Methods and compositions for treating autoimmune diseases

Abstract

Methods and compositions related to the treatment of autoimmune diseases using anti-CD45 antibody drug conjugates (ADCs) are disclosed. Described herein are methods and compositions for improving autologous hematopoietic stem cell transplantation (autoHSCT). Provided herein are methods and compositions for addressing known challenges in the field of autoHSCT, including antibodies and / or antibody drug conjugates (ADCs) that selectively target CD45 to clear autoimmune cells and enable autoHSCT as a potential one-time curative treatment for patients with autoimmune diseases.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefits of U.S. Provisional Application No. 62 / 857,232, filed June 4, 2019; U.S. Provisional Application No. 62 / 863,141, filed June 18, 2019; U.S. Provisional Application No. 62,882,310, filed August 2, 2019; U.S. Provisional Application No. 62 / 933279, filed November 8, 2019; U.S. Provisional Application No. 62 / 944988, filed December 6, 2019; U.S. Provisional Application No. 62 / 968870, filed January 31, 2020; and U.S. Provisional Application No. 63 / 030860, filed May 27, 2020, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list submitted via EFS-Web in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy was created on June 4, 2020, named M103034_2180WO_0511_2_SL.txt, and is 83,868 bytes in size. Technical Field

[0005] This invention relates to the field of treating autoimmune diseases. Background Technology

[0006] Autologous hematopoietic stem cell transplantation (autoHSCT) is a highly effective treatment for selected patients with autoimmune diseases. AutoHSCT can induce long-term remission (up to 15 years) in patients with relapsed / refractory and secondary progressive multiple sclerosis isoforms, with a progression-free survival rate of 70-80% (Muraro 2017), superior to the standard of care in randomized studies (Burt 2019). Similarly, autoHSCT has achieved excellent results in scleroderma patients in two randomized studies (Tyndall 2014, Sullivan 2018). These results are achieved by eliminating autoreactive immune cells and rebuilding the autoimmune system tolerating its own immune response (i.e., immune system reset). However, only a small percentage of eligible patients receive autoHSCT, partly due to toxicities associated with current conditioning regimens. Invention Overview

[0008] Described herein are methods and compositions for improving autologous hematopoietic stem cell transplantation (autoHSCT). Provided herein are methods and compositions comprising antibodies and / or antibody drug conjugates (ADCs) that selectively target CD45 to deplete autoimmune cells and allow autoHSCT as a potential one-time curative treatment for patients with autoimmune diseases that address known challenges in the field of autoHSCT. This method of conditioning prior to autoHSCT can increase the number of autoimmune patients eligible for transplantation and significantly reduce side effects associated with current conditioning regimens.

[0009] Thus, in one aspect, the present application provides a method of depleting a CD45+ cell population in a human patient with an autoimmune disease, the method comprising administering to the patient with an autoimmune disease an effective amount of an anti-CD45 antibody or antigen-binding portion thereof. In another aspect, the present application provides a method of depleting a CD45+ cell population in a human patient with an autoimmune disease, the method comprising administering to the patient with an autoimmune disease an effective amount of an antibody drug conjugate (ADC) comprising an anti-CD45 antibody or antigen-binding portion thereof.

[0010] In another aspect, the present application provides a method of conditioning a human patient with an autoimmune disease to receive a hematopoietic stem cell (HSC) transplant, the method comprising administering to the patient with an autoimmune disease an anti-CD45 antibody or antigen-binding portion thereof. In another aspect, the present application provides a method of conditioning a human patient with an autoimmune disease to receive a hematopoietic stem cell (HSC) transplant, the method comprising administering to the patient with an autoimmune disease an antibody drug conjugate (ADC) comprising an anti-CD45 antibody or antigen-binding portion thereof.

[0011] In some embodiments, the autoimmune disease is inflammatory arthritis (e.g., rheumatoid arthritis), autoimmune encephalitis, scleroderma, multiple sclerosis, type 1 diabetes, or systemic sclerosis.

[0012] In some embodiments, the method further comprises administering to the patient a transplant comprising hematopoietic stem cells (HSCs). In certain embodiments, the HSC transplant is an autologous HSC transplant (autoHSCT).

[0013] In some embodiments, the anti-CD45 ADC is administered to the patient as a single dose.

[0014] In some embodiments, the patient does not require treatment (e.g., long-term treatment) for an autoimmune disease after transplantation. For example, in some embodiments, the patient has multiple sclerosis, and the patient does not require treatment with natalizumab, dimethyl fumarate, or monomethyl fumarate after transplantation. In other embodiments, the patient has arthritis, and the patient does not require treatment with a TNF inhibitor (e.g., an anti-TNFa antibody, such as etanercept, infliximab, adalimumab, certolizumab pegol, or golimumab) after transplantation.

[0015] In some embodiments, the patient enters remission for at least one year after transplantation. In some embodiments, the patient enters remission for at least 2 years after transplantation. In some embodiments, the patient enters remission for at least 5 years after transplantation. In some embodiments, the patient enters remission for at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years. In some embodiments, the patient enters remission for at least 1-3 years, at least 3-5 years, at least 5-7 years, at least 7-9 years, or at least 8-10 years. In some embodiments, the remission is clinical remission. In other embodiments, the remission is biochemical remission. In still further embodiments, the remission is histological remission.

[0016] In some embodiments, the anti-CD45 ADC is administered to the patient about three days before the patient receives a transplant comprising HSCs.

[0017] In some embodiments, the HSC transplant is administered to the human patient after the anti-CD45 ADC has been substantially cleared from the human patient’s blood.

[0018] In another aspect, provided herein is a method of treating a patient having scleroderma (also known as systemic sclerosis), the method comprising administering an anti-CD45 antibody drug conjugate (ADC) to the patient having scleroderma to treat the scleroderma, wherein the anti-CD45 ADC comprises an anti-CD45 antibody or fragment thereof conjugated to a cytotoxin via a linker.

[0019] In some embodiments of any of the foregoing aspects, the anti-CD45 antibody is a chimeric antibody or a humanized antibody.

[0020] In some embodiments, the anti-CD45 antibody is a human antibody.

[0021] In some embodiments, the anti-CD45 antibody is intact.

[0022] In some embodiments, the anti-CD45 antibody or antigen-binding portion thereof is selected from the group consisting of a monoclonal antibody or antigen-binding portion thereof, a polyclonal antibody or antigen-binding portion thereof, a bispecific antibody or antigen-binding portion thereof, a dual variable immunoglobulin domain, a single chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment, a F(ab’)2 molecule, and a tandem di-scFv.

[0023] In some embodiments, the anti-CD45 antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE. In certain embodiments, the anti-CD45 antibody comprises a human IgGl, IgG2, IgG3, or IgG4 isotype Fc domain.

[0024] In some embodiments, the anti-CD45 antibody or antigen-binding fragment thereof comprises an Fc domain, and wherein the anti-CD45 antibody or antigen-binding fragment thereof is conjugated to a cytotoxin via a cysteine residue in the Fc domain. In certain embodiments, the cysteine residue is introduced into the Fc domain by way of an amino acid substitution. In particular embodiments, the amino acid substitution is D265C (EU numbering).

[0025] In some embodiments of any of the foregoing aspects, the anti-CD45 antibody or antigen-binding fragment thereof is conjugated to a cytotoxin. In some embodiments, the cytotoxin is selected from the group consisting of Pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin, maytansine, a maytansinoid, an auristatin, an anthracycline, calicheamicin, irinotecan, SN-38, a duocarmycin, a pyrrolobenzodiazepine pyrrolobenzodiazepine dimer, an indolinobenzodiazepine and an indolinobenzodiazepine dimer.

[0026] In some embodiments, the cytotoxin is an auristatin. In certain embodiments, the auristatin is MMAE or MMAF.

[0027] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine or a pyrrolobenzodiazepine dimer.

[0028] In some embodiments, the cytotoxin is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is an amatoxin.

[0029] In certain embodiments, the amatoxin is of the formula (IA):

[0030]

[0031] wherein R1is H, OH, OR A or OR C ;

[0032] R2is H, OH, OR B or OR C ;

[0033] R A and R B , taken together with the oxygen atom to which they are bound, form an optionally substituted 5-membered heterocycloalkyl group;

[0034] R3is H, R C or R D ;

[0035] R4, R5, R6, and R7are each independently H, OH, OR C , OR D , R C or R D ;

[0036] R8is OH, NH2, OR C , OR D , NHR C or NR C R D ;

[0037] R9is H, OH, OR C or OR D ;

[0038] X is -S-, -S(O)-, or -SO2-;

[0039] R C is -L-Z;

[0040] R D is optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6heteroalkenyl, optionally substituted C2-C6alkynyl, optionally substituted C2-C6heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0041] L is optionally substituted C1-C6 alkylene, optionally substituted C1-C6 heteroalkylene, optionally substituted C2-C6 alkenylene, optionally substituted C2-C6 heteroalkenylene, optionally substituted C2-C6 alkynylene, optionally substituted C2-C6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene; and

[0042] Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within the antibody or antigen-binding fragment thereof, wherein Am comprises only one R C substituent.

[0043] In some embodiments, the anti-CD45 ADC has the following formula:

[0044]

[0045]

[0046] wherein Ab represents an anti-CD45 antibody.

[0047] In some embodiments, the anti-CD45 ADC has the following formula:

[0048] wherein Ab represents an anti-CD45 antibody.

[0049] In some embodiments, the RNA polymerase inhibitor is an amanitin.

[0050] Also provided are methods of depleting a CD45+ cell population in a human patient having an autoimmune disease, the method comprising administering to the patient having an autoimmune disease an effective amount of an engineered toxin body targeting CD45. In some embodiments, the engineered toxin body comprises an antibody or antigen-binding fragment thereof that specifically binds CD45. In some embodiments, the engineered toxin body comprises an scFv that specifically binds CD45. In some embodiments, the engineered toxin body comprises a protein-based toxin. In some embodiments, the protein-based toxin is a protein synthesis inhibitor, e.g., a ribosome inactivating protein. In some embodiments, the protein-based toxin is selected from the group consisting of Shiga toxin, Shiga-like toxin A subunit, saporin, ricin, and mutants, fragments, and derivatives thereof.

[0051] In some embodiments of any of the foregoing aspects, the anti-CD45 antibody or antigen-binding fragment thereof is conjugated to a cytotoxin via a linker.

[0052] In some embodiments of any of the foregoing aspects, the anti-CD45 antibody is a bispecific or biparatopic antibody.

[0053] In some embodiments of any of the foregoing aspects, the anti-CD45 antibody or antigen binding fragment thereof is administered to the patient in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent comprises an antibody or antigen binding portion thereof. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figures 1A-1E Results of in vitro studies showing that anti-CD45 ADCs successfully opsonize mice that are syngeneic transplanted are depicted. Figure 1A is a schematic depicting the in vivo design of the study in which C57BL / 6 (CD45.2) mice were opsonized with a single intravenous dose of anti-mouse CD45-ADC (0.3 mg / kg, 1 mg / kg, or 3 mg / kg) or isotype-ADC (3 mg / kg) 48 hours prior to transplantation. Figure 1B The percentage of donor chimerism in the peripheral blood of mice in each treatment group at the indicated time points (i.e., week 0, week 4, week 8, week 12, and week 16) is graphically depicted. Figures 1C-1E The percentage of donor myeloid cells ( Figure 1C ), B cells ( Figure 1D ), and T cells ( Figure 1E ) in mice in each treatment group at the indicated time points (i.e., week 0, week 4, week 8, week 12, and week 16) is graphically depicted.

[0055] Figure 2A and 2B Results of in vitro cell killing assays showing that anti-CD45 ADCs preferentially kill proliferating T cells in vitro are graphically depicted. The number of total T cells ( Figure 2A ) or the percentage of proliferating T cells (Ki67+; Figure 2B ) is shown as a function of the indicated ADC concentration (anti-mouse anti-CD45 ADC or isotype-ADC).

[0056] Figures 3A-3E Results of in vivo cell depletion assays showing that anti-CD45-ADCs preferentially kill proliferating T cells in an in vivo model of sclerodermic graft versus host disease (scGvDH) that represents scleroderma are graphically depicted. Figure 3Ais a schematic depicting an in vivo study design in which Balb / C (CD45.1) mice were conditioned with 6.5 Gy TBI 24 hours prior to allogeneic (Allo) adoptive cell transfer of DBA / 2 (CD45.2) splenocytes. Host animals (CD45.1+) were treated with a single intravenous dose (3 mg / kg) of anti-mouse CD45-ADC or isotype-ADC 7 days after initiation of scGvHD (CD45.2+ cells). Figure 3B and 3C Flow cytometry analysis of spleen ( Figure 3B ) or peripheral blood ( Figure 3C ) harvested from mice administered with isotype-ADC or anti-CD45-ADC and analyzed for allogeneic cells, T cells, and proliferating T cells. Figure 3D and 3E Graphical depiction of results for number and percentage of allogeneic T cells (total T cells and proliferating Ki-67+ T cells) in spleen ( Figure 3D ) or peripheral blood ( Figure 3E ) harvested from mice administered with isotype-ADC or anti-CD45-ADC.

[0057] Figures 4A-4D Graphical depiction of results from an in vivo study showing that autologous bone marrow transplant (BMT) improves murine experimental autoimmune encephalitis (EAE). Animals were conditioned with 9 Gy TBI on days 8 and 13 post-EAE induction. Conditioned mice were transplanted with 10 7 donor cells 24 hours after TBI. Figure 4A Graphical depiction of EAE clinical scores as a function of days post-induction under the indicated treatment conditions (no BMT, BMT on day 9, or BMT on day 14). Figure 4B Graphical depiction of the percentage of donor chimerism in peripheral blood of mice of the indicated treatment groups (PBS control, BMT on day 9, or BMT on day 14) harvested at the indicated time points (4 weeks post-transplant and 8 weeks post-transplant). Figure 4C and 4D Graphical depiction of a comparison of host and donor cells in peripheral blood ( Figure 4C ) and lymph nodes ( Figure 4D ) harvested from control mice (untransplanted) and treated mice (BMT on day 9).

[0058] Figures 5A-5H Graphical depiction of results from in vivo and in vitro studies using CD45-ADC in the context of experimental autoimmune encephalitis (EAE) and multiple sclerosis (MS). Figures 5A-5GResults from in vivo studies are described showing that administration of CD45-ADC enabled autologous hematopoietic stem cell transplantation (autoHSCT) and significantly reduced pathology in a murine experimental autoimmune encephalitis (EAE) model. Figure 5A is a schematic depicting the study design in which EAE was initiated in C57BL / 6 ("B6") mice via immunization with MOG in complete Freund's adjuvant (CFA) on day 0 35-55 and with pertussis toxin ("PTX") on days 0 and 1. Figure 5B The degree of total donor chimerism, myeloid chimerism, and T cell chimerism in graft recipients in the indicated treatment groups is graphically depicted. Figure 5C and 5D The relative number of splenic T cells Figure 5C and the relative number of splenic IL-1 A+ T cells (i.e., IL-17A-producing effector cells) Figure 5D are graphically depicted. Groups of immunized mice were conditioned ("Tx") with 3 mg / kg of CD45-ADC followed by syngeneic bone marrow transplantation ("BMT"; 2 x 10 7 BM cells (CD45.1 + ) from B6.SJL) on day 5 Figure 5E or day 10 Figure 5F post-immunization. All animals were scored daily starting on day 5 post-immunization. Vehicle and FTY-720 treatment groups are shown as controls. Figure 5E and 5F EAE clinical scores over time (from day 0 to day 56 post-immunization) in mice treated with CD45-ADC on day 5 Figure 5E or day 10 Figure 5F are graphically depicted. Figure 5G EAE clinical scores from day 0 to day 28 post-immunization in mice treated with CD45-ADC on day 5 are graphically depicted, along with results obtained with mice treated with naked CD45 Ab (3 mg / kg, single dose) or isotype-ADC plus BMT for comparison. Figure 5H Results from in vitro studies comparing human anti-CD45-ADC-mediated killing of PBMCs from MS patients and healthy donors are graphically depicted.

[0059] Figures 6A-6I Results from in vitro and in vivo studies showing potent killing of primary human hematopoietic cells and primary cynomolgus hematopoietic cells by anti-human CD45-ADC in vitro and in humanized NSG mice are graphically depicted. Figures 6A-6C Results from in vitro killing assays showing human PBMC killing (Figure 6A ), the lethal effect of cynomolgus monkey PBMCs ( Figure 6B or human CD34+CD90+ cell killing effect ( Figure 6C and Figure 6D The degree of EC50 is used as a function of the specified ADC concentration (anti-human anti-CD45 ADC or allotype-ADC). The corresponding EC50 values ​​are summarized in Table 4. Figure 6E The percentage of human hematopoietic cells (human β2M+ cells) in the peripheral blood of mice treated with PBS, allotype-ADC (3 mg / kg), or anti-human CD45 ADC (1 mg / kg, 3 mg / kg, or 6 mg / kg) on ​​days 0, 7, or 14 post-treatment was graphically depicted. Figure 6F and 6G The percentage of human CD34+ cells in mouse bone marrow 14 days after administration of PBS, allotype-ADC (3 mg / kg), or anti-human CD45 ADC (1 mg / kg, 3 mg / kg, or 6 mg / kg) is graphically depicted. Figure 6F ) and the number of human CD34+ cells ( Figure 6G ). Figure 6H and 6I The results of similar studies, in which CD45 ADCs were administered at doses of 1 mg / kg, 2 mg / kg, or 3 mg / kg, are depicted graphically.

[0060] Figures 7A-7H The results of in vivo studies are depicted graphically, showing that a single dose of anti-human CD45-ADC is well tolerated and causes depletion of lymphocytes and HSCs in non-human primates (NHP). Figure 7A Flow cytometry analysis of peripheral blood harvested from NHP peripheral blood 72 hours after administration of anti-CD45-ADC at doses of 0.5 mg / kg, 1 mg / kg, or 2 mg / kg was described, and myeloid cells, T cells, B cells, or lymphocytes were analyzed. Figure 7B The peripheral lymphocyte levels of NHP were graphically depicted three days after administration of anti-CD45-ADC at doses of 0.5 mg / kg, 1 mg / kg, or 2 mg / kg. Figure 7C Flow cytometry analysis of bone marrow harvested from NHP bone marrow 6 days after administration of anti-CD45-ADC at doses of 0.5 mg / kg, 1 mg / kg, or 2 mg / kg was described, and HSCs were analyzed. Figure 7D (include Figure 7D cont. The levels of white blood cells (WBC), HSCs, and lymphocytes in NHP were graphically depicted 6 days after administration of anti-CD45-ADC at doses of 0.5 mg / kg, 1 mg / kg, or 2 mg / kg, and HSCs were analyzed. Figure 7E and7F (including Figure 7F cont. ) graphically depicts the levels of ALT, bilirubin, AST, and / or ALP of NHPs as a function of days after administration of CD45-ADC at 2 mg / kg. Figure 7G Graphically depicts the levels of peripheral lymphocytes in NHPs after administration of anti-CD45-ADC at 0.5 mg / kg, 1 mg / kg, or 2 mg / kg. Figure 7H Graphically depicts the levels of CD34+CD90+CD45RA-HSCs of NHPs after administration of anti-CD45-ADC at 0.5 mg / kg, 1 mg / kg, or 2 mg / kg.

[0061] Figures 8A-8C Graphically depicts the results of an in vivo study showing that treatment with murine CD45-ADC rendered quiescent T cells incapable of mounting an allogeneic response. Cells were isolated from peripheral blood (d2 and d7) and spleen (d7) at various times post transfer and assessed for CTV staining intensity Figure 8A ), absolute number of T cells in peripheral blood as a function of days post transfer Figure 8B ), and absolute number of T cells in spleen 7 days post transfer Figure 8C ).

[0062] Figures 9A-9E Graphically depicts the results of an in vivo study showing that therapeutic treatment with CD45-ADC can enable immune reset via syngeneic BMT and results in cessation of disease progression in a murine model of rheumatoid arthritis (proteoglycan-induced arthritis). Figure 9A is a schematic showing the study design in which Balb / c mice (CD45.2+) were given 3 immunizations with recombinant human core G1 proteoglycan (60 μg in 2 mg DDA) on study days 0, 21, and 42. Figure 9B Graphically depicts the extent of total donor chimerism, neutrophil chimerism, B cell chimerism, and T cell chimerism in peripheral blood of transplant recipients in the indicated treatment groups at three weeks post transplant. Figure 9C Graphically depicts the extent of total donor chimerism and donor HSCs in bone marrow of transplant recipients in the indicated treatment groups at three weeks post transplant. Figure 9D Images showing mice treated with control or mice treated with CD45-ADC. Figure 9E Graphically depicts the cumulative arthritis score in the indicated treatment groups as a function of days post final immunization.

[0063] Figures 10A-10FResults of in vivo studies are graphically depicted showing that treatment with a single dose of anti-human CD45 ADC can eliminate human effector cells in a xenoGVHD sclerodermatous model and improve disease. Figure 10A Percentages of human hematopoietic cells (human β2Μ+ cells) in peripheral blood of mice treated with PBS, isotype-ADC (3 mg / kg), or anti-human CD45 ADC (1 mg / kg or 3 mg / kg) on day 0, day 7, or day 14 post-treatment are graphically depicted. Figure 10B Numbers of human hematopoietic cells (human β2Μ+ cells) per femur in bone marrow of mice treated with PBS, isotype-ADC (3 mg / kg), or anti-human CD45 ADC (1 mg / kg or 3 mg / kg) are graphically depicted. Figure 10C Numbers of human CD34+CD38 neg cells per femur in bone marrow of mice treated with PBS, isotype-ADC (3 mg / kg), or anti-human CD45 ADC (1 mg / kg or 3 mg / kg) are graphically depicted. Figure 10D Images of mice treated with isotype-ADC or ADC targeting anti-human CD45 before ADC treatment (pre-ADC) and 14 days post-ADC administration are shown. Figure 10E Clinical GVHD scores (based on size and appearance of skin lesions) as a function of days post-ADC administration for mice treated with isotype-ADC or anti-CD45 ADC are graphically depicted. Figure 10F Extent of peripheral human T cell depletion in animals treated with anti-human CD45 ADC on day 0, day 7, day 14, and day 62 are graphically depicted.

[0064] Figure 11A and 11B Study design and results of in vivo studies are graphically depicted showing that treatment with a single dose of CD45-ADC and syngeneic HSC transplant results in prevention of disease in a Type I diabetes adoptive transfer model. Figure 11A Adoptive transfer of OT-I and OT-II into RIP-OVA mice is depicted. Figure 11B Diabetes incidence in mice in the indicated treatment groups is graphically depicted. DETAILED DESCRIPTION

[0066] Disclosed herein are methods and compositions involving anti-CD45 antibody drug conjugates (ADCs) that are useful as therapeutic agents (e.g., for treating autoimmune disorders) or as conditioning agents for transplantation. Accordingly, included herein are anti-hematopoietic cell antibodies (anti-CD45 antibodies) that are useful in hematopoietic stem cell therapy. For example, the antibodies or ADCs herein are useful in conditioning procedures in which a patient is prepared to receive a transplant comprising hematopoietic stem cells. Such procedures facilitate engraftment of the hematopoietic stem cell transplant. According to the methods described herein, a patient can be conditioned for hematopoietic stem cell transplant therapy by administering to the patient an anti-CD45 ADC, an antibody or antigen-binding fragment thereof capable of binding CD45 (e.g., CD45 expressed by hematopoietic cells (e.g., hematopoietic stem cells or mature immune cells (e.g., T cells))). As described herein, the anti-CD45 antibodies can be covalently conjugated to a cytotoxin to form an antibody drug conjugate (ADC). Administration of an ADC capable of binding CD45 to a patient having an autoimmune disease can facilitate engraftment of a hematopoietic stem cell transplant.

[0067] The subsections below provide an overview of anti-CD45 ADCs, which can be administered to a patient, such as a patient having an autoimmune disease, to facilitate engraftment of a hematopoietic stem cell transplant, as well as an overview of methods for administering such therapeutic agents to a patient (e.g., prior to a hematopoietic stem cell transplant). The methods described herein provide a means for eliminating self-reactive immune cells (CD45+ cells) and reestablishing a self-tolerant immune system, i.e., immune system reset. The methods described herein provide a conditioning therapy for undergoing autoHSCT.

[0068] Definitions

[0069] The following terms and phrases, as used herein, are intended to have the following meanings, unless otherwise indicated.

[0070] As used herein, the term“about” refers to a value that is within 5% higher or lower than the stated value.

[0071] As used herein, the term“allogeneic” when used in the context of transplantation is used to define cells (or tissues or organs) that are transplanted from a donor to a recipient of the same species.

[0072] As used herein, the term“autologous” refers to a cell or graft in which the donor and recipient are the same subject.

[0073] As used herein, the term“xenogeneic” refers to a cell in which the donor and recipient are of different species.

[0074] As used herein, the term “immune cell” is intended to include, but is not limited to, cells that are of hematopoietic origin and that play a role in the immune response. Immune cells include, but are not limited to, T cells and natural killer (NK) cells. Natural killer cells are well known in the art. In some embodiments, natural killer cells include cell lines, such as NK-92 cells. NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells. Immune cells can be allogeneic or autologous.

[0075] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen. Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0076] Generally, antibodies comprise heavy and light chains that contain an antigen binding region. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into a number of hypervariable regions (known as complementarity determining regions (CDRs)), interspersed with regions that are more conserved (known as framework regions (FRs)). Each of VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. According to some aspects, the CDRs are according to the Kabat, Chothia, AbM, Contact, or IMGT definition. See, e.g., Dondelinger et al., “Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition”, Front. Immunol. 9:2278 (15 pages) (2018), which is incorporated by reference herein in its entirety. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0077] As used herein, the term "bispecific antibody" refers to an antibody, including but not limited to a monoclonal antibody, e.g., a deimmunized or humanized antibody, that is capable of binding two different epitopes, which can be on the same or different antigens. For example, a first epitope bound by a bispecific antibody can be on a first cell surface antigen expressed by a hematopoietic stem cell (such as CD45), and a second epitope bound by the bispecific antibody can be on a second cell surface antigen, where the first and second cell surface antigens are different. In some embodiments, the second cell surface antigen can be expressed by a hematopoietic cell (such as a hematopoietic stem cell, a hematopoietic progenitor cell, or a mature hematopoietic cell). In some embodiments, the binding specificities can be directed to distinct, non-overlapping epitopes on the same target antigen (i.e., a biparatopic antibody).

[0078] As used herein, the term "antigen binding fragment" refers to one or more portions of an antibody that retain the ability to specifically bind to a target antigen. The antigen binding function of an antibody can be performed by fragments of a full-length antibody. Antibody fragments can be, for example, Fab, F(ab')2, scFv, diabodies, triabodies, affibodies, nanobodies, aptamers, or domain antibodies. Examples of binding fragments encompassed by the term "antigen binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb comprising a VH and VL domain; (vi) a dAb fragment that consists of a VH domain (see, e.g., Ward et al., Nature 341 :544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more (e.g., two, three, four, five, or six) isolated CDRs, which can optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for use in the same manner as are intact antibodies. Antigen binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some cases by chemical peptide synthesis techniques known in the art.

[0079] As used herein, an "intact" antibody or "full-length" antibody refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides that are inter-connected by disulfide bonds.

[0080] As used herein, the term "specifically binds" refers to the ability of an antibody (or ADC) to recognize and bind a particular protein structure (epitope) and not broadly recognize and bind proteins. If an antibody is specific for epitope "A", the presence of a molecule comprising epitope A (or free, unlabelled A) in a reaction comprising labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody. If an antibody specifically binds to SEQ ID NO: "A", the antibody can bind all or some (i.e., a portion) of the residues in SEQ ID NO: "A". For example, an antibody "specifically binds" a target if it can be competed away from the target when labeled by a corresponding unlabeled antibody. In some embodiments, an antibody specifically binds a target if the K D is at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M or less (less meaning less than 10 -12 M, for example, 10 -13 M). In some embodiments, the K D is determined according to standard bio-layer interferometry (BLI). It will be appreciated, however, that an antibody can be able to specifically bind two or more antigens that are related in sequence. For example, in some embodiments, an antibody can specifically bind both the human and non-human (e.g., mouse or non-human primate) homolog of an antigen (e.g., CD45).

[0081] In some embodiments, as used herein, the term "binds specifically to CD45" or "specifically binds CD45" refers to an antibody that binds CD45 and has a dissociation constant (K -7 ) of 1.0 x 10 D M or less, as determined according to standard bio-layer interferometry (BLI). In some embodiments, the K D (M) is determined according to standard bio-layer interferometry (BLI). In some embodiments, the K off (M) is determined according to standard bio-layer interferometry (BLI). In some embodiments, the K(1 / s). It will be understood, however, that an antibody can be capable of specifically binding two or more antigens that are related in sequence. For example, in some embodiments, an antibody can specifically bind both the human and non-human (e.g., mouse or non-human primate) homologs of CD45. Thus, as used herein, an antibody that "specifically binds human CD45" is intended to mean an antibody that binds human CD45 (and possibly CD45 from one or more non-human species) but does not substantially bind non-CD45 proteins. Preferably, the antibody binds human CD45 with a Kd of 1 x 10 -7 M or less D , 5 x 10 -8 M or less D , 3 x 10 -8 M or less D , 1 x 10 -8 M or less D , or 5 x 10 -9 M or less D human CD45.

[0082] As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone, and is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies useful in the present disclosure can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0083] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin (such as a rat or mouse antibody) and human immunoglobulin constant regions (typically selected from a human immunoglobulin template). Methods of producing chimeric antibodies are known in the art. See, e.g., Morrison, 1985, Science 229(4719): 1202-7; Oi et al., 1986, BioTechniques 4:214-221; Gillies et al., 1985, J. Immunol. Methods 125: 191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397.

[0084] As used herein, the terms“Fc region,”“Fc domain,” and“Fc domain” refer to the portion of an immunoglobulin (e.g., an IgG molecule) that is associated with the crystallizable fragment obtained by papain digestion of the IgG molecule. The Fc region can be of an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE. The Fc region comprises the C-terminal half of both heavy chains (e.g., of an IgG molecule) connected by disulfide bonds. It has no antigen binding activity, but contains binding sites for complement and Fc receptors (including the FcRn receptor) (see below). For example, the Fc domain comprises the entire second constant domain CH2 (residues at EU positions 231-340 of IgG) and the third constant domain CH3 (residues at EU positions 341-447 of human IgG1). As used herein, the Fc domain includes the“lower hinge region” (residues at EU positions 233-239 of IgG1).

[0085] Fc can refer to this region in isolation, or to this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at multiple positions in the Fc domain, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, so there can be minor differences between the sequences presented in this application and those known in the art. Thus,“wild-type IgG Fc domain” or“WT IgG Fc domain” refers to any naturally occurring IgG Fc region (i.e., any allele). The heavy chain genes for human IgG1, IgG2, IgG3, and IgG4 can be found in many genetic repositories, for example, under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG1_HUMAN) of the Uniprot database (www.uniprot.org), respectively.

[0086] As used herein, the term "modified Fc region" or "variant Fc region" refers to an IgG Fc domain comprising one or more amino acid substitutions, deletions, insertions, or modifications introduced at any position within the Fc domain. In certain aspects, the variant IgG Fc domain includes one or more amino acid substitutions that result in reduced or ablated binding affinity for FcyR and / or Clq, as compared to a wild-type Fc domain that does not comprise the one or more amino acid substitutions. In addition, Fc binding interactions are important for various effector functions and downstream signaling events, including but not limited to antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, in certain aspects, an antibody comprising a variant Fc domain (e.g., an antibody, fusion protein, or conjugate) can exhibit altered binding affinity for at least one or more Fc ligands (e.g., FcyR), relative to a corresponding antibody having the same amino acid sequence but not comprising the one or more amino acid substitutions, deletions, insertions, or modifications (e.g., an unmodified Fc region comprising a native amino acid residue at the corresponding position in the Fc region).

[0087] Variant Fc domains are defined according to the amino acid modifications that make them up. For all of the amino acid substitutions discussed herein with respect to Fc regions, numbering is always according to the EU index in Kabat. Thus, for example, D265C is an Fc variant that substitutes the aspartic acid (D) at EU position 265 with a cysteine (C), relative to the parent Fc domain. The substitutions are presented in random order.

[0088] As used herein, the term "Fcy receptor" or "FcyR" refers to any member of the family of proteins that bind the IgG antibody Fc region and are encoded by the FcyR genes. In humans, this family includes, but is not limited to, FcyRI (CD64), including isoforms FcyRIa, FcyRIb, and FcyRIc; FcyRII (CD32), including isoforms FcyRIIa (including allotypes H131 and R131), FcyRIIb (including FcyRIIb-l and FcyRIIb-2), and FcyRIIc; and FcyRIII (CD16), including isoforms FcyRIIIa (including allotypes V158 and F158) and FcyRIIIb (including allotypes FcyRIIIb-NA1 and FcyRIIIb-NA2), as well as any undiscovered human FcyR or FcyR isoform or allotype. The FcyR can be from any organism, including but not limited to human, mouse, rat, rabbit, and monkey. Mouse FcyRs include, but are not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and FcyRIII-2 (CD16-2), as well as any undiscovered mouse FcyR or FcyR isoform or allotype.

[0089] As used herein, the term "effector function" refers to biochemical events that result from the interaction of an Fc domain with an Fc receptor. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. As used herein, "effector cell" means an immune system cell that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include, but are not limited to, monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and gd T cells, and can be from any organism, including but not limited to human, mouse, rat, rabbit, and monkey.

[0090] As used herein, the term "silent / silenced" refers to an antibody having an Fc region modified as described herein that has reduced binding to an Fc gamma receptor (Fc gamma R) relative to the same antibody comprising an unmodified Fc region, e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% less binding to an Fc gamma receptor (Fc gamma R) relative to the same antibody comprising an unmodified Fc region when measured by, e.g., BLI. In some embodiments, the Fc silent antibody has no detectable binding to an Fc gamma R. Binding of an antibody having a modified Fc region to an Fc gamma R can be determined using various techniques known in the art, for example, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al., Analytical Biochemistry, Vol. 373: 52-60, 2008; or radioimmunoassay (RIA)), or by surface plasmon resonance assays or other kinetic determination-based mechanisms (e.g., BIACORE.RTM. analysis or Octet® analysis (forteBIO)), among other methods, such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods can utilize a tag on one or more of the components being examined and / or employ various detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic tags. Detailed descriptions of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, Fourth Edition, Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. One example of a competitive binding assay is a radioimmunoassay, which involves incubating a labeled antigen with the target antibody in the presence of increasing amounts of unlabeled antigen, and detecting the antibody bound to the labeled antigen. The affinity of the target antibody for the particular antigen and the rate of dissociation of the binding can be determined from the data by scatchard plot analysis. Radioimmunoassay can also be used to determine competition with a second antibody. In this case, the antigen and the target antibody conjugated to a labeled compound are incubated in the presence of increasing amounts of unlabeled second antibody.

[0091] As used herein, the term "the same antibody comprising an unmodified Fc region" refers to an antibody that lacks the stated amino acid substitution (e.g., D265C, H435A) but has the same amino acid sequence as the Fc modified antibody to which it is being compared.

[0092] ​The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which a polypeptide comprising an Fc domain (e.g., an antibody) binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., primarily NK cells, neutrophils, and macrophages) and enables these cytotoxic effector cells to specifically bind "target cells" bearing antigen and subsequently kill the target cells with cytotoxins. (Hogarth et al., Nature review Drug Discovery 2012, 11:313) It is contemplated that other polypeptides comprising an Fc domain (e.g., Fc fusion proteins and Fc conjugate proteins) in addition to antibodies and fragments thereof that have the ability to specifically bind target cells bearing antigen will be able to effect cell-mediated cytotoxicity.

[0093] For simplicity, cell-mediated cytotoxicity resulting from the activity of a polypeptide comprising an Fc domain is also referred to herein as ADCC activity. The ability of any particular polypeptide of the disclosure to mediate lysis of target cells by ADCC can be determined. To assess ADCC activity, the polypeptide of interest (e.g., an antibody) is added to a combination of target cells and immune effector cells, resulting in lysis of the target cells. Lysis is typically detected by release of label (e.g., radioactive substrate, fluorescent dye, or a native intracellular protein) from lysed cells. Effector cells that can be used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Particular examples of in vitro ADCC assays are described in Bruggemann et al., J. Exp. Med. 166: 1351 (1987); Wilkinson et al., J. Immunol. Methods 258: 183 (2001); Patel et al., J. Immunol. Methods 184: 29 (1995). Alternatively, or additionally, ADCC activity of an antibody of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA 95: 652 (1998).

[0094] As used herein, the term "conditioning" refers to a process of preparing a patient to receive a transplant (e.g., a transplant comprising hematopoietic stem cells). Such procedures facilitate engraftment of a hematopoietic stem cell transplant (e.g., as inferred from a sustained increase in the number of viable hematopoietic stem cells in blood samples isolated from the patient following the conditioning procedure and a subsequent hematopoietic stem cell transplant). According to the methods described herein, a patient can be conditioned for hematopoietic stem cell transplant therapy by administering to the patient an ADC, antibody, or antigen-binding fragment thereof capable of binding CD45 expressed by hematopoietic stem cells. As described herein, the antibody can be covalently conjugated to a cytotoxin to form a drug-antibody conjugate. Administration of an ADC or antibody, or antigen-binding fragment thereof, capable of binding one or more of the foregoing antigens to a patient in need of hematopoietic stem cell transplant therapy can facilitate engraftment of a hematopoietic stem cell transplant, e.g., by selectively depleting endogenous hematopoietic stem cells, thereby creating space to be filled by an exogenous hematopoietic stem cell transplant.

[0095] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of a therapeutic agent (e.g., an anti-CD45 ADC) sufficient to achieve a desired result or have some effect on an autoimmune disease in a human patient.

[0096] As used herein, the term "half-life" refers to the time it takes for the plasma concentration of an antibody drug to decrease by one-half or 50% in a subject (e.g., a human subject). This 50% reduction in plasma concentration reflects the amount of drug circulating.

[0097] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis or mutations introduced by gene recombination during maturation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Human antibodies can be produced in human cells (e.g., by recombinant expression) or by non-human animals or prokaryotic or eukaryotic cells that are capable of expressing functional rearranged human immunoglobulin (such as heavy and / or light chain) genes. When human antibodies are single chain antibodies, they can include linker peptides not found in native human antibodies. For example, an Fv can comprise a linker peptide connecting a heavy chain variable region and a light chain variable region, e.g., two to about eight glycine or other amino acid residues. Such linker peptides can be considered human-derived. Human antibodies can be manufactured by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. Human antibodies can also be produced using transgenic mice capable of producing a human immunoglobulin gene but not an endogenous functional immunoglobulin (see, e.g., PCT Publication Nos. WO 1998 / 24893; WO 1992 / 01047; WO 1996 / 34096; WO 1996 / 33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598).

[0098] "Humanized" forms of non-human antibodies (e.g., murine or rat antibodies) are chimeric immunoglobulins which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody can also incorporate at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin consensus sequence. Methods for humanizing antibodies are known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Patents 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370; EP 239400; PCT publication WO 91 / 09967; U.S. Patent 5,225,539; EP 592106; EP 519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91 :969-973; and U.S. Patent 5,565,332.

[0099] Also provided are "conservative sequence modifications" of the sequences set forth in SEQ ID NOs described herein, i.e., nucleotide and amino acid sequence modifications that do not abrogate the binding of the antibody encoded by that nucleotide sequence or the antibody comprising that amino acid sequence to the antigen. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as conservative nucleotide and amino acid additions and deletions. For example, modifications can be introduced into SEQ ID NOs by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions that do not abrogate the binding of the antibody to the antigen. As will be appreciated by those skilled in the art, the substitutions of certain amino acids for other amino acids can result in the conservative modification of an antibody. Such modifications can be introduced into SEQ ID NOs by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitution groups include: (1) aliphatic: glycine, alanine, serine, threonine, valine, isoleucine, leucine; (2) polar: glycine, alanine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, asparagine, glutamine; (3) positively charged: lysine, arginine, histidine; (4) negatively charged: aspartic acid, glutamic acid; and (5) aromatic: phenylalanine, tyrosine, tryptophan. Substitution of like groups can be made without abrogating the binding of the antibody to the antigen. Methods of identifying conservative nucleotide and amino acid substitutions that do not abrogate antigen binding are known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0100] As used herein, the term "engraftment potential" is used to refer to the ability of hematopoietic stem cells and hematopoietic progenitor cells to repopulate a tissue, whether such cells are naturally circulating or provided by engraftment. The term includes all events surrounding or leading to engraftment, such as tissue homing of the cells and colonization of the cells within the target tissue. Engraftment efficiency or engraftment rate can be assessed or quantified using any clinically acceptable parameter known to one of skill in the art, and can include, for example, assessing competitive repopulating units (CRUs); adding or expressing a marker in the tissue in which the stem cells have homed, colonized, or engrafted; or by assessing the subject's progress in terms of disease progression, hematopoietic stem cell and progenitor cell survival, or recipient survival. Engraftment can also be determined by measuring white blood cell counts in the peripheral blood during the post-transplant period. Engraftment can also be assessed by measuring the extent to which donor cells repopulate bone marrow cells in a bone marrow aspirate sample.

[0101] As used herein, the term "hematopoietic stem cell" ("HSC") refers to immature blood cells with the ability to self-renew and differentiate into mature blood cells of multiple lineages, including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Such cells can include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are believed to comprise a subset of cells with the stem cell properties as defined above, but in mice, HSCs are CD34-. Moreover, HSCs also refer to long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). LT-HSCs and ST-HSCs are differentiated based on functional potential and based on cell surface marker expression. For example, human HSCs are CD34+, CD38, CD45RA, CD90+, CD49F+, and lin- (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD1 1B, CD19, CD20, CD56, CD235A). In mice, bone marrow LT-HSCs are CD34, SCA-1+, C-kit+, CD135, Slamfl / CD150+, CD48, and lin- (negative for mature lineage markers including Ter119, CD1 1b, Gr1, CD3, CD4, CD8, B220, IL7ra), while ST-HSCs are CD34+, SCA-1+, C-kit+, CD135, Slamfl / CD150+, and lin- (negative for mature lineage markers including Ter119, CD1 1b, Gr1, CD3, CD4, CD8, B220, IL7ra). Moreover, under homeostatic conditions, ST-HSCs are less quiescent and more proliferative than LT-HSCs. However, LT-HSCs have greater self-renewal potential (i.e., they can survive the entire adult life and can be serially transplanted through successive recipients), while ST-HSCs have limited self-renewal (i.e., they exist for only a limited time and do not have the potential for serial transplantation). Any of these HSCs can be used in the methods described herein. ST-HSCs are particularly useful because they are highly proliferative and thus can more rapidly generate differentiated progeny.

[0102] As used herein, the term "hematopoietic stem cell functional potential" refers to the functional properties of a hematopoietic stem cell, which include: 1) multipotency (which refers to the ability to differentiate into multiple different blood lineages, including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, T cells, and B cells), 2) self-renewal (which refers to the ability of a hematopoietic stem cell to generate daughter cells with equal potential to the parent cell, and this ability can occur repeatedly throughout the life of an individual without exhaustion), and 3) the ability of a hematopoietic stem cell or its progeny to be re-introduced into a transplant recipient, whereby they home to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis.

[0103] As used herein, the terms "subject" and "patient" refer to an organism (such as a human) who is receiving treatment for a particular disease or condition as described herein.

[0104] As used herein, the term "recipient" refers to a patient who receives a transplant, such as a transplant comprising a population of hematopoietic stem cells. The transplanted cells administered to the recipient can be, for example, autologous, syngeneic, or allogeneic cells.

[0105] As used herein, "to treat" or "treatment" refers to any improvement in the consequences of a disease, such as an increase in survival, a decrease in morbidity, and / or a decrease in side effects that are byproducts of alternative treatment modalities; as is readily appreciated in the art, complete eradication of a disease is preferred but not necessary for a treatment to be a treatment. Beneficial and desirable clinical outcomes include, but are not limited to, promoting engraftment of exogenous hematopoietic cells in a patient following conditioning therapy with an antibody as described herein and subsequent hematopoietic stem cell transplant therapy. Other beneficial outcomes include an increase in cell count or relative concentration of hematopoietic stem cells in a patient following conditioning therapy and subsequent administration of an exogenous hematopoietic stem cell graft to the patient. Beneficial outcomes of the therapies described herein can also include an increase in cell count or relative concentration of one or more cells of the hematopoietic cell lineage, such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, or B lymphocytes, following conditioning therapy and subsequent hematopoietic stem cell transplant therapy. Other beneficial outcomes can include a decrease in the number of pathogenic cell populations, such as autoimmune cell populations (e.g., CD45+ autoimmune lymphocytes, such as CD45+ T cells expressing T cell receptors cross-reactive with self-antigens). Insofar as the methods of the application are intended to prevent a disorder, it will be appreciated that the term "prevent" does not require complete prevention of the disease state. Rather, as used herein, the term prevent refers to the ability of one skilled in the art to identify a population susceptible to a disorder so that the compounds of the application can be administered prior to the onset of the disease. The term does not imply complete avoidance of the disease state.

[0106] As used herein, a patient in need of hematopoietic stem cell transplantation includes a patient suffering from an autoimmune disease described herein. Hematopoietic stem cells generally exhibit: 1) multipotency, and thus the ability to differentiate into multiple different blood cell lineages, including but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells), 2) self-renewal, and thus the ability to generate daughter cells of equal potential to their parent cell, and 3) the ability to rehome into a transplant recipient, whereby they home to the hematopoietic stem cell niche and reestablish productive and sustained hematopoiesis. Hematopoietic stem cells can thus be administered to a patient deficient or lacking in one or more cell types of the hematopoietic lineage in order to reconstitute the deficient or lacking cell population in vivo. In some embodiments, the subject has an autoimmune disease, such as rheumatoid arthritis (represented by proteoglycan-induced arthritis in mice (PGIA)), autoimmune encephalitis, graft-versus-host disease, scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, type I diabetes, or other autoimmune pathologies described herein. In some embodiments, the subject is in need of chimeric antigen receptor T cell (CART) therapy. In some embodiments, the subject has or is afflicted with a metabolic storage disease. The subject can have or be afflicted with a metabolic disease selected from the group consisting of glycogen storage disease, mucopolysaccharidosis, Gaucher's Disease, Hurler's Disease, sphingolipidosis, metachromatic leukodystrophy, or any other disease or condition that can benefit from the treatments or therapies described herein, and including but not limited to, severe combined immunodeficiency, Wiscott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and those described in "Bone Marrow Transplantation for Non-Malignant Disease," ASH Education Book, 1 :319-338 (2000), the disclosure of which is incorporated by reference herein as it relates to pathologies that can be treated by administration of hematopoietic stem cell transplant therapy.Additionally or alternatively, a patient "in need of hematopoietic stem cell transplantation" can be a patient who has or does not have one of the aforementioned pathologies, but who still exhibits a reduced level of one or more endogenous cells within the hematopoietic lineage (e.g., as compared to the level of other healthy subjects), such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes. One of skill in the art can readily determine whether a person has a reduced level of one or more of the aforementioned cell types or other blood cell types relative to other healthy subjects by, for example, flow cytometry and fluorescence-activated cell sorting (FACS) methods, as well as other methods known in the art.

[0107] As used herein, the phrase "stem cell disorder" refers broadly to any disease, disorder, or condition that can be treated or cured by conditioning a target tissue of a subject and / or by removing a population of endogenous stem cells from a target tissue (e.g., removing a population of endogenous hematopoietic stem cells or hematopoietic progenitor cells from the bone marrow of a subject) and / or by implanting or transplanting stem cells into a target tissue of a subject. Other diseases that can be treated using the patient conditioning and / or hematopoietic stem cell transplantation methods described herein include genetic blood disorders (e.g., sickle cell anemia) and autoimmune disorders (such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease). Other diseases that can be treated using the conditioning and / or transplantation methods described herein include myelodysplastic syndrome. In some embodiments, the subject has or otherwise is affected by a metabolic storage disorder. For example, the subject can have or be affected by a metabolic disease selected from the group consisting of glycogen storage disease, mucopolysaccharidosis, Gaucher's disease, Hurler's disease, sphingolipidosis, metachromatic leukodystrophy, or any other disease or disorder that can benefit from the treatments and therapies disclosed herein, including but not limited to, severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi syndrome, hereditary lymphohistiocytosis, osteosclerosis, osteogenesis imperfecta, storage disease, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and those described in "Bone Marrow Transplantation for Non-Malignant Disease," ASH Education Book, 1 :319-338 (2000), the disclosure of which is incorporated by reference herein in its entirety, particularly as it relates to pathologies that can be treated by administration of hematopoietic stem cell transplantation therapy.

[0108] As used herein, the term "vector" includes nucleic acid vectors, such as plasmids, DNA vectors, RNA vectors, viruses, or other suitable replicons. The expression vectors described herein can comprise polynucleotide sequences as well as other sequence elements, e.g., for expressing proteins and / or for integrating these polynucleotide sequences into the genome of a mammalian cell. Certain vectors useful for expressing the antibodies and antibody fragments of the application include plasmids containing regulatory sequences that direct transcription of the gene, such as promoter and enhancer regions. Other vectors useful for expressing the antibodies and antibody fragments include polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of mRNA from transcription of the genes. These sequence elements can include, e.g., 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of the genes carried on the expression vectors. The expression vectors described herein can also include polynucleotides that encode markers for selecting cells that comprise such vectors. Examples of suitable markers include genes that encode resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.

[0109] As used herein, the term "conjugate" or "antibody drug conjugate" or "ADC" refers to an antibody linked to a cytotoxin. An ADC is formed by the chemical coupling of a reactive functional group of one molecule, such as an antibody or antigen-binding fragment thereof, to a suitable reactive functional group of another molecule, such as a cytotoxin described herein. A conjugate can include a linker positioned between the two molecules that are coupled to one another, e.g., a linker positioned between an antibody and a cytotoxin. Examples of linkers that can be used to form conjugates include peptide-containing linkers, such as those comprising natural amino acids or non-natural amino acids, such as D-amino acids. Linkers can be prepared using various strategies described herein and known in the art. Depending on the reactive components therein, a linker can be cleaved by, e.g., enzymatic hydrolysis, thermolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide bond reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0110] As used herein, the "drug / antibody ratio" or "DAR" refers to the number of drugs (e.g., amatoxins) linked to an antibody in a conjugate. The DAR of an ADC can range from 1 to 8, although higher payloads are feasible depending on the number of attachment sites on the antibody. In certain embodiments, a conjugate has a DAR of 1, 2, 3, 4, 5, 6, 7, or 8.

[0111] As used herein, the term "microtubule binding agent" refers to a compound that exerts its effect by disrupting microtubular networks that are vital for mitotic cellular functions and interphase cellular functions in cells. Examples of microtubule binding agents include, but are not limited to, maytansinoids, and derivatives thereof, such as those described herein or known in the art; vinca alkaloids, such as vinblastine, vinblastine sulfate, vincristine, vincristine sulfate, vindesine, and vinorelbine; taxoids, such as docetaxel and paclitaxel; and macrolide antibiotics, such as discodermolide, colchicine, and epothilones, and derivatives thereof, such as epothilone B or derivatives thereof.

[0112] As used herein, the term "amatoxin" refers to a member of the amatoxin family of peptides produced by Amanita phalloides mushrooms or derivatives thereof, such as variants or derivatives thereof that are capable of inhibiting RNA polymerase II activity. Amatoxins that can be used in conjunction with the compositions and methods described herein include compounds described herein, e.g., a-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amaninonol, amanullin, or proamanullin. As described herein, amatoxins can be conjugated to an antibody or antigen-binding fragment thereof, e.g., via a linker moiety (L) (thereby forming an ADC). Exemplary amatoxin conjugation methods and linkers that can be used in such methods are described infra. Exemplary linker-containing amatoxins that can be used in conjugation with an antibody or antigen-binding fragment thereof according to the compositions and methods are also described infra.

[0113] As used herein, the term "acyl" refers to -C(=0)R, wherein R is hydrogen ("aldehyde"), C1-C12alkyl, C2-C12alkenyl, C2-C12alkynyl, C3-C7carbocyclyl, C6-C20aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl, as defined herein. Non-limiting examples include formyl, acetyl, propionyl, benzoyl, and propenoyl.

[0114] As used herein, the term "C1-C12alkyl" refers to a straight or branched chain, saturated hydrocarbon radical having from 1 to 12 carbon atoms. Representative C1-C12alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while branched C1-C12alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl. C1-C12alkyl groups can be unsubstituted or substituted.

[0115] As used herein, the term "alkenyl" refers to a C2-C12hydrocarbon group containing primary, secondary, or tertiary carbon atoms with at least one site of unsaturation (i.e., a carbon-carbon sp2double bond). Examples include, but are not limited to, ethylene or vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, and the like. The alkenyl group can be unsubstituted or substituted.

[0116] As used herein, "alkynyl" refers to a C2-C12hydrocarbon group containing primary, secondary, or tertiary carbon atoms with at least one site of unsaturation (i.e., a carbon-carbon sp triple bond). Examples include, but are not limited to, ethynyl and propynyl. The alkynyl group can be unsubstituted or substituted.

[0117] As used herein, "aryl" refers to a C6-C20carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthryl. The aryl group can be unsubstituted or substituted.

[0118] As used herein, "arylalkyl" refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom (typically a terminal carbon atom or is a sp3carbon atom) is replaced by an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenyleth-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthyleth-1-yl, 2-naphthylethen-1-yl, naphthobenzyl, 2-naphthobenzy-1-yl, and the like. The arylalkyl group contains 6-20 carbon atoms, e.g., the alkyl portion of the arylalkyl group (including alkyl, alkenyl, or alkynyl groups) is 1-6 carbon atoms and the aryl portion is 5-14 carbon atoms. The alkylaryl group can be unsubstituted or substituted.

[0119] As used herein, "cycloalkyl" refers to a saturated carbocyclic group, which can be monocyclic or bicyclic. The cycloalkyl group contains a ring having 3-7 carbon atoms as a monocyclic ring or 7-12 carbon atoms as a bicyclic ring. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The cycloalkyl group can be unsubstituted or substituted.

[0120] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic group, which can be monocyclic or bicyclic. The cycloalkenyl group contains a ring having 3-6 carbon atoms as a monocyclic ring or 7-12 carbon atoms as a bicyclic ring. Examples of monocyclic cycloalkenyl groups include 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl. The cycloalkenyl group can be unsubstituted or substituted.

[0121] As used herein, "heteroarylalkyl" refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom (typically the terminal carbon atom or an sp3carbon atom) is replaced by a heteroaryl group. Typical heteroarylalkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furyl ethyl, and the like. Heteroarylalkyl groups contain 6-20 carbon atoms, e.g., the alkyl portion of the heteroarylalkyl group (including alkyl, alkenyl, or alkynyl groups) is 1-6 carbon atoms, and the heteroaryl portion is 5-14 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S. The heteroaryl portion of the heteroarylalkyl group can be a monocyclic ring having 3-7 ring members (2-6 carbon atoms) or a bicyclic ring having 7-10 ring members (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S), e.g., a bicyclic [4,5], [5,5], [5,6], or [6,6] system.

[0122] As used herein, "heteroaryl" and "heterocycloalkyl" refer to aromatic or non-aromatic ring systems, respectively, in which one or more ring atoms are heteroatoms (e.g., nitrogen, oxygen, and sulfur). Heteroaryl or heterocycloalkyl groups contain 2-20 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S. Heteroaryl or heterocycloalkyl groups can be monocyclic rings having 3-7 ring members (2-6 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S) or bicyclic rings having 7-10 ring members (4-9 carbon atoms and 1-3 heteroatoms selected from N, O, P, and S), e.g., a bicyclic [4,5], [5,5], [5,6], or [6,6] system. Heteroaryl and heterocycloalkyl groups can be unsubstituted or substituted.

[0123] Heteroaryl and heterocycloalkyl groups are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W. A. Benjamin, New York, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), specifically Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.

[0124] Examples of heteroaryl groups include, for example and without limitation, pyridyl, thiazolyl, tetrahydrothienyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthene, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenoxazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, benzotriazolyl, benzisoxazolyl, and isatinoyl.

[0125] Examples of heterocycloalkyl groups include, for example and without limitation, dihydropyridinyl, piperidyl, tetrahydrothienyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinuclidinyl, and morpholinyl.

[0126] For example, but without limitation, carbon-bound heteroaryl and heterocycloalkyl groups are bound at the 2, 3, 4, 5, or 6 position of a pyridine, the 3, 4, 5, or 6 position of a pyridazine, the 2, 4, 5, or 6 position of a pyrimidine, the 2, 3, 5, or 6 position of a pyrazine, the 2, 3, 4, or 5 position of a furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, the 2, 4, or 5 position of an oxazole, imidazole, or thiazole, the 3, 4, or 5 position of an isoxazole, pyrazole, or isothiazole, the 2 or 3 position of an acridine, the 2, 3, or 4 position of an azetidine, the 2, 3, 4, 5, 6, 7, or 8 position of a quinoline, or the 1, 3, 4, 5, 6, 7, or 8 position of an isoquinoline. Still more typically, carbon-bound heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

[0127] For example, but not by way of limitation, nitrogen-bound heteroaryl and heterocycloalkyl groups are bound at the following positions: 1 -position of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole 1 -position, isoindole or isoindoline 2-position, morpholine 4-position, and carbazole or β-carboline 9-position. Still more typically, nitrogen-bound heterocycles include 1-aziridinyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0128] As used herein, and when applied to the above alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocycloalkyl, and the like, "substituted" means that one or more hydrogen atoms are each independently replaced with a substituent. Typical substituents include, but are not limited to, -X, -R, -OH, -OR, -SH, -SR, NH2, -NHR, -N(R)2, -N+(R)3, -CX3, -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO2, -N3, -NC(=O)H, -NC(=O)R, -C(=O)H, -C(=O)R, -C(=O)NH2, -C(=O)N(R)2, -SO3", -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NH2, -S(=O)2N(R)2, -S(=O)R, -OP(=O)(OH)2, -OP(=O)(OR)2, -P(=O)(OR)2, -PO3, -PO3H2, -C(=O)X, -C(=S)R, -CO2H, -CO2R, -CO2", -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NH2, -C(=O)N(R)2, -C(=S)NH2, -C(=S)N(R)2, -C(=NH)NH2, and -C(=NR)N(R)2; wherein each X is independently selected for each occurrence from F, Cl, Br, and I; each R is independently selected for each occurrence from C1-C12 alkyl, C6-C20 aryl, C3-C14 heterocycloalkyl or heteroaryl, a protecting group, and a prodrug moiety. When a group is described as being "optionally substituted" that group can be independently substituted with one or more of the above substituents for each occurrence.

[0129] It should be understood that certain group naming conventions can include either a monovalent group or a divalent group depending on the context. For example, when a substituent requires two points of attachment to the remainder of the molecule, it should be understood that the substituent is a divalent group. For example, substituents of alkyl groups that are identified as requiring two points of attachment include divalent groups such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, and the like. Other group naming conventions make it clear that the group is divalent, such as “alkylene,” “alkenylene,” “arylene,” “heterocycloalkylene,” and the like.

[0130] When a substituent is depicted as a divalent group (i.e., having two points of attachment to the remainder of the molecule), it should be understood that the substituent can be constructed with the attachment in either direction, unless otherwise noted.

[0131] “Isomerism” denotes compounds that have the same molecular formula but differ in the sequence of bonding of their atoms or in the spatial arrangement of their atoms. Isomers that differ in the spatial arrangement of their atoms are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed “diastereomers” those that are mirror images of one another are termed “enantiomers” or sometimes “optical isomers.”

[0132] A carbon atom bonded to four different substituents is termed a “chiral center.” “Chiral isomers” denote compounds having at least one chiral center. Compounds having more than one chiral center can exist as individual diastereomers or as mixtures of diastereomers (termed “diastereomeric mixtures”). When there is one chiral center, the stereoisomer can be characterized by the absolute configuration at that chiral center (R or S). Absolute configuration refers to the spatial arrangement of substituents bonded to the chiral center. The substituents bonded to the chiral center under consideration are ordered according to the sequence rules of Cahn, Ingold, and Prelog (Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116). A mixture containing equal amounts of individual enantiomeric forms having opposite chirality is termed a “racemic mixture.”

[0133] The compounds disclosed in the specification and claims can contain one or more asymmetric centers and can exist in different enantiomeric and / or diastereomeric forms. Unless otherwise specified, any compound described herein is intended to include all enantiomers, diastereomers, and mixtures thereof. Additionally, unless otherwise specified, any compound described herein is intended to include both individual enantiomers and mixtures of enantiomers, including rac mixtures, or other mixtures, as well as any

[0134] If possible, the compounds of any formula described herein include the compounds themselves and salts and solvates thereof. For example, salts can be formed between anions and groups on the compounds of the disclosure that carry a positive charge (e.g., amino groups). Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate). The term “pharmaceutically acceptable anion” refers to an anion suitable for use in forming a pharmaceutically acceptable salt. Similarly, salts can be formed between cations and groups on the compounds of the disclosure that carry a negative charge (e.g., carboxylate groups). Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and ammonium ion (such as tetramethylammonium ion). Examples of some suitable substituted ammonium ions are those in which the substituents are selected from the group consisting of ethyl, diethyl, dicyclohexyl, triethyl, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. The compounds of the disclosure also include those salts that contain a quaternary nitrogen ion.

[0135] Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, propionic, pyroglutamic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid or carboxymethyl cellulose.

[0136] Additionally, the compounds of the present disclosure (e.g., salts of the compounds) can exist in hydrated or unhydrated (anhydrous) form, or can exist as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, and the like. Non-limiting examples of solvates include ethanol solvates, acetone solvates, and the like. A "solvate" refers to a solvent addition form of a compound that retains the chemical integrity of the compound. This form is generally characterized by a substantially fixed melting point and a regular crystalline shape. Some compounds have a tendency, in one or more crystalline states, to trap a fixed molar ratio of solvent molecules in the solvent addition form as molecular complexes. If the solvent is water, the solvate formed is a hydrate; if the solvent is alcohol, the solvate formed is an alcoholate. Hydrates are formed by the combination of water molecules with one molecule of a substance, where the water retains its molecular state, i.e., H2O. Hydrates refer to, for example, monohydrates, dihydrates, trihydrates, and the like.

[0137] Further, for a compound represented by a chemical formula disclosed herein, or a salt thereof, polymorphism can exist. It is noted that any crystalline form, mixture of crystalline forms, or anhydride or hydrate thereof is encompassed within the scope of the present disclosure.

[0138] Anti-CD45 Antibodies

[0139] The present disclosure includes uses of anti-CD45 antibodies or antigen-binding portions thereof. Some embodiments include uses of anti-CD45 ADCs containing anti-CD45 antibodies or antigen-binding portions thereof. The present invention is based, in part, on the discovery that anti-CD45 antibodies and / or ADCs can be used as therapeutic agents to: (i) treat autoimmune diseases characterized by CD45+ cells, and (ii) promote engraftment of transplanted hematopoietic stem cells in patients in need of transplant therapy. These therapeutic activities can arise, for example, from the binding of anti-CD45 antibodies or ADCs to CD45-expressing foreign cells, thereby depleting the autoimmune cells. In this way, transplanted hematopoietic stem cells can successfully engraft in a patient, such as a human patient suffering from an autoimmune disease, as the patient will have targeted immune depletion, leading to reconstitution of the self-tolerant system.

[0140] CD45 is a hematopoietic cell-specific transmembrane protein tyrosine phosphatase that is important for T cell and B cell antigen receptor-mediated signaling. CD45 comprises a large extracellular domain and a cytoplasmic domain containing the phosphatase. CD45 can act both as a positive regulator and as a negative regulator, depending on the nature of the stimulus and the cell type involved. Although there can be a large number of permutations in the CD45 gene, only six isoforms have traditionally been recognized in humans. The isoforms are RA, RO, RB, RAB, RBC, and RABC (Hermiston et al., 2003“CD45: a critical regulator of signaling thresholds in immune cells.” Annu Rev Immunol. 2: 107-137.). CD45RA is expressed on naive T cells, and CD45RO is expressed on activated T cells and memory T cells, some B cell subsets, activated monocytes / macrophages, and granulocytes. CD45RB is expressed on peripheral B cells, naive T cells, thymocytes, weakly on macrophages and dendritic cells.

[0141] Antibodies and antigen-binding fragments capable of binding human CD45 (mRNA NCBI Reference Sequence: NM_080921.3, Protein NCBI Reference Sequence: NP_563578.2), including those capable of binding the isoform CD45RO, can be used in conjunction with the compositions and methods disclosed herein, such as for promoting engraftment of hematopoietic stem cell transplants in patients in need of hematopoietic stem cell transplant therapy. Multiple isoforms of CD45 arise from alternative splicing of 34 exons in the primary transcript. Splicing of exons 4, 5, 6, and possibly 7, leads to multiple CD45 variants.

[0142] Alternative splicing can result in the expression of individual exons or combinations of exons in various isoforms of the CD45 protein (e.g., CD45RA, CD45RAB, CD45RABC). In contrast, CD45RO lacks the expression of exons 4-6 and is produced from the combination of exons 1-3 and 7-34. There is evidence that exon 7 can also be excluded from the protein, resulting in the splicing together of exons 1-3 and 8-34. This protein, designated E3-8, has been detected at the mRNA level, but has not yet been identified by flow cytometry.

[0143] CD45RO is the only CD45 isoform now known to be expressed on hematopoietic stem cells. CD45RA and CD45RABC have not been detected or excluded from the phenotype of hematopoietic stem cells. There is evidence in studies performed on mice that CD45RB is expressed in fetal hematopoietic stem cells, but it is not present in adult bone marrow hematopoietic stem cells. Notably, CD45RC has a high rate of polymorphism in exon 6, which is found in approximately 25% of the Japanese population. This polymorphism results in high expression of CD45RO and reduced levels of CD45RA, CD45RB, and CD45RC. Additionally, CD45RA variants, such as CD45RAB and CD45RAC, show exon 4 polymorphisms, which are associated with autoimmune disease.

[0144] The presence of CD45RO on hematopoietic stem cells and its relatively limited expression on other immune cells, such as T lymphocyte subsets and B lymphocyte subsets, as well as various myeloid cells, makes CD45RO a particularly suitable target for conditioning therapy for patients in need of hematopoietic stem cell transplantation. Because CD45RO lacks only the expression of exons 4, 5, and 6, its use as an immunogen enables the screening of pan-CD45 antibodies and CD45RO-specific antibodies.

[0145] Anti-CD45 antibodies that can be used in conjunction with the patient conditioning methods described herein include anti-CD45 antibodies and their antigen-binding portions. The antigen-binding portion of an antibody is well known in the art and can be readily constructed based on the antigen-binding region of the antibody. In exemplary embodiments, the anti-CD45 antibody used in conjunction with the conditioning methods described herein can be a monoclonal antibody or its antigen-binding fragment, a polyclonal antibody or its antigen-binding fragment, a humanized antibody or its antigen-binding fragment, a fully human antibody or its antigen-binding fragment, a chimeric antibody or its antigen-binding fragment, a bispecific antibody or its antigen-binding fragment, a dual variable immunoglobulin domain, a single-chain Fv molecule (scFv), a double-chain antibody, a triple-chain antibody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment, an F(ab')2 molecule, or a tandem bi-scFv. Exemplary anti-CD45 antibodies that can be used in whole or in part in the ADCs or methods described herein are provided below.

[0146] In some implementations, the anti-CD45 antibody is derived from or is derived from clone HI30 or its humanized variant, which can be obtained commercially. (San Diego, CA). Humanization of antibodies can be performed according to procedures known in the art (e.g., as described in Example 7 below) by replacing the framework and constant region residues of a non-human antibody with those of a human germline antibody. Other anti-CD45 antibodies that can be used in conjunction with the methods described herein include anti-CD45 antibodies ab10558, EP322Y, MEM-28, ab10559, O.N.125, F10-89-4, HIE-1, 2B11, YTH24.5, PD7 / 26 / 16, F10-89-4, 1B7, ab154885, B-A11, phosphor S1007, ab170444, EP350, Y321, GA90, D3 / 9, X1 6 / 99, and LT45 and their humanized variants, all of which are commercially available. (Cambridge, MA). Other anti-CD45 antibodies that can be used in conjunction with the patient conditioning procedures described herein include the anti-CD45 antibody HPA000440 and its humanized variants, which are commercially available from SIGMA. (St. Louis, MO). Other anti-CD45 antibodies that can be used in conjunction with the patient conditioning methods described herein include the murine monoclonal antibody BC8 and humanized variants thereof, which are described, for example, in Matthews et al., Blood 78: 1864-1874, 1991, the disclosure of which is incorporated herein by reference for its teachings related to anti-CD45 antibodies. Other anti-CD45 antibodies that can be used in conjunction with the methods described herein include the monoclonal antibody YAML568 and humanized variants thereof, which are described, for example, in Glatting et al., J. Nucl. Med. 8: 1335-1341, 2006, the disclosure of which is incorporated herein by reference for its teachings related to anti-CD45 antibodies. Other anti-CD45 antibodies that can be used in conjunction with the patient conditioning procedures described herein include the monoclonal antibodies YTH54.12 and YTH25.4 and humanized antibodies thereof, which are described, for example, in Brenner et al., Ann. N.Y. Acad. Sci. 996: 80-88, 2003, the disclosure of which is incorporated herein by reference for its teachings related to anti-CD45 antibodies. Other anti-CD45 antibodies for use with the patient conditioning methods described herein include UCHL1, 2H4, SN130, MD4.3, MBI, and MT2 and humanized variants thereof, which are described, for example, in Brown et al., Immunology 64: 331-336, 1998, the disclosure of which is incorporated herein by reference for its teachings related to anti-CD45 antibodies. Other anti-CD45 antibodies that can be used in conjunction with the methods described herein include those produced and released from American Type Culture Collection (ATCC) Accession Nos. RA3-6132, RA3-2C2, and TIB122, as well as the monoclonal antibodies C363.16A and 13 / 2, which are described, for example, in Johnson et al., J. Exp. Med. 169: 1179-1184, 1989, the disclosure of which is incorporated herein by reference for its teachings related to anti-CD45 antibodies. Other anti-CD45 antibodies that can be used in conjunction with the patient conditioning methods described herein include the monoclonal antibodies AHN-12.1, AHN-12, AHN-12.2, AHN-12.3, AHN-12.4, HLe-1, and KC56 (T200), which are described, for example, in Harvath et al., J. Immunol. 146: 949-957, 1991, the disclosure of which is incorporated herein by reference for its teachings related to anti-CD45 antibodies.

[0147] Other anti-CD45 antibodies that can be used in conjunction with the patient conditioning methods described herein include those described, e.g., in U.S. Patent Nos. 7,265,212 (which describes, e.g., anti-CD45 antibodies 39E11, 16C9, and 1G10, and other clones); 7,160,987 (which describes, e.g., anti-CD45 antibodies produced and released by ATCC Accession No. HB-11873, such as monoclonal antibody 6G3); and 6,099,838 (which describes, e.g., anti-CD45 antibody MT3, and antibodies produced and released by ATCC Accession Nos. HB220 (also designated as MB23G2) and HB223), as well as US 2004 / 0096901 and US 2008 / 0003224 (which describe, e.g., anti-CD45 antibodies produced and released by ATCC Accession No. PTA-7339, such as monoclonal antibody 17.1), the disclosures of each of the foregoing are incorporated herein by reference for their disclosure of anti-CD45 antibodies.

[0148] Other anti-CD45 antibodies that can be used in conjunction with the patient conditioning methods described herein include antibodies produced and released by ATCC Accession Nos. MB4B4, MB23G2, 14.8, GAP 8.3, 74-9-3, I / 24.D6, 9.4, 4B2, M1 / 9.3.4.HL.2, and humanized variants and / or affinity matured variants thereof. Affinity maturation can be performed, e.g., using in vitro display techniques described herein or known in the art (such as phage display), as described in Example 6 below.

[0149] Other anti-CD45 antibodies that can be used in conjunction with the patient conditioning methods described herein include anti-CD45 antibody T29 / 33, which is described, e.g., in Morikawa et al., Int. J. Hematol. 54:495-504, 1991, the disclosure of which is incorporated herein by reference for its disclosure of anti-CD45 antibodies.

[0150] In certain embodiments, the anti-CD45 antibody is selected from apamistamab (also known as 90Y-BC8, Iomab-B, BC8; as described, e.g., in US20170326259, WO2017155937, and Orozco et al., Blood. 127.3 (2016): 352-359), or BC8-B10 (as described, e.g., in Li et al., PloS one 13.10 (2018): e0205135), each of which is incorporated by reference. Other anti-CD45 antibodies have been described, e.g., in WO2003 / 048327, WO2016 / 016442, US2017 / 0226209, US2016 / 0152733, US9,701,756; US2011 / 0076270, or US7,825,222, each of which is incorporated by reference in its entirety.

[0151] In some embodiments, the anti-CD45 antibody or antigen-binding fragment thereof specifically binds human CD45 at a region comprising the amino acid sequence RNGPHERYHLEVEAGNT (SEQ ID NO: 38). For example, in certain embodiments, the anti-CD45 antibody or antigen-binding fragment thereof specifically binds human CD45 at amino acid residues 486R, 493Y, and 502T of SEQ ID NO: 37 (corresponding to a fragment of the CD45 isoform of NP_002829.3), or at residues corresponding thereto in other human CD45 isoforms, that comprise the sequence RNGPHERYHLEVEAGNT (SEQ ID NO: 38; bolded residues indicate binding sites). In some embodiments, the anti-CD45 antibody or antigen-binding fragment thereof specifically binds the fibronectin domain (e.g., the fibronectin d4 domain) of human CD45.

[0152] In some embodiments, the isolated anti-CD45 antibody or antigen-binding portion thereof specifically binds an epitope of human CD45 comprising residues 486R, 493Y, and 502T of SEQ ID NO: 37, and also binds cynomolgous CD45 and / or rhesus CD45.

[0153] In some embodiments, the isolated anti-CD45 antibody or antigen-binding portion thereof specifically binds an epitope of human CD45 comprising the amino acid sequence RNGPHERYHLEVEAGNT (SEQ ID NO: 38), and also binds cynomolgous CD45 and rhesus CD45.

[0154] In some embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, specifically binds to an epitope of human CD45 comprising the amino acid sequence CRPPRDRNGPHERYHLEVEAGNTLVRNESHK (SEQ ID NO: 39), and binds to cynomolgus monkey CD45 and rhesus monkey CD45.

[0155] In some embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, specifically binds to an epitope of human CD45 comprising SEQ ID NO: 37; binds to at least one other amino acid, at least two other amino acids, at least three other amino acids, at least four other amino acids, or at least five other amino acids in the peptide comprising RNGPHERYHLEVEAGNT (SEQ ID NO: 38), wherein the other amino acid residues are not residues 486R, 493Y, and 502T of SEQ ID NO: 37; and the isolated anti-CD45 antibody, or antigen-binding portion thereof, also binds to cynomolgus monkey CD45 and rhesus monkey CD45.

[0156] In some embodiments, the present application provides an anti-CD45 antibody or antigen-binding fragment thereof comprising binding regions, e.g., CDRs or variable regions, corresponding to those of AbA. The heavy chain variable region (VH) amino acid sequence of AbA is set forth in SEQ ID NO: 1. The VH CDR domain amino acid sequences of AbA are set forth in SEQ ID NO: 2 (VH CDR1); SEQ ID NO: 3 (VH CDR2); and SEQ ID NO: 4 (VH CDR3). The light chain variable region (VL) amino acid sequence of AbA is set forth in SEQ ID NO: 5. The VL CDR domain amino acid sequences of AbA are set forth in SEQ ID NO: 6 (VL CDR1); SEQ ID NO: 7 (VL CDR2); and SEQ ID NO: 8 (VL CDR3). Thus, in certain embodiments, the anti-CD45 antibodies or antigen-binding fragments thereof provided herein comprise a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 1 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the anti-CD45 antibodies comprise a heavy chain comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NO: 2, 3, and 4, and a light chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NO: 6, 7, and 8.

[0157] In some embodiments, the present application provides an anti-CD45 antibody or antigen-binding fragment thereof comprising binding regions, e.g., CDRs or variable regions, corresponding to those of AbB. The heavy chain variable region (VH) amino acid sequence of AbB is set forth in SEQ ID NO: 9. The VH CDR domain amino acid sequences of AbB are set forth in SEQ ID NO: 10 (VH CDR1); SEQ ID NO: 11 (VH CDR2); and SEQ ID NO: 12 (VH CDR3). The light chain variable region (VL) amino acid sequence of AbB is set forth in SEQ ID NO: 13. The VL CDR domain amino acid sequences of AbB are set forth in SEQ ID NO: 14 (VL CDR1); SEQ ID NO: 15 (VL CDR2); and SEQ ID NO: 16 (VL CDR3). Thus, in certain embodiments, an anti-CD45 antibody or antigen-binding fragment thereof provided herein comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 9 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, an anti-CD45 antibody comprises a heavy chain comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NO: 10, 11, and 12, and a light chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NO: 14, 15, and 16.

[0158] In some embodiments, the present application provides an anti-CD45 antibody, or antigen-binding fragment thereof, comprising binding regions, e.g., CDRs or variable regions, corresponding to those of AbC. The heavy chain variable region (VH) amino acid sequence of AbC is set forth in SEQ ID NO: 17 (see Table 6). The VH CDR domain amino acid sequences of AbC are set forth in SEQ ID NO: 18 (VH CDR1); SEQ ID NO: 19 (VH CDR2); and SEQ ID NO: 20 (VH CDR3). The light chain variable region (VL) amino acid sequence of AbC is set forth in SEQ ID NO: 21 (see Table 6). The VL CDR domain amino acid sequences of AbC are set forth in SEQ ID NO: 22 (VL CDR1); SEQ ID NO: 23 (VL CDR2); and SEQ ID NO: 24 (VL CDR3). Thus, in certain embodiments, an anti-CD45 antibody, or antigen-binding fragment thereof, provided herein comprises a heavy chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 17 and a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, an anti-CD45 antibody comprises a heavy chain comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NO: 18, 19, and 20, and a light chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NO: 22, 23, and 24.

[0159] In certain embodiments, the antibody comprises a modified heavy chain (HC) variable region comprising a HC variable domain described in Table 6 or a variant of the HC variable region in Table 6 that: i) differs from a HC variable domain described in Table 6 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from a HC variable domain described in Table 6 by at most 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from a HC variable domain described in Table 6 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions; and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a HC variable domain described in Table 6, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions.

[0160] In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising a LC variable domain described in Table 6, or a variant thereof: (i) differing from a LC variable domain described in Table 6 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differing from a LC variable domain described in Table 6 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differing from a LC variable domain described in Table 6 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions; and / or (iv) comprising an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a LC variable domain described in Table 6, wherein in any of (i) to (iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions.

[0161] In certain embodiments, the anti-CD45 antibody comprises a CDR described herein in Table 6, wherein the CDR comprises one conservative amino acid substitution (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 binding specificity of the antibody (i.e., similar to the specificity of AbA, AbB, or AbC).

[0162] In certain embodiments, the anti-CD45 antibody is a deimmunized antibody based on the AbA, AbB, or AbC antibody or antigen-binding portion thereof. Deimmunized antibodies are antibodies whose V regions have been selected or altered to lack T cell epitopes or to remove T cell epitopes, thereby minimizing or eliminating the potential for the antibody to be immunogenic. In certain embodiments, the anti-CD45 antibody is deimmunized by selecting or engineering the framework domains to be free of T cell epitopes that, if present in the antibody sequence, would enable a human subject to mount a HAHA / HAMA response against the anti-CD45 antibody, resulting in an immune-mediated, adverse event- or treatment-effect-reducing reaction in the human subject. The antibodies disclosed herein (i.e., the AbA, AbB, and AbC variable and CDR sequences) can be used as the parent sequence from which a deimmunized antibody can be derived.

[0163] Other anti-CD45 antibodies are provided in International Patent Application Nos. PCT / US2019 / 058973 and PCT / US2019 / 058971, the entire contents of each of which are incorporated herein by reference.

[0164] Other anti-CD45 antibodies and antigen-binding portions thereof can be generated using art-recognized methods, including but not limited to methods that recognize the antibodies described herein (e.g., high-throughput screening of antibody libraries, phage display, computer modeling, etc.).

[0165] The disclosures of each of the publications herein are hereby incorporated by reference in their entirety.

[0166] As known in the art, the antibodies or antigen-binding fragments thereof described herein and used in the ADCs described herein can include modifications and / or mutations that alter the properties of the antibodies and / or fragments, such as those that increase half-life, increase or decrease ADCC, etc.

[0167] In some embodiments, the ADC comprises a bispecific antibody or antigen-binding portion thereof that specifically binds CD45 and another target antigen. Some embodiments comprise a bispecific binding agent comprising an antigen-binding portion comprising an anti-CD45 antibody or antigen-binding portion thereof, e.g., a half-antibody, scFv, Fab, Fab', Fab, Fab', bis-scFv, tandem bis-scFv, tri-scFv, tandem tri-scFv, Fv, disulfide linked Fv, DART, single domain antibody (sdAb), diabody, tandem diabody, triabody, or tandem triabody, or portions thereof. In some embodiments, the anti-CD45 antibody or antigen-binding portion thereof is a deimmunized anti-CD45 antibody or antigen-binding portion thereof. In some embodiments, the anti-CD45 antibody or antigen-binding portion thereof is a chimeric anti-CD45 antibody or antigen-binding portion thereof. In other embodiments, the anti-CD45 antibody or antigen-binding portion thereof is a humanized anti-CD45 antibody or antigen-binding portion thereof. In other embodiments, the anti-CD45 antibody or antigen-binding portion thereof is a fully human anti-CD45 antibody or antigen-binding portion thereof. In some embodiments, the anti-CD45 binding agents described herein or portions thereof can be incorporated into an anti-CD45 bispecific binding agent. For example, in some embodiments, the anti-CD45 bispecific binding agent can comprise the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region sequence of SEQ ID NO: 5. In some embodiments, the anti-CD45 bispecific binding agent can comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, and a light chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 6, 7, and 8.

[0168] In some embodiments, the anti-CD45 bispecific binding agent can comprise a heavy chain variable region sequence of SEQ ID NO: 9 and a light chain variable region sequence of SEQ ID NO: 13. In some embodiments, the anti-CD45 bispecific binding agent can comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 10, 11, and 12, and a light chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 14, 15, and 16. In some embodiments, the anti-CD45 bispecific binding agent can comprise a heavy chain variable region sequence of SEQ ID NO: 17 and a light chain variable region sequence of SEQ ID NO: 21. In some embodiments, the anti-CD45 bispecific binding agent can comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 18, 19, and 20, and a light chain variable region comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences set forth in SEQ ID NOs: 22, 23, and 24.

[0169] Additionally or alternatively, other anti-CD45 antibodies or binding agents known in the art or identified using the methods described herein can also be used in the bispecific binding agents described herein.

[0170] Fc-modified antibodies

[0171] In certain embodiments, the antibodies and / or ADCs used in the present application can comprise an anti-CD45 antibody or antigen-binding portion thereof having an Fc modification, wherein the Fc modification allows for Fc silencing. The Fc-modified antibodies or ADCs described herein not only allow for selective depletion of endogenous hematopoietic stem cells, but also reduce the cytotoxic effects on exogenous hematopoietic stem cell grafts, thereby further facilitating engraftment of the hematopoietic stem cell grafts.

[0172] The antibodies or binding fragments described herein can also include modifications and / or mutations that alter the properties of the antibodies and / or fragments, such as those that increase half-life or increase or decrease ADCC.

[0173] In some embodiments, antibodies comprising one or more radiolabeled amino acids are provided. Radiolabeled antibodies can be used for both diagnostic and therapeutic purposes (conjugation with radiolabeled molecules is another possible feature). Non-limiting examples of labels for polypeptides include, but are not limited to,3H,14C,15N,35S,90Y,99Tc, and125I,131I, and186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, e.g., Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686 (2nd ed. Chafner and Longo eds. Lippincott Raven (1996)), and U.S. Pat. No. 4,681,581, U.S. Pat. No. 4,735,210, U.S. Pat. No. 5,101,827, U.S. Pat. No. 5,102,990 (U.S. RE 35,500), U.S. Pat. No. 5,648,471, and U.S. Pat. No. 5,697,902. For example, a radioisotope can be conjugated by the chloroamine-T method.

[0174] In some embodiments, the anti-CD45 ADC comprises an anti-CD45 antibody or binding fragment thereof comprising a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification relative to a wild-type Fc region such that the molecule has altered affinity for or binds to a Fcgamma R (FcγR). Certain amino acid positions within the Fc region are known to be in direct contact with FcγRs through crystallographic studies. 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). (See Sondermann et al., 2000 Nature, 406:267-273). The antibodies described herein can comprise a variant Fc region comprising at least one residue modification that is in direct contact with a FcγR based on structural and crystallographic analysis. In some embodiments, the Fc region of the anti-CD45 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index of Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH 1, MD (1991), which is expressly incorporated by reference. "EU index as in Kabat" refers to the numbering of the human IgGl EU antibody. In some embodiments, the Fc region comprises a D265A mutation. In some embodiments, the Fc region comprises a D265C mutation. In some embodiments, the Fc region of the antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index as in Kabat.

[0175] In some embodiments, the Fc region comprises a mutation at the amino acid position of D265, V205, H435, I253, and / or H310. For example, specific mutations at these positions include D265C, V205C, H435A, I253A, and / or H310A.

[0176] In some embodiments, the Fc region comprises a L234A mutation. In some embodiments, the Fc region of the anti-CD45 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 235 according to the EU index as in Kabat. In some embodiments, the Fc region comprises a L235A mutation. In still another embodiment, the Fc region comprises L234A and L235A mutations. In a further embodiment, the Fc region comprises D265C, L234A, and L235A mutations. In still a further embodiment, the Fc region comprises D265C, L234A, L235A, and H435A mutations. In a further embodiment, the Fc region comprises D265C and H435A mutations.

[0177] In some embodiments, the anti-CD45 antibodies herein comprise an Fc region comprising one of the following modifications or combinations of modifications: D265A, D265C, D265C / H435A, D265C / LALA, D265C / LALA / H435A, D265C / N297G, D265C / N297G / H435A, D265C(IgG2*), D265C(IgG2) / H435A, D265C / N297Q / H435A, D265C / N297Q, EPLVLAdelG / H435A, N297A, N297G, or N297Q.

[0178] Binding or affinity between a modified Fc region and an Fc gamma receptor can be determined using various techniques known in the art, such as, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al., Analytical Biochemistry, Vol. 373: 52-60, 2008; or radioimmunoassay (RIA)), or by surface plasmon resonance assay or other kinetic-based determination mechanisms (e.g., Biacore®, analytical or OCTET TM analytical (forteBIO)), as well as other methods such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods can utilize a tag on one or more of the components being examined and / or employ various detection methods, including but not limited to chromogenic, fluorescent, luminescent, or isotopic tags. Detailed descriptions of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Edition, Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions. One example of a competitive binding assay is a radioimmunoassay, which involves incubating a labeled antigen with the target antibody in the presence of increasing amounts of unlabeled antigen, and detecting the antibody bound to the labeled antigen. The affinity of the target antibody for the particular antigen and the rate of dissociation of the binding can be determined from the data by scatchard plot analysis. Radioimmunoassay can also be used to determine competition with a second antibody. In this case, the antigen and the target antibody conjugated to a labeled compound are incubated in the presence of increasing amounts of unlabeled second antibody.

[0179] In some embodiments, an antibody having an Fc modification described herein (e.g., D265C, L234A, L235A, and / or H435A) has at least 70% reduction, at least 80% reduction, at least 90% reduction, at least 95% reduction, at least 98% reduction, at least 99% reduction, or 100% reduction in binding to an Fc gamma receptor relative to the binding of the same antibody comprising an unmodified Fc region (e.g., as assessed by biolayer interferometry (BLI)).

[0180] Fc region binding interactions with Fc gamma receptors are important for various effector functions and downstream signaling events, including but not limited to antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Thus, in certain aspects, antibodies comprising a modified Fc region (e.g., comprising L234A, L235A, and / or D265C mutations) have significantly reduced or ablated effector functions. Effector functions can be determined using various methods known in the art, e.g., by measuring cellular responses (e.g., mast cell degranulation or cytokine release) in response to the subject antibody. For example, using standard methods in the art, the ability of an Fc-modified antibody to trigger mast cell degranulation or its ability to trigger cytokine release (e.g., cytokine release by human peripheral blood mononuclear cells) can be determined.

[0181] Thus, in some embodiments, the Fc region comprises a mutation that results in a reduced half-life (e.g., relative to an antibody having an unmodified Fc region). Antibodies having a short half-life can be advantageous in certain situations where the antibody is intended to be a short-term therapeutic, e.g., in the context of the conditioning steps described herein, where HSCs are administered after the antibody. Ideally, the antibody will be substantially cleared prior to delivery of the HSCs, which typically also express the target antigen (e.g., CD45), but unlike the endogenous stem cells, are not targets of the anti-CD45 antibody. In some embodiments, the Fc region comprises a mutation at position 435 (EU index according to Kabat). In some embodiments, the mutation is a H435A mutation.

[0182] In some embodiments, an anti-CD45 described herein has a half-life (e.g., in humans) that is equal to or less than 24 hours, equal to or less than 23 hours, equal to or less than 22 hours, equal to or less than 21 hours, equal to or less than 20 hours, equal to or less than 19 hours, equal to or less than 18 hours, equal to or less than 17 hours, equal to or less than 16 hours, equal to or less than 15 hours, equal to or less than 14 hours, equal to or less than 13 hours, equal to or less than 12 hours, or equal to or less than 11 hours.

[0183] In some embodiments, the anti-CD45 antibodies described herein have a half-life of 1-5 hours, 5-10 hours, 10-15 hours, 15-20 hours, or 20-25 hours (e.g., in humans).

[0184] In some aspects, the Fc region comprises two or more mutations that confer reduced half-life and reduced effector function of the antibody. In some embodiments, the Fc region comprises a mutation that results in reduced half-life and a mutation of at least one residue that can directly contact an FcyR (e.g., based on structural and crystallographic analysis). In some embodiments, the Fc region comprises a H435A mutation, a L234A mutation, and a L235A mutation. In some embodiments, the Fc region comprises a H435A mutation and a D265C mutation. In some embodiments, the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, and a D265C mutation.

[0185] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a cytotoxin (e.g., an amatoxin) via a cysteine residue in the Fc domain of the antibody or antigen-binding fragment thereof. In some embodiments, the cysteine residue is introduced via a mutation in the Fc domain of the antibody or antigen-binding fragment thereof. For example, the cysteine residue can be selected from the group consisting of Cys118, Cys239, and Cys265. In some embodiments, the Fc region of the anti-CD45 antibody (or fragment thereof) comprises an amino acid substitution at amino acid 265 according to the EU index as in Kabat. In some embodiments, the Fc region comprises a D265C mutation. In some embodiments, the Fc region comprises a D265C and a H435A mutation. In some embodiments, the Fc region comprises a D265C, a L234A, and a L235A mutation. In some embodiments, the Fc region comprises a D265C, a L234A, a L235A, and a H435A mutation.

[0186] It is noted that, unless otherwise specified, Fc amino acid positions are referenced to the EU numbering index.

[0187] The disclosure of each of the aforementioned publications is hereby incorporated by reference for its pertinent description, as it pertains to Fc regions. Antibodies and antigen-binding fragments that can be used in conjunction with the compositions and methods described herein include the antibodies described above and antigen-binding fragments thereof, as well as variants of those non-human antibodies and antigen-binding fragments described above and those antibodies or antigen-binding fragments that assess, by competitive antigen-binding assays, to bind to the same epitope as the antibodies or antigen-binding fragments described above.

[0188] Methods for engineering antibodies to include any of the Fc modifications herein are well known in the art. These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a manufactured DNA molecule encoding the antibody, or at least the constant region of the antibody. Site-directed mutagenesis is well known in the art (see, e.g., Carter et al., Nucleic Acids Res., 13:4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA, 82:488 (1987)). PCR mutagenesis is also suitable for making amino acid sequence variants of the starting polypeptide. See Higuchi, in PCR Protocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17:723-733 (1989). Another method for making sequence variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene, 34:315-323 (1985).

[0189] Methods of identifying antibodies

[0190] High-throughput screening methods of libraries of antibodies or antibody fragments capable of binding CD45 expressed by hematopoietic stem cells can be used to identify anti-CD45 antibodies that can be used to treat autoimmune diseases and condition patients in need of hematopoietic stem cells, e.g., human patients, as described herein. Such methods include in vitro display technologies known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display, among others.

[0191] The use of phage display for the isolation of antibodies or antigen-binding fragments that bind to a molecule of interest has been reviewed, for example, in Felici et al., Biotechnol. Annual Rev. 1 : 149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26: 27-45, 1997; and Hoogenboom et al., Immunotechnology 4: 1-20, 1998, the disclosures of each of which are incorporated herein by reference for their teachings relating to in vitro display technology. Random combinatorial peptide libraries have been constructed to select polypeptides that bind to cell surface antigens, as described in Kay, Perspect. Drug Discovery Des. 2: 251-268, 1995 and Kay et al., Mol. Divers. 1 : 139-140, 1996, the disclosures of each of which are incorporated herein by reference for their teachings relating to the discovery of antigen-binding molecules. Proteins, such as multimeric proteins, have been successfully displayed as functional molecules by phage (see, e.g., EP 0349578; EP 4527839; and EP 0589877, and Chiswell and McCafferty, Trends Biotechnol. 10: 80-84 1992, the disclosures of each of which are incorporated herein by reference for their teachings relating to the use of in vitro display technology for the discovery of antigen-binding molecules. In addition, functional antibody fragments, such as Fab and scFv fragments, have been expressed in in vitro display formats (see, e.g., McCafferty et al., Nature 348: 552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88: 7978-7982, 1991; and Clackson et al., Nature 352: 624-628, 1991, the disclosures of each of which are incorporated herein by reference for their teachings relating to in vitro display platforms for the discovery of antigen-binding molecules). Human anti-CD45 antibodies can also be generated in, for example, HUMAB- or XENOMOUSE TM In addition to these, these technologies can be used to identify and improve the affinity of antibodies that are capable of binding CD45 expressed by hematopoietic stem cells for use in depleting endogenous hematopoietic stem cells in a patient (e.g., a human patient) in need of hematopoietic stem cell transplant therapy.

[0192] In addition to in vitro display techniques, computer modeling techniques can be used to design and identify antibodies that are capable of binding to an antigen expressed by hematopoietic stem cells or immune cells (e.g., CD45). For example, using computer modeling techniques, one of skill in the art can screen a library of antibodies or antibody fragments in a silicon chip to find molecules that are capable of binding to a specific epitope on an antigen expressed by hematopoietic stem cells or immune cells (e.g., CD45), such as an extracellular epitope of the antigen.

[0193] Other techniques can be used to identify antibodies or antibody fragments that are capable of binding to CD45 expressed by hematopoietic stem cells and are internalized by the cells (via receptor-mediated endocytosis). For example, the in vitro display techniques described above can be adapted to screen for antibodies or antibody fragments that bind to CD45 and are subsequently internalized. Phage display represents one such technique that can be used in conjunction with such a screening modality. To identify anti-CD45 antibodies or antibody fragments that are internalized by hematopoietic stem cells or immune cells, one of skill in the art can use the phage display techniques described in Williams et al., Leukemia 19: 1432-1438, 2005, the disclosure of which is incorporated by reference in its entirety. For example, using mutagenesis methods known in the art, one can produce a recombinant phage library encoding antibodies, antibody fragments (such as scFv fragments, Fab fragments, diabodies, triabodies, and 10 Fn3 domains, etc.) or ligands comprising random amino acid boxes (e.g., in one or more or all CDRs or equivalent regions thereof or antibodies or antibody fragments). The framework regions, hinge regions, Fc domains, and other regions of the antibodies or antibody fragments can be designed to be non-immunogenic in humans, e.g., by having human germline antibody sequences or sequences that show only minor variations relative to human germline antibodies.

[0194] Using phage display techniques described herein or known in the art, a phage library containing random antibodies or antibody fragments covalently bound to phage particles can be incubated with CD45, for example, by first incubating the phage library with a blocking agent (e.g., e.g., milk protein, bovine serum albumin, and / or IgG) to remove phage encoding antibodies or antibody fragments that display non-specific protein binding and phage encoding antibodies or fragments thereof that bind the Fc domain, followed by incubating the phage library with a population of cells expressing CD45 (e.g., a population of hematopoietic stem cells). The phage library can be incubated with the hematopoietic stem cells for a time sufficient to allow anti-CD45 antibodies or antibody fragments to bind to the cognate cell surface antigen and then be internalized by the hematopoietic stem cells (e.g., 30 minutes to 6 hours of incubation at 4°C, such as 1 hour of incubation at 4°C). Phage comprising antibodies or antibody fragments that do not display sufficient affinity for CD45 to allow binding to and internalization by the hematopoietic stem cells can then be removed by washing the cells, e.g., with cold (4°C) 0.1 M glycine buffer at pH 2.8. Phage bound to antibodies or antibody fragments that have been internalized by the hematopoietic stem cells can be identified by lysing the cells and recovering the internalized phage from the cell culture medium. The phage can then be amplified in bacterial cells, e.g., by incubating bacterial cells with the recovered phage in 2 x YT medium using methods known in the art. Phage recovered from the medium can then be characterized, e.g., by determining the nucleic acid sequence of the gene encoding the antibody or antibody fragment inserted into the phage genome. The encoded antibody or antibody fragment can then be prepared de novo by chemical synthesis (e.g., chemical synthesis of antibody fragments such as svFv fragments) or by recombinant expression (e.g., recombinant expression of full-length antibodies).

[0195] The internalization capacity of the prepared antibodies or antibody fragments can be assessed, e.g., using radionuclide internalization assays known in the art. For example, a radioisotope such as 18 F、 75 Br、 77 Br、 122 I、 123 I、 124 I、 125 I、 129 I、 131 I、 211 At、 67 Ga、 111 In、 99 Tc、 169 Yb、 186 Re、 64 Cu、 67 Cu、 177 Lu、 77 As、 72 As、86 Y, 90 Y, 89 Zr, 212 Bi, 213 Bi or 225 Ac to identify anti-CD45 antibodies or antibody fragments identified using in vitro display techniques described herein or known in the art. For example, a radioactive halogen such as 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 At can be added to the antibody or antibody fragment. The radiolabeled antibody, fragment thereof, or ADC can be incubated with the hematopoietic stem cells for a time sufficient to allow internalization (e.g., 30 minutes to 6 hours at 4°C, e.g., 1 hour at 4°C). The cells are then washed to remove uninternalized antibody or fragment thereof (e.g., washed using cold (4°C) 0.1 M glycine buffer at pH 2.8). Internalized antibody or antibody fragment can be identified by detecting the radiation (e.g., gamma-radiation) emitted by the resulting hematopoietic stem cells compared to the radiation (e.g., gamma-radiation) emitted by the recovered wash buffer. The internalization assay above can also be used to characterize ADCs.

[0196] Antibodies can be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In some embodiments, an isolated nucleic acid encoding an anti-CD45 antibody described herein is provided. Such a nucleic acid can encode an amino acid sequence comprising the VL of an antibody and / or an amino acid sequence comprising the VH of an antibody (e.g., a light chain and / or a heavy chain of an antibody). In further embodiments, one or more vectors (e.g., expression vectors) comprising such a nucleic acid are provided. In further embodiments, a host cell comprising such a nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody. In some embodiments, the host cell is a eukaryotic animal cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In some embodiments, a method of making an anti-CLL-1 antibody is provided, wherein the method comprises culturing a host cell provided above comprising a nucleic acid encoding an antibody under conditions suitable for expression of the antibody, and optionally, recovering the antibody from the host cell (or host cell culture medium).

[0197] For recombinant production of an anti-CD45 antibody, a nucleic acid encoding the antibody as described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are specific to the heavy and light chain of the antibody).

[0198] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237; 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell paste as a soluble fraction and can be further purified.

[0199] Vertebrate cells can also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293 cells, e.g., as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney (BHK); mouse Sertoli cells (TM4 cells, e.g., as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980) and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). In some embodiments, the host cell is a eukaryotic animal cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cells).

[0200] In certain embodiments, an antigen binding protein (a moiety that targets an antigen), such as a ligand or a functionally active fragment thereof, can be used in the conjugates or fusion proteins described herein. For example, stem cell factor (SCF) is a ligand for CD117, where SCF can be conjugated or fused to a toxin to achieve the opsonization methods described herein.

[0201] In certain embodiments, antibody mimetics are used as the antigen-targeting moiety in the compositions and methods described herein. Examples of antibody mimetics include, but are not limited to, adnectins, affibodies, afflins, affimers, affitins, and alphabodies and anticalins, aptamers, armadillo repeat protein-based scaffolds, atrimers, avimers, DARpins, fynomers, knottins, Kunitz domain peptides, monobodies, and nanofitins.

[0202] Antibody drug conjugates (ADCs)

[0203] The antibodies or antigen-binding fragments thereof described herein can be conjugated (linked) to a cytotoxin via a linker. In some embodiments, the cytotoxic molecule is conjugated to the intracellularly internalizing antibodies or antigen-binding fragments thereof disclosed herein such that, upon cellular uptake of the antibody or fragment thereof, the cytotoxin can access its intracellular target and mediate hematopoietic cell death.

[0204] Cytotoxins

[0205] Various cytotoxins can be conjugated to the anti-CD45 antibodies via a linker for use in the therapies described herein. Specifically, the anti-CD45 ADCs include an anti-CD45 antibody (or antigen-binding fragment thereof) conjugated (i.e., covalently linked by a linker) to a cytotoxic moiety (or cytotoxin).

[0206] In various embodiments, the cytotoxic moiety exhibits reduced or no cytotoxicity when bound in the conjugate, but reverts to cytotoxicity after cleavage from the linker. In various embodiments, the cytotoxic moiety retains cytotoxicity without cleavage from the linker. In some embodiments, the cytotoxic molecule is conjugated to the intracellularly internalizing antibodies or antigen-binding fragments thereof disclosed herein such that, upon cellular uptake of the antibody or fragment thereof, the cytotoxin can access its intracellular target and, for example, mediate T cell death.

[0207] Accordingly, the ADCs of the present application can have the general formula Ab-(Z-L-D) n ,

[0208] wherein the antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently linked) to a linker (L) through a chemical moiety (Z), and is linked to a cytotoxic moiety (“drug”, D).

[0209] Accordingly, the anti-CD45 antibody or antigen-binding fragment thereof can be conjugated to a plurality of drug moieties, as indicated by the integer n, which represents the average number of cytotoxins per antibody, which can range, for example, from about 1 to about 20. In some embodiments, n is 1-4. In some embodiments, n is 1. The average number of drug moieties per antibody in the process of preparing the ADC from the conjugation reaction can be characterized by conventional means, such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of ADCs in terms of n can also be determined. In some cases, separation, purification, and characterization of homogenous ADCs, where n is a certain value with other drug loads, can be achieved by, for example, reverse phase HPLC or electrophoresis.

[0210] For some anti-CD45 ADCs, there can be a limitation on the number of attachment sites on the antibody. For example, when the attachment is a cysteine thiol, the antibody can have only one or a few cysteine thiol groups, or can have only one or a few sufficiently reactive thiol groups available for attachment of the linker. Typically, an antibody will not contain many free and reactive cysteine thiol groups that can be attached to a drug moiety; primarily, cysteine thiol residues in antibodies exist as disulfide bridges. In certain embodiments, the antibody can be reduced with a reducing agent, such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partial or full reducing conditions to generate reactive cysteine thiol groups. In certain embodiments, a higher drug load (DAR), e.g., n > 5, can result in aggregation, insolubility, toxicity, or loss of cell permeability of certain antibody drug conjugates.

[0211] In certain embodiments, less than the theoretical maximum of drug moieties are conjugated to the antibody in the conjugation reaction. The antibody can contain, for example, lysine residues that do not react with the drug-linker intermediate or linker reagent, as discussed below. Only the most reactive lysine groups can react with the amine-reactive linker reagent. In certain embodiments, the antibody is subjected to denaturing conditions to reveal reactive affinity groups, such as lysines or cysteines.

[0212] The loading (drug / antibody ratio) of the ADC can be controlled in different ways, for example, by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to the antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reduction conditions for cysteine thiol modification, (iv) engineering the amino acid sequence of the antibody by recombinant techniques to modify the number and location of cysteine residues to control the number and / or location of linker-drug attachments.

[0213] Cytotoxins suitable for use in the compositions and methods described herein include DNA intercalators (e.g., anthracyclines), agents capable of disrupting mitotic spindle apparatus (e.g., vinca alkaloids, maytansinoids, and derivatives thereof), RNA polymerase inhibitors (e.g., amatoxins (e.g., a-amanitin) and derivatives thereof), and agents capable of disrupting protein biosynthesis (e.g., agents displaying rRNA N-glycosidase activity, such as saporin and ricin A-chain), and other agents known in the art.

[0214] In some embodiments, the cytotoxin is a microtubule binding agent (e.g., a maytansinoid or a derivative thereof), an amatoxin, a Pseudomonas exotoxin A, deBouganin, diphtheria toxin, saporin, an auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, a pyrrolobenzodiazepine pyrrolobenzodiazepine dimer, an indolinobenzodiazepine indolinobenzodiazepine dimer, or a variant thereof, or another cytotoxic compound described herein or known in the art.

[0215] In some embodiments, the cytotoxin of the antibody drug conjugate is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is an amatoxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody drug conjugates disclosed herein is an amatoxin or a derivative thereof, such as a-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amadinone, amaninol, amanullin, or a derivative thereof.

[0216] Further details of cytotoxins that can be used in the anti-CD45 ADCs useful in the compositions and methods of the application are described infra.

[0217] Amatoxin

[0218] In some embodiments, the cytotoxin of the antibody drug conjugate is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is an amatoxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody drug conjugates disclosed herein is an amatoxin or a derivative thereof, such as a-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amadinone, amaninol, amanullin, or a derivative thereof.

[0219] In some embodiments, the cytotoxin of the antibody drug conjugate is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is an amatoxin or a derivative thereof.

[0220] In some embodiments, the cytotoxin is an amatoxin or derivative thereof, such as a-amanitin, b-amanitin, g-amanitin, e-amanitin, amanin, amaninamide, amanullin, hemi-synthetic amanin, and proamanullin. The structures of various natural amatoxins are represented by Formula III and Table 1, and are disclosed in, e.g., Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459.

[0221]

[0222] Table 1. Table of natural amatoxin structures.

[0223] Name [R1] [R2] [R3, R4] [R5] [R6, R7] [R8] [R9] α-Amanitin OH OH H OH H <![CDATA[NH2]]> OH β-Amanitin OH OH H OH H OH OH γ-Amanitin OH H H OH H <![CDATA[NH2]]> OH ε-Amanitin OH H H OH H OH OH Amanin OH OH H H H OH OH Amaninamide OH OH H H H <![CDATA[NH2]]> OH Amanullin H H H OH H <![CDATA[NH2]]> OH OH H H H Amanullinic Acid H OH OH OH H H H Proamanullin H ​ H

[0224] Amatoxins can be isolated from various mushroom species (e.g., Amanita phalloides, Galerina marginata, Lepiota brunneo-incarnata), or can be prepared semi-synthetically and synthetically. One member of the family, a-amanitin, is described in Wieland, Int. J. Pept. Protein Res. 1983, 22(3):257-276. Derivatives of amatoxins can be obtained by chemical modification of the natural compounds (“semi-synthetically”), or can be obtained from wholly synthetic sources. Synthetic routes to various amatoxin derivatives are disclosed in, e.g., U.S. Patent No. 9,676,702 and Perrin et al., J. Am. Chem. Soc. 2018, 140, pp. 6513-6517, each of which is incorporated by reference herein in its entirety, for synthetic methods of making and derivatizing amatoxins.

[0225] Many positions on an amatoxin or derivative thereof can be used as the site for covalently binding a linking moiety L, thereby linking an antibody or antigen-binding fragment thereof. In some embodiments, the cytotoxin in the ADCs disclosed herein is an amatoxin or derivative thereof represented by Formula (IV):

[0226]

[0227] wherein R1is H, OH, or OR A ;

[0228] R2is H, OH, or OR B ;

[0229] R A and R BR3is H or R

[0230] R3is H or R D ;

[0231] R4is H, OH, OR D or R D ;

[0232] R5is H, OH, OR D or R D ;

[0233] R6is H, OH, OR D or R D ;

[0234] R7is H, OH, OR D or R D ;

[0235] R8is OH, NH2or OR D ;

[0236] R9is H, OH or OR D ;

[0237] X is -S-, -S(O)-, or -SO2-; and

[0238] 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.

[0239] For example, in one embodiment, the amatoxins useful with the compositions and methods described herein include compounds according to formula (IVA):

[0240]

[0241] wherein R4, R5, X, and R8are each as defined above.

[0242] For example, in one embodiment, the amatoxins useful with the compositions and methods described herein include compounds according to formula (IVB) as follows:

[0243]

[0244] R1is H, OH, or OR A ;

[0245] R2is H, OH, or OR B ;

[0246] R A and R B , when present, combine with the oxygen atom to which they are bound to form an optionally substituted 5-membered heterocycloalkyl group;

[0247] R3is H or R D ;

[0248] R4is H, OH, OR D or R D ;

[0249] R5is H, OH, OR D or R D ;

[0250] R6is H, OH, OR D or R D ;

[0251] R7is H, OH, OR D or R D ;

[0252] R8is OH, NH2, or OR D ;

[0253] R9is H, OH, or OR D ;

[0254] X is -S-, -S(O)-, or -SO2-; and

[0255] 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.

[0256] In one embodiment, the amatoxins useful with the compositions and methods described herein also include compounds according to Formula (IVC) as follows:

[0257]

[0258] R1is H, OH, or OR A ;

[0259] R2is H, OH, or OR B ;

[0260] R A and R B , when present, combine with the oxygen atom to which they are bound to form an optionally substituted 5-membered heterocycloalkyl group;

[0261] R3is H or R D ;

[0262] R4is H, OH, OR D or R D ;

[0263] R5is H, OH, OR D or R D ;

[0264] R6is H, OH, OR D or R D ;

[0265] R7is H, OH, OR D or R D ;

[0266] R8is OH, NH2, or OR D ;

[0267] R9is H, OH, or OR D ;

[0268] X is -S-, -S(O)-, or -SO2-; and

[0269] 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.

[0270] As described herein, amatoxins can be conjugated to an antibody or antigen-binding fragment thereof, e.g., via a linker moiety. Exemplary methods of amatoxin conjugation and linkers useful in such methods are described below in the subsection entitled “Linkers for Chemical Conjugation” and in Table 2. Exemplary linker-containing amatoxins useful for conjugation to an anti-CD45 antibody or antigen-binding fragment according to the compositions and methods described herein are illustrated by structural formulas (I), (IA), (IB), (II), (IIA), and (IIB) as described herein.

[0271] For example, the antibodies and antigen-binding fragments described herein can be conjugated to an amatoxin to form a conjugate represented by the formula Ab-Z-L-Am, wherein Ab is an antibody or antigen-binding fragment thereof, L is a linker, Z is a chemical moiety, and Am is an amatoxin. Many positions on an amatoxin or derivative thereof can be used as a site for covalent attachment of a linker L, thereby attaching an antibody or antigen-binding fragment thereof. In some embodiments, Am-L-Z is represented by formula (I):

[0272]

[0273] wherein R1is H, OH, OR A , or OR C ;

[0274] R2is H, OH, OR B , or OR C ;

[0275] R A and R B , when present, combine with the oxygen atom to which they are bound to form an optionally substituted 5-membered heterocycloalkyl group;

[0276] R3is H, R C , or R D ;

[0277] R4is H, OH, OR C , OR D , R C , or R D ;

[0278] R5is H, OH, OR C , OR D , R C , or R D ;

[0279] R6is H, OH, OR C , OR D , R C , or R D ;

[0280] R7is H, OH, OR C , OR D , R C , or R D ;

[0281] R8is OH, NH2, OR C , OR D , NHR C , or NR C R D ;

[0282] R9is H, OH, OR C or OR D ;

[0283] X is -S-, -S(O)-, or -SO2-;

[0284] R C is -L-Z;

[0285] 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;

[0286] L is a linker, such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, a peptide (e.g., a dipeptide), -(C=O)-, a disulfide, a hydrazone, a -(CH2CH2O) p - group (where p is an integer from 1-6), ((CH2) m O) n (CH2) m - group (where n and each m are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10); or a combination thereof; and

[0287] Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent Z’ present within an antibody or antigen binding fragment thereof that binds a target antigen (e.g., CD45).

[0288] In some embodiments, Am contains only one R C substituent.

[0289] In some embodiments, the linker-reactive substituent group, collectively L-Z’, has the following structure prior to conjugation to an antibody or antigen binding fragment thereof:

[0290]

[0291] wherein the wavy line indicates the point of attachment to the substituent on amatoxin. Alternatively, the linker reactive substituent group L-Z' can be referred to as N-β-maleimidopropyl-Val-Ala-p-aminobenzyl (BMP-Val-Ala-PAB). The wavy line at the end of the linker indicates the point of attachment to amatoxin.

[0292] In some embodiments, the linker L and chemical moiety Z together as L-Z-Ab after conjugation to the antibody, which has the following structure:

[0293]

[0294] wherein S is a sulfur atom representing a reactive substituent present within the CD45 antibody or antigen binding fragment thereof (e.g., a -SH group from a cysteine residue). The wavy line at the end of the linker indicates the point of attachment to amatoxin.

[0295] In some embodiments, the conjugate is as shown in Formula V, Formula VA, or Formula VB:

[0296]

[0297]

[0298] wherein X is S, SO, or S02, and Ab is an antibody (e.g., an anti-CD45 antibody).

[0299] In some embodiments, Am-L-Z-Ab is

[0300]

[0301] In some embodiments, Am-L-Z-Ab is

[0302]

[0303] In some embodiments, Am-L-Z-Ab is

[0304]

[0305] In some embodiments, the Am-L-Z precursor is

[0306]

[0307] wherein the maleimide reacts with a thiol group found on a cysteine within the antibody.

[0308] In some embodiments, the Am-L-Z precursor is

[0309]

[0310] wherein the maleimide reacts with a thiol group found on a cysteine in the antibody.

[0311] In some embodiments, Am-L-Z is as shown in formula (IA):

[0312]

[0313] wherein R1is H, OH, OR A or OR C ;

[0314] R2is H, OH, OR B or OR C ;

[0315] R A and R B , when present, combine with the oxygen atom to which they are bound to form an optionally substituted 5-membered heterocycloalkyl group;

[0316] R3is H, R C or R D ;

[0317] R4is H, OH, OR C , OR D , R C or R D ;

[0318] R5is H, OH, OR C , OR D , R C or R D ;

[0319] R6is H, OH, OR C , OR D , R C or R D ;

[0320] R7is H, OH, OR C , OR D , R C or R D ;

[0321] R8is OH, NH2, OR C , OR D , NHR C or NR C R D ;

[0322] R9is H, OH, OR C or OR D;

[0323] X is -S-, -S(O)-, or -SO2-;

[0324] R C is -L-Z;

[0325] 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;

[0326] L is a linker, such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, a peptide, a dipeptide, -(C=0)-, or a combination thereof;

[0327] Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within the antibody or antigen-binding fragment thereof that binds CD45; and

[0328] wherein Am contains only one R C substituent.

[0329] In some embodiments, L-Z is

[0330]

[0331] In some embodiments, Am-L-Z is according to formula (IB):

[0332]

[0333] wherein R1is H, OH, OR A , or OR C ;

[0334] R2is H, OH, OR B , or OR C ;

[0335] R A and RB When present, they combine with the oxygen atoms they are bonded to to form optionally substituted 5-membered heterocyclic alkyl groups;

[0336] R3 represents H and R. C or R D ;

[0337] R4 represents H, OH, or OR. C OR D R C or R D ;

[0338] R5 represents H, OH, or OR. C OR D R C or R D ;

[0339] R6 represents H, OH, or OR. C OR D R C or R D ;

[0340] R7 represents H, OH, or OR. C OR D R C or R D ;

[0341] R8 represents OH, NH2, or OR. C OR D NHR C or NR C R D ;

[0342] R9 represents H, OH, or OR. C OR D ;

[0343] X is -S-, -S(O)-, or -SO2-;

[0344] R C -LZ;

[0345] R D The substituted alkyl group (e.g., C1-C6 alkyl), the substituted heteroalkyl group (e.g., C1-C6 heteroalkyl), the substituted alkenyl group (e.g., C2-C6 alkenyl), the substituted heteroalkenyl group (e.g., C2-C6 heteroalkenyl), the substituted alkynyl group (e.g., C2-C6 alkynyl), the substituted heteroalkynyl group (e.g., C2-C6 heteroalkynyl), the substituted cycloalkyl group, the substituted heterocycloalkyl group, the substituted aryl group, or the substituted heteroaryl group.

[0346] L is a linker, such as optionally substituted alkylene (e.g., C1-C6 alkylene), optionally substituted heteroalkylene (C1-C6 heteroalkylene), optionally substituted alkenylene (e.g., C2-C6 alkenylene), optionally substituted heteroalkenylene (e.g., C2-C6 heteroalkenylene), optionally substituted alkynylene (e.g., C2-C6 alkynylene), optionally substituted heteroalkynylene (e.g., C2-C6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, a peptide, a dipeptide, -(C=0)-, a disulfide, a hydrazone, -(CH2CH20) p - group (where p is an integer from 1-6), ((CH2) m O) n (CH2) m - group (where n and each m are each independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10); or a combination thereof; and

[0347] Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent Z' present within the antibody or antigen binding fragment thereof that binds CD45; and

[0348] where Am contains only one R C substituent.

[0349] In some embodiments, L-Z-Ab is

[0350]

[0351] In some embodiments, R A and R B when present, combine with the oxygen atom to which they are bound to form a 5-membered heterocycloalkyl group of the formula:

[0352]

[0353] where Y is -(C=0)-, -(C=S)-, -(C=NR E )-, or -(CR E R E’ )-; and

[0354] R E and R E’ are each independently optionally substituted C1-C6 alkylene-R C , optionally substituted C1-C6 heteroalkylene-R C , optionally substituted C2-C6 alkenylene-R C , optionally substituted C2-C6 heteroalkenylene-R C , optionally substituted C2-C6 alkynylene-RC , optionally substituted C2-C6 heteroalkynylene-R C , optionally substituted cycloalkylene-R C , optionally substituted heterocycloalkylene-R C , optionally substituted arylene-R C , or optionally substituted heteroarylene-R C .

[0355] In some embodiments, Am-L-Z is as shown in Formula (IA) or Formula (IB),

[0356] wherein R1is H, OH, OR A , or OR C ;

[0357] R2is H, OH, OR B , or OR C ;

[0358] R A and R B , when present, combine with the oxygen atom to which they are bound to form:

[0359]

[0360] R3is H or R C ;

[0361] R4is H, OH, OR C , OR D , R C , or R D ;

[0362] R5is H, OH, OR C , OR D , R C , or R D ;

[0363] R6is H, OH, OR C , OR D , R C , or R D ;

[0364] R7is H, OH, OR C , OR D , R C , or R D ;

[0365] R8is OH, NH2, OR C , or NHR C ;

[0366] R9is H or OH;

[0367] X is -S-, -S(O)-, or -SO2-; and

[0368] wherein R C and R D are each as defined above.

[0369] In some embodiments, Am-L-Z is as shown in formula (IA) or formula (IB),

[0370] wherein R1is H, OH, OR A , or OR C ;

[0371] R2is H, OH, OR B , or OR C ;

[0372] R A and R B , when present, combine with the oxygen atom to which they are bound to form:

[0373]

[0374] R3is H or R C ;

[0375] R4and R5are each independently H, OH, OR C , R C , or OR D ;

[0376] R6and R7are each H;

[0377] R8is OH, NH2, OR C , or NHR C ;

[0378] R9is H or OH;

[0379] X is -S-, -S(O)-, or -SO2-; and

[0380] wherein R C is as defined above.

[0381] In some embodiments, Am-L-Z is as shown in formula (IA) or formula (IB),

[0382] wherein R1is H, OH, or OR A ;

[0383] R2is H, OH, or OR B ;

[0384] R A and R B , when present, combine with the oxygen atom to which they are bound to form:

[0385]

[0386] R3, R4, R6, and R7 are each H;

[0387] R5 is OR C ;

[0388] R8 is OH or NH2;

[0389] R9 is H or OH;

[0390] X is -S-, -S(O)-, or -SO2-; and

[0391] wherein R C are as defined above. Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2016 / 0002298, the disclosure of which is incorporated by reference herein in its entirety.

[0392] In some embodiments, Am-L-Z is as shown in Formula (IA) or Formula (IB),

[0393] wherein R1and R2are each independently H or OH;

[0394] R3 is R C ;

[0395] R4, R6, and R7 are each H;

[0396] R5 is H, OH, or OC1-C6 alkyl;

[0397] R8 is OH or NH2;

[0398] R9 is H or OH;

[0399] X is -S-, -S(O)-, or -SO2-; and

[0400] wherein R C are as defined above. Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated by reference herein in its entirety.

[0401] In some embodiments, Am-L-Z is as shown in Formula (IA) or Formula (IB),

[0402] wherein R1and R2are each independently H or OH;

[0403] R3, R6, and R7 are each H;

[0404] R4 and R5 are each independently H, OH, OR C or R C;

[0405] R8is OH or NH2;

[0406] R9is H or OH;

[0407] X is -S-, -S(O)-, or -SO2-; and

[0408] wherein R C As defined above. Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated by reference herein in its entirety.

[0409] In some embodiments, Am-L-Z is as shown in Formula (IA) or Formula (IB),

[0410] wherein R1and R2are each independently H or OH;

[0411] R3, R6, and R7are each H;

[0412] R4and R5are each independently H or OH;

[0413] R8is OH, NH2, OR C or NHR C ;

[0414] R9is H or OH;

[0415] X is -S-, -S(O)-, or -SO2-; and

[0416] wherein R C As defined above. Such amatoxin conjugates are described, for example, in U.S. Patent Nos. 9,233,173 and 9,399,681 and US 2016 / 0089450, the disclosure of each of which is incorporated by reference herein in its entirety.

[0417] In some embodiments, Am-L-Z is

[0418]

[0419] Other amatoxins that can be used in conjugation with an antibody or antigen-binding fragment thereof according to the compositions and methods described herein are described, for example, in WO 2016 / 142049; WO 2016 / 071856; WO 2017 / 149077; WO 2018 / 115466; and WO 2017 / 046658, the disclosure of each of which is incorporated by reference herein in its entirety.

[0420] In some embodiments, Am-L-Z is as shown in Formula (II), Formula (IIA), or Formula (IIB):

[0421]

[0422] wherein X is S, SO, or SO2; R1is H or a linker covalently bound to the antibody or antigen binding fragment thereof through a chemical moiety Z, which linker is formed through a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the antibody or antigen binding fragment thereof; R2is H or a linker covalently bound to the antibody or antigen binding fragment thereof through a chemical moiety Z, which linker is formed through a coupling reaction between a reactive substituent present on the linker and a reactive substituent present within the antibody or antigen binding fragment thereof; wherein when R1is H, R2is the linker, and when R2is H, R1is the linker.

[0423] In some embodiments, the linker reactive substituent group, collectively as L-Z’, has the following structure prior to conjugation to the antibody or antigen binding fragment thereof:

[0424]

[0425] Alternatively, the linker reactive substituent group can be referred to as 1-n-hexyl- maleimide, which can be a non-cleavable linker. The wavy line at the end of the linker represents the point of attachment to the amatoxin. In some embodiments, the linker L and the chemical moiety Z, collectively as L-Z, after conjugation to the antibody, has the following structure:

[0426]

[0427] wherein S is a sulfur atom, which represents a reactive substituent present within the antibody or antigen binding fragment thereof that binds CD45 (e.g., a -SH group from a cysteine residue).

[0428] In some embodiments, Am-L-Z-Ab is:

[0429]

[0430] In some embodiments, Am-L-Z-Ab is:

[0431]

[0432] In some embodiments, the cytotoxin is a-amanitin. In some embodiments, the a-amanitin is linked to the antibody (e.g., anti-CD45 antibody) via a linker L. In some embodiments, the linker comprises a hydrazine, a disulfide bond, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the linker comprises a PAB-Cit-Val moiety. In some embodiments, the linker comprises a PAB-Ala-Val moiety. In some embodiments, the linker comprises a -(C=0)(CH2)n- unit, where n is an integer from 1-6. n In some embodiments, the linker is -(CH2)n- unit, where n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2)n-. In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2)n-.

[0433] In some embodiments, the linker comprises a -(CH2)n- unit, where n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2)n-. In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2)n-. n In some embodiments, the linker is -(CH2)n- unit, where n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2)n-. In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2)n-. n In some embodiments, the linker is -(CH2)n- unit, where n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2)n-. In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2)n-. n In some embodiments, the linker L and the chemical moiety Z together form L-Z-Ab after conjugation to the CD45 antibody, which is

[0434]

[0435] In some embodiments, the cytotoxin is a-amanitin. In some embodiments, the a-amanitin is linked to the antibody (e.g., anti-CD45 antibody) via a linker L. In some embodiments, the linker comprises a hydrazine, a disulfide bond, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the linker comprises a PAB-Cit-Val moiety. In some embodiments, the linker comprises a PAB-Ala-Val moiety. In some embodiments, the linker comprises a -(C=0)(CH2)n- unit, where n is an integer from 1-6. n In some embodiments, the linker is -(CH2)n- unit, where n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2)n-. In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2)n-.

[0436] In some embodiments, the linker comprises a -(CH2)n- unit, where n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2)n-. In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2)n-. n In some embodiments, the linker is -(CH2)n- unit, where n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2)n-. In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2)n-. n In some embodiments, the linker is -(CH2)n- unit, where n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2)n-. In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2)n-. n

[0437] ​In some embodiments, the linker L and the chemical moiety Z together form L-Z-Ab after conjugation to the CD45 antibody, which is

[0438]

[0439] In some embodiments, the cytotoxin is γ-amanitin. In some embodiments, the γ-amanitin is linked to the anti-CD45 antibody via a linker L. In some embodiments, the linker comprises a hydrazine, a disulfide bond, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the linker comprises a PAB-Cit-Val moiety. In some embodiments, the linker comprises a PAB-Ala-Val moiety. In some embodiments, the linker comprises a -(C=0)(CH2) n unit, wherein n is an integer from 1-6.

[0440] In some embodiments, the linker comprises a -(CH2) n unit, wherein n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2) n In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2) n In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2)

[0441] In some embodiments, the linker L and the chemical moiety Z together form L-Z-Ab after conjugation to the CD45 antibody, which is

[0442]

[0443] In some embodiments, the cytotoxin is ε-amanitin. In some embodiments, the ε-amanitin is linked to the anti-CD45 antibody via a linker L. In some embodiments, the linker comprises a hydrazine, a disulfide bond, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the linker comprises a PAB-Cit-Val moiety. In some embodiments, the linker comprises a PAB-Ala-Val moiety. In some embodiments, the linker comprises a -(C=0)(CH2) n unit, wherein n is an integer from 1-6.

[0444] In some embodiments, the linker comprises a -(CH2) n- units, where n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2) n -. In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2) n -.

[0445] In some embodiments, the linker L and the chemical moiety Z together form L-Z-Ab after conjugation to the antibody, which is

[0446]

[0447] In some embodiments, the cytotoxin is amanitin. In some embodiments, the amanitin is linked to the anti-CD45 antibody via a linker L. In some embodiments, the linker comprises a hydrazine, a disulfide bond, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a p- aminobenzyl group (PAB). In some embodiments, the linker comprises a PAB-Cit-Val moiety. In some embodiments, the linker comprises a PAB-Ala-Val moiety. In some embodiments, the linker comprises a -(C=0)(CH2) n - units, where n is an integer from 1-6.

[0448] In some embodiments, the linker comprises a -(CH2) n - units, where n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2) n -. In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2) n -.

[0449] In some embodiments, the linker L and the chemical moiety Z together form L-Z-Ab after conjugation to the antibody, which is

[0450]

[0451] In some embodiments, the cytotoxin is a trichothecene amide. In some embodiments, the trichothecene amide is attached to the anti-CD45 antibody via a linker L. In some embodiments, the linker comprises a hydrazine, a disulfide bond, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the linker comprises a PAB-Cit-Val moiety. In some embodiments, the linker comprises a PAB-Ala-Val moiety. In some embodiments, the linker comprises a -(C=0)(CH2) n unit, wherein n is an integer from 1-6.

[0452] In some embodiments, the linker comprises a -(CH2) n unit, wherein n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-(C=0)(CH2) n In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2) n .

[0453] In some embodiments, the linker L and the chemical moiety Z together form L-Z-Ab, which is

[0454]

[0455] In some embodiments, the cytotoxin is an amatoxin. In some embodiments, the amatoxin is linked to the anti-CD45 antibody via a linker L. In some embodiments, the linker comprises a hydrazine, a disulfide bond, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the linker comprises a PAB-Cit-Val moiety. In some embodiments, the linker comprises a PAB-Ala-Val moiety. In some embodiments, the linker comprises a -((C=0)(CH2) n unit, wherein n is an integer from 1-6.

[0456] In some embodiments, the linker comprises a -(CH2) n unit, wherein n is an integer from 2-6. In some embodiments, the linker is -PAB-Cit-Val-((C=0)(CH2) n In some embodiments, the linker is -PAB-Ala-Val-((C=0)(CH2) n .

[0457] In some embodiments, the linker L and the chemical moiety Z, after conjugation with the antibody, together form LZ-Ab, which is...

[0458]

[0459] In some embodiments, the cytotoxin is amaminic acid. In some embodiments, amaminic acid is linked to an anti-CD45 antibody via a linker L. In some embodiments, the linker comprises hydrazine, a disulfide bond, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the linker comprises a PAB-Cit-Val moiety. In some embodiments, the linker comprises a PAB-Ala-Val moiety. In some embodiments, the linker comprises -(C=O)(CH2). n - Unit, where n is an integer from 1 to 6.

[0460] In some embodiments, the connector includes -(CH2) n - Unit, where n is an integer from 2 to 6. In some implementations, the connector is -PAB-Cit-Val-(C=O)(CH2). n - In some embodiments, the connector is -PAB-Ala-Val-(C=O)(CH2). n -

[0461] In some embodiments, the linker L and the chemical moiety Z, after conjugation with the antibody, together form LZ-Ab, which is...

[0462]

[0463] In some embodiments, the cytotoxin is a proamanita nontoxic cyclic peptide. In some embodiments, the proamanita nontoxic cyclic peptide is linked to an anti-CD45 antibody via a linker L. In some embodiments, the linker comprises hydrazine, a disulfide bond, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the linker comprises a PAB-Cit-Val moiety. In some embodiments, the linker comprises a PAB-Ala-Val moiety. In some embodiments, the linker comprises -(C=O)(CH2). n - Unit, where n is an integer from 1 to 6.

[0464] In some embodiments, the connector includes -(CH2) n - Unit, where n is an integer from 2 to 6. In some implementations, the connector is -PAB-Cit-Val-(C=O)(CH2).n - In some embodiments, the linker is -PAB-Ala-Val-(C=0)(CH2) n -.

[0465] In some embodiments, the linker L and the chemical moiety Z together form L-Z-Ab after conjugation to the antibody, which is

[0466]

[0467] Synthetic methods for preparing amatoxins are described in U.S. Patent No. 9,676,702, which is incorporated herein by reference.

[0468] Antibodies and antigen-binding fragments used with the compositions and methods described herein can be conjugated to amatoxins (such as a- amanitin or variants thereof) using conjugation techniques known in the art or described herein. For example, antibodies and antigen-binding fragments thereof that recognize and bind to a target antigen (e.g., an anti-CD45 antibody) can be conjugated to amatoxins (such as a- amanitin or variants thereof) as described in US 2015 / 0218220, the disclosure of which is incorporated herein by reference as it relates to, for example, amatoxins (such as a- amanitin and variants thereof) and covalent linkers that can be used for covalent conjugation.

[0469] OH

[0470] Antibodies and antigen-binding fragments thereof described herein can be conjugated to a cytotoxin that is an auristatin (U.S. Patent Nos. 5,635,483; 5,780,588). Auristatins are antimitotic agents that interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anticancer activity (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965) (U.S. Patent Nos. 5,635,483; 5,780,588). The auristatin drug moiety can be attached to an antibody through the N (amino) terminus or C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172).

[0471] Exemplary auristatin embodiments include N-terminally linked monomethyl auristatin drug moieties DE and DF (MMAE and MMAF, respectively) disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Vol. 45, Abstract No. 623, March 28, 2004, the disclosure of which is incorporated herein by reference in its entirety.

[0472] An exemplary auristatin embodiment is MMAE, wherein the wavy line indicates the point of covalent attachment to the linker of an antibody-linker conjugate (-L-Z-Ab or -L-Z', as described herein),

[0473]

[0474] wherein the wavy line indicates the point of covalent attachment to the linker of an antibody-drug conjugate or antibody-linker conjugate (-L-Z-Ab or -L-Z', as described herein).

[0475] Another exemplary auristatin embodiment is MMAF:

[0476]

[0477] wherein the wavy line indicates the point of covalent attachment to the linker of an antibody-linker conjugate (-L-Z-Ab or -L-Z', as described herein), as disclosed in US 2005 / 0238649.

[0478] Auristatins can be prepared according to the methods of U.S. Patent No. 5,635,483; U.S. Patent No. 5,780,588; Pettit et al., (1989) J. Am. Chem. Soc. 111 :5463-5465; Pettit et al., (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R. et al., Synthesis, 1996, 719-725; Pettit et al., (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784.

[0479] Auristatin

[0480] The antibodies and antigen-binding fragments thereof described herein can be conjugated to a cytotoxin that is a microtubule-binding agent. In some embodiments, the microtubule-binding agent is a maytansinoid, a class of maytansinoids, or a maytansinoid analog. Maytansinoids are mitotic inhibitors that bind to microtubules and act by inhibiting tubulin polymerization. Maytansine was originally isolated from the bark of the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and its derivatives and analogs are disclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533. Maytansinoid drug moieties are attractive drug moieties in antibody drug conjugates because they: (i) are relatively easy to make by fermentation or by chemical modification, derivatization of fermentation products, (ii) are readily derivatizable with functional groups suitable for conjugation to antibodies through non-disulfide linkers, (iii) are stable in plasma, and (iv) are effective against a variety of tumor cell lines.

[0481] Examples of suitable maytansinoids include esters of maytansinol, synthetic maytansinol, and maytansinol analogs and derivatives. Any cytotoxins that inhibit microtubule formation and are highly toxic to mammalian cells, such as maytansinoids, maytansinol, and maytansinol analogs and derivatives, are encompassed herein.

[0482] Examples of suitable maytansinol esters include those having modifications to the aromatic ring and those having modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patent Nos. 4,137,230; 4,151,042; 4,248,870; 4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,424,219; 4,450,254; 4,322,348; 4,362,663; 4,371,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 7,276,497; and 7,473,796, the disclosure of each of which is incorporated herein by reference as it relates to maytansinoids and derivatives thereof.

[0483] In some embodiments, the antibody drug conjugates (ADC) of the present disclosure utilize a thiol-containing maytansinoid (DM1) (formally named N 2 '-deacetyl-N 2 '-(3-mercapto-1-oxopropyl)-maytansinol) as the cytotoxic agent. DM1 is represented by the following structural formula VI:

[0484]

[0485] In another embodiment, the conjugates of the present invention utilize a thiol-containing maytansinoid N 2 '-deacetyl-N 2 '(4-methyl-4-mercapto-1-oxopentyl)-maytansinol (e.g., DM4) as the cytotoxic agent. DM4 is represented by the following structural formula VII:

[0486]

[0487] Another maytansinoid containing a side chain with a sterically hindered thiol linkage is N 2 '-deacetyl-N 2 '(4-mercapto-1-oxopentyl)-maytansinol (named DM3) is represented by the following structural formula VII:

[0488]

[0489] Each of the maytansines taught in U.S. Patent Nos. 5,208,020 and 7,276,497 may also be used in the conjugates of this disclosure. In this regard, the entire disclosure of 5,208,020 and 7,276,497 is incorporated herein by reference.

[0490] Many unknown sites on maytansine can be used as covalently binding linkers, thereby binding antibodies or their antigen-binding fragments (-LZ-Ab or -L-Z', as described herein). For example, the C-3 position with a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with a hydroxyl group, and the C-20 position with a hydroxyl group are all expected to be usable. In some embodiments, the C-3 position is used as the site for covalently linking two linker sites, and in some specific embodiments, the C-3 position of maytansine is used as the site for covalently linking two linker sites. Many linker groups are known in the art for preparing antibody-matansine conjugates, including, for example, those disclosed in U.S. Patent Nos. 5,208,020, 6,441,163 and European Patent No. 0425235B1; Chari et al., Cancer Research 52:127-131 (1992); and US2005 / 0169933 A1, the disclosures of which are expressly incorporated herein by reference. Other linking groups are as described and illustrated herein.

[0491] This invention also includes various isomers and mixtures of maytansine-like compounds and conjugates. Certain compounds and conjugates of this invention can exist in various stereoisomeric, enantiomeric, and diastereomeric forms. Some descriptions of the production of such antibody-maytansine-like conjugates are described in U.S. Patent Nos. 5,208,020, 5,416,064, 6,333,410, 6,441,163, 6,716,821, and 7,368,565, each of which is incorporated herein by reference in its entirety.

[0492] Maytansinoids

[0493] In other embodiments, the antibodies and antigen-binding fragments thereof described herein can be conjugated to a cytotoxin that is an anthracycline drug molecule. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Studies have shown that anthracyclines can be used to kill cells through a variety of different mechanisms, including: 1) insertion of the drug molecule into the DNA of the cell, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) production of free radical drugs that subsequently react with cellular macromolecules resulting in cellular damage; or 3) interaction of the drug molecule with the cell membrane [see, e.g., C. Peterson et al., "Transport and Storage Of Anthracycline In Experimental Systems and Human Leukemia", in Anthracycline Antibiotics In Cancer Therapy; N. R. Bachur, "Free Radical Damage" id., pp. 97-102]. Because of their cytotoxic potential, anthracyclines have been used to treat a number of cancers, such as leukemias, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcomas [see, e.g., P. H- Wiernik, in Anthracycline: Current Status and New Developments, p. 11]. Commonly used anthracyclines include doxorubicin, epirubicin, idarubicin, and daunorubicin. In some embodiments, the cytotoxin is an anthracycline selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin. Representative examples of anthracyclines include, but are not limited to, daunorubicin (Cerubidine; Bedford Laboratories), doxorubicin (Adriamycin® (Adriamycin®- RDF; Pharmacia & Upjohn), also known as doxorubicin hydrochloride, hydroxydoxorubicin, and rubidomycin) ( Bedford Laboratories; also known as doxorubicin hydrochloride, hydroxydoxorubicin, and rubidomycin), epirubicin (Ellence® (Ellence®- RDF; Pharmacia & Upjohn), also known as 4-epi-doxorubicin, 5,6- epidaunorubicin, and 5,6-epidoxorubicin), and idarubicin (Idamycin® (Idamycin®- RDF; Pharmacia & Upjohn), also known as 4-demethoxydaunorubicin, 4-epi-doxorubicin, 5,6-epidaunorubicin, and 5,6-epidoxorubicin).

[0494] Doxorubicin is believed to interact with DNA by intercalating and inhibiting the progression of topoisomerase II, an enzyme that unwinds DNA for transcription. Doxorubicin stabilizes the topoisomerase II complex after it has broken the DNA strand for replication, preventing the DNA double helix from resealing and thus stopping the replication process. Doxorubicin and daunorubicin (DAUNOMYCIN) are prototype cytotoxic natural product anthracycline chemotherapeutics (Sessa et al. (2007) Cardiovasc. Toxicol. 7:75-79).

[0495] ​​​One non-limiting example of a suitable anthracycline for use herein is PNU-159682 ("PNU"). PNU exhibits cytotoxicity more than 3000-fold greater than the parent drug, nemorubicin (Quintieri et al., Clinical Cancer Research 2005, 11, 1608-1617). PNU is represented by the following structural formula:

[0496]

[0497] Multiple positions on anthracyclines, such as PNU, can be used as sites for covalent attachment of a linking moiety, thereby attaching the bispecific binding agents described herein. For example, the linker can be introduced via modification of the hydroxymethyl ketone side chain.

[0498] In some embodiments, the cytotoxin is a PNU derivative represented by the following structural formula:

[0499]

[0500] wherein the wavy line indicates the point of covalent attachment to a linker of an ADC described herein.

[0501] In some embodiments, the cytotoxin is a PNU derivative represented by the following structural formula:

[0502]

[0503] wherein the wavy line indicates the point of covalent attachment to a linker of an ADC described herein.

[0504] Anthracyclines Benzodiazepine

[0505] The anti-CD45 antibodies and antigen-binding fragments thereof described herein, including, for example, bispecific antibodies and biparatopic antibodies, can be conjugated to a cytotoxin comprising a benzodiazepine moiety, such as a PBD or IGN, as described herein.

[0506] Pyrrolobenzodiazepine (PBD)

[0507] PBDs have the following general structure:

[0508]

[0509] diazepine ring ("A") and the pyrrolo ("C") ring, and the degree of saturation of the C ring. In the diazepine In the B ring, there is an imine (N=C), a methanamine (NH-CH(OH)), or a methanamine methylether (NH-CH(OMe)) at the N10-C11 position. This position is the electrophilic moiety responsible for DNA alkylation. All known natural product PBDs have the (S)-configuration at the chiral C11a position, which gives them a right-handed twist when viewed from the C ring towards the A ring. This provides the proper three-dimensional shape for the isohelicity of the minor groove of B-form DNA, resulting in a tight fit at the binding site (Kohn, in Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Ace. Chem. Res., 19, 230-237 (1986)). The ability of PBDs to form adducts in the minor groove allows them to interfere with DNA procession, leading to antitumor activity.

[0510] It has previously been disclosed that the biological activity of these molecules can be enhanced by joining two PBD units together via a flexible alkylene linker through their C8-hydroxyl functional groups (Bose, D.S. et al., J. Am. Chem. Soc, 114, 4939-4941 (1992); Thurston, D.E. et al., J. Org. Chem., 61, 8141-8147 (1996)). The PBD dimers are thought to form sequence-selective DNA lesions, such as interstrand crosslinks of palindromic 5'-Pu-GATC-Py-3' strands (Smellie, M. et al., Biochemistry, 42, 8232-8239 (2003); Martin, C. et al., Biochemistry, 44, 4135-4147), which is thought to be the main cause of their biological activity. Advantageously, dimeric pyrrolobenzodiazepine The compound has been described by Gregson et al. (Chem. Commun. 1999, 797-798; "Compound 1") and by Gregson et al. (J. Med. Chem. 2001, 44, 1161-1174; "Compound 4a"). The compound is also known as SG2000, and has the following structural formula:

[0511]

[0512] Typically, modification of the pyrrolidine olefin moiety provides a handle for covalently attaching a linking moiety, and thus an antibody or antigen-binding fragment thereof (L-Z' and -L-Z-Ab, respectively, described herein). Alternatively, the linker can be attached at position N10.

[0513] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine dimer:

[0514]

[0515] wherein n is an integer from 2 to 5. The compound of this formula wherein n is 3 is known as DSB-120 (Bose et al., J. Am. Chem. Soc. 1992, 114, 4939-4941).

[0516] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine dimer:

[0517]

[0518] wherein n is an integer from 2 to 5. The compound of this formula wherein n is 3 is known as SJG-136 (Gregson et al., J. Med. Chem. 2001, 44, 737-748). The compound of this formula wherein n is 5 is known as DRG-16 (Gregson et al., Med. Chem. 2004; 47: 1161-1174).

[0519] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine dimer:

[0520]

[0521] wherein the wavy line indicates the point of covalent attachment to a linker of an ADC described herein. PBD-based ADCs are disclosed, for example, in Sutherland et al., Blood 2013 122: 1455-1463, which is incorporated herein by reference in its entirety.

[0522] In some embodiments, the cytotoxin is a PBD dimer of structural formula (I):

[0523]

[0524] wherein n is 3 or 5, and wherein the wavy line indicates the point of covalent attachment to a linker of an ADC described herein.

[0525] In some embodiments, the cytotoxin is a PBD dimer of structural formula (I):

[0526]

[0527] wherein the wavy line indicates the point of covalent attachment to a linker of an ADC described herein.

[0528] Indolinobenzodiazepine (IGN)

[0529] In some embodiments, the antibody or antigen-binding fragment thereof that binds CD45 as described herein can be conjugated to a cytotoxin that is an indolinobenzodiazepine ("IGN") or a cytotoxin comprising an IGN. In some embodiments, the IGN cytotoxin is an indolinobenzodiazepine dimer or an indolinobenzodiazepine pseudo-dimer.

[0530] indolinobenzodiazepine dimer represents a relatively new class of chemotherapeutics with high in vitro potency (low pM range IC 50 values) against cancer cells. Similar to the PBD dimer SJG-136, IGN dimers bind to the minor groove of DNA and covalently bind guanine residues via two imine functionalities in the dimer, resulting in cross-linking of the DNA. IGN dimers (IGN 6; with a phenyl ring replacing the methylene group of the PBD moiety) show ~10-fold higher potency in vitro than SJG-136, likely due to the faster rate of DNA IGN adduct formation (see, e.g., Miller et al., "A New Class of Antibody-Drug Conjugates with Potent DNA Alkylating Activity" Mol. Cancer Ther. 2016, 15(8), 1870-1878). In contrast, IGN pseudo-dimers contain a single reactive indolinobenzodiazepine imine; the second indolinobenzodiazepine in the dimeric cytotoxin is present in the reduced (amine) form. Thus, IGN pseudo-dimers alkylate DNA via the single imine moiety present in the dimer and do not cross-link DNA.

[0531] In some embodiments, the cytotoxin is an IGN pseudo-dimer having the structure of:

[0532]

[0533] where the wavy line indicates the point of attachment of the linker.

[0534] In some embodiments, the cytotoxin-linker conjugate (collectively Cy-L-Z') that is conjugated to the antibody prior to and comprises the reactive substituent Z' has the following structure:

[0535]

[0536] The cytotoxin-linker conjugate is referred to herein as DGN549 and is present in ADC IMGN632, both of which are disclosed, for example, in International Patent Application Publication No. WO2017004026, which is incorporated herein by reference.

[0537] In some embodiments, the cytotoxin is an indolinobenzodiazepine having the following structure Pseudodimer:

[0538]

[0539] wherein the wavy line indicates the point of attachment of the linker. The IGN pseudodimer cytotoxin is referred to herein as DGN462 and is disclosed, for example, in U.S. Patent Application Publication No. 20170080102, which is incorporated herein by reference.

[0540] In some embodiments, the cytotoxin-linker conjugate (collectively Cy-L-Z) prior to conjugation to the antibody and comprising the chemical moiety Z has the following structure:

[0541]

[0542] wherein the wavy line indicates the point of attachment to the antibody (e.g., an anti-CD45 antibody or fragment thereof). The cytotoxin-linker conjugate is present in ADC IMGN779 and is disclosed, for example, in U.S. Patent Application Publication No. 20170080102, which is incorporated herein by reference.

[0543] (Benzodiazepine) Cytotoxins

[0544] In other embodiments, the antibodies and antigen-binding fragments thereof described herein can be conjugated to a cytotoxin that is an enediyne antitumor antibiotic (e.g., a calicheamicin, ozogamicin). The antibiotic calicheamicin family of drugs is capable of producing double- strand DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,296, all to American Cyanamid Company. Structural analogues of calicheamicin that can be used include, but are not limited to, those described in, for example, Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the U.S. Patents to American Cyanamid Company previously described.

[0545] An exemplary calicheamicin is designated γΐ, which is simply referred to herein as γ, and has the following structural formula:

[0546]

[0547] In some embodiments, the calicheamicin is a gamma-calicheamicin derivative or an N-acetyl-gamma-calicheamicin derivative. Structural analogues of calicheamicin that can be used include, but are not limited to, those described in, for example, Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the aforementioned U.S. Patents. Calicheamicin comprises a methyltrithio moiety which can react with appropriate mercaptans to form a disulfide while introducing a functional group that can be used to attach the calicheamicin derivative to a bispecific binding agent described herein via a linker. For the preparation of conjugates of the calicheamicin family, see U.S. Patents Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,296 (all to American Cyanamid Company). Structural analogues of calicheamicin that can be used include, but are not limited to, those described in, for example, Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the aforementioned U.S. Patents issued to American Cyanamid Company.

[0548] In some embodiments, the cytotoxin of the ADCs disclosed herein is a calicheamicin disulfide derivative of the following formula:

[0549]

[0550] wherein the wavy line indicates the point of attachment of the linker.

[0551] Calicheamicin

[0552] In other embodiments, the antibodies and their antigen-binding fragments described herein may be conjugated to cytotoxins other than those disclosed herein or those not disclosed herein. Other cytotoxins suitable for use with the compositions and methods described herein include, but are not limited to, 5-ethynyluracil, abiraterone, acylfulvin, adenocyclopentanol, adorexin, aldehyde-interleukin, hexamethylmelamine, ammonimustine, amidox, amifostine, aminolevulinic acid, amrubicin, acridine, anagrelide, anastrozole and andrographolide, angiogenesis inhibitors, antarelix, anti-dorsal morphogenetic protein-1, anti-androgens, prostate cancer, anti-estrogens, anti-antineoplaston, antisense oligonucleotides, glycine afedipine, apoptosis gene regulators, apoptosis regulators, apurinic nucleic acids, and asulacrine. Atamitan, Amustine, Axinastatin 1, Axinastatin 2, Axinastatin 3, Azacillon, Azatoxin, Azatyrosine, Berry Gibberellin III Derivative, Balanol, Balmasstat, BCR / ABL Antagonist, Benzodihydroporphyrin, Benzoylsterosporin, β-lactam Derivative, β-alethine, β-aclamycin B, Betulinic Acid, bFGF Inhibitor, Bicalutamide, Bisantrene, Diaziridinyl Spermine, Diphenoxylate, Bistratene A. Blezygosine, Breflate, Bleomycin A2, Bleomycin B2, Brompirimine, Butonide, Butylthionine Sulfide, Calcipotriol, Calp-hostin; C. Camptothecin derivatives (e.g., 10-hydroxy-camptothecin), Capecitabine, Formamide-amino-triazole, Carboxyamidotriazole, Calzygosine, Casein kinase inhibitors, Sulphurine, Antimicrobial peptide B, Cetrolec, Dihydroporphyrin, Chloroquinoxaline Sulfonamide, Cicalprost, Cis-porphyrin, Clardribine, Clomiphene and its analogs, Clotrimazole, Collimmycin A, Collimmycin B, Compressorin A4, Compressorin analogs, Conagenin, Crambescidin 816, Krestorin, Nostocin 8, Nostocin A derivatives, CuracinA, cyclopentanone, cycloplatam, cypemycin, cytarabine ocfosfate, cytolysin, cytostatin, dalixizumab, decitabine, dehydroleucovibine, 2'-deoxycoformycin (DCF), deslorelin, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didox, diethylnorspermidine, dihydro-5-azacytidine, dihydrobrellin, dioxamycin, diphenylspiromustine, discodermolide, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, edelfosine, eflomithine, eleutherobin, elliptinium acetate, epothilone, epoxomicin, epristeride, estramustine and its analogs, etoposide, etoposide 4'-phosphate (also known as etopofos), exemestane, fadrazole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol, fluazuridin, fluasterone, fludarabine, fluoroacetol, flosinomycin, fluridil, fostestron, fostestron, fostriecin, formestane, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, homoharringtonolide (HHT), hypericin, ibandronic acid, idoxifene, idomastine, ilmofosine, ilomastat, imidazoacridones, imiquimod, immunostimpeptides, iobenguane, iododoxorubicin, ipomeanol, irinotecan, iroplax, isobengazole, jasplakinolide, kahalalide F, triacetate discodermolide-N, lanreotide, leinamycin, lentinan sulfate, leptolstatin, letrozole, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol, lonidamide, losoxantrone, loxoribine, letoxantrone, letoxantrone,Tetraphyrin, Lysofylline, Masoronol, Serin, Matrix Metalloproteinase Inhibitors, Menolidin, Merbarone, Meterelin, Methionin, Metoclopramide, MIF Inhibitors, Mifepristone, Mitefosine, Milistatin, Mitoxacin, Mitoxaphene, Dibromoceramide, Mitomycin and its analogues, Mitonaphthylamine, Mitoxaphene, Mofarotin, Moraxillin, Mycaperoxide B, Myriaporone, N-acetyl-B-N-methylphenidate, N-substituted benzamide, Nagrestip, Napavin, Naphterpin, Natostatin, Nedaplatin, Nemorubicin, Neridonic acid, Nilumet, Nisamycin, Ni Nitrullyn, octreotide, okicenone, ondansetron, oracin, oxaliplatin, oxaunomycin, paclitaxel and its analogues, palauamine, palmitoyl lizoxin, pamidronate, ginsenoside triol, panomiphen, paracoccin, pazelliptine, pegaspargase, peldesine, sodium lignosulfonate, pentostatin, pentrozole, perfluorobromoalkyl, pephosphatamide, phenazinomycin, picibanil, pirarubicin, piprotoxine, podophyllotoxin, purine nucleoside phosphorylase inhibitors, raltitrexate, lizoxin, lotamethasone, roxithromycin, rubiginone B1, ruboxyl, safingo, saintopin, muscle chlorophyll A, saxaglastine, sobuzosen, sonamamine, sparfosic acid, spiramycin D, spiromosine, stipiamide, sulfinosine, tamustine, tegafur, temozolomide, teniposide, thaliblastine, thiocoraline, teirazamin, topsentin, trimethoprim, trimethoprim, veratramide, vinorelbine, vinxaltine, voroxazole, zebuline, and vitamin C, etc.

[0553] In some embodiments, the cytotoxin linked to the antibody, antibody fragment, or other antigen binding agent (e.g., a ligand, such as a stem cell factor) is a protein-based toxin. An example of a protein-based toxin is a Shiga toxin. Thus, in some embodiments, the cytotoxin linked to the antibody, antibody fragment, or other antigen binding agent (e.g., a ligand, such as a stem cell factor) is a Shiga toxin or a mutant, fragment, or derivative thereof, e.g., a Shiga-like toxin A subunit and mutants, fragments, and derivatives thereof. In some embodiments, the cytotoxin linked to the antibody, antibody fragment, or other antigen binding agent is conjugated to a Shiga-like toxin (e.g., SLT I, SLT II, SLT IIV, LT toxin, or C3 toxin).

[0554] In certain embodiments, the cytotoxin is part of a fusion protein comprising a protein-based toxin and an antigen binding protein. For example, the fusion protein in certain embodiments is an engineered toxophore comprising an antibody fragment (e.g., a scFv) and a protein-based toxin, e.g., a protein synthesis inhibitor, e.g., a ribosome inactivating protein, e.g., a Shiga toxin, a Shiga-like toxin A subunit, a saporin, a ricin, and mutants, fragments, and derivatives thereof, and the like.

[0555] Linker

[0556] As used herein, the term "linker" means a bivalent chemical moiety comprising a covalent bond or chain of atoms that covalently links the anti-CD45 antibody drug conjugate (ADC) of Formula I. Suitable linkers have two reactive ends, one for conjugation to the antibody and the other for conjugation to the cytotoxin. The antibody conjugation reactive end of the linker (reactive moiety, Z') is typically a site capable of conjugation to the antibody through a cysteine thiol or lysine amino group on the antibody, and thus is typically a thiol-reactive group (such as a double bond (such as in a maleimide) or a leaving group (such as a chloro, bromo, iodo, or R-sulfonyl group)) or an amine-reactive group (such as a carboxyl group); and the cytotoxin conjugation reactive end of the linker is typically a site capable of conjugation to the cytotoxin through formation of an amide bond with a basic amine or carboxyl group on the cytotoxin, and thus is typically a carboxyl or basic amine group. When the term "linker" is used to describe a conjugated form of the linker, one or both of the reactive ends will be absent (such as the reactive moiety Z' which has been converted to the chemical moiety Z) or incomplete (such as only the carboxyl of a carboxylic acid), as a bond has formed between the linker and / or the cytotoxin and between the linker and / or the antibody or antigen binding fragment thereof. Such conjugation reactions are further described herein below.

[0557] Various linkers can be used to conjugate the antibodies or antibody fragments described herein to cytotoxic molecules. In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In still other embodiments, the linker unit is not cleavable, and the drug is released by degradation of the antibody. Linkers useful in the ADCs of the application are preferably stable extracellularly, prevent aggregation of the ADC molecule, and maintain the solubility of the ADC in aqueous media and monomeric state. Prior to delivery or transport into a cell, the ADC is preferably stable and remains intact, i.e., the antibody remains linked to the drug moiety. The linker is stable extracellularly in the target cell, and can be cleaved with some efficiency inside the cell. An efficient linker will: (i) maintain the specific binding properties of the antibody; (ii) allow intracellular delivery of the conjugate or drug moiety; (iii) remain stable and intact, i.e., not cleaved, until the conjugate is delivered or transported to its target site; and (iv) maintain the cytotoxic, cell-killing, or cell-inhibiting effects of the cytotoxic moiety. Stability of the ADC can be measured according to standard analytical techniques, such as mass spectrometry, HPLC, and separation / analysis techniques LC / MS. Covalent linkage of an antibody to a drug moiety requires that the linker have two reactive functional groups, i.e., bivalency in the sense of reactivity. Bivalent linker reagents useful for linking two or more functional moieties or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter genes, are known, and methods have been described for the resulting conjugates (Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New York, pp. 234-242).

[0558] Suitable cleavable linkers include those that can be cleaved, e.g., by enzymatic hydrolysis, thermolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide bond reduction, nucleophilic cleavage, or metallophilic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated herein by reference as it relates to linkers suitable for covalent conjugation). Suitable cleavable linkers can include, e.g., chemical moieties such as a hydrazine, a disulfide bond, a thioether, or a dipeptide.

[0559] Linkers that can be hydrolyzed under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitamides, orthoesters, acetals, ketals, and the like. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661, the disclosures of each of which are incorporated herein by reference in their entireties as they relate to linkers suitable for covalent conjugation. Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but are unstable at pH 5.5 or 5.0 or below, the appropriate pH of lysosomes.

[0560] Linkers that can be cleaved under reducing conditions include, for example, disulfide bonds. Various disulfide bond linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB, and SMPT (see, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., in Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel, ed., Oxford U. Press, 1987; see also U.S. Patent No. 4,880,935, the disclosures of each of which are incorporated herein by reference in their entireties as they relate to linkers suitable for covalent conjugation).

[0561] Linkers that are susceptible to enzymatic hydrolysis can be, for example, peptide-containing linkers that are cleaved by intracellular peptidases or proteases, including but not limited to lysosomal proteases or endosomal proteases. One advantage of using intracellular proteolytic release of a therapeutic agent is that the agent is typically attenuated upon conjugation, and the serum stability of the conjugate is typically high. In some embodiments, the peptide-based linker is at least two amino acids long or at least three amino acids long. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Examples of suitable peptides include those containing amino acids such as valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine. The amino acid residues that make up the components of the amino acid linkers include which natural amino acids as well as trace amino acids and unnatural amino acid analogs such as citrulline. Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). In some embodiments, the linker includes a dipeptide such as Val-Cit, Ala-Val, or Phe-Lys, Val-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Phe-Arg, or Trp-Cit. Linkers containing dipeptides such as Val-Cit or Phe-Lys are disclosed, for example, in U.S. Patent No. 6,214,345, the disclosure of which is incorporated by reference herein in its entirety as it relates to linkers suitable for covalent conjugation. In some embodiments, the linker includes a dipeptide selected from Val-Ala and Val-Cit.

[0562] Linkers suitable for conjugating an antibody or antibody fragment described herein to a cytotoxic molecule include those that are capable of releasing the cytotoxin through a 1,6-elimination process. Chemical moieties capable of undergoing such an elimination process include para-aminobenzyl (PAB) groups, 6-maleimidocaproic acid, pH-sensitive carbonates, and other agents as described in Jain et al., Pharm. Res. 32:3526-3540, 2015, the disclosure of which is incorporated by reference herein in its entirety as it relates to suitable covalent conjugation.

[0563] In some embodiments, the linker comprises a "self-immolative" group, such as PAB or PABC (p-aminobenzyloxy carbonyl), as described previously, which is disclosed, for example, in Carl et al., J. Med. Chem. (1981) 24:479-480; Chakravarty et al., (1983) J. Med. Chem. 26:638-644; US 6214345; US20030130189; US20030096743; US6759509; US20040052793; US6218519; US6835807; US6268488; US20040018194; W098 / 13059; US20040052793; US6677435; US5621002; US20040121940; W02004 / 032828. Other such chemical moieties capable of undergoing this process ("self-immolative linkers") include methylene carbamate and heteroaryl groups (such as aminothiazoles, aminimidazoles, aminopyrimidines, etc.). Linkers containing such heterocyclic self-immolative groups are disclosed, for example, in U.S. Patent Publication Nos. 20160303254 and 20150079114, and U.S. Patent No. 7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237; US 2005 / 0256030; de Groot et al., (2001) J. Org. Chem. 66:8815-8830; and US 7223837. In some embodiments, a dipeptide is used in combination with a self-immolative linker.

[0564] Suitable linkers for use herein can also include one or more groups selected from C1-C6 alkylene, C1-C6 heteroalkylene, C2-C6 alkenylene, C2-C6 heteroalkenylene, C2-C6 alkynylene, C2-C6 heteroalkynylene, C3-C6 cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which can be optionally substituted. Non-limiting examples of such groups include (CH2) 6环 p p p units, where p is an integer from 1-6, independently selected for each instance.

[0565] Suitable linkers can comprise groups having solubility-enhancing properties. For example, a linker can comprise one or more (CH2CH2O) p ​​​Connectors to the unit (polyethylene glycol, PEG) can enhance solubility, as can alkyl chains substituted with amino, sulfonic, phosphonic, or phosphate residues. Connectors including such portions are disclosed, for example, in U.S. Patent Applications Nos. 8,236,319 and 9,504,756, the disclosures of which are incorporated herein by reference because they relate to connectors suitable for use in joining.

[0566] A suitable linker can contain groups that enhance solubility. For example, it could contain (CH2CH2O). p Linkers in the unit (polyethylene glycol, PEG) can enhance solubility, as can alkyl chains substituted with amino, sulfonic, phosphonic, or phosphate residues. Linkers including such portions are disclosed, for example, in U.S. Patent Applications Nos. 8,236,319 and 9,504,756, the disclosures of which are incorporated herein by reference because they relate to linkers suitable for use in conjugation. Other solubility-enhancing groups include, for example, acyl and carbamoyl sulfonamide groups having the following structures:

[0567]

[0568] Where a is 0 or 1; and

[0569] R 10 Choose from the following groups: hydrogen, C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C1-C 24 (Hetero)aryl groups, C1-C 24 alkyl(hetero)aryl groups and C1-C 24 (Hetero)arylalkyl group, the C1-C 24 Alkyl groups, C3-C 24 Cycloalkyl groups, C2-C 24 (Hetero)aryl group, C3-C 24 alkyl(hetero)aryl groups and C3-C 24 Each of the (hetero)arylalkyl groups may be optionally substituted and / or optionally selected from one or more of O, S, and NR. 11 R 12 The heteroatom is interrupted, where R 11 and R 12 Independently selected from the group consisting of hydrogen and C1-C4 alkyl groups; or R 10toxin, wherein the cytotoxin is optionally linked to N via a spacer moiety. Linkers containing such groups are disclosed in, e.g., U.S. Patent No. 9,636,421 and U.S. Patent Application Publication No. 2017 / 0298145, the disclosures of which are incorporated herein by reference as they relate to linkers suitable for covalent conjugation to cytotoxins and antibodies or antigen-binding fragments thereof.

[0570] In some embodiments, the linker can comprise a hydrazine, a disulfide bond, a thioether, a dipeptide, a p-aminobenzyl (PAB) group, a heterocyclyl self-elimination group, an optionally substituted C1-C6alkyl, an optionally substituted C1-C6heteroalkyl, an optionally substituted C2-C6alkenyl, an optionally substituted C2-C6heteroalkenyl, an optionally substituted C2-C6alkynyl, an optionally substituted C2-C6heteroalkynyl, an optionally substituted C3-C6cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, a solubility-enhancing group, an acyl group, -(C=O)-, or -(CH2CH2O) p One of skill in the art will appreciate that one or more of the recited groups can exist in a bivalent (diradical) species, e.g., C1-C6alkylene, etc.

[0571] In some embodiments, the linker L comprises the moiety *-L1L2-**, wherein:

[0572] L1is absent or -(CH2) m NR 13 C(=O)-, -(CH2) m NR 13 -, -(CH2) m X3(CH2) m -,

[0573]

[0574] L2is absent or -(CH2) m -, -NR 13 (CH2) m -, -(CH2) m NR 13 C(=O)(CH2) m -, -X4, -(CH2) m NR 13 C(=O)X4, -(CH2) m NR 13 C(=O)-, -((CH2) m O) n (CH2) m -, -((CH2)m O) n (CH2) m X3(CH2) m -、-NR 13( (CH2) m O) n X3(CH2) m -、-NR 13( (CH2) m O) n (CH2) m X3(CH2) m -、-X1X2C(=O)(CH2) m -、-(CH2) m (O(CH2) m ) n -、-(CH2) m 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)X2X1C(=O)-、-(CH2) m C(=O)NR 13 (CH2) m -、-(CH2) m C(=O)NR 13 (CH2) m X3(CH2) m -、-(CH2) m X3(CH2) m NR 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)-、-(CH2) m (O(CH2) m ) n X3(CH2) m -、-(CH2) m X 3( (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 -, -(CH2) 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)X2X1C(=O)-, -C(R 13 )2(CH2) m NR 13 C(=O)(CH2) m -, -(CH2) m C(=O)NR 13 (CH2) m C(R 13 )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 -、-(CH2) m X3(CH2) m NR 13 -、-(CH2) m X3(CH2) m (O(CH2) m ) n NR 13 -、-(CH2) m NR 13 -、-(CH2) m C(=O)NR 13 (CH2) m (O(CH2) m ) n NR 13 -、-(CH2) m (O(CH2) m ) n NR 13 -、-(CH2CH2O) n (CH2) m -、-(CH2) m (OCH2CH2) n ;-(CH2) m O(CH2) m -、-(CH2) m S(=O)2-、-(CH2) m C(=O)NR 13 (CH2) m S(=O)2-、-(CH2) m X3(CH2) m S(=O)2-、-(CH2) m X2X1C(=O)-、-(CH2) m (O(CH2) m ) nC(=O)X2X1C(=O)-, -(CH2) m (O(CH2) m ) n X2X1C(=O)-, -(CH2) m X3(CH2) m X2X1C(=O)-, -(CH2) m X3(CH2) m (O(CH2) m ) n X2X1C(=O)-, -(CH2) m X3(CH2) m C(=O)NR 13 (CH2) m NR 13 C(=O)-, -(CH2) m X3(CH2) m C(=O)NR 13 (CH2) m C(=O)-, -(CH2) m X3(CH2) m C(=O)NR 13 (CH2) m (O(CH2) m ) n C(=O)-, -(CH2) m C(=O)X2X1C(=O)NR 13 (CH2) m -, -(CH2) m X3(O(CH2) m ) n C(=O)-, -(CH2) m NR 13 C(=O)((CH2) m O) n (CH2) m -, -(CH2) m (O(CH2) m ) n C(=O)NR 13 (CH2) m -, -(CH2) m NR 13 C(=O)NR 13 (CH2) m - or -(CH2) m X3(CH2) m NR 13 C(=O)-;

[0575] wherein

[0576] X1is

[0577]

[0578] X2is

[0579]

[0580] X3is

[0581]

[0582] X4is

[0583]

[0584] wherein

[0585] R 13 is independently selected for each occurrence from H and C1-C6 alkyl;

[0586] m is independently selected for each occurrence from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0587] n is independently selected for each occurrence from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14; and

[0588] wherein the single asterisk (*) denotes the point of attachment to a cytotoxin (e.g., an amatoxin), and the double asterisk (**) denotes the point of attachment to a reactive substituent Z’ or chemical moiety Z, provided that L1and L2are not both absent.

[0589] In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the p-aminobenzyl group is disposed between the cytotoxic drug and a protease cleavage site in the linker. In some embodiments, the p-aminobenzyl group is part of a p-aminobenzyloxy carbonyl unit. In some embodiments, the p-aminobenzyl group is part of a p-aminobenzyl amide unit.

[0590] In some embodiments, the linker comprises PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0591] In some embodiments, the linker comprises a peptide, an oligosaccharide, -(CH2) p -, -(CH2CH2O) p- a combination of one or more of PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0592] In some embodiments, the linker comprises -(C=0)(CH2)p- units, where p is an integer from 1 to 6. p - units, where p is an integer from 1 to 6.

[0593] In one particular embodiment, the linker comprises the following structure:

[0594]

[0595] where the wavy line indicates the point of attachment to the cytotoxin and the reactive moiety Z'. In another particular embodiment, the linker comprises the following structure:

[0596]

[0597] where the wavy line indicates the point of attachment to the cytotoxin and the reactive moiety Z'. Such PAB-dipeptide-propionyl linkers are disclosed, for example, in patent application publication no. WO 2017 / 149077, which is incorporated by reference herein in its entirety. Also, the cytotoxins disclosed in WO 2017 / 149077 are incorporated by reference herein.

[0598] In certain embodiments, the linker of the ADC is maleimidocaproyl-Val-Ala-para- aminobenzyl (mc-Val-Ala-PAB).

[0599] In certain embodiments, the linker of the ADC is maleimidocaproyl-Val-Cit-para- aminobenzyl (mc-vc-PAB).

[0600] In some embodiments, the linker comprises

[0601]

[0602] In some embodiments, the linker comprises MCC (4-[N-maleimidomethyl]cyclohexane- 1-carboxylate).

[0603] Those skilled in the art will appreciate that any one or more of the chemical groups, moieties, and features disclosed herein can be combined in a variety of ways to form linkers useful in the conjugation of the antibodies and cytotoxins disclosed herein. Additional linkers that can be used with the compositions and methods described herein are described in, e.g., U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0604] In some embodiments, the linker can comprise a hydrazine, a disulfide bond, a thioether, a dipeptide, a p-aminobenzyl (PAB) group, a heterocyclyl self-elimination group, an optionally substituted C1-C6alkyl, an optionally substituted C1-C6heteroalkyl, an optionally substituted C2-C6alkenyl, an optionally substituted C2-C6heteroalkenyl, an optionally substituted C2-C6alkynyl, an optionally substituted C2-C6heteroalkynyl, an optionally substituted C3-C6cycloalkyl, an optionally substituted heterocycloalkyl, an optionally substituted aryl, an optionally substituted heteroaryl, a solubility-enhancing group, an acyl group, -(C=0)-, or -(CH2CH20) p - groups (where p is an integer from 1-6). Those skilled in the art will appreciate that one or more of the listed groups can exist in a bivalent (diradical) species, e.g., C1-C6alkylene, etc.

[0605] In some embodiments, the linker comprises a p-aminobenzyl group (PAB). In some embodiments, the p-aminobenzyl group is disposed between a protease cleavage site in the cytotoxic drug and the linker. In some embodiments, the p-aminobenzyl group is part of a p-aminobenzyloxy carbonyl unit. In some embodiments, the p-aminobenzyl group is part of a p-aminobenzyl amido unit.

[0606] In some embodiments, the linker comprises PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0607] In some embodiments, the linker comprises a combination of one or more of a peptide, an oligosaccharide, -(CH2) p - -(CH2CH20) p - PAB, Val-Cit-PAB, Val-Ala-PAB, Val-Lys(Ac)-PAB, Phe-Lys-PAB, Phe-Lys(Ac)-PAB, D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn-PAB, or Ala-PAB.

[0608] In some embodiments, the linker comprises -(C=0)(CH2) p - units, where p is an integer from 1-6.

[0609] In one particular embodiment, the linker comprises the following structure:

[0610]

[0611] where the wavy line indicates the point of attachment to the cytotoxin and the reactive moiety Z'. In another particular embodiment, the linker comprises the following structure:

[0612]

[0613] where the wavy line indicates the point of attachment to the cytotoxin and the reactive moiety Z'. Such PAB-dipeptide-propionyl linkers are disclosed, for example, in Patent Application Publication No. WO 2017 / 149077, which is incorporated by reference herein in its entirety. Also, the cytotoxins disclosed in WO 2017 / 149077 are incorporated by reference herein.

[0614] Those skilled in the art will appreciate that any one or more of the chemical groups, moieties, and features disclosed herein can be combined in a variety of ways to form linkers useful for conjugation of the antibodies and cytotoxins disclosed herein. Other linkers that can be used with the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated by reference herein in its entirety.

[0615] In certain embodiments, an intermediate, which can be a precursor to a linker, is reacted with a drug moiety under suitable conditions. In certain embodiments, a reactive group is used on the drug and / or the intermediate or linker. The product of the reaction between the drug and the intermediate, or a derivatized drug, is then reacted with an antibody or antigen-binding fragment under suitable conditions. Alternatively, the linker or intermediate can first be reacted with the antibody or derivatized antibody, and then reacted with the drug or derivatized drug. Such conjugation reactions will now be described more fully.

[0616] A number of different reactions can be used to covalently link a linker or drug-linker conjugate to an antibody or antigen binding fragment thereof. Suitable attachment points on the antibody molecule include the amine groups of lysine, the free carboxylic acid groups of glutamic acid and ascorbic acid, the thiol groups of cysteine, and various moieties of the aromatic amino acids. For example, non-specific covalent attachment can be performed using a carbodiimide reaction to link a carboxy (or amino) group on a compound to an amino (or carboxy) group on the antibody moiety. Additionally, bifunctional reagents such as dialdehydes or imidoesters can also be used to link an amino group on a compound to an amino group on the antibody moiety. Also useful for linking drugs to binding agents is the Schiff base reaction. This method involves periodate oxidation of a drug containing diol or hydroxyl groups, thereby forming an aldehyde, which is then reacted with the binding agent. The linkage is made by formation of a Schiff base with an amino group of the binding agent. Isothiocyanates can also be used as coupling agents to covalently link drugs to binding agents. Other techniques are known to the skilled artisan and are within the scope of the present disclosure.

[0617] Linkers that can be used in conjugation with the antibodies or antigen binding fragments described herein include, but are not limited to, linkers containing a chemical moiety Z formed by a coupling reaction as described in Table 2 below. The wavy line indicates the point of attachment to the antibody or antigen binding fragment or cytotoxic molecule.

[0618] Table 2. Exemplary chemical moieties Z formed by coupling reactions in the formation of antibody drug conjugates

[0619]

[0620]

[0621]

[0622]

[0623] The skilled artisan will appreciate that the reactive substituent Z' attached to the linker and the reactive substituent of the antibody or antigen binding fragment thereof combine in a covalent coupling reaction to produce the chemical moiety Z, and will appreciate the reactive moiety Z'. Thus, antibody drug conjugates that can be used in conjunction with the methods described herein can be formed by reaction of an antibody or antigen binding fragment thereof with a linker or cytotoxin-linker conjugate described herein, the linker or cytotoxin-linker conjugate comprising a reactive substituent Z' that is suitable for reaction with a reactive substituent on the antibody or antigen binding fragment thereof to form the chemical moiety Z.

[0624] In some embodiments, Z' is -NR 13 C(=O)CH=CH2, -N3, -SH, -S(=O) 2(CH=CH2), -(CH2)2S(=0) 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)CH2Br, -NHC(=O)CH2I, -ONH2, -C(O)NHNH2, -CO2H, -NH2, -NH(C=O), -NC(=S),

[0625]

[0626]

[0627] wherein

[0628] R 13 is independently selected for each occurrence from H and C1-C6 alkyl;

[0629] R 14 is -S(CH2) n CHR 15 NHC(=O)R 13 ;

[0630] R 15 is R 13 or -C(=O)OR 13 ;

[0631] R 16 is independently selected for each occurrence from H, C1-C6 alkyl, F, Cl, and -OH;

[0632] R 17 is independently selected for each occurrence from H, C1-C6 alkyl, F, Cl, -NH2, -OCH3, -OCH2CH3, -N(CH3)2, -CN, -NO2, and -OH; and

[0633] R 18 is independently selected for each occurrence from H, C1-C6 alkyl, F, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C1-C4 alkoxy substituted with -C(=O)OH, and C1-C4 alkyl substituted with -C(=O)OH;

[0634] m is independently selected for each occurrence from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; and

[0635] n is independently selected for each instance from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.

[0636] As shown in Table 2, examples of substituents on the linker and antibody or antigen-binding fragment thereof having suitable reactivity include nucleophile / electrophile pairs (e.g., thiol / haloalkane pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, etc.), diene / diophile pairs (e.g., azide / alkyne pairs or diene / α,β-unsaturated carbonyl pairs, etc.), and the like. Reactions between reactive substituents forming chemical moiety Z include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine or hydroxylamine condensation, hydrazine formation, amidation, esterification, disulfide bond formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, etc.), nucleophilic aromatic substitution, electrophilic aromatic substitution, and other reaction patterns known in the art or described herein. Preferably, the linker comprises an electrophilic functional group for reaction with a nucleophilic functional group on the antibody or antigen-binding fragment thereof.

[0637] Reactive substituents that can be present within an antibody or antigen-binding fragment thereof disclosed herein include, but are not limited to, nucleophilic groups such as (i) N-terminal amine groups, (ii) side chain amine groups, e.g., lysine, (iii) side chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Reactive substituents that can be present within an antibody or antigen-binding fragment thereof disclosed herein include, but are not limited to, the hydroxyl moieties of serine, threonine, and tyrosine residues; the amino moieties of lysine residues; the carboxyl moieties of aspartic acid and glutamic acid residues; and the thiol moieties of cysteine residues, as well as the propargyl, azido, haloaryl (e.g., fluoroaryl), halo heteroaryl (e.g., appended heteroaryl), haloalkyl, and halo heteroalkyl moieties of unnatural amino acids. In some embodiments, the reactive substituents present within an antibody or antigen-binding fragment thereof disclosed herein include, are, amino or amine or thiol moieties. Certain antibodies have reducible intra-chain disulfide bonds, i.e., cysteine bridges. An antibody can be rendered reactive for conjugation with a linker reagent by treatment with a reducing agent, such as DTT (dithiothreitol). Thus, each cysteine bridge will theoretically form two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into an antibody by reaction of lysine with 2-iminothiolane (Traut's reagent), thereby converting an amine to a thiol. Reactive thiol groups can be introduced into an antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues, e.g., by making a mutant antibody comprising one or more non-native cysteine amino acid residues. U.S. Patent No. 7,521,541 teaches engineering of antibodies by introduction of reactive cysteine amino acids.

[0638] In some embodiments, the reactive moiety Z' attached to the linker is a nucleophilic group that is reactive with an electrophilic group present on the antibody. Useful electrophilic groups on the antibody include, but are not limited to, aldehyde carbonyl groups and ketone carbonyl groups. The heteroatom of the nucleophilic group can react with the electrophilic group on the antibody and form a covalent bond with the antibody. Useful nucleophilic groups include, but are not limited to, hydrazides, oximes, amines, hydroxyls, hydrazines, thiosemicarbazones, formic acid hydrazides, and aryl hydrazides.

[0639] In some embodiments, Z is the product of a reaction between a reactive nucleophilic substituent (such as an amine and thiol moiety) present within the antibody or antigen binding fragment thereof and a reactive electrophilic substituent Z'. For example, Z' can be a Michael acceptor (e.g., a maleimide), an activated ester, an electron-deficient carbonyl compound, and an aldehyde, among others.

[0640] For example, linkers suitable for use in the synthesis of ADCs include, but are not limited to, reactive substituents Z' such as maleimides or haloalkyl groups. These can be attached to the linker by, for example, reagents described in Liu et al., 18:690-697, 1979 (e.g., succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among others), the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation.

[0641] In some embodiments, the reactive substituent Z' attached to the linker L is a maleimide, an azide, or an alkyne. An example of a maleimide-containing linker is a non-cleavable maleimidocaproyl-based linker that is particularly useful for conjugation of microtubule disruptors such as auristatins. Such linkers are described by Doronina et al., Bioconjugate Chem. 17: 14-24, 2006, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation.

[0642] In some embodiments, the reactive substituent Z' is -(C=0)- or -NH(C=0)-, such that the linker can be joined to the antibody or antigen binding fragment thereof through an amide moiety or a urea moiety, respectively, from the reaction of the -(C=0)- or -NH(C=0)- group with an amino group of the antibody or antigen binding fragment thereof.

[0643] In some embodiments, the reactive substituent is an N-maleimido group, a halogenated N-alkylamido group, a sulfonyloxy N-alkylamido group, a carbonate group, a sulfonyl halide group, a thiol group or derivative thereof, an alkynyl group containing an internal triple bond, a (hetero)cycloalkynyl group, a bicyclo[6.1.0]non-4-yn-9-yl group, an alkenyl group containing an internal carbon-carbon double bond, a cycloalkenyl group, a tetrazine group, an azido group, a phosphine group, an nitrile oxide group, a nitroso ketone group, a nitrile imine group, a diazo group, a ketone group, an (O-alkyl)hydroxylamino group, a hydrazine group, a halogenated N-maleimido group, a 1,1-bis(sulfonylmethyl)methylcarbonyl group or an elimination derivative thereof, a halogenated carbonyl group, or an allenamide group, each of which can be optionally substituted. In some embodiments, the reactive substituent comprises a cycloalkene group, a cycloalkyne group, or an optionally substituted (hetero)cycloalkynyl group.

[0644] Non-limiting examples of amatoxin-linker conjugates containing a reactive substituent Z' suitable for reaction with a reactive residue on an antibody or antigen-binding fragment thereof include, but are not limited to: 7'C-(4-(6-(maleimidocaproyl)piperazin-l-yl)-amatoxin; 7'C-(4-(6-(maleimidocaproamide)piperidin-l-yl)-amatoxin; 7'C-(4-(6-(6-(maleimidocaproamide)caproyl)piperazin-l-yl)-amatoxin; 7'C-(4-(4-((maleimidyl))cyclohexanecarbonyl)piperazin-l-yl)-amatoxin; 7'C-(4-(6-(4-((maleimidyl)methyl)cyclohexanecarboxamido)caproyl)piperazin-l-yl)-amatoxin; 7'C-(4-(2-(6-(maleimidocaproamide)ethyl)piperidin-l-yl)-amatoxin; 7'C-(4-(2-(6-(6-(maleimidocaproamide)caproylamido)ethyl)piperidin-l-yl)-amatoxin; 7'C-(4-(2-(4-((maleimidyl)methyl)cyclohexanecarboxamido)ethyl)piperidin-l-yl)-amatoxin; 7'C-(4-(2-(6-(4-((maleimidyl)methyl)cyclohexanecarboxamido)caproylamido)ethyl)piperidin-l-yl)-amatoxin; 7'C-(4-(2-(3-carboxypropanamido)ethyl)piperidin-l-yl)-amatoxin; 7'C-(4-(2-(2-bromoacetamido)ethyl)piperidin-l-yl)-amatoxin; 7'C-(4-(2-(3-(pyridin-2- yldisulfanyl)propanamido)ethyl)piperidin-l-yl)-amatoxin; 7'C-(4-(2-(4- (maleimidyl)butanamido)ethyl)piperidin-l-yl)-amatoxin; 7'C-(4-(2- (maleimidyl)acetyl)piperazin-l-yl)-amatoxin; 7'C-(4-(3-(maleimidyl)propanoyl)piperazin-l-yl)-amatoxin; 7'C-(4-(4-(maleimidyl)butanoyl)piperazin-l-yl)-amatoxin; 7'C-(4-(2-(6-(4-((maleimidyl)methyl)cyclohexanecarboxamido)ethyl)piperidin-l-yl)-amatoxin; 7'C-(3-((6-(maleimidocaproamide)methyl)piperidin-l-yl)-amatoxin; 7'C-(3-((6-(6-(maleimidocaproamide)caproylamido)methyl)piperidin-l-yl)-amatoxin; 7'C-(3-((4-((maleimidyl)methyl)cyclohexanecarboxamido)methyl)piperidin-l-yl)-amatoxin; 7'C-(3-((6-((4-(maleimidyl)methyl)cyclohexanecarboxamido)caproylamido)methyl)piperidin-l-yl)-amatoxin;7'C-(4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-l-yl)-amanitin; 7'C-(4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-l-yl)-amanitin; 7'C-(4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-l-yl)-amanitin; 7'C-(4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazin-l-yl)-amanitin; 7'C-((4-(6-(maleimido)hexanamido)piperidin-l-yl)methyl)-amanitin; 7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-l-yl)methyl)-amanitin; 7'C-((4-(6-(maleimido)hexanoyl)piperazin-l-yl)methyl)-amanitin; (R)-7'C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-l-yl)methyl)- amanitin; (S)-7'C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-l-yl)methyl)- amanitin; 7'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-l- yl)methyl)-amanitin; 7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-l- yl)methyl)-amanitin; 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-l- yl)methyl)-amanitin; 7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-l-yl)methyl)- amanitin; 7'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-l- yl)methyl)-amanitin; 7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-l- yl)methyl)-amanitin; 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperazin-l- yl)methyl)-amanitin; 7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-S-methyl)pyrrolidin-l- yl)methyl)-amanitin; 7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-R-methyl)pyrrolidin-l- yl)methyl)-amanitin; 7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-l- yl)methyl)-amanitin;7'C-((3-((4-((maleimidomethyl)cyclohexanecarbamido)-R-methyl)pyrrolidin-1- yl)methyl)-amanitin; 7'C-((3-((6-(4-((maleimidomethyl)cyclohexanecarbamido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amanitin; 7'C-((4-(2-(3-carboxypropanamido)ethyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(6-(6-(maleimidomethyl)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(6-(4-((maleimidomethyl)cyclohexanecarbamido)hexanoyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(2-(maleimidomethyl)acetyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(3-(maleimidomethyl)propanoyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(4-(maleimidomethyl)butanoyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(2-(2-(maleimidomethyl)acetamido)ethyl)piperidin-1-yl)methyl)-amanitin; 7'C-((4-(2-(4-(maleimidomethyl)butanamido)ethyl)piperidin-1-yl)methyl)-amanitin; 7'C-((4-(2-(6-(4-((maleimidomethyl)cyclohexanecarbamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amanitin; 7'C-((3-((6-(maleimidomethyl)hexanamido)methyl)azetidin-1-yl)methyl)-amanitin; 7'C-((3-(2-(6-(maleimidomethyl)hexanamido)ethyl)azetidin-1-yl)methyl)-amanitin; 7'C-((3-((4-((maleimidomethyl)cyclohexanecarbamido)methyl)azetidin-1-yl)methyl)-amanitin; 7'C-((3-(2-(4-((maleimidomethyl)cyclohexanecarbamido)ethyl)azetidin-1-yl)methyl)-amanitin; 7'C-((3-(2-(6-(4-((maleimidomethyl)cyclohexanecarbamido)hexanamido)ethyl)azetidin-1-yl)methyl)-amanitin; 7'C-(((2-(6-(maleimidomethyl)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amanitin; 7'C-(((4-(6-(maleimidomethyl)-N-methylhexanamido)butyl(methyl)amino)methyl)-amanitin; 7'C-((2-(2-(6-(maleimidomethyl)hexanamido)ethyl)aziridin-1-yl)methyl)-amanitin; 7'C-((2-(2-(6-(4-((maleimidomethyl)cyclohexanecarbamido)hexanamido)ethyl)aziridin-1-yl)methyl)-amanitin;7'C-((4-(6-(6-(2-(aminooxy)acetamido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azahexadecan-17-oyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(2-(2-(aminooxy)acetamido)acetyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(3-(2-(aminooxy)acetamido)propionyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amanitin; 7'C-((4-(2-(2-(2-(aminooxy)acetamido)acetamido)ethyl)piperidin-1-yl)methyl)-amanitin; 7'C-((4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)methyl)-amanitin; 7'C-((4-(20-(aminooxy)-4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazicosa-yl)piperidin-1-yl)methyl)-amanitin; 7'C-(((2-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amanitin; 7'C-(((4-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)butyl)(methyl)amino)methyl)-amanitin; 7'C-((3-((6-(4-((maleimidomethyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-S-methyl)-amanitin; 7'C-((3-((6-(4-((maleimidomethyl)cyclohexanecarboxamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amanitin; 7'C-((4-(2-(2-bromoacetamido)ethyl)piperazin-1-yl)methyl)-amanitin; 7'C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)methyl)-amanitin; 7'C-((4-(2-(3-(pyridin-2-yl disulfanyl)propanamido)ethyl)piperidin-1-yl)methyl)-amanitin; 6'O-(6-(6-(maleimidomethyl)hexanamido)hexyl)-amanitin; 6'O-(5-(4-((maleimidomethyl)cyclohexanecarboxamido)pentyl)-amanitin; 6'O-(2-((6-(maleimidomethyl)oxy)-2-oxoethyl)-amanitin; 6'O-((6-(maleimidomethyl)carbamoyl)-amanitin;6Ό-(6-(4-((maleimidomethyl)cyclohexanecarboxamido)hexyl)carbamoyl)- amatoxin; 6Ό-(6-(2-bromoacetamido)hexyl)-amatoxin; 7Ό-(4-(6-(azido)hexanamido)piperidin- 1 -yl)-amatoxin; 7Ό-(4-(hex-5-ynoylaminopiperidin-1-yl)-amatoxin; 7Ό-(4-(2-(6-(maleimidomethyl)hexanamido)ethyl)piperazin-1-yl)-amatoxin; 7Ό-(4-(2-(6-(6- (maleimidomethyl)hexanamido)ethyl)piperazin-1-yl)-amatoxin; 6Ό-(6-(6-(11,12-didehydro-5,6- dihydro-dibenzo[b,f]azocine-5-yl)-6-oxohexanamido)hexyl)-amatoxin; 6Ό-(6-(hex-5-ynoylaminohexyl)- amatoxin; 6Ό-(6-(2-(aminooxy)acetylaminohexyl)-amatoxin; 6Ό-((6-aminooxy)hexyl)-amatoxin; and 6Ό-(6-(2-iodoacetamido)hexyl)-amatoxin.

[0645] In some embodiments, the amatoxins disclosed herein are conjugated to a linker- reactive moiety-L-Z' having the following formula:

[0646]

[0647] In some embodiments, the amatoxins disclosed herein are conjugated to a linker- reactive moiety-L-Z' having the following formula:

[0648] In some embodiments, the amatoxins disclosed herein are conjugated to a linker- reactive moiety-L-Z' having the following formula:

[0649]

[0650] The foregoing linker moieties and amatoxin-linker conjugates, etc., useful in the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220 and Patent Application Publication No. WO 2017 / 149077, each of which is incorporated by reference herein in its entirety.

[0651] The foregoing linker moieties and amatoxin-linker conjugates, etc., useful in the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220 and Patent Application Publication No. WO 2017 / 149077, each of which is incorporated by reference herein in its entirety.

[0652] Preparation of antibody drug conjugates

[0653] In the ADCs of Formula I disclosed herein, the anti-CD45 antibody or antigen-binding fragment thereof can be conjugated to one or more cytotoxic drug moieties (D) through the linkers L and chemical moieties Z disclosed herein, e.g., from about 1 to about 20 drug moieties per antibody. The ADCs of the disclosure can be prepared by several routes employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a reactive substituent of the antibody or antigen-binding fragment thereof with a bivalent linker reagent to form Ab-Z-L as described herein above, followed by reaction with a drug moiety D; or (2) reaction of a reactive substituent of a drug moiety with a bivalent linker reagent to form D-L-Z', followed by reaction with a reactive substituent of the antibody or antigen-binding fragment thereof as described herein above. Other methods for preparing ADCs are described herein.

[0654] In another aspect, the anti-CD45 antibody or antigen-binding fragment thereof has one or more lysine residues that can be chemically modified to introduce one or more thiol groups. Subsequent conjugation through the sulfur atom of the thiol group forms the ADC as described herein above. Reagents useful for modifying lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).

[0655] In another aspect, the anti-CD45 antibody or antigen-binding fragment thereof can have one or more carbohydrate groups that can be chemically modified to have one or more thiol groups. Subsequent conjugation through the sulfur atom of the thiol group forms the ADC as described herein above.

[0656] In still another aspect, the anti-CD45 antibody can have one or more carbohydrate groups that can be oxidized to provide an aldehyde (-CHO) group (see, e.g., Laguzza et al., J. Med. Chem. 1989, 32(3), 548-55). Subsequent conjugation through the corresponding aldehyde aldehyde forms the ADC as described herein above. Other methods for modifying proteins to attach or associate cytotoxins are described in Coligan et al., Current Protocols in Protein Science, Vol. 2, John Wiley & Sons (2002), which is hereby incorporated by reference in its entirety.

[0657] Methods for conjugating linker-drug moieties to proteins that target cells, such as antibodies, immunoglobulins, or fragments thereof, can be found, e.g., in U.S. Patent No. 5,208,020; U.S. Patent No. 6,441,163; WO2005037992; WO2005081711; and WO2006 / 034488, all of which are expressly incorporated by reference herein in their entirety.

[0658] Alternatively, fusion proteins comprising an antibody and a cytotoxic agent can be prepared, e.g., by recombinant techniques or peptide synthesis. The DNA can comprise regions encoding each portion of the conjugate, adjacent to each other or separated by a region encoding a linking peptide that does not destroy the physical properties of the conjugate.

[0659] Therapeutic methods

[0660] CD45 is an important cell surface molecule that is widely expressed throughout the hematopoietic and immune systems. Described herein are anti-CD45 antibodies and anti-CD45 ADCs that can be used to treat patients with autoimmune diseases such as arthritis (or diseases represented by proteoglycan-induced arthritis (PGIA)), autoimmune encephalitis, graft versus host disease, multiple sclerosis, or scleroderma. In addition, there is a current need for compositions and methods for promoting engraftment of exogenous hematopoietic stem cell implants in order to preserve the multipotency and hematopoietic function of these cells after transplantation. The compositions disclosed herein also provide a solution to this challenging problem.

[0661] Therefore, this article discloses methods for treating various autoimmune diseases, such as rheumatoid arthritis (represented by PGIA in mouse models), autoimmune encephalitis, type 1 diabetes, or scleroderma graft-versus-host disease. The compositions and methods described herein can (i) directly consume pathological cell populations, such as autoimmune cell populations (e.g., self-reactive T cell populations), and / or (ii) consume endogenous hematopoietic stem cell populations to facilitate the engraftment of transplanted hematopoietic stem cells by providing niches into which transplanted cells can be incorporated. The above activities can be achieved by administering an ADC, antibody, or antigen-binding fragment thereof capable of binding to antigens expressed by endogenous pathogenic cells or hematopoietic stem cells. In the case of directly treating the disease, such administration can lead to a reduction in the number of cells causing the target pathology. In the case of preparing a patient for hematopoietic stem cell transplantation therapy, such administration can lead to the selective consumption of endogenous hematopoietic stem cell populations, thereby creating gaps in hematopoietic tissues (such as bone marrow) that can subsequently be filled by transplanted exogenous hematopoietic stem cells. This invention is based in part on the discovery that ADCs, antibodies, or antigen-binding fragments thereof capable of binding to CD45 expressed by hematopoietic stem cells can be administered to patients to affect both of these activities. ADCs, antibodies, or antigen-binding fragments thereof binding to antigens (e.g., CD45) expressed by hematopoietic stem cells and / or autoimmune cells can be administered to patients suffering from autoimmune diseases (e.g., rheumatoid arthritis (represented by PGIA in mouse models), autoimmune encephalitis, type 1 diabetes, or scleroderma) to directly deplete the autoimmune cell population. The aforementioned agents can also condition patients to receive grafts containing hematopoietic stem cells and can be administered to patients requiring hematopoietic stem cell transplantation therapy to promote the survival and engraftment potential of transplanted hematopoietic stem cells.

[0662] As described herein, hematopoietic stem cell transplantation therapy can be administered to subjects requiring treatment (e.g., treatment for autoimmune diseases or conditions) to populate or repopulate one or more blood cell types. Hematopoietic stem cells typically exhibit pluripotency and can therefore differentiate into a variety of different blood cell lineages, including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), coagulation cells (e.g., promegakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Furthermore, hematopoietic stem cells are capable of self-renewal and thus can produce daughter cells with the same potential as their parent cells, and are also characterized by the ability to reintroduce them into the graft recipient, thereby returning them to the hematopoietic stem cell niche and re-establishing productive and sustained hematopoiesis.

[0663] The compositions and methods described herein can be used to treat autoimmune disorders, such as rheumatoid arthritis (represented by PGIA in a mouse model), autoimmune encephalitis, type 1 diabetes, or scleroderma. For example, an antibody or antigen-binding fragment thereof can be administered to a subject, such as a human patient, who is afflicted with an autoimmune disorder, in order to kill CD45+ immune cells. For example, the CD45+ immune cells can be autoreactive lymphocytes, such as T cells expressing T cell receptors that specifically bind to and are immunoreactive against self-antigens. By depleting autoreactive CD45+, the compositions and methods described herein can be used to treat autoimmune pathologies, such as those described herein. Additionally or alternatively, the compositions and methods described herein can be used to treat autoimmune diseases by depleting the endogenous hematopoietic stem cell population prior to hematopoietic stem cell transplant therapy, in which case the transplanted cells can repopulate the niche created by the endogenous cell depletion step and establish productive hematopoiesis. This in turn can reconstitute the cell population depleted during the autoimmune cell elimination process.

[0664] In some embodiments, the anti-CD45 antibody or ADC can be administered to a subject in combination with a second therapeutic agent.

[0665] Autoimmune diseases that can be treated using the compositions and methods described herein include, but are not limited to, PGIA, autoimmune encephalitis, scleroderma, psoriasis, psoriatic arthritis, type I diabetes mellitus, rheumatoid arthritis (RA), human systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), lymphocytic colitis, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid antibody syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac sprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus, familial dysautonomia, endometriosis, mixed cold agglutinin disease, fibromyalgia-fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki's disease, lichen planus, Lyme disease, Meniere's disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, ophthalmoplegia-mylajia-syndrome (OMS), optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndromes, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, Takayasu's arteritis, temporal arteritis (also known as "giant cell arteritis"), ulcerative colitis, collagenous colitis, uveitis, vasculitis, vitiligo, vulvodynia ("vulvar vestibulitis"), and Wegener's granulomatosis. In certain embodiments, the autoimmune disease is rheumatoid arthritis (represented by PGIA in mouse models). In certain embodiments, the autoimmune disease is inflammatory arthritis. Non-limiting examples of inflammatory arthritis include rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, juvenile idiopathic arthritis, and systemic lupus erythematosus. In other embodiments, the autoimmune disease is autoimmune encephalitis. In still further embodiments, the autoimmune disease is scleroderma graft versus host disease.

[0666] In some embodiments, the transplant is allogeneic. In some embodiments, the transplant is autologous.

[0667] In some embodiments, the transplant is a bone marrow transplant, a peripheral blood transplant, or a cord blood transplant.

[0668] In some embodiments, the transplant comprises hematopoietic cells (e.g., hematopoietic stem cells).

[0669] In any of the embodiments described herein, the transplant can be any solid organ transplant or skin transplant. In some embodiments, the transplant is selected from the group consisting of a kidney transplant, a heart transplant, a liver transplant, a pancreas transplant, a lung transplant, an intestinal transplant, and a skin transplant.

[0670] In some embodiments, the compositions and methods described herein can be effective to cause immune reset in a patient with an autoimmune disease. In some such embodiments, a patient with an autoimmune disease who undergoes treatment according to the methods of the application does not require additional treatment (e.g., chronic treatment) for the autoimmune disease after transplantation. For example, in some embodiments, the patient has multiple sclerosis, and the patient does not require treatment with natalizumab, dimethyl fumarate, or monomethyl fumarate after treatment with an anti-CD45 ADC and transplantation according to the methods herein. In other embodiments, the patient has arthritis, and the patient does not require treatment with a TNF inhibitor (e.g., an anti-TNFa antibody such as etanercept, infliximab, adalimumab, certolizumab pegol, or golimumab) after treatment with an anti-CD45 ADC and transplantation according to the methods herein. In certain embodiments, a single dose of an anti-CD45 ADC can be effective to achieve immune reset.

[0671] In some cases, a patient with an autoimmune disease who undergoes treatment according to the methods provided herein can enter remission, such as clinical remission, biochemical remission, or histological remission. For example, in some embodiments, the patient enters remission for at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years. In some embodiments, the patient enters remission for at least 1-3 years, at least 3-5 years, at least 5-7 years, at least 7-9 years, or at least 8-10 years.

[0672] Routes of administration and dosing

[0673] The antibodies, antigen-binding fragments, or ADCs described herein can be administered to a patient (e.g., a human patient afflicted with an autoimmune disease or in need of hematopoietic stem cell transplant therapy) in various dosage forms. For example, the ADCs described herein can be administered to a patient afflicted with an autoimmune disease or in need of hematopoietic stem cell transplant therapy in the form of an aqueous solution, such as an aqueous solution containing one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients for use with the compositions and methods described herein include viscosity modifiers. The aqueous solutions can be sterilized using techniques known in the art.

[0674] Some embodiments include administering to a subject an anti-CD45 antibody or antigen-binding portion thereof. Some embodiments include administering to a subject an ADC comprising an anti-CD45 antibody or antigen-binding portion thereof.

[0675] Some embodiments include administering to a subject a combination of an anti-CD45 antibody (or antigen-binding portion thereof) and a second therapeutic agent. Some embodiments include administering to a subject an ADC comprising an anti-CD45 antibody (or antigen-binding portion thereof) in combination with a second therapeutic agent. Some embodiments include administering to a subject a bispecific antibody, antigen-binding portion thereof, or ADC that specifically binds CD45 and a second antigen. Some aspects include administering to a subject an anti-CD45 antibody (or antigen-binding portion thereof) or ADC in combination with two or more other therapeutic agents.

[0676] Pharmaceutical formulations comprising an anti-CD45 antibody or conjugate thereof (e.g., an ADC described herein) are prepared by mixing such antibody or ADC in admixture with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16thEdition, Osol, A. Ed. (1980)) in either a lyophilized or aqueous solution form. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including sucrose, mannitol, or dextrins; chelating agents, such as EDTA; sugars such as sucrose, xylitol, trehalose or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG).

[0677] The ADCs described herein can be administered by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular, or parenteral routes. The most suitable route of administration in any given case will depend on the specific antibody or antigen-binding fragment administered, the patient, the method of formulation, the method of administration (e.g., the time and route of administration), the age, weight, sex, severity of disease, consciousness of the patient, and / or the rate of excretion of the patient.

[0678] An effective amount of an antibody or antigen-binding fragment thereof described herein can range from about 0.001 to about 100 mg / kg body weight in a single (e.g., single) administration, multiple administrations, or continuous administration, or can range to achieve optimal serum concentrations of the antibody or antigen-binding fragment thereof (e.g., serum concentrations of 0.0001-5000 pg / mL). Dosing can be administered to a patient affected by an autoimmune disease or to a patient undergoing conditioning therapy in preparation for receiving a hematopoietic stem cell transplant one or more times (e.g., 2-10 times) per day, week, or month.

[0679] In certain embodiments, the anti-CD45 antibody or ADC is used as a single dose to the patient. In other embodiments, the anti-CD45 antibody or ADC is administered to the patient as a split dose, wherein the dose of the anti-CD45 antibody or ADC is split and administered to the subject at an interval time. For example, in a split dosing regimen, the dose of the anti-CD45 antibody or ADC can be split into two, three, four, five, six, seven, eight, nine, or ten portions, and each portion is administered to the subject at an interval time. In some embodiments, the time is separated by 1 hour, 3 hours, 6 hours, 9 hours, 12 hours, 15 hours, 18 hours, 21 hours, 24 hours, 36 hours, 48 hours, 72 hours, 96 hours, 120 hours, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks. In one embodiment, the anti-CD45 antibody or ADC described herein is administered to the patient as a split dose, wherein two portions are administered to the patient. In one embodiment, the anti-CD45 antibody or ADC described herein is used as a split dose to the patient, wherein three portions are administered to the patient.

[0680] In some embodiments, the dose of the antibody or ADC administered to a human patient is about 0.001 mg / kg to 10 mg / kg, about 0.01 mg / kg to 9.5 mg / kg, about 0.1 mg / kg to 9 mg / kg, about 0.1 mg / kg to 8.5 mg / kg, about 0.1 mg / kg to 8 mg / kg, about 0.1 mg / kg to 7.5 mg / kg, about 0.1 mg / kg to 7 mg / kg, about 0.1 mg / kg to 6.5 mg / kg, about 0.1 mg / kg to 6 mg / kg, about 0.1 mg / kg to 5.5 mg / kg, about 0.1 mg / kg to 5 mg / kg, about 0.1 mg / kg to 4.5 mg / kg, about 0.1 mg / kg to 4 mg / kg, about 0.5 mg / kg to 3.5 mg / kg, about 0.5 mg / kg to 3 mg / kg, about 1 mg / kg to 10 mg / kg, about 1 mg / kg to 9 mg / kg, about 1 mg / kg to 8 mg / kg, about 1 mg / kg to 7 mg / kg, about 1 mg / kg to 6 mg / kg, about 1 mg / kg to 5 mg / kg, about 1 mg / kg to 4 mg / kg, or about 1 mg / kg to 3 mg / kg.

[0681] In some embodiments, the dose of the antibody or ADC administered to a human patient is about 0.1 mg / kg to about 0.3 mg / kg.

[0682] In some embodiments, the dose of the antibody or ADC administered to a human patient is about 0.15 mg / kg to about 0.3 mg / kg.

[0683] In some embodiments, the antibody or ADC is administered to the human patient at a dose of about 0.15 mg / kg to about 0.25 mg / kg.

[0684] In some embodiments, the antibody or ADC is administered to the human patient at a dose of about 0.2 mg / kg to about 0.3 mg / kg.

[0685] In some embodiments, the antibody or ADC is administered to the human patient at a dose of about 0.25 mg / kg to about 0.3 mg / kg.

[0686] In some embodiments, the antibody or ADC is administered to the human patient at a dose of about 1 mg / kg to about 3 mg / kg.

[0687] In some embodiments, the antibody or ADC is administered to the human patient at a dose of about 0.1 mg / kg.

[0688] In some embodiments, the antibody or ADC is administered to the human patient at a dose of about 0.2 mg / kg.

[0689] In some embodiments, the antibody or ADC is administered to the human patient at a dose of about 0.3 mg / kg.

[0690] In some embodiments, the antibody or ADC is administered to the human patient at a dose of about 1 mg / kg.

[0691] In some embodiments, the antibody or ADC is administered to the human patient at a dose of about 2 mg / kg.

[0692] In some embodiments, the antibody or ADC is administered to the human patient at a dose of about 3 mg / kg.

[0693] In some embodiments, the antibody or ADC is administered to the human patient at a dose of about 1 mg / kg.

[0694] Using the methods disclosed herein, a skilled medical practitioner can administer an ADC, antibody, or antigen-binding fragment thereof that is capable of binding to CD45 expressed by hematopoietic stem cells to a human patient in need of hematopoietic stem cell transplant therapy. In this manner, the endogenous hematopoietic stem cell population can be depleted prior to administration of an exogenous hematopoietic stem cell implant, thereby facilitating engraftment of the hematopoietic stem cell implant. The antibody can be covalently conjugated to a toxin, such as a cytotoxic molecule described herein or known in the art. For example, the anti-CD45 antibody or antigen-binding fragment thereof can be covalently conjugated to a cytotoxin, such as Pseudomonas exotoxin A, deBouganin, diphtheria toxin, amatoxin (such as a-amanitin, β-amanitin), saporin, maytansinoid, maytansinoid, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine pyrrolobenzodiazepine dimer, indolinobenzodiazepine indolinobenzodiazepine dimer, or a variant thereof. Such conjugation can be performed using covalent bond formation techniques described herein or known in the art. The antibody, antigen-binding fragment thereof, or drug-antibody conjugate can be subsequently administered to the patient, e.g., by intravenous administration, prior to transplanting exogenous hematopoietic stem cells (such as autologous, syngeneic, or allogeneic hematopoietic stem cells) to the patient.

[0695] The anti-CD45 antibody, antigen-binding fragment thereof, or ADC can be administered prior to hematopoietic stem cell transplant therapy in an amount sufficient to reduce the number of endogenous hematopoietic stem cells, e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. Reduction in hematopoietic stem cell count can be detected using routine techniques in the art, such as by FACS analysis of cells expressing characteristic hematopoietic stem cell surface antigens in blood samples drawn from the patient at various time intervals during conditioning therapy. For example, a skilled medical practitioner can draw blood samples from the patient at various time points during conditioning therapy and determine the extent of reduction in endogenous hematopoietic stem cells by FACS analysis using antibodies that bind to hematopoietic stem cell marker antigens, thereby elucidating the relative concentration of hematopoietic stem cells in the sample. According to some embodiments, the medical practitioner can end conditioning therapy when the hematopoietic stem cell concentration reaches a minimum in response to conditioning therapy with the anti-CD45 antibody, antigen-binding fragment thereof, or ADC, and can begin preparing the patient for hematopoietic stem cell transplant therapy.

[0696] The anti-CD45 antibody, antigen-binding fragment thereof, or ADC can be administered to the patient in the form of an aqueous solution comprising one or more pharmaceutically acceptable excipients, such as a viscosity modifier. The aqueous solution can be sterilized using techniques described herein or known in the art. The antibody, antigen-binding fragment thereof, or drug-antibody conjugate can be administered to the patient at a dose of, for example, 0.001 mg / kg to 100 mg / kg prior to implanting the hematopoietic stem cell implant into the patient. The anti-CD45 antibody, antigen-binding fragment thereof, or ADC can be administered to the patient at a time that optimally promotes engraftment of the exogenous hematopoietic stem cells, for example, 1 hour to 1 week (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) or more prior to administering the exogenous hematopoietic stem cell graft.

[0697] After the conditioning therapy is completed, the patient can subsequently receive an infusion (e.g., intravenous infusion) of exogenous hematopoietic stem cells, such as from the same physician who performed the conditioning therapy or from a different physician. The physician can administer autologous, syngeneic, or allogeneic hematopoietic stem cells to the patient, for example, at 1 x 105 / kg to 1 x 107 / kg. 3 to 1 x 107 / kg. 9A dose of 1 x 106hematopoietic stem cells / kg. The physician can detect engraftment of the hematopoietic stem cell graft, for example, by drawing a blood sample from the patient after administration of the graft and determining an increase in the concentration of hematopoietic stem cells or hematopoietic lineage cells (such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes). This analysis can be performed 1 hour to 6 months or more after the hematopoietic stem cell transplant therapy (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, or more) after the transplant therapy. A finding that the concentration of hematopoietic stem cells or hematopoietic lineage cells has increased (e.g., by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 500%, or more) relative to the concentration of the respective cell type prior to the transplant therapy provides a clue that treatment with the anti-CD45 antibody or antigen-binding fragment thereof or ADC has successfully facilitated engraftment of the transplanted hematopoietic stem cell engraftment.

[0698] Engraftment of hematopoietic stem cell transplants due to administration of an anti-CD45 antibody, antigen-binding fragment thereof, or ADC can be manifested in various empirical measures. For example, engraftment of transplanted hematopoietic stem cells can be assessed by the number of competitive repopulating units (CRUs) present in a patient's bone marrow after administration of an anti-CD45 antibody or antigen-binding fragment thereof and subsequent administration of a hematopoietic stem cell transplant. In addition, one can observe engraftment of a hematopoietic stem cell transplant by adding a reporter gene (such as an enzyme that catalyzes a chemical reaction that produces a fluorescent, chromogenic, or luminescent product) to a vector that has been transduced with donor hematopoietic stem cells and subsequently monitoring for the corresponding signal in tissues (such as bone marrow) where the hematopoietic stem cells have engrafted. One can also observe hematopoietic stem cell engraftment by assessing the number and survival time of hematopoietic stem cells and hematopoietic progenitor cells (e.g., as determined by fluorescence-activated cell sorting (FACS) analysis methods known in the art). Engraftment can also be determined by measuring white blood cell counts in peripheral blood during the post-transplant period, and / or by measuring bone marrow cell recovery through bone marrow aspirate samples of donor cells. EMBODIMENTS

[0699] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein are made and evaluated, and are intended to be purely exemplary of the application and are not intended to limit the scope of what the inventors regard as their application.

[0700] Example 1. Anti-CD45 ADC successfully conditions mice for syngeneic transplantation

[0701] To address the challenges associated with autologous HSC transplantation, an antibody drug conjugate (ADC) targeting CD45, a target expressed throughout the hematopoietic system, was developed that enables simultaneous myeloablation and lymphodepletion prior to autologous transplantation in autoimmune disease. To model this strategy, an anti-mouse CD45-ADC comprising an anti-CD45 antibody conjugated to pyrrolobenzodiazepine (PBD) was developed that achieved complete myeloablation in mice with a single dose administration. (PBD) conjugated to an anti-CD45 antibody that achieved complete myeloablation in mice with a single dose administration.

[0702] C57BL / 6 (CD45.2) and C57BL / 6 (CD45.1) mice were purchased from Jackson Laboratories. C57BL / 6 (CD45.2) mice were conditioned with a single intravenous dose of anti-mouse CD45-ADC (0.3 mg / kg, 1 mg / kg, or 3 mg / kg) or isotype-ADC (isotype control mAb conjugated to PBD) (3 mg / kg) 48 h prior to transplantation (see Other CytotoxinsDepiction of Study Design). 5 Gy TBI was used as a positive control for conventional conditioning administered 24 h prior to transplant. Conditioned mice were transplanted with 2 x 10 7 7 35-55 As shown in -9 a dose-responsive donor chimerism was observed with CD45-ADC conditioning. Dose-responsive donor myeloid cells, B cells, and T cells -10 were observed with CD45-ADC conditioning over a 16 week time course. These results indicate that anti-CD45-ADC (3 mg / kg, iv, single dose) conditioning enables full donor chimerism after syngeneic transplantation. -10 50 Example 2. Anti-CD45-ADC preferentially kills proliferating T cells in vitro b g7 Mouse PBMC were cultured with plate-bound aCD3 clone 145-2C11C to promote survival and expansion of murine T cells. T cells were cultured with anti-mouse CD45-ADC (anti-CD45 mAb conjugated to PBD) or isotype-ADC and the total T cell number and percentage of proliferating (Ki67+) T cells were assessed. d + As shown in + anti-mouse CD45-ADC (versus isotype-ADC) treatment significantly reduced the number of T cells. As shown in -10 there was a dose-responsive decrease in proliferating T cells (Ki67+) with increasing doses of anti-mouse CD45-ADC compared to isotype-ADC. These results show that anti-mouse CD45-ADC preferentially kills circulating murine T cells in vitro. + -10 Example 3. Anti-CD45-ADC preferentially kills proliferating T cells in the scGvDH in vivo model 7

[0001] Next, anti-CD45-ADC (anti-CD45 mAb conjugated to PBD) was evaluated in a sclerodermic graft versus host disease (scGVHD) model of scleroderma (also known as systemic sclerosis).

[0002] ​DBA / 2 and Balb / c (CD45.1) mice were purchased from Jackson Laboratories. Balb / C (CD45.1) mice were conditioned with 6.5 Gy total body irradiation (TBI) 24 hours prior to allogeneic (Allo) adoptive cell transfer of DBA / 2 (CD45.2) splenocytes (see Figure 2B Study design in

[0710] Host animals (CD45.1+) were treated with a single intravenous dose (3 mg / kg) of anti-mouse CD45-ADC or isotype-ADC 7 days after initiation of scGvHD (CD45.2+ cells). Animals were euthanized at day 12 after initiation (5 days after ADC administration). Allogeneic cells, T cells and proliferating T cells in tissues (blood, spleen) were analyzed Figure 3A and 3C As shown in Figure 3B and 3E , greater T cell depletion was observed in the spleen or peripheral blood with CD45-ADC treatment compared to isotype-ADC. These results indicate that CD45-ADC selectively depletes a greater percentage of proliferating allogeneic T cells compared to isotype-ADC.

[0711] Example 4. Temporal improvement of murine EAE disease after syngeneic transplantation is achievable by total body irradiation (TBI).

[0712] Next, the murine experimental autoimmune encephalomyelitis (EAE) model of autoimmune disease was evaluated. Conditioning by total body irradiation (TBI) was tested in the EAE model.

[0713] Experimental autoimmune encephalomyelitis (EAE) model: C57BL / 6 (CD45.2) mice were induced with MOG35-55 peptide in complete Freund’s adjuvant, followed by administration of pertussis toxin (PTX) at 4h post MOG35-55 and 24h post initial dose of PTX. Disease onset was approximately 9-12 days post induction. Animals were scored twice daily for the duration of 80 days post EAE induction. EAE clinical score scale: 1 - tail weakness; 2 - partial hind limb paralysis; 3 - complete hind limb paralysis; 4 - complete hind limb paralysis and partial front limb paralysis; 5 - moribund bound. Animals were euthanized at 2 consecutive scores of 4. Terminal analysis of tissues (peripheral blood, lymph nodes) was evaluated to determine chimerism and immune components of relapsing EAE mice. (S) lymph node cells were stimulated with PMS / Ionomycin (50 ng / mL) and Brefeldin A (1 pg / mL) or (NS) lymph node cells were not stimulated and T cell chimerism (donor, host) and IL-17 production were evaluated.

[0714] Animals were conditioned with 9 Gy TBI at day 8 and day 13 post-EAE induction. Conditioned mice were transplanted with 10 7 donor cells 24 hours after TBI. Data show correlation of transient disease improvement or delay of disease progression when transplanted at low clinical score Figure 3D ). Data also show that full donor chimerism was achieved at 4 weeks post-transplantation and was maintained for 8 weeks Figure 4A Figure 4B and 4D show a comparison of host and donor cells in peripheral blood Figure 4C ) and lymph nodes Figure 4C ) harvested from control mice (untransplanted) and treated mice (BMT at day 9). More than 95% donor chimerism was observed in end analysis of total cells and >90% donor T cells in peripheral blood or lymph nodes. IL-17 is a proinflammatory cytokine that plays a key role in the development of EAE. Residual host IL-17 producing CD4 T cells in the lymph nodes can drive disease relapse.

[0715] Taken together, these results show that in the EAE model, syngeneic transplantation after conditioning with irradiation (9 Gy) leads to transient improvement of disease.

[0716] Example 5. A single dose of CD45-targeted ADC safely conditions for autologous transplantation and improves disease in multiple autoimmune disease models

[0717] The following example describes the use of a single dose of an anti-CD45 ADC for autologous transplantation, wherein the anti-CD45 ADC improves disease in multiple mouse models of autoimmune diseases.

[0718] To address the challenges associated with autologous HSC transplantation, an antibody drug conjugate (ADC) that selectively targets CD45 was tested to determine whether an anti-CD45 ADC can deplete autoimmune cells and enable autologous HSCT as a potentially one-time curative treatment for patients with autoimmune diseases.

[0719] ​To model this approach in mice, an ADC targeting murine CD45 (anti-CD45 mAb conjugated to PBDs) was generated as a single conditioning agent for murine syngeneic grafts. The murine CD45 ADC specifically targets the CD45.2 isoform of mouse CD45 and was engineered to ablate effector function, allowing for site-specific conjugation of the linker payload and rapid clearance. The payload of this murine tool ADC is potent and preferentially kills dividing cells. In a murine syngeneic transplant model, the ability of the anti-mouse CD45 ADC to condition the recipient was evaluated following a single myeloablative dose. The ability of this ADC to ablate pathogenic host-reactive cells and enable immune reset of the recipient was further assessed in the context of multiple mouse models of autoimmune disease, including MOG-induced experimental autoimmune encephalitis (EAE; murine model of multiple sclerosis), proteoglycan-induced arthritis (PGIA), type 1 diabetes, and sclerodermic graft versus host disease (scGVHD).

[0720] To induce EAE in the murine model, mice were immunized with MOG in complete Freund’s adjuvant 35-55 Mice were induced with peptide followed by administration of pertussis toxin (PTX). Immunized mice were conditioned against CD45.1+ syngeneic grafts (B6.SJL donors) on day 5 post-immunization and transplanted 48 hours post-conditioning. A schematic of the study design is shown in Figure 4D

[0721] A single dose of tool anti-mouse CD45-ADC at 3 mg / kg achieved complete myeloablation in recipient mice and enabled full donor chimerism (>99% depletion of LT-HSCs (Lin-Kit+Sca-1+CD150+CD48-) in the syngeneic mouse transplant model). Due to anticipated hematopoietic failure, non-transplanted mice were euthanized approximately 12 days post-dosing. Transplanted mice achieved complete engraftment of syngeneic BMT (>90% chimerism at week 16). Specifically, end analysis of tissues (bone marrow, peripheral blood, spleen, lymph nodes) demonstrated that conditioning with CD45-ADC enabled full donor chimerism Figure 5A The disease-modifying effects of the ADC were associated with a global reduction in host splenic T cells Figure 5B and a reduction in IL-17A-producing effector cells in the spleen Figure 5C

[0722] ​​In EAE, conditioning with a myeloablative dose of CD45-ADC followed by syngeneic transplantation prior to disease onset resulted in full donor chimerism, significantly delayed disease onset (mean time to onset 38 days), reduced overall disease severity, and reduced incidence of disease (3 / 12, 25%) Figure 5D and 5G ). In contrast, vehicle or CD45 antibody treated animals ( Figure 5E and Figure 5E ) developed disease at approximately 9-12 days post induction.

[0723] Figure 5G It is shown that, following treatment with 1 mg / kg CD45-ADC, disease onset was at 21 days with a peak disease score of 2.1; following treatment with 3 mg / kg CD45-ADC + BMT, disease onset was at 42 days with a peak disease score of 0.75; and for mice treated with vehicle, disease onset was at 11 days with a peak disease score of 3.1. Figure 5E Similar findings associated with 3 mg / kg CD45-ADC + BMT treatment are shown, and additionally, the effect of isotype-ADC or naked CD45 antibody treatment is shown. Less pronounced effects on donor chimerism and disease amelioration were observed in animals treated with isotype-ADC (28 days to disease onset, 5 / 12 (40%) incidence of disease), a result consistent with known platform toxicity of murine ADC payloads against rapidly dividing cells in immune models. Table 3 summarizes Figure 5G the mean time to EAE onset and incidence of disease for each treatment group in

[0724] Table 3: Mean time to EAE onset

[0725]

[0726] In active EAE, treatment with 3 mg / kg anti-mouse CD45-ADC on day 10 or day 13 followed by syngeneic transplantation prevented the progression of disease activity ( Figure 5G , there was no increase in disease score at the time of treatment; peak disease scores were 0.75 and 2.3, respectively). The effect of syngeneic transplantation observed with CD45-ADC treatment on day 13 was comparable to that achieved with the use of the clinically validated standard of care FTY-720 (a S1P1 antagonist approved equivalent to Gilenya) on day 13, which also arrested disease with a peak score of 2.3.

[0727] Compared to previous studies (mice treated with 9 Gy TBI and homologous BMT on day 9), this study demonstrated superior disease control. These data show that CD45-ADC modulation followed by homologous transplantation effectively resets the immune system and improves disease in an EAE model. Specifically, immune reset via anti-mouse CD45 modulation enables homologous transplantation, delays EAE onset, reduces EAE morbidity, and kills disease-mediating effector T cells in vivo. Furthermore, the disease-improving effect of anti-mouse CD45-ADC modulation in EAE demonstrates efficacy comparable to clinically validated modulation therapies.

[0728] To translate these encouraging preclinical data, an anti-human CD45 ADC with cross-reactivity with non-human primates (NHP) was developed. This human-targeting CD45 ADC contains a mature mAb with affinity targeting epitopes present on all human CD45 isotypes, cross-reactivity with NHP CD45, and conjugation to amatoxins (a potent payload for killing both resting and circulating cells). This ADC was engineered to eliminate Fc-mediated effector function, enabling site-specific conjugation of the linker / payload and rapid clearance. Its ability to deplete hematopoietic and immune cells was evaluated in vivo and in vitro in humanized NSG (hNSG) mice and NHP.

[0729] Anti-human CD45-ADC showed resistance to human BM CD34+ (EC50 2.44x10) -9 M) and peripheral CD3+ cells from normal donors (EC50 7.6x10) -10 M) and peripheral CD3+ cells from MS patients (EC50 1.5x10 -10 Effective killing of M) (Fig. 5H). Furthermore, anti-human CD45-ADC effectively killed human and cynomolgus monkey PBMCs in vitro. Figure 5F and 6B ) and human CD34+CD90+ cells ( Figure 6A Anti-human CD45-Amatoxin ADCs effectively kill human CD34+CD90+ human stem cells. Figure 6C Table 4 summarizes the EC5-mediated killing effects of CD45-ADC in representative experiments. 50 These results indicate that anti-human CD45-ADCs effectively kill primary human and cynomolgus monkey hematopoietic cell types in vitro, including CD34+ hematopoietic stem cells and hematopoietic progenitor cells (HSPCs) as well as CD3+ T cells from both healthy donors and MS patients.

[0730] A single dose of CD45-ADC was well tolerated in hNSG in vivo and resulted in extensive (>95%) depletion of human cells (see also Example 8). A single dose of CD45-ADC depleted human hematopoietic-derived cells in a dose-dependent manner in the peripheral ( Figure 6D ) and bone marrow ( Figure 6E , 6G , 6H and 61) of humanized NSG mice.

[0731] Table 4: EC50 values of anti-human CD45-ADC

[0732]

[0733] In NHPs, a single dose of anti-human CD45-ADC achieved >90% peripheral lymphocyte depletion ( Figure 6F and 7B ) and >80% hematopoietic stem cell depletion (HSC; Figure 7A and 7D ). See also Figure 7C , which shows that a single dose of anti-human CD45-ADC resulted in immune cell depletion, and Figure 7G , which shows that a single dose of anti-human CD45-ADC resulted in CD34+CD90+CD45RA-HSC depletion. In addition, as shown in Figure 7H and 7F , clinical chemistry values for alanine aminotransferase (ALT), total bilirubin (TBIL), alkaline phosphatase (ALP), and aspartate aminotransferase (AST) indicated that a single dose of anti-human CD45-ADC was well tolerated in NHPs. There were no changes in clinical chemistry values for gamma-glutamyl transferase (GGT), albumin, blood urea nitrogen (BUN), creatinine, glucose, and prothrombin (PT).

[0734] These results indicate that targeted immune depletion with a single treatment using CD45-ADC can be sufficient for auto-HSCT and allows for immune reset and reestablishment of immune tolerance. Targeted CD45-ADC can represent a safer and better tolerated way of conditioning patients prior to immune reset by auto-HSCT and can significantly reduce side effects associated with existing conditioning regimens.

[0735] Example 6. Treatment with murine CD45-ADC renders quiescent T cells incapable of mounting an allogeneic response

[0736] B6 mice (H-2 b) with vehicle, isotype-ADC or CD45-ADC (anti-CD45 mAb conjugated to PBDs). Seven days later, splenocytes were isolated, labeled with CellTrace Violet and adoptively transferred into naive, immunodeficient NSG hosts (H-2 g7 , H-2 d ). Cells were isolated from peripheral blood (d2 and d7) and spleen (d7) at various times post transfer and assessed for CTV staining intensity Figure 7E ), absolute number of T cells in peripheral blood as a function of days post transfer Figure 8A ), and absolute number of T cells in spleen 7 days post transfer Figure 8B .

[0737] As described in Figure 8C , T cells from untreated mice or isotype-ADC treated mice expanded or accumulated after adoptive transfer into immunodeficient allogeneic transplant recipients, while T cells from CD45-ADC treated mice were unable to proliferate. These results show that treatment with murine CD45-ADC rendered quiescent (i.e., resting) T cells unable to mount an allogeneic response.

[0738] These results indicate that anti-mouse tool CD45-ADC has a dual mechanism of action in a mouse model of autoimmune disease by (a) preferentially killing allogeneic T cells in vivo compared to quiescent T cells (see Example 3), and (b) rendering the remaining T cells unable to mount an allogeneic response. These results show that CD45-ADC mediated targeted elimination and neutralization of CD45 + effectors.

[0739] Example 7. Anti-mouse AD45-ADC conditioning and immune reset after BMT improves disease in a murine model of rheumatoid arthritis

[0740] Conditioning with anti-mouse CD45-ADC (anti-CD45 mAb conjugated to PBDs) prior to bone marrow transplantation was evaluated in mouse proteoglycan-induced arthritis (PGIA), a murine model of rheumatoid arthritis. As shown in Figures 8A-8C Balb / c mice (CD45.2+) were immunized 3 times with recombinant human core G1 proteoglycan (aggrecan) (60 μg in 2 mg DDA) (day 0, day 21 and day 42 of study). Animals were treated 11 days after the final immunization (day 53 of study) and 48 hours later conditioned animals were transplanted with Balb / c CD45.1+ syngeneic BM. Animals treated with a neutralizing monoclonal antibody to murine TNFα received 500 μg / mouse IP weekly starting on day 53 of study.

[0741] AsFigure 9A and 9C Treatments with 2 mg / kg of CD45-ADC but not with isotype-ADC allowed the formation of full donor chimerism in peripheral blood ( Figure 9B ) and bone marrow ( Figure 9B ) 3 weeks post-transplantation. Animals were scored using the model clinical scoring system developed by Glantt and Mikecz (Methods in Molecular Medicine, Vol. 102(17): 313-338) and examples of scores for control treated animals and animals treated with CD45-ADC are shown in Figure 8C . Scores over time for the treatment groups are shown in Figure 9D .

[0742] In the PGIA model, therapeutic intervention with CD45-ADC and BMT (11 days post 3rdimmunization) prevented disease progression, similar to clinically validated TNFa neutralization approaches. These results indicate that therapeutic treatment with CD45-ADC enables immune reset via syngeneic BMT in a murine model of rheumatoid arthritis and results in the halt of disease progression.

[0743] Example 8. Anti-human CD45-ADC eliminates effector cells and reduces disease in a xenoGVHD scleroderma-like model

[0744] In this study, an anti-human CD45 ADC (anti-CD45 mAb conjugated to amanitin) was used to evaluate its ability to deplete hematopoietic and immune cells in vivo in humanized NSG (hNSG) mice. hNSG mice that developed chronic xenoGVHD with skin involvement after adoptive transfer of human PBMCs were given a single dose of isotype-ADC or an anti-human CD45-targeted ADC. A single dose of anti-human CD45-ADC was well tolerated and eliminated human hematopoietic cells in the periphery ( Figure 9E ) and progenitors in the BM ( Figure 10A and 10C ). As shown in Figure 10B , in images of selected animals, there was clear resolution of skin pathology and hair regrowth in animals administered CD45-ADC 14 days post-treatment, while animals treated with isotype-ADC did not improve. In Figure 10D clinical scores of animals based on size and appearance of skin lesions were summarized. The animal with the highest score in the isotype-ADC treatment group had to be euthanized on day 14 due to its symptoms. In addition, durable depletion of peripheral human T cells was achieved in animals treated with anti-human CD45 ADC ( Figure 10E ).

[0745] As in Example 5 and Figure 10FAs stated above, in vitro, CD45-ADC showed susceptibility to human peripheral CD3 from healthy donors. + Cells (EC50 7.6x10) -10 M) and human peripheral CD3 from MS patients + Cells (EC50 1.5x10) -10 Effective killing of human effector cells (M). This example demonstrates that, in vivo, a single dose of CD45-ADC in hNSG mice is well tolerated and leads to significant depletion of both lymphocytes and hematopoietic stem cells (HSCs). A single dose of CD45-ADC, rather than allotype-ADC, resulted in the elimination of pathogenic T cells and reversal of clinical symptoms in a scleroderma-like chronic xenoGVHD model. These results indicate that treatment with a single dose of anti-human CD45 ADC in a hNSG mouse model of xenoGVHD leads to the elimination of human effector cells and disease improvement.

[0746] Example 9. Treatment with a single dose of CD45-ADC and homologous HSCT resulted in disease prevention in an adoptive transfer model of type 1 diabetes.

[0747] The study evaluated the use of a single dose of CD45-ADC (an anti-CD45 mAb conjugated with PBD) followed by homologous HSC transplantation in a type 1 diabetic adoptive transfer mouse model.

[0748] exist Figure 5H Table 5 summarizes the design of studies utilizing the RIP-OVA / OT-I / II system. RIP-OVA mice are B6 mice that express chicken ovalbumin in the β-islet cells of the pancreas. A combination of central and peripheral tolerance mechanisms enables the adaptive immune system to recognize OVA as itself. OT-I (MHC I-restricted) and OT-II (MHC II-restricted) are two TCR Tg lines that specifically recognize the OVA epitope. They are maintained in the B6 background.

[0749] Adoptive transfer of OT-I and OT-II cells into RIP-OVA mice resulted in β-islet cell destruction and the onset of diabetes. Diabetes development was monitored by blood glucose levels (tested on test strips via tail vein incision; glucose >14.3 mmol / L). In this model, mice developed diabetes 5–12 days after CD8+ OT-I cell transfer (two consecutive measurements at 250 mg / dL, Drujont PLoS One 2014). Mice were peeled off on day 6 post-adoptive transfer (n=4 / treatment). Flow cytometry was performed to detect Vβ5 and Vα2 in peripheral blood, spleen, dLN, and pancreas to measure the number of transferred cells. Subsequently, 2 days post-treatment, mice (n=6 mice / group) were transplanted with 2x10-1 cells. 7cells from B6 CD45.1. Body weight, BCS, and blood glucose were measured for two weeks as endpoints.

[0750] Table 5. Study design

[0751]

[0752] RIP-OVA mice were treated with vehicle control, anti-Thyl.2 (500 ug), 9 Gy TBI, 1 mg / kg or 3 mg / kg isotype-ADC (Iso-PBD), or 1 mg / kg or 3 mg / kg CD45-ADC (CD45-PBD). As shown in Figure 11A Figure 11B approximately 40% of vehicle-treated mice developed diabetes at week 4 post- engraftment. In contrast, none of the 6 mice treated with CD45-ADC at 1 mg / kg or 3 mg / kg developed diabetes. These results indicate that treatment with a single dose of CD45-ADC and syngeneic HSC engraftment leads to disease prevention in a model of adoptive transfer of type 1 diabetes.

[0753] Table 6: Sequence Listing

[0754]

[0755]

[0756]

[0757]

[0758]

[0759]

[0760]

[0761]

[0762]

[0763] Other Embodiments

[0764] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0765] While the application has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains and fall within the scope of the appended claims.

[0766] Other embodiments are within the claims. SEQUENCE LISTING <110> Merdama Therapeutics, Inc. <120> Methods and compositions for treating autoimmune diseases <130> M103034 2180WO(0511.2) <140> <141> <150> 63 / 030,860 <151> 2020-05-27 <150> 62 / 968,870 <151> 2020-01-31 <150> 62 / 944,988 <151> 2019-12-06 <150> 62 / 933,279 <151> 2019-11-08 <150> 62 / 882,310 <151> 2019-08-02 <150> 62 / 863,141 <151> 2019-06-18 <150> 62 / 857,232 <151> 2019-06-04 <160> 39 <170> PatentIn version 3.5 <210> 1 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic Polypeptide <400> 1 Glu Val Gin Leu Val Glu Ser Gly Gly Asp Arg Val Gin Pro Gly Arg 1 5 10 15 Ser Leu Thr Leu Ser Cys Val Thr Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Trp Met Thr Trp He Arg Gin Val Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ser He Ser Ser Ser Gly Gly Ser He Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gin Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Arg Trp Ala Gly Ala Met Asp Ala Trp Gly Gin Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic peptide <400> 2 Phe Thr Phe Asn Asn Tyr Trp Met Thr 1 5 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Note: Synthetic Peptide <400> 3 Ser lie Ser Ser Ser Gly Gly Ser lie Tyr Tyr Pro Asp Ser Val Lys 1 5 10 15 Gly <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Note: Synthetic Peptide <400> 4 Ala Arg Asp Glu Arg Trp Ala Gly Ala Met Asp Ala 1 5 10 <210> 5 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence Note: Synthetic Polypeptide <400> 5 Asp lie Gin Met Thr Gin Ser Pro Pro Val Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Leu Ser Cys Lys Ala Ser Gi...

Claims

1. Use of an antibody drug conjugate (ADC) in the manufacture of a medicament for depleting a CD45+ cell population in a human patient having an autoimmune disease, the use comprising administering the ADC to the human patient having an autoimmune disease to effect immune reset, wherein the ADC is an anti-CD45 ADC comprising an anti-CD45 antibody or antigen binding portion thereof conjugated to a cytotoxin via a linker, wherein the cytotoxin is a pyrrolobenzodiazepine having the following formula dimer: wherein the wavy line indicates the point of attachment of the cytotoxin to the linker.

2. Use of an ADC in the manufacture of a medicament for conditioning a human patient having an autoimmune disease to receive a hematopoietic stem cell (HSC) transplant, the use comprising administering the ADC to the human patient having an autoimmune disease to effect immune reset, wherein the ADC is an anti-CD45 ADC comprising an anti-CD45 antibody or antigen binding portion thereof conjugated to a cytotoxin via a linker, wherein the cytotoxin is a pyrrolobenzodiazepine having the following formula dimer: wherein the wavy line indicates the point of attachment of the cytotoxin to the linker.

3. The use of claim 1 or 2, wherein the autoimmune disease is rheumatoid arthritis, autoimmune encephalitis, scleroderma, multiple sclerosis, type 1 diabetes, or systemic sclerosis.

4. The use of claim 1 or 2, further comprising administering to the patient a transplant comprising HSCs.

5. The use of claim 4, wherein the HSC transplant is an autologous HSC transplant.

6. The use of claim 4, wherein the anti-CD45 ADC is administered to the patient three days prior to the patient receiving the transplant comprising HSCs.

7. The use of claim 4, wherein the HSC transplant is administered to the patient after the anti-CD45 ADC has been cleared from the blood of the human patient.

8. Use of an anti-CD45 ADC in the manufacture of a medicament for treating a patient having scleroderma graft versus host disease (scGVHD) or multiple sclerosis, the use comprising administering an anti-CD45 ADC to a patient having scGVHD or multiple sclerosis such that immune reset is effected and the scGVHD or multiple sclerosis is treated, wherein the anti-CD45 ADC comprises an anti-CD45 antibody or fragment thereof conjugated to a cytotoxin via a linker, wherein the cytotoxin is a pyrrolobenzodiazepine having the following formula dimer: wherein the wavy line indicates the point of attachment of the cytotoxin to the linker.

9. The use of claim 1, 2, or 8, wherein the anti-CD45 ADC is administered to the patient as a single dose or as divided doses.

10. The use of claim 1, 2, or 8, wherein the patient does not require treatment for the autoimmune disease after transplantation.

11. The use of claim 1, 2, or 8, wherein the patient has multiple sclerosis, and wherein the patient does not require treatment with natalizumab, dimethyl fumarate, or monomethyl fumarate after transplantation.

12. The use of claim 1, 2, or 8, wherein the patient has arthritis, and wherein the patient does not require treatment with a TNF inhibitor after transplantation.

13. The use of claim 12, wherein the TNF inhibitor is an anti-TNFa antibody.

14. The use of claim 1, 2, or 8, wherein the patient enters remission for at least 1 year after transplantation.

15. The use of claim 14, wherein the remission is clinical remission.

16. The use of claim 14, wherein the remission is biochemical remission.

17. The use of claim 14, wherein the remission is histological remission.

18. The use of claim 1, 2, or 8, wherein the anti-CD45 antibody is a chimeric antibody or a humanized antibody.

19. The use of claim 1, 2, or 8, wherein the anti-CD45 antibody is a human antibody.

20. The use of claim 1, 2, or 8, wherein the anti-CD45 antibody is intact.

21. The use of claim 1, 2, or 8, wherein the anti-CD45 antibody or antigen-binding portion thereof is selected from the group consisting of a monoclonal antibody or antigen-binding portion thereof, a polyclonal antibody or antigen-binding portion thereof, a bispecific antibody or antigen-binding portion thereof, a dual variable immunoglobulin domain, a single chain Fv molecule (scFv), a diabody, a triabody, a nanobody, an antibody-like protein scaffold, an Fv fragment, a Fab fragment, a F(ab’)2 molecule, and a tandem di-scFv.

22. The use of claim 1, 2, or 8, wherein the anti-CD45 antibody has an isotype selected from the group consisting of IgG, IgA, IgM, IgD, and IgE.

23. The use of claim 22, wherein the anti-CD45 antibody contains a human IgG1, IgG2, IgG3, or IgG4 isotype Fc domain.

24. The use of claim 1, 2, or 8, wherein the anti-CD45 antibody or antigen-binding portion thereof comprises an Fc domain, and wherein the anti-CD45 antibody or antigen-binding portion thereof is conjugated to the cytotoxin through a cysteine residue in the Fc domain.

25. The use of claim 24, wherein the cysteine residue is introduced into the Fc domain by way of an amino acid substitution.

26. The use of claim 25, wherein the amino acid substitution is D265C according to EU numbering.

27. The use of claim 1, 2, or 8, wherein the patient enters remission for at least 2 years after transplantation.

28. The use of claim 1, 2, or 8, wherein the patient enters remission for at least 5 years after transplantation.

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