can be produced by a variety of methods, see, for example, Songsivilai and Lachman n, 1990, Clin.Exp.Immunol.79:315-321, Ko stelny et al., 1992, J. Immunol.148:1547-1 See page 553. The two binding sites of a bispecific antibody may be directed to the same or different protein targets. For example, one of the binding specificities can be a CD40 binding specificity. 2, and the other can be directed against a different T cell surface antigen or another Cell surface proteins, particularly receptors involved in signal transduction pathways that enhance cell proliferation Or it can be directed to a receptor subunit or the like.

[0065] As used herein, the term "complementarity determining region" (CDR) refers to the light chain complementarity determining region of an antibody. The term "hypervariable region" refers to the hypervariable region found in both the heavy chain variable domain and the heavy chain variable domain. The more highly conserved parts are called framework regions (FR). The amino acid positions that delineate the regions will vary depending on the context and various definitions known in the art. Some positions within the variable domain may vary depending on the It can be considered to be within the hypervariable region, but under different criteria it is considered to be outside the hypervariable region. These positions can be considered hybrid hypervariable positions in that they can be considered One or more of the following may also be found in the extended hypervariable region. These hybrid hypervariable positions may contain modifications. Each of these amino acids contains four framework regions that are primarily β-sheet structures and three CDRs. They are connected by loops that connect the β-sheet structure and in some cases The CDRs in each chain form part of the FR1-CDR1-FR2-CDR2- The sequences are arranged in the order FR3-CDR3-FR4, and are closely spaced by the framework regions. These CDRs are conserved and, together with the CDRs from the other antibody chains, contribute to the formation of the target binding site of the antibody (Ka bat et al., Sequences of Proteins of Immuno Logical Interest, National Institutes of Health, Bethesda, MD, 19 As used herein, the numbering of immunoglobulin amino acid residues is Unless otherwise specified, the numbers are based on the immunoglobulin amino acid residue numbering system of Kabat et al. This is done according to the audit system.

[0066] As used herein, "condition" and "conditioning" The term refers to the process of preparing a patient to receive a graft containing hematopoietic stem cells. Such procedures promote hematopoietic stem cell transplant engraftment (e.g., conditioning procedures and Viable hematopoietic stem cells in blood samples isolated from patients following subsequent hematopoietic stem cell transplantation The methods described herein demonstrate that T cell-mediated immune responses are associated with a sustained increase in the number of T cells. an antibody or antigen capable of binding to an antigen expressed by the Administration of antigen-binding fragments to patients allows patients to be considered for hematopoietic stem cell transplantation therapy. As described herein, anti-CD2 antibodies or anti- The CD5 antibody is covalently linked to a cytotoxin to form an antibody-drug conjugate (ADC). Antibodies capable of binding to one or more of the aforementioned antigens may be covalently conjugated. and the antigen-binding fragment or antibody-drug conjugate thereof for hematopoietic stem cell transplantation therapy. Administration to a patient in need thereof may involve, for example, the administration of one or more non-self antibodies expressed by hematopoietic stem cells. CD2+ T cells (CD4+ and and / or CD8+ T cells) and / or CD2+ NK cells or CD5+ N Endogenous immune cells such as K cells (e.g., CD4+ and / or CD8+ T cells) Selective depletion can enhance the survival of hematopoietic stem cell grafts. Selective depletion of immune cells in the brain is achieved through exogenous (e.g., autologous, allogeneic, or syngeneic) hematopoietic stem cell transplantation. Prevent or reduce the likelihood of graft rejection after transplantation.

[0067] As used herein, the term "conjugate" refers to an antibody or its antigen-binding The reactive functional group of one molecule, such as a fragment, and the reactive functional group of another molecule, such as a cytotoxin described herein. A compound formed by chemical bonding with an appropriate reactive functional group on a molecule. The antibody uses a linker between two molecules (e.g., an anti-CD2 antibody and a cytotoxin) that are bound to each other. Examples of linkers that can be used to form the conjugates include naturally occurring amino peptides, including those containing non-naturally occurring amino acids such as D-amino acids Linkers include those described herein and known in the art. Depending on the reactive moiety therein, linkers can be prepared using a variety of strategies. For example, enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, and hydrolysis under basic conditions can be used. can be cleaved by hydrolysis, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (e.g., For example, Leriche et al., Bioorg.Med.Chem., 20:571 (See page 582, 2012.) Note that the term "conjugate" (when referring to a compound) The terms "drug conjugate," "antibody drug conjugate," or "antibody drug conjugate" are used herein. They are also referred to interchangeably as "ADC."

[0068] As used herein, the term "coupling reaction" refers to reactions in which two or more molecules are suitable for reacting with each other. Two or more bonded substituents react to form molecular fragments attached to each substituent (e.g., covalent bonds). Coupling reactions involve chemical reactions that form chemical moieties that link (bond) together. Cytotoxins, such as those known in the art or described herein. The reactive group attached to the fragment is an antibody, an antigen-binding fragment thereof, or an antigen-binding fragment thereof. The antibody may be, for example, an antibody, an antigen-binding fragment thereof, or any other antibody known in the art. or a fragment thereof that is an antibody specific for CD2 or CD5 as described herein. Also included are those that react with suitably reactive substituents. Examples of suitably reactive substituents include nucleophile / electrophile pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, among others) thiol pairs, or thiol / α,β-unsaturated carbonyl pairs), diene / dienophile pairs (e.g. Coupling reactions include, among others, azide / alkyne pairs. However, thiol alkylation, hydroxyl alkylation, amine alkylation, and amine alkylation are not performed. Condensation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+ 2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition), nucleophilic aromatic substitution , electrophilic aromatic substitution, and other reactions known in the art or described herein. Response styles are included.

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

[0070] As used herein, "drug-to-antibody ratio" or "DAR" refers to the ratio of the drug-to-antibody ratio bound to the antibody of an ADC. This refers to the number of cytotoxins (e.g., amatoxins) bound to the antibody. The DAR of the ADC can range from 1 to 8, although higher loads are possible. In certain embodiments, the ADCs described herein comprise 1, 2, 3, 4, 5, 6, 7, or 8 It has a DAR of

[0071] As used herein, the term "donor" refers to the donor from which one or more cells are derived. refers to a human or animal from which the cell or its progeny is isolated prior to administration to a recipient. Or the plurality of cells can be, for example, a population of hematopoietic stem cells.

[0072] As used herein, the term "diabody" refers to a molecule comprising two polypeptide chains. refers to a bivalent antibody having a VH and VL domain on the same peptide chain, each polypeptide chain A linker that is too short (e.g., a linker consisting of five amino acids) to allow intramolecular association of the This configuration forms a homodimer, which contains a VH and a VL domain linked by a VL anchor. Each domain is paired with a complementary domain on another polypeptide chain to form a complex structure. Thus, the term "triabody" refers to a trivalent antibody containing three peptide chains, Each of these allows intramolecular association of VH and VL domains within the same peptide chain. are connected by a very short linker (e.g., a linker consisting of 1-2 amino acids) The VH and VL domains are fused together and fold into their native structure. Therefore, peptides constructed in this way typically have VH and VH sequences in adjacent peptide chains. The VL domains are trimerized to place them in close spatial proximity to each other (e.g., Holt et al., 2001). iger et al., Proc. Natl. Acad. Sci. USA 90:6 (See pp. 444-48, 1993).

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

[0074] The term "effective amount" refers to an amount that prevents or reduces at least one or more signs or symptoms of pain. the amount of therapeutic agent, e.g., anti-CD5 ADC or anti-CD2 ADC, required to treat or alleviate For example, to treat a subject with cancer, a sufficient amount of The effective amount includes a dose that prevents or delays the onset of disease symptoms and slows the progression of disease symptoms. Altering the course of a disease (e.g., slowing the progression of symptoms of a disease) or reversing symptoms of a disease It also contains sufficient amounts.

[0075] As used herein, the term "endogenous" refers to a gene that is endogenous to a human patient, e.g., a gene that is endogenous to a human subject, e.g., ... molecules found naturally in certain organisms, such as human patients undergoing hematopoietic stem cell transplantation therapy, Material such as cells, tissues, or organs (e.g., hematopoietic stem cells or cells of the hematopoietic lineage, e.g., Megakaryocytes, thrombocytes, platelets, red blood cells, Mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts cells, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes ( For example, CD4+ or CD8+ T lymphocytes, or B lymphocytes, etc.

[0076] As used herein, the term "engraftment potential" refers to hematopoietic stem and progenitor cells. It is used to refer to the ability of cells to remodel tissue, and is used when such cells are naturally circulating. The term refers to the tissue homing of cells, whether delivered by transplantation or by endothelial cell endothelial cell. The mechanisms surrounding or leading to engraftment, such as cell migration and colonization of the cells within the tissue of interest, The engraftment efficiency or engraftment rate can be measured by any clinical trial known to those skilled in the art. can be assessed or quantified using clinically accepted parameters, e.g., competitive Evaluation of the reconstituting unit (CRU), stem cell homing, colony formation, or engraftment It can include the incorporation or expression of a marker in a tissue or tissues, and may be due to disease progression, hematopoietic stem and progenitor cell survival, or recipient survival. Engraftment is assessed by measuring peripheral blood white blood cell counts during the post-transplant period. Engraftment can also be determined by the number of donor cells in the bone marrow aspirate sample. It can also be assessed by measuring cell recovery.

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

[0078] As used herein, the term "exogenous" refers to a substance that is native to a particular organism, such as a human patient. molecules, cells, tissues, or organs (e.g., T cells, hematopoietic stem cells) that are not found in , or cells of the hematopoietic lineage, such as megakaryocytes, thrombocytes, platelets ( platelets), red blood cells, mast cells, myeloblasts, basophils, neutrophils, eosinophils, myelophils Macroglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, Exogenous substances include external These include those provided to the organism from the source or to the culture extracted therefrom.

[0079] As used herein, the term "framework region" or "FW region" refers to a It comprises the amino acid residues adjacent to the CDRs of an antibody or antigen-binding fragment thereof. The domain residues may be, for example, those found in human antibodies, humanized antibodies, monoclonal antibodies, antibody fragments, among others. fragment, Fab fragment, single chain antibody fragment, scFV fragment, antibody domains, and bispecific antibodies.

[0080] The terms "full length antibody" and "intact antibody" refer to an antibody in its substantially intact form. are used interchangeably to refer to antibody fragments and do not refer to antibody fragments as defined herein. In one embodiment, the ADCs described herein are antibodies that bind to intact antibodies, e.g., anti-CD 5 or anti-CD2 intact antibodies. The body comprises two heavy chains, each containing a variable region, a constant region, and an Fc region; More specifically, intact IgG contains two light chains that contain a constant region and a constant region. Each contains two light chains, each containing a light chain variable region (VL) and a light chain ground region (CL), Each contains 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 cell" ("HSC") refers to a cell that is self-renewing and Possess the ability to differentiate into mature blood cells of diverse lineages, including but not limited to: Refers to immature blood cells: granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryocytes, blood 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 It is believed to contain a subpopulation of cells with stem cell properties as defined above, but in mice, H SCs are CD34-. Furthermore, HSCs can be divided into long-term repopulating HSCs (LT-HSCs) and Also referred to as short-term repopulating HSCs (ST-HSCs). LT-HSCs and ST-HSCs are functionally They are differentiated based on their ability and cell surface marker expression. For example, human HSCs are differentiated based on their CD3 4+, CD38-, CD45RA-, CD90+, CD49F+, and lin-(CD 2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, C Negative for mature lineage markers including CD56, CD235A, and CD235A. Bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, and Sl amf1 / CD150+, CD48- and lin- (Ter119, CD11b, Gr 1, mature lineage markers including CD3, CD4, CD8, B220, and IL7ra ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamf1 / CD150+, and lin- (Ter119, CD11 Mature lineage markers including b, Gr1, CD3, CD4, CD8, B220, and IL7ra Furthermore, ST-HSCs are more potent than LT-HSCs under homeostatic conditions. However, LT-HSCs have a greater ability to self-replicate. are capable of producing (i.e., they survive to maturity and are able to survive through a series of recipients) can be serially transplanted), whereas ST-HSCs have limited self-renewal (i.e. (However, they survive for only a limited period of time and do not have the capacity for serial transplantation.) These can also be used in the methods described herein. ST-HSCs are highly proliferative and This is particularly useful because it allows the production of more differentiated progeny more quickly. do.

[0082] As used herein, the term "functional potential as a hematopoietic stem cell" means Refers to the functional properties of hematopoietic stem cells, including: 1) pluripotency (granulocytes (e.g., promyelocytes, neutrophils) leukocytes (e.g., eosinophils, basophils), red blood cells (e.g., reticulocytes, erythrocytes), thrombocytes ocytes) (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets) , monocytic cells (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and Lymphocytes (e.g., multiple cells, including but not limited to NK cells, B cells, and T cells) 2) self-renewal (the ability of hematopoietic stem cells to differentiate into different blood lineages), and This refers to the ability of a cell to give rise to competent daughter cells, and this ability continues throughout the life of an individual without being depleted. can recur throughout a person's life), and 3) hematopoietic stem cells or their progeny The offspring are reintroduced into the transplant recipient, where they home to the hematopoietic stem cell niche, Ability to restore productive and sustained hematopoiesis.

[0083] As used herein, "major histocompatibility complex antigens" ("MHC", in the human context) The term "human leukocyte antigen" (also called "HLA") refers to a group of antigens that are unique to a cell. Refers to proteins expressed on the cell surface that confer antigen identity. MHC / HL A antigen is expressed by T cells and NK cells from the same hematopoietic stem cell source as immune effector cells. Hematopoietic reconstituting cells may be derived from the hematopoietic stem cells themselves ("autologous") or from another source ("non-autologous"). Two major classes of HLA antigens are recognized: HLA class I antigens (A, B, C in humans) and HLA class II antigens (HLA class I and HLA class II) ) enable each cell to be recognized as "self," while HLA class II antigens (DR, DP, DQ) are involved in the reaction between lymphocytes and antigen-presenting cells. 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) are present in different alleles. HLA alleles are designated by a number and a subscript. For example, two unrelated individuals share class I HLA-B, genes B5, and B The allelic products may each carry one of the following sequences in the α and / or β domains: Use a large panel of specific antibodies or nucleic acid reagents to identify clusters that differ in more than one amino acid. Leukocytes expressing class I and class II molecules are used to tag an individual's HLA haplotype. The genes commonly used for HLA typing are the six MHC class I and and class II proteins, HLA-A, HLA-B, and HLA-DR, respectively. There are two alleles for HLA. The HLA gene is a "superficial allele" located at chromosome location 6p21. The six classical HLA genes and the immune system are clustered in a single locus. and in the regulation of several other fundamental molecular and cellular processes. It encodes at least 132 protein-coding genes that play important roles. The complete locus is approximately 3.6 Mb and contains at least 224 loci. One effect of tarring is the creation of "haplotypes," or single chromosomes inherited from one parent. The set of alleles present in the body tends to be inherited as a group. The set of alleles inherited from each parent forms a haplotype, within which some pairs Alleles tend to be associated together. Some alleles and haplotypes are associated with other alleles. are more common than other disorders, and they occur with different frequencies in different racial and ethnic groups. Because haplotypes are distributed, identifying a patient's haplotype increases the chances of finding a matched donor. This helps predict and aids in the development of search strategies.

[0084] As used herein, the term "HLA-matched" refers to a patient in need of hematopoietic stem cell transplantation therapy. donor and recipient, such as a donor providing a hematopoietic stem cell graft to a recipient This refers to a donor-recipient pair in which there is no mismatch in any of the HLA antigens between the donors. T cells and NK cells are less likely to recognize the foreign graft as foreign, and therefore HLA-matched (i.e., six alleles) Donor-recipient pairs (all genetically identical) have a reduced risk of graft rejection. do.

[0085] As used herein, the term "HLA incompatibility" refers to HLA-A, HLA- For B, HLA-C, and HLA-DR, at least one HLA antigen is present in the hematopoietic stem cells. Donors who provide hematopoietic stem cell grafts to recipients who require cell transplantation therapy refers to a donor-recipient pair where there is a mismatch between the donor and recipient. In some embodiments, one haplotype is matched and the other is mismatched. In the case of a donor-recipient pair, endogenous T cells and NK cells differentiate into the foreign graft. Therefore, such T cells and NK cells are more likely to recognize the graft as a target. HLA-mismatched donor-recipients are more likely to mount an immune response against pairs have a higher risk of graft rejection compared with HLA-matched donor-recipient pairs It can become.

[0086] As used herein, the term "human antibody" refers to an antibody that is produced using human germline immunoglobulin sequences. and antibody regions, such as variable and constant regions or domains, that substantially correspond to the sequences. Human antibodies refer to antibodies produced in human cell lines (e.g., by recombinant expression), non-human animals, or The gene encoding the functionally rearranged human immunoglobulin (heavy and / or light chain) genes Human antibodies can be produced by prokaryotic or eukaryotic cells capable of expressing single chain antibodies. If the antibody is a human antibody, it may contain a linker peptide not found in naturally occurring human antibodies. For example, an Fv may be composed of two to about eight linker peptides, such as glycine or other amino acid residues. The variable region of the heavy chain may comprise a linker linking the variable region of the light chain. Car peptides are believed to be of human origin. Human antibodies are derived from human immunoglobulin sequences. methods known in the art, including phage display methods using antibody libraries derived from Human antibodies can also be produced by a variety of methods, including those that express functional endogenous immunoglobulins. Transgenes that cannot express human immunoglobulin genes but can express human immunoglobulin genes can be produced using a transgenic mouse (see, e.g., WO 1998 / 24893, Nos. O1992 / 01047, 1996 / 34096, and 1996 / 3373 No. 5, U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,42 No. 5, No. 5, 569, 825, No. 5, 661, 016, No. 5, 545, 806, No. Nos. 5, 814, 318, 5, 885, 793, 5, 916, 771, and 5, (See, e.g., 939,598). In one embodiment, the human antibody is The recombinant method is used to generate a polypeptide that has a different amino acid sequence from an antibody with the same sequence if it occurs naturally. It is made using.

[0087] As used herein, the term "humanized" antibody refers to an antibody that is composed of non-human CDRs and human frames. In one embodiment, the term "antibody" refers to a chimeric antibody, which generally comprises amino acid sequences from the framework region. In humanized antibodies, residues from the recipient CDRs are modified to provide the desired specificity, affinity, and / or or non-human animals such as mice, rats, rabbits, or non-human primates that have the capacity to Generally, humanized antibodies are human antibodies in which the CDR residues of the target species are replaced by residues from the CDRs of that species. The antibody comprises substantially all of at least one, and typically two, variable domains, wherein C All or substantially all of the DR region corresponds to that of a non-human immunoglobulin. All or substantially all of the region may also be of human immunoglobulin sequences. A recombinant antibody contains at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. It may also include that of the Blin consensus sequence. Methods for humanizing antibodies are well known in the art. It is known, for example, that Riechmann et al., Nature 332:32 pp. 3-7, 1988, U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370 It is written in the following way.

[0088] As used herein, the term "immune cell" refers to a cell that is involved in the innate or adaptive immune response. Immune cells are cells of the immune system involved in initiating and maintaining the immune response. Immune cells include those capable of targeting an antigen of interest, e.g. In the case of autoimmune cells, receptors specifically bind to autoantigens and initiate an immune response against them. Exemplary immune cells include mast cells, basophils, and neutrophils. , eosinophils, microglia, granulocytes, monocytes, antigen-presenting cells, macrophages, dendritic cells, These include neural killer cells, T lymphocytes, and B lymphocytes.

[0089] As used herein, a patient "in need" of a hematopoietic stem cell transplant includes one or more These include patients who exhibit defects or deficiencies in any of the blood cell types, as well as patients suffering from stem cell disorders. Hematopoietic stem cells generally exhibit 1) pluripotency and therefore can give rise to granulocytes (e.g., promyelocytes, eosinophils, etc.). neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, erythrocytes), thrombocytes bocytes) (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets) ), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and and lymphocytes (e.g., NK cells, B cells, and T cells), but are not limited to these. 2) can differentiate into multiple different blood lineages; and 3) exhibit self-renewal and thus 3) can give rise to daughter cells of equivalent potency, and 4) can repopulate the transplant recipient. demonstrated the ability to be introduced into the hematopoietic stem cell niche, where they homed to the hematopoietic stem cell niche and became productive and persistent. restores continuous hematopoiesis, thus replacing defective or deficient cell populations in vivo To reconstitute hematopoietic stem cells, hematopoietic stem cells are derived from cells that are defective or deficient in one or more cell types of the hematopoietic lineage. For example, the patient may be suffering from cancer, and the deficiency may be due to administration of chemotherapeutic or other agents that selectively or non-specifically deplete cancerous cell populations; Additionally or alternatively, the patient may have sickle cell anemia, thalassemia, phagocytosis, Defects in one or more blood cell types, such as Nkoni anemia, and Wiskott-Aldrich syndrome You may have a non-malignant hemoglobinopathy that can cause a defect or deficiency of Elephants are susceptible to Adenosine Deaminase Severe Combined Immunodeficiency (ADA SCID), HIV / AIDS, DS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwama The subject may have a genetic blood disorder (e.g., leukemia, leukemia-associated ... may have or be affected by an autoimmune disorder (e.g., sickle cell anemia) Additionally or alternatively, the subject may have a malignancy, e.g., a blood cancer (e.g., leukemia, lymphoma, from the group consisting of: myeloma, multiple myeloma, or myelodysplastic syndrome) and neuroblastoma May have or be affected by selected malignancies. In embodiments, the subject has or is otherwise affected by a metabolic disorder. For example, the subject may suffer from or otherwise be affected by: : Glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidoses, metachromatosis metabolic disorders selected from the group consisting of: leukodystrophies, or Other diseases that may benefit from the treatments and therapies disclosed herein include: or disorders: severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmune globulinemia IgM syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis , osteogenesis imperfecta, storage disease, thalassemia major, sickle cell disease, systemic sclerosis, systemic erythrocyte sequestration Irritability, multiple sclerosis, juvenile rheumatoid arthritis, and "Bone Marrow" Transplantation for Non-Malignant Disease ASH Education Book, 1:319-338 (2000) Disclosure of a disease or disorder that is treatable by the administration of hematopoietic stem cell transplantation therapy The entire contents of which are incorporated herein by reference as they relate to the medical conditions that may be caused by the use of the method. Alternatively, a patient who "needs" a hematopoietic stem cell transplant may have one of the aforementioned conditions. Those with or without the disease, but still have megakaryocytes, thrombocytes ), platelets, red blood cells, mast cells, myeloblasts, basophils, neutrophils , eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells One within the hematopoietic lineage, such as leukocytes, natural killer cells, T lymphocytes, and B lymphocytes Decreased levels of the above endogenous cell types (e.g., compared to levels in an otherwise healthy subject) Those skilled in the art can perform one or more of the above procedures for an otherwise healthy subject. Whether the levels of a particular cell type or other blood cell types are reduced can be determined, for example, by methods known in the art. Among other known procedures, flow cytometry and fluorescence-activated cell sorting This can be readily determined using FACS methods.

[0090] When used in the context of a protein such as an antibody, the term "isolated" refers to the Not related to the naturally occurring components that accompany it in its natural state because of its source or origin Proteins, proteins substantially free of other proteins of the same species, proteins produced by cells of different species It refers to a protein that is expressed by a gene or that does not occur in nature. Proteins that are chemically synthesized or synthesized in a cell system other than the cell from which they are naturally derived Such a protein would be "isolated" from its naturally associated components. Isolation using conventional protein purification techniques ensures that the protein is substantially free of naturally associated components. It is also possible to avoid including it.

[0091] The term "monoclonal antibody" or "mAb" refers to a population of substantially homogeneous antibodies. That is, each antibody constituting the population is a mutant antibody, e.g., a monoclonal antibody. Except for naturally occurring mutants or variants that arise during the production of the lonal antibody preparations, are identical and / or bind the same epitope, and such variants are present in low amounts. Polyclonal antibody preparations, which usually contain different antibodies against different determinants (epitopes), are used. In contrast to antibody preparations, each mAb is directed against a single determinant on the antigen. "Clonal" should not be construed as requiring production of the antibody by any particular method. do not have.

[0092] As used herein, the term "patient at risk for GVHD" refers to a patient who has GVHD. This refers to patients who have one or more risk factors for developing the disease. Risk factors include mismatched human leukocyte antigens. Allogeneic donor transplants (e.g., bone marrow transplants) including original (HLA) donors and gender-mismatched donors Hematopoietic stem cell transplantation from donor and donor hematopoietic stem cells, T cell-replete stem cell transplantation ... donor and donor hematopoietic stem cell transplantation, and recipient age, donor or host cytomegalovirus (CMV) status, or the presence or absence of CMV antibodies, increased total body irradiation (TBI), condition Intensity of the treatment regimen, prophylaxis of acute GVHD, lack of a protective environment, splenectomy, immunoglobulin use, underlying diseases, ABO compatibility, history of herpes virus exposure, donor transfusion, performance These include, but are not limited to, urinary score, antibiotic bowel irrigation, and post-allotransplant blood transfusion. stomach.

[0093] As used herein, the term "patient at risk for autoimmune disease" refers to a patient at risk for autoimmune disease. It refers to patients who have one or more risk factors for developing an epidemic. Risk factors include age (younger age, Age group (young to middle-aged), gender (female), ethnicity (African American, American Indian) American or Latino), family history of autoimmune disease, exposure to environmental factors, past These include infection, chronic inflammation, and donor transplantation (e.g., transplantation of hematopoietic stem cells from bone marrow transplantation). These include, but are not limited to:

[0094] As used herein, the term "pharmaceutically acceptable" means a compound that does not cause excessive toxicity, irritation, breastfeeding without allergic reactions and other complications, with a reasonable benefit / risk ratio Compounds, materials, compositions suitable for contact with tissue of a subject, such as an animal (e.g., a human), and / or refers to the dosage form.

[0095] As used herein, the term "pharmaceutical composition" refers to, among other things, a pharmaceutical composition for treating autoimmune disorders, Preventing or treating certain diseases or conditions that affect mammals, such as cancer or blood disorders A therapeutic compound administered to a mammalian subject, e.g., a human, to treat or regulate means a mixture comprising:

[0096] As used herein, the term "recipient" refers to a recipient of a transplant containing a population of hematopoietic stem cells. A patient receiving a transplant, such as a graft, may be administered to a recipient, e.g., The cells may be autologous, syngeneic, or allogeneic.

[0097] As used herein, the term "rejection" in the context of a transplant, such as a hematopoietic stem cell transplant, The term refers to the process by which the recipient mounts an immune response against the incoming transplant, thereby causing the transplant A process that reduces the ability of transplanted material (e.g., hematopoietic stem cells) to survive in the recipient. Rejection of a graft, such as a hematopoietic stem cell graft, can occur, for example, from a patient at different times after transplantation. Quantification by measuring the amount or concentration of transplanted cells in various isolated samples The amount or concentration of transplanted cells in a sample isolated from a patient can be measured over time. , for example, about 20%, about 25%, about 30%, about 35%, about 40%, about 56%, about 50%, Approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, A reduction of approximately 95% or more indicates that the patient is suffering from graft rejection. Conversely, the amount or concentration of transplanted cells in a sample isolated from a patient may be significantly different from, e.g. , less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than that. The finding that the graft remains stable over time indicates that the patient is not suffering from graft rejection. Alternatively, graft rejection may be due to the presence of erythrocytes isolated from patients at different times after transplantation. Immune cells in various samples that cross-react with the MHC antigens expressed by the transplanted cells ( These can be quantified by measuring the amount or concentration of T cells and / or NK cells. The MHC antigens expressed by the transplanted cells in the sample isolated from the patient can be interacted with. The amount or concentration of differentially responding immune cells (such as T cells and / or NK cells) changes over time. , for example, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, Approximately 56%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, Approximately 85%, approximately 90%, approximately 95%, approximately 100%, approximately 200%, approximately 300%, or more An increase in the level of graft rejection indicates that the patient is suffering from graft rejection. Immunoglobulins in samples isolated from the recipient that cross-react with MHC antigens expressed by the transplanted cells The amount or concentration of cells (such as T cells and / or NK cells) may increase over time, for example, to about 2 0%, approx. 25%, approx. 30%, approx. 35%, approx. 40%, approx. 56%, approx. 50%, approx. 55%, approx. 6 0%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 95% or Any further decrease indicates that the patient is not suffering from graft rejection.

[0098] As used herein, the term "sample" refers to a specimen (e.g., a sample) taken from a subject. , 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.

[0099] As used herein, the term "scFv" refers to a fragment of heavy and light chain variable domains derived from an antibody. A single-chain Fv antibody is one in which the fragments are linked to form a single chain. is a variable region (VL) of an antibody light chain (e.g., CDR-L1) separated by a linker. , CDR-L2 and / or CDR-L3) and the variable region (VH) of an antibody heavy chain (e.g., a single polypeptide chain comprising CDR-H1, CDR-H2 and / or CDR-H3) The linker connecting the VL and VH regions of the scFv fragment contains a protein. Alternatively, the linker may be a peptide linker consisting of proteolytic amino acids. to increase the resistance of the fragment to proteolysis (e.g., by including D-amino acids). linker), to increase the solubility of the scFV fragment (e.g., polyethylene Glycol-containing linkers or polypeptides containing repeated glycine and serine residues hydrophilic linkers such as amides), to improve the biophysical stability of the molecule (e.g., linkers containing cysteine ​​residues that form intra- or intermolecular disulfide bonds), or or scFV fragments (e.g., containing glycosylation sites) The variable regions of the scFV molecules described herein can be It is also known that the antibody molecules can be modified to have different amino acid sequences than those from which they are derived. Those skilled in the art will understand that, for example, conservative substitutions or nucleotides resulting in changes of amino acid residues may be used. peptide or amino acid substitutions (e.g., in CDR and / or framework residues) (in some cases), retain or enhance the ability of the scFV to bind to the antigen recognized by the corresponding antibody. It can be implemented to strengthen the

[0100] With respect to the interaction of an antibody or antigen-binding fragment with a second chemical species, the term "specific "Binding" or "specifically binding" refers to an interaction that targets a specific structure on a chemical species (e.g., an antigen). This means that the antibody depends on the presence of a specific determinant (epitope). The antibody recognizes and binds to a specific protein structure, not the protein itself. If specific, it contains epitope A in a reaction involving labeled "A" and antibody. The presence of the molecule (or free unlabeled A) reduces the amount of labeled A bound to the antibody. In one embodiment, the antibody has a KD for its target of at least about 10 -4 M, approx. 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 means that the antibody is In one embodiment, the term "specifically binds to CD2" as used herein refers to a The terms "specific binding" or "specific binding to CD2" refer to binding to CD2 and binding to a surface protein. The dissociation constant (KD) determined by Schottky resonance is 1.0 × 10 -7 Antibodies with M or less In another embodiment, "specific binding to CD5" or "specific binding to CD5" refers to The term "antibody" refers to a molecule that binds to CD5 and has a dissociation constant determined by surface plasmon resonance. (KD) is 1.0 x 10 -7 In one embodiment, KD refers to an antibody that has a KD equal to or less than the target KD. The antibody is determined by standard biolayer interferometry (BLI). It should be understood that the antibody may be capable of specifically binding to more than one antigen. For example, in one embodiment In some embodiments, the antibody may be of both human and non-human (e.g., murine or non-human primate) origin. In another embodiment, the antibody is capable of specifically binding to an ortholog of CD2. to orthologs of CD5 in both human and non-human (e.g., mouse or non-human primate) Capable of specifically binding. Specific binding may also refer to ADCs that include antibodies.

[0101] As used herein, the terms "subject" and "patient" refer to any of the compounds described herein. refers to a mammal, such as a human, undergoing treatment for a particular disease or condition, such as The patient, such as a human autoimmune patient, may be a patient suffering from an autoimmune disease as described herein, and (i) populations of autoimmune cells (e.g., populations of autoimmune CD2+ T cells and / or NK cells, or autoimmune CD5+ T cell, B cell and / or NK cell populations) and / or (ii) non-self antigens expressed by hematopoietic stem cells (e.g., A population of CD2+ immune cells that cross-react with non-self MHC antigens expressed by blood stem cells (e.g., CD2+ T cells and / or NK cells) or populations of CD5+ immune cells (e.g., For example, to deplete CD5+ T cells, B cells and / or NK cells, The anti-CD2 antibody or antibody-drug conjugate or the anti-CD5 antibody or antibody-drug conjugate according to Inducate may be administered to reduce the likelihood of graft rejection prior to hematopoietic stem cell transplantation therapy. Prevent or reduce.

[0102] As used herein, the phrase "substantially removed from the blood" refers to a therapeutic agent (antibody) At some point after administration of the antibody (anti-CD2 antibody or anti-CD5 antibody) to the patient, the antibody is isolated from the patient. The concentration of the therapeutic agent in the collected blood sample is such that the therapeutic agent can be detected by conventional means. When the therapeutic agent is not readily available (e.g., when the therapeutic agent is not readily available in the device or application used to detect the therapeutic agent). (when the signal is not detected above the noise threshold of the assay) or the ELISA-based detection assays described herein. A variety of techniques known in the art can be used to detect antibodies or antibody fragments. Additional assays that can be used to detect antibodies and antibody fragments include those described in the art. These include immunoprecipitation techniques and immunoblot assays, among others known in the art. .

[0103] As used herein, the phrase "stem cell disorder" refers to a condition that conditions a target tissue in a subject. by ablating endogenous T cell populations in the target tissue and / or by engrafting or transplanting stem cells into the target tissue of the subject. It broadly refers to any disease, disorder, or condition that can be treated or cured. For example, type 1 diabetes These patients have been shown to be cured by hematopoietic stem cell transplantation, and the combinations described herein may benefit from conditioning according to the compositions and methods of the present invention. Additional disorders that can be treated using the compositions and methods described herein include sickle cell anemia, Thalassemia, Fanconi anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, These conditions include, but are not limited to, glaucoma, glaucoma syndrome, and Schwabman-Diamond syndrome. Have an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune disorder, or In addition to or instead of the above, The animals are those with hematological cancers (e.g., leukemia, lymphoma, multiple myeloma, or myelodysplastic syndromes) and have a malignant tumor, such as a malignant tumor selected from the group consisting of glioma and neuroblastoma, or In some embodiments, the subject may be suffering from a metabolic disorder. For example, a subject may suffer from: or may be affected by: glycogen storage diseases, mucopolysaccharidoses, A group consisting of leukodystrophy, sphingolipidosis, Hurler's disease, and metachromatic leukodystrophy or a metabolic disorder selected from the disclosed treatments and methods, including but not limited to: and other diseases or disorders that may benefit from therapy: severe combined immunodeficiency, viral Cott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chédiak syndrome Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia Sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile onset Rheumatoid arthritis, and Bone and Marrow Transplantation for Non-Malignant Disease” ASH Education Book, 1:319-338 (2000), The disclosure is provided in its entirety as it relates to conditions that may be treated by the administration of hematopoietic stem cell transplantation therapy. is incorporated herein by reference.

[0104] As used herein, "transfection" means The term refers to methods such as electroporation, lipofection, calcium phosphate precipitation, DE AE-dextran transfection, etc., is used to introduce exogenous DNA into prokaryotes or eukaryotes. "Transgenic" refers to any of a wide variety of techniques commonly used to introduce a gene into a host cell.

[0105] As used herein, the terms "treat" or "treatment" refer to the treatment of a disease in which the purpose is to to prevent or slow down (reduce) undesirable physiological changes or disorders, or refers to a therapeutic treatment that aims to promote a beneficial phenotype in the patient receiving the treatment. The beneficial or desired clinical outcome will depend on the disorder being treated and may include a reduction in tumor burden, autoimmune disease, When directly treating a disease, autoimmune cells present in a sample isolated from a patient, e.g., autologous a population of CD2+ T cells and / or NK cells (or CD5 + T cell, B cell and / or NK cell populations), or anti-CD and the administration of anti-CD2 or anti-CD5 antibodies, and hematopoietic stem cell transplantation. If so, non-self antigens expressed by hematopoietic stem cells (e.g., non-self antigens) should be identified before hematopoietic stem cell transplantation. Further beneficial results include, but are not limited to, a reduction in the amount of MHC antigens. , conditioning therapy and subsequent Increase in cell number or relative concentration of hematopoietic stem cells after administration of exogenous hematopoietic stem cell grafts to patients The beneficial results of the treatments described herein include conditioning regimens and After subsequent hematopoietic stem cell transplantation, megakaryocytes, thrombocytes, and microparticles Platelets, red blood cells, mast cells, myeloblasts, basophils, neutrophils, eosinophils , microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, One or more cells of the hematopoietic lineage, such as natural killer cells, T lymphocytes, or B lymphocytes In certain embodiments, the patient is diagnosed with a disorder or an increase in the number or relative concentration of cells. The patient is diagnosed and then treated with a therapeutic agent, e.g., an anti-CD5 ADC. In this case, patients are at risk of developing disorders such as GVHD, so Treatment is provided as a preventative measure to reduce the risk or alleviate the symptoms of the disorder.

[0106] As used herein, the terms "variant" and "derivative" are used interchangeably. and the use of natural, synthetic compounds, peptides, proteins, or other substances described herein. The compounds, peptides, proteins, and the like described herein are intended to be synthetic and semi-synthetic analogs. Variants or derivatives of other substances may retain or improve the biological activity of the original material. There may be cases where it is better.

[0107] As used herein, the term "vector" includes plasmids, DNA vectors, a nucleic acid vector, such as a plasmid, RNA vector, virus, or other suitable replicon The expression vectors described herein contain a polynucleotide sequence, as well as a vector containing a target gene, e.g. , protein expression and / or genomic DNA synthesis of these polynucleotide sequences in mammalian cells. The antibodies and antibody flags of the present invention may contain additional sequence elements that are used for integration into the system. The particular vector that can be used to express the nucleotides contains a promoter that directs gene transcription. and plasmids containing regulatory sequences such as enhancer regions. Other useful vectors for expression of fragments include those that enhance the translation rate of these genes. or polynucleotides that improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements may be used in, for example, a gene carried in an expression vector. Contains 5' and 3' untranslated regions and polyadenylation signals to direct efficient transcription of the gene. The expression vectors described herein can be used in cells containing such vectors. The gene may contain a polynucleotide encoding a marker for selecting the desired gene. Examples include ampicillin, chloramphenicol, kanamycin, and nourceotrichin. Examples include genes that encode resistance to antibiotics such as cyclosporin.

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

[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 alkylenes include methylene, ethylene, propylene, isopropylene, and the like.

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

[0111] As used herein, the term "heteroalkylene" refers to a straight or branched chain heteroalkylene. The divalent positions may be the same or different atoms within the heteroalkyl chain. The divalent positions may be one or more heteroatoms.

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

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

[0114] As used herein, the term "heteroalkenyl" refers to heteroalkenyl groups, e.g., groups having 2 to 2 alkyl groups in the chain. 0 carbon atoms and in addition one or more heteroatoms (e.g., oxygen, nitrogen, among others) in the chain. refers to straight or branched chain alkenyl groups containing (atomic atoms, carbon atoms, or sulfur atoms).

[0115] As used herein, the term "heteroalkenylene" refers to a straight or branched chain It refers to a divalent heteroalkenyl group. The divalent positions may be the same or different positions within the heteroalkenyl chain. The divalent positions can be on one or more heteroatoms.

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

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

[0118] As used herein, the term "heteroalkynyl" refers to heteroalkynyl groups, e.g., groups having 2 to 2 alkyl groups in the chain. 0 carbon atoms and in addition one or more heteroatoms (e.g., acid refers to straight or branched chain alkynyl groups containing an alkynyl group (carbon, nitrogen, or sulfur).

[0119] As used herein, the term "heteroalkynylene" refers to a straight or branched chain heteroalkynylene. It refers to a divalent heteroalkynyl group. The divalent positions may be the same or different positions within the heteroalkynyl chain. The divalent positions can be on one or more heteroatoms.

[0120] As used herein, the term "cycloalkyl" refers to a saturated group, e.g., a group having 3 to 12 carbon atoms. refers to a monocyclic, or fused, bridged, or spiro polycyclic ring structure having 10 carbon ring atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[3.1.0]hexane, etc. Examples include:

[0121] As used herein, the term "cycloalkylene" refers to a divalent cycloalkyl The divalent positions can be on the same or different atoms within the ring structure. Examples of cyclopentylene include cyclopropylene, cyclobutylene, cyclopentylene, and cyclohexylene. Examples include:

[0122] As used herein, the term "heterocycloalkyl" refers to a saturated group, e.g., For example, carbon atoms and heteroatoms selected from, for example, nitrogen, oxygen, and sulfur, among others. monocyclic, or fused, bridged, having 3 to 12 ring atoms per ring structure selected from or spiropolycyclic ring structures. Ring structures may include, for example, rings of carbon, nitrogen, or sulfur. Examples of heterocycloalkyl include, but are not limited to, cycloalkyl groups, which may contain one or more oxo groups on one or more rings. Examples include dihydropyridyl, tetrahydropyridyl (piperidyl), tetrahydropyridyl, Hydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl Tetrahydrofuranyl, tetrahydropyranyl, bistetrahydropyranyl, tetra Hydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroquinolinyl Includes isoquinolinyl, piperazinyl, quinuclidinyl, and morpholinyl.

[0123] As used herein, the term "heterocycloalkylene" refers to a divalent heterocycloalkylene. The divalent positions can be on the same or different atoms within the ring structure. As used herein, the term "aryl" refers to an aryl group having, for example, 6 to 19 carbon atoms. Aryl groups include phenyl, fluorenyl, and the like. , naphthyl, etc. The divalent position may be one or more The heteroatom may be:

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

[0125] As used herein, the term "heteroaryl" refers to one or more ring atoms is a heteroatom, such as nitrogen, oxygen, or sulfur, or a monocyclic heteroaromatic or bicyclic Heteroaryl groups include: Examples include: pyridyl, pyrrolyl, furyl, thienyl, imidazolyl, oxazolyl, isooxyl Thiazolyl, 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-triazolyl Azinyl, 1,2,3-triazinyl, benzofuryl, [2,3-dihydro]benzofuryl Isobenzofuryl, benzothienyl, benzotriazolyl, isobenzothienyl, iodobenzofuryl, 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, etc.

[0126] As used herein, the term "heteroarylene" refers to a divalent heteroaryl group The divalent positions can be on the same or different atoms. There may be multiple heteroatoms.

[0127] Unless otherwise limited by the definition of an individual substituent, the aforementioned chemical moieties, e.g., "arylene" alkyl, alkylene, heteroalkyl, heteroalkylene, alkenyl "Alkenylene", "heteroalkenyl", "heteroalkenylene", "alkynyl" "alkynylene," "heteroalkynyl," "heteroalkynylene," "cycloalkynyl," "cycloalkylene," "heterocycloalkyl," "heterocycloalkylene" "aryl", "arylene", "heteroaryl", and "heteroarylene" groups is, for example, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl alkylaryl, alkylheteroaryl, alkylcycloalkyl, alkylheteroaryl Tetracycloalkyl, amino, ammonium, acyl, acyloxy, acylamino, acyl Aminocarbonyl, alkoxycarbonyl, ureido, carbamate, aryl, heteroaromatic Aryl, sulfinyl, sulfonyl, alkoxy, sulfanyl, halogen, carboxy , trihalomethyl, cyano, hydroxy, mercapto, nitro, etc. 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, -CO2 R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O )NH 2、 -C(=O)N(R)2, -C(=S)NH 2、 -C(=S)N(R)2, - -C(=NH)NH2 and -C(=NR)N(R)2, but are not limited to these. Each X, in each occurrence, is independently selected from F, Cl, Br, and I. and each R is independently selected in each occurrence from alkyl, aryl, heterocycloalkyl, and a protecting group and a prodrug moiety, each independently selected from alkyl or heteroaryl. Whenever a group is described as "optionally substituted," that group may be optionally substituted, respectively. may be independently substituted with one or more of the above substituents. Ring closure, etc., where adjacent substituents undergo ring closure, e.g., lactams, lactams formed by ring closure, acetals, cyclic anhydrides, acetals, hemiacetals, thioacetals, aminals, and hemiacetals. This may include situations where a minor is formed, for example to provide a protecting group.

[0128] Certain radical naming conventions include either monoradicals or diradicals depending on the context. For example, it is understood that a substituent may have two points of attachment to the remainder of the molecule. Where necessary, the substituent is understood to be a diradical. For example, Substituents identified as alkyl include -CH2-, -CH2CH2-, -CH Other radical naming conventions include diradicals such as 2CH(CH3)CH2-. The alkyl groups are "alkylene," "alkenylene," "arylene," and "heterocycloalkylene." This clearly shows that it is a diradical such as "n".

[0129] When a substituent is shown as a diradical (i.e., with two bonds to the rest of the molecule), If the substituent has a point, the substituent may be attached in any orientation unless otherwise specified. I would like it to be understood that.

[0130] Anti-CD2 antibody drug conjugate The compositions and methods described herein may be used to treat or prevent the development of CD2+ ADCs, e.g., CD2+ cancer cells. (e.g., CD2+ leukemia cells) and CD2+ autoimmune cells (e.g., CD2+ autoimmune The ability of such agents to kill immune T cells and / or NK cells (immune T cells and / or NK cells) can be used to treat cancer and This is based in part on the discovery that steroids can be used to directly treat autoimmune diseases. The anti-CD2 antibody is then conjugated to a cytotoxin via a linker. Where an anti-CD2 antibody is mentioned, its conjugates are also contemplated unless otherwise specified. can be.

[0131] The compositions and methods described herein also provide an ADC capable of binding to CD2 that binds to immune cells. By preventing or reducing the likelihood of graft rejection via The discovery that IFN-γ-γ can be used as a therapeutic agent to promote the engraftment of transplanted hematopoietic stem cells in patients with IFN-γ-γ syndrome is part of For example, anti-CD2 antibodies and antigen-binding fragments are derived from hematopoietic stem cells. one or more non-self hematopoietic stem cells, such as one or more non-self MHC antigens expressed by immune cells, such as T cells or NK cells, that cross-react with cellular antigens and initiate an immune response against them Such antibodies and antibodies capable of binding to cell surface CD2 expressed by a cell. Binding of the antigen-binding fragment to hematopoietic stem cell-specific CD2+ immune cells can be, for example, antibody-dependent. by reactive cell-mediated cytotoxicity or by conjugation with antibodies or their antigen-binding fragments. The action of the conjugated cytotoxic agent can induce the death of the bound immune cells. Therefore, depletion of the population of CD2+ immune cells that cross-react with non-autologous hematopoietic stem cells is essential for the development of immune cells. by weakening the recipient's immune system's ability to mount an immune response against the incoming graft. This may promote hematopoietic stem cell transplant engraftment in patients who require hematopoietic stem cell transplantation. In this way, defective or deficient cell lineages can be repopulated in a subject. In addition, hematopoietic stem cell transplantation can be provided to a subject, thereby providing a stem cell disorder as described herein, Patients suffering from cancer, autoimmune diseases, or other blood disorders can be treated. For example, it was administered to a subject with the goal of eradicating cancerous cells, but in the process destroyed healthy hematopoietic cells. The subject may have a depleted cell population due to chemotherapy that also depletes the cells.

[0132] For example, in certain embodiments, a polypeptide capable of binding to an antigen expressed by a T cell may be The administration of an antibody or its antigen-binding fragment that can inhibit the engraftment of transplanted hematopoietic stem cells is The present invention provides compositions and methods for promoting the expression of CD4+ and CD8+ T cells. This selective depletion of T cells can be prevented by exogenous factors. Preventing graft rejection after transplantation of a heterologous (e.g., autologous, allogeneic, or syngeneic) hematopoietic stem cell graft. For example, the anti-CD2 antibodies, antigen-binding fragments, and antibody- CD4+ and / or CD4+ antibody-drug conjugates Selective depletion of CD8+ T cells may occur in transplanted hematopoietic stem cell grafts. The compositions and methods described herein can attenuate CD4+-mediated immune responses. Antibodies and antigen-binding fragments thereof capable of binding to 2 are used to treat transplanted hematopoietic stem cells. Patients requiring hematopoietic stem cell transplantation therapy to promote cell survival and engraftment potential This is based in part on the discovery that the compound can be administered to a subject.

[0133] The survival of hematopoietic stem cell grafts treated with anti-CD2 antibodies or their antigen-binding fragments is unclear. This can be determined by various empirical measurements, such as the engraftment of transplanted hematopoietic stem cells. The present invention relates to a method for treating rheumatoid arthritis by administering an antibody or antigen-binding fragment thereof capable of binding to CD2, Following administration of a hematopoietic stem cell graft, competitive reconstituting units (C) present in the patient's bone marrow are used. It can be evaluated by measuring the amount of fluorophores, colorimetric or A reporter gene, such as an enzyme that catalyzes a chemical reaction that produces a luminescent product, is transfected into donor hematopoietic stem cells. The corresponding signal is then introduced into the transfected vector, and the resulting signal is then expressed in the bone marrow. By monitoring the tissues to which hematopoietic stem cells are homing, The engraftment of the cell graft can be monitored, for example, by methods known in the art. Hematopoietic stem cells, as determined by fluorescence-activated cell sorting (FACS) analysis The engraftment of hematopoietic stem cell grafts can be monitored by assessing the quantity and survival of stem and progenitor cells. Engraftment can also be assessed by measuring the number of leukocytes in the peripheral blood during the post-transplant period. and / or by measuring bone marrow cell recovery by donor cells in bone marrow aspirate samples. Therefore, it can also be determined.

[0134] The following sections discuss how to use hematopoietic stem cell transplantation therapy to promote hematopoietic stem cell transplant engraftment. Antibodies or antigen-binding fragments thereof that can be administered to patients in need thereof, as well as hematopoietic stem cells. Methods for administering such therapeutic agents to patients prior to cell transplantation are described.

[0135] Anti-CD2 antibody The compositions and methods described herein include antibodies or their derivatives that specifically bind to human CD2. Human CD2 contains a fragment of the T cell surface antigen T11 / Leu-5, T11, C Also called D2 antigen (p50), and sheep red blood cell receptor (SRBC). Two isoforms of human CD2 have been identified. Group 1 contains 351 amino acids and is described in Seed, B. et al. (1987) 84:3 pp. 365-69 (see also Sewell et al. (1986) 83:8718-22). Reference) 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 referred to herein as the NCBI reference sequence: Identified as P_001315538.1.

[0136] T cells and NK cells have been shown to express CD2, a cell adhesion molecule and is a specific marker for such lymphocytes. For example, CD2 is a It interacts with other adhesion molecules, such as lymphocyte-associated antigen-3 (LFA-3 / CD58), to promote T cell proliferation. Antibodies and antigen-binding fragments thereof capable of binding to CD2 enhance activation. inhibits T cell activation, for example by inhibiting the interaction between CD2 and LFA-3. This cell surface antigen can suppress the T cell-mediated immune response to hematopoietic stem cell grafts. Antibodies and antigen-binding fragments thereof that are compatible with the antibody have been identified using immunization, computational modeling techniques, and Phage display and cell-based display platforms, such as those described below, Any in vitro selection method known in the art and described herein, including These can be identified using techniques currently available.

[0137] As used herein, a CD2 polypeptide, e.g., a human CD2 polypeptide, is specifically bound to the CD2 polypeptide. Exemplary antibodies and antigen-binding fragments thereof, and uses thereof, are described. In embodiments, an antibody or antigen-binding fragment thereof that specifically binds to a CD2 polypeptide is The component comprises 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 having the amino acid sequence of SEQ ID NO: 1. In one embodiment, the heavy chain variable region comprises CDR1. In one embodiment, the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, the heavy chain variable region comprises a VH CDR3 having the sequence of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In one embodiment, the heavy chain variable region comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In one embodiment, the VH CDRs comprise two or more VH CDRs selected from the group consisting of sequence no. 3. The heavy chain variable region comprises a VH CDR1 having SEQ ID NO: 1, a VH CDR2 having SEQ ID NO: 2, 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 having the amino acid sequence of SEQ ID NO:4. In one embodiment, the light chain variable region comprises CDR1. In one embodiment, the light chain variable region comprises a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, the light chain variable region comprises a VL CDR3 having the sequence of SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In one embodiment, the light chain variable region comprises SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. In one embodiment, the VL CDRs comprise two or more VL CDRs selected from the group consisting of sequence no. 6. , the light chain variable region comprises a VL CDR1 having SEQ ID NO: 4, a VL CDR2 having SEQ ID NO: 5 2, and a VL CDR3 having SEQ ID NO:6.

[0140] In an exemplary embodiment, the antibody or antigen-binding fragment thereof has SEQ ID NO:1. a VH CDR1 having SEQ ID NO:2, a VH CDR2 having SEQ ID NO:3, a heavy chain variable region comprising CDR3 having SEQ ID NO: 4, a VL CDR1 having SEQ ID NO: 5, and a light chain variable region comprising a VL CDR2 having SEQ ID NO: 6, and a VL CDR3 having SEQ ID NO: 7. .

[0141] In certain embodiments, one or more of the CDRs (i.e., one having SEQ ID NOs: 1-3) one or more heavy chain CDRs, and / or one or more light chain CDRs having SEQ ID NOs: 4-6 The heavy chain CDRs of the antibody are those of the CD2 specificity (i.e., heavy chain CDRs of SEQ ID NOs: 1-3 and SEQ ID NO: (similar specificity to an antibody or its antigen-binding fragment containing 4-6 light chain CDRs) Conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) may include (conversion).

[0142] In one embodiment, the antibody or antigen-binding fragment thereof is an antibody having the antigen binding domain set forth in SEQ ID NO:7. In another embodiment, the antibody or its antigen comprises a heavy chain variable region containing the amino acid sequence A binding fragment has at least 95% identity to SEQ ID NO:7, e.g., at least 95% identity to SEQ ID NO:7. Amino acids having at least 95%, 96%, 97%, 98%, 99% or 100% identity In certain embodiments, the antibody comprises a heavy chain variable region having the sequence of SEQ ID NO:7. or a modified heavy chain (HC) variable region comprising an HC variable domain having a variant of SEQ ID NO: 7. The variants include (i) 1, 2, 3, 4 or 5 amino acid substitutions, additions, or deletion from SEQ ID NO: 7; (ii) up to 5, 4, 3, 2, or 1 amino acid sequence (iii) 1 to 5, 1 The sequence may be modified by substitution, addition, or deletion of up to 3, 1 to 2, 2 to 5, or 3 to 5 amino acids. and / or (iv) at least about 75%, 80%, or Amino acid sequences that are 85%, 90%, 95%, 96%, 97%, 98% or 99% identical In any of (i) to (iv), the amino acid substitution is a conservative amino acid substitution or or non-conservative amino acid substitutions, and the modified heavy chain variable region maintains the CD2 binding specificity of the antibody. i.e., has similar binding to an antibody or antigen-binding fragment thereof comprising SEQ ID NO: 7. The heavy chain variable region of SEQ ID NO: 7 has enhanced biological activity while retaining specificity. In one embodiment, the antibody or antigen-binding fragment thereof may be The heavy chain variable region may differ from the amino acid sequence described above by 1, 2, 3, or 4 amino acids. For example, the antibody or antigen-binding fragment thereof may have a nucleotide sequence at positions 12, 13, 28, and / or or 48 positions, one, two, three, or four of which contain the amino acid sequence set forth in SEQ ID NO: 7. In one embodiment, the heavy chain variable region may comprise a heavy chain variable region that is different from the sequence of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, , 28 and 48 from the amino acid sequence set forth in SEQ ID NO: 7. The heavy chain variable region may have one, two, or three of the following substitutions in the sequence set forth in SEQ ID NO: 7: or including four: K12Q, K13R, T28I, and M48V. wherein the heavy chain variable region is SEQ ID NO: 7 with substitutions K12Q, K13R, T28I, and and M48V.

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

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

[0145] In one embodiment, the antibody or antigen-binding fragment thereof is an antibody having the antigen binding domain set forth in SEQ ID NO: 9. In another embodiment, the antibody or its antigen comprises a heavy chain variable region containing the amino acid sequence A binding fragment has at least 95% identity to SEQ ID NO:9, e.g., at least 95% identity to SEQ ID NO:9. at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity In an exemplary embodiment, the antibody or or an antigen-binding fragment thereof, has at least 95% identity to SEQ ID NO: 9, e.g., sequence Column number 9 and at least about 95%, about 96%, about 97%, about 98%, about 99% or 100 a heavy chain variable region containing an amino acid sequence having at least 1% identity with SEQ ID NO: 10; about 95% identity, e.g., at least about 95%, about 96%, about 97% identity with SEQ ID NO: 10, A light chain variable region containing an amino acid sequence having about 98%, about 99%, or 100% identity In one embodiment, the antibody or antigen-binding fragment thereof comprises the region of SEQ ID NO:9. and a light chain variable region comprising SEQ ID NO: 10. In one embodiment, the antibody comprises a heavy chain variable region comprising SEQ ID NO: 9 and a light chain variable region comprising SEQ ID NO: 10. It is an Ab1 antibody containing the chain variable region.

[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 V comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15. In one embodiment, the heavy chain variable region comprises a VH CDR2 containing the sequence SEQ ID NO: 1. In one embodiment, the heavy chain variable region comprises a VH CDR3 comprising ... is one or more 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 one or more VH CDRs. two or more VH CDRs selected from the group consisting of SEQ ID NO: 15, and SEQ ID NO: 16 In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO: 14, VH CDR2 containing SEQ ID NO: 15, and VH CDR3 containing SEQ ID NO: 16 include.

[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 V comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 15. In one embodiment, the heavy chain variable region comprises a VH CDR2 containing the sequence SEQ ID NO: 1. In one embodiment, the heavy chain variable region comprises a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 7. is one or more 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 multiple VH CDRs. comprising two or more VH CDRs selected from the group consisting of SEQ ID NO: 15 and SEQ ID NO: 17 In one embodiment, the heavy chain variable region comprises a VH CDR1 comprising SEQ ID NO: 14, the sequence It comprises a VH CDR2 containing sequence number 15 and a VH CDR3 containing sequence number 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 C region comprising the amino acid sequence of SEQ ID NO: 18. In one embodiment, the light chain variable region comprises the amino acid sequence of SEQ ID NO: 19. In one embodiment, the light chain variable region comprises a VL CDR2 having the amino acid sequence of SEQ ID NO: 20. In one embodiment, the light chain variable region comprises a VL CDR3 containing the amino acid sequence One or more VL 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 SEQ ID NO: 18, SEQ ID NO: 19, and and two or more VL CDRs selected from the group consisting of SEQ ID NO: 20. wherein 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 SEQ ID NO: 14. VH CDR1 containing SEQ ID NO: 15 and VH CDR2 containing SEQ ID NO: 16 a heavy chain variable region comprising a VH CDR3 having the sequence SEQ ID NO: 18; a VL CDR1 having the sequence SEQ ID NO: 19; VL CDR2 containing sequence number 19 and VL CDR3 containing sequence number 20 and a light chain variable region.

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

[0151] In certain embodiments, one or more of the CDRs (i.e., having SEQ ID NOs: 14-17) and / or one or more heavy chain CDRs having SEQ ID NOs: 18-19. The heavy chain CDRs and light chain CDRs of the antibody have the CD2 specificity (i.e., the heavy chain CDRs and sequences of SEQ ID NOs: 14-16). The same specificity as an antibody or antigen-binding fragment thereof containing the light chain CDRs of sequences 18-20 Conservative amino acid substitutions (or two, three, four, or five amino acid substitutions).

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

[0153] In one embodiment, the antibody or antigen-binding fragment thereof is In another embodiment, the antibody or its anti- The original binding fragment has at least about 95% identity to SEQ ID NO: 22, e.g., SEQ ID NO: 8, having at least about 95%, 96%, 97%, 98%, 99% or 100% identity to In certain embodiments, the antibody comprises a heavy chain variable region containing an amino acid sequence corresponding to Modified heavy chains comprising an HC variable domain containing a variant of SEQ ID NO: 21 or SEQ ID NO: 22 (HC) variable region, the variant comprising: (i) 1, 2, 3, 4 or 5 amino acids (ii) differs from SEQ ID NO: 22 in substitution, addition, or deletion of up to 5, 4, 3, differing from SEQ ID NO: 22 in two or one amino acid substitution, addition, or deletion, iii) 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or (iv) differs from SEQ ID NO: 22 in a deletion or deletion; and / or (iv) differs from SEQ ID NO: 22 in at least one of the following: Approximately 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% The amino acid sequence is identical to that of the first amino acid sequence, and in any of (i) to (iv), the amino acid substitution is , conservative amino acid substitutions or non-conservative amino acid substitutions, and the modified heavy chain variable region may comprise: Retains the CD2 binding specificity of the antibody, i.e., an antibody comprising SEQ ID NO: 22 or its antigen binding fragment, but compared to the heavy chain variable region of SEQ ID NO: 22 It may have enhanced biological activity.

[0154] In one embodiment, the antibody or antigen-binding fragment thereof is In another embodiment, the antibody or its anti- The original binding fragment has at least about 95% identity to SEQ ID NO: 23, e.g., SEQ ID NO: 23 at least about 95%, 96%, 97%, 98%, 99% or 100% identity In certain embodiments, the antibody comprises a light chain variable region comprising an amino acid sequence having: A modified heavy chain comprising an LC variable domain containing SEQ ID NO: 23 or a variant of SEQ ID NO: 23. The variants include a nucleotide sequence (LC) variable region, and the variants include (i) 1, 2, 3, 4, or 5 amino acids (ii) differs from SEQ ID NO: 23 by up to 5, 4, 3, or 4 amino acid substitutions, additions, or deletions; , which differs from SEQ ID NO: 23 in two or one amino acid substitution, addition, or deletion; (iii) 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, or (iv) differs in a sequence or deletion from SEQ ID NO:23; and / or (v) differs in a sequence or deletion from SEQ ID NO:23 At least approximately 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% % identical amino acid sequence, and in any of (i) to (iv), an amino acid substitution may be conservative or non-conservative amino acid substitutions, and the modified heavy chain variable region and retain the CD2 binding specificity of the antibody, i.e., an antibody comprising SEQ ID NO: 23 or its antigen binding While having similar binding specificity to the heavy chain variable region of SEQ ID NO: 23, The compound may have enhanced biological activity.

[0155] In an exemplary embodiment, the antibody or antigen-binding fragment thereof has the sequence identified as SEQ ID NO:21. At least about 95% identity, e.g., at least about 95%, about 96%, about A heavy chain containing an amino acid sequence having 97%, about 98%, about 99% or 100% identity The chain variable region has at least about 95% identity with SEQ ID NO: 23, e.g., at least about 95% identity with SEQ ID NO: 23. at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identity and a light chain variable region comprising an amino acid sequence having the amino acid sequence The antigen-binding fragment comprises a heavy chain variable region containing SEQ ID NO: 21, and a heavy chain variable region containing SEQ ID NO: 2 The light chain variable region contains 3.

[0156] In an exemplary embodiment, the antibody or antigen-binding fragment thereof has the sequence identified as SEQ ID NO: 22. At least about 95% identity, e.g., at least about 95%, about 96%, to SEQ ID NO: 22; containing amino acid sequences with about 97%, about 98%, about 99%, or 100% identity and a heavy chain variable region having at least about 95% identity with SEQ ID NO: 23, e.g., SEQ ID NO: 23 at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% identical to and a light chain variable region comprising an amino acid sequence having the same structure as that of the antibody. or an antigen-binding fragment thereof, comprising a heavy chain variable region containing SEQ ID NO: 22, and The light chain variable region contains SEQ ID NO:23.

[0157] Anti-CD2 Antibodies that can be used in combination with the compositions and methods described herein includes those having one or more or all of the following CDRs: a. CDR-H1 having the amino acid sequence EYYMY (SEQ ID NO: 1); b. CDR-H2 having 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. 6,849,258, and anti-CD2 antibodies and antigen-binding fragments thereof. No. 6,299,499, the disclosure of which is incorporated herein by reference.

[0159] LO-CD2a, BTI-322 and deposited under ATCC accession number HB 11423 Antibodies produced by hybridoma cell lines (e.g., ATCC Deposit No. HB The antibody LO-CD2a isolated from the hybridoma cell line deposited as 11423 an antibody or antigen-binding fragment thereof comprising one or more or all of the CDR sequences; U.S. Patent Nos. 5,730,979, 5,817,311, 5,951,983 and and 7,592,006 are used herein. The compositions and methods described herein may be used in conjunction with the compositions and methods disclosed herein. Exemplary antibodies that can be used in conjunction with the method include those deposited under ATCC deposit number HB 11423. One or more or all of the CDR sequences of the antibody isolated from the deposited hybridoma cell line MEDI-507 is a humanized antibody containing the above A humanized anti-CD2 monoclonal antibody comprising the CDR-H and CDR-L sequences (a) to (f) above. Branco et al., Transplantation 68: 1588-1596 (1999). MEDI-507 is an international Patent Publications Nos. 99 / 03502A1 and 1994 / 020619A1, U.S. Pat. Nos. 7,59 ... 2, 006, 6, 849, 258, 5, 951, 983, 5, 817, 31 1 and 5,730,979, and U.S. Patent Application Publication No. 2011 / 02808 68, 2004 / 0265315 and 2011 / 0091453 and each of these disclosures discloses the use of anti-CD2 antibodies, such as the anti-CD2 antibody MEDI-507. The present invention is incorporated herein by reference as it relates to antibodies and antigen-binding fragments thereof. In one embodiment, the anti-CD2 antibody is siplizumab or its derivatives. is an antigen-binding fragment of

[0160] The disclosures in the aforementioned scientific journal articles and U.S. patents relate to anti-CD2 antibodies and their antigen-binding fragments. and is hereby incorporated by reference.

[0161] Other anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, For example, those described in U.S. Pat. No. 6,541,611 and U.S. Pat. No. 7,250,167. Each of these disclosures describes an anti-CD2 antibody and its antigen-binding domain. Fragments, such as the anti-CD2 antibody LO-CD2b and ATCC deposit number PTA-8 02 and therefore relates to the antibody produced by the hybridoma cell line deposited as reference These compounds may be used in conjunction with the compositions and methods described herein. Exemplary antibodies include the hybridoma cell line deposited under ATCC Accession No. PTA-802. The present invention also includes humanized antibodies containing one or more or all of the CDR sequences of an antibody isolated from a cell line. It can be enjoyed.

[0162] Other anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, For example, those described in U.S. Pat. No. 5,795,572 and U.S. Pat. No. 5,807,734. Each of these disclosures describes an anti-CD2 antibody and its antigen-binding domain. Fragments, such as the hybrid deposited under ATCC Accession No. HB 69277 The present invention is incorporated by reference as it relates to anti-CD2 antibodies produced by tumor cell lines. For example, anti-CD2 antibodies and and antigen-binding fragments thereof include EPKSSDKTHTSPPSP (SEQ ID NO: 287 ) a hinge region having the amino acid sequence scFv fragment containing a hinge region having the amino acid sequence PPSP (SEQ ID NO: 287) The incorporation of a hinge region having the amino acid sequence of SEQ ID NO: 287 is Reactions that can promote undesired oxidative dimerization of single-chain antibody fragments, such as Fv fragments This hinge motif is similar to the wild-type hinge region sequence, potentially eliminating a critical cysteine ​​residue. It can be informative because it is mutated compared to the sequence.

[0163] Other anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, For example, the anti-CD2 antibody TS2 / 18 and the antibody H. cerevisiae deposited under ATCC accession number HB-195. Antibodies produced by hybridoma cell lines, such as those described in U.S. Pat. No. 6,764,688, The disclosure of U.S. Patent No. 6,764,688 includes anti-CD2 antibodies. D2 antibody and antigen-binding fragments thereof, which are incorporated herein by reference. do.

[0164] Other anti-CD2 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, For example, U.S. Patent No. 6,162,432, U.S. Patent No. 6,558,662, U.S. Patent No. 7 ,408,039, U.S. Patent No. 7,332,157, U.S. Patent No. 7,638,121 No., U.S. Patent No. 7,939,062 and U.S. Patent No. 7,115,259, U.S. Patent Publication No. 2006 / 0084107, U.S. Patent Application Publication No. 2014 / 0369974 No. 2002 / 0051784 and U.S. Patent Application Publication No. 2013 / 0183322, and PCT Publication No. WO1992 / 016563. Each of these disclosures provides a description of the anti-CD2 antibody and its antigen-binding fragment. and is hereby incorporated by reference as it relates to the present invention.

[0165] Antibodies and fragments thereof for use in conjunction with the methods described herein include those comprising Fc Variants of the above antibodies, such as antibody fragments containing or lacking the domain, and One or more or all of the CDRs or fragments thereof of the antibodies or antibody fragments described herein Humanized variants of the non-human antibodies and antibody-like proteins described herein, including equivalent regions of Protein scaffolds (e.g., 10 Fn3 domain). Suitable antigen-binding fragments include, inter alia, dual variable immunoglobulin domains, single chain F scFv molecules, diabodies, triabodies, nanobodies, antibody-like protein skin fragments (scFv) Fv fragments, Fab fragments, F(ab')2 molecules, and Examples include di-scFv.

[0166] In one embodiment, the anti-CD2 antibody or binding fragment thereof comprises an altered Fc region. The modified Fc region comprises at least one amino acid sequence different from that of the wild-type Fc region. and a nucleotide modification, such that the molecule has increased affinity for FcgammaR (FcγR). Specific amino acid positions within the Fc region have been identified by crystallographic studies as altered Fc binding. It is known to directly contact γR. Specifically, amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C' / E loop), and amino acids 327-332 (F / G) loop (Sondermann e (See, e.g., 2000 Nature 406:267-273). The described antibodies comprise a variant Fc region, the variant Fc region having structural and crystallographic properties. The modification comprises altering at least one residue that directly contacts FcγR based on quantitative analysis. In this embodiment, the Fc region of an anti-CD2 antibody (or fragment thereof) is herein incorporated by reference. Kabat et al., Sequences of Proteins, expressly incorporated herein by reference. ins of Immunological Interest, 5th Edition Public H Health Service, NH1, Maryland (1991) EU Index Contains an amino acid substitution at amino acid 265 according to the "EU Index of Kabat" refers to the numbering of a human IgG1 EU antibody. In one embodiment, the Fc region is D2 In one embodiment, the Fc region comprises a D265A mutation. In an embodiment, the Fc region of the antibody (or fragment thereof) is In one embodiment, the Fc region contains an amino acid substitution at amino acid 234 according to the Fc region index. In some embodiments, the anti-CD2 antibody (or its The Fc region of the IgG1A-specific antibody fragment (IgG1A-specific antibody fragment) is located at amino acid 235 according to the EU index of Kabat. In one embodiment, the Fc region comprises an amino acid substitution at L235A. In another embodiment, the Fc region comprises the L234A and L235A mutations. In an embodiment, the Fc region comprises D265C, L234A, and L235A mutations. In a further embodiment, the Fc region comprises the amino acid sequence D265C, L234A, L235A, and In a further embodiment, the Fc region comprises a D265C and H435A mutation. Contains the 35A mutation.

[0167] Antibodies as used herein may be used in any of the methods described, for example, in Dall'Acqua et al. 2006) J Biol Chem 281:23514-24), (Zalevsky e (2010) Nat Biotechnol 28:157-9), (Hi nton et al. (2004) J Biol Chem 279:6213-6) , (Hinton et al. (2006) J Immunol 176:346-56 Page), (Shields et al. (2001) J Biol Chem 276:6 pp. 591-604), (Petkova et al. (2006) Int Immun ol 18:1759-69), (Datta-Mannan et al. (2007 ) Drug Metab Dispos 35:86-94), (Vaccaro et al. (2005) Nat Biotechnol 23:1283-8), (Ye ung et al. (2010) Cancer Res 70:3269-77) and (Kim et al. (1999) Eur J Immunol 29:2819-2 Antibody half-life can be improved by introducing additional Fc mutations, such as those described on page 5. Further manipulations are made to further adjust, 250, 252, 253, 254, 256, 25 It may include positions 7, 307, 376, 380, 428, 434 and 435. Exemplary mutations that can be made in combination are T250Q, M252Y, 1253A, S2 54T, T256E, P2571, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H4 It is a 35R mutation.

[0168] In some embodiments, the anti-CD2 antibody or antigen-binding fragment thereof is an antibody or its antigen-binding fragment via a cysteine ​​residue in the Fc domain of a cytotoxin ( In some embodiments, the system is conjugated to a cytosine-3-phosphate dehydrogenase (C3P) ... The in-residues are mediated by mutations in the Fc domain of an antibody or its antigen-binding fragment. For example, cysteine ​​residues are introduced at Cys118, Cys239, and Cys26. 5. In one embodiment, the anti-CD2 antibody (or a fragment thereof) The Fc region of the IgG1 antibody (IgG1A) is located at amino acid 265 according to the EU index of Kabat. In one embodiment, the Fc region comprises a D265C mutation. In this case, the Fc region contains D265C and H435A mutations.

[0169] Thus, in one embodiment, the Fc region contains a mutation that results in a decreased half-life. Antibodies with short half-lives have the advantage that they are expected to function as short-lived therapeutic agents. In certain cases, for example, the antibody is administered and then the HSCs are subsequently treated with the conditions described herein. Ideally, the antibody is substantially Unlike endogenous stem cells, HSCs generally also express CD2, but anti-CD2 antibodies In one embodiment, the Fc region is not a target of position 435 (EU IgE according to Kabat). In one embodiment, the mutation is a H435A mutation.

[0170] The aforementioned anti-CD2 antibodies or antigen-binding fragments thereof can be used to treat, for example, CD2 infection in human subjects. In various aspects of the invention described herein, including methods for depletion of D2+ cells, The aforementioned anti-CD2 antibodies or antigen-binding fragments thereof can also be used as described herein. As described in obtain.

[0171] Anti-CD5 antibody drug conjugate The present disclosure provides a method for treating a leukemia, encephalopathy, or rheumatoid arthritis (LEC), in which an anti-CD5 antibody or antigen-binding fragment thereof, or an anti-CD5 ADC is , CD5+ cancer cells (e.g., CD5+ leukemia cells) and CD5+ autoimmune cells (e.g., , CD5+ autoimmune T cells, B cells and / or NK cells) The ability of the agent to be directly used in the treatment of cancers, including T-cell malignancies and autoimmune diseases Anti-CD5 ADCs are also thought to be effective in preventing graft-versus-host disease (GVHD). It can also be used to treat patients at risk of

[0172] In particular, the anti-CD5 antibodies described herein can be conjugated to a cytotoxin via a linker. Therefore, where anti-CD5 antibodies are mentioned, they are used unless otherwise stated. Also contemplated are conjugates of

[0173] Embodiments of the disclosure described herein further include antibodies capable of binding to CD5 or antibody binding thereto. The fragments may prevent or reduce the likelihood of immune cell-mediated graft rejection. , as a therapeutic agent to promote engraftment of transplanted hematopoietic stem cells in patients requiring transplantation therapy. For example, anti-CD5 antibodies and antigen-binding flag A ment is one of the antigens expressed by hematopoietic stem cells, such as one or more non-self MHC antigens. or T cells that cross-react with and initiate an immune response against multiple non-self hematopoietic stem cell antigens. binds to cell surface CD5 expressed by immune cells such as cytoplasmic reticulocytes, B cells, or NK cells Such antibodies and antigen-binding fragments can be used to inhibit hematopoietic stem cell-specific CD5+ Binding to immune cells can be achieved, for example, by antibody-dependent cell-mediated cytotoxicity or by binding to antibodies or The antigen-binding fragment binds to the cytotoxic agent conjugated to the bound antibody. It can induce the death of immune cells. Therefore, C Depletion of the D5+ immune cell population may contribute to the recipient's initiation of an immune response against the incoming graft. It weakens the immune system of the patient, thereby improving hematopoietic function in patients requiring hematopoietic stem cell transplantation. The engraftment of stem cells can be promoted. In this way, defective or missing stem cells can be detected in a subject. Hematopoietic stem cell transplants can be provided to subjects to repopulate deficient cell lineages. Therefore, patients suffering from a stem cell disorder, cancer, autoimmune disease, or other blood disorder described herein may For example, a subject can be administered a therapeutic agent with the intent of eradicating cancerous cells. The subjects were given chemotherapy, but the chemotherapy also depleted healthy hematopoietic cells in the process, resulting in the cells becoming may be lacking.

[0174] For example, as used herein, an antibody capable of binding to an antigen expressed by a T cell is administration of the antibody or antigen-binding fragment thereof, or ADC, The present invention provides compositions and methods for promoting cell engraftment. This selective depletion of endogenous T cell populations, such as T cells, can lead to the selective depletion of endogenous T cell populations. Thirst is a common cause of graft rejection after transplantation of exogenous (e.g., autologous, allogeneic, or syngeneic) hematopoietic stem cell grafts. For example, the anti-CD5 antibodies described herein, antigen-binding fragments, CD4+ and CD4+ antibodies using antibodies, antibody-drug conjugates, or antibody-drug conjugates Selective depletion of CD8+ T cells and / or CD8+ T cells occurs in transplanted hematopoietic stem cell grafts. The compositions and methods described herein can attenuate T cell-mediated immune responses that may occur. The method comprises grafting an antibody and an antigen-binding fragment thereof capable of binding to CD5. To promote the survival and engraftment potential of hematopoietic stem cells, hematopoietic stem cell transplantation therapy is required. This is based in part on the discovery that the compound can be administered to patients in need thereof.

[0175] The survival of hematopoietic stem cell grafts treated with anti-CD5 antibody or its antigen-binding fragment is unclear. This can be determined by various empirical measurements, such as the engraftment of transplanted hematopoietic stem cells. The present invention relates to a method for treating rheumatoid arthritis by administering an antibody or antigen-binding fragment thereof capable of binding to CD5, Following administration of a hematopoietic stem cell graft, competitive reconstituting units (C) present in the patient's bone marrow are used. It can be evaluated by measuring the amount of fluorophores, colorimetric or A reporter gene, such as an enzyme that catalyzes a chemical reaction that produces a luminescent product, is transfected into donor hematopoietic stem cells. The corresponding signal is then introduced into the transfected vector, and the resulting signal is then expressed in the bone marrow. By monitoring the tissues to which hematopoietic stem cells are homing, The engraftment of the cell graft can be monitored, for example, by methods known in the art. Hematopoietic stem cells, as determined by fluorescence-activated cell sorting (FACS) analysis The engraftment of hematopoietic stem cell grafts can be monitored by assessing the quantity and survival of stem and progenitor cells. Engraftment can also be assessed by measuring the number of leukocytes in the peripheral blood during the post-transplant period. and / or by measuring bone marrow cell recovery by donor cells in bone marrow aspirate samples. Therefore, it can also be determined.

[0176] The following sections discuss various therapeutic options for treating autoimmune diseases, cancer, graft-versus-host disease (GVHD), and other conditions. ) or to promote hematopoietic stem cell transplant engraftment. an antibody or antigen-binding fragment thereof that can be administered to a patient in need of cell transplant therapy; and methods of administering such therapeutic agents to patients prior to hematopoietic stem cell transplantation.

[0177] Anti-CD5 antibody The compositions and methods described herein include antibodies or their derivatives that specifically bind to human CD5. Human CD5 is also called LEU1 or T1. , a type I transmembrane glycoprotein found on the surface of thymocytes, T lymphocytes, and a subset of B lymphocytes Two isoforms of human CD5 have been identified. M1 contains 438 amino acids and is described in Jones et al. (1988) Nature 3 23(6086), pp. 346-349 and the following (NCBI reference sequence: NP_00133 3385.1) states: MVCSQSWGRS SKQWEDPSQASKVCQRLNCG VPLSLGPFLV TYTPQSSIICYGQL GSFSNCSHSRNDMCHS LGLTC LEPQKTTPPTTRPPPTTTPEPTAPP RLQLVAQSGG QHCAGVVEFYSGSLGGTISY EAQDKTQDLE NFLCNNLQCG SFLKHLPETE AGRAQDPGEP REHQPLPIQWKIQNSSCTSL EHCFRKIKPQ KSGRVLALLC SGFQPKVQSR LVGG SSICEG TVEVRQGAQWAALCDSSSAR SSLRWEEVCR EQQCGSVNSY RVLDAGDPTS RGLFCPHQKL SQCHELWE RNSYCKKVFVTCQDPNPAGLAAGTVASIILAL VLLVVLLVVC GPLAYKKLVK KFRQKKQRQWIGPTGMNQNM SFHR NHTATV RSHAENPTAS HVDNEYSQPP RNSHLSAYPA LEGALHRSSMQPDNSSDSDY DLHGAQRL (SEQ ID NO: 286)

[0178] T cells have been shown to express CD5, a cell adhesion molecule that mediates activation. It is involved in both T cell proliferation and T cell helper function. It functions by interacting with CD72, a cell surface protein exclusive to B cells, It has also been shown that antibodies that bind to CD5 and their The antigen-binding fragment may be, for example, a fragment that inhibits the interaction between CD5 and CD72. This may suppress T cell activation and T cell-mediated immune responses to hematopoietic stem cell grafts. Antibodies and antigen-binding fragments thereof that bind to D5 may be used to treat cytotoxins (as described herein) or known in the art) to By conjugating or recruiting complement proteins to T cells, By using unconjugated antibodies or antigen-binding fragments thereof that can It can also be used to directly kill D5+ T cells.

[0179] Furthermore, a subset of activated B cells has been shown to express CD5; 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 is expressed on a subset of NK cells. It has also been shown to be expressed by low-density CD5+ cells, particularly in patients with multiple myeloma. It has been shown that there exists a population of (CD5LOW+) NK cells, and this surface antigen is expressed on NK cells. It is believed to be involved in the activation of ATP (Ishiyama et al., Anticancer Research 14:725-730 (1994), the entire disclosure of which is incorporated herein by reference. Therefore, antibodies or antigen-binding fragments thereof that specifically bind to CD5 can be used. By using CD5-binding antibodies, the activation of B cells and NK cells can be suppressed. and antigen-binding fragments thereof, can be cytotoxins (as described herein or in the art). Conjugating an antibody or its antigen-binding fragment to a soluble antibody (as known in the art) or by non-human resident macrophages that can recruit complement proteins to B cells or NK cells. By using a conjugated antibody or antigen-binding fragment thereof, CD5+ It can also be used to directly kill B cells and NK cells.

[0180] As used herein, a CD5 polypeptide, e.g., a human CD5 polypeptide, is specifically bound to the CD5 polypeptide. Exemplary antibodies and antigen-binding fragments thereof, and uses thereof, are described. In embodiments, an antibody or antigen-binding fragment thereof that specifically binds to a CD5 polypeptide is The component comprises 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 having the amino acid sequence of SEQ ID NO: 42. In one embodiment, the heavy chain variable region comprises CDR1 of the amino acid sequence of SEQ ID NO: 43. In one embodiment, the heavy chain variable region comprises a VH CDR2 having the sequence of SEQ ID NO: 44. In one embodiment, the heavy chain variable region comprises a VH CDR3 having the amino acid sequence One or more 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 the VH CDRs of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ 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 having the amino acid sequence of SEQ ID NO: 45. In one embodiment, the light chain variable region comprises CDR1 of the amino acid sequence of SEQ ID NO: 46. In one embodiment, the light chain variable region comprises a VL CDR2 having the sequence of SEQ ID NO: 47. In one embodiment, the light chain variable region comprises a VL CDR3 having the amino acid sequence One or more 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 the VL CDRs of SEQ ID NO: 45, SEQ ID NO: 46, and two or more VL CDRs selected from the group consisting of SEQ ID NO: 47. In an embodiment, the light chain variable region comprises a VL CDR1 having SEQ ID NO: 45, a VL CDR2 having SEQ ID NO: 4 6 and a VL CDR3 having SEQ ID NO:46.

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

[0184] In certain embodiments, one or more of the CDRs (i.e., having SEQ ID NOs: 42-44) one or more heavy chain CDRs having SEQ ID NOs: 45-47; The CD5 specificity of the antibody (i.e., the heavy chain CDRs of SEQ ID NOs: 42-44 and and an antibody or antigen-binding fragment thereof comprising the light chain CDRs of SEQ ID NOs: 45 to 47. Conservative amino acid substitutions (or two, three, four, or 5 amino acid substitutions).

[0185] In certain embodiments, the anti-CD5 antibody or antigen-binding fragment thereof is a murine anti-CD5 antibody. The murine antibody 5D7 binds to human CD5, or a humanized version thereof. and is described in U.S. Patent Publication No. 20008 / 0245027, and the The contents relating to the antibody sequences listed in Table 5 are incorporated herein by reference. Nos. 54 to 59 correspond to the CDRs of the mouse anti-CD5 antibody 5D7. The humanized versions are SEQ ID NO: 282 (humanized heavy chain variable region) and SEQ ID NO: 283 (human In one embodiment, the ADCs and and uses thereof include antibodies comprising the CDRs set forth in SEQ ID NOS: 54 to 59. In some embodiments, the ADCs and uses thereof described herein may comprise the polypeptides of SEQ ID NOs: 282 and 283, respectively. and 283.

[0186] In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of: In another embodiment, the antibody or its An antigen-binding fragment has at least 95% identity to SEQ ID NO: 282, e.g., SEQ ID NO: At least 95%, 96%, 97%, 98%, 99% or 100% identical to No. 282 In certain embodiments, the antibody comprises a heavy chain variable region having an amino acid sequence having: Modifications comprising an HC variable domain having SEQ ID NO: 282 or a variant of SEQ ID NO: 282 The variants comprise a heavy chain (HC) variable region, and the variants include (i) 1, 2, 3, 4, or 5 amino acids. (ii) differs from SEQ ID NO: 282 in substitution, addition, or deletion of up to 5, 4 282 in the substitution, addition, or deletion of one, two, or three amino acids. (iii) 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acid substitutions, additions, (iv) a sequence identical to SEQ ID NO: 282, which sequence differs from SEQ ID NO: 282 in addition, deletion, or deletion; 82 and at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, and The amino acid substitutions may be conservative or non-conservative amino acid substitutions, and the modified heavy chain The variable region is identical to the heavy chain variable region of SEQ ID NO: 282 while retaining the CD5 binding specificity of the antibody. and the like, i.e., an antibody comprising SEQ ID NO: 282 or an antibody thereof. The antigen-binding fragment of the present invention has the same binding specificity as that of the antigen-binding fragment of the present invention.

[0187] In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of: In another embodiment, the antibody or its anti- The original binding fragment has at least 95% identity to SEQ ID NO: 283, e.g., SEQ ID NO: 283 with at least 95%, 96%, 97%, 98%, 99% or 100% identity In certain embodiments, the antibody comprises a light chain variable region having an amino acid sequence having the sequence A modified light chain comprising an LC variable domain having SEQ ID NO: 283 or a variant of SEQ ID NO: 283. The variants include a nucleotide sequence (LC) variable region, and the variants include (i) 1, 2, 3, 4, or 5 amino acids (ii) differs from SEQ ID NO: 283 in substitution, addition, or deletion of up to 5, 4, 283 differing from SEQ ID NO: 283 in 3, 2 or 1 amino acid substitution, addition, or deletion. (iii) substitutions, additions, or substitutions of 1 to 5, 1 to 3, 1 to 2, 2 to 5, or 3 to 5 amino acids , or a deletion, differs from SEQ ID NO: 283, and / or (iv) SEQ ID NO: 28 3 and at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence, and in any of (i) to (iv), The amino acid substitutions may be conservative or non-conservative amino acid substitutions, and the modified light chain may The variable region retains the CD5 binding specificity of the antibody, i.e., an antibody comprising SEQ ID NO: 283 or The light chain variable fragment of SEQ ID NO: 283 has similar binding specificity to the antigen-binding fragment. The region may have enhanced biological activity compared to the region.

[0188] In an exemplary embodiment, the antibody or antigen-binding fragment thereof is SEQ ID NO: 282 At least 95% identical to, for example, at least about 95%, about 96% identical to, for example, SEQ ID NO: 282 , about 97%, about 98%, about 99% or 100% identity to the amino acid sequence. and a heavy chain variable region having at least about 95% identity with SEQ ID NO: 283, e.g., SEQ ID NO: 2 83 and at least about 95%, about 96%, about 97%, about 98%, about 99% or 100% and a light chain variable region containing an amino acid sequence having identity to the antibody. The antibody or antigen-binding fragment thereof comprises a heavy chain variable region having SEQ ID NO: 282, and a sequence It contains a light chain variable region having sequence number 283.

[0189] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof is SEQ ID NO: 28 8 and a light chain variable region containing SEQ ID NO:289.

[0190] In one embodiment, the anti-CD5 antibody or antigen-binding fragment thereof is SEQ ID NO: 29 1 and a light chain variable region comprising SEQ ID NO:290.

[0191] In another embodiment, the anti-CD5 antibody or antigen-binding fragment thereof is SEQ ID NO:5 In one embodiment, the heavy chain variable region may comprise a VH CDR1 comprising the amino acid sequence of SEQ ID NO:4. wherein the heavy chain variable region comprises a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 55. In one embodiment, the heavy chain variable region comprises a VH comprising the amino acid sequence of SEQ ID NO:56. In one embodiment, the heavy chain variable region comprises CDR3. and SEQ ID NO: 56. In embodiments, the heavy chain variable region is selected from SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56. In one embodiment, the heavy chain variable The regions are VH CDR1 containing SEQ ID NO: 54, 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 VV comprising the amino acid sequence of SEQ ID NO: 57. In one embodiment, the light chain variable region comprises a CDR1 of the amino acid sequence set forth in SEQ ID NO: 58. In one embodiment, the light chain variable region comprises a VL CDR2 containing the sequence SEQ ID NO:5. In one embodiment, the light chain variable region comprises a VL CDR3 containing a 96 amino acid sequence. The region is one or more sequences 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 one or more VL CDRs. comprising two or more VL CDRs selected from the group consisting of SEQ ID NO: 58 and SEQ ID NO: 59. In one embodiment, the light chain variable region comprises a VL CDR1 comprising SEQ ID NO: 57, a VL CDR2 comprising SEQ ID NO: 58, a VL CDR3 comprising SEQ ID NO: 59, a VL CDR4 comprising SEQ ID NO: 60, a VL CDR5 comprising SEQ ID NO: 61, a VL CDR6 comprising SEQ ID NO: 62, a VL CDR7 comprising SEQ ID NO: 63, a VL CDR VL CDR2 containing SEQ ID NO: 58 and VL CDR3 containing SEQ ID NO: 59 nothing.

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

[0194] In certain embodiments, one or more of the CDRs (i.e., having SEQ ID NOs: 54-56) and / or one or more heavy chain CDRs having SEQ ID NOs: 57-59. The heavy chain CDRs and light chain CDRs of the antibody are identical to those of the CD5 specificity of the antibody (i.e., the heavy chain CDRs and sequences of SEQ ID NOs: 54-56). The antibody or antigen-binding fragment thereof containing the light chain CDRs of sequences 57 to 59 has the same specificity as the antibody or antigen-binding fragment thereof. Conservative amino acid substitutions (or two, three, four, or five amino acid substitutions).

[0195] Antibodies and antigen-binding fragments thereof capable of binding to the CD5 antigen are useful in immunological studies. computational modeling techniques, and the phage display and cell-based display techniques described below. In vitro selection methods such as the Ray platform are known in the art. , can be identified using the techniques described herein.

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

[0197] The aforementioned V L and V H Antibodies and antigen-binding fragments thereof comprising the sequence are described, for example, in U.S. The anti-CD5 antibody and its antigen-binding fragment are described in Japanese Patent No. 5,869,619. The disclosure of which is incorporated herein by reference as it relates to the present invention. In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof is selected from the group consisting of SEQ ID NO: 26 and SEQ ID NO: 27. Number 27 V L and V H In some embodiments, the anti-CD5 antibody and The antigen-binding fragments thereof include the V sequences of SEQ ID NO: 26 and SEQ ID NO: 27. L and V H including chains In some embodiments, the anti-CD5 antibody and its antigen-binding fragments comprise CDRs having the following structure: The fragments are V of SEQ ID NO: 26 and SEQ ID NO: 27. L and V H CDRs contained in the chain Including V L and V H The remainder of the sequence is SEQ ID NO: 26 and SEQ ID NO: 27. L and V H At least 85% (e.g., 85%, 90%, 95%, 97%, 98%) of the sequence , with 99% or more sequence identity.

[0198] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof comprises: The following CDRs are included: 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: has one or both of the following variable regions, or has at least 85% sequence identity: amino acid sequence with similarity (e.g., 85%, 90%, 95%, 97%, 98%, 99% or or higher 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 NTHYGEPTYADSFKGTRTFSLDDSKN TAYLQINSLRAEDTAVYFCTRRGYDW YFDVWGQGGTTVTVSS (SEQ ID NO: 35)

[0200] In some embodiments, the aforementioned V L and V H Antibodies containing the sequences and their antigen binding The conjugated fragments are described, for example, in U.S. Pat. No. 5,869,619 and are intended to be used in the manufacture of anti-CD40 antibodies. 5 antibodies and antigen-binding fragments thereof, e.g., the he3 antibody, as disclosed therein. In some embodiments, an anti-CD5 antibody or The antigen-binding fragments include the V of SEQ ID NO: 28 and SEQ ID NO: 29. L and V H Contained in the chain V L and V H The remainder of the sequence is SEQ ID NO: 28 and SEQ ID NO: 29. L and V H At least 85% (e.g., 85%, 90%, 95%, 97%) of the sequence , 98%, 99% or more) sequence identity.

[0201] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof is Contains CDRs of: 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 aforementioned CDR sequences are described, for example, in U.S. Pat. 5,869,619, and anti-CD5 antibodies and antigen-binding fragments thereof. No. 6,299,499, the disclosure of which is incorporated herein by reference.

[0203] Other anti-CD5 antibodies that can be used in combination with the compositions and methods described herein include: See, for example, U.S. Patent Nos. 5,766,886, 5,770,196, 7,153,9 No. 32, No. 5, 621, 083, No. 6, 649, 742, No. 6, 146, 631, No. 5, 756, 699, No. 5, 744, 580, No. 6, 376, 217, No. 5, 8 37,491 and 6,146,850, and anti-CD5 antibodies and The disclosure of which is incorporated herein by reference as it relates to antigen-binding fragments of

[0204] Other anti-CD5 antibodies that can be used in combination with the compositions and methods described herein include: For example, the antibody deposited as ATCC CRL 8000 (anti-CD5 mouse antibody OKT1) Such antibodies include those produced by hybridoma cell lines. Nos. 4,515,894, 4,657,760, and 4,363,799 and its disclosure relates to anti-CD5 antibodies and antigen-binding fragments thereof. is incorporated herein by reference.

[0205] Additional anti-CD5 antibodies that can be used in combination with the compositions and methods described herein No. 8,679,500 and International Patent Application Publication No. 2005 / 0109994, such as the anti-CD5 antibody MAT304. Anti-CD5 antibodies and their derivatives include those described in Publication No. 2010 / 145895. The disclosures of which are incorporated herein by reference as they relate to antigen-binding fragments of .

[0206] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, For example, the hybridoma (produced by the hybridoma deposited under ATCC Accession No. CRL-8023) (Issued on 2001) U.S. Pat. No. 4,675,386; Manske et al. (J. Immunol. unol 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. (JImmunol 125:725-73 1 (1980)). US Patent No. 4,675,375 relates to CD5 antibodies and antigen-binding fragments thereof. The disclosure of No. 86 is incorporated herein by reference.

[0207] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, For example, the hybridoma produced by the hybridoma cells deposited under ATCC HB9285 (anti-CD5 conjugate zolimomab aritox-ricin protein Such antibodies include those described in WO 1989006968 No., Henslee-Downey et al., Transplantation 61:738-45, 1996, Henslee et al., Transplan t.Proc.21:3004-3007, 1989, Przepiorka et a I., Ther. Immunol. 1:77-82, 1994, and anti-CD 5 antibodies and antigen-binding fragments thereof, the disclosures of each of which are incorporated herein by reference. and is incorporated herein by reference.

[0208] Additional anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include: U.S. Application No. 20110250203 and International Publication No. 2010 / 022737 Anti-CD5 antibodies described in Koefoed et al., Br. J. Hae See also the teachings of Koefoed et al., 2013. Anti-C US Application No. 201102 relating to the D5 antibody and antigen-binding fragments thereof 50203 and WO 2010 / 022737 and Koefoed et al. al., the disclosure of which is incorporated herein by reference.

[0209] Anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include those listed below. DR may include one or more or all of the following: 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. 8,679,500, and anti-CD5 antibodies and antigen-binding fragments thereof. No. 6,299,499, the disclosure of which is incorporated herein by reference.

[0211] Anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include those listed below. R may contain one or more or all of: 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 U.S. Pat. 8, 679, and 500.

[0213] Anti-CD5 antibodies that may be used in conjunction with the compositions and methods described herein include those listed below. R may contain one or more or all of: 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 aforementioned CDR sequences are described, for example, in U.S. Pat. The anti-CD5 antibody and its antigen binding agent are described in Patent Publication No. 2008 / 0254027. The disclosure of which is incorporated herein by reference as it relates to fusion fragments.

[0215] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, For example, the hybrids deposited at the Pasteur Institute on January 10, 1991 under numbers 1-1025 are Anti-CD5 antibodies produced by doma cell lines, etc., WO 1992 / 014491 Anti-CD5 antibodies and their antigen-binding fragments are also included. The disclosure of WO 1992 / 014491 is incorporated herein by reference in its entirety for purposes of clarity. Reference is made to the following.

[0216] Other anti-CD5 antibodies that can be used in conjunction with the compositions and methods described herein include, for example, See, for example, U.S. Patent Nos. 6,010,902 and 7,192,736, U.S. Patent Application Publication No. Nos. 2011 / 0250203 and 2017 / 0129128, and the International Patent Nos. 2016 / 172606, 1994 / 023747 and 1996 / 041 608, and the anti-CD5 antibodies and antigen-binding fragments thereof. No. 6,299,499, the disclosure of which is incorporated herein by reference.

[0217] In some embodiments, it may be used in conjunction with the compositions and methods described herein. The anti-CD5 antibodies include CDR-H1, CDR-H2, CDR-H3, and CDR-H4 shown in Table 1 below. These include those with a combination of CDR-L1, CDR-L2 and CDR-L3 regions. .

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

[0219] Antibodies and antigen-binding fragments thereof comprising the aforementioned CDR sequences of Table 1 are available from, for example, Anti-CD5 antibodies and their use are described in Patent Application Publication No. 2011 / 0250203. As it relates to antigen-binding fragments, the disclosure of which is incorporated herein by reference.

[0220] Antibodies and fragments thereof for use in conjunction with the compositions and methods described herein The antibodies include variants of the above antibodies, such as antibody fragments that contain or lack the Fc domain. and one or more or all of the C of the antibodies or antibody fragments described herein. Humanized variants of the non-human antibodies described herein, comprising a DR or equivalent region thereof; and Antibody-like protein scaffolds (e.g., 10 Fn3 domain) as mentioned above. Exemplary antigen-binding fragments of antibodies include, inter alia, bivariable immunoglobulin domain fragments. Fv molecules (scFv), diabodies, triabodies, nanobodies, antibody-like proteins Protein scaffold, Fv fragment, Fab fragment, F(ab')2 fragment di-scFv, and tandem di-scFv.

[0221] The aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can be used to treat, for example, CD5 in human subjects. The present invention is used in various embodiments described herein, including methods for depletion of D5+ cells. The aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can also be used in the methods described herein. The compound may be conjugated to a drug, e.g., a cytotoxin such as an amatoxin, so that the compound can be effectively inhibited. Additional anti-CD5 antibodies that may be used in the composition and method embodiments described herein No. 8,679,500 and U.S. Patent Application Publication No. 2011 / 0250203. and U.S. Patent Application Publication No. 2008 / 0254027, each of which The entire contents of which are incorporated herein by reference. Compositions and methods described herein Additional anti-CD5 antibodies that can be used in this embodiment include, for example, those described in Dillman et al. al., J. Clin. Oncol. (1984), 2(8):881-891 The monoclonal antibody T101 and the antibody described by Miller et al., Blood (1983), 62(5)988-95. Includes:

[0222] In one embodiment, the anti-CD5 antibody or binding fragment thereof comprises an altered Fc region. The modified Fc region comprises at least one amino acid sequence different from that of the wild-type Fc region. and a nucleotide modification, such that the molecule has increased affinity for FcgammaR (FcγR). Specific amino acid positions within the Fc region have been identified by crystallographic studies as altered Fc binding. It is known to directly contact γR. Specifically, amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C' / E loop), and amino acids 327-332 (F / G) loop (Sondermann e (See, e.g., 2000 Nature 406:267-273). The described antibodies comprise a variant Fc region, and the variant Fc region has structural and crystalline Based on analytical analysis, the modification of at least one residue that directly contacts FcγR is included. In this embodiment, the Fc region of an anti-CD2 antibody (or fragment thereof) is herein incorporated by reference. Kabat et al., Sequences of Proteins, expressly incorporated herein by reference. eins of Immunological Interest, 5th Edition Public Health Service, NH1, Maryland (1991) EU Indicator Contains an amino acid substitution at amino acid 265 according to the EU Index of Kabat. " refers to the numbering of a human IgG1 EU antibody. In one embodiment, the Fc region is D In one embodiment, the Fc region comprises a D265A mutation. In another embodiment, the Fc region comprises a D265C mutation. In an embodiment, the Fc region of the antibody (or a fragment thereof) is In one embodiment, the Fc In some embodiments, the region comprises a L234A mutation. The Fc region of the IgG1A-specific antibody fragment (Fc region) is located at amino acid 23 according to the EU index of Kabat. In one embodiment, the Fc region comprises an amino acid substitution at position 5. In another embodiment, the Fc region comprises a L235A mutation. In yet another embodiment, the Fc region comprises L234A and L235A mutations. In some embodiments, the Fc region comprises D265C, L234A, and L235A mutations. In a further embodiment, the Fc region comprises the amino acids D265C, L234A, L235A, and In a further embodiment, the Fc region comprises a D265C and H435A mutation. Contains the H435A mutation.

[0223] Antibodies as used herein may be used in any of the methods described, for example, in Dall'Acqua et al. 2006) J Biol Chem 281:23514-24), (Zalevsky e (2010) Nat Biotechnol 28:157-9), (Hi nton et al. (2004) J Biol Chem 279:6213-6) , (Hinton et al. (2006) J Immunol 176:346-56 Page), (Shields et al. (2001) J Biol Chem 276:6 pp. 591-604), (Petkova et al. (2006) Int Immun ol 18:1759-69), (Datta-Mannan et al. (2007 ) Drug Metab Dispos 35:86-94), (Vaccaro et al. (2005) Nat Biotechnol 23:1283-8), (Ye ung et al. (2010) Cancer Res 70:3269-77) and (Kim et al. (1999) Eur J Immunol 29:2819-2 Antibody half-life can be improved by introducing additional Fc mutations, such as those described on page 5. Further manipulations are made to further adjust, 250, 252, 253, 254, 256, 25 It may include positions 7, 307, 376, 380, 428, 434 and 435. Exemplary mutations that can be made in combination are T250Q, M252Y, 1253A, S2 54T, T256E, P2571, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H4 It is a 35R mutation.

[0224] In some embodiments, the anti-CD5 antibody or antigen-binding fragment thereof is an antibody or its antigen-binding fragment via a cysteine ​​residue in the Fc domain of a cytotoxin ( In some embodiments, the system is conjugated to a cytosine-3-phosphate dehydrogenase (C3P) ... The in-residues are mediated by mutations in the Fc domain of an antibody or its antigen-binding fragment. For example, cysteine ​​residues are introduced at Cys118, Cys239, and Cys2 65. In one embodiment, the anti-CD5 antibody (or a fragment thereof) The Fc region of the IgG1 fragment begins 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. The Fc region contains the D265C and H435A mutations.

[0225] Thus, in one embodiment, the Fc region contains a mutation that results in a decreased half-life. Antibodies with short half-lives have the advantage that they are expected to function as short-lived therapeutic agents. In certain cases, for example, the antibody is administered and then the HSCs are subsequently treated with the conditions described herein. Ideally, the antibody is substantially HSCs, unlike endogenous stem cells, generally also express CD2 but not anti-CD5 antibodies. In one embodiment, the Fc region is not a target of position 435 (EU IgE according to Kabat). In one embodiment, the mutation is a H435A mutation.

[0226] The aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can be used to treat, for example, CD5 in human subjects. In various aspects of the invention described herein, including methods for depletion of D5+ cells, The aforementioned anti-CD5 antibodies or antigen-binding fragments thereof can also be used as described herein. As described in obtain.

[0227] Methods for identifying anti-CD2 and anti-CD5 antibodies High-throughput antibody or antibody fragment library binding to CD2 or CD5 The method for input screening involves administering to a patient in need of hematopoietic stem cell therapy (e.g., conditioning of the patient) and / or the cancer or autologous Used to identify and mature the affinity of drugs useful for the direct treatment of immune diseases. Such methods include, inter alia, phage display, bacterial display, yeast display, display, mammalian cell display, ribosome display, mRNA display and in vitro display, known in the art, such as cDNA display. Antibody or antigen binding to biologically relevant molecules. The use of phage display to isolate fragments is described, for example, in Felici et al., Biotechnol.Annual Rev.1:149-183, 1 995, Katz, Annual Rev. Biophys. Biomol. Stru. ct.26:27-45, 1997, and Hoogenboom et al., I Reviewed in mmunotechnology 4:1-20, 1998. The disclosures of each of these are incorporated herein by reference as they relate to in vitro display technology. Kay, Perspective. Drug Discovery De s.2:251-268, 1995 and Kay et al., Mol. Dive rs.1:139-140, 1996, binds to cell surface antigens. To select polypeptides that and US Pat. No. 6,299,099, 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, can be successfully synthesized as functional molecules. It is often displayed on phage (e.g., EP0349578, EP452783 9, and EP0589877, and Chiswell and McCaffert y, Trends Biotechnol. 10:80-84, 1992; Each of these disclosures describes in vitro display techniques for the discovery of antigen-binding molecules. (These fragments are incorporated herein by reference in their entirety for purposes related to the use of Fab fragments.) Functional antibody fragments, such as scFV fragments, can be expressed in vitro. are expressed in prey format (e.g., McCafferty et al., Nature 348:552-554, 1990, Barbas et al., P roc.Natl.Acad.Sci.USA 88:7978-7982 pages, 1991 and Clackson et al., Nature 352:624-628, 1991, each of which discloses in vitro methods for the discovery of antigen-binding molecules. (These are incorporated herein by reference as they relate to the use of ro ​​display technology.) This technology involves, inter alia, the detection of antibodies and antigen-binding fragments thereof that bind to CD2 or CD5. can be used to identify and improve the affinity of target cells, which can then be used in hematopoietic stem cell transplantation therapy. and / or suffering from a cancer or autoimmune disease described herein (e.g., For example, CD2+ T cells and / or NK cells (or CD5+ T cells) in a human patient These may be used to deplete immune cells (cells and / or NK cells).

[0228] Binds to CD2 or CD5 on the surface of cells (e.g., T cells, B cells, or NK cells) For example, antibodies or other antibodies that are taken up by the cells by receptor-mediated endocytosis. Further techniques can be used to identify antigen-binding fragments of For example, antibodies and their receptors that bind to CD2 on the surface of T cells or NK cells and are subsequently internalized. To screen for antigen-binding fragments of the For example, the in vitro display technology described above can be applied. The technology binds to CD2 or CD5 on the surface of T cells, B cells, or NK cells, and then Adapted for screening internalized antibodies and antigen-binding fragments thereof Phage display can be used in conjunction with this screening paradigm. This represents one such technique that can be used to bind to CD2 or CD5 and subsequently Anti-CD2 antibodies and their fragments, which are taken up by CD4+, CD8+, CD9+, CD10+, CD12+, CD2+, CD3+, CD4+, CD5+, CD6+, CD8+, CD9+, CD12+, CD14+, CD16+, CD18+, CD2 ... To identify fragments, one skilled in the art can use methods such as those described in Williams et al., Leuke mia 19:1432-1438, 2005. can be used, the entire disclosure of which is incorporated herein by reference. Mutagenesis techniques known in the art may be used to specifically target antibodies, antibody fragments, e.g. scFv fragments, Fab fragments, diabodies, triabodies, and 10 Fn3 domain, or a randomized amino acid cassette (e.g., a CDR or its or one or more, or all, of the equivalent regions, or an antibody or antibody fragment A recombinant phage library can be generated that encodes antibodies having the following structure: The framework region, hinge, Fc domain, and other regions of the antibody or antibody fragment , e.g., human germline antibody sequences or slight variations relative to human germline antibodies It can be designed to be non-immunogenic in humans by having a sequence that only displays the do.

[0229] using phage display techniques described herein or known in the art. and phage containing randomized antibodies or antibody fragments covalently bound to the phage particle. The library can be incubated with CD2 or CD5 antigens, which For example, first, the phage library is blocked with a blocking agent (e.g., milk protein, Incubate with serum albumin and / or IgG to remove nonspecific proteins. Phage encoding antibodies or fragments thereof that exhibit protein binding and those that bind to the Fc domain phage encoding antibodies or fragments thereof that bind to the antibody are removed, and then the phage are The cells are then combined with a population of CD2+ (or CD5+) T cells, B cells, or NK cells. by incubating with a CD2-specific antibody or its antigen-binding fragment (CD5 specific antibody or antigen-binding fragment thereof) binds to cell surface CD2 or CD5, Thereafter, the antibody is allowed to react with the target antigen for a sufficient period of time to allow it to be taken up by 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. (e.g., 30 min to 6 h at 4°C, e.g., 1 h at 4°C). Sufficient CD2 or C to allow binding to and uptake by NK cells Phage containing antibodies or fragments thereof that do not show affinity for D5 were Afterwards, cells are washed with cold (4°C) 0.1 M glycine buffer, for example, pH 2.8. Antibodies taken up by T cells, B cells and / or NK cells can be removed by Phages bound to the antibody or fragments thereof can, for example, lyse the cell and induce internalization. The phage can be identified by recovering them from the cell culture medium. The phage can be prepared, for example, by transfecting bacterial cells with phage recovered in 2xYT medium, as known in the art. It can be amplified in bacterial cells by incubation using methods known to The phage recovered from this medium can then be used to, for example, insert the phage genome. The nucleic acid sequence of the gene(s) encoding the antibody or fragment thereof The encoded antibody, or fragment thereof, can be characterized by determining , followed by chemical synthesis (e.g., of antibody fragments such as scFV fragments) or They can be prepared de novo by recombinant expression (eg, of a full-length antibody).

[0230] Anti-CD2 or anti-CD5 antibodies for use in conjunction with the compositions and methods described herein An exemplary method for the in vitro evolution of phage is phage display. The display library is designed to target CDRs of antibodies or similar regions of antibody-like scaffolds. (for example, 10 designed within the coding sequence of the Fn3 domain (BC, CD, and DE loops) These mutations can be generated by making a series of selected mutations or alterations. The template antibody-encoding sequence into which mutations are introduced may 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 is identical to the template except for one or more amino acid mutations. Retroviral and phage display vectors encode the corresponding antibodies. They can be engineered using standard vector construction techniques known in the art. The P3 phage display vector was used in conjunction with a protein expression vector for antibody diversification. Phage display vectors for this purpose can be generated.

[0231] The mutated DNA provides sequence diversity, and each transformant phage is coded by its DNA. The amino acid sequence of the original template is displayed as a single variation, and a large number of different but structurally related sequences are generated. This results in a phage population (library) displaying a large number of amino acid sequences. Due to the well-defined structure of the region, the amino acid mutations introduced in the phage display screen were , the binding properties of the binding peptide or domain without significantly altering its overall molecular structure. is expected to change.

[0232] In a typical screen, the phage library is screened against CD2 or CD5 or other The epitopes of the IgG can be contacted and bound to the IgG. To facilitate separation of the target, it is convenient to immobilize it on a solid support. Phage bearing CD2 or CD5 binding moieties form complexes with targets on a solid support The unbound phage remain in solution and can be washed away with excess buffer. Then, by changing the buffer to an extreme pH (pH 2 or pH 10), by changing the ionic strength of the solution, by adding denaturants, or by other known means. , the bound phage can be released from the target.

[0233] The recovered phage can then be amplified through infection of bacterial cells, A new pool in which the gated antibodies are depleted and enriched for antibodies that bind to CD2 or CD5 The screening process can be repeated using the same method. Even if only a few rounds are required, this is sufficient to amplify the phage for subsequent rounds of screening. After selection, antibodies or their derivatives derived from the selected phage clones in the binding pool are isolated. The gene sequence encoding the antigen-binding fragment of the The peptide sequence that confers binding affinity of the phage to the target is revealed. During the process, the sequence diversity of the population is reduced by selection until the desired peptide-binding antibodies remain. The sequence is reduced with each round of sequencing. The sequence converges to a small number of related antibodies or their antigen-binding fragments. The increase in the number of phage recovered in each round of selection indicates the size of the library. This is an indication that convergence has occurred within the screening.

[0234] Another method for identifying anti-CD2 or anti-CD5 antibodies is, for example, by the following procedure. This includes using humanized non-human antibodies that bind to CD2 or anti-CD2 antibodies. A non-human antibody that binds to D2 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. (e.g., "VBASE" human germline sequence database, Kabat et al. .Sequences of Proteins of Immunological In interest, 5th ed., US Department of Health and Human man Services, NIH Publication No.91 -3242, 1 991, 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. (These are incorporated herein by reference as they relate to heavy and light chain sequences.) Using procedures, one skilled in the art can identify the variable domain framework residues and The CDRs can be identified (e.g., by sequence alignment). one of the heavy and / or light chain variable domains of a consensus human antibody to produce These CDRs can be combined with one or more corresponding CDRs of a non-human antibody that binds to CD2 or CD5. This CDR exchange can be carried out by any method described herein or in the art. This can be done using gene editing techniques known in the art.

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

[0236] To create a humanized antibody, one or more variable region CDRs are 5. The consensus sequence of any one of the above subunits is replaced with one or more variable region CDR sequences of a non-human antibody that binds to 5. A polynucleotide encoding the CD2 or C sequence can be recombinantly expressed. Since the affinity of the antibody for D5 is mainly determined by the CDR sequences, the resulting humanized antibody It is believed that the humanized antibody exhibits approximately the same CD2 or CD5 affinity 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, those described herein. ELISA-based techniques described in and known in the art, as well as surface plasmon These include fluorescein resonance, fluorescence anisotropy, and isothermal titration calorimetry, among others.

[0237] The uptake capacity of the prepared antibodies or fragments thereof can be determined, for example, by methods known in the art. This can be assessed using radionuclide uptake assays that are well known in the art, for example, as described herein. In vitro display techniques described in or known in the art Anti-CD2 antibodies or fragments thereof (or anti-CD5 antibodies or is its fragment) 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, 16 9 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 A group of radioisotopes such as Ac For example, 18 F, 75 Br, 77 Br, 122 I, 12 3 I, 124 I, 125 I, 129 I, 131 I, 211 Radioactive halogens such as At, Electrophilic halogen reagents (e.g., iodinated beads, Thermo Fisher Scientific) Beads such as polystyrene beads containing The antibody or fragment thereof can be incorporated using the same. The fragments may be used to interact with T cells, B cells, and / or NK cells, and / or to allow uptake. Incubate for a sufficient time to allow the cells to grow (e.g., 30 min to 6 h at 4°C, e.g., 1 h at 4°C). The cells can then be washed to remove any unincorporated antibodies or Fragments can be removed (e.g., by adding cold (4 °C) 0.1 M glycerol at pH 2.8). The resulting antibody or fragment is then Radiation (e.g., gamma rays) emitted by T cells, B cells, and / or NK cells that are circulating The collected wash buffer is then compared with the radiation (e.g., gamma rays) emitted by the wash buffer. can be identified.

[0238] For recombinant production of anti-CD2 or anti-CD5 antibodies, the antibodies can be co-produced, e.g., as described above. The nucleic acid to be loaded is isolated and used for further cloning and / or expression in a host cell. Such nucleic acids may be inserted into one or more vectors using conventional procedures (e.g., as an anti- oligonucleotides capable of specifically binding to genes encoding the heavy and light chains of the human body; These can be easily isolated and sequenced (by using a nucleotide probe).

[0239] Suitable host cells for cloning or expressing antibody-encoding vectors include those described herein. For example, antibodies may be prepared by, among other things, glycosylation and Fc If effector functions are not required, they can be produced in bacteria. and for expression of polypeptides, see, e.g., U.S. Pat. No. 5,648,237, U.S. Pat. See U.S. Patent Nos. 5,789,199 and 5,840,523 ( Charlton, Meth., describing the expression of antibody fragments in E. coli. ods in Molecular Biology, Vol. 248 (edited by BKC Lo) , Humana Press, Totowa, NJ, 2003), 245-2 (See also page 54.) After expression, the antibody is isolated in a soluble fraction from the bacterial cell paste and further purified. It can be manufactured.

[0240] Vertebrate cells can also be used as hosts, e.g., cells grown in suspension. Adapted mammalian cell lines may be useful. Other examples of useful mammalian host cell lines include S. V40 (COS-7) transformed monkey kidney CV1 line, human embryonic kidney line (e.g. In Graham et al., J. Gen Virol. 36:59 (1977). 293 or 293 cells), baby hamster kidney (BHK) cells, mouse Sertoli cells (e.g., Mather, Biol. Reprod. 23:243-25 TM4 cells described in page 1 (1980), monkey kidney cells (CV1), African migratory Dorisal kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MD CK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human liver Hep G2 cells, mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals NY Acad.Sci.383:44-68 (19 These are TRI cells, MRC 5 cells, and FS4 cells, which were described in

[82] . Useful mammalian host cell lines include Chinese hamster egg yolk, including DHFR-CHO cells. nest (CHO) cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and Y0, NS0, Sp2 / 0, etc. For a review of specific mammalian host cell lines suitable for antibody production, see For details, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (ed. BKC Lo, Humana Press, Toto) See, for example, WA, NJ, pp. 255-268 (2003). In some embodiments, the host cell is a eukaryote, such as a Chinese hamster ovary (CHO) cell or are lymphoid cells (Y0, NS0, Sp20 cells, etc.).

[0241] Antibody-drug conjugates (ADCs) Anti-CD5 or CD2 antibody drug conjugates that may be used in the methods described herein Gates were determined by measuring the activity of anti-CD5 or anti-CD2 antibodies conjugated to cytotoxins via a linker. Anti-CD5 antibodies or anti-CD2 antibodies that may be used in the methods described herein The cytotoxins, linkers and conjugates are known in the art and are described above. The method of jugation is described below.

[0242] cytotoxin (i) to directly treat cancer or an autoimmune disease as described herein, or (ii) Rejection of hematopoietic stem cells during transplantation into patients (e.g., human patients) in need of hematopoietic stem cell transplantation therapy To deplete endogenous immune cells so as to prevent or reduce the likelihood of death, the present invention provides The antibodies and antigen-binding fragments thereof described herein (e.g., those that recognize and bind to CD2 or CD5) Antibodies and antigen-binding fragments thereof, Pseudomonas exotoxin A, deBouganin , diphtheria toxin, amatoxins such as α-amanitin, saporin, maytansine, maytansine, Tansinoids, auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine 2 indolinobenzodiazepines, indolinobenzodiazepine dimers, or or variants thereof, or any of the cytotoxins described herein or in the art. It can be conjugated to other known cytotoxic compounds. In this embodiment, after cellular uptake of the antibody or antigen-binding fragment, the cytotoxin is transferred to the cell. to access internal targets and kill endogenous T cells, B cells, and / or NK cells. In addition, the cytotoxic molecule is conjugated to the antibody or antigen-binding fragment thereof that is then incorporated. Suitable cytotoxins for use with the compositions and methods described herein include: DNA intercalators (e.g., anthracite), among others known in the art, drugs that can disrupt the spindle apparatus (e.g., vinca alkaloids, meitans, maytansin, maytansinoids, and their derivatives), RNA polymerase inhibitors (e.g., amatoxins such as α-amanitin and their derivatives), which disrupt protein biosynthesis Agents that can disrupt rRNA N-glycosylation (e.g., saporin and ricin A chain) Drugs that exhibit cosidase activity.

[0243] In some embodiments, the cytotoxin of the antibody-drug conjugate inhibits RNA polymerase activity. In some embodiments, the RNA polymerase inhibitor is an amphotericin It is a toxin or a derivative thereof.

[0244] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof, e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, flaxseed Amanurinamide, amanulin, amanurinic acid, and proamanuline. The structure of the amatoxin is represented by Formula III and is described, for example, in Zanotti et al. l., Int.J.Peptide Protein Res.30, 1987, 450 -Disclosed on page 459.

[0245] In one embodiment, the cytotoxin is amanitin. or antigen-binding fragment thereof, comprising a conjugate represented by the formula Ab-ZL-Am Ab may be an antibody or its antigen-binding fragment. fragment, L is a linker, Z is a chemical moiety, and Am is an amatoxin. Many positions on the amatoxin or its derivatives are linked to the linking moiety L and thus to the antibody or It can serve as a site for covalently attaching the antigen-binding fragment. The antibodies and antigen-binding fragments described herein are represented by the formula Ab-ZL-Am. and Ab may be bound to an amatoxin to form a conjugate, or an antigen-binding fragment thereof, wherein Z is a chemical moiety, L is a linker, and A In some embodiments, Am-LZ has 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 atoms 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 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 optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl or optionally substituted heteroaryl; L is optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene, quinylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene arylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, a linker that is -C(=O)-, a peptide, or a combination thereof; Z is a compound selected from the group consisting of a reactive substituent present on L and an antibody or a compound thereof that binds to CD2 or CD5. formed from a coupling reaction between reactive substituents present in the antigen-binding fragment It is a chemical moiety.

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

[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 RA and RB, when present, together with the oxygen atoms to which they are attached, and, when joined, 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 D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2 -C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), ... optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (e.g., C1-C6 heteroalkylene), optionally substituted alkyl alkenylene (e.g., C-C alkenylene), optionally substituted heteroalkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C 2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 alkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, alkylene, optionally substituted arylene, optionally substituted heteroarylene, diphenyl peptide, -C(=O)-, peptide, disulfide, hydrazone or a combination thereof A linker such as a-(CH2CH2O) p - group, and p is an integer of 1 to 6. , ((CH2) m O) n (CH2) m - group, wherein n and m are each independently 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or combinations thereof. Z is a compound selected from the group consisting of a reactive substituent present on L and an antibody or a compound thereof that binds to CD2 or CD5. formed from a coupling reaction between reactive substituents present in the antigen-binding fragment is a chemical moiety that Am contains exactly one RC substituent.

[0251] In some embodiments, the linker is -((CH) n where n is 6. In some embodiments, LZ is as follows: [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 R A and RB If present, together with the oxygen atom to which they are attached and, when joined, 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 D is optionally substituted alkyl (e.g., C1-C6 alkyl), optionally substituted heteroalkyl (e.g., C1-C6 heteroalkyl), optionally substituted alkenyl (e.g., C2-C6 alkenyl), optionally substituted heteroalkenyl (e.g., C2- C6 heteroalkenyl), optionally substituted alkynyl (e.g., C2-C6 alkynyl ), optionally substituted heteroalkynyl (e.g., C2-C6 heteroalkynyl), optionally optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl or optionally substituted heteroaryl; L is optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene, quinylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene arylene, optionally substituted arylene, optionally substituted heteroarylene, dipeptide, -C(=O)-, peptide, disulfide, hydrazone, -(CH2CH2O) p -Group( p is an integer from 1 to 6), ((CH2) m O) n (CH2) m -group (n and each m are independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) or combinations thereof It is a linker such as Z is a reactive substituent present on L and an antibody or an antibody thereof that binds to CD2 or CD5. formed from a coupling reaction between reactive substituents present in the original bonded fragment is the chemical part, 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 are identical to the oxygen atoms to which they are bonded. together, and join 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 alkyl Lukenylene-R C , optionally substituted C2-C6 heteroalkenylene-RC , optionally replaced C2-C6 alkynylene-R C , optionally substituted C2-C6 heteroalkynylene -R C、 optionally substituted cycloalkylene-R C , optionally substituted heterocycloalkanes Kiren-R C , optionally substituted arylene-R C or optionally substituted heteroaryl Ren-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, Forming the bottom, [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 D are 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 Accomplished, [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 Btogether with the oxygen atoms to which they are attached, Forming the bottom, [ka] R3, R4, R6 and R7 are each H; R5 is OR C and 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 / 002494. No. 002298, the entire disclosure of which is incorporated herein by reference.

[0264] In some embodiments, Am-LZ is represented by formula (IA) or (IB), and wherein R1 and R2 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 This is described, for example, in U.S. Patent Application Publication No. 2014 / 0294865, The entire disclosure 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 Cor R C and R8 is OH or NH2, R9 is H or OH; X and R C is as defined above. Such amatoxin conjugates This is described, for example, in U.S. Patent Application Publication No. 2015 / 0218220. The entire disclosure is incorporated herein by reference.

[0266] In some embodiments, Am-LZ is represented by formula (IA) or (IB), and wherein R1 and R2 are each independently H or OH; R3, R6 and R7 are each H; R4 and R5 are each independently H or OH; R8 is OH, NH2, OR C or NHR C and R9 is H or OH; R C is as defined above. Such amatoxin conjugates are described, for example, in U.S. Pat. No. 9,233,173. and 9,399,681, and U.S. Patent Application Publication No. 2016 / 0089450 No. 6,239,593, the entire disclosure of which is incorporated herein by reference.

[0267] In accordance with the compositions and methods described herein, antibodies or antigen-binding fragments thereof may be Additional amatoxins that can be used for conjugation are described, for example, in WO 201 Nos. 6 / 142049, 2016 / 071856, and 2017 / 046658 and JP 2003-1026634, the entire disclosures of each of which are incorporated herein by reference.

[0268] In some embodiments, Am-LZ is represented by formula (II), (IIA), or (IIB ): [ka] is expressed as wherein X is S, SO or SO2, R1 is H or an antibody or its derivatives linked via chemical moiety Z. A linker covalently attached to the antigen-binding fragment, wherein the linker has a reactive substituent. Coupling between the group and reactive substituents present in the antibody or antigen-binding fragment thereof R2 is linked to the antibody or its antigen-binding flag via H or chemical moiety Z. a linker covalently attached to the antibody or formed from a coupling reaction between reactive substituents present within the antigen-binding fragment and when R1 is H, R2 is a linker, and when R2 is H, R1 is a linker. .

[0269] In some embodiments, the linker is -(CH2) n― where n is an integer from 2 to 6. In some embodiments, R1 is a linker and R2 is H, and the linker and The chemical moieties are both LZ and are as follows: [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. III, α-amanitin, is an antibody or a ligand that binds to CD2 or CD5 via linker L. The linker L can be attached to the antigen-binding fragment at several possible positions, e.g. Ba, R 1 ~R 9 or any one of the following) linked to α-amanitin of formula III by one of the following formula: I, IA, IB, II, IIA, or IIB α-amanitin-linker conjugates In some embodiments, the linker can be R 1 Positionally bonded In some embodiments, the linker is R 2 Some implementations In the form, the linker is R 3 In some embodiments, the linker - is R 4 In some embodiments, the linker is attached at the R 5 Positionally bonded In some embodiments, the linker is R 6 Some implementations In the form, the linker is R 7 In some embodiments, the linker - is R 8 In some embodiments, the linker is attached at the R 9 Positionally bonded In some embodiments, the linker is a hydrazine, a disulfide, a thioether, or a hydroxyl group. In some embodiments, the linker comprises a tert- or dipeptide. a and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). The linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker In some embodiments, the linker comprises the moiety PAB-Ala-Val. -((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 -PAB-Cit-Val-((C=O)(CH2) n - Some implementations In the form, the linker is -PAB-Ala-Val-((C=O)(CH) n -in In some embodiments, the linker L and chemical moiety Z are collectively referred to as LZ. The following is a summary: [ka]

[0273] Antibodies and antigen-binding flags for use in conjunction with the compositions and methods described herein The compost may be prepared using conjugation techniques known in the art or described herein. conjugated to an amatoxin such as α-amanitin or its variants. 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 are α- The disclosure of the present invention relates to a compound that can be conjugated to an amatoxin, such as amanitin, or a variant thereof. The present invention relates to amatoxins, such as α-amanitin, and variants thereof, as well as covalently linked For information regarding covalent linkers that can be used for conjugation, see Synthetic methods for making amatoxins are described, for example, in U.S. Pat. 676,702, which is incorporated herein by reference in its entirety with respect to the synthetic methods disclosed therein. The present application is incorporated by reference.

[0274] Antibody or antigen-binding flags for use in conjunction with the compositions and methods described herein The compost may be prepared using conjugation techniques known in the art or described herein. conjugated to an amatoxin such as α-amanitin or its variants. For example, as described in U.S. Patent Application Publication No. 2015 / 0218220 The antibodies or antigen-binding fragments thereof that recognize and bind to CD2 or CD5 are α- The disclosure of the present invention relates to a compound that can be conjugated to an amatoxin, such as amanitin, or a variant thereof. The present invention relates to amatoxins, such as α-amanitin, and variants thereof, as well as covalently linked For information regarding covalent linkers that can be used for conjugation, see No. 6,029,499, filed on Oct. 1, 2004, which is incorporated herein

[0275] Exemplary antibody-drug conjugates useful in conjunction with the methods described herein include antibodies or and its antigen-binding fragment and a reactive residue on the antibody or antigen-binding fragment. and an amatoxin conjugated to a linker containing a substituent suitable for reaction with a group The antibody or antigen-binding fragment thereof described herein may be formed by reaction. Amatoki conjugated to a linker containing a substituent suitable for reaction with a reactive residue Synthons include, but are not limited to: 7'C-(4-(6-(Male (mido)hexanoyl)piperazin-1-yl)-amatoxine, 7'C-(4-(6-( Maleimido)hexanamido)piperidin-1-yl)-amatoxin, 7'C-(4- (6-(6-(maleimido)hexanoyl)piperazin-1-yl)- Amatoxin, 7'C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl (1-yl)piperazin-1-yl)-amatoxin, 7'C-(4-(6-(4-((maleimine De)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)- Amatoxin, 7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)pi Peridine-1-yl)-amatoxin, 7'C-(4-(2-(6-(6-(maleimide )hexanamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine, 7'C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamide) ethyl)piperidin-1-yl)-amatoxine, 7'C-(4-(2-(6-(4-( (Maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidine Lysin-1-yl)-amatoxin, 7'C-(4-(2-(3-carboxypropanediol) (4-(2-(2-methyl-2-isopropyl)-2-methyl-1-pyridin-1-yl)-amatoxine, 7'C-( ...1-pyridin-1-yl)-amatoxine, 7'C-(4-(2 bromoacetamido)ethyl)piperidin-1-yl)-amatoxine, 7'C-(4-( 2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidine -1-yl)-amatoxin, 7'C-(4-(2-(4-(maleimido)butanamide )ethyl)piperidin-1-yl)-amatoxine, 7'C-(4-(2-(maleimide) )acetyl)piperazin-1-yl)-amatoxine, 7'C-(4-(3-(maleimine 7'C-(4-(4-((propanoyl)piperazin-1-yl)-amatoxin, 7'C-(4-(2-(trimethylimido)butanoyl)piperazin-1-yl)-amatoxin (6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamide )ethyl)piperidin-1-yl)-amatoxine, 7'C-(3-((6-(maleimine hexanamido)methyl)pyrrolidin-1-yl)-amatoxine, 7'C-(3- ((6-(6-(maleimido)hexanamido)hexanamido)methyl)pyrrolidine- 1-yl)-amatoxine, 7'C-(3-((4-((maleimido)methyl)cyclohexane Xanthanecarboxamido)methyl)pyrrolidin-1-yl)-amatoxine, 7'C-(3 -((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanea (4-(2-(6-( ... 2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl) -Amatoxin, 7'C-(4-(2-(4-(2-(aminooxy)acetamido)butanol Tanamido)ethyl)piperidin-1-yl)-amatoxin, 7'C-(4-(4-( 2-(Aminooxy)acetamido)butanoyl)piperazin-1-yl)-amatoxy 7'C-(4-(6-(2-(aminooxy)acetamido)hexanoyl)piperane (4-(6-(maleimido)hexaneamine)-7'C-( ... 7'C-((4-(2-(6-( Maleimido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin , 7'C-((4-(6-(maleimido)hexanoyl)piperazin-1-yl)methyl )-Amatoxin, (R)-7'C-((3-((6-(maleimido)hexanamide) (S)-7'C-((3-(methyl)pyrrolidin-1-yl)methyl)-amatoxine (6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amanamido Toxin, 7'C-((4-(2-(6-(6-(maleimido)hexanamide)hexa 7'C-((4-((amino)ethyl)piperidin-1-yl)methyl)-amatoxine (2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin 7'C-((4-(2-(6-(4-((mamatoxin-1-yl)methyl)-amatoxin methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidinyl 7'C-((4-(2-(6-(maleimide) Hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine, 7'C-( (4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)pi Perazin-1-yl)methyl)-amatoxin, 7'C-((4-(2-(4-((male Imido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl 7'C-((4-(2-(6-(4-((maleimido)methyl)silyl)amino)-amatoxin, Cyclohexanecarboxamido)hexanamido)ethyl)piperazin-1-yl)methyl )-Amatoxin, 7'C-((3-((6-(6-(maleimido)hexanamide) Xanamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxine, 7'C -((3-((6-(6-(maleimido)hexanamide)hexanamide)-R-methyl 7'C-((3-((4-(((pyrrolidin-1-yl)methyl)-amatoxine, Cyclohexanecarboxamido)-S-methyl)pyrrolidine-1-yl 7'C-((3-((4-((maleimido)methyl)cyclohexyl)methyl)-amatoxine, (R-methyl)pyrrolidin-1-yl)methyl)-amatoki Synthon, 7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarbo oxamido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(3-carboxypropanamido)ethyl)piperazine-1-yl) 7'C-((4-(6-(6-(maleimido)hexane) (4'C)-((4' ... -(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl )piperazin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(maleimine (de)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)-amanamido Toxin, 7'C-((4-(2-(2-(maleimido)acetamido)ethyl)piperidine (2-(4-(maleimide) ) butanamido) ethyl) piperidin-1-yl) methyl) - amatoxine, 7'C-( (4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamide) Xanamido)ethyl)piperidin-1-yl)methyl)-amatoxine, 7'C-(( 3-((6-(maleimido)hexanamido)methyl)azetidin-1-yl)methyl) -Amatoxin, 7'C-((3-(2-(6-(maleimido)hexanamido)ethyl )azetidin-1-yl)methyl)-amatoxine, 7'C-((3-((4-((male Imido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl 7'C-((3-(2-(4-((maleimido)methyl)cyclohexane Xanthanecarboxamido)ethyl)azetidin-1yl)methyl)-amatoxine, 7'C -((3-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamide )Hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxine, 7'C- (((2-(6-(maleimido)-N-methylhexanamido)ethyl)(methyl)amido 7'C-(((4-(6-(maleimido)N-methylhexyl)-amatoxine Sanamido)butyl(methyl)amino)methyl)-amatoxin, 7'C-((2-(2 -(6-(maleimido)hexanamido)ethyl)aziridin-1-yl)methyl)-aziridin-1-yl 7'C-((2-(2-(6-(4-((maleimido)methyl)cyclohexane (Hexanamide) Ethyl) Aziridin-1-yl) Methyl) Aza Toxin, 7'C-((4-(6-(6-(2-(aminooxy)acetamido)hexa 7'C-(hexanoyl)piperazin-1-yl)methyl)-amatoxine (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)piperazine-1 -yl)methyl)-amatoxin, 7'C-((4-(3-(2-(aminooxy)acetate (isopropyl)propanoyl)piperazin-1-yl)methyl)-amatoxine, 7'C-( (4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl )methyl)-amatoxin, 7'C-((4-(2-(6-(2-(aminooxy)acetate hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxine, 7'C-((4-(2-(2-(2-(aminooxy)acetamido)acetamido)e 7'C-((4-(2-(4- (2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl) methyl)-amatoxin, 7'C-((4-(20-(aminooxy)-4,19-di (6,9,12,15-tetraoxa-3,18-diazaicosyl)piperidine-1 -yl)methyl)-amatoxin, 7'C-(((2-(6-(2-(aminooxy)amatoxin Cetamido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amino Toxin, 7'C-(((4-(6-(2-(aminooxy)acetamido)-N-methyl hexanamido)butyl)(methyl)amino)methyl)-amatoxine, 7'C-(( 3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexane Amido)methyl)pyrrolidin-1-yl)-S-methyl)-amatoxine, 7'C-(( 3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexane Amido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxin, 7'C-(( 4-(2-(2-bromoacetamido)ethyl)piperazin-1-yl)methyl)-aman Toxin, 7'C-((4-(2-(2-bromoacetamido)ethyl)piperidine-1 -yl)methyl)-amatoxin, 7'C-((4-(2-(3-(pyridin-2-yl)methyl)-amatoxin, Disulfanyl)propanamido)ethyl)piperidin-1-yl)methyl)-Amatoki Syn, 6'O-(6-(6-(maleimido)hexanamido)hexyl)-amatoxin , 6'O-(5-(4-((maleimido)methyl)cyclohexanecarboxamide)pentaerythritol 6'O-(2-((6-(maleimido)hexyl)oxy)-2 -oxoethyl)-amatoxine, 6'O-((6-(maleimido)hexyl)carbamo yl)-amatoxine, 6'O-((6-(4-((maleimido)methyl)cyclohexa (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)-amatoxine, 7'C- (4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)- Amatoxin, 7'C-(4-(2-(6-(6-(maleimido)hexanamide)hexadecyl) Sanamido)ethyl)piperazin-1-yl)-amatoxin, 6'O-(6-(6-( 11,12-didehydro-5,6-dihydro-dibenzo[b,f]azocin-5-yl) -6-oxohexanamido)hexyl)-amatoxin, 6'O-(6-(hexa-5 -O-(6-(2-aminooxy)-amino-2-methyl-2-oxo ... Acetylamido)hexyl)-amatoxin, 6'O-((6-aminooxy)hexyl )-amatoxin, and 6'O-(6-(2-iodoacetamido)hexyl)-amatoxin Toxins. The foregoing linkers are useful, among other things, in conjunction with the compositions and methods described herein. and is described, for example, in U.S. Patent Application Publication No. 2015 / 0218220. the entire disclosure of which is incorporated herein by reference.

[0276] Anti-CD5 or CD2 antibodies and their antigen binding, including those described herein The fragment can be conjugated to a cytotoxin, the cytotoxin being an auristatin (US (Patent Nos. 5,635,483 and 5,780,588). Auristatins have the ability to bind to microtubules. It is an antimitotic agent that interferes with the dynamics of ATP, GTP hydrolysis, nuclear and cell division (W oyke et al(2001)Antimicrob.Agents and Ch emother.45(12):3580-3584), anticancer drugs (U.S. Patent No. 5,66 3, No. 149) and antifungal activity (Pettit et al. (1998) Antimi crob. Agents Chemother. 42:2961-2965) (U.S. Pat. (Nos. 5,635,483, 5,780,588). Auristatin Drug Division The peptide drug moiety is attached to the antibody via the N-terminus or C-terminus. (WO 02 / 088172).

[0277] Exemplary auristatin embodiments include an N-terminally linked monomethyl auristatin drug. The compounds contain the compound DE and DF (MMAE and MMAF, respectively), and are used in cancer research. Disclosed in Proceedings, Vol. 45, Abstract No. 623, submitted May 28, 2004 No. 6,239,999, the entire disclosure of which is incorporated herein by reference.

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

[0279] Another exemplary auristatin embodiment is MMAF: [ka] where the wavy lines represent anti- The covalent attachment point of the antibody-linker conjugate to the linker (as described herein) is shown. -LZ-Ab or -L-Z').

[0280] Auristatins are disclosed in U.S. Patent Nos. 5,635,483, 5,780,588, Pe ttit et al (1989) J.Am.Chem.Soc.111:5463-5 Page 465, Pettit et al (1998) Anti-Cancer Drug Design 13:243-277, Pettit, GR, et al. Synt. hesis, 1996, 719-725, Pettit et al (1996) J. Chem.Soc.Perkin Trans.15:859-863, and Doro Nina (2003) Nat.Biotechnol.21(7):778-784 It can be prepared according to the method.

[0281] Maytansinoids The anti-CD5 or CD2 antibodies and antigen-binding fragments thereof described herein inhibit the activity of microtubules. In some embodiments, the binding agent can be a cytotoxin. The binding agent is maytansine, a maytansinoid, or a maytansinoid analog. Tansinoids act by binding to microtubules and inhibiting tubulin polymerization in mitosis Maytansine is a plant inhibitor originally from the East African shrub Maytans serrata. (U.S. Patent No. 3,896,111). Subsequently, certain microorganisms also isolated maytans. It has been discovered that the α-amylinositol esters maytansinol and C-3 maytansinol are produced in the α-amylinositol esters. (U.S. Patent No. 4,151,042) Synthetic maytansinol and its derivatives The compounds and analogs are described, for example, in U.S. Pat. Nos. 4,137,230 and 4,248,870. , No. 4, 256, 746, No. 4, 260, 608, No. 4, 265, 814, No. 4 , 294, 757, 4, 307, 016, 4, 308, 268, 4, 308 , No. 269, No. 4, 309, 428, No. 4, 313, 946, No. 4, 315, 929 No. 4, 317, 821, 4, 322, 348, 4, 331, 598, No. 4, 361, 650, 4, 364, 866, 4, 424, 219, 4, 45 0,254, 4,362,663, and 4,371,533. Maytansinoid drug moieties are produced by (i) fermentation or chemical modification, or by derivatization of the fermentation products; (ii) It can be attached to an antibody via a non-disulfide linker. (iii) be stable in plasma; and (iv) its efficacy against various tumor cell lines. This makes it an attractive drug moiety for conjugates.

[0282] Examples of suitable maytansinoids include esters of maytansinol, synthetic maytansinol, Maytansinol and analogs and derivatives thereof are included herein. Maytansinoids, maytansinol, maytansinol analogues and derivatives , any cytotoxin that inhibits microtubule formation and is highly toxic to mammalian cells.

[0283] Examples of suitable maytansinol esters include those with modified aromatic rings and others. Such suitable maytansinoids include those having modifications at positions 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 39, 38, 39, 40, 41, 42, 43, License No. 4, 137, 230, 4, 151, 042, 4, 248, 870, 4, 256, 746, 4, 260, 608, 4, 265, 814, 4, 294, No. 757, No. 4, No. 307, No. 016, No. 4, 308, No. 268, No. 4, 308, 269 , No. 4, 309, 428, No. 4, 313, 946, No. 4, 315, 929, No. 4 , 317, 821, 4, 322, 348, 4, 331, 598, 4, 361 , No. 650, No. 4, No. 362, No. 663, No. 4, 364, No. 866, No. 4, 424, 219 No. 4, 450, 254, 4, 322, 348, 4, 362, 663, No. No. 4, 371, 533, No. 5, 208, 020, No. 5, 416, 064, No. 5, 47 5, 092, 5, 585, 499, 5, 846, 545, 6, 333, 41 0, 7,276,497, and 7,473,796, each of which The disclosures herein are incorporated by reference as they relate to maytansinoids and their derivatives. do.

[0284] In some embodiments, the antibody-drug conjugates (ADCs) of the present disclosure are As a toxic agent, officially known as N 2 '-Deacetyl-N 2 '-(3-mercapto-1-oxopeptide It utilizes a thiol-containing maytansinoid called (DM1)-maytansine. DM1 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)-mei A cytotoxic agent is a dansin (e.g., DM4), which has the following structural formula VII: Represented: [ka]

[0286] Another maytansinoid with a side chain containing a sterically hindered thiol bond has the structure N represented by formula VIII 2 '-Deacetyl-N 2 '(4-mercapto-1-oxopentane Chil) Meitanshi (also called D3). [ka]

[0287] Each of the methods taught in U.S. Pat. Nos. 5,208,020 and 7,276,497 Itansinoids may also be used in the conjugates of the present disclosure. , U.S. Patent Nos. 5,208,020 and 7,276,497, the entire disclosures of which are incorporated herein. , incorporated by reference.

[0288] Many positions on the maytansinoid are suitable for binding to the linking moiety and, therefore, the antibody or its antigen-binding fragment. and a position to covalently attach a segment (-LZ-Ab or -L-Z' as described herein) to the For example, the C-3 position, which has a hydroxyl group, C-14 modified with hydroxyl, C-15 modified with hydroxyl, and All C-20 positions are expected to be useful. Position 3 serves as a point for covalently attaching a linker moiety, and in some particular embodiments The C-3 position of maytansinol serves as the site for covalent attachment of the linking moiety. -many linking groups known in the art for making maytansinoid conjugates For example, U.S. Pat. Nos. 5,208,020 and 6,441,163, and European Pat. No. 0425235 B1, Chari et al., Cancer Research h 52:127-131 (1992), USA US2005 / 0169933 A1, which are expressly incorporated herein by reference. The groups are described and exemplified herein.

[0289] The present invention also relates to various isomers and mixtures of maytansinoids and conjugates. Certain compounds and conjugates of the present invention may be present in various stereoisomers, enantiomers, Such antibody-maytansinoid complexes may exist in stereoisomeric and diastereomeric forms. Descriptions of how to prepare conjugates are found in U.S. Pat. Nos. 5,208,020 and 5,416,796. , No. 064, No. 6, 333, 410, No. 6, 441, 163, No. 6, 716, 821 Nos. 7,368,565, each of which is incorporated herein by reference in its entirety. To quote.

[0290] Anthracyclines In other embodiments, the anti-CD5 or CD2 antibodies and their antigen-binding fragments described herein The anthracycline fragment can be attached to a cytotoxin that is an anthracycline molecule. Cyclins are antibiotic compounds that exhibit cytotoxic activity. Phosphorus is a compound that acts as a nucleotide analogue of 1) DNA-dependent nucleic acid receptors by intercalating drug molecules into cellular DNA. 1) inhibiting the synthesis of free radicals, which then react with cellular macromolecules and cause cell damage. or 3) the interaction of drug molecules with cell membranes (e.g., C. Peter son et al., "Transport and Storage of Anthracyclines in Experimental Systems and Human Leukemia." , Anthracycline Antibodies in Cancer Therapy, N. RBachur, "Free Radicals damage,” pp. 97–102). It has been shown that it can act to kill cancer. Lacycline is effective against many cancers, including leukemia, breast cancer (carcinoma), lung cancer, and egg It is used to treat focal adenocarcinomas and sarcomas (e.g., anthracyclines P. H. Wie rnik, in Experimental Systems and Human l See Eukemia: Current Status and New Developments p11. Clinicals include doxorubicin, epirubicin, idarubicin, and daunomycin In some embodiments, the cytotoxin is daunorubicin, doxorubicin, epilepsy, or The anthracycline is selected from the group consisting of bicine, and idarubicin. A typical example of tracycline is daunorubicin (Cerubicin, Bedford Research Institute) Bedford Research Institute), doxorubicin (ADRIAMYCIN®, Bedford Research Institute, Also known as doxorubicin hydrochloride, hydroxydaunorubicin, and RUBEX® (Epilubicin), epirubicin (ELLENCE®, Pfizer), and idarubicin These include, but are not limited to, Idamaycin (Pfizer Inc.) It will not be done.

[0291] Doxorubicin (ADRIAMYCIN®), an anthracycline analog ) is the intercalation of the enzyme topoisomerase II, which unwinds DNA for transcription. Doxorubicin is thought to interact with DNA by inhibiting progression. It stabilizes the topoisomerase II complex after DNA strand cleavage and prevents DNA duplex formation. It prevents the strands from recombining, thereby halting the replication process. Daunorubicin and daunorubicin are the prototype anthracycline chemotherapeutic agents. It is a cytotoxic natural product (Sessa et al., (2007) Cardiovasc). Sychor. 7:75-79).

[0292] One non-limiting example of an anthracycline suitable for use herein is PNU-15 9682 ("PNU"). The PNU is over 3000 times higher than that of the parent nemorubicin. (Quintieri et al., Clinical Cancer Research 2005, 11, 1 608-1617). PNU has the structural formula: [ka] is expressed by

[0293] Many positions on anthracyclines, such as PNU, are designated as linking moieties, and thus, a site for covalently binding an anti-CD45 antibody or antigen-binding fragment thereof as described in For example, the linker can be attached via a modification to a hydroxymethyl ketone side chain. It can be introduced as follows.

[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 (e.g., bispecific (including isotopic and biparatopic antibodies) may be used in combination with other antibodies, such as PBDs and IGNs, as described herein. , can be conjugated to a cytotoxin that contains a benzodiazepine moiety. Pyrrolobenzodiazepines (PBDs) A PBD has the general structure: [ka]

[0297] They are found in both their aromatic ("A") and pyrrolo ("C") rings. , and the degree of saturation of the C ring differs in the number, type, and position of substituents. The diazepine B ring contains imine (N=C) and carbinolamine (NH) at the N10-C11 positions. -CH(OH)), or carbinolamine metal ether (NH-CH(OMe)) This position is the ionizable group responsible for DNA alkylation. Known natural product PB All of D have the (S) configuration at the chiral C11a position, which is directed from the C ring to the A ring. This gives them a right-handed twist when viewed from the side. This is in line with the helicity of the minor groove of B-form DNA. It provides a suitable three-dimensional shape and fits snugly into the binding site (Kohn, In Antibiotic s III. Springer-Verlag, New York, pp.3-11 ( 1975), Hurley and Needham-VanDevanter, Acc. ) Chemistry Institute, 19, 230-237 (1986) PBD forms an adduct in the minor groove This ability to disrupt DNA processing allows them to have anti-cancer activity. vinegar.

[0298] The biological activity of these molecules is mediated by the connection of two PBs via a flexible alkylene phosphorus linking device. It has been previously disclosed that this is possible by combining D units (Bose, DS, et al., J.Am.Chem.Soc., 114, 4939-4941 (1992), Thurston, DE, et al., J.Org.Chem ., 61, 8141-8147 (1996)). The PBD dimer is a palindrome 5'- Pu-GATC-Py-3' interstrand cross-link (Smellie, M., et al., Biochemistry, 42, 8232-8239(2003), Martin, C., et al., Biochemistry, 44, 4135-4147) They are thought to form sequence-selective DNA lesions, which are primarily responsible for their biological activity. It is thought that Gregson et al. (Chem.Commun.1999, 7 97-798, "Complex 1" and Gregson et al. (J. Med.Ch em.2001, 44, 1161-1174, "Complex 4a") is a favorable dimeric pyrrolo This complex is also known as SG2000. The structural formula is as follows: [ka]

[0299] In general, modifications to pyrrolidine alkenes provide handles for homovalent attachment of linkage moieties. and therefore, their antigen-binding fragments (-L-Z' and -LZ) -Ab, as described herein). Alternatively, a linker at position N10 can also be attached.

[0300] 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. The compound of this formula where n is 3 is 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 SJG- 136 (Gregson et al., J. Med. Chem. 2 001, 44, 737-748). When n is 5, the compound of this formula is DRG-16. (Gregson et. Al., Med. Chem. 2004, 47 :1161-1174).

[0302] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine having the formula: It is a dimer. [ka] where the wavy line indicates the point of covalent attachment of the ADC described herein to the linker. PBD-based ADCs have been described, for example, in Sutherland et al., Blood 2 013 122:1455-1463, the entire disclosure of which is incorporated herein by reference. To quote.

[0303] In some embodiments, the cytotoxin is a PBD having the formula: [ka] where n is 3 or 5 and the wavy line represents the covalent bond to the linker of the ADC described herein. This shows the consensus.

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

[0305] Indolinobenzodiazepines (IGNs) In some embodiments, the antibodies that bind CD45 or antigens thereof described herein The binding fragment may be an indolinobenzodiazepine ("IGN") or an IGN-containing molecule. In some embodiments, the IGN cytotoxin can be conjugated to an IGN cytotoxin. The compound is an indolinobenzodiazepine dimer or an indolinobenzodiazepine pseudodimer. be.

[0306] Indolinobenzodiazepine dimers have high in vitro efficacy against cancer cells ( Low pM range IC 50 (value) is a relatively new chemical class of cytotoxins. Similar to the target molecule SJG-136, the IGN dimer binds to the minor groove of DNA and is contained within the dimer. It covalently binds to guanine residues via two imine functional groups and crosslinks DNA. The structure (IGN6, where the methylene group in the PBD moiety is replaced with a phenyl ring) is compared with SJG-136. It showed approximately 10 times the potency in vivo, due to the increased rate of adduct formation with DNA IGN. This is thought to be due to the speed (e.g., Miller et al., "A New Class s of Antibody-Drug Conjugates with Poten t DNA Alkylating Activity”Mol.Cancer The r.2016, 15(8), 1870-1878). On the other hand, the IGN pseudo-second The dimer consists of a single reactive indolinobenzodiazepine imine and is present in the dimeric cytotoxin The second indolinobenzodiazepine exists in the reduced (amine) form. Thus, I The GN pseudodimer alkylates DNA via a single imine site in the dimer, resulting in D It does not cross-link NA.

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

[0308] In some embodiments, the reactive substituent Z' prior to conjugation to the antibody is The cytotoxin-linker conjugates are summarized as Cy-L-Z' and have the following structure: It has. [ka]

[0309] This cytotoxin-linker conjugate is referred to herein as DGN549, ADC IMGN632, both of which are described, for example, in WO 201700 No. 4026, which is incorporated herein by reference.

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

[0311] In some embodiments, prior to conjugation to the antibody, the compound comprises the chemical moiety Z: The cytotoxin-linker conjugate 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 the ADC IMGN779, e.g. As disclosed in U.S. Patent Application Publication No. 20170080102, previously incorporated by reference herein. It has been done.

[0312] Kalicare sewing machine In other embodiments, the antibodies and antigen-binding fragments thereof described herein The compound is an enediyne antitumor compound (e.g., calicheamicin, ozogamicin). The calicheamicin family of antibiotics has sub-picomolar activity. It is possible to cause cleavage of the non-stranded DNA at high concentrations. See U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,1 No. 16, No. 5, 767, 285, No. 5, 770, 701, No. 5, 770, 710, Nos. 5,773,001 and 5,877,296 (all American Cy See the Calicheamicin Company. Analogs include, for example, Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer R Research 58:2925-2928 (1998) and the American These include, but are not limited to, those disclosed in U.S. Patent No. 6,229,623. do not have.

[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 N-acetyl gamma-calicheamicin derivatives. Structural analogs include those described, for example, in Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer R Research 58:2925-2928 (1998) and the above-mentioned U.S. patents. Calicheamicin can be reacted with the appropriate thiol. and at the same time, comprising a methyl trisulfide moiety capable of forming a disulfide in response to The calicheamicin derivative is attached to a bispecific binding agent, such as those described herein, via a linker. The functional groups are useful for conjugating to calicheamicin family conjugates. For the preparation of 739, 116, 5, 767, 285, 5, 770, 701, 5, 770, Nos. 710, 5, 773, 001, and 5, 877, 296 (all American See the Cyanamid Company. Structural analogs of thiamin include those described, for example, in Hinman et al., Cancer Research rch 53:3336-3342 (1993), Lode et al., Cance r Research 58:2925-2928 (1998) and the American These and other compounds are disclosed in U.S. patents including, but not limited to, those disclosed in U.S. patents for Can Cyanamid. It will not be done.

[0315] In some embodiments, the cytotoxin of the ADCs disclosed herein is represented by the formula: It is a calicheamicin disulfide derivative that is used as a steroid. [ka] In the formula, the wavy line indicates the point of attachment of the linker.

[0316] Directly treat cancer, autoimmune conditions, or prepare for hematopoietic stem cell transplantation therapy Recognizing and using CD2 or CD5 for use in conditioning a patient (e.g., a human patient). and additional cytotoxins that can be conjugated to antibodies and antigen-binding fragments thereof. include, but are not limited to: 5-ethynyluracil, abirate Ron, acylfulvene, adecipenol, adzelesin, aldesleukin, altreta Min, Ambamustine, Amidox, Amifostine, Aminolevulinic Acid, Amruvisine methicone, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis antagonists Anti-, Antarelix, Anti-thoracic vertebrate morphogenetic protein-1, Anti-androgen, Prostate cancer, Anti Estrogen, anti-neoplaston, antisense oligonucleotide, aphidicolin Glycinate, apoptotic gene regulator, apoptotic inhibitor, apurinic acid, ara-C DP-DL-PTBA, arginine deaminase, asulaculin, atamestane, atoli Mustine, Axinastatin 1, Axinastatin 2, Axinastatin 3, Azacetro Azatoxin, azatyrosine, bacactin III derivatives, balanol, batimastat , BCR / ABL antagonists, benzochlorins, benzoylstaurosporines, beta-lactams derivatives, beta-arretin, beta-clomicin B, betulinic acid, bFGF inhibitors, bica Lutamide, Bisantrene, Bisatirizinylsulfoermine, Bisnafide, Bistraten A, bizelesin, bleflate, bleomycin A2, bleomycin B2, bropiromi Buthionine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives (e.g., 10-hydroxy-camptothecin), capecitabine, Ruboxamido-amino-triazole, carboxyamidotriazole, carzelesin, Casein kinase inhibitors, castanospermine, cecropin B, cetrorelix, cloprid Phosphorus, chloroquinoxaline sulphonamide, cicaprost, cis-porphyrin, cladoxane Livin, clomiphene analogues, clotrimazole, colismycin A, colismycin Combrestatin B, combrestatin A4, combrestatin analogues, conagenin, clambecid 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentaanthraquinone, cycloplatam, sipemycin, cytarabine ocfos Fate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidem Nin B, 2'-deoxycoformycin (DCF), deslorelin, dexphosphamide , dexrazoxane, dexverapamil, diatiquone, didemnin B, didox, Diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxa mycin, diphenylspiromustine, discodermolide, docosanol, dolastero doxifluridine, droloxifene, dronabinol, doucarmycin SA, Ebselen, Ecomustine, Edelfosine, Edrecolomab, Eflornithine, Elemene , Emitefur, Epothilone, Epithilon, Epristeride, Estramustine-related compounds Etoposide, etoposide 4'-phosphate (also known as etopophos), exemestane , fadrozole, fazarabine, fenretinide, filgrastim, finasteride , flavopiridol, flezelastine, fluasterone, fludarabine, fluoroda hydrochloride Unolnisin, Forfenimex, Formestane, Fostriecin, Fortemus tin, gadolinium texaphyrin, gallium nitrate, gallocitabine, ganirelix, di Ferratinase inhibitors, gemcitabine, glutathione inhibitors, hepzulfam, homohalin Tonin (HHT), hypericin, ibandronic acid, idarubicin, idoxifene, id Lamantone, Ilmofosine, Ilomastat, Imidazoacridone, Imiquimod, Immuno Stimulatory peptides, iobenguane, iododoxorubicin, ipomeanol, irinotecan , Ilopract, Irsogladine, Isobengazole, Jasplakinolide, Kahalalide F, Lamellarin N Triacetate, Lanreotide, Leinamycin, Lenogucrastin, Lentinan sulfate, leptolstatin, letrozole, lipophilic platinum compounds, lysocrinami Do7, lobaplatin, lometrexol, lonidamine, losoxantrone, loxoribine , lurtotecan, lutetium texaphyrin, lisofylline, natansin, mazoproko ol, maspin, matrix metalloproteinase inhibitors, menogaril, merbaron , meterelin, methioninase, metoclopramide, mifepristone, miltefosine , millimostin, mithracin, mitoguazone, mitolactol, mitomycin analogues substances, mitonafide, mitoxantrone, mofalotene, mofgramostim, micapero Oxide B, Myriaporone, N-acetyldynaline, N-substituted benzamide, Nafarelli Nagressip, Navavin, Nafterpin, Nartograstim, Nedaplatin, Ne Morubicin, neridronic acid, neutral endopeptidase, nilutamiud, octreotide, Oxenon, onapristone, ondansetron, oracin, ormaplatin, oxaliplatin Platin, oxaunomycin, paclitaxel analogues, palaumin, palmitoyl Rhizoxin, pamidronate, panaxytriol, panomyphen, parabactin, pazelle Putin, Pegaspargase, Perdecin, Sodium Pentosan Polysulfate, Pen Intostatin, pentrozole, perflubron, periosufamide, phenazinomycin Picibanil, pirarubicin, piriterexim, podophyllotoxin, porphyromonas Isin, purine nucleoside phosphorylase inhibitors, raltitrexed, rhizoxin, Gretimide, Rohitukine, Rubiginone B1, Ruboxil, Safingol, Saitopine , Sarcophytol A, Sargramostim, Sobuzoxane, Sonermin, Spahisinic acid, Spicamycin D, spiromustine, stipiramide, sulfinosine, talimustine, te Gafoor, temozolomide, teniposide, saliblastine, thiocoraline, tirapazamine, Topotecan, topsentin, triciribine, trimetrexate, veramine, vinorelbine vinxartine, vorozole, zeniplatin and zilascorub.

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

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

[0319] As used herein, the term "linker" refers to an anti-CD5 or CD2 antibody or its derivatives. The fragment (Ab) is covalently attached to the drug moiety (D) to form the antibody-drug conjugate of the present disclosure. Covalent bond or chain of atoms forming a conjugate (ADC, Ab-ZLD, D is a cytotoxin) A suitable linker has two reactive ends, one of which is an anti- one for conjugation to the body and the other for conjugation to a cytotoxin. The reactive end of the linker for antibody conjugation (reactive moiety, Z) is typically a Sites available for conjugation to antibodies via cysteine ​​thiol or lysine amine groups and usually contains a double bond (e.g., maleimide) or a leaving group (e.g., chloro, bromo, iodo, R -sulfanyl groups), or carboxyl groups On the other hand, the reactive end of the linker for antibody conjugation is an amine-reactive group such as a methyl group. The end is usually attached via amide bond formation with a basic amine or carboxyl group on the cytotoxin. A site available for conjugation to a cytotoxin, typically a carboxyl or base group. The term "linker" describes the conjugated form of the linker. When used in describing the linker and / or cytotoxin, and / or reactive fragments for the formation of bonds between antibodies or antigen-binding fragments thereof. One or both ends may be missing (e.g., reactive moiety Z converted to chemical moiety Z) or incomplete (e.g., Such conjugation reactions are described in the present invention. Further details are provided below in the specification.

[0320] A variety of linkers can be used to link the antibodies or antibody fragments described herein to cytotoxic In some embodiments, the linker can be cleaved. The linker is designed to be capable of releasing the drug unit from the antibody under intracellular conditions by In yet other embodiments, the linker unit is not cleavable and is The linker useful in the present ADCs is preferably These compounds are stable outside the cell, prevent aggregation of ADC molecules, and allow the ADC to be easily absorbed into aqueous media and monomers. Prior to transport or delivery to cells, the ADC is preferably stable and remains freely soluble in water. and remains intact, i.e., the antibody remains linked to the drug moiety. An effective linker is stable outside the target cell and can be cleaved at an effective rate inside the cell. (i) maintain the specific binding properties of the antibody, and (ii) maintain the integrity of the conjugate or drug moiety. (iii) allowing the conjugate to be delivered or transported to its target site (iv) the cytotoxic moiety remains stable and intact (i.e., not cleaved) until maintains cytotoxic, cytocidal or cytostatic effects. The stability of the ADC is determined by the mass Analysis, measured by standard analytical techniques such as HPLC and separation / analysis techniques LC / MS Covalent attachment of an antibody to a drug moiety can be achieved by providing a linker having two reactive functional groups, In other words, it is necessary to have bivalency in terms of reactivity. Two or more functional or biologically active moieties such as nucleotides, haptens, and reporter groups Bivalent linker reagents useful for linking the resulting conjugates are known, and methods are known to those skilled in the art. Gates are described (Hermanson, GT (1996) Bioc Onjugate Techniques, Academic Press: New York , pp. 234-242).

[0321] The linker may be, for example, hydrolyzed by enzymes, photolysis, hydrolysis under acidic conditions, or hydrolysis under basic conditions. Cleavage by hydrolysis, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage under Examples include those that can be used for the detection of HIV-1-associated ... d. Chem., 20:571-582, 2012, the disclosure of which is based on covalent bonds. (which are incorporated herein by reference as they relate to linkers suitable for conjugation) .

[0322] Linkers that are hydrolyzable under acidic conditions include, for example, hydrazones, semicarbazones, Thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketters For example, U.S. Patent No. 5,122,368, U.S. Patent No. 5,824, No. 805, U.S. Patent No. 5,622,929, Dubowchik and Walker , 1999, Pharm.Therapeutics 83:67-123, Nev ille et al., 1989, Biol.Chem.264:14653-146 See page 61. Each of these disclosures provides linkers suitable for covalent conjugation. For purposes of relevance, the entire contents of which are incorporated herein by reference. It is relatively stable under neutral pH conditions, such as in the lysosomal pH range. It is unstable at pH 5.5 or below 5.0.

[0323] Linkers that are cleavable under reducing conditions include, for example, disulfides. ATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-S-acetylthioacetate) N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyl Benzyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)toluene Various disulfides, including those that can be formed using SMPT, SPDB, and SMPT Linkers are known in the art, see, e.g., Thorpe et al., 1987 Cancer Res. 47:5924-5931, Wawrzynczak e t al., In Immunoconjugates: Antibody Conju gates in Radioimagery and Therapy of Cancer (See C.W. Vogel, ed., Oxford University Press, 1987. Also See also U.S. Pat. No. 4,880,935, each of which discloses covalent conjugates. and the like, which is incorporated herein by reference in its entirety as it relates to linkers suitable for fusion.

[0324] Examples of linkers useful in the synthesis of drug-antibody conjugates include, among others, Michael acceptors. derivatives (e.g., maleimides), activated esters, electron-deficient carbonyl compounds, and aldehydes. Amine or thiol sites present in an antibody or antigen-binding fragment, such as a hydroxyl group These include those containing electrophiles suitable for reaction with nucleophilic substituents such as drug- Suitable linkers for the synthesis of antibody conjugates include, but are not limited to, 4- (N-Maleimidomethyl)-cyclohexane-L-carboxylate succinimidyl (SMC C), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleic acid Imidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MB S, and succinimidyl iodoacetate, e.g., Liu et al. 18:690-697, 1979, the disclosure of which is incorporated herein by reference. The present invention relates to gating linkers and is hereby incorporated by reference. These include undisclosed ligands that are particularly useful for conjugating microtubule-disrupting agents such as auristatins. Examples of the cleavable maleimidocaproyl linker include Doronina et al., Bio conjugate Chem. 17:14-24, 2006, The disclosure of which relates to linkers for chemical conjugation is incorporated herein by reference. Additional linkers suitable for the synthesis of the drug-antibody conjugates described herein are Some of these are capable of releasing cytotoxins by a 1,6-elimination process ("self-immolative"). " group), for example, p-aminobenzyl alcohol (PABC), 6-maleimidohexane Acid, pH-sensitive carbonate, and Jain et al., Pharm.Res.32:35 26-3540, 2015, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the linker is a PAB or or PABC (paraaminobenzyloxycarbonyl), which contain self-immolative groups. See, e.g., Carl et al., J. Med. Chem. (1981) 24:479 -480 pages, Chakravarty etal (1983) J.Med.Chem. 26:638-644, U.S. Patent No. 6,214,345, U.S. Patent Application Publication No. 20030 No. 130189, U.S. Patent Application Publication No. 20030096743, U.S. Patent No. 67595 No. 09, 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. 200 40018194, WO 98 / 13059, U.S. Patent Application Publication No. 200400 No. 52793, U.S. Patent No. 6677435, U.S. Patent No. 5621002, U.S. Patent No. Disclosed in Patent Publication No. 20040121940 and International Publication No. 2004 / 032828 Other such chemical moieties ("self-immolative linkers") that are capable of this process include , methylene carbamate and aminothiazole, aminoimidazole, aminopyrimidine, etc. Linkers containing such heterocyclic self-immolative groups include, for example, See, for example, U.S. Patent Publication Nos. 20160303254 and 20150079114, and U.S. Patent No. 7,754,681, Hay et al. (1999) Biomaterials g. Med. Chem. Lett. 9:2237 pages, US2005 / 0256030 , de Groot et al (2001) J.Org.Chem.66:8815- 8830, and in US Pat. No. 7,223,837.

[0325] Linkers that are susceptible to enzymatic hydrolysis include, for example, those that are hydrolyzed by lysosomal or endosomal proteins. by intracellular peptidase or protease enzymes, including but not limited to ATPases The linker may be a peptide-containing linker that is cleaved by intracellular proteolytic release of the therapeutic agent. One advantage of using conjugated drugs is that they are generally attenuated and conjugated. In some embodiments, the serum stability of the peptidyl ligase is generally high. The anchor is at least two amino acids in length or at least three amino acids in length. The amino acid linker may comprise a dipeptide, tripeptide, tetrapeptide, or pentapeptide. Examples of suitable peptides include valine, alanine, citrulline (Cit), phenylalanine, and the like. These include those containing amino acids such as riboflavin, lysine, leucine, and glycine. The amino acid residues comprising the acid linker moiety include naturally occurring as well as minor amino acids. Exemplary dipeptides include: Valine-citrulline (vc or val-cit) and alanine-phenylalanine ( Exemplary tripeptides include glycine-valine. -Citrulline (gly-val-cit) and glycine-glycine-glycine (gly In some embodiments, the linker includes Val-Cit ,Ala-Val,Phe-Lys,Val-Lys,Ala-Lys,Phe-Cit , dipeptides such as Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit Linkers containing dipeptides such as Val-Cit or Phe-Lys For example, U.S. Pat. No. 6,214,345 discloses covalent bonds. As they relate to linkers suitable for conjugation, they are incorporated herein by reference in their entirety. In some embodiments, the linker is Val-Ala and Val-C. In some embodiments, the dipeptide is a self-immolative dipeptide selected from the group consisting of: Used in combination with a linker.

[0326] Suitable for conjugating the antibodies or antibody fragments described herein with cytotoxic molecules The selected linkers are capable of releasing cytotoxins by a 1,6-elimination process. Chemical moieties that enable this release process include p-aminobenzyl (PAB) groups. , 6-maleimidohexanoic acid, pH-sensitive carbonate, and Jain et al., Pha Other reagents described in R.M. Res. 32:3526-3540, 2015 are also included. , the entire disclosure of which is incorporated herein by reference, particularly as it relates to linkers suitable for covalent attachment. To quote.

[0327] In some embodiments, the linker is a PAB or PABC (para-amino acid) benzyloxycarbonyl), which are described, for example, in Carl et al. al., J. Med. Chem. (1981) 24:479-480, Chakra varty 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 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 No. 6,268,488, U.S. Patent Application Publication No. 20040018194, International Publication No. 98 / 13059, U.S. Patent Application Publication No. 20040052793, U.S. Pat. No. 6,677,435, U.S. Patent No. 5,621,002, U.S. Patent Application Publication No. 20040121 This process is disclosed in International Publication No. 2004 / 032828. Other such chemical moieties ("self-immolative linkers") that can be used include methylene carbamates and Heteroaryl groups such as aminothiazole, aminoimidazole, and aminopyrimidine Linkers containing such heterocyclic self-immolative groups are described, for example, in U.S. Pat. Nos. 160303254 and 20150079114, and U.S. Pat. 4, No. 681, Hay et al. (1999) Bioorg.Med.Chem. Lett. 9:2237 pages, US2005 / 0256030, de Groot et a l (2001) J. Org. Chem. 66:8815-8830, and U.S. Pat. In some embodiments, the dipeptide is self-immolating. It is used in combination with a hydroxyl linker.

[0328] Suitable linkers may contain groups with solubility enhancing properties, for example: (CH2CH2O) p Linkers containing units (polyethylene glycol, PEG) are soluble The activity of the amino, sulfonic acid, phosphonic acid or phosphate residue-substituted amino acids can be increased. The same applies to alkyl chains. Linkers containing such moieties are described, for example, in U.S. Pat. , 319 and 9,504,756, which disclose covalent conjugates. The entire contents of which are incorporated herein by reference as they relate to suitable linkers. Examples of the solubility enhancing group include acyl and carbamoyl sulfonates having the following structures: An example is an amide group. [ka] In the formula, a is 0 or 1; R 10 is hydrogen, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C1-C2 4 (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 group , C2-C 24 (Hetero)aryl groups, C3-C 24 Alkyl (hetero)aryl groups and and C3-C 24(hetero)arylalkyl groups, each of which is selected from the group consisting of O, S, NR 11 R 12 and / or may be optionally interrupted, R 11 and R 12 is hydrogen and C1-C4 alkyl or R 10 is a cytotoxin, The linker may be, for example, See, for example, U.S. Patent No. 9,636,421 and U.S. Patent Application Publication No. 2017 / 029814 No. 5, and these disclosures relate to the use of cytotoxins and antibodies or their antigen-binding fragments. and the like, which are incorporated herein by reference in their entirety as they relate to linkers suitable for covalent attachment to fragments. This is incorporated by reference as follows.

[0329] In some embodiments, the linker is a hydrazine, a disulfide, a thioether, Dipeptide, p-aminobenzyl (PAB) group, heterocyclic autoimmune group, optionally substituted C 1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2 -C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C 2-C6 alkynyl, optionally substituted C2-C6 heteroalkynyl, optionally substituted C3-C6 cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, solubility enhancing groups, acyl, -(C=O)-, or -(CH2CH2O) p - groups (wherein p is an integer from 1 to 6). Those skilled in the art will appreciate that one or more of the listed groups may be divalent (diradical) species (e.g., It will be recognized that the alkylene may exist in the form of alkylene, 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 1 3 -, -(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)X 4, - (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 NR 13 (CH2) m -、-(CH2) m NR 13 C(=O)(CH2) m X3(CH2) m -、-(CH2) m C(=O)NR 13 (CH2) m NR 13 C(= O)(CH2) m -、-(CH2) m C(=O)-、-(CH2) m NR 13 (CH2) m C(=O)X2X1C(=O)-、-(CH2) m X3(CH2) m C(=O)X2X 1C(=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 N R 13 C(=O)(CH2) m -、-(CH2) m X3(CH2) m C(=O)NR 13 (CH2) m -、-(CH2) m O) n (CH2) m NR 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 NR 13 (CH2) m C(=O )-、-(CH2) m C(=O)NR 13 (CH2) m NR 13 C(=O)-、-(CH 2) 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 NR 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)NR 13 (CH2) m C(=O )-、-(CH2) m C(=O)NR 13 (CH2)m (O(CH2) m ) n C(=O) -、-((CH2) m O) n (CH2) m NR 13 C(=O)(CH2) m -、-(CH 2) m C(=O)NR 13 (CH2) m C(=O)NR 13 (CH2) m -、-(CH2 ) m NR 13 C(=O)(CH2) m NR 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 NR 13 -、-(CH2) m C(= O)NR 13 (CH2) m NR 13 C(=O)NR 13 -、-(CH2) m C(=O)X 2X1C(=O)-、-C(R 13 )2(CH2) m NR 13 C(=O)(CH2) m - 、-(CH2) m C(=O)NR 13 (CH2) m C(R13 )2NR 13 -、-C(R 13 )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 -、-(CH 2) 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 (CH 2) 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(CH 2) 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 O) n (CH2)m -, -(CH2) m (O(CH2) m ) n C(=O)NR 13 (CH2) m -, -(CH 2) 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 from H and C1-C6 alkyl and selected, m is independently 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 for each case and selected, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 is selected independently for each case, where a single asterisk (*) indicates the point of attachment to a cytotoxin (e.g., amatoxin). and the double asterisk (**) indicates the point of attachment to the reactive substituent Z' or chemical moiety Z. However, neither L1 nor L2 exists.

[0331] In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the p-aminobenzyl group is a cytotoxic drug and a proton in the linker. In one embodiment, the p-aminobenzyl group is located between the protease cleavage site. , is part of a p-aminobenzyloxycarbonyl unit. , the p-aminobenzyl group is part of a p-aminobenzylamide unit.

[0332] In some embodiments, the linker is 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, A Contains la-Ala-Asn-PAB or Ala-PAB.

[0333] In some embodiments, the linker is a peptide, an oligosaccharide, -(CH2)p-, -(CH2CH2O)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-A sn-PAB, or Ala-PAB.

[0334] In some embodiments, the linker comprises a -(C=O)(CH)- unit, Here, 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 reactive moiety Z'. The anchor comprises the following 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- It is para-aminobenzyl (mc-vc-PAB).

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

[0339] In some embodiments, the linker is MCC (4-[N-maleimidomethyl]cyclohexyl Contains hexane-1-carboxylate).

[0340] Any one or more of the chemical groups, moieties and features disclosed herein may be used herein. to form linkers useful for conjugating antibodies and cytotoxins, such as Those skilled in the art will recognize that the various aspects of the invention may be combined in a variety of ways. Additional linkers useful in conjunction with the compositions and methods described in, for example, U.S. Pat. Publication No. 2015 / 0218220, the entire disclosure of which is incorporated herein by reference. Use.

[0341] Linkers suitable for use herein include C1-C6 alkylene, C1-C6 hetero Alkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, Alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocyclo selected from alkylene, arylene, heteroarylene, and combinations thereof It may further comprise one or more groups, each of which may be optionally substituted. Non-limiting examples include (CH2) n , (CH2CH2O) n , and -(C=O)(CH2) n - units, where n is an integer from 1 to 6, and It is selected.

[0342] In some embodiments, the linker is a hydrazine, a disulfide, a thioether , dipeptide, p-aminobenzyl (PAB) group, heterocyclic self-immolative group, optionally substituted C 1-C6 alkyl, optionally substituted C1-C6 heteroalkyl, optionally substituted C2 -C6 alkenyl, optionally substituted C2-C6 heteroalkenyl, optionally substituted C 2-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 -(CH 2CH2O) n -group, where n is an integer from 1 to 6. If one or more of the listed groups is a divalent (diradical) species, e.g., C1-C6 alkyl, It will be appreciated that the compound may exist in various forms such as chiral and cyclohexyl groups.

[0343] In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In one embodiment, the p-aminobenzyl group is a cytotoxic drug and a protease in the linker. In one embodiment, the p-aminobenzyl group is located between the p-aminobenzyl group and the enzyme cleavage site. In one embodiment, the p-aminobenzyl group is part of a benzyloxycarbonyl unit. , which is part of the p-aminobenzylamide unit.

[0344] In some embodiments, the linker is 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, Al Contains a-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-As Contains one or more combinations of n-PAB or Ala-PAB.

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

[0347] In some embodiments, the linker is -(CH) n -units, where n is An integer between 2 and 6.

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

[0349] Used to conjugate antibodies or antigen-binding fragments thereof to cytotoxic agents. Possible linkers include a cytotoxic agent covalently attached at one end of the linker and a cytotoxic agent at the other end of the linker. and a reactive substituent present on the linker and an antibody or antibody thereof that binds to CD2 or CD5. The antigen-binding fragment of the present invention is formed from a coupling reaction between the reactive substituents present in the antigen-binding fragment of the present invention and the Examples include antibodies or other antibodies that bind to CD2 or CD5 and contain chemical moieties that bind to CD2 or CD5. Reactive substituents that may be present in an antigen-binding fragment of include, but are not limited to: Non-limiting examples include the hydroxyl moieties of serine, threonine, and tyrosine residues, lysine residues the amino moiety of the hydroxyl group, the carboxyl moiety of aspartic acid and glutamic acid residues, and The thiol moiety of the stein residue, as well as propargyl, azido, haloaryl (e.g., fluoroaryl), haloheteroaryl (e.g., fluoroheteroaryl), halo and haloheteroalkyl moieties of non-naturally occurring amino acids.

[0350] Examples of linkers useful in the synthesis of drug-antibody conjugates include, among others, Michael acceptors. derivatives (e.g., maleimides), activated esters, electron-deficient carbonyl compounds, and aldehydes. Amine or thiol moieties present in antibodies or antigen-binding fragments, such as hydroxyl groups These include those containing electrophiles suitable for reaction with nucleophilic substituents such as drug- Suitable linkers for the synthesis of antibody conjugates include, but are not limited to, 4- (N-Maleimidomethyl)-cyclohexane-L-carboxylate succinimidyl (SMC C), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleic acid Imidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MB S, and succinimidyl iodoacetate, e.g., Liu et al. 18:690-697, 1979, the disclosure of which is incorporated herein by reference. The present invention relates to linkers for fusion, and is hereby incorporated by reference. These include undisclosed ligands that are particularly useful for conjugating microtubule-disrupting agents such as auristatins. Examples of such linkers include cleavable maleimidocaproyl linkers, and Doronina et al., Bi oconjugate Chem. 17:14-24, 2006, The disclosure of which relates to linkers for chemical conjugation is incorporated herein by reference. Use it in writing.

[0351] Any one or more of the chemical groups, moieties and features disclosed herein may be combined in multiple ways. In combination, the antibodies disclosed herein and linkers useful for conjugation of cytotoxins are It will be appreciated by those skilled in the art that the compositions and compositions described herein may form anchors. Additional linkers useful in conjunction with the methods are described, for example, in U.S. Patent Application Publication No. 2015 / 021 No. 8220, the entire disclosure of which is incorporated herein by reference.

[0352] Linkers useful in combination with the antibody-drug conjugates described herein include: The present invention also includes a linker containing a chemical moiety formed by a coupling reaction as shown in Table 2. The wavy lines indicate points of attachment to the antibody or antigen-binding fragment. , and the point of attachment 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 the reactive substituent Z attached to the linker and the antibody or its antigen-binding fragment and a reactive substituent on the cation that participates in a covalent coupling reaction to form the chemical moiety Z. and will recognize the reactive substituent Z. Antibody-drug conjugates useful in conjunction with the methods include antibodies or The reaction of the antigen-binding fragment with a linker or a cytotoxin-linker conjugate is The linker or cytotoxin-linker conjugate may be formed by chemical reaction reacting with a reactive substituent on an antibody or antigen-binding fragment thereof to form moiety Z The compound contains a reactive substituent Z suitable for the reaction.

[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)CH2R 14 ,- NR 13 C(=O)CH2Br, -NR 13 C(=O)CH2I, -NHC(=O)CH 2Br, -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 independently selected from H, C1-C6 alkyl, F, Cl and -OH, Selected, R 17 is H, C1-C6alkyl, F, Cl, -NH2, -OCH3, -OCH2 CH3, -N(CH3)2, -CN, -NO2 and -OH, independently in each case Selected, R 18is benzyloxy substituted with H, C1-C6 alkyl, F, -C(=O)OH , benzyl substituted with -C(=O)OH, C1-C4 alkyl substituted with -C(=O)OH alkoxy, and C1-C4 alkyl substituted by -C(=O)OH, as appropriate Independently selected, m is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, And, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 It is selected independently on a case-by-case basis.

[0356] As shown in Table 2, linkers and suitable antibodies or antigen-binding fragments thereof Examples of reactive substituents include nucleophile / electrophile pairs (e.g., thiol / haloalkyl, among others). pair, amine / carbonyl pair, or thiol / α,β-unsaturated carbonyl pair), diene / Dienophile pairs (e.g., azide / alkyne pairs or diene / α,β-unsaturated carboxylates, among others) The coupling between reactive substituents to form the chemical moiety Z is Ring-linking reactions include, but are not limited to, thiol alkylation, hydroxyl alkylation, Amine alkylation, amine or hydroxylamine condensation, hydrazine purification, amidation, Esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels- Alder cycloaddition, [3+2] Huisgen cycloaddition), nucleophilic aromatic substitution, electrophilic aromatic substitution and other reaction modes known in the art or described herein. Preferably, the linker reacts with a nucleophilic functional group on the antibody or antigen-binding fragment thereof. The compound contains an electrophilic functional group for reacting with the hydroxy group.

[0357] As disclosed herein, the antigen present in an antibody or antigen-binding fragment thereof Reactive substituents include, but are not limited to, (i) an N-terminal amine group, (ii) a side chain amine group (e.g., lysine), (iii) side chain thiol groups (e.g., cysteine), and (iv) sugar hydroxyl groups or an amino group (where the antibody is glycosylated). However, the hydroxyl moieties of serine, threonine, and tyrosine residues, and the alkyl groups of lysine residues amino moieties, the carboxyl moieties of aspartic acid and glutamic acid residues, and propionyl groups aryl, azido, haloaryl (e.g., fluoroheteroaryl), haloalkyl, non- The thiol moiety of the haloheteroalkyl moiety of a natural amino acid is also included. In some embodiments, reactive substituents present in the antibodies disclosed herein, or their antigen binding The fragments contain amine or thiol moieties. Certain antibodies have reducible interchain disulfides. Antibodies have sulfide, i.e., cysteine ​​bridges. They are also cleaved with DTT (dithiothreitol). can be made reactive for conjugation with a linker reagent by treatment with a reducing agent such as Thus, each cysteine ​​bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by reaction of lysines with 2-iminothiolane (Traut's reagent). The amine can be converted to a thiol by introducing one reactive thiol group. , by introducing two, three, four, or more cysteine ​​residues (e.g., preparing a variant antibody containing one or more non-native cysteine ​​amino acid residues; ) can be introduced into an antibody (or fragment thereof). No. 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 target molecule present on the antibody. Nucleophilic groups that are electrophilic and reactive. Useful electrophiles on antibodies include aldehydes and The heteroatom of the nucleophilic group may be an antibody. These nucleophilic groups can react with the electrophilic groups above to form covalent bonds with antibodies. Hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine These include, but are not limited to, carboxylates, and aryl hydrazides. In some embodiments, Z is a reactive nucleophilic substituent present in an antibody, or its antigen. A binding fragment (e.g., an amine moiety and a thiol moiety) and a reactive electrophilic substituent Z For example, Z may be, among other things, a ...

Claims

1. 1. A method of depleting T cells in a subject suffering from an autoimmune disease, the method comprising administering an anti-CD5 antibody An effective amount of the drug conjugate (ADC) or anti-CD2 ADC is administered to a subject suffering from an autoimmune disease. administering to the subject The ADC may comprise an anti-CD5 antibody or antibody conjugated to a cytotoxin via a linker. or an antigen-binding fragment thereof, or an anti-CD2 antibody or an antigen-binding fragment thereof A method including:

2. The effective amount substantially depletes endogenous CD5+ or CD2+ T cells in the thymus of the subject.

10. The method of claim 1, wherein the amount is sufficient to

3. The subject has multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus (SLE), or 3. The method of claim 1 or 2, wherein the patient is suffering from systemic sclerosis (SSc).

4. Have or have developed steroid-resistant graft-versus-host disease (GVHD) 2. A method of treating a subject at risk of developing steroid-resistant GVHD, the method comprising: The subject is administered an anti-CD2 ADC or a steroid-resistant GVHD inhibitor such that the steroid-resistant GVHD is treated. or an anti-CD5 ADC, wherein the ADC is linked to a cytotoxin via a linker. Conjugated anti-CD5 antibodies or antigen-binding fragments thereof, or anti-CD 2 antibody or antigen-binding fragment thereof.

5. The method according to claim 4, wherein the steroid-resistant GVHD is steroid-resistant acute GVHD. How to post.

6. The method of claim 4 or 5, wherein the subject has previously undergone an allogeneic HSC transplant.

7. The subjects were treated with steroid-resistant acute GVHD grade 2 to grade 4 (Mount Sinai Acute GVHD).

7. The method according to claim 5 or 6, wherein the method has the Multi-Area Graft-Vs-Heart Disease International Consortium (MAGIC) standard. method.

8. After administration of the anti-CD2 ADC or anti-CD5 ADC, G according to the MAGIC criteria 8. The method of claim 7, wherein the VHD grade is reduced by one grade.

9. 1. A method of treating a subject suffering from a T-cell malignancy, comprising administering to a subject an effective amount of an anti-CD2 ADC or administering an anti-CD5 ADC to the subject, wherein the ADC is linked to cells via a linker. an anti-CD5 antibody or antigen-binding fragment thereof conjugated to a cytotoxin; comprises an anti-CD2 antibody or antigen-binding fragment thereof.

10. 10. The method of claim 9, wherein the T-cell malignancy is lymphoma.

11. The T-cell malignancies include T-cell acute lymphoblastic lymphoma (T-ALL), T-cell adenomatous lymphoma (TALL), 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 or peripheral T-cell lymphoma (PTCL).

12. 10 or 11, wherein the T-cell malignancy is a relapsed and refractory T-cell malignancy.

1. The method according to claim 1.

13. The ADC according to any one of claims 1 to 12, wherein the ADC comprises a humanized or human antibody. How to do it.

14. The antibody is selected from the group consisting of IgG, IgA, IgM, IgD and IgE. The method according to any one of claims 1 to 13, wherein the antibody has an isotype.

15. 15. The method of claim 14, wherein the IgG isotype is IgG1 or IgG4.

16. The method of any one of claims 1 to 15, wherein the ADC is an anti-CD5 ADC.

17. The method of any one of claims 1 to 15, wherein the ADC is an anti-CD2 ADC.

18. The cytotoxins include Pseudomonas exotoxin A, deboganin, diphtheria toxin, amatoxin, saporin, maytansine, maytansinoids, auristatins, anthracyclines , calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine zepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, or indolinobenzodiazepine 18. The compound according to claim 1, wherein the compound is selected from the group consisting of benzodiazepine dimers. The method described.

19. The method according to any one of claims 1 to 17, wherein the cytotoxin is an RNA polymerase inhibitor. How to post.

20. 20. The method of claim 19, wherein the RNA polymerase inhibitor is an RNA polymerase II inhibitor. The method described.

21. 20. The method of claim 19, wherein the RNA polymerase inhibitor is an amatoxin.

22. The ADC is represented by the formula Ab-ZL-Am, where Ab is the anti-CD5 antibody or its equivalent. wherein L is a linker, Z is a chemical moiety, and Am is an ama is a toxin having the formula (I): 【Chemical 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 If present, they are bonded to any oxygen atom. forming 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 Haitai alkyl, optionally substituted C ２ -C ６ Alkenyl, optionally substituted C ２ -C ６ Haitai alkenyl, optionally substituted C ２ -C ６ Alkynyl, optionally substituted C ２ -C ６ F cycloalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkynyl alkyl, optionally substituted aryl or optionally substituted heteroaryl; L is an optionally substituted C １ -C ６ Alkylene, optionally substituted C １ -C ６ Haitai C alkylene, 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 heteroalkynylene, cycloalkylene, optionally substituted arylene, optionally substituted heteroarylene , a dipeptide, —C(═O)—, a peptide, or a combination thereof; Z is a reactive substituent present on L and the antibody or antigen-binding fragment thereof. is a chemical moiety formed from a coupling reaction between a reactive substituent present in 18. The compound according to claim 1, wherein Am contains exactly one RC substituent. How to do it.

23. Am-L-Z is a compound represented by formula (IA): 【Chemistry 2】 is expressed as 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, they are optionally substituted with the oxygen atom to which they are attached. forming a substituted 5-membered heterocycloalkyl group, R ３ is H, R Ｃ or R Ｄ and R ４ , R ５ , R ６ and R ７ are each independently H, OH, or OR Ｃ , OR Ｄ , R Ｃ Also is R Ｄ and R ８ OH, NH ２ , OR Ｃ , OR Ｄ , N.H.R. Ｃ or NR Ｃ R Ｄ and R ９ is H, OH, OR Ｃ or OR Ｄ and X is -S-, -S(O)- or -SO ２ - and R Ｃ is -L-Z, R Ｄ is an optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ Heteroal Kill, optionally replaced C ２ -C ６ Alkenyl, optionally substituted C ２ -C ６ Heteroal phenyl, optionally substituted C ２ -C ６ Alkynyl, optionally substituted C ２ -C ６ Heteroa alkynyl, 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 Heteroal Xylene, optionally substituted C ２ -C ６ Alkenylene, optionally substituted C ２ -C ６ Hetero Alkenylene, optionally substituted C ２ -C ６ Alkynylene, optionally substituted C ２ -C ６ Heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene chloroalkylene, optionally substituted arylene, optionally substituted heteroarylene, diaryl peptide, -C(=O)-, peptide, or a combination thereof; Z is a reactive substituent present on L and a group present in an antibody or antigen-binding fragment thereof. is a chemical moiety formed from a coupling reaction between reactive substituents present, 23. The method of claim 22, wherein Am contains exactly one R.sup.C substituent.

24. Am-L-Z is a compound represented by formula (IB): 【Chemistry 3】 is expressed as 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, they are optionally substituted with the oxygen atom to which they are attached. forming a substituted 5-membered heterocycloalkyl group, R ３ is H, R Ｃ or R Ｄ and R ４ , R ５ , R ６ and R ７ are each independently H, OH, or OR Ｃ , OR Ｄ , R Ｃ Also is R Ｄ and R ８ OH, NH ２ , OR Ｃ , OR Ｄ , N.H.R. Ｃ or NR Ｃ R Ｄ and R ９ is H, OH, OR Ｃ or OR Ｄ and X is -S-, -S(O)- or -SO ２ - and R Ｃ is -L-Z, R Ｄ is an optionally substituted C １ -C ６ Alkyl, optionally substituted C １ -C ６ Heteroal Kill, optionally replaced C ２ -C ６ Alkenyl, optionally substituted C ２ -C ６ Heteroal phenyl, optionally substituted C ２ -C ６ Alkynyl, optionally substituted C ２ -C ６ Heteroa alkynyl, 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 Heteroal Xylene, optionally substituted C ２ -C ６ Alkenylene, optionally substituted C ２ -C ６ Hetero Alkenylene, optionally substituted C ２ -C ６ Alkynylene, optionally substituted C ２ -C ６ Heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene chloroalkylene, optionally substituted arylene, optionally substituted heteroarylene, diaryl peptide, -C(=O)-, peptide, or a combination thereof; Z is a reactive substituent present on L and a group present in an antibody or antigen-binding fragment thereof. is a chemical moiety formed from a coupling reaction between reactive substituents present, 24. The method of claim 23, wherein Am contains exactly one Rc substituent.

25. The ADC is represented by the formula Ab-ZL-Am, where Ab is an antibody or its antigen-binding fragment. Z is a chemical moiety, L is a linker, Am is an amatoxin, The toxin-linker conjugate Am-L-Z is represented by formula (II), formula (IIA), or Formula (IIB): 【Chemistry 4】 is expressed as In the formula, X is S, SO or SO ２ and R １ is H, or a reactive substituent present on the linker and a reactive substituent present in the antibody. A chemical moiety formed from a coupling reaction between a substituent or an antigen-binding fragment thereof a linker covalently attached to the antibody or antigen-binding fragment thereof via moiety Z; R ２ is H, or a reactive substituent present on the linker and a reactive substituent present in the antibody. A chemical moiety formed from a coupling reaction between a substituent or an antigen-binding fragment thereof a linker covalently attached to the antibody or antigen-binding fragment thereof via moiety Z; R １ is H, then R ２ is a linker, and R ２ is H, then R １ is the linker be, The method according to any one of claims 1 to 17.

26. 18. Any one of claims 1 to 17, wherein the cytotoxin of the ADC is a maytansinoid. The method described below.

27. 27. The method of claim 26, wherein the maytansinoid is DM1 or DM4.

28. 18. The method of claim 1, wherein the cytotoxin of the ADC is an auristatin. The method described.

29. The auristatin may be monomethyl auristatin E (MMAE) or monomethyl auristatin E (MMAE).

29. The method of claim 28, wherein the compound is ristatin F (MMAF).

30. 18. The method according to claim 1, wherein the cytotoxin of the ADC is an anthracycline. The method described in paragraph .

31. The anthracycline may be daunorubicin, doxorubicin, epirubicin, or ibuprofen.

31. The method of claim 30, wherein the compound is darbicine.

32. The cytotoxin of the ADC has formula (IV): 【Chemistry 5】 Any one of claims 1 to 17, which is a pyrrolobenzodiazepine dimer derivative represented by The method described below.

33. The ADC is internalized by CD5+ or CD2+ immune cells after administration to a patient. The method according to any one of claims 1 to 32,

34. the ADC is internalized by CD5+ immune cells after administration to the patient.

33. The method of any one of 1 to 32.

35. the ADC is internalized by CD2+ immune cells after administration to the patient.

33. The method of any one of 1 to 32.

36. 35. The method of claim 34, wherein the immune cell is a malignant T cell.

37. The method of any one of claims 1 to 36, wherein the subject is a human.

38. The anti-CD5 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO: 282 and a heavy chain comprising the variable region set forth in SEQ ID NO:

283.

38. The method of any one of claims 1 to 37, comprising a light chain comprising a variable region comprising:

39. The anti-CD5 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO: 288 and a heavy chain comprising the variable region set forth in SEQ ID NO:

289.

39. The method of any one of claims 1 to 38, comprising a light chain comprising a variable region comprising:

40. The anti-CD5 antibody or antigen-binding fragment thereof has the amino acid sequence of SEQ ID NO:

291. and a heavy chain comprising the variable region set forth in SEQ ID NO:

290.

40. The method of any one of claims 1 to 39, comprising a light chain comprising a variable region comprising:

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