Anti-CD45 antibodies and their conjugates

Abstract

Targeting hematopoietic stem cells (HSCs) or immune cells that express CD45 prior to transplantation and (b) administering to the patient a therapeutically effective amount of an anti-CD45 antibody or antibody-drug conjugate (ADC) useful in a therapeutic method comprising administering to the patient a therapeutically effective amount of an anti-CD45 antibody or antibody-drug conjugate (ADC).

Description

[Technical field]

[0001] [CROSS REFERENCE TO RELATED APPLICATIONS] This application claims priority to U.S. Provisional Application No. 62 / 753,002, filed October 30, 2018, U.S. Provisional Application No. 62 / 774,006, filed November 30, 2018, and U.S. Provisional Application No. 62 / 807,582, filed February 19, 2019. The contents of each priority application are incorporated by reference into this application.

[0002] [Technical field] The present invention relates to the field of anti-CD45 antibodies or antibody drug conjugates thereof. The present invention further relates to the treatment of patients suffering from various pathologies, such as hematological diseases, metabolic disorders, cancer, and autoimmune diseases, among others, by administration of anti-CD45 antibodies or antibody drug conjugates (ADCs), where said antibodies or ADCs are capable of binding to CD45 on the surface of either hematopoietic stem cells or immune cells. [Background technology]

[0003] CD45 is a member of the protein tyrosine phosphatase (PTP) family and is a transmembrane glycoprotein of 180-240 kD. CD45 is a signaling molecule that regulates many cellular processes, including cell proliferation and differentiation. CD45 is a type I transmembrane protein found in various isoforms on the surface of differentiated hematopoietic cells and has been shown to play an important role in T-cell and B-cell receptor signaling. In general, cells of hematopoietic origin express CD45, except for mature erythrocytes and platelets.

[0004] Different isoforms of CD45 exist due to multiple splicing of its exons. The various isoforms of human CD45 differ in their extracellular domains and are differentially expressed on subpopulations of B- and T-cell lymphocytes. At least six different human isoforms of CD45 mRNA have been isolated, which contain all three exons (ABC isoforms), two of the three exons (AB and BC isoforms), only one exon (A and B isoforms), or no exon (O isoform) (Hermiston et al. (2003) Annu Rev Immunol. 21, 107-137.). The suffixes RA, RB, or RO indicate the requirement of amino acid residues corresponding to exon A (RA), exon B (RB), or the lack of amino acid residues corresponding to exons A, B, and C (RO) for CD45 antigen expression.

[0005] Despite advances in medical technology, there remains a need to treat pathologies of the hematopoietic system, such as diseases of certain blood cells, metabolic disorders, cancer, and autoimmune conditions, among others. While hematopoietic stem cells (HSCs) have great therapeutic potential, they have limitations that prevent their clinical use due to the difficulty in reliably engrafting HSC grafts in the host. In particular, hematopoietic stem cell therapies that include antibodies targeting cell surface antigens on endogenous HSCs can induce undesirable immune stimulatory and effector functions that prevent exogenous HSC grafts from engrafting. There remains a need in the art for targeted therapies that affect both HSCs and immune cells to improve engraftment, such as therapies targeting CD45 as an antigen that can condition patients for HSC transplantation. Summary of the Invention [Problem to be solved by the invention]

[0006] This application describes anti-CD45 antibodies (anti-human CD45 9 (hCD45) antibodies) and antibody drug conjugates (ADCs) that bind to hematopoietic stem cells (HSCs) and are useful, for example, as conditioning agents for transplantation. In particular, the anti-CD45 antibodies and ADCs described herein can be used to specifically deplete host HSCs, immune cells, and disease-causing cells. Additionally, the anti-CD45 antibodies and ADCs described herein may be used to treat patients with leukemia or lymphoma, as well as to treat patients with autoimmune diseases such as multiple sclerosis and scleroderma. The anti-CD45 antibodies and ADCs described herein fill a need for compositions and methods to promote the engraftment of exogenous hematopoietic stem cell grafts such that the multi-potency and hematopoietic functionality of these cells is preserved after transplantation. [Means for solving the problem]

[0007] In one embodiment, the isolated anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising a variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO:2, a CDR2 having the amino acid sequence set forth in SEQ ID NO:3, and a CDR3 having the amino acid sequence set forth in SEQ ID NO:4, and a light chain comprising a variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO:6, a CDR2 having the amino acid sequence set forth in SEQ ID NO:7, and a CDR3 having the amino acid sequence set forth in SEQ ID NO:8.

[0008] In one embodiment, the isolated anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1 and / or comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:5.

[0009] In one embodiment, the isolated anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising a variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO:10, a CDR2 having the amino acid sequence set forth in SEQ ID NO:11, and a CDR3 having the amino acid sequence set forth in SEQ ID NO:12, and a light chain comprising a variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO:14, a CDR2 having the amino acid sequence set forth in SEQ ID NO:15, and a CDR3 having the amino acid sequence set forth in SEQ ID NO:16.

[0010] In one embodiment, the isolated anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9 and / or comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:13.

[0011] In one embodiment, the isolated anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising a variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 18, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 20, and a light chain comprising a variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 22, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 23, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 24.

[0012] In one embodiment, the isolated anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:17 and / or comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:21.

[0013] In certain embodiments, the isolated anti-CD45 antibody, or antigen binding portion thereof, comprises an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising a heavy chain comprising a variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 2, CDR2 having the amino acid sequence set forth in SEQ ID NO: 3, CDR3 having the amino acid sequence set forth in SEQ ID NO: 4, and a light chain comprising a variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 6, CDR2 having the amino acid sequence set forth in SEQ ID NO: 7, CDR3 having the amino acid sequence set forth in SEQ ID NO: 8; an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1 and / or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5; an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 having the amino acid sequence set forth in SEQ ID NO: an isolated anti-CD45 antibody, or antigen-binding portion thereof, comprising a heavy chain comprising a variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 11, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 12, and a light chain comprising a variable region comprising a CDR1 having the amino acid sequence set forth in SEQ ID NO: 14, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 16; an isolated anti-CD45 antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9 and / or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13;or an isolated anti-CD45 antibody, or antigen-binding portion thereof, comprising a heavy chain comprising a variable region that includes a CDR1 having the amino acid sequence set forth in SEQ ID NO: 18, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 20, and a light chain comprising a variable region that includes a CDR1 having the amino acid sequence set forth in SEQ ID NO: 22, a CDR2 having the amino acid sequence set forth in SEQ ID NO: 23, and a CDR3 having the amino acid sequence set forth in SEQ ID NO: 24;

[0014] In one embodiment, the isolated anti-CD45 antibody, or antigen binding portion thereof, specifically binds to an epitope on human CD45 comprising residues 486R, 493Y, and 502T of SEQ ID NO: 37 and binds to cynomolgus and rhesus CD45; or specifically binds to an epitope on human CD45 comprising the amino acid sequence RNGPHARYHLEVEAGNT (SEQ ID NO: 38) and binds to cynomolgus and rhesus CD45; or specifically binds to an epitope on human CD45 comprising the amino acid sequence CRPPRDRNGPHERYHLEVEAGNTLVRNESHK (SEQ ID NO: 39) and binds to cynomolgus and rhesus CD45.

[0015] In another embodiment, the isolated anti-CD45 antibody, or antigen binding portion thereof, specifically binds to an epitope of human CD45 comprising residues 486R, 493Y, and 502T of SEQ ID NO: 37; binds to at least one additional amino acid, at least two additional amino acids, at least three additional amino acids, at least four additional amino acids, or at least five additional amino acids in a peptide comprising RNGPHERYHLEVEAGNT (SEQ ID NO: 38), where the additional amino acid residues are not residues 486R, 493Y, and 502T of SEQ ID NO: 37; and / or binds to cynomolgus and rhesus CD45.

[0016] In another embodiment, provided herein is an anti-CD45 antibody, or antigen-binding portion thereof, that specifically binds to an epitope of human CD45 that includes residues 486R, 493Y, and 502T of SEQ ID NO: 37, and that binds to cynomolgus and rhesus monkey CD45.

[0017] In yet another embodiment, provided herein is an anti-CD45 antibody, or antigen-binding portion thereof, that specifically binds to an epitope of human CD45 that includes the amino acid sequence RNGPHARYHLEVEAGNT (SEQ ID NO:38) and binds to cynomolgus and rhesus monkey CD45.

[0018] In a further embodiment, provided herein is an anti-CD45 antibody, or antigen-binding portion thereof, that specifically binds to an epitope of human CD45 comprising the amino acid sequence CRPPRDRNGPHERYHLEVEAGNTLVRNESHK (SEQ ID NO:39) and binds to cynomolgus and rhesus monkey CD45.

[0019] In one embodiment, the antibody competes with antibody AbA for binding to human CD45.

[0020] In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is a de-immunized antibody as described herein.

[0021] In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is a chimeric antibody (eg, chimeric AbA, AbB, or AbC).

[0022] In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is a humanized antibody (eg, humanized AbA, AbB, or AbC).

[0023] In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is an intact antibody (eg, an intact IgG antibody).

[0024] In another embodiment, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is an isolated deimmunized monoclonal anti-CD45 antibody, or antigen-binding portion thereof, that specifically binds to human CD45 and cynomolgus CD45, wherein the anti-CD45 antibody, or antigen-binding portion thereof, of any one of claims 1 to 15 is a parent antibody, or antigen-binding portion thereof, of said deimmunized anti-CD45 antibody.

[0025] In one embodiment, the isolated anti-CD45 antibody, or antigen-binding portion thereof, comprises an Fc region comprising at least one amino acid substitution that is H435 or I235 / H310 / H435 (EU numbering) (e.g., H435A (EU numbering), I235A / H310A / H435A (EU numbering)).

[0026] In certain embodiments, the isolated anti-CD45 antibody, or antigen-binding portion thereof, is an IgG (eg, IgG1 or IgG4).

[0027] In one embodiment, the invention features an antibody drug conjugate (ADC) comprising an anti-CD45 antibody disclosed herein attached via a linker to a cytotoxin.

[0028] In some embodiments, the cytotoxin is an amatoxin, Pseudomonas exotoxin A, de bouganin, diphtheria toxin, saporin, maytansine, maytansinoid, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, or indolinobenzodiazepine dimer.

[0029] In some embodiments, the cytotoxin is a microtubule binding agent or an RNA polymerase inhibitor, hi one embodiment, the cytotoxin is an RNA polymerase inhibitor.

[0030] In one embodiment, the RNA polymerase inhibitor is an amatoxin.

[0031] In some embodiments, the amatoxin is α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, or proamanutin.

[0032] In one embodiment, the amatoxin is represented by formula (IV): [ka] Here, R 1 H, OH, OR A OR C is; R 2 H, OH, OR B OR C is; R A and R Btogether with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R 3 , H, R C , or R D is; R 4 , R 5 , R 6, and R 7 are independently H, OH, or OR C , OR D , R C , or R D is; R 8 OH, NH 2 , OR C , OR D , N.H.R. C , or NR C R D is; R 9 H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO 2 - is; R C is -LZ; R D is an optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 Heteroalkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 Heteroalkenyl, optionally substituted C 2 -C 6 Alkynyl, optionally substituted C 2 -C 6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is optionally substituted C 1 -C6 Alkylene, optionally substituted C 1 -C 6 Heteroalkylene, optionally substituted C 2 -C 6 Alkenylene, optionally substituted C 2 -C 6 Heteroalkenylene, optionally substituted C 2 -C 6 Alkynylene, optionally substituted C 2 -C 6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, a peptide, a dipeptide, -(C=O)-, or a combination thereof; and Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in the antibody or antigen-binding fragment.

[0033] In one embodiment, the amatoxin-linker conjugate (Am-LZ) is represented by formula (IA): [ka] Where: R 1 H, OH, OR A OR C is; R 2 H, OH, OR B OR C is; R A and R B together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R 3 , H, R C , or R D is; R 4 , R 5 , R 6,and R 7 are independently H, OH, or OR C , OR D , R C , or R D is; R 8 OH, NH 2 , OR C , OR D , N.H.R. C , or NR C R D is; R 9 H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO 2 - is; R C is -LZ; R D is an optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 Heteroalkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 Heteroalkenyl, optionally substituted C 2 -C 6 Alkynyl, optionally substituted C 2 -C 6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is optionally substituted C 1 -C 6 Alkylene, optionally substituted C 1 -C 6 Heteroalkylene, optionally substituted C 2 -C 6 Alkenylene, optionally substituted C 2 -C 6 Heteroalkenylene, optionally substituted C2 -C 6 Alkynylene, optionally substituted C 2 -C 6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene, a peptide, a dipeptide, -(C=O)-, or a combination thereof; and Z is a chemical moiety formed from a coupling reaction between a reactive substituent Z' present on L and a reactive substituent present in the antibody or antigen-binding fragment. where Am is exactly one R C Contains substituents.

[0034] In some embodiments, the LZ [ka] It is.

[0035] In one embodiment, the ADC has the formula Am-LZ-Ab, represented as follows: [ka] Here, Ab represents the position that binds to the anti-CD45 antibody.

[0036] In some embodiments, the linker-chemical moiety LZ is [ka] It is.

[0037] In some embodiments, the LZ is [ka] It is.

[0038] In one embodiment, the ADC has the formula Am-LZ-Ab, represented as follows: [ka] Here, Ab represents the position that binds the anti-CD45 antibody.

[0039] In one embodiment, the RNA polymerase inhibitor is amanitin.

[0040] In one embodiment, the cytotoxin is selected from the group consisting of Pseudomonas exotoxin A, de bouganin, diphtheria toxin, saporin, maytansine, maytansinoid, auristatin, anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolinobenzodiazepines, and indolinobenzodiazepine dimers or indolinobenzodiazepine pseudodimers.

[0041] In some embodiments, the cytotoxin is a microtubule binding agent or an auristatin. In some embodiments, the microtubule binding agent is maytansine. In some embodiments, the microtubule binding agent is a maytansinoid. In some embodiments, the maytansinoid is selected from the group consisting of DM1, DM3, and DM4, and maytansinol.

[0042] In one embodiment, the auristatin is MMAE or MMAF.

[0043] In some embodiments, the cytotoxin is an anthracycline selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin.

[0044] In one embodiment, the antibody is conjugated to the toxin through a cysteine ​​residue in the Fc domain of the antibody.

[0045] In one embodiment, the cysteine ​​residue is introduced by amino acid substitution in the Fc domain of the antibody.

[0046] In one embodiment, the amino acid substitutions are D265C and / or V205C (EU numbering).

[0047] In some embodiments, the ADC has a drug-to-antibody ratio (DAR) of 1, 2, 3, 4, 5, 6, 7, or 8.

[0048] Also provided is a pharmaceutical composition comprising an antibody or ADC described in the present application and a pharma- ceutically acceptable carrier.

[0049] Further provided is a method of reducing the population of hematopoietic stem cells (HSCs) in a human patient, the method comprising administering to the patient an effective amount of an antibody or ADC described herein, hi one embodiment, the method further comprises administering to the patient a graft comprising hematopoietic stem cells.

[0050] In one embodiment, the graft is allogeneic.

[0051] In one embodiment, the graft is autologous.

[0052] Also provided is a method comprising administering to a human patient a graft comprising hematopoietic stem cells, wherein the patient has previously been administered an antibody or ADC described herein in an amount sufficient to reduce the population of hematopoietic stem cells in the patient.

[0053] In one embodiment, the hematopoietic stem cells are CD45+ cells.

[0054] In certain embodiments, the patient has a blood disease, a metabolic disorder, cancer, or an autoimmune disease, or severe combined immunodeficiency disease (SCID).

[0055] Also disclosed is a method of treating leukemia in a human patient, the method comprising administering to a human patient with leukemia an antibody or ADC described in the present application.

[0056] Further provided is a method comprising administering to a human patient a graft comprising hematopoietic stem cells, wherein the patient has previously been administered an antibody or ADC described herein in an amount sufficient to reduce the population of immune cells in the patient.

[0057] In one embodiment, the immune cells are CD137+, CD2+, or CD5+ cells.

[0058] In one embodiment, the immune cell is a T cell. [Brief description of the drawings]

[0059] [Figure 1] Figures 1A, 1B, 1C, 1D and 1E graphically depict the results of experiments demonstrating that anti-CD45 amanitin antibody drug conjugate (CD45-Am) enables in vitro and in vivo depletion of lymphocytes and hematopoietic stem cells (HSCs). [Diagram 2] Figures 2A and 2B graphically depict the results of an in vitro cytotoxicity assay demonstrating that anti-CD45-AM is highly effective in killing human HSCs in vitro. In Figure 2A, human PBMCs were cultured for 4 days in the presence of the indicated anti-CD45-amanitin conjugates (antibody AbB-amanitin; "CD45-AM") or control non-targeting isotype-matched ADCs ("isotype-AM"), and cell viability was measured by Celltiter Glo in luminescence (RLU; y-axis) at varying antibody concentrations (x-axis). In Figure 2B, primary human CD34+ bone marrow cells were cultured for 5 days with CD45-AM or isotype-AM, and viable CD34+CD90+ HSCs were counted by flow cytometry at varying antibody concentrations (x-axis) (y-axis). [Diagram 3]Figures 3A and 3B show graphical results of in vivo survival studies using xenograft mouse models inoculated with human leukemia cells derived from immortalized CD45-expressing cell lines (5x106 cells / mouse, luciferase-labeled CD45-expressing ALL cell lines). They show that anti-CD45-AM (AbB conjugated to amanitin) doubles median survival to 40 days in the REH-Luc xenograft model. Figure 3A shows that a single injection of 1 mg / kg anti-CD45-AM 5 days after inoculation with ALL resulted in a median survival of 15 days longer compared to vehicle (PBS)-treated control or unconjugated anti-CD45 antibody (n=10 mice / group, p<0.0001). Figure 3B shows representative pseudocolor images of bioluminescence signals acquired using an IVIS Imaging System (Perkin Elmer) at day 19 post-implantation for the anti-CD45-Am and isotype-AM treated groups. [Figure 4] Figures 4A-4H show graphical results of in vivo survival studies using three AML patient-derived xenografts generated from FLT-3+NPM1+ AML samples, demonstrating that a single dose of anti-CD45 antibody AbB conjugated to amanitin ("anti-CD45-AM"; 1 mg / kg) effectively reduces human leukemia cells and extends survival in mouse models. Figure 4A shows the results of a flow cytometry assay assessing cell surface expression of CD45 on CD33+ splenocytes from diseased mice. Figures 4B-4G show survival curves for various PDX AML mice (4-5 mice / group / AML-PDX model) treated with a single intravenous dose of anti-CD45-AM, IgG1 isotype-AM, or vehicle (PBS) when 2-5% blasts were present in the blood. Figure 4H shows that cell surface expression of CD45 on splenocytes from diseased mice was assessed by flow cytometry. [Diagram 5]Figures 5A-5C graphically depict the results of in vivo cell depletion assays, showing that CD45-AM ADCs selectively deplete human HSCs and immune cells in humanized NSG mice. Figure 5A is a schematic of the in vivo humanized NSG model. Figure 5B shows the absolute number of human CD45+ cells in peripheral blood 14 days after injection with anti-CD45-amanitin ("CD45-AM"), unconjugated anti-CD45 antibody ("CD45-naked"), or control non-targeting isotype-matched ADC ("isotype-AM"). Figure 5C shows the absolute number of human CD34+ cells in bone marrow of humanized NSG mice 14 days after administration. Similar results were obtained for CD34+CD90+ (data not shown). *p<0.05 when comparing CD45-AM to both control groups. [Figure 6] Figures 6A-6C show the design and results of an in vivo pharmacokinetic study showing that the CD45-AM ADC has a fast half-life pharmacokinetic profile in cynomolgus monkeys. Figure 6A illustrates an overview of the in vivo cynomolgus monkey model and study design (n = 3 / group, except for 1 mg / kg where n = 2). Figures 6B and 6C graphically show the pharmacokinetics of anti-CD45-amanitin ("CD45-AM") in cynomolgus monkeys. These show that CD45-AM exhibits non-linear PK, with a short half-life suitable for transplantation, and target-mediated drug disposition (TMDD) PK. As used in Figure 6B, "TAb" refers to the whole antibody used to detect anti-hIgG1, while ADC refers to detect anti-amanitin. [Figure 7]Figures 7A, 7B and 7C graphically show the results of an in vivo depletion assay, which demonstrate that anti-CD45 amanitin antibody drug conjugate (CD45-Am) is capable of depleting peripheral lymphocytes in vivo (Figure 7A) and hematopoietic stem cells (HSC), T- and B-cells in the bone marrow of cynomolgus monkeys, as assessed 14 days after administration of CD45-AM. The data in Figure 7C represent data from the time course described in Figure 7A, specifically 1 day after administration. [Figure 8] Figures 8A-8D graphically show the results of assays detecting red blood cell count (Figure 8A; units of 106 / µL), platelet count (Figure 8B; units of 103 / µL), plasma alanine aminotransaminase levels (Figure 8C; units of U / L), and plasma bilirubin levels (Figure 8D; units of U / mL) compared to vehicle control (i.e., PBS) versus days after administration of various doses of a fast half-life anti-CD45 amanitin antibody drug conjugate (CD45-Am). [Figure 9] Figures 9A and 9B show the results of a CD45 epitope mapping study with AbA. Figure 9A shows the AbA interaction site on CD45 along with the amino acid sequence of the region containing the AbA epitope (SEQ ID NO: 39). Contact residues are highlighted as 486R, 493Y, and 502T (numbering see SEQ ID NO: 37). Figure 9B shows the CD45 PDB structure (5FMV) with the epitope site (corresponding to amino acid residues 486-502 of CD45 (RNGPHERYHLEVEAGNT; SEQ ID NO: 38)) in black. Figure 9B shows ribbon / plane representations of the front view (A), back view (B), side view 1 (C), side view 2 (D), and top view (E). [Figure 10]10 shows a sequence alignment of the fibronectin D1-D4 regions of human CD45R0 (SEQ ID NO: 40) with the corresponding regions in cynomolgus monkey (cyno) CD45 (SEQ ID NO: 41). The epitope regions of human CD45 to which AbA binds and the corresponding regions in cynomolgus monkey CD45 are shown in bold. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] [Detailed Description] Disclosed herein are anti-CD45 antibodies and conjugates thereof (antibody drug conjugates; ADCs) that are useful as therapeutic agents, for example, in treating leukemia or acting as conditioning agents for transplantation. Accordingly, included within the present application are anti-hematopoietic cell antibodies (anti-CD45 antibodies) that are useful for hematopoietic stem cell therapy. For example, the antibodies or ADCs of the present application are useful in conditioning procedures that prepare patients for receiving a graft that includes hematopoietic stem cells. Such procedures promote the engraftment of hematopoietic stem cell transplants. According to the methods described herein, a patient may be conditioned for hematopoietic stem cell transplantation therapy by administering to the patient an anti-CD45 ADC, antibody, or antigen-binding fragment thereof that can bind to 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 may be covalently conjugated to a cytotoxin to form an antibody drug conjugate (ADC). Administration of an ADC capable of binding CD45 to a patient in need of hematopoietic stem cell transplantation therapy may promote engraftment of hematopoietic stem cell grafts, for example, by selectively depleting endogenous hematopoietic stem cells, thereby creating vacancies that are filled by the exogenous hematopoietic stem cell graft.

[0061] The following section provides a description of anti-CD45 antibodies or conjugates thereof that may be administered to patients, such as patients suffering from cancer or an autoimmune disease, or patients in need of hematopoietic stem cell transplantation therapy, to promote hematopoietic stem cell graft engraftment, as well as methods of administering such therapeutics to patients (e.g., prior to hematopoietic stem cell transplantation).

[0062] definition As used in this application, the term "about" refers to a value that is 5% higher or lower than the stated value.

[0063] As used in this application, the term "allogeneic" when used in the context of transplantation is used to define cells (or tissues or organs) that are transplanted from a genetically dissimilar donor into a recipient of the same species.

[0064] As used in this application, the term "autologous" refers to cells or grafts where the donor and recipient are the same subject.

[0065] As used in this application, the term "xenogeneic" refers to cells where the donor and recipient species are different.

[0066] As used herein, the term "immune cells" is intended to include, but is not limited to, cells that are of hematopoietic origin and 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 certain embodiments, natural killer cells include cell lines such as NK-92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells. Immune cells can be allogeneic or autologous.

[0067] 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), genetically engineered antibodies, and other modified forms of antibodies (e.g., but are not limited to, de-immunized antibodies, chimeric antibodies, humanized antibodies, heteroconjugate antibodies (e.g., bi-, tri-, and quad-specific antibodies, diabodies, triabodies, and tetrabodies), and antibody fragments (i.e., antigen-binding fragments of antibodies) [e.g., Fab', F(ab') 2 , Fab, Fv, rlgG, and scFv fragments, etc., as long as they exhibit the desired antigen-binding activity.

[0068] Generally, an antibody comprises a heavy chain and a light chain, which contain an antigen-binding region. Each heavy chain is composed of a heavy chain variable region (abbreviated in this application as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains (CH1, CH2 and CH3). Each light chain is composed of a light chain variable region (abbreviated in this application as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability (called complementarity determining regions (CDRs)), interspersed with more conserved regions (called framework regions (FRs)). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains comprise the binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1q).

[0069] 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 a fragment of a full-length antibody. Such antibody fragments include, for example, Fab, F(ab') 2 , scFv, diabody, triabody, affibody, nanobody, aptamer, or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab') 2(iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an 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 consisting 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., 2, 3, 4, 5, or 6) isolated CDRs, optionally linked by a synthetic linker. Furthermore, although the two domains of an Fv fragment (VL and VH) are encoded by separate genes, they can be joined by a linker using recombinant methods to form a single protein chain (where the VL and VH regions pair to form a monovalent molecule) (known as single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those of skill in the art, and the fragments can be screened for utility in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, by chemical peptide synthesis procedures known in the art.

[0070] As used in this application, an "intact" or "full length" antibody refers to an antibody having two heavy (H) chain polypeptides and two light (L) chain polypeptides interconnected by disulfide bonds.

[0071] The term "specifically binds," as used in this application, refers to the ability of an antibody (or ADC) to recognize and bind to a specific protein structure (epitope), rather than proteins in general. If an antibody is specific for epitope "A," then in a reaction involving labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce the amount of labeled A that binds to the antibody. For example, an antibody (if labeled) "specifically binds" to a target if it can be competed away from its target by a corresponding unlabeled antibody. In one embodiment, an antibody specifically binds to a target (e.g., an antigen expressed by hematopoietic stem cells [e.g., CD45]); the antibody specifically binds to a target (e.g., an antigen expressed by hematopoietic stem cells [e.g., CD45]); the antibody specifically binds to a target (e.g., an antigen expressed by hematopoietic stem cells [e.g., CD45]) at least about 10 -4 M, about 10 -5 M, about 10 -6 M, about 10 -7 M, about 10 -8 M, about 10 -9 M, about 10 -10 M, about 10 -11 M, about 10 -12 M or less (less than about 10 -12 means a number less than, e.g. 10 -13 ) for the target D In one embodiment, K D is measured according to standard Bio-Layer Interferometry (BLI). However, it should be understood that the antibody may specifically bind to more than one antigen that is related in sequence. For example, in one embodiment, an antibody may specifically bind to both human and non-human (e.g., mouse or non-human primate) orthologues of an antigen (e.g., CD45). Thus, as used in this application, an antibody that "specifically binds to human CD45" is intended to refer to an antibody that binds to human CD45 (and possibly CD45 from one or more non-human species) but does not substantially bind to non-CD45 proteins. Preferably, the antibody has a specific binding capacity of 1x10 -7 K below M D , 5x10 -8 K below M D, 3x10 -8 K below M D , 1x10 -8 K below M D , or 5x10 -9 K below M D and binds to human CD45.

[0072] As used herein, the terms "anti-CD45 antibody" or "antibody that binds CD45" refer to an antibody that can bind to CD45 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent that targets CD45. In one embodiment, the anti-CD45 antibody (or fragment thereof) is an anti-human CD45 (hCD45) antibody (or fragment thereof).

[0073] The antibodies of the present invention are generally isolated or recombinant. "Isolated" as used in this application refers to a polypeptide (e.g., an antibody) that has been identified and separated and / or recovered from a cell or cell culture that expresses it. Usually, an isolated antibody is prepared by at least one purification step. Thus, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities. For example, an isolated antibody that specifically binds to CD45 is substantially free of antibodies that specifically bind to antigens other than CD45.

[0074] The term "monoclonal antibody" as used in this application refers to an antibody obtained by any means available or known in the art from a single clone, including any eukaryotic, prokaryotic, or phage clone, and is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies useful in this disclosure can be prepared using a wide variety of techniques known in the art, including using hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0075] As used herein, the term "diabody" refers to a bivalent antibody comprising two polypeptide chains, each of which comprises a VH and a VL domain linked by a linker (e.g., a linker consisting of 5 amino acids) that is too short to allow intramolecular association of the VH and VL domains on the same peptide chain. In this configuration, each domain pairs with a complementary domain on another polypeptide chain to form a homodimeric structure. Thus, the term "triabody" refers to a trivalent antibody comprising three peptide chains, each of which comprises one VH domain and one VL domain linked by a linker (e.g., a linker consisting of 1-2 amino acids) that is too short to allow intramolecular association of the VH and VL domains on the same peptide chain. In order for a peptide constructed in this manner to fold into its native structure, it typically trimerizes such that the VH and VL domains of adjacent peptide chains are brought into spatial proximity to one another (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-48, 1993).

[0076] As used herein, the term "bispecific antibody" refers to an antibody (e.g., a monoclonal, e.g., deimmunized or humanized antibody) that can bind to two different epitopes that can be on the same or different antigens. For example, one of the binding specificities can be directed to a hematopoietic stem cell surface antigen, e.g., CD45, and the other can specifically bind to a different hematopoietic stem cell surface antigen or another cell surface protein, such as a receptor or receptor subunit involved in a signaling pathway that enhances cell proliferation, among others. In some embodiments, the binding specificities can be directed to unique, non-overlapping epitopes on the same target antigen (i.e., a biparatopic antibody).

[0077] As used in this application, the term "complementarity determining region" (CDR) refers to the hypervariable regions found in both the light and heavy chain variable domains of an antibody. The more highly conserved portions of the variable domains are called framework regions (FR). The amino acid positions that represent the hypervariable regions of an antibody may vary depending on the context and the various definitions known in the art. Some positions within the variable domain may be considered hybrid hypervariable positions, which may be considered to be within the hypervariable region under one set of criteria, while being outside the hypervariable region under another set of criteria. One or more of these positions may also be found in extended hypervariable regions. The antibodies described in this application may contain modifications at these hybrid hypervariable positions. Natural heavy and light chain variable domains each comprise four framework regions that adopt a predominantly β-sheet structure and are connected by three CDRs (the CDRs form loops that connect, and in some cases form part of, the β-sheet structure). The CDRs in each chain are grouped together in close proximity by a framework region in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with the CDRs from the other antibody chain, contribute to the formation of the antibody target binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD., 1987). In certain embodiments, immunoglobulin amino acid residue numbering is done according to the immunoglobulin amino acid residue numbering system of Kabat et al. (although any antibody numbering scheme may be used, including but not limited to IMGT and Chothia, etc.), unless otherwise specified.

[0078] Also provided are "conservative sequence modifications" of the sequences set forth in the SEQ ID NOs described herein, i.e., modifications to the nucleotide and amino acid sequences that do not abolish the binding of an antibody encoded by or containing the nucleotide or amino acid sequence to an antigen. Such conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. Modifications may be introduced into the SEQ ID NOs described herein by routine techniques known in the art, such as, for example, site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, preferably, a predicted non-essential amino acid residue in an anti-CD45 antibody is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art (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)).

[0079] The terms "Fc", "Fc region", "Fc domain" and "IgG Fc domain" as used herein refer to a portion of an immunoglobulin (e.g., an IgG molecule) that is related to a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region comprises the C-terminal halves of the two heavy chains of an IgG molecule linked by disulfide bonds. It does not have antigen binding activity but contains carbohydrate moieties and binding sites for Fc receptors, including complement and the FcRn receptor (see below). For example, the Fc region comprises the second constant domain CH2 (e.g., residues at EU positions 231-340 of human IgG1) and the third constant domain CH3 (e.g., residues at EU positions 341-447 of human IgG1). As used herein, the Fc region comprises the "lower hinge region" (e.g., residues at EU positions 233-239 of human IgG1).

[0080] Fc may refer to this region in isolation or in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at many positions within the Fc domain, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, and therefore slight differences may exist 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 sequences of human IgG1, IgG2, IgG3, and IgG4 heavy chains can be found in many sequence databases, for example in the Uniprot database (www.uniprot.org) under accession numbers P01857 (IGHG1_human), P01859 (IGHG2_human), P01860 (IGHG3_human), and P01861 (IGHG1_human), respectively.

[0081] As used herein, the term "altered Fc region" or "variant Fc region" refers to an IgG Fc domain that contains one or more amino acid substitutions, deletions, insertions or modifications introduced at any position within the Fc region. In certain embodiments, the variant IgG Fc domain contains one or more amino acid substitutions that result in reduced or abolished binding affinity to FcgammaR and / or C1q compared to a wild-type Fc domain that does not contain the one or more amino acid substitutions. Furthermore, Fc binding interactions are essential 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 (e.g., an antibody, fusion protein, or conjugate) comprising a variant Fc domain may have altered binding affinity for at least one or more Fc ligands (e.g., Fc gamma R) compared to a corresponding antibody having an otherwise identical amino acid sequence that does not contain one or more amino acid substitutions, deletions, insertions, or modifications (e.g., an unaltered Fc region comprising naturally occurring amino acid residues at the corresponding positions in the Fc region).

[0082] The variant Fc domains described in this application are defined according to the amino acid modifications that compose them. For all amino acid substitutions discussed in this application with respect to the Fc region, the numbering always follows the EU index as in Kabat. Thus, for example, D265C is an Fc variant in which an aspartic acid (D) at EU position 265 is replaced with a cysteine ​​(C) relative to the parent Fc domain. Note that the order in which the substitutions are indicated is arbitrary. Similarly, for example, D265C / L234A / L235A defines an Fc variant with substitutions at EU positions 265 (D to C), 234 (L to A), and 235 (L to A) relative to the parent Fc domain. Variants may also be designated according to their final amino acid composition at the mutated EU amino acid positions. For example, the L234A / L235A mutation may be referred to as "LALA". As a further example, the E233P.L234V.L235A.delG236 (deletion of 236) mutation may be referred to as "EPLVLAdelG." As yet another example, the I253A.H310A.H435A mutation may be referred to as "IHH." Note that the order in which the substitutions are listed is arbitrary.

[0083] The term "Fc gamma receptor" or "Fc gamma R" as used herein refers to any member of a family of proteins that bind to the IgG antibody Fc region and are encoded by the Fc gamma R gene. In humans, this family includes, but is not limited to, Fc gamma RI (CD64) (e.g., isoforms Fc gamma RIa, Fc gamma RIb, and Fc gamma RIc); Fc gamma RII (CD32) (e.g., isoforms Fc gamma RIIa [e.g., allotypes H131 and R131], Fc gamma RIIb [e.g., Fc gamma RIIb-1 and Fc gamma RIIb-2], and Fc gamma RIIc); and Fc gamma RIII (CD16) (e.g., isoforms Fc gamma RIIIa [e.g., allotypes V158 and F158] and Fc gamma RIIIb [e.g., allotypes Fc gamma RIIIb-NA1 and Fc gamma RIIIb-NA2], as well as any undiscovered human Fc gamma R or isoforms or allotypes of Fc gamma R. Fc gamma R may be derived from any organism, such as, but not limited to, human, mouse, rat, rabbit, and monkey. Mouse Fc gamma R includes, but is not limited to, Fc gamma RI (CD64), Fc gamma RII (CD32), Fc gamma RIII (CD16), and Fc gamma RIII-2 (CD16-2), as well as any undiscovered mouse Fc gamma R or isoforms or allotypes of Fc gamma R.

[0084] The term "effector function" as used herein refers to a biochemical event resulting 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" refers to a cell of the immune system 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 gamma delta T cells, and may be derived from any organism (e.g., but not limited to, human, mouse, rat, rabbit, and monkey).

[0085] As used herein, the terms "silent," "silenced," or "silencing" refer to an antibody having an altered Fc region as described herein that has reduced binding to Fc gamma receptors (FcγRs) compared to the binding of the same antibody comprising an unaltered Fc region to the FcγR (e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% reduced binding to FcγRs compared to the binding of the same antibody comprising an unaltered Fc region to the FcγR, e.g., as measured by BLI). In some embodiments, an Fc silenced antibody does not exhibit detectable binding to FcγRs. Binding of antibodies with altered Fc regions to FcγRs can be assayed using a variety of techniques known in the art, including, but not limited to, equilibrium methods [e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; radioimmunoassay (RIA)], or kinetic-based assays such as surface plasmon resonance assays or other mechanisms [e.g., BIACORE TM Analysis or OctetTM Analysis (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), etc. These and other methods may utilize labels on one or more of the components being evaluated and / or may use a variety of detection methods, including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. Binding affinity and kinetics are described in detail in Paul, WE, ed., Fundamental Immunology, 4th Ed., 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 antibody of interest in the presence of increasing amounts of unlabeled antigen, and detecting antibody bound to the labeled antigen. From the data, the affinity and binding off-rate of the antibody of interest for a particular antigen may be determined by Scatchard plot analysis. Competition with a second antibody may also be measured using radioimmunoassays, in which the antigen is incubated in the presence of the antibody of interest conjugated to a labeled compound and increasing amounts of an unlabeled second antibody.

[0086] As used herein, the term "same antibody comprising an unaltered Fc region" refers to an antibody that lacks the recited amino acid substitutions (e.g., D265C, H435A, L234A, and / or L235A) but otherwise has the same amino acid sequence as the compared Fc modified antibody.

[0087] The term "antibody dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which a polypeptide containing an Fc domain (e.g., an antibody) binds to an Fc receptor (FcR) present on certain cytotoxic cells (e.g., mainly NK cells, neutrophils, and macrophages), and these cytotoxic effector cells become specifically bound to an antigen-bearing "target cell" and subsequently kill said target cell with their cytotoxins (Hogarth et al., Nature review Drug Discovery 2012, 11:313). In addition to antibodies and fragments thereof, other polypeptides containing an Fc domain (e.g., Fc fusion proteins and Fc conjugate proteins) that have the ability to specifically bind to antigen-bearing target cells are contemplated to effect cell-mediated cytotoxicity.

[0088] For simplicity, cell-mediated cytotoxicity resulting from the activity of a polypeptide containing an Fc domain is also referred to as ADCC activity in this application. The ability of any particular polypeptide of the present disclosure to mediate lysis of target cells by ADCC can be assayed. To assess ADCC activity, a polypeptide of interest (e.g., an antibody) is added to target cells together with immune effector cells, resulting in cytolysis of the target cells. Cytolysis is generally detected by the release of a label (e.g., a radioactive substrate, a fluorescent dye, or a natural intracellular protein) from the lysed cells. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. 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 the antibody of interest may 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)).

[0089] As used herein, the terms "conditioning" and "conditioning" refer to the process of preparing a patient for receiving a graft (e.g., a graft containing hematopoietic stem cells). Such procedures promote the engraftment of hematopoietic stem cell grafts (e.g., as inferred from a sustained increase in the amount of viable hematopoietic stem cells in blood samples isolated from the patient after the conditioning procedure and subsequent hematopoietic stem cell transplantation). According to the methods described herein, a patient may be conditioned for hematopoietic stem cell transplantation therapy by administering to the patient an ADC, antibody, or antigen-binding fragment thereof capable of binding to CD45 expressed by hematopoietic stem cells. As described herein, the antibody may be covalently conjugated to a cytotoxin to form a drug-antibody conjugate (also referred to interchangeably herein as an antibody drug conjugate (ADC)). Administration of an ADC, antibody, or antigen-binding fragment thereof capable of binding to one or more of the above antigens to a patient in need of hematopoietic stem cell transplantation therapy may promote engraftment of hematopoietic stem cell grafts, for example, by selectively depleting endogenous hematopoietic stem cells, thereby creating vacancies that are filled by the graft of exogenous hematopoietic stem cells.

[0090] As used in this application, the term "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic agent, e.g., an anti-CD45 ADC, sufficient to achieve a desired result or to have an effect against an autoimmune disease or cancer in a human patient.

[0091] As used in this application, the term "half-life" refers to the time it takes for the plasma concentration of an antibody drug in a subject, e.g., a human subject, to decrease by half or by 50%. This 50% decrease in serum concentration reflects the amount of drug in circulation.

[0092] 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 may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by gene rearrangement or somatic mutation in vivo). However, as used herein, the term "human antibody" 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 may be produced in human cells (e.g., by recombinant expression) or by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. If the human antibody is a single chain antibody, it may contain a linker peptide not found in naturally occurring human antibodies. For example, an Fv may include a linker peptide, such as two to about eight glycine or other amino acid residues, that links the variable region of the heavy chain and the variable region of the light chain. Such a linker peptide is considered to be of human origin. Human antibodies may be made 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 which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes (see, e.g., PCT Publication Nos. WO 1998 / 24893; WO 1992 / 01047; WO 1996 / 34096; WO 1996 / 33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598).

[0093] The term "chimeric antibody" is intended to refer to an antibody whose variable region sequences are derived from one species and whose constant region sequences are derived from another species (e.g., an antibody whose variable region sequences are derived from a rat antibody and whose constant region sequences are derived from a human antibody). Methods for producing chimeric antibodies are known in the art. See, for example, 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; US Pat. Nos. 5,807,715; 4,816,567; and 4,816,397.

[0094] The term "de-immunized" or "de-immunization" as used herein refers to modifying the original wild-type construct (or parent antibody) by making it non-immunogenic or less immunogenic in humans. A deimmunized antibody comprises portions of non-human origin (e.g., framework regions and / or CDRs). As used herein, the term "deimmunized antibody" refers to an antibody that has been deimmunized by mutation so that it does not activate the immune system of a subject (e.g., Nanus et al., J. Urology 170:S84-S89, 2003; WO98 / 52976; WO00 / 34317).

[0095] "Humanized" forms of non-human (e.g., mouse or rat) antibodies are immunoglobulins that contain minimal sequences derived from non-human immunoglobulins. Generally, a humanized antibody will contain 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. A humanized antibody may also contain at least a portion of an immunoglobulin constant region (Fc), typically at least a portion 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 to Queen et al.; 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; See also U.S. Patent 5,565,332.

[0096] As used herein, the term "engraftment potential" is used to refer to the ability of hematopoietic stem and progenitor cells to repopulate tissues, whether such cells are naturally circulating or provided by transplantation. The term encompasses all events surrounding or leading to engraftment, such as tissue homing and colonization of cells in the tissue of interest. Engraftment efficiency or rate can be assessed or quantified using any clinically accepted parameter known to those of skill in the art, including, for example, assessing competitive repopulating units (CRUs); marker uptake or expression in the stem cell-homed, colonized, or engrafted tissue; or assessing subject progression by disease progression, hematopoietic stem and progenitor cell survival, or recipient survival. Engraftment can also be determined by measuring peripheral blood white blood cell counts after transplantation. Engraftment can also be assessed by measuring bone marrow cell recovery by donor cells in bone marrow aspirate samples.

[0097] As used herein, the term "hematopoietic stem cell" ("HSC") refers to an immature blood cell that has the ability to self-renew and differentiate into mature blood cells of diverse 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 express the CD34 + May contain CD34 cells. +The cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are believed to comprise a subpopulation of cells with the characteristics of stem cells defined above, whereas in mice, HSCs are CD34-. Furthermore, HSCs are also referred to as long-term repopulating HSCs (LT-HSCs) and short-term repopulating HSCs (ST-HSCs). LT-HSCs and ST-HSCs are differentiated based on their functional potential and expression of cell surface markers. For example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, CD235A, etc.). In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, CD48-, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra, etc.), whereas ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, and lin- (negative for mature lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, IL7ra, etc.). In addition, ST-HSCs are less quiescent and more proliferative than LT-HSCs under homeostatic conditions. However, LT-HSCs have a higher self-renewal capacity (i.e., LT-HSCs survive throughout adulthood and can be serially transplanted through successive generations of recipients), whereas ST-HSCs have a limited self-renewal capacity (i.e., ST-HSCs survive only for a limited period of time and cannot be serially transplanted). Any of these HSCs may be used in the methods described in this application. ST-HSCs are particularly useful because they are highly proliferative and therefore can give rise to differentiated progeny more quickly.

[0098] As used herein, the term "functional potential of hematopoietic stem cells" refers to any of the following: 1) multi-potency (which may include, but is 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., erythrocytes ... These characteristics refer to the functional properties of hematopoietic stem cells, including: 1) the ability to differentiate into multiple different blood lineages, including multiple myeloid cells (e.g., NK cells, T cells, and B cells); 2) self-renewal, which refers to the ability of hematopoietic stem cells to give rise to daughter cells with potential equal to that of the parent cell, and which may occur repeatedly throughout an individual's lifetime without exhaustion; and 3) the ability of hematopoietic stem cells or their progeny, when reintroduced into a transplant recipient, to home to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis.

[0099] As used herein, the terms "subject" and "patient" refer to an organism, such as a human, receiving treatment for a particular disease or condition as described herein. For example, a patient, such as a human patient, may receive treatment prior to hematopoietic stem cell transplantation therapy to promote engraftment of exogenous hematopoietic stem cells.

[0100] As used herein, the term "donor" refers to a human or animal from which one or more cells have been isolated prior to administration of the cells or their progeny to a recipient. The one or more cells may be, for example, a population of hematopoietic stem cells.

[0101] As used herein, the term "recipient" refers to a patient who receives a transplant, such as a transplant that includes a population of hematopoietic stem cells. The transplanted cells administered to the recipient can be, for example, autologous, syngeneic, or allogeneic cells.

[0102] As used herein, the term "endogenous" describes a substance such as a molecule, cell, tissue, or organ that is naturally found in a particular organism, such as a human patient (e.g., a hematopoietic stem cell, or a cell of the hematopoietic lineage, such as a megakaryocyte, thrombocyte, platelet, red blood cell, mast cell, myeoblast, basophil, neutrophil, eosinophil, microglial cell, granulocyte, monocyte, osteoclast, antigen presenting cell, macrophage, dendritic cell, natural killer cell, T-lymphocyte, or B-lymphocyte).

[0103] As used in this application, the term "sample" refers to a specimen taken from a subject (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or dermis), pancreatic juice, chorionic villus samples, and cells).

[0104] As used herein, the phrase "substantially cleared from the blood" refers to a time point after administration of a therapeutic agent (e.g., an anti-CD45 antibody, or antigen-binding fragment thereof) to a patient, where the concentration of the therapeutic agent in a blood sample isolated from the patient is such that the therapeutic agent is not detectable by conventional methods (e.g., the therapeutic agent is not detectable above the noise threshold of a device or assay used to detect the therapeutic agent). Antibodies, antibody fragments, and protein ligands may be detected using a variety of techniques known in the art, such as ELISA-based detection assays known in the art or described herein. Additional assays that may be used to detect antibodies or antibody fragments include immunoprecipitation and immunoblot assays, among others, known in the art.

[0105] As used herein, the term "to treat" or "treatment" refers to reducing the severity and / or frequency of disease symptoms, eliminating disease symptoms and / or the underlying cause of said symptoms, reducing the frequency or likelihood of disease symptoms and / or their underlying causes, and ameliorating or repairing damage caused directly or indirectly by the disease, any improvement in any outcome of the disease (e.g., longer survival, less morbidity and / or fewer side effects that are a by-product of alternative therapeutic modalities, etc.); as will be readily appreciated in the art, complete eradication of the disease is preferred, but is not a requirement of therapeutic intervention. Beneficial or desired clinical outcomes include, but are not limited to, enhanced engraftment of exogenous hematopoietic cells in patients following antibody conditioning therapy and subsequent hematopoietic stem cell transplantation therapy as described herein. Further beneficial outcomes include an increased cell number or relative concentration of hematopoietic stem cells in a patient in need of hematopoietic stem cell transplantation after a conditioning regimen and subsequent administration of an exogenous hematopoietic stem cell graft to said patient. Beneficial outcomes of the therapies described herein may also include an increased cell number or relative concentration of one or more cells of the hematopoietic lineage (such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglial cells, granulocytes, monocytes, osteoclasts, antigen presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, or B lymphocytes) after a conditioning regimen and subsequent hematopoietic stem cell transplantation therapy. Further beneficial outcomes may include a reduction in the amount of disease-causing cell populations, such as populations of cancer cells (e.g., CD45+ leukemia cells) or autoimmune cells (e.g., CD45+ autoimmune lymphocytes, such as CD45+ T-cells expressing a T-cell receptor that cross-reacts with an autoantigen). To the extent that the methods of the present disclosure are directed to preventing a disorder, it is understood that the term "preventing" does not require that the disease state be completely averted.Rather, as used in this application, the term prevent refers to the ability of one of skill in the art to identify a population susceptible to a disorder, administer a compound of the present disclosure prior to the onset of the disease, etc. The term does not imply that the disease state is completely avoided.

[0106] As used in this application, a patient "in need" of a hematopoietic stem cell transplant includes patients who exhibit a defect or deficiency in one or more blood cell types, as well as patients with a stem cell disorder, autoimmune disease, cancer, or other condition described in this application. Hematopoietic stem cells generally exhibit: 1) multi-potency, thus capable of differentiating 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, B-cells, and T-cells); 2) self-renewal, thus capable of giving rise to daughter cells with potential equivalent to that of the parent cell; and 3) the ability to home to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis when reintroduced into a transplant recipient. Thus, hematopoietic stem cells may be administered to a patient with a defect or deficiency in one or more cell types of the hematopoietic lineage to reconstitute the defective or deficient cell population in vivo. For example, the patient may be afflicted with cancer, and the deficiency may be caused by administering a chemotherapeutic or other agent that selectively or non-specifically reduces the cancerous cell population. Additionally or alternatively, the patient may be afflicted with a hemoglobinopathy (e.g., non-malignant hemoglobinopathy), such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome. The subject may be a subject with adenosine deaminase severe combined immunodeficiency (ADA SCID), HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome. The subject may have or be affected by an inherited blood disorder (e.g., sickle cell anemia) or an autoimmune disease. Additionally, or alternatively, the subject may have or be affected by a malignancy, such as neuroblastoma or a blood cancer. For example, the subject may have leukemia, lymphoma, or myeloma.In some embodiments, the subject has acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. In some embodiments, the subject has myelodysplastic syndrome. In some embodiments, the subject has an autoimmune disease, such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, type I diabetes, or another autoimmune condition 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 affected by a metabolic storage disorder. The subject may be suffering from or affected by a metabolic disorder selected from the group consisting of glycogen storage disease, mucopolysaccharidoses, Gaucher's disease, Hurler's disease, sphingolipidosis, metachromatic leukodystrophy, or any other disease or disorder that may benefit from the treatments and therapies disclosed herein (including but not limited to severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M 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), as well as diseases or disorders described in "Bone Marrow Transplantation for Non-Malignant disease", ASH Education Book, 1:319-338 (2000), the disclosure of which relates to conditions that may be treated by prescribing hematopoietic stem cell transplantation therapy, which is incorporated herein by reference in its entirety).Additionally or alternatively, a patient "in need of" hematopoietic stem cell transplantation may be a patient who is or is not afflicted with one of the aforementioned pathologies (but who nevertheless exhibits reduced levels (e.g., compared to levels in an otherwise healthy subject) of one or more endogenous cell types within the hematopoietic lineage, such as megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes). One of skill in the art can readily determine whether levels of one or more of the aforementioned cell types or other blood cell types are reduced relative to an otherwise healthy subject, for example, by flow cytometry and fluorescence activated cell sorting (FACS) methods, among other procedures 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 endogenous stem cell populations in the target tissue (e.g., by removing endogenous hematopoietic stem cell or hematopoietic progenitor cell populations from the bone marrow tissue of a subject) and / or by engrafting or transplanting stem cells into the target tissue of a subject. For example, type I diabetes has been shown to be cured by hematopoietic stem cell transplantation and can benefit from conditioning according to the compositions and methods described herein. Additional disorders that can be treated using the compositions and methods described herein include, but are not limited to, sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, Wiskott-Aldrich syndrome, ADA SCID, HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Shwachman-Diamond syndrome. Additional diseases that may be treated using the patient conditioning and / or hematopoietic stem cell transplantation methods described herein include inherited blood disorders (e.g., sickle cell anemia) and autoimmune diseases such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease. Additional diseases that may be treated using the conditioning and / or transplantation methods described herein include malignant tumors (e.g., neuroblastoma), or blood cancers (e.g., leukemia, lymphoma, and myeloma). For example, the cancer may be acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Additional diseases that may be treated using the conditioning and / or transplantation methods described herein include myelodysplastic syndromes. In some embodiments, the subject has or is affected by a metabolic storage disorder.For example, the subject may be a patient suffering from a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidosis, metachromatic leukodystrophy, or any other disease or disorder that may benefit from the treatments and therapies disclosed herein (including, but not limited to, severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis), as well as a patient suffering from a disease or disorder that may benefit from the treatments and therapies disclosed herein (including, but not limited to, severe combined immunodeficiency, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage disease, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, ... Book, 1:319-338 (2000), the disclosure of which relates to conditions that may be treated by prescribing hematopoietic stem cell transplantation therapy and which is incorporated by reference in its entirety into this application.

[0108] As used in this application, the term "vector" includes nucleic acid vectors, such as plasmids, DNA vectors, plasmids, RNA vectors, viruses, or other suitable replicons. The expression vectors described in this application may contain polynucleotide sequences and additional sequence elements that are used, for example, to express proteins and / or to integrate these polynucleotide sequences into the genome of mammalian cells. Certain vectors that can be used to express the antibodies and antibody fragments of the invention include plasmids that contain regulatory sequences, such as promoter regions and enhancers that direct gene transcription. Other useful vectors for expressing antibodies and antibody fragments include polynucleotide sequences that enhance the translation rate of these genes or improve the stability or nuclear export of mRNA resulting from gene transcription. These sequence elements may include, for example, 5' and 3' untranslated regions and polyadenylation signal sites that allow efficient transcription of genes located on the expression vector. The expression vectors described in this application may also include a polynucleotide that encodes a marker for selecting cells that contain such a vector. Examples of suitable markers include genes encoding resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.

[0109] As used in this application, the term "conjugate" or "antibody drug conjugate" or "ADC" refers to an antibody bound to a cytotoxin. An ADC is formed by chemically binding a reactive functional group of one molecule, such as an antibody, or an antigen-binding fragment thereof, to a suitable reactive functional group of another molecule, such as a cytotoxin as described in this application. The conjugate may include a linker between the two molecules that are attached to each other (e.g., between the antibody and the cytotoxin). Examples of linkers that can be used to form the conjugate include peptide-containing linkers (e.g., linkers containing naturally occurring or non-naturally occurring amino acids, e.g., D-amino acids). Linkers may be prepared using a variety of strategies, described in this application and known in the art. Depending on the reactive moieties therein, the linker may be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0110] As used herein, "drug-to-antibody ratio" or "DAR" refers to the number of drugs (e.g., amatoxins) attached to the antibody of a conjugate. Depending on the number of attachment sites on the antibody, higher loadings are possible, but the DAR of an ADC can range from 1 to 8. In certain embodiments, the 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 acts by disrupting the microtubule network, which is essential for mitosis and interphase cell functions in cells. Examples of microtubule binding agents include, but are not limited to, maytasine, 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, taxanes such as docetaxel and paclitaxel, macrolides such as discodermolide, cochicine, and epothilones, and derivatives thereof (such as epothilone B or its derivatives).

[0112] As used herein, the term "amatoxin" refers to a member of the amatoxin family of peptides produced by the Amanita phalloides mushroom, or a variant or derivative thereof (e.g., a variant or derivative thereof capable of inhibiting RNA polymerase II activity). Amatoxins useful in conjunction with the compositions and methods described herein include, for example, but are not limited to, compounds of formula (IV), (IVA), (IVB), and (IVC), or other amatoxins (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanulin, amanurin, amanuric acid, or proamanutin, etc.). As described herein, amatoxins may be conjugated to an antibody, or an antigen-binding fragment thereof (thereby forming an ADC), for example, via a linker moiety (L). Exemplary amatoxin conjugation methods and linkers useful for such processes are described below. Exemplary linker-containing amatoxins useful for conjugation to antibodies, or antigen-binding fragments, according to the compositions and methods are also described in this application.

[0113] The term "acyl" as used herein refers to -C(=O)R, where R is hydrogen (an "aldehyde"), C is an alkyl group,1 -C 12 Alkyl, C 2 -C 12 Alkenyl, C 2 -C 12 Alkynyl, C 3 -C 7 Carbocyclyl, C 6 -C 20 aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl. Non-limiting examples include formyl, acetyl, propanoyl, benzoyl, and acryloyl.

[0114] As used in this application, the term " 1 -C 12 "Alkyl" refers to a straight or branched chain saturated hydrocarbon having 1 to 12 carbon atoms. Typical C 1 -C 12 Alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while branched C 1 -C 12 Alkyl includes, but is not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl. 1 -C 12 The alkyl group may be unsubstituted or substituted.

[0115] The term "alkenyl" as used herein means an alkyl group having at least one site of unsaturation, i.e., carbon-carbon, sp 2 C containing normal, secondary, or tertiary carbon atoms with double bonds 2 -C 12 It refers to a hydrocarbon. Examples include, but are not limited to, ethylene or vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, etc. Alkenyl groups can be unsubstituted or substituted.

[0116] As used herein, "alkynyl" refers to a C alkyl group containing normal, secondary, or tertiary carbon atoms with at least one site of unsaturation, i.e., a carbon-carbon, sp triple bond. 2 -C 12 Refers to a hydrocarbon. Examples include, but are not limited to, acetylene and propargyl. Alkynyl groups can be unsubstituted or substituted.

[0117] As used herein, "aryl" refers to 6 -C 20 It refers to a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and anthracenyl. Aryl groups can be unsubstituted or substituted.

[0118] As used herein, "arylalkyl" refers to an alkyl group consisting of a carbon atom (typically a terminal or sp 3 A non-cyclic alkyl radical in which one of the hydrogen atoms bonded to the aryl group (carbon atom) is replaced with an aryl radical. Exemplary arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethene-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like. The arylalkyl group contains 6 to 20 carbon atoms, e.g., the alkyl portion of the arylalkyl group (e.g., alkanyl, alkenyl, or alkynyl groups, etc.) is 1 to 6 carbon atoms, and the aryl portion is 5 to 14 carbon atoms. The alkaryl group can be unsubstituted or substituted.

[0119] As used herein, "cycloalkyl" refers to a saturated carbocyclic radical, which may be monocyclic or bicyclic. Cycloalkyl groups include rings having 3 to 7 carbon atoms as a monocycle, or rings having 7 to 12 carbon atoms as a bicycle. Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups may be unsubstituted or substituted.

[0120] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic radical, which may be monocyclic or bicyclic. Cycloalkenyl groups include rings having 3 to 6 carbon atoms as a monocycle, or rings having 7 to 12 carbon atoms as a bicycle. 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. Cycloalkenyl groups may be unsubstituted or substituted.

[0121] As used herein, a "heteroaralkyl" refers to an alkyl group consisting of a carbon atom (typically a terminal or sp 3A heteroaryl alkyl group refers to an acyclic alkyl radical in which one of the hydrogen atoms bonded to the aryl group (carbon atom) is replaced with a heteroaryl radical. Exemplary heteroaryl alkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furylethyl, and the like. The heteroaryl alkyl group contains 6-20 carbon atoms, e.g., the alkyl portion of the heteroaryl alkyl group (including, e.g., alkanyl, 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 heteroaryl alkyl 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., bicyclic [4,5], [5,5], [5,6], or [6,6] systems).

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

[0123] Heteroaryl and heterocycloalkyl groups are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), especially 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), especially volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.

[0124] Examples of heteroaryl groups include, but are not limited to, pyridyl, thiazolyl, tetrahydrothiophenyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl, isoquinolinyl, benzimidazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl, 4H- Examples include quinolidinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, benzotriazolyl, benzisoxazolyl, and isatinoyl.

[0125] Examples of heterocycloalkyl include, but are not limited to, dihydropyridyl, tetrahydropyridyl (piperidyl), tetrahydrothiophenyl, piperidinyl, 4-piperidonyl, pyrrolidinyl, 2-pyrrolidonyl, tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, piperazinyl, quinuclidinyl, and morpholinyl.

[0126] By way of example and not limitation, carbon-linked heteroaryls and heterocycloalkyls are bonded 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 aziridine, 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. More typically, carbon-linked 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] By way of example and not limitation, nitrogen-linked heteroaryls and heterocycloalkyls are bonded at the 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, 2-position of isoindole or isoindoline, 4-position of morpholine, and 9-position of carbazole or beta-carboline. More typically, nitrogen-linked heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

[0128] As used herein, and as applied to any of the above alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, etc., "substituted" means that one or more hydrogen atoms are each independently replaced with a substituent. Exemplary substituents include, but are not limited to, -X, -R, -OH, -OR, -SH, -SR, NH 2 , -NHR, -N(R) 2 , -N + (R) 3 , -CX 3 , -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO 2 , -N 3 , -NC(=O)H, -NC(=O)R, -C(=O)H, -C(=O)R, -C(=O)NH 2 , -C(=O)N(R) 2 , -SO 3 -, -SO 3 H, -S(=O) 2 R, -OS(=O) 2 OR, -S(=O) 2 NH 2, -S(=O) 2 N(R) 2 , -S(=O)R, -OP(=O)(OH) 2, -OP(=O)(OR)2 , -P(=O)(OR) 2 , -PO 3 , -PO 3 H 2 , -C(=O)X, -C(=S)R, -CO 2 H, -CO 2 R, -CO 2 -, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NH 2, -C(=O)N(R) 2 , -C(=S)NH 2, -C(=S)N(R) 2 , -C(=NH)NH 2, and -C(=NR)N(R) 2 wherein each X is independently selected at each occurrence from F, Cl, Br, and I; and each R is C 1 -C 12 Alkyl, C 6 -C 20 Aryl, C 3 -C 14 is independently selected at each occurrence from heterocycloalkyl or heteroaryl, a protecting group, and a prodrug moiety. Wherever a group is described as "optionally substituted," that group may be substituted, independently at each occurrence, with one or more of the above substituents.

[0129] It should be understood that the naming convention for a radical may include either a mono-radical or a di-radical, depending on the context. For example, if a substituent requires two points of attachment to the remainder of the molecule, the substituent is understood to be a di-radical. For example, a substituent identified as an alkyl requiring two points of attachment is -CH 2 -, -CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2-, etc. Other radical naming conventions explicitly state that the radical is a di-radical, such as "alkylene," "alkenylene," "arylene," "heterocycloalkylene," etc.

[0130] Wherever a substituent is shown as a di-radical (i.e., having two points of attachment to the remainder of the molecule), it is understood that said substituent can be attached in any directional orientation unless otherwise specified.

[0131] Anti-CD45 antibody Disclosed herein are anti-CD45 antibodies capable of binding to both human and cynomolgus CD45 and that can be used as therapeutic agents alone or as antibody drug conjugates (ADCs) to, for example, (i) treat cancer and autoimmune diseases characterized by CD45+ cells, and (ii) promote the engraftment of transplanted hematopoietic stem cells in patients requiring transplantation therapy. These therapeutic activities can be triggered, for example, by the binding of isolated anti-CD45 antibodies, antigen-binding fragments thereof, which bind to CD45 expressed on the surface of cells (such as cancer cells, autoimmune cells, or hematopoietic stem cells), followed by induction of cell death. The depletion of endogenous hematopoietic stem cells can provide a niche to which transplanted hematopoietic stem cells can home, and productive hematopoiesis can then be established. In this way, transplanted hematopoietic stem cells can successfully engraft in patients (such as human patients suffering from stem cell disorders described herein).

[0132] CD45 is a hematopoietic cell-specific transmembrane protein tyrosine phosphatase essential for signaling through the T- and B-cell antigen receptor. CD45 has a large extracellular domain and a cytoplasmic domain that contains the phosphatase. CD45 can act as both a positive and negative regulator, depending on the nature of the stimulus and the cell type involved. The CD45 gene exists in a variety of permutations, but only six isoforms have been identified in humans to date. The isoforms are RA (Uniprot Accession No.: P08575-8; SEQ ID NO: 31), RO (NCBI Accession No.: NP_563578.2; SEQ ID NO: 32), RB (NCBI Accession No.: XP_006711537.1; SEQ ID NO: 33), RAB (NCBI Accession No.: XP_006711535.1; SEQ ID NO: 34), RBC (NCBI Accession No.: XP_006711536.1; SEQ ID NO: 35), and RABC (NCBI Accession No.: NP_002829.3; SEQ ID NO: 36) (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 and memory T cells, some B cell subsets, activated monocytes / macrophages, and granulocytes. CD45RB is expressed on peripheral B cells, naive T cells, thymocytes, and weakly on macrophages and dendritic cells.

[0133] As described below, novel anti-human CD45 (hCD45) antibodies and fragments thereof were identified by immunizing rats with human CD45. The identified antibodies have diagnostic and therapeutic properties. Antibody A (AbA), antibody B (AbB), and antibody C (AbC) were identified in this screening. These antibodies cross-react with human CD45 and rhesus monkey CD45. Furthermore, these antibodies disclosed in the present application can bind to the extracellular domains of various isoforms of human CD45. Thus, in certain embodiments, the antibodies of the present application are pan-specific anti-CD45 antibodies (i.e., antibodies that bind to all six human CD45 isoforms). Furthermore, AbA, AbB, and AbC (or antibodies having the binding regions or specificities of these antibodies) disclosed in the present application can also bind to cynomolgus monkey CD45. Identifying both cross-reactive cyno / human anti-CD45 antibodies and antibodies pan-specific for the multiple human isoforms of CD45 was a challenge but was met by the screen described in Example 1 below.

[0134] The amino acid sequences of the various binding regions of anti-CD45 antibodies AbA, AbB, and AbC are set forth in Table 3. Humanized and chimeric anti-CD45 antibodies based on antibodies AbA, AbB, or AbC (e.g., comprising the CDRs set forth in Table 3) are included in the present invention.

[0135] In one embodiment, the invention provides an anti-CD45 antibody, or antigen-binding fragment thereof, that comprises a binding region (e.g., CDR, variable region) corresponding to the binding region of AbA. The amino acid sequence of the heavy chain variable region (VH) of Ab is set forth in SEQ ID NO: 1 (see Table 3). The amino acid sequence of the VH CDR domain of AbA is set forth in SEQ ID NO: 2 (VH CDR1); SEQ ID NO: 3 (VH CDR2) and SEQ ID NO: 4 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of AbA is set forth in SEQ ID NO: 5 (see Table 3). The amino acid sequence of the VL CDR domain of AbA is 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 antibody, or antigen-binding fragment thereof, provided herein comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 1, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the anti-CD45 antibody comprises a heavy chain 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.

[0136] Anti-human CD45 antibodies, or fragments thereof, that bind to an epitope on human CD45 bound by any one of antibodies AbA, AbB, or AbC (or antibodies having the binding region of AbA, AbB, or AbC) are also contemplated in the present application. Anti-human CD45 antibodies, or antigen-binding fragments thereof, that compete with any one of antibodies AbA, AbB, or AbC (or antibodies having the binding region of AbA, AbB, or AbC) are further contemplated.

[0137] In some embodiments, the anti-CD45 antibody, or antigen-binding fragment thereof, specifically binds to 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 to human CD45 at amino acid residues 486R, 493Y, and 502T of SEQ ID NO: 37 (a fragment of the CD45 isoform corresponding to NP_002829.3), or at the sequence in other human CD45 isoforms. [ka] In some embodiments, the anti-CD45 antibody, or antigen-binding fragment thereof, specifically binds to a fibronectin domain (e.g., a fibronectin d4 domain) of human CD45.

[0138] In one embodiment, the isolated anti-CD45 antibody, or antigen binding portion thereof, specifically binds to an epitope of human CD45 that includes residues 486R, 493Y, and 502T of SEQ ID NO: 37, and also binds to cynomolgus and / or rhesus CD45.

[0139] In one embodiment, the isolated anti-CD45 antibody, or antigen binding portion thereof, specifically binds to an epitope of human CD45 that includes the amino acid sequence RNGPHARYHLEVEAGNT (SEQ ID NO:38), and also binds to cynomolgus and rhesus CD45.

[0140] In one embodiment, the isolated anti-CD45 antibody, or antigen binding portion thereof, specifically binds to an epitope of human CD45 that includes the amino acid sequence CRPPRDRNGPHERYHLEVEAGNTLVRNESHK (SEQ ID NO:39) and binds to cynomolgus and rhesus monkey CD45.

[0141] In one embodiment, the isolated anti-CD45 antibody, or antigen binding portion thereof, specifically binds to an epitope of human CD45 that includes residues 486R, 493Y, and 502T of SEQ ID NO: 37; binds to at least one additional amino acid, at least two additional amino acids, at least three additional amino acids, at least four additional amino acids, or at least five additional amino acids in a peptide that includes RNGPHARYHLEVEAGNT (SEQ ID NO: 38), where the additional amino acid residues are not residues 486R, 493Y, and 502T of SEQ ID NO: 37; and also binds to cynomolgus and rhesus CD45.

[0142] In one embodiment, the invention provides an anti-CD45 antibody, or antigen-binding fragment thereof, that comprises a binding region (e.g., CDR, variable region) corresponding to the binding region of AbB. The amino acid sequence of the heavy chain variable region (VH) of AbB is set forth in SEQ ID NO: 9 (see Table 3). The amino acid sequence of the VH CDR domain of AbB is set forth in SEQ ID NO: 10 (VH CDR1); SEQ ID NO: 11 (VH CDR2) and SEQ ID NO: 12 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of AbB is set forth in SEQ ID NO: 13 (see Table 3). The amino acid sequence of the VL CDR domain of AbB is 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, the anti-CD45 antibody, or antigen-binding fragment thereof, provided herein comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the anti-CD45 antibody comprises a heavy chain 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.

[0143] In one embodiment, the invention provides an anti-CD45 antibody, or antigen-binding fragment thereof, that comprises a binding region (e.g., CDR, variable region) corresponding to the binding region of AbC. The amino acid sequence of the heavy chain variable region (VH) of AbC is set forth in SEQ ID NO: 17 (see Table 3). The amino acid sequence of the VH CDR domain of AbC is set forth in SEQ ID NO: 18 (VH CDR1); SEQ ID NO: 19 (VH CDR2) and SEQ ID NO: 20 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of AbC is set forth in SEQ ID NO: 21 (see Table 3). The amino acid sequence of the VL CDR domain of AbC is 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, the anti-CD45 antibody, or antigen-binding fragment thereof, provided herein comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 17, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the anti-CD45 antibody comprises a heavy chain 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.

[0144] In certain embodiments, the antibody comprises an altered heavy chain (HC) variable region comprising an HC variable domain set forth in Table 3, or a variant of an HC variant region of Table 3, which variant (i) differs from an HC variable domain set forth in Table 3 in 1, 2, 3, 4 or 5 amino acid substitutions, additions or deletions; (ii) differs from an HC variable domain set forth in Table 3 in at most 5, 4, 3, 2 or 1 amino acid substitutions, additions or deletions; (iii) differs from an HC variable domain set forth in Table 3 in 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 SEQ ID NO:1, wherein in any of (i)-(iv), the amino acid substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions.

[0145] In certain embodiments, the antibody comprises an altered light chain (LC) variable region comprising an LC variable domain set forth in Table 3, or a variant thereof, which variant (i) differs from an LC variable domain set forth in Table 3 in 1, 2, 3, 4 or 5 amino acid substitutions, additions or deletions; (ii) differs from an LC variable domain set forth in Table 3 in at most 5, 4, 3, 2 or 1 amino acid substitutions, additions or deletions; (iii) differs from an LC variable domain set forth in Table 3 in 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 an LC variable domain set forth in Table 3, wherein in any of (i)-(iv), the amino acid substitutions may be conservative amino acid substitutions or non-conservative amino acid substitutions.

[0146] In certain embodiments, an anti-CD45 antibody comprises a CDR set forth in Table 3 of the present application, wherein the CDR comprises a conservative amino acid substitution (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., AbA, AbB, or AbC-like specificity).

[0147] In certain embodiments, the anti-CD45 antibody is a de-immunized antibody based on an AbA, AbB or AbC antibody, or antigen-binding portion thereof, whose V regions have been selected to lack or modified to remove T-cell epitopes, thereby minimizing or eliminating the possibility of the antibody being immunogenic. In certain embodiments, the anti-CD45 antibody is de-immunized by selecting or engineering the framework domain so that it does not contain T-cell epitopes that, if present in the antibody sequence, would cause a human subject to potentially elicit a HAHA / HAMA response against the anti-CD45 antibody, resulting in an immune-mediated response that would cause adverse events or reduce therapeutic efficacy in the human subject. The antibodies disclosed in the present application (i.e., the variable and CDR sequences of AbA, AbB, and AbC set forth in Table 3) can serve as parent sequences from which deimmunized antibodies can be derived.

[0148] Humanized anti-CD45 antibody The present invention encompasses humanized anti-CD45 antibodies based on antibodies AbA, AbB, or AbC described in this application. Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody may have one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, and are typically taken from an "import" variable domain. Humanization may be performed essentially according to the method of Winter and coworkers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species (U.S. Patent No. 4,816,567). In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0149] The choice of human variable domains (both light and heavy chains) used in making humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework for the humanized antibody (Sims et al. (1993) J Immunol. 151:2296; Chothia et al. (1987) J Mol. Biol. 196:901). Another method uses a specific framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same scaffold may be used for several different humanized antibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0150] It is further important that antibodies be humanized while retaining high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs are available which illustrate and display possible three-dimensional conformational structures for selected candidate immunoglobulin sequences. By inspecting these representations, the likely role of the residues in the functioning of the candidate immunoglobulin sequence can be analyzed (i.e., analysis of residues which influence the ability of the candidate immunoglobulin to bind its antigen). In this way, FR residues can be selected and combined from the recipient and import sequences such that the desired antibody characteristic (e.g., increased affinity for its target antigen) is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0151] Fc-engineered antibodies The present disclosure is based in part on the discovery that antibodies, or antigen-binding fragments thereof, having Fc modifications that allow Fc silencing and capable of binding to antigens expressed by hematopoietic cells (e.g., CD45, etc.) can be used as sole therapeutic agents or as ADCs to (i) treat cancers and autoimmune diseases characterized by CD45+ hematopoietic stem cells, and (ii) promote the engraftment of transplanted hematopoietic stem cells in patients requiring transplantation therapy. These therapeutic activities can be triggered, for example, by the binding of anti-CD45 antibodies, or antigen-binding fragments thereof, that bind to CD45 expressed by blood cells (e.g., hematopoietic stem cells, or mature immune cells (e.g., T cells)), to, for example, cancer cells, autoimmune cells, or hematopoietic stem cells, and then inducing cell death. Reducing endogenous hematopoietic stem cells can provide a niche to which transplanted hematopoietic stem cells can home, and then productive hematopoiesis can be established. In this manner, the transplanted hematopoietic stem cells can successfully engraft in the patient (e.g., a human patient suffering from a stem cell disorder described herein.) The Fc-modified antibodies and ADCs of the present application not only selectively deplete endogenous hematopoietic stem cells, but also reduce the cytotoxic effects of exogenous hematopoietic stem cell grafts, thereby further promoting the engraftment of hematopoietic stem cell grafts.

[0152] The antibodies, or binding fragments described in this application may contain modifications and / or mutations that alter the properties of the antibodies and / or fragments (e.g., modifications and / or mutations that increase half-life, or increase or decrease ADCC).

[0153] In some embodiments, an antibody is provided that comprises one or more radiolabeled amino acids. Radiolabeled antibodies can be used for both diagnostic and therapeutic purposes (another feature is that they can be conjugated to radiolabeled molecules). Non-limiting examples of labels for polypeptides include, but are not limited to, 3H, 14C, 15N, 35S, 90Y, 99Tc and 125I, 131I and 186Re. Methods for preparing radiolabeled amino acid and related peptide derivatives are known in the art (see, for example, Junghans et al., Cancer Chemotherapy and Biotherapy 655-686, 2nd Edition, Chafner and Longo, Eds., Lippincott Raven (1996) and U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 [US RE35,500], 5,648,471, 5,697,902). For example, radioisotopes may be conjugated by the chloramine T method.

[0154] In certain embodiments, an anti-CD45 antibody, or binding fragment thereof, comprises an altered Fc region, wherein the altered Fc region comprises at least one amino acid modification relative to a wild-type Fc region such that the molecule has altered affinity or binding to Fc gamma R (FcγR). Certain amino acid positions within the Fc region are known by crystallographic studies to be in direct contact with FcγR, 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.) In some embodiments, the antibodies described herein may comprise a variant Fc region in which at least one residue that directly contacts FcγR has been altered based on structural and crystallographic analysis. In one embodiment, 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 et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, NH1, MD (1991), expressly incorporated by reference herein. "EU index as in Kabat" refers to the numbering of the human IgG1 EU antibody. In one embodiment, the Fc region comprises a D265A mutation. In one embodiment, the Fc region comprises a D265C mutation. In some embodiments, the Fc region of the antibody (or fragment thereof) comprises an amino acid substitution at amino acid 234 according to the EU index as in Kabat, for example.

[0155] In one embodiment, the Fc region comprises mutations at amino acid positions D265, V205, H435, I253, and / or H310. For example, specific mutations at these positions include D265C, V205C, H435A, I253A, and / or H310A.

[0156] In one embodiment, 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 one embodiment, the Fc region comprises a L235A mutation. In yet another embodiment, the Fc region comprises L234A and L235A mutations. In a further embodiment, the Fc region comprises D265C, L234A, and L235A mutations. In yet 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.

[0157] In yet another embodiment, the Fc region comprises an L234A and an L235A mutation (also referred to in the present application as "L234A.L235A" or "LALA"). In another embodiment, the Fc region comprises an L234A and an L235A mutation, wherein the Fc region does not comprise a P329G mutation. In a further embodiment, the Fc region comprises a D265C, an L234A, and an L235A mutation (also referred to in the present application as "D265C.L234A.L235A"). In another embodiment, the Fc region comprises a D265C, an L234A, and an L235A mutation, wherein the Fc region does not comprise a P329G mutation. In yet a further embodiment, the Fc region comprises D265C, L234A, L235A, and H435A mutations (also referred to in the present application as "D265C.L234A.L235A.H435A"). In another embodiment, the Fc region comprises D265C, L234A, L235A, and H435A mutations, wherein the Fc region does not comprise a P329G mutation. In a further embodiment, the Fc region comprises D265C and H435A mutations (also referred to in the present application as "D265C.H435A"). In yet another embodiment, the Fc region comprises D265A, S239C, L234A, and L235A mutations (also referred to in the present application as "D265A.S239C.L234A.L235A"). In yet another embodiment, the Fc region comprises D265A, S239C, L234A, and L235A mutations, wherein the Fc region does not comprise a P329G mutation. In another embodiment, the Fc region comprises D265C, N297G, and H435A mutations (also referred to in the present application as "D265C.N297G.H435A"). In another embodiment, the Fc region comprises D265C, N297Q, and H435A mutations (also referred to in the present application as "D265C.N297Q.H435A"). In another embodiment, the Fc region comprises E233P, L234V, L235A, and delG236 (deletion of 236) mutations (also referred to in the present application as "E233P.L234V.L235A.delG236" or "EPLVLAdelG"). In another embodiment, the Fc region comprises E233P, L234V, L235A, and delG236 (deletion of 236) mutations, wherein the Fc region does not comprise a P329G mutation.In another embodiment, the Fc region comprises E233P, L234V, L235A, delG236 (deletion of 236), and H435A mutations (also referred to in the present application as "E233P.L234V.L235A.delG236.H435A" or "EPLVLAdelG.H435A"). In another embodiment, the Fc region comprises E233P, L234V, L235A, delG236 (deletion of 236), and H435A mutations, wherein the Fc region does not comprise a P329G mutation. In another embodiment, the Fc region comprises L234A, L235A, S239C, and D265A mutations. In another embodiment, the Fc region comprises L234A, L235A, S239C, and D265A mutations, wherein the Fc region does not comprise a P329G mutation. In another embodiment, the Fc region comprises H435A, L234A, L235A, and D265C mutations. In another embodiment, the Fc region comprises H435A, L234A, L235A, and D265C mutations, wherein the Fc region does not comprise a P329G mutation.

[0158] In some embodiments, the antibody has an altered Fc region such that the antibody has reduced effector function by decreasing binding to an Fc receptor (FcR) in an in vitro effector function assay, as compared to binding of the same antibody comprising an unmodified Fc region to the FcR. In some embodiments, the antibody has an altered Fc region such that the antibody has reduced effector function by decreasing binding to an Fc gamma receptor (FcγR) in an in vitro effector function assay, as compared to binding of the same antibody comprising an unmodified Fc region to the FcγR. In some embodiments, the FcγR is FcγR1. In some embodiments, the FcγR is FcγR2A. In some embodiments, the FcγR is FcγR2B. In some embodiments, the FcγR is FcγR2C. In some embodiments, the FcγR is FcγR3A. In some embodiments, the FcγR is FcγR3B. In other embodiments, the reduction in binding is at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, at least a 95% reduction, at least a 98% reduction, at least a 99% reduction, or a 100% reduction in antibody binding to FcγR compared to binding of the same antibody comprising an unmodified Fc region to the FcγR. In other embodiments, the reduction in binding is at least a 70%-100% reduction, at least an 80%-100% reduction, at least a 90%-100% reduction, at least a 95%-100% reduction, or at least a 98%-100% reduction in antibody binding to FcγR compared to binding of the same antibody comprising an unmodified Fc region to the FcγR.

[0159] In some embodiments, the antibody has an altered Fc region such that the antibody exhibits a reduction in cytokine release in an in vitro cytokine release assay, with at least a 50% reduction in cytokine release compared to the cytokine release of the same antibody comprising an unmodified Fc region. In some embodiments, the reduction in cytokine release is at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, at least a 95% reduction, at least a 98% reduction, at least a 99% reduction, or a 100% reduction in cytokine release compared to the cytokine release of the same antibody comprising an unmodified Fc region. In some embodiments, the reduction in cytokine release is at least a 70%-100% reduction, at least an 80%-100% reduction, at least a 90%-100% reduction, or at least a 95%-100% reduction in cytokine release compared to the cytokine release of the same antibody comprising an unmodified Fc region. In certain embodiments, the cytokine release is by immune cells.

[0160] In some embodiments, the antibody has an altered Fc region such that the antibody reduces mast cell degranulation in an in vitro mast cell degranulation assay by at least 50% reduction in mast cell degranulation compared to the mast cell degranulation of the same antibody with an unmodified Fc region. In some embodiments, the reduction in mast cell degranulation is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% reduction in mast cell degranulation compared to the mast cell degranulation of the same antibody with an unmodified Fc region. In some embodiments, the reduction in mast cell degranulation is at least 70%-100%, at least 80%-100%, at least 90%-100%, or at least 95%-100% reduction in mast cell degranulation compared to the mast cell degranulation of the same antibody with an unmodified Fc region.

[0161] In some embodiments, the antibody has an altered Fc region such that the antibody reduces or avoids antibody dependent cell phagocytosis (ADCP) by at least 50% reduction in ADCP in an in vitro ADCP assay compared to the ADCP of the same antibody comprising an unmodified Fc region. In some embodiments, the reduction in ADCP is at least a 70% reduction, at least an 80% reduction, at least a 90% reduction, at least a 95% reduction, at least a 98% reduction, at least a 99% reduction, or a 100% reduction in cytokine release compared to the cytokine release of the same antibody comprising an unmodified Fc region.

[0162] In some embodiments, the anti-CD45 antibodies described herein comprise an Fc region that comprises one of the following modifications or combinations of modifications: D265A, D265C, D265C / H435A, D265C / LALA, D265C / LALA / H435A, D265A / S239C / L234A / L235A / H435A, D265A / S239C / L234A / L235A, D265C / N297G, D265C / N297G / H435A, D265C (EPLVLAdelG*), D265C (EPLVLAdelG) / H435A, D265C / N297Q / H435A, D265C / N297Q, EPLVLAdelG / H435A, EPLVLAdelG / D265C, EPLVLAdelG / D265A, N297A, N297G, or N297Q.

[0163] The binding or affinity between the modified Fc region and the Fc gamma receptor may be measured using a variety of techniques known in the art, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al. Analytical Biochemistry, Vol. 373:52-60, 2008; radioimmunoassay (RIA)), or surface plasmon resonance assays or other mechanism-based kinetic assays (e.g., BIACORE® analysis or Octet® analysis (forteBIO)), as well as other methods (e.g., indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration), etc.). These and other methods may utilize a label on one or more of the components being evaluated and / or may use a variety of detection methods (e.g., but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels). Binding affinity and kinetics are described in detail in Paul, WE, ed., Fundamental Immunology, 4th Ed., 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 an antibody of interest in the presence of increasing amounts of unlabeled antigen and detecting antibody bound to the labeled antigen. From the data, the affinity and binding off-rate of the antibody of interest for a particular antigen may be determined by Scatchard plot analysis. Competition with a second antibody may also be measured using a radioimmunoassay, in which the antigen is incubated with the antibody of interest conjugated to a labeled compound in the presence of increasing amounts of an unlabeled second antibody.

[0164] In certain embodiments, an antibody having the Fc modifications described herein (e.g., D265C, L234A, L235A, and / or H435A) has binding to an Fc gamma receptor that is at least 70% reduced, at least 80% reduced, at least 90% reduced, at least 95% reduced, at least 98% reduced, at least 99% reduced, or 100% reduced compared to binding to said Fc gamma receptor of the same antibody comprising an unmodified Fc region (e.g., as assessed by biolayer interferometry (BLI)).

[0165] Without wishing to be bound by any theory, Fc region binding interactions with Fc gamma receptors are believed to be essential for a variety of 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 embodiments, antibodies comprising altered Fc regions (e.g., comprising L234A, L235A, and / or D265C mutations) have substantially reduced or eliminated effector function. Effector function may be assayed using a variety of methods known in the art (e.g., by measuring cellular responses to the antibody of interest, e.g., mast cell degranulation or cytokine release). For example, Fc modified antibodies may be assayed for their ability to induce mast cell degranulation in vitro, or to induce cytokine release (e.g., released by human peripheral blood mononuclear cells) using standard methods in the art.

[0166] The antibodies of the present disclosure may be further modified to form fusion proteins as described in, for example, (Dall'Acqua et al. (2006) J Biol Chem 281: 23514-24), (Zalevsky et al. (2010) Nat Biotechnol 28: 157-9), (Hinton et al. (2004) J Biol Chem 279: 6213-6), (Hinton et al. (2006) J Immunol 176: 346-56), (Shields et al. (2001) J Biol Chem 276: 6591-604), (Petkova et al. (2006) Int Immunol 18: 1759-69), (Datta-Mannan et al. (2007) Drug Metab Dispos 35: The half-life of the antibody may be further modulated by introducing Fc mutations such as those described in (Vaccaro et al. (2005) Nat Biotechnol 23: 1283-8), (Yeung et al. (2010) Cancer Res 70: 3269-77), and (Kim et al. (1999) Eur J Immunol 29: 2819-25), and may include positions 250, 252, 253, 254, 256, 257, 307, 376, 380, 428, 434, and 435. Exemplary mutations that may be made alone or in combination are T250Q, M252Y, 1253A, S254T, T256E, P2571, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations.

[0167] Thus, in one embodiment, the Fc region comprises a mutation that results in a reduced half-life (e.g., compared to an antibody having an unmodified Fc region). An antibody with a short half-life may be beneficial in certain instances (e.g., in the conditioning step described in this application, where the antibody is administered followed by administration of HSCs) where the antibody is expected to function as a short-lived therapeutic agent. Ideally, the antibody should be substantially cleared before administering HSCs, which, unlike endogenous stem cells, generally also express a target antigen (e.g., CD45) but are not the target of the anti-CD45 antibody. In one embodiment, the Fc region comprises a mutation at position 435 (EU index according to Kabat). In one embodiment, the mutation is an H435A mutation.

[0168] In certain embodiments, the anti-CD45 antibodies described in the present application have a half-life (e.g., in humans) of about 24 hours or less, about 23 hours or less, about 22 hours or less, about 21 hours or less, about 20 hours or less, about 19 hours or less, about 18 hours or less, about 17 hours or less, about 16 hours or less, about 15 hours or less, about 14 hours or less, about 13 hours or less, about 12 hours or less, or about 11 hours or less.

[0169] In certain embodiments, the anti-CD45 antibodies described herein have a half-life (e.g., in humans) of about 1-5 hours, about 5-10 hours, about 10-15 hours, about 15-20 hours, or about 20-25 hours. In certain embodiments, the half-life of the anti-CD45 antibodies is about 5-7 hours; about 5-9 hours; about 5-11 hours; about 5-13 hours; about 5-15 hours; about 5-20 hours; about 5-24 hours; about 7-24 hours; about 9-24 hours; about 11-24 hours; about 12-22 hours; about 10-20 hours; about 8-18 hours; or about 14-24 hours.

[0170] In some aspects, the Fc region comprises two or more mutations that decrease half-life and reduce effector function of the antibody. In some embodiments, the Fc region comprises a mutation that results in a decrease in half-life and a mutation in at least one residue that can directly contact an FcγR (e.g., based on structural and crystallographic analysis). In some embodiments, the Fc region comprises an H435A mutation, an L234A mutation, and an L235A mutation. In some embodiments, the Fc region comprises an H435A mutation and a D265C mutation. In some embodiments, the Fc region comprises an H435A mutation, an L234A mutation, an L235A mutation, and a D265C mutation.

[0171] 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 by a mutation in the Fc domain of the antibody, or antigen-binding fragment thereof. For example, the cysteine ​​residue may be selected from the group consisting of Cys118, Cys239, and Cys265. In 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 H435A mutations. In some embodiments, the Fc region comprises a D265C, L234A, and L235A mutations. In one embodiment, the Fc region comprises D265C, L234A, L235A, and H435A mutations. In one embodiment, the Fc region of the anti-CD45 antibody, or antigen-binding fragment thereof, comprises an amino acid substitution at amino acid 239 according to the EU index as in Kabat. In one embodiment, the Fc region comprises a S239C mutation. In one embodiment, the Fc region comprises a L234A mutation, a L235A mutation, a S239C mutation, and a D265A mutation. In another embodiment, the Fc region comprises a S239C and H435A mutation. In another embodiment, the Fc region comprises a L234A mutation, a L235A mutation, and a S239C mutation. In yet another embodiment, the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, and a S239C mutation. In yet another embodiment, the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, a S239C mutation, and a D265A mutation.

[0172] In particular, amino acid positions in Fc refer to the EU numbering index unless otherwise specified.

[0173] The disclosure of each of the above publications is incorporated by reference into this application as it relates to anti-CD45 antibodies. Antibodies and antigen-binding fragments that may be used in combination with the compositions and methods described in this application include the above antibodies and antigen-binding fragments thereof, as well as humanized variants of the above non-human antibodies and antigen-binding fragments, and antibodies or antigen-binding fragments that bind to the same epitope as the above antibodies and antigen-binding fragments thereof (e.g., as assessed by competitive antigen binding assays).

[0174] Methods for engineering antibodies to include any of the Fc modifications of the present application 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 prepared DNA molecules encoding the antibody or at least the constant region of the antibody. Site-directed mutagenesis is well known in the art (see, for example, 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 generating amino acid sequence variants of a 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 preparing sequence variants (cassette mutagenesis) is based on the technique described in Wells et al., Gene, 34:315-323 (1985).

[0175] Methods for identifying antibodies Methods for high throughput screening of libraries of antibodies, or antibody fragments, capable of binding to CD45 expressed by hematopoietic stem cells may be used to identify anti-CD45 antibodies useful for treating cancer, autoimmune diseases, and for conditioning patients (e.g., human patients) in need of hematopoietic stem cell therapy as described herein. Such methods may be used to identify improved AbA, AbB, and AbC antibodies. Such methods include in vitro display techniques known in the art, such as phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display, among others.

[0176] The use of phage display to isolate antibodies, or antigen-binding fragments, that bind biologically relevant molecules has been reviewed in, for example, Felici et al., Biotechnol. Annual Rev. 1:149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26:27-45, 1997; and Hoogenboom et al., Immunotechnology 4:1-20, 1998, the disclosures of each of which are incorporated by reference into this application as they relate to in vitro display techniques. Randomized combinatorial peptide libraries have also been constructed to select polypeptides that bind to cell surface antigens, as described in Kay, Perspect. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, 1996, the disclosures of each of which are incorporated herein by reference as they relate to the discovery of antigen-binding molecules. Phage display of proteins (e.g., multimeric proteins) as functional molecules has been successfully achieved (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 as they relate to the use of in vitro display techniques to discover antigen-binding molecules).Additionally, functional antibody fragments (e.g., Fab and scFv fragments) have been expressed in in vitro display formats (see, e.g., McCafferty et al., Nature 348:552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991; and Clackson et al., Nature 352:624-628, 1991, the disclosures of each of which are incorporated herein by reference as they relate to in vitro display platforms for discovering antigen-binding molecules). Human anti-CD45 antibodies can be expressed, for example, in HuMAb-mice. 登録商標 or XenoMouse TM Among other things, these techniques may be used to identify and improve the affinity of antibodies, antibodies, or fragments that can bind to CD45 expressed by hematopoietic stem cells and then be used to deplete endogenous hematopoietic stem cells in patients (e.g., human patients) in need of hematopoietic stem cell transplantation therapy.

[0177] In addition to in vitro display techniques, computational modeling techniques may be used to design and identify antibodies capable of binding to hematopoietic stem cell expressed antigens (e.g., CD45). For example, using computational modeling techniques, one skilled in the art may screen in silico a library of antibodies, or antibody fragments, for molecules capable of binding to a specific epitope (e.g., an extracellular epitope of the antigen) on a hematopoietic stem cell expressed antigen (e.g., CD45).

[0178] Further techniques may be used to identify antibodies or antibody fragments that can bind to CD45 expressed by hematopoietic stem cells and that can be internalized by the cells, for example, by receptor-mediated endocytosis. For example, the in vitro display techniques described above may be adapted to screen for antibodies or antibody fragments thereof that bind to and are subsequently internalized by CD45. Phage display represents one such technique that may be used in combination with this screening paradigm. To identify anti-CD45 antibodies or antibody fragments that can be internalized into hematopoietic stem cells, one of skill in the art may use the phage display technique described in Williams et al., Leukemia 19:1432-1438, 2005, the disclosure of which is incorporated herein by reference in its entirety. For example, mutagenesis methods known in the art may be used to identify antibodies, antibody fragments (e.g., scFv fragments, Fab fragments, diabodies, triabodies, and antibodies, among others). 10 Recombinant phage libraries can be produced that encode antibodies, polypeptides, or ligands (which contain randomized amino acid cassettes (e.g., in the CDRs or equivalent regions, or in one or more or all of the antibodies or antibody fragments)). The framework regions, hinges, Fc domains, and other regions of the antibodies or antibody fragments may be designed to be non-immunogenic in humans, for example, by having human germline antibody sequences or sequences that are slightly different compared to human germline antibodies.

[0179] Using phage display techniques described herein or known in the art, a phage library comprising randomized antibodies or antibody fragments covalently attached to phage particles is incubated with CD45 (e.g., first incubating the phage library with a blocking agent (e.g., milk protein, bovine serum albumin, and / or IgG, etc.) to remove phage encoding antibodies or antibody fragments that exhibit non-specific protein binding and to remove phage encoding antibodies or fragments thereof that bind to Fc domains, and then incubating the phage library with a population of cells (e.g., hematopoietic stem cells) that express CD45). The phage library may be incubated with the hematopoietic stem cells for a sufficient time (e.g., 30 minutes to 6 hours at 4° C., e.g., 1 hour at 4° C.) for the anti-CD45 antibodies or antibody fragments to bind to cell surface cognate antigens and subsequently be internalized by the hematopoietic stem cells. Phage containing antibodies or antibody fragments that do not exhibit sufficient affinity for the CD45 to bind to and be internalized by hematopoietic stem cells may then be removed, for example, by washing with cold (4° C.) 0.1 M glycine buffer, pH 2.8. Phage that bound to the internalized antibodies or antibody fragments thereof may be identified, for example, by lysing the cells and recovering the internalized phage from the cell culture medium. The phage may then be amplified in bacterial cells, for example, by incubating bacterial cells with the recovered phage in 2×YT medium, using methods known in the art. Phage recovered from the medium may then be identified, for example, by determining the nucleic acid sequence of the antibody or antibody fragment-encoding gene inserted into the phage genome.The encoded antibody or antibody fragment may then be prepared de novo by chemical synthesis (e.g., for the antibody fragment, e.g., an scFv fragment) or by recombinant expression (e.g., for a full-length antibody).

[0180] Additionally, the epitope regions described herein may be used as peptides to screen for additional anti-hCD45 antibodies using screening techniques disclosed herein and known in the art.

[0181] How to make production Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid is provided encoding an anti-CD45 antibody described herein. Such a nucleic acid may encode an amino acid sequence comprising the VL of the antibody and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the 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, the host cell comprises (e.g., is transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-CLL-1 antibody is provided, the method comprising culturing a host cell comprising nucleic acid encoding an antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0182] To produce an anti-CD45 antibody recombinantly, nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody). Nucleic acids that may be used to express the antibodies, or fragments, disclosed in the present application include those set forth in SEQ ID Nos: 25 to 30 of Table 3.

[0183] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly where glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody may be isolated from the bacterial cell paste as a soluble fraction and further purified.

[0184] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to grow in suspension can be useful. Other examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 line (COS-7); human embryonic kidney cell lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells 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 carcinoma (MMT 060562); TRI cells (e.g., Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells (including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines (e.g., 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 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). In certain embodiments, the host cell is eukaryotic, e.g., Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20 cells).

[0185] The capacity of the anti-CD45 antibody, or antibody fragment, to be incorporated may be assessed using, for example, radionuclide internalization assays known in the art. For example, an anti-CD45 antibody, or antibody fragment, identified using in vitro display techniques described in this application or known in the art, may be fused to a radioisotope (e.g., 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 make it functional. For example, radioactive halogens (e.g. 18 F, 75 Br, 77 Br, 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211A radioactively labeled antibody, fragment thereof, or ADC may be incorporated into the antibody or antibody fragment using beads (e.g., polystyrene beads) containing an electrophilic halogen reagent (e.g., iodized beads, Thermo Fisher Scientific, Inc., Cambridge, MA). The radioactively labeled antibody, fragment thereof, or ADC may be incubated with the hematopoietic stem cells for a time sufficient for incorporation (e.g., 30 minutes to 6 hours at 4°C, e.g., 1 hour at 4°C). The cells are then washed (e.g., with cold (4°C) 0.1 M glycine buffer, pH 2.8) to remove any antibody or fragment thereof that has not been incorporated. Incorporated antibody or antibody fragment may be identified by detecting radiation (e.g., gamma radiation) emitted from the resulting hematopoietic stem cells and comparing it to radiation (e.g., gamma radiation) emitted from the recovered wash buffer. The internalization assay described above may be used to characterize the ADC.

[0186] Antibody Drug Conjugates (ADCs) The anti-CD45 antibodies, or antigen-binding fragments thereof, described herein may be conjugated to a cytotoxin via a linker. In some embodiments, the cytotoxic molecule may be conjugated to an antibody, or antigen-binding fragment thereof, that is internalized in a cell as disclosed herein, such that after the antibody, or fragment thereof, is internalized in the cell, the cytotoxin may access its intracellular target and mediate the death of the hematopoietic cell.

[0187] cytotoxin A variety of cytotoxins can be conjugated to anti-CD45 antibodies via linkers for use in the therapies described herein. In particular, anti-CD45 ADCs include an anti-CD45 antibody (or antigen-binding fragment thereof) conjugated (i.e., covalently attached by a linker) to a cytotoxic moiety (or cytotoxin). In various embodiments, the cytotoxic moiety has reduced or no cytotoxicity when attached as a conjugate. However, after cleavage from the linker, it regains cytotoxicity. In various embodiments, the cytotoxic moiety retains cytotoxicity without cleavage from the linker. In some embodiments, the cytotoxic molecule is conjugated to an antibody, or antigen-binding fragment thereof, as described herein, that is internalized into a cell, such that after the antibody, or fragment thereof, is internalized into a cell, the cytotoxin may have access to its intracellular target and, for example, mediate T cell death.

[0188] Thus, the ADCs of the invention have the general formula Ab-(ZLD): n where an antibody or antigen-binding fragment thereof (Ab) is conjugated (covalently bonded) to a linker (L) and, through a chemical moiety (Z), to a cytotoxic moiety ("drug", D).

[0189] Thus, the anti-CD45 antibody or antigen-binding fragment thereof may be conjugated to multiple drug moieties as indicated by the integer n, where the integer n represents the average number of cytotoxins per antibody, where the integer n may range, for example, from about 1 to about 20. In some embodiments, n is 1 to 4. In some embodiments, n is 1. The average number of drug moieties per antibody in a preparation of ADC from a conjugation reaction can be determined by conventional means such as mass spectrometry, ELISA assay, and HPLC. The quantitative distribution of the ADC with respect to n can also be determined. In some cases, homogenous ADCs with a particular value of n can be separated, purified, and characterized from ADCs with other drug loads by means such as reverse phase HPLC or electrophoresis.

[0190] For some anti-CD45 ADCs, n may be limited by the number of binding sites on the antibody. For example, if the linkage is a cysteine ​​thiol, the antibody has only one or a few cysteine ​​thiol groups, or only one or a few thiol groups that are sufficiently reactive for the linker to be attached. In general, antibodies do not contain many free and reactive cysteine ​​groups that can be attached to a drug moiety; primarily, the cysteine ​​thiol residues in antibodies are present as disulfide bridges. In certain embodiments, the antibody may be reduced with a reducing agent, such as dithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), to generate reactive cysteine ​​thiol groups under partial or total reducing conditions. In certain embodiments, higher drug loading (DAR) (e.g., n>5) may cause aggregation, insolubility, toxicity, or loss of cell permeability for certain antibody-drug conjugates.

[0191] In certain embodiments, fewer drug moieties than the theoretical maximum are conjugated to the antibody during the conjugation reaction. The antibody may contain lysine residues that do not react with the drug-linker intermediate or linker reagent, for example, as discussed below. Only the most reactive lysine groups can react with amine-reactive linker reagents. In certain embodiments, the antibody is subjected to denaturing conditions to expose reactive nucleophilic groups such as lysine or cysteine.

[0192] The loading (drug / antibody ratio) of ADCs may be controlled in a variety of ways, such as by (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody, (ii) limiting the time or temperature of the conjugation reaction, (iii) using partial or limiting reducing conditions for thiol modification of cysteines, and (iv) recombinantly engineering the amino acid sequence of the antibody to alter the number and position of cysteine ​​residues to control the number and / or position of linker-drug bonds.

[0193] Cytotoxins suitable for use with the compositions and methods described in this application include, among others, those known in the art, DNA-intercalating agents (e.g., anthracyclines), agents capable of disrupting the spindle apparatus (e.g., vinca alkaloids, maytansine, maytansinoids, and derivatives thereof), RNA polymerase inhibitors (e.g., amatoxins such as α-amanitin, and derivatives thereof), and agents capable of disrupting protein biosynthesis (e.g., agents exhibiting rRNA N-glycosidase activity, such as saporin and ricin A chain).

[0194] In some embodiments, the cytotoxin is a microtubule binding agent (e.g., maytansine or a maytansinoid), an amatoxin, Pseudomonas exotoxin A, de bouganin, diphtheria toxin, saporin, auristatin, an anthracycline, a calicheamicin, irinotecan, SN-38, a duocarmycin, a pyrrolobenzodiazepine, a pyrrolobenzodiazepine dimer, an indolinobenzodiazepine, an indolinobenzodiazepine dimer, an indolinobenzodiazepine pseudodimer, or variants thereof, or other cytotoxic compounds described herein or known in the art.

[0195] 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 conjugate disclosed herein is an amatoxin or a derivative thereof (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, proamanuline, or a derivative thereof, etc.).

[0196] Further details regarding cytotoxins that may be used in the anti-CD45 ADCs useful in the compositions and methods of the invention are provided below.

[0197] Amatoxins In some embodiments, the RNA polymerase inhibitor is an amatoxin or a derivative thereof. In some embodiments, the cytotoxin of the antibody-drug conjugate disclosed herein is an amatoxin or a derivative thereof (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, proamanuline, or a derivative thereof, etc.).

[0198] In some embodiments, the cytotoxin of the antibody-drug conjugate is an RNA polymerase inhibitor, hi some embodiments, the RNA polymerase inhibitor is an amatoxin or a derivative thereof.

[0199] In some embodiments, the cytotoxin is an amatoxin or a derivative thereof (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, amanuric acid, and proamanutin). The structures of various naturally occurring amatoxins are disclosed, for example, in Zanotti et al., Int. J. Peptide Protein Res. 30, 1987, 450-459.

[0200] Amatoxins useful in conjunction with the compositions and methods described in the present application include, but are not limited to, compounds according to formula (IV): [ka] Here, R 1 is H, OH, or OR A is; R 2 is H, OH, or OR B is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R 3 is H or R D is; R 4 H, OH, OR D , or R D is; R 5 H, OH, OR D , or R D is; R 6 H, OH, OR D , or R D is; R 7 H, OH, OR D , or R D is; R 8 OH, NH 2 OR D is; R 9 is H, OH, or OR D is; X is -S-, -S(O)-, or -SO 2 - is; and R D is an optionally substituted alkyl (e.g., C 1 -C 6 alkyl), optionally substituted heteroalkyl (e.g., C 1 -C 6 heteroalkyl), optionally substituted alkenyl (e.g., C 2 -C 6 alkenyl), optionally substituted heteroalkenyl (e.g., C 2 -C 6 heteroalkenyl), optionally substituted alkynyl (e.g., C 2 -C 6 alkynyl), optionally substituted heteroalkynyl (e.g., C 2 -C 6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0201] For example, in certain embodiments, amatoxins useful in conjunction with the compositions and methods described herein include compounds according to formula (IVA): [ka] Here, R 4 , R 5 , X, and R 8 are each defined above.

[0202] For example, in certain embodiments, amatoxins useful in conjunction with the compositions and methods described in the present application include compounds according to formula (IVB): [ka] Here, R 1 is H, OH, or OR A is; R 2 is H, OH, or OR B is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R 3 is H or R D is; R 4 H, OH, OR D , or R D is; R 5 H, OH, OR D , or R D is; R 6 H, OH, OR D , or R D is; R 7 H, OH, OR D , or R D is; R 8 OH, NH 2 OR D is; R 9 is H, OH, or OR D is; X is -S-, -S(O)-, or -SO 2 - is; and R D is an optionally substituted alkyl (e.g., C 1 -C 6alkyl), optionally substituted heteroalkyl (e.g., C 1 -C 6 heteroalkyl), optionally substituted alkenyl (e.g., C 2 -C 6 alkenyl), optionally substituted heteroalkenyl (e.g., C 2 -C 6 heteroalkenyl), optionally substituted alkynyl (e.g., C 2 -C 6 alkynyl), optionally substituted heteroalkynyl (e.g., C 2 -C 6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0203] In certain embodiments, amatoxins useful in conjunction with the compositions and methods described in this application include compounds according to the following formula (IVC): [ka] where: R 1 is H, OH, or OR A is; R 2 is H, OH, or OR B is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R 3 is H or R D is; R 4 H, OH, OR D , or R D is; R 5 H, OH, OR D , or R D is; R 6 H, OH, OR D , or R D is; R 7 H, OH, OR D , or R D is; R 8 OH, NH 2 OR D is; R 9 is H, OH, or OR D is; X is -S-, -S(O)-, or -SO 2 - is; and R D is an optionally substituted alkyl (e.g., C 1 -C 6 alkyl), optionally substituted heteroalkyl (e.g., C 1 -C 6 heteroalkyl), optionally substituted alkenyl (e.g., C 2 -C 6 alkenyl), optionally substituted heteroalkenyl (e.g., C 2 -C 6 heteroalkenyl), optionally substituted alkynyl (e.g., C 2 -C 6 alkynyl), optionally substituted heteroalkynyl (e.g., C 2 -C 6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0204] Synthetic methods for producing amatoxins are described in US Pat. No. 9,676,702, which is incorporated by reference into this application.

[0205] For example, the antibodies and antigen-binding fragments described in this application may be conjugated to an amatoxin (e.g., of formula IV, IVA, IVB, or IVC) to form a conjugate represented by the formula Ab-ZL-Am, where Ab is an antibody, or an 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 serve as positions for covalent attachment to a linking moiety L, and thus to the antibody, or an antigen-binding fragment thereof.

[0206] In some embodiments, the amatoxin-linker conjugate Am-LZ is represented by formula (I): [ka] where: R 1 H, OH, OR A OR C is; R 2 H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R 3 , H, R C , or R D is; R 4 H, OH, OR C , OR D , R C , or R D is; R 5 H, OH, OR C , OR D , R C , or R D is; R 6H, OH, OR C , OR D , R C , or R D is; R 7 H, OH, OR C , OR D , R C , or R D is; R 8 OH, NH 2 , OR C , OR D , N.H.R. C , or NR C R D is; R 9 H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO 2 - is; R C is -LZ; R D is an optionally substituted alkyl (e.g., C 1 -C 6 alkyl), optionally substituted heteroalkyl (e.g., C 1 -C 6 heteroalkyl), optionally substituted alkenyl (e.g., C 2 -C 6 alkenyl), optionally substituted heteroalkenyl (e.g., C 2 -C 6 heteroalkenyl), optionally substituted alkynyl (e.g., C 2 -C 6 alkynyl), optionally substituted heteroalkynyl (e.g., C 2 -C 6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C 1 -C 6alkylene), optionally substituted heteroalkylene (e.g., C 1 -C 6 heteroalkylene), optionally substituted alkenylene (e.g., C 2 -C 6 alkenylene), optionally substituted heteroalkenylene (e.g., C 2 -C 6 heteroalkenylene), optionally substituted alkynylene (e.g., C 2 -C 6 alkynylene), optionally substituted heteroalkynylene (e.g., C 2 -C 6 a linker such as an optionally substituted aryl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, an optionally substituted aryl group, an optionally substituted aryl group, an optionally substituted heteroaryl group, a peptide, a dipeptide, -C(=O)-, a disulfide, a hydrazone, or a combination thereof; and Z is a chemical moiety formed from a coupling reaction between a reactive substituent Z' present on L and a reactive substituent present in an antibody, or antigen-binding fragment thereof, that binds to a target antigen (e.g., CD45).

[0207] In some embodiments, Am is exactly one R C Contains substituents.

[0208] In some embodiments, the LZ is [ka] Here, S is a sulfur atom representing a reactive substituent present in an antibody, or antigen-binding fragment thereof, that binds to the target antigen (e.g., resulting from the -SH group of a cysteine ​​residue).

[0209] In some embodiments, the conjugate Am-LZ-Ab is represented by one of formulas III, IIIA, or IIIB: [ka] where X is S, SO or SO 2 and Ab is shown to indicate the position of binding to Ab.

[0210] In some embodiments, Am-LZ-Ab is [ka] where Ab is shown to indicate the position of binding to Ab.

[0211] In some embodiments, Am-LZ-Ab is [ka] where Ab is shown to indicate the position of binding to Ab.

[0212] In some embodiments, Am-LZ-Ab is [ka] where Ab is shown to indicate the position of binding to Ab.

[0213] In some embodiments, the Am-LZ-Ab precursor, Am-L-Z′, is [ka] where the maleimide reacts with a thiol group on a cysteine ​​in the antibody.

[0214] In some embodiments, the Am-LZ-Ab precursor, Am-L-Z′, is [ka] where the maleimide reacts with a thiol group on a cysteine ​​in the antibody.

[0215] In some embodiments, Am-LZ is represented by formula (IA): [ka] where: R 1 H, OH, OR A OR C is; R 2 H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R 3 , H, R C , or R D is; R 4 H, OH, OR C , OR D , R C , or R D is; R 5 H, OH, OR C , OR D , R C , or R D is; R 6 H, OH, OR C , OR D , R C , or R D is; R 7 H, OH, OR C , OR D , R C , or R D is; R 8 OH, NH 2 , ORC , OR D , N.H.R. C , or NR C R D is; R 9 H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO 2 - is; R C is -LZ; R D is an optionally substituted alkyl (e.g., C 1 -C 6 alkyl), optionally substituted heteroalkyl (e.g., C 1 -C 6 heteroalkyl), optionally substituted alkenyl (e.g., C 2 -C 6 alkenyl), optionally substituted heteroalkenyl (e.g., C 2 -C 6 heteroalkenyl), optionally substituted alkynyl (e.g., C 2 -C 6 alkynyl), optionally substituted heteroalkynyl (e.g., C 2 -C 6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C 1 -C 6 alkylene), optionally substituted heteroalkylene (e.g., C 1 -C 6 heteroalkylene), optionally substituted alkenylene (e.g., C 2 -C 6 alkenylene), optionally substituted heteroalkenylene (e.g., C 2 -C 6 heteroalkenylene), optionally substituted alkynylene (e.g., C 2-C 6 alkynylene), optionally substituted heteroalkynylene (e.g., C 2 -C 6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, a peptide, a dipeptide, -C(=O)-, a disulfide, a hydrazone, or a combination thereof; Z is a chemical moiety formed from a coupling reaction between a reactive substituent Z′ present on L and a reactive substituent present in an antibody, or antigen-binding fragment thereof, that binds to CD45; and where Am is exactly one R C Contains substituents.

[0216] In some embodiments, the LZ is [ka] It is.

[0217] In some embodiments, Am-LZ is represented by formula (IB): [ka] where: R 1 H, OH, OR A OR C is; R 2 H, OH, OR B OR C is; R A and R B when present, together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R 3 , H, R C , or R D is; R4 H, OH, OR C , OR D , R C , or R D is; R 5 H, OH, OR C , OR D , R C , or R D is; R 6 H, OH, OR C , OR D , R C , or R D is; R 7 H, OH, OR C , OR D , R C , or R D is; R 8 OH, NH 2 , OR C , OR D , N.H.R. C , or NR C R D is; R 9 H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO 2 - is; R C is -LZ; R D is an optionally substituted alkyl (e.g., C 1 -C 6 alkyl), optionally substituted heteroalkyl (e.g., C 1 -C 6 heteroalkyl), optionally substituted alkenyl (e.g., C 2 -C 6 alkenyl), optionally substituted heteroalkenyl (e.g., C 2 -C 6 heteroalkenyl), optionally substituted alkynyl (e.g., C 2 -C 6alkynyl), optionally substituted heteroalkynyl (e.g., C 2 -C 6 heteroalkynyl), optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is an optionally substituted alkylene (e.g., C 1 -C 6 alkylene), optionally substituted heteroalkylene (e.g., C 1 -C 6 heteroalkylene), optionally substituted alkenylene (e.g., C 2 -C 6 alkenylene), optionally substituted heteroalkenylene (e.g., C 2 -C 6 heteroalkenylene), optionally substituted alkynylene (e.g., C 2 -C 6 alkynylene), optionally substituted heteroalkynylene (e.g., C 2 -C 6 heteroalkynylene), optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, a peptide, a dipeptide, -C(=O)-, a disulfide, a hydrazone, or a combination thereof; Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in an antibody, or antigen-binding fragment thereof, that binds to CD45; and where Am is exactly one R C Contains substituents.

[0218] In some embodiments, the LZ [ka] It is.

[0219] In some embodiments, R A and R B when present, together with the oxygen atom to which they are attached form a 5-membered heterocycloalkyl group of the formula: [ka] Here, Y is -C(=O)-, -C(=S)-, -C(=NR E )-, or -C(R E R E’ )-; and R E and R E' each independently represents an optionally substituted C 1 -C 6 Alkylene-R C , optionally substituted C 1 -C 6 Heteroalkylene-R C , optionally substituted C 2 -C 6 Alkenylene-R C , optionally substituted C 2 -C 6 Heteroalkenylene-R C , optionally substituted C 2 -C 6 Alkynylene-R C , optionally substituted C 2 -C 6 Heteroalkynylene-R C , optionally substituted cycloalkylene-R C , optionally substituted heterocycloalkylene-R C、 Optionally substituted arylene-R C、 or optionally substituted heteroarylene-R C , is.

[0220] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): Here, R 1 H, OH, OR A OR C is; R 2 H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached form: [ka] R 3 is H or R C is; R 4 H, OH, OR C , OR D , R C , or R D is; R 5 H, OH, OR C , OR D , R C , or R D is; R 6 H, OH, OR C , OR D , R C , or R D is; R 7 H, OH, OR C , OR D , R C , or R D is; R 8 OH, NH 2 , OR C , or NHR C is; R 9 is H or OH; X is -S-, -S(O)-, or -SO 2 - is; and Here, R C and R D are each as defined above.

[0221] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): Here, R 1 H, OH, OR A OR C is; R 2 H, OH, OR B OR C is; R A and R B When present, together with the oxygen atom to which they are attached form: [ka] R 3 is H or R C is; R 4 and R 5 are independently H, OH, or OR C , R C OR D is; R 6 and R 7 are H, respectively; R 8 OH, NH 2 , OR C , or NHR C is; R 9 is H or OH; X is -S-, -S(O)-, or -SO 2 - is; and Here, R C is as defined above.

[0222] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): Here, R 1 is H, OH, or OR A is; R 2 is H, OH, or OR B is; R A and R BWhen present, together with the oxygen atom to which they are attached form: [ka] R 3 , R 4 , R 6 , and R 7 are H, respectively; R 5 OR C is; R 8 is OH or NH 2 is; R 9 is H or OH; X is -S-, -S(O)-, or -SO 2 - is; and Here, R C Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2016 / 0002298, the disclosure of which is incorporated herein by reference in its entirety.

[0223] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): Here, R 1 and R 2 are each independently H or OH; R 3 is R C is; R 4 , R 6 , and R 7 are H, respectively; R 5 is H, OH, or OC 1 -C 6 is alkyl; R 8 is OH or NH 2 is; R 9 is H or OH; X is -S-, -S(O)-, or -SO 2 - is; and Here, R C Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2014 / 0294865, the disclosure of which is incorporated herein by reference in its entirety.

[0224] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): Here, R 1 and R 2 are each independently H or OH; R 3 , R 6 , and R 7 are H, respectively; R 4 and R 5 are independently H, OH, or OR C , or R C is; R 8 is OH or NH 2 is; R 9 is H or OH; X is -S-, -S(O)-, or -SO 2 - is; and Here, R C Such amatoxin conjugates are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0225] In some embodiments, Am-LZ is represented by formula (IA) or formula (IB): where: R 1 and R 2 are each independently H or OH; R 3 , R 6 , and R 7 are H, respectively; R 4 and R 5are each independently H or OH; R 8 OH, NH 2 , OR C , or NHR C is; R 9 is H or OH; X is -S-, -S(O)-, or -SO 2 - is; and Here, R C Such amatoxin conjugates are described, for example, in U.S. Patent Nos. 9,233,173 and 9,399,681, and in US 2016 / 0089450, the disclosures of each of which are incorporated by reference in their entireties into this application.

[0226] In some embodiments, Am-L-Z′ is [ka] It is.

[0227] Additional amatoxins that can be used for conjugation to antibodies, or antigen-binding fragments thereof, in accordance with the compositions and methods described herein are described, for example, in WO 2016 / 142049; WO 2016 / 071856; WO 2017 / 149077; WO 2018 / 115466; and WO 2017 / 046658, the disclosures of each of which are incorporated by reference in their entireties into this application.

[0228] In some embodiments, Am-LZ is represented by formula (II), formula (IIA), or formula (IIB): [ka] where X is S, SO, or SO 2 R 1is a linker covalently attached to the antibody or antigen-binding fragment thereof via H or a chemical substructure Z, which is formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the antibody or antigen-binding fragment thereof; and R 2 is a linker covalently attached to the antibody or antigen-binding fragment thereof via H or a chemical substructure Z, which is formed from a coupling reaction between a reactive substituent present on the linker and a reactive substituent present in the antibody or antigen-binding fragment thereof; 1 If H, then R 2 is the linker, and R 2 If H, then R 1 is the linker.

[0229] In some embodiments, R 1 is a linker, and R 2 is H, and the linker and chemical moiety together as LZ are [ka] It is.

[0230] In some embodiments, R 1 is a linker, and R 2 is H, and the linker and chemical moiety together as LZ are [ka] It is.

[0231] In one embodiment, Am-LZ-Ab is: [ka] It is.

[0232] In one embodiment, Am-LZ-Ab is: [ka] It is.

[0233] In some embodiments, the Am-LZ-Ab precursor (i.e., Am-L-Z') is one of the following: [ka] where the maleimide reacts with a thiol group on a cysteine ​​in the antibody.

[0234] In some embodiments, the cytotoxin is α-amanitin. In some embodiments, the α-amanitin is attached to an anti-CD45 antibody via a linker, L. In some embodiments, the α-amanitin is a compound of formula IV. The linker, L, is located at any one of several possible positions of the α-amanitin of formula IV (e.g., R 1 -R 9 or any of the moieties (C-(CH)-(CH). In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker comprises the moiety -((C=O)(CH 2 ) n - units, where n is an integer from 1 to 6.

[0235] In some embodiments, the linker is -(CH 2 ) n- units, where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH 2 ) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH 2 ) n In some embodiments, the linker L and chemical moiety Z, taken together as LZ, are [ka] It is.

[0236] In some embodiments, the cytotoxin is β-amanitin. In some embodiments, the β-amanitin is attached to an anti-CD45 antibody via a linker, L. In some embodiments, the β-amanitin is a compound of formula IV. The linker, L, may be located at any one of several possible positions of the β-amanitin of formula IV (e.g., R 1 -R 9 (Any of the moieties) to provide β-amanitin-linker conjugates of formula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker comprises the moiety -((C=O)(CH 2 ) n - units, where n is an integer from 1 to 6.

[0237] In some embodiments, the linker is -(CH 2 ) n- units, where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH 2 ) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH 2 ) n In some embodiments, the linker L and chemical moiety Z, taken together as LZ, are [ka] It is.

[0238] In some embodiments, the cytotoxin is γ-amanitin. In some embodiments, the γ-amanitin is attached to an anti-CD45 antibody via a linker L. In some embodiments, the γ-amanitin is a compound of formula IV. The linker L may be located at any one of several possible positions of the γ-amanitin of formula IV (e.g., R 1 -R 9 or any of the following) to provide a γ-amanitin-linker conjugate of formula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker comprises the moiety -((C=O)(CH 2 ) n - units, where n is an integer from 1 to 6.

[0239] In some embodiments, the linker is -(CH 2 ) n- units, where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH 2 ) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH 2 ) n In some embodiments, the linker L and chemical moiety Z, taken together as LZ, are [ka] It is.

[0240] In some embodiments, the cytotoxin is ε-amanitin. In some embodiments, the ε-amanitin is attached to an anti-CD45 antibody via a linker L. In some embodiments, the ε-amanitin is a compound of formula IV. The linker L may be located at any one of several possible positions of the ε-amanitin of formula IV (e.g., R 1 -R 9 or any of the moieties (C-(CH)-Ala) to provide an ε-amanitin-linker conjugate of formula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker comprises the moiety -((C=O)(CH 2 ) n - units, where n is an integer from 1 to 6.

[0241] In some embodiments, the linker is -(CH 2 ) n- units, where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH 2 ) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH 2 ) n In some embodiments, the linker L and chemical moiety Z, taken together as LZ, are [ka] It is.

[0242] In some embodiments, the cytotoxin is amanin. In some embodiments, the amanin is attached to an anti-CD45 antibody via a linker L. In some embodiments, the amanin is a compound of formula IV. The linker L can be attached to any one of several possible positions of the amanin of formula IV (e.g., R 1 -R 9 (Any of the moieties) to provide amanine-linker conjugates of formula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker comprises the moiety -((C=O)(CH 2 ) n - units, where n is an integer from 1 to 6.

[0243] In some embodiments, the linker is -(CH 2 ) n- units, where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH 2 ) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH 2 ) n In some embodiments, the linker L and chemical moiety Z, taken together as LZ, are [ka] It is.

[0244] In some embodiments, the cytotoxin is amaninamide. In some embodiments, the amaninamide is attached to an anti-CD45 antibody via a linker, L. In some embodiments, the amaninamide is a compound of formula IV. The linker, L, may be attached to any one of several possible positions of the amaninamide of formula IV (e.g., R 1 -R 9 or any of the moieties) to provide an amaninamide-linker conjugate of formula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker comprises the moiety -((C=O)(CH 2 ) n - units, where n is an integer from 1 to 6.

[0245] In some embodiments, the linker is -(CH 2 ) n- units, where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH 2 ) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH 2 ) n In some embodiments, the linker L and chemical moiety Z, taken together as LZ, are [ka] It is.

[0246] In some embodiments, the cytotoxin is amanuline. In some embodiments, the amanuline is attached to an anti-CD45 antibody via a linker L. In some embodiments, the amanuline is a compound of formula IV. The linker L may be attached at any one of several possible positions of the amanuline of formula IV (e.g., R 1 -R 9 (Any of the moieties) to provide an amanulin-linker conjugate of formula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker comprises the moiety -((C=O)(CH 2 ) n - units, where n is an integer from 1 to 6.

[0247] In some embodiments, the linker is -(CH 2 ) n- units, where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH 2 ) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH 2 ) n In some embodiments, the linker L and chemical moiety Z, taken together as LZ, are [ka] It is.

[0248] In some embodiments, the cytotoxin is amanurinic acid. In some embodiments, the amanurinic acid is attached to an anti-CD45 antibody via a linker, L. In some embodiments, the amanurinic acid is a compound of formula IV. The linker, L, may be attached to any one of several possible positions of the amanurinic acid of formula IV (e.g., R 1 -R 9 amanurinic acid-linker conjugate of formula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker comprises the moiety -((C=O)(CH 2 ) n - units, where n is an integer from 1 to 6.

[0249] In some embodiments, the linker is -(CH 2 ) n- units, where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH 2 ) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH 2 ) n In some embodiments, the linker L and chemical moiety Z, taken together as LZ, are [ka] It is.

[0250] In some embodiments, the cytotoxin is proamanuline. In some embodiments, the proamanuline is attached to an anti-CD45 antibody via a linker L. In some embodiments, the proamanuline is a compound of formula IV. The linker L may be attached to any one of several possible positions of the proamanuline of formula IV (e.g., R 1 -R 9 (Any of the moieties) to provide proamanulin-linker conjugates of formula I, IA, IB, II, IIA, or IIB. In some embodiments, the linker comprises a hydrazine, a disulfide, a thioether, or a dipeptide. In some embodiments, the linker comprises a dipeptide selected from Val-Ala and Val-Cit. In some embodiments, the linker comprises a para-aminobenzyl group (PAB). In some embodiments, the linker comprises the moiety PAB-Cit-Val. In some embodiments, the linker comprises the moiety PAB-Ala-Val. In some embodiments, the linker comprises the moiety -((C=O)(CH 2 ) n - units, where n is an integer from 1 to 6.

[0251] In some embodiments, the linker is -(CH 2 ) n- units, where n is an integer from 2 to 6. In some embodiments, the linker is -PAB-Cit-Val-((C=O)(CH 2 ) n In some embodiments, the linker is -PAB-Ala-Val-((C=O)(CH 2 ) n In some embodiments, the linker L and chemical moiety Z, taken together as LZ, are [ka] It is.

[0252] Antibodies and antigen-binding fragments for use with the compositions and methods described herein can be conjugated to amatoxins, such as α-amanitin or variants thereof, using conjugation techniques known in the art or described herein. For example, antibodies and antigen-binding fragments thereof that recognize and bind to a target antigen (anti-CD45 antibodies) can be conjugated to amatoxins, such as α-amanitin or variants thereof, as described in US 2015 / 0218220, the disclosure of which is incorporated herein by reference as it relates to amatoxins, such as α-amanitin and variants thereof, and covalent linkers that can be used for covalent conjugation.

[0253] Auristatins The anti-CD45 antibodies or antigen-binding fragments thereof described in this application may be conjugated to cytotoxins that are auristatins (U.S. Patent Nos. 5,635,483; 5,780,588). Auristatins are anti-mitotic agents that interfere with microtubule dynamics, GTP hydrolysis, nuclear division and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), as well as anti-cancer (U.S. Patent No. 5,663,149) and anti-fungal (Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). (U.S. Patent Nos. 5,635,483; 5,780,588). The auristatin drug moiety may be attached to the antibody via the N-(amino) terminus or the C-(carboxyl) terminus of the peptidic drug moiety (WO02 / 088172).

[0254] Exemplary auristatin embodiments include the N-terminally linked monomethyl auristatin drug moieties DE and DF, which are disclosed in Senter et al., Proceedings of the American Association for Cancer Research, Volume 45, Abstract Number 623, March 28, 2004, the disclosure of which is incorporated by reference in its entirety into this application.

[0255] An exemplary auristatin embodiment is MMAE, where the wavy line indicates the position of covalent attachment to the linker of the antibody-linker conjugate (-LZ-Ab or -L-Z', as described herein). [ka]

[0256] Another exemplary auristatin embodiment is MMAF, where the wavy line indicates the position of covalent attachment to the linker of an antibody-linker conjugate (-LZ-Ab or -L-Z', as described herein), as disclosed in US 2005 / 0238649. [ka]

[0257] Auristatins can be prepared according to the methods of U.S. Pat. No. 5,635,483; U.S. Pat. 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, GR, et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin Trans. 15:859-863; and Doronina (2003) Nat. Biotechnol. 21(7):778-784.

[0258] Maytansinoids The antibodies and antigen-binding fragments thereof described in this application may be conjugated to a cytotoxin that is a microtubule-binding agent. In some embodiments, the microtubule-binding agent is maytansine, a maytansinoid, or a maytansinoid analog. Maytansinoids are mitotic inhibitors that act by binding to microtubules and inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids (e.g., maytansinol and C-3 maytansinol esters) (U.S. Pat. No. 4,151,042). Synthetic maytansinol, as well as its derivatives and analogs, are disclosed in, for example, U.S. Patents 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 Maytansinoid drug moieties are attractive drug moieties for antibody-drug conjugates because they are: (i) relatively accessible to preparation by fermentation or chemical modification or derivatization of the fermentation products, (ii) amenable to derivatization with functional groups suitable for conjugation to antibodies via non-disulfide linkers, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.

[0259] Examples of suitable maytansinoids include esters of maytansinol, synthetic maytansinol, and maytansinol analogues and derivatives. Any cytotoxin that inhibits microtubule formation and is highly toxic to mammalian cells, such as maytansinoids, maytansinol, and maytansinol analogues and derivatives, is included in this application.

[0260] Examples of suitable maytansinol esters include those with modified aromatic rings, and those with modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patents 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; Nos. 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, each of which is incorporated by reference into this application as relating to maytansinoids and derivatives thereof.

[0261] In some embodiments, the antibody-drug conjugates (ADCs) of the disclosure contain, as a cytotoxic drug, a thiol-containing maytansinoid (DM1) (formerly known as N 2 '-Deacetyl-N 2 DM1 is represented by the following structural formula V: [ka] .

[0262] In another embodiment, the conjugate of the invention comprises a thiol-containing maytansinoid N 2 '-Deacetyl-N 2'(4-methyl-4-mercapto-1-oxopentyl)-maytansine (e.g., DM4) is utilized as a cytotoxic drug. DM4 is represented by the following structural formula VI: [ka] .

[0263] Another maytansinoid that contains a side chain containing a sterically hindered thiol bond is N 2 '-Deacetyl-N- 2 '(4-mercapto-1-oxopentyl)-maytansine (referred to as DM3), which is represented by the following structural formula VII: [ka] .

[0264] The maytansinoids taught in U.S. Patent Nos. 5,208,020 and 7,276,497 may also be used in the conjugates of the present invention, the entire disclosures of which are incorporated by reference in this regard.

[0265] Many positions on maytansinoids can serve as a position for covalent attachment to a linking moiety, and therefore to the antibody or antigen-binding fragment thereof (-LZ-Ab or -L-Z', as described herein). For example, the C-3 position bearing a hydroxyl group, the C-14 position modified with a hydroxymethyl, the C-15 position modified with a hydroxy, and the C-20 position bearing a hydroxy group are all expected to be useful. In some embodiments, the C-3 position serves as a position for covalent attachment to a linker moiety, and in some particular embodiments, the C-3 position of maytansinol serves as a position for covalent attachment to a linking moiety. There are many linking groups known in the art for making antibody-maytansinoid conjugates, including, for example, those disclosed in U.S. Patent Nos. 5,208,020, 6,441,163, and European Patent No. 0425235 B1; Chari et al., Cancer Research 52:127-131 (1992); and US 2005 / 0169933 A1, the disclosures of which are expressly incorporated by reference into this application. Additional linking groups are described and exemplified herein.

[0266] The present invention also includes various isomers and mixtures of maytansinoids and conjugates. Certain compounds and conjugates of the present invention may exist in various stereoisomeric, enantiomeric, and diastereomeric forms. Producing such antibody-maytansinoid conjugates is described in U.S. Patents 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 in its entirety.

[0267] Anthracyclines In other embodiments, the antibodies and antigen-binding fragments thereof described herein may be conjugated to a cytotoxin that is an anthracycline molecule. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Studies have shown that anthracyclines can damage cells by a number of different mechanisms, including: 1) the drug molecule intercalates into the cell's DNA, thereby inhibiting DNA-dependent nucleic acid synthesis; 2) the drug produces free radicals, which then react with cellular macromolecules to cause damage to the cell; or 3) the drug molecule interacts 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; See NR Bachur, "Free Radical Damage" id. at pp.97-102.] Because of their potential for cytotoxicity, anthracyclines are used in the treatment of a number of cancers, such as leukemia, breast cancer, lung cancer, ovarian adenocarcinoma, and sarcoma [see, e.g., PH-Wiernik, in Anthracycline: Current Status And New Developments [See p 11]. Commonly used anthracyclines include doxorubicin, epirubicin, idarubicin, and daunomycin. In some embodiments, the cytotoxin is an anthracycline selected from the group consisting of daunorubicin, doxorubicin, epirubicin, and idarubicin. Representative examples of anthracyclines include, but are not limited to, daunorubicin (Cerubidine; Bedford Laboratories), doxorubicin (Adriamycin; Bedford Laboratories; also known as doxorubicin hydrochloride, hydroxy-daunorubicin, and Rubex), epirubicin (Ellence; Pfizer), and idarubicin (Idamycin; Pfizer).

[0268] Doxorubicin (ADRIAMYCINO), an anthracycline analog, is believed to interact with DNA by intercalation and inhibit the progression of the enzyme topoisomerase II, which 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 reclosing, thereby halting the replication process. Doxorubicin and daunorubicin (DAUNOMYCIN) are the prototypic cytotoxic natural product anthracycline chemotherapy agents (Sessa et al., (2007) Cardiovasc. Toxicol. 7:75-79).

[0269] A non-limiting example of an anthracycline suitable for use in the present application is PNU-159682 ("PNU"). PNU is more than 3000 times more cytotoxic than the parent nemorubicin (Quintieri et al., Clinical Cancer Research 2005, 11, 1608-1617). PNU is represented by the following structural formula: [ka] .

[0270] Several positions on anthracyclines, such as PNU, can serve as positions for covalent attachment to the linking moiety, and therefore to an anti-CD45 antibody or antigen-binding fragment thereof, as described herein. For example, a linker may be introduced via modification to a hydroxymethyl ketone side chain.

[0271] In some embodiments, the cytotoxin is a PNU derivative having the following structural formula: [ka] Here, the wavy line indicates the position of covalent attachment to a linker of an ADC as described in this application.

[0272] In some embodiments, the cytotoxin is a PNU derivative having the following structural formula: [ka] Here, the wavy line indicates the position of covalent attachment to a linker of an ADC as described in this application.

[0273] Pyrrolobenzodiazepines (PBDs) In other embodiments, the anti-CD45 antibodies, or antigen-binding fragments thereof, described herein may be conjugated to a cytotoxin that is a pyrrolobenzodiazepine (PBD) or that includes a PBD. PBDs are natural products produced by certain actinomycetes and have been shown to be sequence-selective DNA alkylating compounds. PBD cytotoxins include, but are not limited to, anthramycin, dimeric PBDs, and those disclosed, for example, in Hartley, JA (2011). "The development of pyrrolobenzodiazepines as antitumour agents." Expert Opin. Inv. Drug, 20(6), 733-744; and Antonow, D.; Thurston, DE (2011) "Synthesis of DNA-interactive pyrrolo[2,1-c][1,4]benzodiazepines (PBDs)." Chem. Rev. 111: 2815-2864.

[0274] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine dimer having the formula: [ka] Here, the wavy line indicates the position of attachment to the linker.

[0275] In some embodiments, the cytotoxin is conjugated to the antibody, or antigen-binding fragment thereof, via a maleimidocaproyl linker.

[0276] In some embodiments, the linker is a peptide, an oligosaccharide, -(CH 2 ) p -, -(CH 2 CH 2 O) q -, -(C=O)(CH 2 ) r -, -(C=O)(CH 2 CH 2 O) t -, -(NHCH 2 CH 2 ) u -, and one or more of: Ala-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, wherein each of p, q, r, t, and u is an integer from 1 to 12 and is independently selected in each occurrence.

[0277] In some embodiments, the linker has a structure of the following formula: [ka] Here, R 1 is CH 3 (Ala) or (CH 2 ) 3 NH(CO)NH 2 (Cit),

[0278] In some embodiments, the linker, prior to conjugation to the antibody, comprises a reactive substituent Z′ and, together as L-Z′, has the following structure: [ka] Here, the wavy line indicates the position of attachment to the cytotoxin (e.g., PBD). 1 CH 3 It is.

[0279] In some embodiments, the cytotoxin-linker conjugate, prior to conjugation to the antibody, comprises a reactive substituent Z', and together, as Cy-L-Z', has the following structural formula: [ka] .

[0280] This particular cytotoxin-linker conjugate is known as Tesilin (SG3249) and is described, for example, in Howard et al., ACS Med. Chem. Lett. 2016, 7(11), 983-987, the disclosure of which is incorporated by reference in its entirety into this application.

[0281] In some embodiments, the cytotoxin is a pyrrolobenzodiazepine dimer having the formula: [ka] Here, the wavy line indicates the position of attachment to the linker.

[0282] In some embodiments, the cytotoxin-linker conjugate, prior to conjugation to the antibody, comprises a reactive substituent Z', and together, as Cy-L-Z', has the following structure: [ka] .

[0283] This particular cytotoxin-linker conjugate is known as talirine and is described, for example, in the context of the ADC vadastuximab talirine (SGN-CD33A) in Mantaj et al., Angewandte Chemie International Edition English 2017, 56, 462-488, the disclosure of which is incorporated by reference in its entirety into this application.

[0284] In some embodiments, the cytotoxin is an indolinobenzodiazepine pseudodimer having the following structural formula: [ka] Here, the wavy line indicates the position of attachment to the linker.

[0285] In some embodiments, the cytotoxin-linker conjugate, prior to conjugation to the antibody, comprises a reactive substituent Z', and together, as Cy-L-Z', has the following structure: [ka] This includes the ADC IMGN632 (disclosed, for example, in International Patent Application Publication No. WO2017004026, which is incorporated by reference into this application).

[0286] Calicheamicin In other embodiments, the antibodies and antigen-binding fragments thereof described herein may be conjugated to cytotoxins that are enediyne antitumor antibiotics (e.g., calicheamicins, ozogamicin). The calicheamicin family of antibiotics are capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates, see U.S. Patent Nos. 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,296 (all to American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, those disclosed in, for example, Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the above-referenced U.S. patents for American Cyanamid.

[0287] An exemplary calicheamicin is gamma 1 These are referred to in this application simply as gamma and have the following structural formula: [ka] .

[0288] In some embodiments, the calicheamicin is a gamma-calicheamicin derivative or an N-acetyl-gamma-calicheamicin derivative. Structural analogs of calicheamicin that may be used include, but are not limited to, those disclosed in Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the U.S. patents cited above. The calicheamicins contain a methyl trisulfide moiety that can react with an appropriate thiol to form a disulfide, which at the same time introduces a functional group useful for coupling the calicheamicin derivative to an anti-CD45 antibody or antigen-binding fragment thereof described herein via a linker. For preparing conjugates of the calicheamicin family, see U.S. Patents 5,712,374; 5,714,586; 5,739,116; 5,767,285; 5,770,701; 5,770,710; 5,773,001; and 5,877,296 (all to American Cyanamid Company). Structural analogs of calicheamicin that may be used include, but are not limited to, those disclosed in Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the above-listed U.S. patents to American Cyanamid.

[0289] In one embodiment, the cytotoxin of the ADC disclosed herein is a calicheamicin disulfide derivative having the following formula: [ka] Here, the wavy line indicates the position of attachment to the linker.

[0290] Additional cytotoxins In other embodiments, the antibodies and antigen-binding fragments thereof described in this application may be conjugated to cytotoxins other than, or in addition to, the cytotoxins disclosed above in this application. Additional cytotoxins suitable for use with the compositions and methods described herein include, but are not limited to, 5-ethynyluracil, abiraterone, acylfulvene, adecypenol, adozelesin, aldesleukin, altretamine, ambamustine, amidox, amifostine, aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole, andrographolide, angiogenesis inhibitors, antarelix, anti-dorsalizing morphogenetic protein-1, protein-1, antiandrogens, prostate cancer, antiestrogens, antineoplastic agents (antineoplastons), antisense oligonucleotides, aphidicolin glycinate, apoptosis gene modulators, apoptosis regulators, apurinic acid, asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2, axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III. derivatives, balanol, batimastat, BCR / ABL antagonists, benzochlorines, benzoylstaurosporine, β-lactam derivatives,beta-alethine, beta-clamycin B, betulinic acid, bFGF inhibitor, bicalutamide, bisantrene, bisaziridinylspermine, bisnafide, bistratene A, bizelesin, breflate, bleomycin A2, bleomycin B2, bropirimine, budotitane, buthionine sulfoximine sulfoximine, calcipotriol, calphostin C, camptothecin derivatives (e.g., 10-hydroxy-camptothecin), capecitabine, carboxamido-amino-triazoles, carboxyamidotriazoles, carzelesin, casein kinase inhibitors, castanospermine, cecropin B, cetrorelix, chlorins, chloroquinoxaline sulfonamides sulfonamide, cicaprost, cis-porphyrin, cladribine, clomiphene and its analogs, clotrimazole, collismycin A, collismycin B, combretastatin A4, combretastatin analogs, conagenin, crambescidin 816, crisnatol, cryptophycin 8, cryptophycin A derivatives, curacin A, cyclopentanthraquinones, cycloplatam, cypemycin,Cytarabine ocfosfate, cytolytic factor, cytostatin, dacliximab, decitabine, dehydrodidemnin B, 2'-deoxycoformycin (DCF), deslorelin, dexifosfamide, dexrazoxane, dexverapamil, diaziquone, didemnin B, didox, diethylnorspermine, dihydro-5-azacytidine, dihydrotaxol, dioxamycin, diphenylspiromustine spiromustine, discodermolide, docosanol, dolasetron, doxifluridine, droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine, edelfosine, edrecolomab, eflornithine, elemene, emitefur, epothilones, epithilones, epristeride, estramustine and its analogs, etoposide, etoposide 4'-phosphate 4'-phosphate) (also called etopophos), exemestane, fadrozole, fazarabine, fenretinide, filgrastim, finasteride, flavopiridol,Flezelastine, fluasterone, fludarabine, fluorodaunorunicin hydrochloride, forfenimex, formestane, fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate, galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, glutathione inhibitors, hepsulfam, homoharringtonine (HHT), hypericin, ibandronic acid acid, idoxifene, idramantone, ilmofosine, ilomastat, imidazoacridone, imiquimod, immune stimulating peptides, iobenguane, iododoxorubicin, ipomeanol, irinotecan, iroplact, irsogladine, isobengazole, jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide, leinamycin, lenograstim, lentinan sulfate sulfate, leptolstatin, letrozole, lipophilic platinum compounds, lissoclinamide 7, lobaplatin, lometrexol, lonidamine, losoxantrone,loxoribine, lurtotecan, lutetium texaphyrin, lysofylline, masoprocol, maspin, matrix metalloproteinase inhibitors, menogaril, merbarone, meterelin, methioninase, metoclopramide, MIF inhibitors, mifepristone, miltefosine, mirimostim im), mithracin, mitoguazone, mitolactol, mitomycin and its analogues, mitonafide, mitoxantrone, mofarotene, molgramostim, mycaperoxide B, myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin (nafarelin), nagrestip, napavin, naphterpin, nartograstim, nedaplatin, nemorubicin, neridronic acid, nilutamide, nisamycin, nitrullyn, octreotide, oxenone, onapristone ristone, ondansetron, oracin, ormaplatin, oxaliplatin, oxaunomycin, paclitaxel and its analogs, palauamine, palmitoylrhizoxin, pamidronic acid, panaxytriol, panomifene,parabactin, pazelliptine, pegaspargase, peldesine, sodium pentosan polysulfate, pentostatin, pentrozole, perflubron, perfosfamide, phenazinomycin, picibanil, pirarubicin, piritrexim, podophyllotoxin, Porfiromycin, purine nucleoside phosphorylase inhibitors, raltitrexed, rhizoxin, rogletimide, rohitukine, rubiginone B1, ruboxyl, safingol, saintopin, sarcophytol A, sargramostim, sobuzoxane, sonermin, sparfosic acid acid, spicamycin D, spiromustine, stipiamide, sulfinosine, tallimustine, tegafur, temozolomide, teniposide, thaliblastine, thiocoraline, tirapazamine, topotecan, topsentin, triciribine, trimetrexate, veramine, vinorelbine, vinxaltine, vorozole, zeniplatin, and zilascorb.

[0291] Linkers The term "linker" as used herein means a bivalent chemical moiety, including a covalent bond or chain of atoms, that is covalently attached to an anti-CD45 antibody-drug conjugate (ADC) of formula I. A suitable linker has two reactive ends, one for conjugation to an antibody and the other for conjugation to a cytotoxin. The reactive end of the linker that is conjugated to an antibody (reactive moiety, Z') is typically a site that can be conjugated to the antibody via a cysteine ​​thiol or lysine amine group on the antibody, and is therefore typically a thiol-reactive group such as a double bond (e.g., maleimide, etc.), or a leaving group such as chloro, bromo, iodo, or an R-sulfanyl group, or a group that is amine-reactive, such as a carboxyl group; whereas the reactive end of the linker that is conjugated to an antibody is typically a site that can be conjugated to a cytotoxin via formation of an amide bond with a basic amine or carboxyl group on the cytotoxin, and is therefore typically a carboxyl or basic amine group. When the term "linker" is used to describe a conjugated form of a linker, one or both of the reactive ends are either absent (e.g., converted to a reactive moiety Z', chemical moiety Z) or unreacted (e.g., only a carbonyl of a carboxylic acid, etc.) as bonds between the linker and / or the cytotoxin, and between the linker and / or the antibody or antigen-binding fragment thereof, are formed. Such conjugation reactions are further described below in this application.

[0292] A variety of linkers can be used to conjugate the antibodies or antibody fragments described herein to cytotoxic molecules. In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In yet other embodiments, the linker unit is not cleavable, and the drug is released, for example, by antibody degradation. Linkers useful in the ADCs of the invention are preferably stable outside the cell, prevent the ADC molecules from aggregating, and keep the ADC soluble in aqueous media and in a monomeric form. Before being transported or delivered into the cell, the ADC is preferably stable and remains intact, i.e., the antibody remains linked to the drug moiety. The linker is stable outside the target cell and may be cleaved at an effective rate inside the cell. An effective linker: (i) maintains the specific binding properties of the antibody; (ii) allows the conjugate or drug moiety to be delivered intracellularly; (iii) remains stable and intact, i.e., is not cleaved, until the conjugate is delivered or transported to its target site; and (iv) maintains the cytotoxic, cytotoxic or cytostatic effect of the cytotoxic moiety. The stability of the ADC can be measured by standard analytical techniques such as mass spectrometry, HPLC, and LC / MS separation / analysis techniques. Covalent attachment of the antibody and the drug moiety requires that the linker has two reactive functional groups (i.e., is bivalent in the reactive sense). Bivalent linker reagents useful for attaching two or more functional or biologically active moieties, such as peptides, nucleic acids, drugs, toxins, antibodies, haptens, and reporter groups, are known, and methods for obtaining these conjugates have been described (Hermanson, GT (1996) Bioconjugate Techniques; Academic Press: New York, p. 234-242).

[0293] Suitable cleavable linkers include, for example, those that can be cleaved by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012, the disclosure of which is incorporated by reference herein as it relates to linkers suitable for covalent conjugation). Suitable cleavable linkers include, for example, chemical moieties such as hydrazines, disulfides, thioethers, or dipeptides.

[0294] Linkers that are hydrolyzable under acidic conditions include, for example, hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, and the like (see, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661, the disclosures of which are incorporated herein by reference in their entirety for their disclosures regarding linkers suitable for covalent conjugation). Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but are unstable below pH 5.5 or 5.0, which is the approximate pH of the lysosome.

[0295] Linkers that can be cleaved under reducing conditions include, for example, disulfides. A variety of disulfide linkers are known in the art, including disulfide linkers that can be formed using, for example, SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene), SPDB and SMPT (see, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel ed., Oxford U. Press, 1987. See also U.S. Patent No. 4,880,935, the disclosures of each of which are incorporated by reference in their entirety into this application for their disclosures regarding linkers suitable for covalent conjugation.

[0296] Linkers susceptible to enzymatic hydrolysis may be, for example, peptide-containing linkers that are cleaved by intracellular peptidase or protease enzymes, including, but not limited to, lysosomal or endosomal proteases. One advantage of using intracellular proteolysis to release a therapeutic agent is that the therapeutic agent is usually attenuated when conjugated, and the serum stability of the conjugate is usually high. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Exemplary amino acid linkers include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Examples of suitable peptides include peptides containing amino acids such as valine, alanine, citrulline (Cit), phenylalanine, lysine, leucine, and glycine. Amino acid residues that comprise amino acid linker components include naturally occurring as well as minor amino acids and non-naturally occurring amino acid analogs (e.g., 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 including dipeptides such as Val-Cit or Phe-Lys are disclosed, for example, in U.S. Pat. No. 6,214,345 (the disclosure of which is incorporated by reference 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.

[0297] Linkers suitable for conjugating antibodies or antibody fragments to cytotoxic molecules described herein include linkers capable of releasing cytotoxins by a 1,6-elimination process. Chemical moieties that enable this process include p-aminobenzyl (PAB) groups, 6-maleimidohexanoic acid, pH-sensitive carbonates, and other reagents described in Jain et al., Pharm. Res. 32:3526-3540, 2015 (the disclosure of which is incorporated herein by reference in its entirety as it relates to linkers suitable for covalent conjugation).

[0298] In some embodiments, the linker includes a "self-immolative" group such as PAB or PABC (para-aminobenzyloxycarbonyl) as described above, e.g., in Carl et al., J. Med. Chem. (1981) 24:479-480; Chakravarty et al (1983) J. Med. Chem. 26:638-644; US 6214345; US20030130189; US20030096743; US6759509; US20040052793; US6218519; US6835807; US6268488; US20040018194; WO98 / 13059; US20040052793; US6677435; US5621002; US20040121940; WO2004 / 032828. Other such chemical moieties ("self-immolative linkers") that enable this process include methylene carbamates and heteroaryl groups, such as aminothiazoles, aminoimidazoles, aminopyrimidines, and the like. Linkers containing such heterocyclic self-immolative groups are disclosed, for example, in U.S. Patent Application Publications 20160303254 and 20150079114, as well as U.S. Patent No. 7,754,681; Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237; US 2005 / 0256030; de Groot et al (2001) J. Org. Chem. 66:8815-8830; and US 7,223837. In some embodiments, a dipeptide is used in conjunction with a self-immolative linker.

[0299] Suitable linkers for use in this application include C 1 -C 6 Alkylene, C 1 -C 6 Heteroalkylene, C 2 -C 6 Alkenylene, C 2 -C 6 Heteroalkenylene, C 2 -C 6 Alkynylene, C 2 -C 6Heteroalkynylene, C 3 -C 6 It may further include one or more groups selected from cycloalkylene, heterocycloalkylene, arylene, heteroarylene, and combinations thereof, each of which may be optionally substituted. Non-limiting examples of such groups include (CH 2 ) p , (CH 2 CH 2 O) p , and -(C=O)(CH 2 ) p - units, where p is an integer from 1 to 6, independently selected for each occurrence.

[0300] Suitable linkers may include groups that have solubility enhancing properties, such as (CH 2 CH 2 O) p Linkers containing units (polyethylene glycol, PEG) can enhance solubility (e.g., alkyl chains can be substituted with amino, sulfonic acid, phosphonic acid, or phosphate residues to enhance solubility). Linkers containing such moieties are disclosed, for example, in U.S. Patent Nos. 8,236,319 and 9,504,756, the disclosures of each of which are incorporated by reference in their entirety into this application as they relate to linkers suitable for covalent conjugation. Further solubility enhancing groups include, for example, acyl and carbamoyl sulfamide groups having the following structures: [ka] where a is 0 or 1; and R 10 is hydrogen, C 1 -C 24 Alkyl group, C 3 -C 24 Cycloalkyl groups, C 1 -C 24 (Hetero)aryl groups, C 1 -C 24Alkyl(hetero)aryl groups, and C 1 -C 24 (Hetero)arylalkyl groups, C 1 -C 24 Alkyl group, C 3 -C 24 Cycloalkyl groups, C 2 -C 24 (Hetero)aryl groups, C 3 -C 24 Alkyl(hetero)aryl groups, and C 3 -C 24 (hetero)arylalkyl groups, each of which is selected from the group consisting of O, S, and NR 11 R 12 wherein R 11 and R 12 is hydrogen and C 1 -C 4 or R 10 is a cytotoxin, where the cytotoxin is optionally attached to N via a spacer moiety. Linkers containing such groups are described, for example, in U.S. Pat. No. 9,636,421 and U.S. Patent Application Publication No. 2017 / 0298145, the disclosures of which are incorporated by reference in their entireties into this application as they relate to linkers suitable for covalent conjugation to cytotoxins and antibodies or antigen-binding fragments thereof.

[0301] In some embodiments, the linker is a hydrazine, a disulfide, a thioether, a dipeptide, a p-aminobenzyl (PAB) group, a heterocyclic self-immolative group, an optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 Heteroalkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 Heteroalkenyl, optionally substituted C2 -C 6 Alkynyl, optionally substituted C 2 -C 6 Heteroalkynyl, optionally substituted C 3 -C 6 Cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, a solubility enhancing group, acyl, -(C=O)-, or -(CH 2 CH 2 O) p - groups, where p is an integer from 1 to 6. Some of skill in the art will appreciate that one or more of the listed groups may be a divalent (diradical) species (e.g., C 1 -C 6 It is recognized that the alkylene may exist in the form of alkylene.

[0302] In some embodiments, the linker L is a moiety *-L 1 L 2 -**Contains, where: L 1 does not exist or -(CH 2 ) m NR 13 C(=O)-, -(CH 2 ) m NR 13 -, -(CH 2 ) m X 3 (CH 2 ) m -, [ka] is; L 2 does not exist or -(CH 2 ) m -, -NR 13 (CH 2 ) m -, -(CH 2 ) m NR 13 C(=O)(CH 2 ) m -, -X4 , -(CH 2 ) m NR 13 C(=O)X 4 , - (CH 2 ) m NR 13 C(=O)-, -((CH 2 ) m O) n (CH 2 ) m -, -((CH 2 ) m O) n (CH 2 ) m X 3 (CH 2 ) m -, - NR 13 ((CH 2 ) m O) n X 3 (CH 2 ) m -, -NR 13 ((CH 2 ) m O) n (CH 2 ) m X 3 (CH 2 ) m -, -X 1 X 2 C(=O)(CH 2 ) m -, - (CH 2 ) m (O(CH 2 ) m ) n -, -(CH 2 ) m NR 13 (CH 2 ) m -, -(CH 2 ) m NR 13 C(=O)(CH 2 ) m X 3 (CH 2 ) m -, - (CH 2 ) m C(=O)NR 13 (CH2 ) m NR 13 C(=O)(CH 2 ) m -, -(CH 2 ) m C(=O)-, - (CH 2 ) m NR 13 (CH 2 ) m C(=O)X 2 X 1 C(=O)-, -(CH 2 ) m X 3 (CH 2 ) m C(=O)X 2 X 1 C(=O)-, - (CH 2 ) m C(=O)NR 13 (CH 2 ) m -, -(CH 2 ) m C(=O)NR 13 (CH 2 ) m X 3 (CH 2 ) m -, -(CH 2 ) m X 3 (CH 2 ) m NR 13 C(=O)(CH 2 ) m -, -(CH 2 ) m X 3 (CH 2 ) m C(=O)NR 13 (CH 2 ) m -, - (CH 2 ) m O) n (CH 2 ) m NR 13 C(=O)(CH 2 ) m -, -(CH 2 ) m C(=O)NR 13(CH 2 ) m (O(CH 2 ) m ) n -, - (CH 2 ) m (O(CH 2 ) m ) n C(=O)-, -(CH 2 ) m NR 13 (CH 2 ) m C(=O)-, -(CH 2 ) m C(=O)NR 13 (CH 2 ) m NR 13 C(=O)-, -(CH 2 ) m (O(CH 2 ) m ) n X 3 (CH 2 ) m -, - (CH 2 ) m X 3 ((CH 2 ) m O) n (CH 2 ) m -, -(CH 2 ) m X 3 (CH 2 ) m C(=O)-, - (CH 2 ) m C(=O)NR 13 (CH 2 ) m O) n (CH 2 ) m X 3 (CH 2 ) m -, - (CH 2 ) m X 3 (CH 2 ) m (O(CH 2 ) m ) n NR 13C(=O)(CH 2 ) m -, -(CH 2 ) m X 3 (CH 2 ) m (O(CH 2 ) m ) n C(=O)-, -(CH 2 ) m X 3 (CH 2 ) m (O(CH 2 ) m ) n -, -(CH 2 ) m C(=O)NR 13 (CH 2 ) m C(=O)-, -(CH 2 ) m C(=O)NR 13 (CH 2 ) m (O(CH 2 ) m ) n C(=O)-, -((CH 2 ) m O) n (CH 2 ) m NR 13 C(=O)(CH 2 ) m -, -(CH 2 ) m C(=O)NR 13 (CH 2 ) m C(=O)NR 13 (CH 2 ) m -, -(CH 2 ) m NR 13 C(=O)(CH 2 ) m NR 13 C(=O)(CH 2 ) -(CH 2 ) m X 3 (CH 2 ) m C(=O)NR 13 -, -(CH2 ) m C(=O)NR 13 -, -(CH 2 ) m X 3 -, -C(R 13 ) 2 (CH 2 ) m -, -(CH 2 ) m C(R 13 ) 2 NR 13 -, -(CH 2 ) m C(=O)NR 13 (CH 2 ) m NR 13 -, - (CH 2 ) m C(=O)NR 13 (CH 2 ) m NR 13 C(=O)NR 13 -, -(CH 2 ) m C(=O)X 2 X 1 C(=O)-, - C(R 13 ) 2 (CH 2 ) m NR 13 C(=O)(CH 2 ) m -, -(CH 2 ) m C(=O)NR 13 (CH 2 ) m C(R 13 ) 2 NR 13 -, - C(R 13 ) 2 (CH 2 ) m X 3 (CH 2 ) m -, -(CH 2 ) m X 3 (CH 2 ) m C(R 13 ) 2 NR 13-, -C(R 13 ) 2 (CH 2 ) m OC(=O)NR 13 (CH 2 ) m -, -(CH 2 ) m NR 13 C(=O)O(CH 2 ) m C(R 13 ) 2 NR 13 -, -(CH 2 ) m X 3 (CH 2 ) m NR 13 -, -(CH 2 ) m X 3 (CH 2 ) m (O(CH 2 ) m ) n NR 13 -, -(CH 2 ) m NR 13 -, -(CH 2 ) m C(=O)NR 13 (CH 2 ) m (O(CH 2 ) m ) n NR 13 -, -(CH 2 ) m (O(CH 2 ) m ) n NR 13 -, -(CH 2 CH 2 O) n (CH 2 ) m -, -(CH 2 ) m (OCH 2 CH 2 ) n; -(CH 2 ) m O(CH 2 ) m -, -(CH2 ) m S(=O) 2 -, - (CH 2 ) m C(=O)NR 13 (CH 2 ) m S(=O) 2 -, -(CH 2 ) m X 3 (CH 2 ) m S(=O) 2 -, -(CH 2 ) m X 2 X 1 C(=O)-, -(CH 2 ) m (O(CH 2 ) m ) n C(=O)X 2 X 1 C(=O)-, -(CH 2 ) m (O(CH 2 ) m ) n X 2 X 1 C(=O)-, - (CH 2 ) m X 3 (CH 2 ) m X 2 X 1 C(=O)-, -(CH 2 ) m X 3 (CH 2 ) m (O(CH 2 ) m ) n X 2 X 1 C(=O)-, - (CH 2 ) m X 3 (CH 2 ) m C(=O)NR 13 (CH 2 ) m NR 13 C(=O)-, -(CH 2 ) m X3 (CH 2 ) m C(=O)NR 13 (CH 2 ) m C(=O)-, -(CH 2 ) m X 3 (CH 2 ) m C(=O)NR 13 (CH 2 ) m (O(CH 2 ) m ) n C(=O)-, -(CH 2 ) m C(=O)X 2 X 1 C(=O)NR 13 (CH 2 ) m -, -(CH 2 ) m X 3 (O(CH 2 ) m ) n C(=O)-, -(CH 2 ) m NR 13 C(=O)((CH 2 ) m O) n (CH 2 ) m -, -(CH 2 ) m (O(CH 2 ) m ) n C(=O)NR 13 (CH 2 ) m -, -(CH 2 ) m NR 13 C(=O)NR 13 (CH 2 ) m - or -(CH 2 ) m X 3 (CH 2 ) m NR 13 is C(=O)-; Herein, X 1teeth, [ka] is; X 2 teeth, [ka] is; X 3 teeth, [ka] is; and X 4 teeth, [ka] is; where R 13 H and C 1 -C 6 independently selected in each occurrence from alkyl; m is independently selected in each occurrence from 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; n is independently selected in each occurrence from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14; and, where one asterisk (*) indicates the position of attachment to a cytotoxin (e.g., an amatoxin) and two asterisks (**) indicate the position of attachment to a reactive substituent Z' or chemical substructure Z (wherein L 1 and L 2 (Provided that it is not the case that both do not exist.)

[0303] 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-aminobenzyloxycarbonyl unit. In some embodiments, the p-aminobenzyl group is part of a p-aminobenzylamide unit.

[0304] 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.

[0305] In some embodiments, the linker is a peptide, an oligosaccharide, -(CH 2 ) p -, -(CH 2 CH 2 O) 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 in combination.

[0306] In some embodiments, the linker is -(C=O)(CH 2 ) p - units, where p is an integer from 1 to 6.

[0307] In a specific embodiment, the linker comprises the following structure: [ka] Here, the wavy lines indicate the positions of attachment of the cytotoxin and the reactive moiety Z'.

[0308] In another specific embodiment, the linker comprises the following structure: [ka] Here, the wavy line indicates the position of attachment to the cytotoxin and the reactive moiety Z'. Such PAB-dipeptide-propionyl linkers are disclosed, for example, in WO 2017 / 149077, which is incorporated herein by reference. Additionally, the cytotoxins disclosed in WO 2017 / 149077 are incorporated herein by reference.

[0309] In certain embodiments, the linker of the ADC is maleimidocaproyl-Val-Ala-para-aminobenzyl (mc-Val-Ala-PAB).

[0310] In certain embodiments, the linker of the ADC is maleimidocaproyl-Val-Cit-para-aminobenzyl (mc-vc-PAB).

[0311] In some embodiments, the linker is [ka] Includes.

[0312] In some embodiments, the linker comprises MCC (4-[N-maleimidomethyl]cyclohexane-1-carboxylate).

[0313] It will be appreciated by one of skill in the art that any one or more of the chemical groups, moieties, and features disclosed herein may be combined in a variety of ways to form linkers useful for conjugating antibodies and cytotoxins as disclosed herein. Additional linkers useful in conjunction with the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220, the disclosure of which is incorporated herein by reference in its entirety.

[0314] In certain embodiments, an intermediate that is a precursor of 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 may be first reacted with the antibody or derivatized antibody and then reacted with the drug or derivatized drug. Such conjugation reactions are now more fully described.

[0315] Many different reactions can be used to covalently attach linkers or drug-linker conjugates to the antibody or antigen-binding fragment thereof. Suitable attachment locations on the antibody molecule include the amine groups of lysines, the free carboxylic acid groups of glutamic and aspartic acids, the sulfhydryl groups of cysteine, and various moieties of aromatic amino acids. For example, non-specific covalent attachment may 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 agents such as dialdehydes or imidoesters may be used to couple amino groups on a compound to amino groups on the antibody moiety. Drugs may also be attached to the binding agent using a Schiff base reaction. This method involves periodate oxidation of a drug containing a glycol or hydroxyl group, thus forming an aldehyde that is then reacted with the binding agent. Attachment occurs via the formation of a Schiff base with the amino group of the binding agent. Isothiocyanates may also be used as coupling agents to covalently attach drugs to binding agents. Other techniques are known to those skilled in the art and are within the scope of the present invention.

[0316] As described in this application, linkers useful for conjugation to an antibody or antigen-binding fragment include, but are not limited to, linkers comprising a chemical moiety Z formed by a coupling reaction, as shown below in Table 1. The curved lines indicate the positions of attachment to the antibody or antigen-binding fragment, and the cytotoxic molecule, respectively.

[0317] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0318] One of skill in the art will recognize that the reactive substituent Z' attached to the linker and the reactive substituent on the antibody or antigen-binding fragment thereof are capable of participating in a covalent coupling reaction to generate chemical moiety Z, and will recognize the reactive moiety Z'. Thus, antibody-drug conjugates useful in conjunction with the methods described herein may be formed by reacting an antibody, or antigen-binding fragment thereof, with a linker or cytotoxin-linker conjugate that includes a reactive substituent Z', etc., suitable for reaction with a reactive substituent on an antibody, or antigen-binding fragment thereof, to form chemical moiety Z, as described herein.

[0319] In some embodiments, Z′ is —NR 13 C(=O)CH=CH 2 , -N 3 , -SH, -S(=O) 2 (CH=CH 2 ), -(CH 2 ) 2 S(=O)2 (CH=CH 2 ), -NR 13 S(=O) 2 (CH=CH 2 ), -NR 13 C(=O)CH 2 R 14 , -NR 13 C(=O)CH 2 Br, -NR 13 C(=O)CH 2 I, -NHC(=O)CH 2 Br, -NHC(=O)CH 2 I, -ONH 2 , -C(O)NHNH 2 , -CO 2 H, -NH 2 , -NH(C=O), -NC(=S), [ka] [ka] is; Where: R 13 H and C 1 -C 6 independently selected in each occurrence from alkyl; R 14 is -S(CH 2 ) n CHR 15 NHC(=O)R 13 is; R 15 is R 13 Or -C(=O)OR 13 is; R 16 , H, C 1 -C 6 independently selected at each occurrence from alkyl, F, Cl, and OH; R 17 , H, C 1 -C 6 Alkyl, F, Cl, -NH 2 , -OCH3 , -OCH 2 CH 3 , -N(CH 3 ) 2 , -CN, -NO 2 and, independently in each occurrence, selected from -OH; and R 18 , H, C 1 -C 6 Alkyl, F, benzyloxy substituted with -C(=O)OH, benzyl substituted with -C(=O)OH, C substituted with -C(=O)OH 1 -C 4 Alkoxy groups and C substituted with -C(=O)OH 1 -C 4 is independently selected in each occurrence from alkyl groups.

[0320] As shown in Table 31, examples of suitable reactive substituents on the linker and antibody, or antigen-binding fragment thereof, include nucleophilic / electrophilic pairs (e.g., thiol / haloalkyl pairs, amine / carbonyl pairs, or thiol / α,β-unsaturated carbonyl pairs, etc.), diene / dienophile pairs (e.g., azide / alkyne pairs, or diene / α,β-unsaturated carbonyl pairs, among others). Coupling reactions between reactive substituents to form chemical moieties Z include, but are not limited to, thiol alkylation, hydroxyl alkylation, amine alkylation, amine or hydroxylamine condensation, hydrazine formation, amidation, esterification, disulfide formation, cycloaddition (e.g., [4+2] Diels-Alder cycloaddition, [3+2] Huisgen cycloaddition, among others), aromatic nucleophilic substitution, aromatic electrophilic substitution, and other reaction modes known in the art or described in this application. Preferably, the linker comprises an electrophilic functional group for reaction with a nucleophilic functional group on the antibody, or antigen-binding fragment thereof.

[0321] As disclosed herein, reactive substituents that may be present in an antibody, or antigen-binding fragment thereof, include nucleophilic groups such as, but are not limited to, (i) an N-terminal amine group, (ii) a side chain amine group (e.g., lysine), (iii) a side chain thiol group (e.g., cysteine), and (iv) a sugar hydroxyl or amino group (where the antibody is glycosylated). As disclosed herein, reactive substituents that may be present in an antibody, or antigen-binding fragment thereof, 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), haloheteroaryl (e.g., fluoroheteroaryl), haloalkyl, and haloheteroalkyl moieties of non-natural amino acids. In some embodiments, as disclosed herein, reactive substituents present in an antibody, or antigen-binding fragment thereof, include an amine or thiol moiety. Certain antibodies have reducible interchain disulfides (i.e., cysteine ​​bridges). Antibodies can be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine ​​bridge thus theoretically forms two reactive thiol nucleophiles. Additional nucleophiles can be introduced into antibodies by reaction of lysines with 2-iminothiolane (Traut's reagent) to convert amines to thiols. Reactive thiol groups may be introduced into antibodies (or fragments thereof) by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing a mutant antibody containing one or more non-existent cysteine ​​amino acid residues). U.S. Patent No. 7,521,541 teaches engineered antibodies by introducing reactive cysteine ​​amino acids.

[0322] In some embodiments, the reactive moiety Z' attached to the linker is a nucleophilic group that is reactive with an electrophilic group present on an antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of the nucleophilic group may react with an electrophilic group on an antibody to form a covalent bond with the antibody. Useful nucleophilic groups include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and aryl hydrazide.

[0323] In some embodiments, Z is the product of a reaction between reactive nucleophilic substituents (e.g., amine and thiol moieties) present in an antibody, or antigen-binding fragment thereof, and a reactive electrophilic substituent Z'. For example, Z' can be a Michael acceptor (e.g., maleimide), an activated ester, an electron-deficient carbonyl compound, and an aldehyde, among others.

[0324] For example, suitable linkers for the synthesis of ADCs include, but are not limited to, reactive substituents Z', such as maleimide or haloalkyl groups, which may be attached to the linker by reagents such as succinimidyl 4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC), N-succinimidyl iodoacetate (SIA), sulfo-SMCC, m-maleimidobenzoyl-N-hydroxysuccinimidyl ester (MBS), sulfo-MBS, and succinimidyl iodoacetate, among others, as described, for example, in Liu et al., 18:690-697, 1979, the disclosure of which is incorporated herein by reference as it relates to linkers for chemical conjugation.

[0325] 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, which is particularly useful for conjugating microtubule disrupting agents such as the auristatins. Such linkers are described in Doronina et al., Bioconjugate Chem. 17:14-24, 2006 (the disclosure of which is incorporated by reference into this application as it relates to linkers for chemical conjugation).

[0326] In some embodiments, the reactive substituent Z' is -(C=O)- or -NH(C=O)-, such that the linker may be attached to an antibody, or antigen-binding fragment thereof, through an amide or urea moiety resulting from reaction of the -(C=O)- or -NH(C=O)- group, respectively, with an amino group of the antibody, or antigen-binding fragment thereof.

[0327] In some embodiments, the reactive substituent is an N-maleimidyl group, a halogenated N-alkylamide group, a sulfonyloxy N-alkylamide group, a carbonate group, a halogenated sulfonyl group, a thiol group or derivatives thereof, an alkynyl group containing an internal carbon-carbon 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 tetrazinyl group, an azide group, a phosphine group, a nitrile oxide group, a nitrone group, a nitrile imine group, a diazo group, a ketone group, an (O-alkyl)hydroxylamino group, a hydrazine group, a halogenated N-maleimidyl group, a 1,1-bis(sulfonylmethyl)methylcarbonyl group or elimination derivatives thereof, a carbonyl halide group, or an allenamide group, each of which may be optionally substituted. In some embodiments, the reactive substituent comprises a cycloalkene group, a cycloalkyne group, or an optionally substituted (hetero)cycloalkynyl group.

[0328] Non-limiting examples of amatoxin-linker conjugates containing a reactive substituent Z' suitable for reaction with a reactive residue on the antibody or antigen-binding fragment thereof include, but are not limited to, 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin; 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin; 7'C-(4-(6-(maleimido)hexanoyl)piperazin-1-yl)-amatoxin; 7'C-(4-(4-((maleimido)methyl)cyclohexanecarbonyl)piperazin-1-yl)-amatoxine;7'C-(4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)-amatoxine;7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine ;7'C-(4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(3-carboxypropanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)-amatoxine C-(4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(4-(2-(maleimido)acetyl)piperazin-1-yl)-amatoxine;7'C-(4-(3-(maleimido)propanoyl)piperazin-1-yl)-amatoxine;7'C-(4-(4-(maleimido)butanoyl)piperazin-1-yl)-amatoxine;7'C-(4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)-amatoxine;7'C-(3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxine;7'C-(3-((6-(6-(maleimido)hexanamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxine;7'C-(3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)pyrrolidin-1-yl)- Amatoxin;7'C-(3-((6-((4-(maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-amatoxin;7'C-(4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)-amatoxin;7'C-(4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)-amatoxin;7'C-(4-(4-(2-(aminooxy)acetamido)butanoyl)piperidine (R)-7'C-((4-(6-(2-(aminooxy)acetamido)hexanoyl)piperazin-1-yl)-amatoxin;7'C-((4-(6-(maleimido)hexanamido)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(6-(maleimido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;(R)-7'C-((3-( (6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;(S)-7'C-((3-((6-(maleimido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperidin-1-yl)methyl)-amatoxin;7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)piperazin-1-yl)methyl)-amatoxine 7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperazin-1-yl)methyl)-amatoxine;7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((3-((6-(6-(maleimido)hexanamido)hexanamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C- ((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-S-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)-R-methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(3-carboxypropanamido)ethyl)piperidine 7'C-((4-(6-(6-(maleimido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(maleimido)acetyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(3-(maleimido)propanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(4-(maleimido)butanoyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(2-(maleimido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(4-(maleimido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C- ((3-((6-(maleimido)hexanamido)methyl)azetidin-1-yl)methyl)-amatoxine;7'C-((3-(2-(6-(maleimido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxine;7'C-((3-((4-((maleimido)methyl)cyclohexanecarboxamido)methyl)azetidin-1-yl)methyl)-amatoxine;7'C-((3-(2-(4-((maleimido)methyl)cyclohexanecarboxamido)ethyl)azetidin-1-yl)methyl)-amatoxine;7' C-((3-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)azetidin-1-yl)methyl)-amatoxine;7'C-(((2-(6-(maleimido)-N-methylhexanamido)ethyl)(methyl)amino)methyl)-amatoxine;7'C-(((4-(6-(maleimido)-N-methylhexanamido)butyl(methyl)amino)methyl)-amatoxine;7'C-((2-(2-(6-(maleimido)hexanamido)ethyl)aziridin-1-yl)methyl) -Amatoxin;7'C-((2-(2-(6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)ethyl)aziridin-1-yl)methyl)-amatoxin;7'C-((4-(6-(6-(2-(aminooxy)acetamido)hexanamido)hexanoyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(1-(aminooxy)-2-oxo-6,9,12,15-tetraoxa-3-azaheptadecan-17-oyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(2-(2-(aminooxy)acetamido)acetyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(3-(2-(aminooxy)acetamido)propanoyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(4-(2-(aminooxy)acetamido)butanoyl)piperazin-1-yl)methyl)-amatoxin;7'C-((4-(2-(6-(2-(aminooxy)acetamido)hexanamido)ethyl)piperidin-1-yl)methyl)-amatoxin ;7'C-((4-(2-(2-(2-(aminooxy)acetamido)acetamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(4-(2-(aminooxy)acetamido)butanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(20-(aminooxy)-4,19-dioxo-6,9,12,15-tetraoxa-3,18-diazaicosyl)piperidin-1-yl)methyl)-amatoxine;7'C-(((2-(6-(2-(aminooxy)amatoxine 7'C-(((4-(6-(2-(aminooxy)acetamido)-N-methylhexanamido)butyl)(methyl)amino)methyl)-amatoxin;7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-S-methyl)-amatoxin;7'C-((3-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexanamido)methyl)pyrrolidin-1-yl)-S-methyl)-amatoxin 7'C-((4-(2-(2-bromoacetamido)ethyl)piperazin-1-yl)methyl)-amatoxine;7'C-((4-(2-(2-bromoacetamido)ethyl)piperidin-1-yl)methyl)-amatoxine;7'C-((4-(2-(3-(pyridin-2-yldisulfanyl)propanamido)ethyl)piperidin-1-yl)methyl)-amatoxine;6'O-(6-(6-(maleimido)hexanamido)hexyl)-amatoxine;6'O-(5-(4-((maleimido)methyl)cyclohexanecarboxamido)pentyl)-amatoxine;6'O-(2-((6-(maleimido)hexyl)oxy)-2-oxoethyl)-amatoxine;6'O-((6-(maleimido)hexyl)carbamoyl)-amatoxine;6'O-((6-(4-((maleimido)methyl)cyclohexanecarboxamido)hexyl)carbamoyl)-amatoxine;6'O-(6-(2-bromoacetamido)hexyl)-amatoxine;7'C-(4-(6-(azido)hexanamido)piperidin-1-yl)-amatoxine;7'C-(4-(hex-5-ynoylamino)piperidin-1-yl)-amatoxine toxin;7'C-(4-(2-(6-(maleimido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;7'C-(4-(2-(6-(6-(maleimido)hexanamido)hexanamido)ethyl)piperazin-1-yl)-amatoxin;6'O-(6-(6-(11,12-didehydro-5,6-dihydro-dibenz[b,f]azocin-5-yl)-6-oxohexanamido)hexyl)-amatoxin;6'O-(6-(hex-5-ynoylamino)hexyl)-amatoxin;6'O-(6-(2-(aminooxy)acetylamido)hexyl)-amatoxin;6'O-((6-aminooxy)hexyl)-amatoxin;and 6'O-(6-(2-iodoacetamido)hexyl)-amatoxin;

[0329] In some embodiments, the chemical substructure Z is selected from Table 1. In some embodiments, the chemical substructure Z is [ka] Here, S is a sulfur atom representing a reactive substituent present in an antibody, or antigen-binding fragment thereof, that binds to CD45 (e.g., resulting from the -SH group of a cysteine ​​residue).

[0330] In some embodiments, the structure L-Z′ of the linker-reactive substituent prior to conjugation to an antibody or antigen-binding fragment thereof is: [ka] .

[0331] In some embodiments, the amatoxins disclosed in the present application are conjugated to a linker-reactive moiety-L-Z′ having the formula: [ka] .

[0332] In some embodiments, the amatoxins disclosed in the present application are conjugated to a linker-reactive moiety-L-Z′ having the formula: [ka] .

[0333] The above-described linker moieties and amatoxin-linker conjugates that are particularly useful in conjunction with the compositions and methods described herein are described, for example, in U.S. Patent Application Publication No. 2015 / 0218220 and Patent Application Publication No. WO2017 / 149077, the disclosures of each of which are incorporated herein by reference in their entireties.

[0334] Preparation of antibody-drug conjugates In the ADCs of formula I disclosed herein, an anti-CD45 antibody, or antigen-binding fragment thereof, is conjugated to one or more cytotoxic drug moieties (D) (e.g., about 1 to about 20 drug moieties per antibody) via a linker L and a chemical moiety Z as disclosed herein. The ADCs of the present disclosure can be prepared by several routes using organic chemistry reactions, conditions, and reagents known to those of skill in the art, including: (1) reacting a reactive substituent of an antibody, or antigen-binding fragment thereof, with a bivalent linker reagent to form Ab-ZL, as described above in the present application, followed by reaction with a drug moiety D; or (2) reacting a reactive substituent of a drug moiety with a bivalent linker reagent to form DL-Z', followed by reaction with a reactive substituent of an antibody or antigen-binding fragment thereof, as described above in the present application. Further methods for preparing ADCs are described herein.

[0335] In another embodiment, 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 sulfhydryl groups. The ADC is then formed by conjugating through the sulfhydryl group's sulfur atom as described above in this application. Reagents that can be used to modify lysines include, but are not limited to, N-succinimidyl S-acetylthioacetate (SATA) and 2-iminothiolane hydrochloride (Traut's reagent).

[0336] In another embodiment, the anti-CD45 antibody, or antigen-binding fragment thereof, may have one or more carbohydrate groups that may be chemically modified to have one or more sulfhydryl groups, and the ADC is then formed by conjugation through the sulfur atom of the sulfhydryl group, as described above in this application.

[0337] In yet another embodiment, the anti-CD45 antibody may have one or more carbohydrate groups that may be oxidized to provide aldehyde (-CHO) groups (see, e.g., Laguzza, et al., J. Med. Chem. 1989, 32(3), 548-55). The ADC is then formed by conjugation via the corresponding aldehyde as described above in this application. Other protocols for modifying proteins to attach or associate with cytotoxins are described in Coligan et al., Current Protocols in Protein Science, vol. 2, John Wiley & Sons (2002), which is incorporated herein by reference.

[0338] Methods for conjugating linker-drug moieties to cell-targeting proteins, such as antibodies, immunoglobulins or fragments thereof, are described, for example, in U.S. Pat. No. 5,208,020; U.S. Pat. No. 6,441,163; WO2005037992; WO2005081711; and WO2006 / 034488, all of which are expressly incorporated by reference in their entirety into this application.

[0339] Alternatively, a fusion protein comprising the antibody and cytotoxic drug may be produced, for example, by recombinant techniques or peptide synthesis. The length of DNA may contain regions encoding the two portions of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0340] The ADCs described herein may be administered to a patient (e.g., a human patient suffering from an autoimmune disease or cancer) in a variety of dosage forms. For example, the ADCs described herein may be administered to a patient suffering from an autoimmune disease or cancer in the form of an aqueous solution, such as an aqueous solution containing one or more pharma- ceutically acceptable excipients. Suitable pharma-ceutically acceptable excipients for use with the compositions and methods described herein include viscosity modifiers. The aqueous solution may be sterilized using techniques known in the art.

[0341] Pharmaceutical formulations comprising the anti-CD45 ADCs as described herein are prepared by mixing such ADCs with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic 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 compounds such as phenylalanine, ... 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 sugars, such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, 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).

[0342] Treatment method CD45 is an important cell surface molecule that is widely expressed throughout the hematopoietic and immune systems. Described in this application are anti-CD45 antibodies and anti-CD45 ADCs that can be used to treat patients with leukemia and lymphoma, as well as autoimmune diseases such as multiple sclerosis and scleroderma. Furthermore, there is currently a need for compositions and methods to promote the engraftment of xenogeneic hematopoietic stem cell grafts such that the multi-potency and hematopoietic functionality of these cells is preserved after transplantation. The compositions disclosed in this application further provide a solution to this difficult problem.

[0343] Thus, methods of treating various disorders (e.g., diseases of cell types in the hematopoietic lineage, cancer, autoimmune diseases, metabolic disorders, stem cell disorders, etc., among others) are further disclosed in this application. The compositions and methods described in this application can (i) directly reduce the population of cells causing the pathology, such as the population of cancer cells (e.g., leukemia cells) and autoimmune cells (e.g., autoreactive T-cells), and / or (ii) reduce the population of endogenous hematopoietic stem cells, thereby promoting the engraftment of transplanted hematopoietic stem cells by providing a niche to which transplanted cells can home. The above activities are obtained by administering ADCs, antibodies, or antigen-binding fragments thereof, capable of binding to antigens expressed by endogenous disease-causing cells or hematopoietic stem cells. In the case of directly treating a disease, this administration can reduce the amount of cells causing the pathology of interest. In the case of preparing a patient for hematopoietic stem cell transplantation therapy, this administration selectively reduces the population of endogenous hematopoietic stem cells, thereby creating a vacancy in hematopoietic tissues such as bone marrow, which may then be filled by transplanted exogenous hematopoietic stem cells. The present 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 a patient to achieve both of the above activities. ADCs, antibodies, or antigen-binding fragments that bind to an antigen expressed by hematopoietic stem cells (e.g., CD45) may be administered to patients suffering from cancer or autoimmune disease to directly reduce the population of cancerous or autoimmune cells, as well as to patients in need of hematopoietic stem cell transplantation therapy to promote the survival and engraftment of transplanted hematopoietic stem cells.

[0344] As described herein, hematopoietic stem cell transplantation therapy may be prescribed to a subject in need of treatment to engraft or regenerate one or more blood cell types. Hematopoietic stem cells generally exhibit multi-potency and thus can 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, B-cells, and T-cells). Hematopoietic stem cells are further characterized by their ability to self-renew, thus giving rise to daughter cells with potential equal to that of the parent cell, and by their ability to be reintroduced into the transplant recipient, home to the hematopoietic stem cell niche, and re-establish productive, sustained hematopoiesis.

[0345] Thus, patients with defects or deficiencies in one or more cell types of the hematopoietic lineage may be administered hematopoietic stem cells to reconstitute the defective or deficient cell population in vivo, thereby treating a condition associated with a defect or deficiency in an endogenous blood cell population. Thus, the compositions and methods described herein may be used to treat non-malignant hemoglobinopathies (e.g., hemoglobinopathies selected from the group consisting of sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome). Additionally or alternatively, the compositions and methods described herein may be used to treat immune deficiencies, such as congenital immune deficiencies. Additionally or alternatively, the compositions and methods described herein may be used to treat acquired immune deficiencies (e.g., acquired immune deficiencies selected from the group consisting of HIV and AIDS). The compositions and methods described in this application may be used to treat a metabolic disorder, e.g., a metabolic disorder selected from the group consisting of glycogen storage diseases, mucopolysaccharidoses, Gaucher disease, Hurler disease, sphingolipidosis, and metachromatic leukodystrophy.

[0346] Additionally or alternatively, the compositions and methods described herein can be used to treat malignancies or proliferative disorders, such as hematological cancers and myeloproliferative disorders. For cancer treatment, the compositions and methods described herein can be administered to patients to deplete the endogenous hematopoietic stem cell population prior to hematopoietic stem cell transplantation therapy, where the transplanted cells can home to the niche created by the depletion step and establish productive hematopoiesis. This, in turn, can reconstitute the population of cells that have been depleted during the eradication of cancer cells, such as during the course of systemic chemotherapy. Exemplary hematological cancers that can be treated using the compositions and methods described herein include, but are not limited to, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma, as well as other cancer conditions, including neuroblastoma.

[0347] Additional diseases that may be treated with the compositions and methods described herein include, but are not limited to, adenosine deaminase deficiency and severe combined immunodeficiency, hyperimmunoglobulin M syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteopetrosis, osteogenesis imperfecta, storage diseases, thalassemia major, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, and juvenile rheumatoid arthritis.

[0348] The antibodies, or antigen-binding fragments thereof, and conjugates described herein may be used to induce solid organ transplant tolerance. For example, the compositions and methods described herein may be used to deplete or deplete a population of cells from a target tissue (e.g., deplete hematopoietic stem cells from the bone marrow stem cell niche). After such depletion of cells from the target tissue, a population of stem or progenitor cells from the organ donor (e.g., hematopoietic stem cells from the organ donor) may be administered to the transplant recipient. Such stem or progenitor cells may then engraft, resulting in a temporary or stable mixed chimerism that allows for long-term transplant tolerance without the need for additional immunosuppressive drugs. For example, the compositions and methods described herein may be used to induce transplant tolerance in solid organ transplant recipients (e.g., kidney, lung, liver, and heart transplants, among others). The compositions and methods described in this application are well suited for use in connection with inducing solid organ transplant tolerance, since, for example, low rates of temporary or stable donor engraftment are sufficient to induce long-term tolerance of the transplanted organ.

[0349] Additionally, the compositions and methods described herein may be used to directly treat cancers, such as cancers characterized by cells that are CD45+. For example, the compositions and methods described herein may be used to treat leukemia (e.g., leukemia in patients exhibiting CD45+ leukemic cells). By reducing CD45+ cancerous cells (e.g., leukemic cells), the compositions and methods described herein may be used to directly treat a variety of cancers. Exemplary cancers that may be treated in this manner include hematological cancers such as acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma.

[0350] Additionally, the compositions and methods described herein may be used to treat autoimmune disorders. For example, an antibody, or an antigen-binding fragment thereof, may be administered to a subject, such as a human patient, suffering from an autoimmune disorder to necrotize CD45+ immune cells. For example, CD45+ immune cells may be autoreactive lymphocytes, such as T-cells, that express T-cell receptors that specifically bind to autoantigens and initiate an immune response against the autoantigen. By reducing autoreactive CD45+ cells, the compositions and methods described herein may be used to treat autoimmune conditions, such as those described below. Additionally or alternatively, the compositions and methods described herein may be used to treat autoimmune diseases by reducing the population of endogenous hematopoietic stem cells prior to hematopoietic stem cell transplantation therapy, where the transplanted cells can home to the niche created by the step of reducing the endogenous cells and establish productive hematopoiesis. This, in turn, allows for reconstitution of the population of cells that was reduced during the eradication of the autoimmune cells.

[0351] Autoimmune diseases that can be treated using the compositions and methods described in this application include, but are not limited to, psoriasis, psoriatic arthritis, type 1 diabetes mellitus (T1DM), rheumatoid arthritis (RA), human systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), lymphocytic colitis, acute disseminated encephalomyelitis (ADEM), Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid syndrome (APS), aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune oophoritis, Baro's disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas disease, chronic fatigue immune deficiency syndrome (CFIDS), chronic inflammation, chronic inflammatory bowel disease (CIADS), chronic inflammation, ... Demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac-sprue dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus erythematosus, autonomic neuropathy, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki disease, lichen planus, Lyme disease, Meniere's disease, mixed connective tissue disease (MCTD), myasthenia gravis, neuromyotonia, opsoclonus-myoclonus syndrome (OMS), optic neuritis, Ord's thyroiditis thyroiditis), pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyendocrine syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff-person syndrome, Takayasu's arteritis, temporal arteritis (also known as giant cell arteritis), ulcerative colitis, collagenous colitis, uveitis, vasculitis, vitiligo, vulvar pain (vulvar vestibulitis), and Wegener's granulomatosis.

[0352] In some embodiments, the graft is allogeneic, hi some embodiments, the graft is autologous.

[0353] In some embodiments, the transplant is a bone marrow transplant, a peripheral blood transplant, or an umbilical cord blood transplant.

[0354] In some embodiments, the graft comprises hematopoietic cells (eg, hematopoietic stem cells).

[0355] In any of the embodiments described herein, the graft may be any solid organ or skin graft, hi some embodiments, the graft is selected from the group consisting of a kidney transplant, a heart transplant, a liver transplant, a pancreas transplant, a lung transplant, a small intestine transplant, and a skin graft.

[0356] Route and method of administration The antibodies, antigen-binding fragments thereof, or ADCs described herein may be administered to patients (e.g., human patients suffering from cancer, autoimmune diseases, or in need of hematopoietic stem cell transplantation therapy) in various dosage forms. For example, the antibodies, antigen-binding fragments thereof, or ADCs described herein may be administered to patients suffering from cancer, autoimmune diseases, or in need of hematopoietic stem cell transplantation therapy in the form of an aqueous solution (e.g., an aqueous solution containing one or more pharma- ceutically acceptable excipients). Pharmaceutically acceptable excipients for use with the compositions and methods described herein include viscosity modifiers. The aqueous solution may be sterilized using techniques known in the art.

[0357] Pharmaceutical formulations comprising anti-CD45 antibodies, or conjugates thereof (e.g., ADCs described herein) are prepared by mixing such antibodies or ADCs with one or more optional pharma- ceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphates, citrates, 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 compounds such as phenylalanine, ... 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 sugars, such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, 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).

[0358] The antibodies, antigen-binding fragments, or ADCs described in this application may be administered by a variety of routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular, or parenteral. The most suitable route of administration in any given case will depend on the particular antibody or antigen-binding fragment being administered, the patient, the pharmaceutical formulation method, the method of administration (e.g., time of administration and route of administration), the patient's age, weight, sex, the severity of the disease being treated, the patient's diet, and the patient's excretion rate.

[0359] An effective dose of an antibody, antigen-binding fragment thereof, or ADC described in the present application may be in the range of about 0.001 to about 100 mg / kg of body weight per single (e.g., bolus), multiple, or continuous administration, which may result in an optimal blood concentration of the antibody, antigen-binding fragment thereof, or ADC (e.g., a blood concentration of about 0.0001 to about 5000 μg / mL). The dose may be administered one or more times (e.g., 2 to 10 times) per day, week, or month to a subject suffering from cancer, an autoimmune disease, or a subject (e.g., a human) undergoing conditioning therapy in preparation for undergoing hematopoietic stem cell transplantation.

[0360] In one embodiment, the dose of anti-CD45 antibody, antigen-binding fragment thereof, or ADC (e.g., an anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to the human patient is about 0.1 mg / kg to about 0.3 mg / kg.

[0361] In one embodiment, the dose of anti-CD45 antibody, antigen-binding fragment thereof, or ADC (e.g., an anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to the human patient is about 0.15 mg / kg to about 0.3 mg / kg.

[0362] In one embodiment, the dose of anti-CD45 antibody, antigen-binding fragment thereof, or ADC (e.g., an anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to the human patient is about 0.15 mg / kg to about 0.25 mg / kg.

[0363] In one embodiment, the dose of anti-CD45 antibody, antigen-binding fragment thereof, or ADC (e.g., an anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to the human patient is about 0.2 mg / kg to about 0.3 mg / kg.

[0364] In one embodiment, the dose of anti-CD45 antibody, antigen-binding fragment thereof, or ADC (e.g., an anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to the human patient is about 0.25 mg / kg to about 0.3 mg / kg.

[0365] In one embodiment, the dose of anti-CD45 antibody, antigen-binding fragment thereof, or ADC (e.g., an anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to the human patient is about 0.1 mg / kg.

[0366] In one embodiment, the dose of anti-CD45 antibody, antigen-binding fragment thereof, or ADC (e.g., an anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to the human patient is about 0.2 mg / kg.

[0367] In one embodiment, the dose of anti-CD45 antibody, antigen-binding fragment thereof, or ADC (e.g., an anti-CD45 antibody conjugated to a cytotoxin via a linker) administered to the human patient is about 0.3 mg / kg.

[0368] In one embodiment, the dose of an anti-CD45 antibody, antigen-binding fragment thereof, or ADC (e.g., an anti-CD45 antibody conjugated to a cytotoxin via a linker) described herein administered to the 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.

[0369] In one embodiment, the anti-CD45 antibody, antigen-binding fragment thereof, or ADC described herein administered to a human patient for treatment or conditioning has a half-life of 24 hours or less, 22 hours or less, 20 hours or less, 18 hours or less, 16 hours or less, 14 hours or less, 13 hours or less, 12 hours or less, 11 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, 7 hours or less, 6 hours or less, or 5 hours or less. In one embodiment, the half-life of the anti-CD45 antibody, antigen-binding fragment thereof, or ADC is 5 hours to 7 hours; 5 hours to 9 hours; 15 hours to 11 hours; 5 hours to 13 hours; 5 hours to 15 hours; 5 hours to 20 hours; 5 hours to 24 hours; 7 hours to 24 hours; 9 hours to 24 hours; 11 hours to 24 hours; 12 hours to 22 hours; 10 hours to 20 hours; 8 hours to 18 hours; or 14 hours to 24 hours.

[0370] In certain embodiments, the methods disclosed herein minimize liver toxicity in patients receiving anti-CD45 antibodies, antigen-binding fragments thereof, or ADCs for conditioning. For example, in certain embodiments, the methods disclosed herein result in liver marker levels remaining below known toxic levels in the patient for more than 24, 48, 72, or 96 hours. In other embodiments, the methods disclosed herein result in liver marker levels remaining within the reference range in the patient for more than 24, 48, 72, or 96 hours. In certain embodiments, the methods disclosed herein result in liver marker levels not increasing more than 1.5-fold above the reference range, more than 3-fold above the reference range, more than 5-fold above the reference range, or more than 10-fold above the reference range for more than 24, 48, 72, or 96 hours. Examples of liver markers that may be used in toxicity testing include alanine aminotransaminase (ALT), lactate dehydrogenase (LDH), and aspartate aminotransaminase (AST). In certain embodiments, administration of an ADC as described herein (i.e., administering two doses instead of a single dose) results in a transient increase in liver markers (e.g., AST, LDH, and / or ALT). In some instances, liver markers indicative of toxicity may reach high levels, but within a certain period of time (e.g., about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, about 7 days, about 7.5 days, or less than one week) the liver marker levels return to normal levels that are not associated with liver toxicity. For example, in humans (average adult males), normal non-toxic levels of ALT are 7-55 units per liter (U / L); normal non-toxic levels of AST are 8-48 U / L. In certain embodiments, at least one of the patient's blood AST, ALT, or LDH levels does not reach a toxic level between administering to the patient a first dose of an ADC and 14 days after administering the first dose.For example, the patient may be administered a first dose, followed by a second, third, fourth, or greater number of doses, e.g., within 5, 10, or 14 days after administering the first dose, but at least one of the patient's blood AST, ALT, or LDH levels does not reach toxic levels between administering the first dose of ADC to the patient and 14 days after administering the first dose.

[0371] In certain embodiments, at least one of the patient's blood AST, ALT, or LDH levels is not elevated above normal levels, not elevated more than 1.5-fold above normal levels, not elevated more than 3-fold above normal levels, not elevated more than 5-fold above normal levels, or not elevated more than 10-fold above normal levels.

[0372] In the case of a conditioning procedure prior to hematopoietic stem cell transplantation, the antibody, or antigen-binding fragment thereof, may be administered to the patient at a time that optimally promotes engraftment of exogenous hematopoietic stem cells, for example, about 1 hour to about 1 week (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days) or more prior to administration of the exogenous hematopoietic stem cell graft. Ranges including the numbers recited in this application are also included within the contemplated methods.

[0373] Using the methods disclosed in this application, a physician skilled in the art may administer to a human patient in need of hematopoietic stem cell transplantation therapy an ADC, antibody, or antigen-binding fragment thereof capable of binding to CD45 expressed by hematopoietic stem cells. In this manner, the population of endogenous hematopoietic stem cells may be depleted prior to administration of an exogenous hematopoietic stem cell graft, such that engraftment of the hematopoietic stem cell graft is promoted. The antibody may be covalently conjugated to a toxin, such as a cytotoxic molecule described in this application or known in the art. For example, an anti-CD45 antibody, or antigen-binding fragment thereof, may be covalently conjugated to a cytotoxin (e.g., Pseudomonas exotoxin A, de bouganin, diphtheria toxin, amatoxins such as gamma-amanitin, alpha-amanitin, saporin, maytansine, maytansinoids, auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycins, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolinobenzodiazepines, and indolinobenzodiazepine dimers, or variants thereof). This conjugation may be performed using covalent bond formation techniques described in this application or known in the art. The antibody, antigen-binding fragment thereof, or drug-antibody conjugate may then be administered to the patient, for example, by intravenous administration, prior to transplantation of exogenous hematopoietic stem cells (e.g., autologous, syngeneic, or allogeneic hematopoietic stem cells, etc.) into the patient.

[0374] The anti-CD45 antibody, antigen-binding fragment thereof, or ADC may be administered prior to hematopoietic stem cell transplantation therapy in an amount sufficient to reduce the amount of endogenous hematopoietic stem cells, for example, by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more. At various intervals during the conditioning therapy, the reduction in the number of hematopoietic stem cells may be monitored using conventional techniques known in the art, such as FACS analysis of cells expressing characteristic hematopoietic stem cell surface antigens in blood samples taken from the patient. For example, a skilled physician may measure the extent to which endogenous hematopoietic stem cells have been reduced by taking blood samples from the patient at various times during the conditioning therapy and performing FACS analysis using antibodies that bind to hematopoietic stem cell marker antigens to determine the relative concentration of hematopoietic stem cells in the samples. According to some embodiments, when the concentration of hematopoietic stem cells reaches a nadir in response to conditioning therapy with an anti-CD45 antibody, antigen-binding fragment thereof, or ADC, the physician may terminate the conditioning therapy and begin preparing the patient for hematopoietic stem cell transplant therapy.

[0375] The anti-CD45 antibody, antigen-binding fragment thereof, or ADC may be administered to the patient in an aqueous solution containing one or more pharma- ceutically acceptable excipients, such as, for example, viscosity modifiers. The aqueous solution may be sterilized using techniques described in this application or known in the art. For example, prior to administering a hematopoietic stem cell graft to the patient, the antibody, antigen-binding fragment thereof, or drug-antibody conjugate may be administered to the patient at, for example, about 0.001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 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 ... may be administered at doses of about 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.The anti-CD45 antibody, antigen-binding fragment thereof, or ADC may be administered to the patient at a time that optimally promotes engraftment of exogenous hematopoietic stem cells, for example, about 1 hour to about 1 week (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days) or more prior to administration of an exogenous hematopoietic stem cell graft.

[0376] In one embodiment, the anti-CD45 antibody, antigen-binding fragment thereof, or ADC is administered to the subject along with an immunosuppressant. In one embodiment, the immunosuppressant is an agent that depletes T cells. The immunosuppressant may be administered to the subject before, simultaneously with, or after administration of the anti-CD45 antibody, antigen-binding fragment thereof, or ADC. Exemplary immunosuppressants include, but are not limited to, anti-CD4 antibodies, anti-CD8 antibodies, total body irradiation, and cytoxan, and combinations thereof.

[0377] In certain embodiments, the anti-CD45 antibody, antigen-binding fragment thereof, or ADC is administered to a subject in combination with total body irradiation (TBI) (e.g., low-dose TBI). In an exemplary embodiment, the subject receives TBI at a dose of 3 Gy or less, e.g., 3 Gy TBI or less, 2.5 Gy TBI or less, 2 Gy TBI or less, 1.5 Gy TBI or less, 1 Gy TBI or less, or 0.5 Gy TBI or less.

[0378] In one embodiment, the anti-CD45 antibody, antigen-binding fragment thereof, or ADC is administered to the subject in combination with a cytoxan.

[0379] After the conditioning regimen is completed, the patient may then receive an injection (e.g., intravenous injection) of exogenous hematopoietic stem cells, for example, from the same physician who administered the conditioning regimen or from a different physician. The physician may administer to the patient an injection of autologous, syngeneic, or allogeneic hematopoietic stem cells, for example, at a dose of 1×10 3 ~1×10 9After administering the graft, the physician may monitor the engraftment of the hematopoietic stem cells by, for example, taking a blood sample from the patient and measuring an increase in the concentration of hematopoietic stem cells or cells of the hematopoietic lineage (e.g., megakaryocytes, thrombocytes, platelets, red blood cells, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presenting cells, macrophages, dendritic cells, natural killer cells, T-lymphocytes, and B-lymphocytes, etc.). This analysis can be performed, for example, 1 hour to 6 months after hematopoietic stem cell transplantation therapy or later (e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, may be performed at about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, or later). Observing that the concentration of hematopoietic stem cells or cells of hematopoietic lineage is increased (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 500% increased, or more) after the transplantation therapy compared to the concentration of the corresponding cell type before the transplantation therapy would indicate that treatment with an anti-CD45 antibody, or antigen-binding fragment thereof, or ADC was successful in promoting engraftment of the transplanted hematopoietic stem cell graft.

[0380] Administration of anti-CD45 antibodies, antigen-binding fragments thereof, or ADCs can demonstrate hematopoietic stem cell graft survival in a variety of experimental measurements. For example, survival of transplanted hematopoietic stem cells can be assessed by assessing the amount of competitive repopulating units (CRUs) present in the patient's bone marrow after administration of anti-CD45 antibodies, or antigen-binding fragments thereof, and subsequent administration of a hematopoietic stem cell graft. Survival of hematopoietic stem cell grafts can also be observed by incorporating a reporter gene, such as an enzyme that catalyzes a chemical reaction that produces a fluorescent, chromogenic, or luminescent product, into a vector transfecting the donor's hematopoietic stem cells, and then monitoring the corresponding signal in the tissue to which the hematopoietic stem cells home, such as the bone marrow. Survival of hematopoietic stem cells can also be observed by assessing the amount and viability of hematopoietic stem cells and hematopoietic progenitor cells, for example, as measured by fluorescence activated cell sorting (FACS) analysis methods known in the art. Engraftment can also be measured during the post-transplant period by measuring peripheral blood white blood cell counts and / or by measuring bone marrow cell recovery by donor cells in bone marrow aspirate samples. EXAMPLES

[0381] The following examples are presented to illustrate to one of ordinary skill in the art how to use, make, and evaluate the compositions and methods described in this application, and are purely illustrative of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0382] Example 1. Preparation of anti-CD45 monoclonal antibodies Anti-human CD45 antibodies were obtained by immunizing mice with human CD45 (specifically the extracellular domain of human CD45 RO). Cell lines expressing human CD45RO, human full-length CD45 (RABC), cynomolgus monkey RABC and mouse CD45 RABC were generated and binding of each clone was assessed by flow cytometry. 216 antibodies were assessed in duplicate by cytotoxicity in secondary Fab-saporin experiments on two cell lines and human CD34 bone marrow cells, using primary antibodies at 10, 100 and 1000 pM in the presence of anti-rat Fab-saporin (10 nM for cell lines and 20 nM for CD34). Staurosporine was used as a 100% death control on each plate. Rat anti-human CD45 mAbs were included for comparison. Cell lines were incubated for 72 hours and CD34 cells were incubated for 120 hours, after which viability was assessed using Cell titer glo. Dose titration toxicity curves were then generated to select clones in REH cell lines and human bone marrow CD34 cells.

[0383] Octet binding experiments were performed to identify mAbs with good cross-reactivity. Clones were selected based on their ability to bind to human CD45RO and CD45RABC, and to non-human primate cynomolgus CD45RABC. Five clones with cross-reactivity to human and cynomolgus CD45 were identified, and among them, only AbA, AbB, and AbC were identified as having good cross-reactivity to human and non-human primate CD45RABC.

[0384] These monoclonal antibodies were cloned and their variable regions were cloned into a vector containing human IgG1. Chimeric antibodies AbA, AbB, and AbC were then expressed as IgG1 antibodies and evaluated for binding and activity to human CD45 cells. The degree of monovalent binding of AbA, AbB, and AbC to human CD45 RABC, cynomolgus CD45 RABC, and rhesus CD45 RABC, as assessed by octet, is listed in Table 2. Binding of AbA, AbB, and AbC was also evaluated by flow cytometry for PBMC binding at 24 hours. In each case, each clone was detected for binding to human and cynomolgus PBMC.

[0385] [Table 2]

[0386] These antibodies were also evaluated as ADCs to determine whether they could be internalized into cells expressing CD45. AbA, AbB, and AbC were each internalized into the cells.

[0387] Fc variant versions of AbA, AbB, and AbC were also made. Specifically, human IgG1 chimeras of AbA, AbB, and AbC were made with or without the Fc mutations D265C and V205C (intended for conjugation to ADCs). Additional Fc variant antibodies were made with or without H435A or I253A / H310A / H435A to provide further differences in half-lives of the antibodies or ADCs.

[0388] The amino acid sequences of the CDRs and variable regions of AbA, AbB, and AbC are shown in Table 3.

[0389] [Table 3-1] [Table 3-2] [Table 3-3]

[0390] Example 2. Non-genotoxic conditioning with amatoxin antibody drug conjugates targeting CD45 is effective in depleting hematopoietic stem cells and immune cells in humans and non-human primates preface Bone marrow transplantation (BMT) is a potentially curative treatment for malignant and non-malignant hematological disorders. Current regimens for preparing or conditioning patients prior to BMT limit the use of this curative procedure due to regimen-associated mortality and morbidity, including organ toxicity, infertility, and risk of secondary malignancies. To safely condition patients for BMT, antibody-drug conjugates (ADCs) have been developed that target CD45, a target expressed throughout the hematopoietic system, resulting in the specific depletion of both hematopoietic stem cells (HSCs) and immune cells prior to transplantation.

[0391] result Anti-CD45 ADCs were generated by conjugating anti-CD45 antibody AbA (chimeric AbA as IgG1) to an amatoxin (AM) payload (RNA polymerase II inhibitor). The resulting anti-CD45 AM ADCs (CD45-AM) potently cytosed primary human bone marrow CD34+CD90+HSC (IC50 67 pM, FIG. 1A) and PBMC immune cells (IC50 55 pM, FIG. 1B) in vitro. The CD45-AM ADCs were then tested in vivo in humanized NSG mice. A single dose of 3 mg / kg CD45-AM depleted >95% of human CD34+ cells in the bone marrow (FIG. 1C; 14 days post-dose) and >95% of human B-, T-, and myeloid cells in the periphery and bone marrow (FIG. 1D). A single dose of 3 mg / kg CD45-AM also depleted >99% of peripheral human CD45 cells (Figure 1E; 14 days after dosing; N = 3 / group). A control non-targeting isotype-matched ADC and anti-CD45 antibody without toxin had minimal effects on HSCs, immune cells, or peripheral human CD45 cells. Because CD45 is highly expressed in many leukemias and lymphomas, we also evaluated the leukemia-reducing activity of CD45-AM in ALL and three PDX-AML models. A single dose of 1 mg / kg CD45-AM more than doubled median survival in all four models (p-value <0.001), with several mice surviving free-of-disease.

[0392] In BMT, ADCs need to be cleared quickly so as not to reduce the incoming graft, so the t 1 / 2 We created a fast half-life CD45-AM variant, , that is a cross-reactive variant of CD45-AM that is fast half-life. Increasing doses of the cross-reactive, fast half-life CD45-AM induced a dose-dependent depletion of peripheral lymphocytes in cynomolgus monkeys. A single dose of 1 mg / kg was well tolerated and induced a >95% depletion of HSCs (CD34+CD90+CD45RA- cells) and a >80% depletion of lymphocytes in the bone marrow 14 days after dosing (Fig. 1D).

[0393] In conclusion, targeting CD45 with amatoxin-bearing ADCs potently depleted human HSCs and immune cells in vitro and in vivo. Anti-CD45 amatoxin ADCs significantly reduced disease burden in multiple leukemia models. Preliminary evaluation of fast half-life ADCs in cynomolgus monkeys efficiently depleted HSCs and immune cells in bone marrow. Amatoxin ADCs targeting CD45 are i) non-genotoxic; ii) amanitin has poor cell permeability as a free toxin, thus avoiding bystander toxicity; and iii) toxic to cycling and non-cycling cells, the latter of which may be essential for effective depletion of HSCs and immune cells. These combined properties may allow for safer targeted conditioning and expanded use of BMT in malignant and non-malignant conditions.

[0394] Example 3. Anti-CD45 antibody drug conjugate (ADC) conditioning agents have in vivo anti-leukemia activity in cell lines and patient-derived xenograft models Allogeneic hematopoietic stem cell transplantation is a potentially curative approach in patients with refractory or high-risk hematologic malignancies. Prior to transplantation, patients are conditioned with high-dose chemotherapy or chemotherapy and total body irradiation, which are associated with substantial risks of early and late morbidity and mortality. As a result, many eligible patients do not consider transplantation, and two out of three transplanted patients may only tolerate reduced-intensity conditioning, which is associated with increased relapse rates. Thus, safer and more effective conditioning agents with improved disease control are urgently needed. To meet this need, we developed a novel antibody-drug conjugate (ADC) using an antibody AbB conjugated to amatoxin (AM) (chimeric AbB in human IgG1) targeting CD45, which is expressed on all lymphohematopoietic cells and almost all hematologic malignancies, except multiple myeloma (Rahul 2018). The goal of this project was to measure the anti-leukemic potential of anti-CD45-AM, an agent previously shown to deplete primary human HSPCs in vitro and in vivo.

[0395] method. The ADC was tested with REH-Luc (a luciferase-labeled, CD45-expressing AML cell line) and with three patient-derived xenografts (PDX) (J000106132, J000106565, J000106134) (Jackson Laboratories) generated from FLT-3+NPM1+ AML samples expressing both CD117 and CD45 and with different growth kinetics (median survival times in vehicle-treated groups were 43, 63, and 82 days post-inoculation).

[0396] result.In the REH-Luc model, a single injection of 1 mg / kg CD45-AM on day 5 post-AML inoculation resulted in longer survival with a median of 15 days compared to vehicle-treated controls or unconjugated anti-CD45 antibody (n=10 mice / group, p<0.0001). Using 4-5 mice / group / AML-PDX model, survival was significantly increased in recipients of 0.1 mg / kg CD45-AM compared to vehicle controls.

[0397] Conclusion. Based on these data in humanized mouse models with established AML, CD45-AM is a potent anti-leukemic agent, in addition to depleting normal host HSPC. Together with previous reports of the efficacy of CD45-AM conditioning agents, these non-genotoxic ADCs may be useful in patients with active disease and in recipients of reduced-dose conditioning who are at high risk for disease relapse.

[0398] Example 4. A single dose of a CD45-targeted antibody drug conjugate (ADC) has potent in vivo anti-leukemia activity and confers survival benefit in a patient-derived AML model Allogeneic bone marrow transplantation (BMT) is a potentially curative approach in patients with refractory or high-risk hematological malignancies, such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Prior to transplant, patients are prepared with non-specific, high-dose chemotherapy alone or in combination with total body irradiation, which are associated with substantial risks of early and late morbidity (e.g., organ toxicity, infertility, secondary malignancies, etc.) and mortality. As a result, many eligible patients do not consider transplantation, and two out of three transplant recipients may only tolerate reduced-intensity conditioning, which is associated with increased relapse rates (Scott et al. Journal of Clinical Oncology 2017, 1154-1161). Thus, safer and more effective conditioning agents with improved disease control are urgently needed. To meet this need, we developed two novel antibody-drug conjugates (ADCs) conjugated to amatoxins (AMs) targeting CD117 (C-KIT, Pearse 2018), which is expressed on hematopoietic stem and progenitor cells and on AML and MDS cells in approximately 80% of patients (Gao et al. PLOS One. 2015), and CD45, which is expressed on all lymphohematopoietic cells and almost all hematologic malignancies except multiple myeloma (Palchaudhuri 2018). The goal of this project was to design non-genotoxic agents with a dual benefit of depleting primary human hematopoietic stem and progenitor cells (HSPCs) while reducing disease burden in leukemia models.

[0399] method The anti-CD45 antibody used in the ADCs in the following examples is an anti-CD45 antibody AbB conjugated (interchain) to amanitin with a drug-to-antibody ratio (DAR) of 4. The ADCs were generated using human leukemia cells from an immortalized cell line (REH-Luc, a luciferase-labeled, CD45-expressing ALL cell line (5x10 6The efficacy and safety of AML was evaluated in xenograft mouse models inoculated with three patient-derived xenografts (PDX) [AML #1 (J000106132), AML #2 (J000106565), and AML #3 (J000106134)] (Jackson Laboratories) made with FLT-3+NPM1+ AML samples that express CD45 and have different proliferation kinetics (median survival times in vehicle-treated groups were 43, 63, and 82 days post-inoculation). All in vivo studies were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council of the National Academies and with approval from the Institutional Animal Care and Use Committee.

[0400] result Effective target reduction in vitro For in vitro cytotoxicity assays with human PBMCs, human PBMCs were cultured for 4 days in the presence of anti-CD45-amatoxin conjugate (antibody AbB-amatoxin; "CD45-AM") or IgG1 isotype conjugated to amatoxin ("isotype-AM") at varying antibody concentrations (x-axis) and cell viability was measured as luminescence (RLU; y-axis) by Celltiter Glo (Figure 2A). For in vitro cytotoxicity assays using human HSCs (i.e., isolated primary human CD34+ selected bone marrow cells (BMCs)), primary human CD34 + Bone marrow cells were cultured with CD45-AM or isotype-AM for 5 days and the counts of live CD34+CD90+ HSC (y-axis) were determined by flow cytometry in the presence of varying antibody concentrations (x-axis).

[0401] The results in Figures 2A and 2B show that CD45-ADCs express CD45-expressing cell lines (e.g., PBMCs; 614 pM; Figure 2A) or primary human CD34 + It was shown to be highly effective in killing CD90+ HSCs (68 pM, FIG. 2B) in vitro.

[0402] Anti-CD45-AM doubles median survival to 40 days in a REH-luciferase ALL xenograft model Human leukemia cells derived from an immortalized cell line expressing CD45 (REH-Luc, luciferase-labeled CD45-expressing ALL cell line (5 × 10 6 The CD45-ADC was tested in a xenograft mouse model inoculated with ALL (1000 cells / mouse). As shown in Figure 3A, a single injection of 1 mg / kg anti-CD45-AM on day 5 after ALL inoculation resulted in a median 15-day longer survival compared to vehicle (PBS)-treated control or unconjugated anti-CD45 antibody (n=10 mice / group, p<0.0001). Furthermore, pseudocolor images of typical bioluminescence signals were acquired on day 19 after transplantation for the anti-CD45-AM and isotype-AM treated groups using an IVIS imaging system (Perkin Elmer) (Figure 3B). These results indicate that anti-CD45-AM doubles the median survival to 40 days in the REH-Luc xenograft model. ...

Claims

1. a heavy chain comprising a variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 2, CDR2 having the amino acid sequence set forth in SEQ ID NO: 3, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 4; and a light chain comprising a variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 6, CDR2 having the amino acid sequence set forth in SEQ ID NO: 7, and CDR3 having the amino acid sequence set forth in SEQ ID NO:

8.

1. An isolated anti-CD45 antibody, or an antigen-binding portion thereof, comprising:

2. a heavy chain comprising a variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 10, CDR2 having the amino acid sequence set forth in SEQ ID NO: 11, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 12; and a light chain comprising a variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 14, CDR2 having the amino acid sequence set forth in SEQ ID NO: 15, and CDR3 having the amino acid sequence set forth in SEQ ID NO:

16.

1. An isolated anti-CD45 antibody, or an antigen-binding portion thereof, comprising:

3. a heavy chain comprising a variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 18, CDR2 having the amino acid sequence set forth in SEQ ID NO: 19, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 20; and a light chain comprising a variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 22, CDR2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDR3 having the amino acid sequence set forth in SEQ ID NO:

24.

1. An isolated anti-CD45 antibody, or an antigen-binding portion thereof, comprising:

4. 2. The anti-CD45 antibody, or antigen-binding portion thereof, of claim 1, wherein The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

1.

5. 3. The anti-CD45 antibody, or antigen-binding portion thereof, of claim 2, wherein The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

9.

6. 4. The anti-CD45 antibody, or antigen-binding portion thereof, of claim 3, wherein The heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

17.

7. 2. The anti-CD45 antibody, or antigen-binding portion thereof, of claim 1, wherein The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

5.

8. 3. The anti-CD45 antibody, or antigen-binding portion thereof, of claim 2, wherein The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

13.

9. 4. The anti-CD45 antibody, or antigen-binding portion thereof, of claim 3, wherein The light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

21.

10. An isolated anti-CD45 antibody, or antigen-binding portion thereof, which is (i) specifically binds to an epitope of human CD45 that includes residues 486R, 493Y, and 502T of SEQ ID NO: 37, and binds to cynomolgus and rhesus monkey CD45; or (ii) specifically binds to an epitope of human CD45 containing the amino acid sequence RNGPHARYHLEVEAGNT (SEQ ID NO:38), and binds to cynomolgus and rhesus monkey CD45;

11. 11. The anti-CD45 antibody, or antigen-binding portion thereof, of claim 10, wherein the antibody, or antigen-binding portion thereof, binds to at least one additional amino acid, at least two additional amino acids, at least three additional amino acids, at least four additional amino acids, or at least five additional amino acids in a peptide comprising the amino acid sequence RNGPHARYHLEVEAGNT (SEQ ID NO:38).

12. 12. The anti-CD45 antibody, or antigen-binding portion thereof, of claim 10 or 11, wherein the antibody competes for binding to human CD45 with an antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:1, and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

5.

13. 12. The anti-CD45 antibody, or antigen-binding portion thereof, of any one of claims 1 to 11, which is (i) a deimmunized antibody, or an antigen-binding fragment thereof; (ii) a humanized antibody, or an antigen-binding fragment thereof; or (iii) a chimeric antibody, or an antigen-binding fragment thereof; It is.

14. 14. An anti-CD45 antibody according to any one of claims 1 to 13, which is an intact monoclonal IgG antibody.

15. 14. The anti-CD45 antibody or antigen-binding portion thereof of claim 1 , comprising an Fc region comprising the amino acid substitutions D265 / L234 / L235, and H435 (EU numbering).

16. 16. An anti-CD45 antibody, or antigen-binding portion thereof, according to any one of claims 1 to 15, wherein the antibody, or antigen-binding portion thereof, is an IgG.

17. 17. An antibody drug conjugate (ADC) comprising the antibody of any one of claims 1 to 16 conjugated to a cytotoxin via a linker.

18. 18. The ADC of claim 17, wherein the cytotoxin is an RNA polymerase inhibitor.

19. 20. The ADC of claim 18, wherein the RNA polymerase inhibitor is amatoxin or amanitin.

20. 20. The ADC of claim 19, wherein the amatoxin is represented by formula (IV): 【Chemistry 1】 Here, R 1 H, OH, OR A OR C is; R 2 H, OH, OR B OR C is; R A and R B together with the oxygen atom to which they are attached form an optionally substituted 5-membered heterocycloalkyl group; R 3 , H, R C , or R D is; R 4 , R 5 , R 6, and R 7 are independently H, OH, or OR C , OR D , R C , or R D is; R 8 OH, NH 2 , OR C , OR D , N.H.R. C , or NR C R D is; R 9 H, OH, OR C OR D is; X is -S-, -S(O)-, or -SO 2 -is; R C is -LZ; R D is an optionally substituted C 1 -C 6 Alkyl, optionally substituted C 1 -C 6 Heteroalkyl, optionally substituted C 2 -C 6 Alkenyl, optionally substituted C 2 -C 6 Heteroalkenyl, optionally substituted C 2 -C 6 Alkynyl, optionally substituted C 2 -C 6 heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl; L is optionally substituted C 1 -C 6 Alkylene, optionally substituted C 1 -C 6 Heteroalkylene, optionally substituted C 2 -C 6 Alkenylene, optionally substituted C 2 -C 6 Heteroalkenylene, optionally substituted C 2 -C 6 Alkynylene, optionally substituted C 2 -C 6 heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, a peptide, a dipeptide, -(C=O)-, or a combination thereof; and Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in the antibody or antigen-binding fragment, where Am is exactly one R C Contains substituents.

21. 18. The ADC of claim 17, wherein the ADC has the formula: 【Chemistry 2】 Here, Ab represents the site of binding of the anti-CD45 antibody.

22. 18. The ADC of claim 17, wherein the ADC has the formula: 【Chemistry 3】 Here, Ab represents the site of binding of the anti-CD45 antibody.

23. 18. The ADC of claim 17, wherein the cytotoxin is selected from the group consisting of Pseudomonas exotoxin A, de bouganin, diphtheria toxin, saporin, maytansine, maytansinoids, auristatins, anthracyclines, calicheamicin, irinotecan, SN-38, duocarmycins, pyrrolobenzodiazepines, pyrrolobenzodiazepine dimers, indolinobenzodiazepines, and indolinobenzodiazepine dimers.

24. 24. The ADC of claim 23, wherein the auristatin is MMAE or MMAF.

25. 25. The ADC of any one of claims 17 to 24, wherein the antibody is conjugated to the toxin through a cysteine ​​residue in the Fc domain of the antibody.

26. 26. The ADC of claim 25, wherein the cysteine ​​residue is introduced by amino acid substitution in the Fc domain of the antibody.

27. 27. A pharmaceutical composition comprising an antibody or ADC described in any one of claims 1 to 26 and a pharma- ceutically acceptable carrier.

28. 27. An anti-CD45 antibody or antibody drug conjugate (ADC) according to any one of claims 1 to 26 for use in a method for reducing the population of hematopoietic stem cells (HSC) in a human patient, said method comprising administering to said patient an effective amount of said anti-CD45 antibody or ADC.

29. 30. The anti-CD45 antibody or antibody drug conjugate (ADC) of claim 28, further comprising administering to the patient a graft comprising hematopoietic stem cells.

30. 30. The anti-CD45 antibody or antibody drug conjugate (ADC) of claim 29, wherein the graft is allogeneic or autologous.

31. 27. The anti-CD45 antibody or antibody drug conjugate (ADC) of any one of claims 1 to 26 for use in a method comprising administering to a human patient a graft comprising hematopoietic stem cells, wherein the patient has previously been administered the anti-CD45 antibody or ADC in an amount sufficient to reduce the population of hematopoietic stem cells or the population of immune cells in the patient.

32. 32. The anti-CD45 antibody or antibody drug conjugate (ADC) of claim 31 , wherein the hematopoietic stem cell is a CD45+ cell, or wherein the immune cell is a CD137+, CD2+, or CD5+ cell, or a T cell.

33. 33. The anti-CD45 antibody or antibody drug conjugate (ADC) of any one of claims 28 to 32, wherein the patient has a hematological disease, a metabolic disorder, cancer, or an autoimmune disease, or severe combined immunodeficiency disease (SCID).

34. 27. An anti-CD45 antibody or antibody drug conjugate (ADC) according to any one of claims 1 to 26 for use in a method of treating leukemia in a human patient, the method comprising administering the anti-CD45 antibody or ADC to a human patient having leukemia.

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