Anti-CD45 antibodies and conjugates thereof

Abstract

Disclosed herein are anti-CD45 antibodies, antigen-binding fragments thereof, and antibody drug conjugates (ADCs) that specifically bind to human CD45. The antibodies and ADCs can be used in therapeutic methods including methods of depleting CD45 + cells in a patient. The compositions and methods described herein can be used for direct treatment of conditions, for example, by depleting CD45 + cancer cells or populations of autoimmune cells. The compositions and methods described herein can also be used to prepare a patient for hematopoietic stem cell transplantation therapy and to improve the implantation of hematopoietic stem cell grafts by selectively depleting endogenous CD45 + cells prior to the transplantation procedure.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080086243.5, filed on October 30, 2020, entitled “Anti-CD45 Antibodies and Conjugates Thereof.”

[0002] Related Applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 929,137, filed November 1, 2019; U.S. Provisional Application No. 62 / 929,194, filed November 1, 2019; U.S. Provisional Application No. 62 / 929,207, filed November 1, 2019; U.S. Provisional Application No. 62 / 929,288, filed November 1, 2019; U.S. Provisional Application No. 62 / 929,601, filed November 1, 2019; U.S. Provisional Application No. 62 / 929,283, filed November 1, 2019; U.S. Provisional Application No. 62 / 929,347, filed November 1, 2019; U.S. Provisional Application No. 62 / 940,742, filed November 26, 2019; U.S. Provisional Application No. 62 / 978,147, filed February 18, 2020; U.S. Provisional Application No. 63 / 015,348, filed April 24, 2020; U.S. Provisional Application No. 63 / 046,046, filed June 30, 2020; U.S. Provisional Application No. 63 / 046,164, filed June 30, 2020; and U.S. Provisional Application No. 63 / 084,903, filed September 29, 2020. The contents of each of the foregoing priority applications are incorporated herein by reference in their entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. This ASCII copy, created on October 29, 2020, is named 2020-10-29_M103034_1525WO_SL_ST25.txt and is 233 kilobytes in size. TECHNICAL FIELD

[0006] Anti-CD45 antibodies, antigen-binding fragments thereof, and antibody drug conjugates thereof are set forth herein. Also set forth are methods of treating patients having a variety of conditions, especially, for example, hematological diseases, metabolic disorders, cancers, and autoimmune diseases, by administering an anti-CD45 antibody, antigen-binding fragment thereof, or antibody drug conjugate (ADC) thereof, wherein the antibody, antigen-binding fragment thereof, or ADC is capable of binding CD45 on a target cell, such as a hematopoietic stem cell, an immune cell, or other type of cell. BACKGROUND

[0007] CD45, also known as protein tyrosine phosphatase receptor type C (PTPRC), is an enzyme encoded in humans by the PTPRC gene (Kaplan et al., PNAS 87:7000-7004 (1990)). CD45 is a member of the protein tyrosine phosphatase (PTP) family, which includes signaling molecules that regulate a variety of cellular processes, including cell growth, differentiation, mitotic cycle, and oncogenic transformation. CD45 contains extracellular, single-pass transmembrane, and two tandem cytoplasmic catalytic domains, and thus belongs to the receptor type PTP family. CD45 is a type I transmembrane protein that exists in multiple isoforms on differentiated hematopoietic cells (except red blood cells and plasma cells) (Holmes, Immunology 117:145-55 (2006)). CD45 has been shown to be a regulator of T cell and B cell antigen receptor signaling. It interacts directly with components of the antigen receptor complex via its extracellular domain (in a co-stimulatory fashion), or acts via its cytoplasmic domains to activate various Src family kinases required for antigen receptor signaling. CD45 also inhibits JAK kinases, and thus serves as a negative regulator of interleukin receptor signaling.

[0008] CD45 is present on the surface of hematopoietic cells, including HSCs, leukocytes, and osteoclasts, which have hematopoietic origin (Shivtiel et al., J Exp Med 205:2381 (2008)). Deletion mutations within CD45 in humans are associated with severe immunodeficiency. This is primarily due to the absence of CD45 on T cells, which is normally abundant and necessary to modulate SFK activity during antigen responses. CD45-deficient (CD45 - / - ) mice have normal numbers of hematopoietic cells in their bone marrow, but the number of most primitive HSCs is reduced, and their mobilization in response to G-CSF is impaired. In part, this deficit is intrinsic to HSCs; without CD45-mediated downregulation of SFK activity, integrin-mediated adhesion is higher and HSCs are more likely to remain in the stem cell niche. CD45 - / -HSCs also lack G-CSF-stimulated mobilization and homing to the chemokine CXCL12 / SDF-1, which negatively impacts cell engraftment post-transplant. These defects can be restored by supplementing with SFK inhibitors, indicating that this role is typically fulfilled by CD45. Likewise, CD45 - / - Recipients also show defective engraftment of normal HSCs and subsequent mobilization, indicating a role for CD45 in the stem cell niche and in HSCs (Shivtiel et al., J Exp Med 205:2381 (2008)).

[0009] Despite advances in the medical field, there remains a need for treating conditions of the hematopoietic system, particularly diseases of specific blood cells, metabolic disorders, cancers, and autoimmune disorders, for example. Despite the significant therapeutic potential of hematopoietic stem cells (HSCs), a limiting factor that hinders their clinical application is the difficulty in ensuring engraftment of HSC transplants in the host. In particular, hematopoietic stem cell therapies involving antibodies that target cell surface antigens on endogenous HSCs can trigger unwanted immune stimulation and effector functions, hindering engraftment of exogenous HSC transplants. Because CD45 is expressed on, for example, HSCs and leukocytes, it presents a target for therapies including conditioning therapies, immune resetting, and disease treatment. SUMMARY

[0010] In view of the important role of CD45 in cell biology, there is a need for anti-CD45 antibodies and fragments thereof. Anti-CD45 antibodies, antigen-binding fragments thereof, and antibody drug conjugates (ADCs) thereof are set forth herein. Anti-CD45 antibodies, antigen-binding fragments thereof, and antibody drug conjugates (ADCs) thereof can bind to hematopoietic stem cells (HSCs) and can be used as conditioning agents for, for example, HSC transplantation. In particular, the anti-CD45 antibodies, antigen-binding fragments thereof, and ADCs thereof described herein can be used to specifically deplete, for example, host HSCs, immune cells (e.g., leukocytes), or pathogenic cells. Additionally, the anti-CD45 antibodies, antigen-binding fragments thereof, and ADCs thereof described herein can be used to treat patients with leukemia or lymphoma, or to treat patients with autoimmune diseases (e.g., multiple sclerosis and scleroderma). The anti-CD45 antibodies, antigen-binding fragments thereof, and ADCs described herein meet the need for compositions and methods that promote engraftment of exogenous hematopoietic stem cell transplants, such that the multi-potency and hematopoietic function of the cells are preserved post-transplant.

[0011] In a first aspect, the present disclosure provides an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising: (a) a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:2, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO:3, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:4; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:6, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO:7; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:8; (b) a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 12, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 13, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 14; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 16, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 17; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 18; (c) a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 22, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 23, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 24; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 26, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 27; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 28; (d) a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 32, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 33, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 34; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 36, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 37; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 38.(e) a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:42, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO:43, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:44; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:46, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO:47, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:48; (f) a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:52, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO:53, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:54; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:56, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO:57, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:58; (g) a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:62, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO:63, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:64; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:66, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO:67, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:68; (h) a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:72, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO:73, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:74; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:76, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO:77, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:78; (i) a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO:82, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO:83, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO:84;(i) a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 86, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 87; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 88; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 96, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 97; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 98; or (j) a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 92, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 93; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 94; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 96, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 97; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 98.

[0012] In another aspect, provided herein is an isolated anti-CD45 antibody or antigen binding portion thereof, comprising: a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 119, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 120; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 121; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 122, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 123; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 124.

[0013] In another aspect, the present disclosure provides an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising: (a) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 1, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 5; (b) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 11, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 15; (c) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 21, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 25; (d) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 31, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 35; (e) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 41, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 45; (f) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 51, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 55; (g) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 61, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 65; (h) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 71, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 75; (i) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 81, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 85; or (j) a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 91, and a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 95.

[0014] In some embodiments of the above-referenced aspects, the isolated anti-CD45 antibody, or antigen binding portion thereof, comprises an Fc region. In certain embodiments, the Fc region is a human IgGl Fc region or a human IgG4 Fc region.

[0015] In some embodiments, the antibody is a monoclonal antibody.

[0016] In some embodiments, the antibody is an intact antibody comprising a constant region.

[0017] In some embodiments, the antibody is an IgG. In particular embodiments, the IgG is an IgGl or an IgG4.

[0018] In some embodiments of the above-mentioned aspects, the antibody comprises a constant region, wherein the constant region comprises at least one, at least two, at least three, at least four, or at least five amino acid substitutions selected from the group consisting of L234A, L235A, D265C, H310A, and H435A (numbering according to EU index). In particular embodiments, the constant region comprises the amino acid substitutions L234A, L235A, and D265C (numbering according to EU index).

[0019] In some embodiments, the isolated anti-CD45 antibody of the present disclosure comprises a constant region, wherein the constant region comprises (a) a heavy chain amino acid sequence as set forth in SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106; and (b) a light chain amino acid sequence as set forth in SEQ ID NO: 101.

[0020] In another aspect, the present disclosure provides an isolated anti-CD45 antibody comprising: (a) a heavy chain amino acid sequence as set forth in SEQ ID NO: 9, and a light chain amino acid sequence as set forth in SEQ ID NO: 10; (b) a heavy chain amino acid sequence as set forth in SEQ ID NO: 19, and a light chain amino acid sequence as set forth in SEQ ID NO: 20; (c) a heavy chain amino acid sequence as set forth in SEQ ID NO: 29, and a light chain amino acid sequence as set forth in SEQ ID NO: 30; (d) a heavy chain amino acid sequence as set forth in SEQ ID NO: 39, and a light chain amino acid sequence as set forth in SEQ ID NO: 40; (e) a heavy chain amino acid sequence as set forth in SEQ ID NO: 49, and a light chain amino acid sequence as set forth in SEQ ID NO: 50; (f) a heavy chain amino acid sequence as set forth in SEQ ID NO: 59, and a light chain amino acid sequence as set forth in SEQ ID NO: 60; (g) a heavy chain amino acid sequence as set forth in SEQ ID NO: 69, and a light chain amino acid sequence as set forth in SEQ ID NO: 70; (h) a heavy chain amino acid sequence as set forth in SEQ ID NO: 79, and a light chain amino acid sequence as set forth in SEQ ID NO: 80; (i) a heavy chain amino acid sequence as set forth in SEQ ID NO: 89, and a light chain amino acid sequence as set forth in SEQ ID NO: 90; or (j) a heavy chain amino acid sequence as set forth in SEQ ID NO: 99, and a light chain amino acid sequence as set forth in SEQ ID NO: 100.

[0021] In another aspect, the present disclosure provides an isolated anti-CD45 antibody, or antigen binding portion thereof, that specifically binds to human CD45 at an epitope located within CD45 fragment 1 (SEQ ID NO: 114), CD45 fragment 2 (SEQ ID NO: 115), CD45 fragment 3 (SEQ ID NO: 116), CD45 fragment 4 (SEQ ID NO: 117), and / or CD45 fragment 5 (SEQ ID NO: 118).

[0022] In some embodiments of the above aspects, the isolated anti-CD45 antibody, or antigen binding portion thereof, specifically binds to (a) an epitope of human CD45 located within CD45 fragment 2, and an epitope of human CD45 located within fragment 4; (b) an epitope of human CD45 located within CD45 fragment 1, and an epitope of human CD45 located within CD45 fragment 3; or (c) an epitope of human CD45 located within CD45 fragment 5.

[0023] In some embodiments, the isolated anti-CD45 antibody, or antigen binding portion thereof, specifically binds to (a) one or more residues selected from the group consisting of 405T, 407K, 419Y, 425K, 481R, 505R, and 509H in human CD45 (numbering with reference to SEQ ID NO: 113); or (b) one or more residues selected from the group consisting of 486R, 493Y, and 502T in human CD45 (numbering with reference to SEQ ID NO: 113).

[0024] In some embodiments, the isolated anti-CD45 antibody or antigen binding portion of the present disclosure specifically binds human CD45 and cross-reacts with cynomolgus CD45.

[0025] In some embodiments, the isolated anti-CD45 antibody or antigen binding portion of the present disclosure binds to human CD45 and has an off-rate (K 解离 ) of 1 x 10 -2 to 1 x 10 -3 , 1 x 10 -3 to 1 x 10 -4 , 1 x 10 -4 to 1 x 10 -5 , 1 x 10 -5 to 1 x 10 -6 , 1 x 10 -6 to 1 x 10 -7 , or 1 x 10 -7 to 1 x 10 -8 , as measured by biolayer interferometry (BLI).

[0026] In some embodiments, the isolated anti-CD45 antibody or antigen-binding portion of the disclosure binds to human CD45 and K D about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 10 nM or less, or about 0.1 nM or less, as determined by biolayer interferometry (BLI).

[0027] In another aspect, provided herein is a pharmaceutical composition comprising an antibody or antigen-binding portion thereof of the present disclosure and a pharmaceutically acceptable carrier.

[0028] In another aspect, provided herein is an isolated nucleic acid comprising a nucleic acid sequence encoding an antibody or antigen-binding portion thereof of the present disclosure.

[0029] In another aspect, provided herein is a vector comprising an isolated nucleic acid of the present disclosure.

[0030] In another aspect, provided herein is a host cell comprising an isolated nucleic acid of the present disclosure or a vector of the present disclosure. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell.

[0031] In another aspect, provided herein is a pharmaceutical composition comprising an antibody or antigen-binding portion thereof of any of the above-mentioned aspects for depleting a population of CD45+ cells in a human patient.

[0032] In another aspect, provided herein is a method of depleting a population of CD45+ cells in a human patient by administering to the human patient an antibody or antigen-binding portion thereof of any of the above-mentioned aspects. In some embodiments, the CD45+ cells are CD3+, CD19+, CD33+, CD34+, or CD45+ / B2M+. In other embodiments, the CD45+ cells are hematopoietic stem cells (HSCs). In other embodiments, the CD45+ cells are HSCs, T cells, B cells, and / or bone marrow cells.

[0033] In some embodiments of the above aspects, the CD45+ cells are depleted from the bone marrow of the patient and / or from the peripheral blood of the patient.

[0034] In some embodiments of the above aspects, the patient is in need of a hematopoietic stem cell transplant. In some embodiments of the above aspects, the method further comprises administering to the patient a transplant comprising hematopoietic stem cells. In certain embodiments, the transplant is allogeneic. In alternative embodiments, the transplant is autologous.

[0035] In another aspect, the present application provides an antibody drug conjugate (ADC) comprising an anti-CD45 antibody or antigen-binding portion thereof conjugated to a cytotoxin via a linker, wherein the anti-CD45 antibody or antigen-binding portion thereof is an antibody or antigen-binding portion thereof of any of the above-mentioned aspects.

[0036] In some embodiments of the above-mentioned aspects, the antibody is conjugated to the cytotoxin by way of a cysteine residue in the constant domain of the antibody. In certain embodiments, the cysteine residue is introduced by way of an amino acid substitution in the constant region of the antibody. In particular embodiments, the amino acid substitution is D265C and / or V205C (EU numbering).

[0037] In some embodiments of the above-mentioned aspects, the drug-to-antibody ratio (DAR) of the ADC is 1, 2, 3, 4, 5, 6, 7, or 8.

[0038] In some embodiments of the above-mentioned aspects, the cytotoxin is an RNA polymerase inhibitor, a DNA intercalator, a DNA alkylator, a DNA cross-linker, an agent that disrupts protein synthesis, an agent that disrupts microtubule dynamics, or an agent that disrupts the mitotic spindle.

[0039] In some embodiments of the above-mentioned aspects, the cytotoxin is selected from the group consisting of an amatoxin, an anthracycline, an auristatin, a calicheamicin, a deBouganin, a diphtheria toxin, a duocarmycin, an indolinobenzodiazepine (IGN), an indolinobenzodiazepine dimer, an irinotecan, a maytansine, a maytansinoid, a Pseudomonas exotoxin A, a pyrrolobenzodiazepine (PBD), a pyrrolobenzodiazepine dimer, a saporin, and SN-38. In certain embodiments, the cytotoxin is an RNA polymerase inhibitor. In certain embodiments, the RNA polymerase inhibitor is an amatoxin.

[0040] In some embodiments, the ADC is represented by the formula Ab-Z-L-Am, wherein Ab is an antibody or antigen-binding portion thereof, L is a linker, Z is a chemical moiety, and Am is an amatoxin. In certain embodiments, Am-L-Z is represented by formula (I):

[0041]

[0042] wherein R1is H, OH, OR A or OR C ;

[0043] R2is H, OH, OR B or OR C ;

[0044] R A and R B , if present, combine with the oxygen atom to which they are bound to form an optionally substituted 5-membered heterocycloalkyl group;

[0045] R3is H, R C or R D ;

[0046] R4, R5, R6, and R7are each independently H, OH, OR C , OR D , R C or R D ;

[0047] R8is OH, NH2, OR C , OR D , NHR C or NR C R D ;

[0048] R9is H, OH, OR C or OR D ;

[0049] X is -S-, -S(O)-, or -SO2-;

[0050] R C is -L-Z;

[0051] R D is optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6heteroalkenyl, optionally substituted C2-C6alkynyl, optionally substituted C2-C6heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0052] L is optionally substituted C1-C6alkylene, optionally substituted C1-C6heteroalkylene, optionally substituted C2-C6alkenylene, optionally substituted C2-C6heteroalkenylene, optionally substituted C2-C6alkynylene, optionally substituted C2-C6heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, optionally substituted heteroarylene, a peptide, a dipeptide, -(C=0)-, a disulfide, a hydrazone, or a combination thereof; and

[0053] Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present in the antibody or antigen-binding fragment thereof,

[0054] wherein Am comprises exactly one R C substituent.

[0055] In some embodiments, L-Z is

[0056]

[0057] or

[0058] .

[0059] In some embodiments, the ADC is represented by one of the following:

[0060]

[0061] (III) (IIIA) (IIIB)

[0062] wherein X is -S-, -S(O)-, or -SO2-.

[0063] In some embodiments, the ADC has the formula

[0064]

[0065] wherein Ab represents the point of attachment of the anti-CD45 antibody.

[0066] In other embodiments, the ADC has the formula

[0067]

[0068] wherein Ab represents the point of attachment of the anti-CD45 antibody.

[0069] In alternative embodiments, the ADC has the formula

[0070]

[0071] wherein Ab represents the point of attachment of the anti-CD45 antibody.

[0072] In other embodiments, the ADC has the formula

[0073]

[0074] wherein Ab represents the point of attachment of the anti-CD45 antibody.

[0075] In other embodiments, the ADC has the formula

[0076] ,

[0077] wherein Ab represents the point of attachment of the anti-CD45 antibody.

[0078] In some embodiments of the ADCs disclosed herein, the cytotoxin is a pyrrolobenzodiazepine (PBD). In certain embodiments, the cytotoxin is a PBD dimer. In particular embodiments, the PBD dimer is represented by the following formula

[0079] ,

[0080] wherein the wavy line indicates the point of attachment to the linker of the ADC.

[0081] In some embodiments of the ADCs disclosed herein, the linker comprises one or more of the following: a peptide, an oligosaccharide, -(CH2) p -, -(CH2CH2O) q -, -(C=O)(CH2) r -, -(C=O)(CH2CH2O) t -, -(NHCH2CH2) u -, -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, independently selected at each occurrence.

[0082] In some embodiments of the ADCs disclosed herein, the linker has the following structure:

[0083]

[0084] wherein R1is CH3(Ala) or (CH2)3NH(CO)NH2(Cit).

[0085] In some embodiments of the ADCs disclosed herein, the linker has the following structure prior to attachment to the anti-CD45 antibody and inclusion of the reactive substituent Z' (together in the form L-Z'):

[0086] .

[0087] In certain embodiments, R1is CH3.

[0088] In some embodiments of the ADCs disclosed herein, the cytotoxin-linker conjugate has the following structure before conjugation to the anti-CD45 antibody and inclusion of the reactive substituent Z' (together in the form Cy-L-Z'):

[0089] .

[0090] In some embodiments, the ADCs disclosed herein have the structure

[0091] ,

[0092] wherein Ab is an anti-CD45 antibody or antigen binding portion thereof, and S represents a sulfur atom present in or introduced into the anti-CD45 antibody or antigen binding portion thereof.

[0093] In some embodiments of the ADCs disclosed herein, the cytotoxin is an indolinobenzodiazepine (IGN). In certain embodiments, the cytotoxin is an IGN dimer or IGN pseudodimer.

[0094] In some embodiments, the cytotoxin is an IGN pseudodimer represented by the following formula:

[0095] ,

[0096] wherein the wavy line indicates the point of covalent attachment to the ADC linker.

[0097] In some embodiments, the linker comprises a dipeptide, a disulfide, a C1-C12 alkyl, C=0, or a combination thereof.

[0098] In some embodiments, the linker comprises

[0099] or

[0100] .

[0101] In some embodiments, the cytotoxin-linker conjugate has the following structure before conjugation to the anti-CD45 antibody or antigen binding portion thereof and inclusion of the reactive substituent Z' (together in the form Cy-L-Z'):

[0102] .

[0103] In another aspect, the present disclosure provides a pharmaceutical composition comprising the ADC described above and a pharmaceutically acceptable carrier.

[0104] In another aspect, provided herein is a method of depleting a population of CD45+ cells in a human patient by administering to the patient an effective amount of an anti-CD45 ADC described above.

[0105] In some embodiments of the above aspects, the CD45+ cell lineage is CD3+, CD19+, CD33+, CD34+, or CD45+ / B2M+. In certain embodiments, the CD45+ cell lineage is a hematopoietic stem cell (HSC). In other embodiments, the CD45+ cell lineage is a HSC, a T cell, a B cell, and / or a bone marrow cell.

[0106] In some embodiments of the above aspects, the CD45+ cell lineage is depleted from the bone marrow of the patient and / or from the peripheral blood of the patient.

[0107] In some embodiments of the above aspects, the patient is in need of a hematopoietic stem cell transplant.

[0108] In some embodiments of the above aspects, the method further comprises administering to the patient a transplant comprising hematopoietic stem cells.

[0109] In another aspect, the present disclosure provides a method of depleting a population of CD45+ cells in a human patient in need of a hematopoietic stem cell (HSC) transplant by administering to the patient an ADC described above prior to the patient receiving a transplant comprising hematopoietic stem cells.

[0110] In another aspect, the present disclosure provides a method comprising: (a) administering to a human patient an amount of an ADC described above sufficient to deplete a population of CD45+ cells in the patient; and (b) subsequently administering to the patient a transplant comprising hematopoietic stem cells.

[0111] In some embodiments, the transplant is allogeneic. In other embodiments, the transplant is autologous.

[0112] In some embodiments, the transplant comprising hematopoietic stem cells is administered to the patient after the concentration of the ADC has been substantially cleared from the blood of the patient.

[0113] In some embodiments, the hematopoietic stem cells or progeny thereof are capable of localizing to hematopoietic tissue and / or reconstituting hematopoiesis after transplantation of the hematopoietic stem cells into the patient.

[0114] In some embodiments, the hematopoietic stem cells, after transplantation into the patient, give rise to a recovery of a population of cells selected from the group consisting of megakaryocytes, thrombocytes, platelets, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

[0115] In some embodiments, the patient has a hematological disease, a metabolic disorder, a cancer, or an autoimmune disorder or a severe combined immunodeficiency disease (SCID). In certain embodiments, the patient has a cancer. In particular embodiments, the cancer is a hematological cancer. In specific embodiments, the hematological cancer is acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, or multiple myeloma.

[0116] In certain embodiments, the patient has an autoimmune disorder. In particular embodiments, the autoimmune disorder is multiple sclerosis, type 1 diabetes, or scleroderma. In other embodiments,The autoimmune disorder is selected from the group consisting of multiple sclerosis, human systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, treating psoriasis, type 1 diabetes mellitus, acute disseminated encephalomyelitis, Addison's disease, alopecia universalis, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune oophoritis, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelinating polyneuropathy, Crohn's disease, cicatricial pemphigoid, celiac sprue-dermatitis herpetiformis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus, dysautonomia, endometriosis, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic and / or acute thrombocytopenic purpura, idiopathic pulmonary fibrosis, IgA neuropathy, interstitial cystitis, juvenile arthritis, Kawasaki's disease, lichen planus, Lyme disease, Meniere's disease, mixed connective tissue disease, myasthenia gravis, neuromyotonia, opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndromes, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis, vitiligo, vulvodynia, and Wegener's granulomatosis.

[0117] In addition, the present application also includes the following embodiments:

[0118] In one embodiment, the present application provides an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising a heavy chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 2, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 3, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 4; and a light chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 7, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 8.

[0119] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 1.

[0120] In other embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 5.

[0121] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 1; and

[0122] a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 5.

[0123] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 9.

[0124] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 10.

[0125] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 9; and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 10.

[0126] In one embodiment, the present application provides an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising a heavy chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 12, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 13, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 14; and a light chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 16, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 17, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 18.

[0127] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 11.

[0128] In other embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 15.

[0129] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 11; and

[0130] a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 15.

[0131] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 19.

[0132] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 20.

[0133] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 19; and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 20.

[0134] In one embodiment, the present application provides an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising a heavy chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 22, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 23, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 24; and a light chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 26, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 27, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 28.

[0135] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 21.

[0136] In other embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 25.

[0137] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 21; and

[0138] a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 25.

[0139] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 29.

[0140] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 30.

[0141] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 29; and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 30.

[0142] In one embodiment, the present application provides an isolated anti-CD45 antibody, or antigen-binding portion thereof, comprising a heavy chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 32, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 33, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 34; and a light chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 36, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 37, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 38.

[0143] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 31.

[0144] In other embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 35.

[0145] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 31; and

[0146] a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 35.

[0147] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 39.

[0148] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 40.

[0149] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 39; and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 40.

[0150] In one embodiment, the present application provides an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising a heavy chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 42, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 43, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 44; and a light chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 46, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 47, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 48.

[0151] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 41.

[0152] In other embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 45.

[0153] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 41; and

[0154] a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 45.

[0155] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 49.

[0156] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 50.

[0157] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 49; and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 50.

[0158] In one embodiment, the present application provides an isolated anti-CD45 antibody, or antigen-binding portion thereof, comprising a heavy chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 52, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 53, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 54; and a light chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 56, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 57, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 58.

[0159] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 51.

[0160] In other embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 55.

[0161] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 51; and

[0162] a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 55.

[0163] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 59.

[0164] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 60.

[0165] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 59; and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 60.

[0166] In one embodiment, the present application provides an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising a heavy chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 62, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 63, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 64; and a light chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 66, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 67, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 68.

[0167] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 61.

[0168] In other embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 65.

[0169] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 61; and

[0170] a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 65.

[0171] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 69.

[0172] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 70.

[0173] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 69; and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 70.

[0174] In one embodiment, the present application provides an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising a heavy chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 72, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 73, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 74; and a light chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 76, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 77, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 78.

[0175] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 71.

[0176] In other embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 75.

[0177] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 71; and

[0178] a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 75.

[0179] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 79.

[0180] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 80.

[0181] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 79; and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 80.

[0182] In one embodiment, the present application provides an isolated anti-CD45 antibody, or antigen binding portion thereof, comprising a heavy chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 82, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 83, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 84; and a light chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 86, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 87, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 88.

[0183] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 81.

[0184] In other embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 85.

[0185] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 81; and

[0186] a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 85.

[0187] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 89.

[0188] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 90.

[0189] In certain embodiments, the anti-CD45 antibody, or antigen binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 89; and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 90.

[0190] In one embodiment, the present application provides an isolated anti-CD45 antibody, or antigen-binding portion thereof, comprising a heavy chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 92, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 93, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 94; and a light chain comprising a variable region comprising a CDR1 having an amino acid sequence as set forth in SEQ ID NO: 96, a CDR2 having an amino acid sequence as set forth in SEQ ID NO: 97, a CDR3 having an amino acid sequence as set forth in SEQ ID NO: 98.

[0191] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 91.

[0192] In other embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 95.

[0193] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 91; and

[0194] a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 95.

[0195] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 99.

[0196] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 100.

[0197] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 99; and a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 100.

[0198] In one embodiment, the anti-CD45 antibody described herein is intact.

[0199] In certain embodiments, the anti-CD45 antibody fragment is selected from the group consisting of a Fab, a F(ab')2, and a scFv.

[0200] In other embodiments, the anti-CD45 antibody or its antigen-binding portion is a human antibody or its binding fragment.

[0201] In other embodiments, the anti-CD45 antibody or its antigen-binding portion comprises an Fc region containing at least one amino acid substitution of H435 or I235 / H310 / H435 (EU designation). In one embodiment, the Fc region contains an H435 amino acid substitution of H435A (EU designation). In other embodiments, the Fc region contains an I235 / H310 / H435 amino acid substitution shown as I235A / H310A / H435A (EU designation).

[0202] In some embodiments, the anti-CD45 antibody or its antigen-binding portion is IgG, such as IgG1 or IgG4.

[0203] This article also describes antibody-drug conjugates (ADCs) containing anti-CD45 antibodies (or antigen-binding fragments), wherein the antibody (or CD45-binding fragment) binds to the cytotoxic agent via a linker.

[0204] In one embodiment, the anti-CD45 ADC comprises an anti-CD45 antibody that binds to a cytotoxin (RNA polymerase inhibitor). In one embodiment, the RNA polymerase inhibitor is amatoxins.

[0205] In one embodiment, the anti-CD45 ADC comprises a cytotoxin, which is a phallotoxin represented by formula (IA).

[0206] (IA)

[0207] Among them, R1 series H, OH, OR A OR C ;

[0208] R2 series H, OH, OR B OR C ;

[0209] R A and R B Together with the oxygen atom it is bonded to, it forms, as appropriate, a substituted 5-membered heterocyclic alkyl group;

[0210] R3 series H, R C Or R D ;

[0211] R4, R5, R6, and R7 each independently generate H, OH, and OR. C OR D R C Or R D ;

[0212] R8is OH, NH2, OR C , OR D , NHR C or NR C R D ;

[0213] R9is H, OH, OR C or OR D ;

[0214] X is -S-, -S(O)-, or -SO2-;

[0215] R C is -L-Z;

[0216] R D is optionally substituted C1-C6alkyl, optionally substituted C1-C6heteroalkyl, optionally substituted C2-C6alkenyl, optionally substituted C2-C6heteroalkenyl, optionally substituted C2-C6alkynyl, optionally substituted C2-C6heteroalkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted aryl, or optionally substituted heteroaryl;

[0217] L is optionally substituted C1-C6alkylene, optionally substituted C1-C6heteroalkylene, optionally substituted C2-C6alkenylene, optionally substituted C2-C6heteroalkenylene, optionally substituted C2-C6alkynylene, optionally substituted C2-C6heteroalkynylene, optionally substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted arylene, or optionally substituted heteroarylene; and

[0218] Z is a chemical moiety formed from a coupling reaction between a reactive substituent present on L and a reactive substituent present within the antibody or antigen binding fragment thereof,

[0219] wherein Am comprises exactly one R C substituent.

[0220] In one embodiment, the anti-CD45 ADC has the formula

[0221] ,

[0222] , or

[0223] ,

[0224] wherein Ab represents the point of attachment of the anti-CD45 antibody.

[0225] In one embodiment, the anti-CD45 ADC has the formula

[0226] or ,

[0227] wherein Ab represents the point of attachment of the anti-CD45 antibody.

[0228] In one embodiment, the anti-CD45 ADC comprises a cytotoxin that is an amanitin (e.g., a-amanitin, β-amanitin, γ-amanitin, ε-amanitin), amanin, amaninamide, amanullin, amanullinic acid, and proto-amanullin.

[0229] In one embodiment, the anti-CD45 ADC comprises a cytotoxin selected from the group consisting of Pseudomonas exotoxin A, gelonin, diphtheria toxin, saporin, maytansinoid, maytansinoid analog, auristatin (e.g., MMAE or MMAF), anthracycline, calicheamicin, irinotecan, SN-38, duocarmycin, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, and indolinobenzodiazepine dimer.

[0230] In one embodiment, the anti-CD45 ADC comprises a cytotoxin comprising a benzodiazepine moiety. In some embodiments, the anti-CD45 ADC comprises a pyrrolobenzodiazepine (“PBD”). In some embodiments, the anti-CD45 ADC comprises an indolinobenzodiazepine (“IGN”).

[0231] In one embodiment, the anti-CD45 ADC comprises an anti-CD45 antibody that is conjugated to a toxin via a cysteine residue in the antibody Fc domain. In one embodiment, the cysteine residue is introduced via an amino acid substitution in the antibody Fc domain. In one embodiment, the amino acid substitution is D265C and / or V205C (EU numbering).

[0232] In certain embodiments, the drug-to-antibody ratio (DAR) of the anti-CD45 ADC is 1, 2, 3, 4, 5, 6, 7, or 8.

[0233] The present disclosure also includes pharmaceutical compositions comprising an anti-CD45 antibody or ADC described herein and a pharmaceutically acceptable carrier.

[0234] The anti-CD45 antibodies, fragments, and ADCs described herein can be used in methods of treatment of human patients, including, but not limited to, conditioning treatment prior to allogeneic or autologous transplantation.

[0235] In one embodiment, disclosed herein is a method of depleting a population of hematopoietic stem cells (HSCs) in a human patient, the method comprising administering to the patient an effective amount of an anti-CD45 antibody, fragment, or ADC described herein. In certain embodiments, the method further comprises administering to the patient a transplant comprising hematopoietic stem cells.

[0236] In other embodiments, disclosed herein is a method comprising administering to a human patient a transplant comprising hematopoietic stem cells (allogeneic or autologous), wherein the patient has been previously administered an amount of an anti-CD45 antibody, fragment, or ADC described herein sufficient to deplete a population of hematopoietic stem cells in the patient. In certain embodiments, the hematopoietic stem cells are CD45+ cells.

[0237] In other embodiments, an anti-CD45 antibody, fragment, or ADC described herein is used to treat a human patient having a hematological disease, a metabolic disorder, a cancer, or an autoimmune disease or a severe combined immunodeficiency disease (SCID).

[0238] In one embodiment, an anti-CD45 antibody, fragment, or ADC described herein is administered to a human patient to treat leukemia in the human patient.

[0239] In other embodiments, disclosed herein is a method comprising administering to a human patient a transplant comprising hematopoietic stem cells, wherein the patient has been previously administered an amount of an anti-CD45 antibody, fragment, or ADC described herein sufficient to deplete a population of immune cells in the patient. In one embodiment, the immune cells are CD137+, CD2+, or CD5+ cells. In other embodiments, the immune cells are T cells. BRIEF DESCRIPTION OF DRAWINGS

[0240] Figure 1 Results of in vitro binding analysis are depicted in graphical format, wherein affinity matured anti-CD45 antibodies and their corresponding parental antibodies (Parentals 1-4) were evaluated for binding to human CD45 or cynomolgus CD45. Parental 1 corresponds to Ab1 described herein. Binding was measured by Bio-Layer Interferometry (BLI) of the indicated purified IgG (sensogram) incubated with purified human CD45 or cynomolgus CD45 extracellular domain.

[0241] Figure 2Results of in vitro cell binding assays to evaluate the binding of the indicated anti-CD45 antibodies (Ab5 and Ab7) to REH cells, human PBMC cells, and cynomolgus monkey ("cyno") PBMC cells are depicted in graphical format. Fc variants of Ab5 and Ab7, Ab5_D265C_LALA_H435A and A7_D265C_LALA_H435A, were used in this study. Non-targeted isotype IgG with Fc modification region (D265C LALA H435A) was evaluated as a control.

[0242] Figure 3 Results of CD45 epitope mapping studies using anti-CD45 antibody Ab5 are depicted. A schematic of the Ab5 interaction site on CD45 is depicted, with the amino acid sequences (SEQ ID NO: 115 and SEQ ID NO: 117) around the Ab5-epitope noted. Contact residues are highlighted as 405T, 407K, 419Y, 425K, 481R, 505R, and 509H, with residue numbering with reference to the human CD45 fragment represented by SEQ ID NO: 113.

[0243] Figure 4 Results of in vitro internalization assays to evaluate the internalization of anti-CD45 antibody-drug conjugates (ADCs) constructed from Ab5 in human CD34+ bone marrow cells are depicted in graphical format. Fc variant of Ab5, Ab5 D265C.LALA.H435A, was conjugated to a venetoclax peptide (venetoclax 1 (AM1)) to form Ab5-AM1 D265C.LALA.H435A. The anti-CD45 ADC was conjugated to a water-soluble, bright, photosensitive, and pH-reactive pHAb dye. Upon internalization, the bound antibody can move to an acidic endosome / lysosome, where the pHAb dye emits at 563 nM and can be detected by flow cytometry. Human bone marrow CD34+ cells were incubated on ice for 2 hours, with the ADC at saturating concentration for 0 hours, 2 hours, 24 hours, 48 hours, or 72 hours. The left panel depicts the pHAb levels over time in graphical format. The bound surface hlgGl was evaluated by flow cytometry using a fluorophore-labeled anti-lgG molecule, from which the percentage of surface IgG over time was calculated, as depicted in the right panel.

[0244] Figure 5A and 5BResults from in vitro cell line killing assays are graphically depicted showing that anti-CD45 ADCs constructed from Ab4 and Ab5 can effectively kill in vitro CD45+ cell lines (Jurkat (acute T-cell leukemia cell line, ATCC number TIB-152), SKNO-1 (acute myeloid leukemia cell line JCRB1170), and REH-1 (B cell non-Hodgkin's lymphoma cell line, ATCC number CRL-3004)). Fc variants of Ab4 and Ab5, Ab4 D265C.LALA.H435A and Ab5 D265C.LALA.H435A, were conjugated to a venetoclax (venetoclax 1 (AM1) or venetoclax 2 (AM2)) to form Ab4-AM2 D265C.LALA.H435A (“Ab4-AM2”) and Ab5-AM1 D265C.LALA.H435A (“Ab5-AM1”). In Figure 5A SKNO1, Jurkat, REH (CD45+) or REH (CD45- / -) cell lines were cultured for 7 days in the presence of Ab5-AM1 or a control non-targeting isotype-matched ADC (“isotype-AM1”), and cell viability was measured as a function of antibody concentration (x-axis) by luminescence (y-axis) by Celltiter Glo. In Figure 5B SKNO1, Jurkat, or REH (CD45+) cell lines were cultured for 7 days in the presence of Ab4-AM2 or a control non-targeting isotype-matched ADC (“isotype-AM2”), and cell viability was measured as a function of antibody concentration (x-axis) by luminescence (y-axis) by Celltiter Glo.

[0245] Figures 6A-6C Results from in vitro primary cell killing assays are graphically depicted showing that anti-CD45 ADCs constructed from Ab2, Ab4, or Ab5 can effectively kill in vitro primary human or cynomolgus monkey peripheral blood mononuclear cells (PBMCs) or human hematopoietic stem cells (HSCs). Fc variants of Ab2, Ab4, and Ab5, Ab2 D265C.LALA.H435A, Ab4 D265C.LALA.H435A, Ab5 D265C.LALA.H435A, were conjugated to a venetoclax (venetoclax 1 (AM1) or venetoclax 2 (AM2)) to form Ab2-AM2 D265C.LALA.H435A (“Ab2-AM2”), Ab4-AM2 D265C.LALA.H435A (“Ab4-AM2”), and Ab5-AM1 D265C.LALA.H435A (“Ab5-AM1”). In Figure 6AIn this study, human PBMCs were cultured for 7 days in either CD45-AM conjugate (Ab5-AM1) or a control non-targeted isotype-matched ADC ("isotype-AM1"), and cell viability (y-axis) was measured as a function of antibody concentration (x-axis) using CelltiterGlo. Figure 6B and Figure 6C In the middle, the original human CD34 + Bone marrow cells and anti-CD45-ADC ( Figure 6B Ab5-AM1 in the middle; Figure 6C The antibodies (Ab2-AM2 or Ab4-AM2) or homo-AM were cultured together for 5 days, and the active CD34+CD90+ HSC count (y-axis) was measured by flow cytometry as the antibody concentration changed (x-axis).

[0246] Figure 7 The results of in vitro primary cell killing analysis are presented graphically, showing that the anti-CD45 ADC constructed from Ab2 can effectively kill unstimulated (non-driven) and stimulated (driven) peripheral blood mononuclear cells (PBMCs) in vitro. The Fc variant of Ab2, Ab2 D265C.LALA.H435A, binds to phallotoxin 2 (AM2) to form Ab2-AM2D265C.LALA.H435A (“Ab2-AM2”). Stimulated and unstimulated PBMCs were cultured for 4 days in the presence of the CD45-AM conjugate (Ab2-AM2) or the control non-targeted isotype-matched ADC (“isotype-AM2”), and cell viability (y-axis) as a function of antibody concentration was measured using Celltiter Glo.

[0247] Figure 8 The results of the in vitro killing assay are presented graphically, showing that the anti-CD45 ADC constructed from Ab6 can effectively kill in vivo macrophages. The Fc variant of Ab6, Ab6 D265C.LALA.H435A, binds to phallotoxin 1 (AM1) to form Ab6-AM1 D265C.LALA.H435A ("Ab6-AM1"). Macrophages were cultured for 6 days in the presence of the CD45-AM conjugate (Ab6-AM1) or the control non-targeted isotype-matched ADC ("isotype-AM1"), and cell viability was measured by Celltiter Glo luminescence (RLU; y-axis) as a function of antibody concentration (x-axis).

[0248] Figure 9Results of a serum stability analysis depicting evaluation of the stability of anti-CD45 ADCs constructed from Ab5 in human serum (top panel) or cynomolgus monkey ("cyno") serum (bottom panel) in a graphical manner. Fc variant Ab5 D265C.LALA.H435A was conjugated to Umbrella Peptide 1 (AM1) to form Ab5-AM1 D265C.LALA.H435A ("Ab5-AM1"). To determine if this ADC is stable in serum, the ADC was pre-incubated in human or cynomolgus monkey (cyno) serum at 37 °C for 0 or 72 hours, then the ADC was evaluated for cell killing against REH cells. % cell viability of REH cells as a function of ADC concentration (x-axis) treated with serum was evaluated by Cell-titer Glo (y-axis).

[0249] Figures 10A-10E Results of an in vivo cell depletion analysis showing that short half-life anti-CD45 ADCs constructed from Ab2, Ab3, Ab5, and Ab7 selectively deplete human HSCs and immune cells in humanized NSG mice. Ab2 D265C.LALA.H435A, Ab3 D265C.LALA.H435A, Ab5 D265C.LALA.H435A, and Ab7 D265C.LALA.H435A (Fc variants of Ab2, Ab3, Ab5, and Ab7) were conjugated to Umbrella Peptide 1 (AM1) or Umbrella Peptide 2 (AM2) to form Ab2 D265C.LALA.H435A-AM2 ("Ab2-AM2"), Ab3 D265C.LALA.H435A-AM2 ("Ab3-AM2"), Ab5 D265C.LALA.H435A-AM1 ("Ab5-AM1"), and Ab7 D265C.LALA.H435A-AM1 ("Ab7-AM1"). PBS treated mice were evaluated as controls. Figure 10A Percentage of human CD3+ T cells, CD19+ B cells, and CD33+ myeloid cells in mice relative to baseline 14 days after administration of anti-CD45-ADCs (Ab2-AM2, Ab3-AM2, AbA-AM2). Figure 10B Percentage of human CD45+ cells in peripheral blood at 0 days, 7 days, and 14 days after administration of anti-CD45-ADCs (Ab2-AM2 or Ab3-AM2). Figure 10C Absolute number of human CD45 cells (left panel) and human HSCs (right panel) in bone marrow of humanized NSG mice 14 days after administration of anti-CD45-ADCs (Ab2-AM2 or Ab3-AM2). Figure 10DThe percentage and absolute number of HSCs (CD34+ cells, CD34+CD38- and CD34+CD117+ cells) in the bone marrow of humanized NSG mice 14 days after administration of anti-CD45 ADCs (Ab2-AM2 and Ab3-AM2) were plotted in a graphical manner. Figure 10E The percentage of human CD45 cells in peripheral blood (relative to baseline), the absolute number of human CD45 cells in bone marrow, and the absolute number of HSCs (CD34+CD38- cells) in bone marrow of humanized NSG mice were plotted in graph form 14 days after administration of anti-CD45-ADC (Ab5-AM1 or Ab7-AM1).

[0250] Figure 11A and 11B The results of in vivo cell depletion analysis are presented graphically, showing that the short half-life anti-CD45-phallothin ADCs constructed from Ab4, Ab5, and Ab7 can effectively deplete HSCs and immune cells in non-human primates in vivo. Ab4 D265C.LALA.H435A, Ab5 D265C.LALA.H435A, and Ab7 D265C.LALA.H435A (Fc variants of Ab4, Ab5, and Ab7) bind to phallothin 1 (AM1) to form Ab4 D265C.LALA.H435A-AM2 (“Ab4-AM1”), Ab5 D265C.LALA.H435A-AM1 (“Ab5-AM1”), and Ab7 D265C.LALA.H435A-AM1 (“Ab7-AM1”). Figure 11A The absolute number of lymphocytes in the peripheral blood of cynomolgus monkeys 72 hours after administration of anti-CD45 ADC (0.5 mg / kg or 2 mg / kg of Ab5-AM1 or Ab7-AM1) is plotted in a graph. Figure 11B The levels of white blood cells, HSCs, and lymphocytes in the bone marrow of cynomolgus monkeys on day 7 after administration of anti-CD45ADC (1 mg / kg Ab4-AM1 or 0.5 or 2 mg / kg Ab5-AM1) were plotted in a graphical format.

[0251] Figure 12Results of pharmacokinetic analysis of short half-life anti-CD45 ADCs constructed from Ab4 in cynomolgus macaques are depicted in graphical format. Fc variant Ab4 D265C.LALA.H435A was conjugated to either umbrella peptide 1 (AM1) or umbrella peptide 2 (AM2) to form Ab4 D265C.LALA.H435A-AM1 ("Ab4-AM1") and Ab4 D265C.LALA.H435A-AM2 ("Ab4-AM2"). Mean plasma drug concentrations (y-axis) for each ADC were monitored as a function of time (x-axis).

[0252] Figure 13 Results of multi-specific reagent (PSR) analysis evaluating binding of humanized clones derived from parental clones A, B, and C of Example 15 to a mixture of membrane and cytoplasmic proteins are depicted in graphical format.

[0253] Figure 14 Results of in vitro cell binding analysis for evaluating binding of anti-CD45 antibodies AbA, AbB, and AbC (identified in Example 15) to human PBMC cells and cynomolgus monkey ("cyno") PBMC cells are depicted in graphical format.

[0254] Figure 15 Results of CD45 epitope mapping studies using anti-CD45 antibody AbA identified in Example 15 are depicted. A schematic of AbA interaction sites on CD45 is depicted, with the amino acid sequence (SEQ ID NO: 117) surrounding the AbA epitope noted. Contact residues are highlighted as 486R, 493Y, and 502T, with residue numbering with reference to human CD45 fragment represented by SEQ ID NO: 113.

[0255] Figure 16Results of an in vitro internalization assay evaluating the internalization of anti-CD45 antibody-drug conjugates (ADCs) constructed from AbA in human CD34+ bone marrow cells are depicted graphically. Fc variant AbA_D265C_LALA_H435A of AbA was conjugated to a venetoclax peptide (venetoclax 1) to form anti-CD45 ADC AbA_D265C_LALA_H435A-AM1. The anti-CD45 ADC was conjugated to a water-soluble, bright, photosensitive, and pH-reactive pHAb dye. Upon internalization, the bound antibody can move to an acidic endosome / lysosome, where the pHAb dye emits at 563 nM and can be detected by flow cytometry. Human bone marrow CD34+ cells were incubated on ice for 2 hours, with a saturating concentration of ADC for 0 hours, 2 hours, 24 hours, 48 hours, or 72 hours. The left panel depicts the pHAb over time in a graph. The bound surface hlgGl was evaluated by flow cytometry using a fluorophore-labeled anti-lgG molecule, from which the percentage of surface IgG over time was calculated, as depicted in the right panel.

[0256] Figure 17A and 17B Results of an in vitro primary cell killing assay showing that anti-CD45 ADCs constructed from AbA can effectively kill in vitro primary human or cynomolgus monkey peripheral blood mononuclear cells (PBMCs) are depicted graphically. Fc variant AbA_D265C_LALA_H435A of AbA was conjugated to one of two venetoclax peptides, venetoclax 1 (AM1) or venetoclax 2 (AM2), to form AbA_D265C_LALA_H435A-AM1 or AbA_D265C_LALA_H435A-AM2. Human PBMCs were cultured for 7 days in the presence of the indicated CD45-AM conjugate (AbA_D265C_LALA-H435A-AM1 Figure 17A ) or AbA_D265C_LALA-H435A-AM2 Figure 17B ) or a control non-targeted isotype-matched ADC (“isotype-AM1” or “isotype-AM2”), and cell viability (y-axis) as a function of antibody concentration (x-axis) was measured by Celltiter Glo.

[0257] Figure 18Results of in vitro primary cell killing assay are depicted in a graph showing that anti-CD45 ADCs constructed from AbA can effectively kill in vitro human hematopoietic stem cells (HSCs). Fc variants of AbA, AbA_D265C_LALA_H435A and AbA_D265C_LALA_IHH, were conjugated to Umbrella Peptide 1 (AM1), Umbrella Peptide 2 (AM2), or PBD to form AbA_D265C_LALA_H435A-AM1, AbA_D265C_LALA_H435A-AM2, or AbA_D265C_LALA_IHH-PBD. Primary human CD34 + Bone marrow cells were cultured for 5 days. Live CD34+CD90+ HSC counts were determined by flow cytometry as a function of antibody concentration (x-axis).

[0258] Figure 19 Results of in vitro killing assay are depicted in a graph showing that anti-CD45 ADCs constructed from AbA can effectively kill in vitro macrophages. Fc variant of AbA, AbA_D265C_LALA_H435A, was conjugated to Umbrella Peptide 1 (AM1) to form CD45 ADC AbA_D265C_LALA_H435A-AM1. Macrophages were cultured for 6 days in the presence of ADC (AbA_D265C_LALA_H435A-AM1) or control non-targeting isotype-matched ADC (“Iso-AM1”) and cell viability was measured as luminescence by CelltiterGlo (RLU; y-axis) as a function of antibody concentration (x-axis).

[0259] Figure 20 Results of in vivo depletion assay in humanized NSG mice treated with anti-CD45 antibody drug conjugate (AbA-PBD) are depicted in a graph, where depletion of human cells in peripheral blood was evaluated. hNSG mice were administered the indicated single dose of vehicle (PBS), isotype control-PBD (“Iso-PBD”), or CD45-PBD (AbA-PBD). Peripheral blood was collected at the indicated time points and assessed for total human hematopoietic cell content (hβ2M + ), bone marrow cell content (CD33 + ), B cell content (CD19 + ), and T cell content (CD3 + ). Results are presented as % depletion normalized to baseline.

[0260] Figure 21Results of in vivo depletion analysis in humanized NSG mice treated with anti-CD45-ADC (AbA-PBD) are graphically depicted, wherein depletion of human cells in the bone marrow was evaluated. hNSG mice were administered the indicated single dose of vehicle (PBS), Iso-PBD or CD45-PBD (AbA-PBD). BM samples were collected at day 14 post-treatment and human progenitor / HSC content was assessed. Results are presented as % of human cells and absolute number / femur.

[0261] Figure 22 Results of in vivo depletion analysis in humanized NSG mice treated with anti-CD45-ADC (AbA-PBD) are graphically depicted, wherein human CD45+ cells, double positive (DP) thymocytes, mature CD4 + single positive (SP) thymocytes or mature CD8 + single positive (SP) thymocytes were evaluated at 14 days post-treatment. hNSG mice were administered the indicated single dose of vehicle (PBS), Iso-PBD or CD45-PBD (AbA-PBD).

[0262] Figure 23 Results of in vivo depletion analysis in humanized NSG mice treated with anti-CD45 antibody drug conjugate (AbA-IGN) are graphically depicted, wherein depletion of human cells in the peripheral blood was evaluated. hNSG mice were administered the indicated single dose of vehicle (PBS), Iso-control-IGN ("Iso-IGN") or CD45-IGN (AbA-IGN). Peripheral blood was collected at the indicated time points and total human hematopoietic cell content (hβ2M + ), bone marrow cell content (CD33 + ), B cell content (CD19 + ) and T cell content (CD3 + ) were assessed. Results are presented as % depletion normalized to baseline.

[0263] Figure 24 Results of in vivo depletion analysis in humanized NSG mice treated with CD45-IGN are graphically depicted, wherein depletion of human cells in the bone marrow was evaluated. hNSG mice were administered the indicated single dose of vehicle (PBS), Iso-IGN or CD45-IGN. BM samples were collected at day 14 post-treatment and human progenitor / HSC content was assessed. Results are presented as % of human cells and absolute number / femur.

[0264] Figure 25Results of in vivo depletion analysis in humanized NSG mice treated with AbA-IGN are depicted in a graphical manner, where human CD45+ cells, double positive (DP) thymocytes, mature CD4 + single positive (SP) thymocytes or mature CD8 + Depletion of single positive (SP) thymocytes. hNSG mice were administered the indicated single dose of vehicle (PBS), isotype-IGN or AbA-IGN.

[0265] Figure 26 Results of in vivo cell depletion analysis showing that anti-CD45-umbrella peptide ADCs constructed from AbA can effectively deplete non-human primate HSCs and immune cells in vivo are depicted in a graphical manner. Fc variants of AbA (introducing modifications in the Fc region to shorten half-life) AbA_D265C_LALA_H435A were conjugated to either Umbrella Peptide 1 (AM1) or Umbrella Peptide 2 (AM2) to form AbA_D265C_LALA_H435A-AM1 (“CD45 ADC-AM1”) or AbA_D265C_LALA_H435A-AM2 (“CD45 ADC-AM2”). Levels of white blood cells, HSCs and immune cells (B cells, T cells and myeloid cells) in cynomolgus monkey bone marrow were measured at the indicated times (day 6, day 14, day 20 and day 28) after a single 1 mg / kg injection of the indicated ADC.

[0266] Figure 27 Results of pharmacokinetic analysis of anti-CD45 ADCs constructed from AbA in cynomolgus macaques are depicted in a graphical manner. Fc variants of AbA (introducing modifications in the Fc region to shorten half-life) AbA_D265C_LALA_H435A were conjugated to either Umbrella Peptide 1 (AM1) or Umbrella Peptide 2 (AM2) to form AbA_D265C_LALA_H435A-AM1 (“CD45 ADC-AM1”) or AbA_D265C_LALA_H435A-AM2 (“CD45 ADC-AM2”). Mean plasma drug concentrations (y-axis) for each ADC were monitored as a function of time (x-axis).

[0267] Figure 28The results of in vivo rodent studies are illustrated graphically, showing that a single dose of AbA-AM ADC has a tumor-reducing effect on patient-derived tumors and extends survival beyond standard care in a set of models simulating untreated and refractory disease. Patient-derived xenograft (PDX) models of AML [AML #1 (derived from untreated patients), AML #2 (derived from highly pretreated relapsed / refractory patients after allogeneic HSCT)] and T-ALL (derived from patients who progressed after DHAP chemotherapy), as well as a cell line-derived ALL model (REH-Luc) of immortalized cell lines, were systemically inoculated into immunodeficient mice (NSG-SGM3 or NSG). Treatment was initiated when the peripheral tumor burden in PDX (n=3-5 mice / group) and CDX mice reached 2-16% of blast cells in the peripheral blood, or on day 5 after implantation in the REH-luc model (n=10 mice / group). This study compared single-dose anti-CD45-ADCs (1 mg / kg, 3 mg / kg, 6 mg / kg, or 10 mg / kg) with mediators (PBS) or homologous anti-AM (6 mg / kg or 10 mg / kg), and was comparable to clinically validated standard of care regimens such as Ara-c (30 mg / kg QD×5, IV), dexamethasone (5 mg / kg Q3D×9, IP), or doxorubicin (3 mg / kg QW×3, IV). Tumor burden during the study period was shown.

[0268] Figures 29A-29C The results of in vivo rodent studies are presented graphically, showing that short half-life CD45-ADC (AbA-AM) increases median survival in the REH-luciferase ALL xenograft model. Figure 29A The survival rate of mice in the indicated treatment group as of days post-implantation is plotted in a graph. Figure 29B The radiation levels of REH-luciferase in the indicated treatment groups during the study period are plotted in a graph (mean ± SEM). Figure 29C These are representative bioluminescent signal false-color images captured on days 22-23 post-implantation in all treatment groups.

[0269] Figure 30A and 30B The results of in vivo mouse studies are presented graphically, showing that in the T-ALL PDX model, short half-life CD45-ADC (AbA-AM) reduces peripheral leukemia cells, thereby causing tumor growth delay. Figure 30A The survival rate of mice in the indicated treatment group as of days post-implantation is plotted in a graph. Figure 30BTumor burden (hCD45+) in peripheral blood of mice as a function of days post-implantation in each indicated treatment group is depicted graphically.

[0270] Figures 31A-31D Results from in vivo murine studies showing that short half-life CD45-DC (AbA-AM) can effectively deplete human leukemia cells in two patient-derived AML models are depicted graphically. Figure 31A and 31B Survival of PDX model AML #1 mice ( Figure 31A ) or PDX model AML #2 mice ( Figure 31B ) as a function of days post-implantation in the indicated treatment groups is depicted graphically. Figure 31A and 31B The inset of each depicts flow cytometric analysis of each AML PDX model to evaluate CD117 and CD45 cell surface expression on splenocytes of diseased mice. Figure 31C and 31D Tumor burden (hCD45+) in peripheral blood of PDX model AML #1 mice ( Figure 31C ) and PDX model AML #2 mice ( Figure 31D ) as a function of days post-implantation in each indicated treatment group is depicted graphically.

[0271] Figure 32 Depicted is a multiple sequence alignment of the heavy chain variable region and light chain variable region of anti-CD45 antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, and Ab7. The CDRs of each variable region are indicated in bold. DETAILED DESCRIPTION

[0272] Disclosed herein are novel anti-CD45 antibodies, antigen-binding fragments thereof, and conjugates thereof (e.g., antibody drug conjugates; ADCs), which are useful, for example, because of their cross-reactivity between human CD45 and non-human primate CD45. In addition, the anti-CD45 antibodies and fragments thereof described herein can be used as therapeutic agents, for example, to treat leukemia or as conditioning agents for transplantation. Accordingly, included herein are anti-hematopoietic cell antibodies (anti-CD45 antibodies) that can be used in hematopoietic stem cell therapy. For example, the antibodies or ADCs herein can be used in conditioning procedures in which a patient is prepared to receive a transplant comprising hematopoietic stem cells. Such procedures facilitate engraftment of a hematopoietic stem cell transplant. According to the methods described herein, a patient can be conditioned for hematopoietic stem cell transplant therapy by administering to the patient an anti-CD45 ADC, antibody, or antigen-binding fragment thereof capable of binding CD45, for example, expressed by hematopoietic cells (e.g., hematopoietic stem cells or mature immune cells (e.g., T cells). As described herein, an anti-CD45 antibody can be covalently bound 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 transplant therapy can facilitate engraftment of a hematopoietic stem cell transplant, for example, by selectively depleting endogenous hematopoietic stem cells, thereby creating a void to be filled by a transplant of exogenous hematopoietic stem cells.

[0273] The following section provides novel anti-CD45 antibodies and fragments thereof having unique properties, for example, cross-reactivity with human and non-human primate CD45. The following section also provides a description of anti-CD45 antibodies or conjugates thereof that can be administered to a patient (e.g., a patient having a cancer or an autoimmune disease or a patient in need of hematopoietic stem cell transplant therapy) to facilitate engraftment of a hematopoietic stem cell transplant, as well as methods of administering such therapeutic agents to a patient (e.g., prior to a hematopoietic stem cell transplant).

[0274] Definitions

[0275] The term "about" as used herein refers to a value that is within 5% of the stated value. For example, the term "about 100 nM" indicates a range of 95-105 nM.

[0276] The term "allogeneic" as used herein in the context of transplantation is used to define a transplant (e.g., a cell, tissue, or organ transplant) from a donor to a recipient, wherein the recipient is a different individual of the same species as the donor.

[0277] The term "autologous" as used herein in the context of transplantation refers to a transplant in which the donor and recipient are the same individual, i.e., the same subject.

[0278] The term "xenogeneic" as used herein in the context of transplantation refers to a transplant in which the donor and recipient are of different species.

[0279] The term "immune cell" as used herein is intended to include, but is not limited to, cells of hematopoietic origin and that play a role in immune responses. 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 one embodiment, natural killer cells include cell lines such as NK-92 cells. Other examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells. Immune cells can be allogeneic or autologous.

[0280] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen. Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, e.g., bispecific antibodies, and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0281] Generally, antibodies comprise heavy and light chains that contain antigen binding regions. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), and regions that are more conserved, termed framework regions (FRs), alternating with one another. Each VHand VLis composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0282] The term "antigen binding fragment" or "antigen binding portion" of an antibody as used herein refers to one or more portions of an antibody that retain the ability to specifically bind to a target antigen. The antigen binding function of an antibody can be performed by fragments of a full-length antibody. Antibody fragments can be, for example, Fab, F(ab')2, scFv, diabodies, triabodies, affibodies, nanobodies, aptamers, or domain antibodies. Examples of binding fragments encompassed by the term "antigen binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb including a VH and VL domain; (vi) a dAb fragment that consists of a VH domain (see, e.g., Ward et al., Nature 341 :544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more (e.g., two, three, four, five, or six) isolated CDRs, which can optionally be linked by a synthetic linker. In addition, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some cases by chemical peptide synthesis procedures known in the art.

[0283] An "intact" or "full-length" antibody as used herein refers to an antibody having two heavy chain (H) polypeptides and two light chain (L) polypeptides interconnected by disulfide bonds. In certain embodiments, a toxin can be conjugated to an intact anti-CD45 antibody having heavy and / or light chain amino acid sequences described herein.

[0284] The term "monoclonal antibody" as used herein refers to an antibody obtained from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any method available in the art or known in the art, and is not limited to antibodies produced via hybridoma technology. Monoclonal antibodies useful in the present disclosure can be prepared using a variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0285] The terms "Fc region," "Fc domain," and "IgG Fc domain" as used herein refer to the portion of an immunoglobulin (e.g., an IgG molecule) associated with the crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region comprises the C-terminal half of both heavy chains of an IgG molecule linked by disulfide bonds. It has no antigen-binding activity, but contains a carbohydrate moiety and binding sites for complement and Fc receptors, including the FcRn receptor (see below). For example, the Fc domain contains the entire second constant domain, CH2 (residues at EU positions 231-340 of IgG1) and the third constant domain, CH3 (residues at EU positions 341-447 of human IgG1). As described herein, the Fc domain includes the "lower hinge region" (residues at EU positions 233-239 of IgG1).

[0286] Fc can refer to this region alone, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at multiple positions in the Fc domain, including but not limited to EU positions 270, 272, 312, 315, 356, and 358, and thus minor differences can exist between the sequences presented in the present application and those known in the art. Thus, "wild-type IgG Fc domain" or "WT IgG Fc domain" refers to any native IgG Fc region (i.e., any allotype). The heavy chain sequences of human IgG1, IgG2, IgG3, and IgG4 can be found in various sequence databases, e.g., on the Uniprot database (www.uniprot.org) under accession numbers P01857 (IGHG1_human), P01859 (IGHG2_human), P01860 (IGHG3_human), and P01861 (IGHG1_human), respectively.

[0287] The term "modified Fc region" or "variant Fc region" as used herein refers to an IgG Fc domain comprising one or more amino acid substitutions, deletions, insertions, or modifications introduced at any position within the Fc domain. In certain aspects, the variant IgG Fc domain comprises one or more amino acid substitutions that reduce or eliminate binding affinity for FcyR and / or Clq, as compared to a wild-type Fc domain that does not comprise the one or more amino acid substitutions. In addition, Fc binding interactions are required 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 can exhibit altered binding affinity for at least one or more Fc ligands (e.g., FcyR), relative to a corresponding antibody that otherwise has the same amino acid sequence but does not comprise the one or more amino acid substitutions, deletions, insertions, or modifications (e.g., an unmodified Fc region containing the native amino acid residues at the corresponding positions of the Fc region).

[0288] A variant Fc domain is defined according to the amino acid modifications that make it up. For all amino acid substitutions discussed herein with respect to Fc regions, numbering is always according to the EU index as in Kabat. Thus, for example, D265C is an Fc variant in which the aspartic acid (D) at EU position 265 is substituted with a cysteine (C), relative to the parent Fc domain. It should be noted that the order in which substitutions are provided is arbitrary.

[0289] The term "Fcy receptor" or "FcyR" as used herein refers to any member of the family of proteins that bind IgG antibody Fc regions and are encoded by FcyR genes. In humans, this family includes, but is not limited to, FcyRI (CD64), including isoforms FcyRIa, FcyRIb, and FcyRIc; FcyRII (CD32), including isoforms FcyRIIa (including allotypes H131 and R131), FcyRIIb (including FcyRIIb-l and FcyRIIb-2), and FcyRIIc; and FcyRIII (CD16), including isoforms FcyRIIIa (including allotypes V158 and F158) and FcyRIIIb (including allotypes FcyRIIIb-NA1 and FcyRIIIb-NA2), as well as any undiscovered human FcyR or FcyR isoform or allotype. FcyR can be from any organism, including but not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcyR include, but are not limited to, FcyRI (CD64), FcyRII (CD32), FcyRIII (CD16), and FcyRIII-2 (CD16-2), as well as any undiscovered mouse FcyR or FcyR isoform or allotype.

[0290] The term "effector function" as used herein refers to biochemical events that result from the interaction of an Fc domain with an Fc receptor. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. "Effector cell" as used herein means 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 can be from any organism, including but not limited to, humans, mice, rats, rabbits, and monkeys.

[0291] The terms "silent," "silenced," or "silencing" as used herein refer to an antibody having a modified Fc region as described herein that has reduced binding to an Fc gamma receptor (FcγR) relative to the binding of the same antibody comprising an unmodified Fc region to an FcγR (e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% less binding to an FcγR relative to the binding of the same antibody comprising an unmodified Fc region to an FcγR, as measured by, e.g., BLI). In some embodiments, the Fc silent antibody has no detectable FcγR binding. Binding of an antibody having a modified Fc region to an FcγR can be determined using a variety of techniques known in the art, such as, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al., Analytical Biochemistry, vol. 373:52-60, 2008; or radioimmunoassay (RIA)) or surface plasmon resonance assays or other kinetic mechanism-based assays (e.g., BIACORE.RTM. assays or Octet assays). In some embodiments, the Fc silent antibody has reduced binding to an FcγR relative to the binding of the same antibody comprising an unmodified Fc region to an FcγR, as measured by, e.g., BLI. TMAnalysis (forteBIO) and other methods, such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods can utilize labels and / or employ detection methods including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels on one or more of the components being examined. Detailed descriptions of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which addresses antibody-immunogen interactions. An example of a competitive binding assay is a radioimmunoassay, which includes incubating labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and detecting antibody bound to the labeled antigen. The affinity and dissociation rate of the antibody of interest for a particular antigen can be determined from the data analyzed by a scatchard plot. Competition with a second antibody can also be determined using a radioimmunoassay. In this case, the antigen is incubated with the antibody of interest bound to a labeled compound in the presence of increasing amounts of unlabeled second antibody.

[0292] The term "identical antibody comprising an unmodified Fc region" as used herein refers to an antibody that lacks the recited amino acid substitution (e.g., D265C, H435A), but otherwise has the same amino acid sequence as the Fc modified antibody with which it is compared.

[0293] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which a polypeptide comprising an Fc domain (e.g., an antibody) binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., primarily NK cells / neutrophils and macrophages) and enables these cytotoxic effector cells to bind specifically to a "target cell" bearing antigen and subsequently kill the target cell with cytotoxins. (Hogarth et al., Nature review Drug Discovery 2012, 11:313) In addition to antibodies and fragments thereof, other polypeptides comprising an Fc domain (e.g., Fc fusion proteins and Fc binding proteins) that have the ability to bind specifically to a target cell bearing antigen will be able to effect cell-mediated cytotoxicity.

[0294] For simplicity, cell-mediated cytotoxicity resulting from the activity of a polypeptide comprising an Fc domain is also referred to herein as ADCC activity. The ability of any particular polypeptide of the disclosure to mediate lysis of target cells by ADCC can be analyzed. To assess ADCC activity, a polypeptide of interest (e.g., an antibody) is added to target cells in combination with immune effector cells, resulting in cytolysis of the target cells. Cytolysis is typically detected by the release of a label (e.g., a radioactive substance, a fluorescent dye, or a native intracellular protein) from the lysed cells. Effector cells that can be used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Particular examples of in vitro ADCC assays are described in Bruggemann et al., J. Exp. Med. 166: 1351 (1987); Wilkinson et al., J. Immunol. Methods 258: 183 (2001); Patel et al., J. Immunol. Methods 184: 29 (1995). Alternatively, or additionally, ADCC activity of an antibody of interest can be assessed in vivo, for example, in an animal model as described in, e.g., Clynes et al., Proc. Natl. Acad. Sci. USA 95: 652 (1998).

[0295] The terms "condition" and "conditioning" as used herein refer to a process of preparing a patient for receiving a transplant, e.g., a transplant containing hematopoietic stem cells. Such procedures promote engraftment of a hematopoietic stem cell transplant, e.g., as inferred from a sustained increase in the amount of viable hematopoietic stem cells in blood samples isolated from patients after conditioning procedures and subsequent hematopoietic stem cell transplantation. According to the methods described herein, a patient can be conditioned for hematopoietic stem cell transplant therapy by administering to the patient an ADC, antibody, or antigen-binding fragment thereof capable of binding CD45 expressed by hematopoietic stem cells. As described herein, an antibody can be covalently bound to a cytotoxin to form a drug-antibody conjugate. Administration of an antibody, antigen-binding fragment thereof, or ADC capable of binding the aforementioned antigen to a patient in need of hematopoietic stem cell transplant therapy can promote engraftment of a hematopoietic stem cell transplant, e.g., by selectively depleting endogenous hematopoietic stem cells, thereby creating a void to be filled by an exogenous hematopoietic stem cell transplant.

[0296] The terms "effective amount" or "therapeutically effective amount" as used herein refer to the amount of a therapeutic agent (e.g., an anti-CD45 ADC) sufficient to effectuate a desired result in the context of treating, preventing, ameliorating, or reducing symptoms of a disease or disorder in a patient. For example, in some embodiments, a therapeutically effective amount of an anti-CD45 antibody or ADC is an amount sufficient to reduce or deplete a population of CD45+ cells in a patient. In other embodiments, a therapeutically effective amount of an anti-CD45 antibody or ADC is an amount sufficient to condition a patient to receive a hematopoietic stem cell transplant. In such embodiments, a therapeutically effective amount can be, for example, an amount sufficient to selectively deplete endogenous hematopoietic stem cells in a patient, and / or an amount sufficient to facilitate engraftment of a hematopoietic stem cell transplant in a patient. In other embodiments, a therapeutically effective amount of an anti-CD45 antibody or ADC is an amount sufficient to effect an immune response against an autoimmune disease or cancer in a human patient.

[0297] The term "half-life" as used herein refers to the time it takes for the plasma antibody drug concentration to decrease by one-half or 50% in an individual (e.g., a human individual) in vivo. This 50% decrease in serum concentration reflects the amount of drug circulating.

[0298] The phrase "substantially cleared from the blood" as used herein refers to the point in time after administration of a therapeutic agent (e.g., an anti-CD45 antibody or antigen-binding fragment thereof) to a patient at which the concentration of the therapeutic agent in a blood sample isolated from the patient is such that the therapeutic agent is not detectable by conventional methods (e.g., such that the therapeutic agent is not detectable above the noise threshold of the device or assay used to detect the therapeutic agent). Antibodies or antibody fragments can be detected using a variety of techniques known in the art, such as ELISA-based detection assays known in the art or described herein. Other assays that can be used to detect antibodies or antibody fragments include, among others, immunoprecipitation techniques and immunoblot assays known in the art.

[0299] The terms "specific binding" or "specifically binding" as used herein refer to the ability of an antibody to recognize and bind to a specific protein structure (antigenic determinant), as opposed to binding non-specifically to any protein. If an antibody is specifically directed against an antigenic determinant "A," the presence of molecules bearing antigenic determinant A (or free, unlabelled A) will reduce the amount of label- ed A bound to the antibody in a reaction containing the labeled A and the antibody. For example, if an antibody can be competed away from its target by a corresponding unlabeled antibody, then the antibody "specifically binds" to the target. In one embodiment, an antibody binds to a target with a Kd of less than about 10 D at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10-9 M, 10 -10 M, 10 -11 M, 10 -12 M or smaller (smaller means less than 10) -12 The value, for example, 10 -13 If the antibody binds specifically to a target, such as CD45, then the antibody binds specifically to that target. In one embodiment, the terms "specific binding to CD45" or "specifically binds to CD45" as used herein refer to binding to CD45 and having a dissociation constant (K0). D The value measured by surface plasma resonance is 1.0 × 10⁻⁶. -7 M or smaller antibodies. In one embodiment, K D (M) is determined based on standard biological layer interferometry (BLI). In one embodiment, K 解离 (1 / s) is determined according to standard biolayer interference (BLI). However, it should be understood that antibodies may be able to specifically bind to two or more sequence-related antigens. For example, in one embodiment, an antibody may specifically bind to human and non-human (e.g., mouse, cynomolgus monkey, or non-human primate) CD45 analogs. Therefore, as used herein, an antibody that "specifically binds to human CD45" refers to an antibody that binds to human CD45 (and possibly from one or more non-human species, such as cynomolgus monkey CD45) but does not substantially bind to non-CD45 proteins. Preferably, the antibody binds to human CD45 and K D 1×10 -7 M or smaller, K D 5×10 -8 M or smaller, K D 3×10 -8 M or smaller, K D 1×10 -8 M or smaller, or K D 5×10 -9 M or smaller.

[0300] The term "human antibody", as used herein, is intended to include antibodies having variable regions in which each domain is derived from human germline immunoglobulin sequences. In those embodiments in which the human antibody comprises a constant region, the constant region can also be derived from human germline immunoglobulin sequences. Human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences. Human antibodies can be produced in human cells (e.g., by recombinant expression), or by non-human animals or prokaryotic or eukaryotic cells that are capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. When human antibodies are single chain antibodies, they can include linker peptides not found in native human antibodies. For example, an Fv can contain a linker peptide joining a heavy chain variable region and a light chain variable region, e.g., two to about eight glycine or other amino acid residues. Human antibodies can be made by a variety of methods including phage display methods or yeast display methods known in the art, using antibody libraries derived from human immunoglobulin sequences. Human antibodies can also be generated using genetically modified mice that are incapable of expressing functional endogenous immunoglobulins, but are capable of expressing human immunoglobulin genes (see, e.g., PCT Publication Nos. WO 1998 / 24893; WO 1992 / 01047; WO 1996 / 34096; WO 1996 / 33735; U.S. Patent 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).

[0301] The term "chimeric antibody" is intended to refer to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a rat antibody and the constant region sequences are derived from a human antibody.

[0302] "Humanized" forms of non-human (e.g., murine or rat) antibodies are immunoglobulins which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody can also comprise all or a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin common sequence. Methods for humanizing antibodies are known in the art. See, e.g., Riechmann et al., 1988, Nature 332:323-7; U.S. Pat. Nos. 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. Pat. No. 5,225,539; EP 592106; EP 519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Pat. No. 5,565,332.

[0303] Conservative sequence modifications of the sequences shown in the SEQ ID NOs described herein are also provided. Conservative sequence modifications include nucleotide and amino acid sequence modifications that do not abrogate the binding of the antibodies or antigen-binding portions thereof comprising the amino acid sequence encoded by the nucleotide sequence to its cognate antigen (e.g., CD45). Such conservative sequence modifications include conservative nucleotide and amino acid substitutions as well as nucleotide and amino acid additions and deletions. For instance, modifications can be introduced into the SEQ ID NOs described herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions, wherein 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, cysteine, tyrosine), 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, a predicted nonessential amino acid residue in an anti-CD73 antibody is preferably replaced with another amino acid residue from the same side chain family. Methods of identifying nucleotide and amino acid conservative substitutions which do not abrogate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0304] The term "engraftment potential" as used herein is used to refer to the ability of hematopoietic stem and progenitor cells to repopulate a tissue, whether the cell line is naturally circulating or provided by transplantation. The term encompasses all events surrounding or leading to engraftment, such as tissue homing of cells and colonization of cells within the tissue of interest. Engraftment efficiency or engraftment rate can be assessed or quantified using any clinically acceptable parameter as known to one of skill in the art and can include, for example, evaluation of competitive repopulating units (CRUs); incorporation or expression of markers in the tissue to which the stem cells have homed, colonized or engrafted; or evaluation of the progress of the individual via disease progression, survival of hematopoietic stem and progenitor cells or survival of the recipient. Engraftment can also be determined by measuring white blood cell counts in the peripheral blood over a period of time post-transplant. Engraftment can also be evaluated by measuring the recovery of donor cells to bone marrow cells in bone marrow aspirate samples.

[0305] The term "hematopoietic stem cell" ("HSC") as used herein refers to an immature blood cell that has the ability to self-renew and differentiate into mature blood cells including different lineages including, but not limited to, granulocytes (e.g., myeloblasts, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, megakaryocytes that produce platelets, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglial cells, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). These cells can include CD34 + cells. CD34 +Cell lines express immature cells that are CD34 cell surface marker positive. In humans, CD34+ cells are thought to include a subset of cells with the properties of stem cells as defined above, while in mice, HSCs are CD34-. In addition, HSCs also refer to long-term population reconstituting HSCs (LT-HSCs) and short-term population reconstituting HSCs (ST-HSCs). LT-HSCs and ST-HSCs are differentiated based on functional potential and cell surface marker expression. For example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for mature lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD1 1 B, CD19, CD20, CD56, CD235A). In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, CD48-, and lin- (negative for mature lineage markers including Ter119, CD1 1 b, Gr1, CD3, CD4, CD8, B220, IL7ra), while ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, and lin- (negative for mature lineage markers including Ter119, CD1 1 b, Gr1, CD3, CD4, CD8, B220, IL7ra). In addition, under steady state conditions, ST-HSCs are less quiescent and more proliferative than LT-HSCs. However, LT-HSCs have greater self-renewal potential (i.e., they survive throughout adulthood and can be serially transplanted via serial recipients), while ST-HSCs have limited self-renewal (i.e., they survive only for a limited period of time and do not have serial transplantation potential). Any of these HSCs can be used in the methods described herein. ST-HSCs are particularly useful because they are highly proliferative and thus can generate differentiated progeny more quickly.

[0306] The term "hematopoietic stem cell functional potential" as used herein refers to the functional properties of a hematopoietic stem cell, which include 1) multi-potency (which refers to the ability to differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), red blood cells (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, megakaryocytes that produce platelets, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, T cells, and B cells), 2) self-renewal (which refers to the ability of a hematopoietic stem cell to generate daughter cells with equivalent potential to the parent cell, and further that this ability can be repeated throughout the life of an individual without exhaustion), and 3) the ability of a hematopoietic stem cell or its daughter to re-introduce into a transplant recipient, then home to the hematopoietic stem cell niche, and re-establish productive and sustainable hematopoiesis.

[0307] The terms "individual" and "patient" as used herein refer to an organism, such as a human, who receives treatment for a particular disease or condition as described herein. In some embodiments, the individual or patient referred to in the methods provided herein is a human individual.

[0308] The term "recipient" as used herein refers to a patient who receives a transplant, such as a transplant containing a population of hematopoietic stem cells. The transplanted cells administered to a recipient can be, for example, autologous, syngeneic, or allogeneic cells.

[0309] As used herein, "treatment" or "treating" refers to any improvement in the disease outcome, e.g., increased survival, decreased morbidity, and / or decreased side effects as a byproduct of an alternative treatment modality; as is readily appreciated in the art, complete eradication of the disease is preferred, but is not a requirement for treatment. For example, treatment can refer to decreasing the severity and / or frequency of disease symptoms, eliminating disease symptoms and / or the underlying cause of such symptoms, decreasing the frequency or likelihood of disease symptoms and / or their underlying cause, and ameliorating or remedying damage caused directly or indirectly by the disease. Beneficial or desired clinical results include, but are not limited to, promoting engraftment of exogenous hematopoietic cells in a patient following conditioning therapy with an antibody as described herein and subsequent hematopoietic stem cell transplant therapy. Other beneficial results include an increase in the cell count or relative concentration of hematopoietic stem cells in a patient in need of a hematopoietic stem cell transplant following conditioning therapy and subsequent administration of a hematopoietic stem cell transplant to the patient. Beneficial results of the therapies described herein can also include an increase in the cell count or relative concentration of one or more hematopoietic lineage cells (e.g., 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) following conditioning therapy and subsequent hematopoietic stem cell transplant therapy. Other beneficial results can include a decrease in the amount of a pathogenic cell population (e.g., a cancer cell population (e.g., CD45+leukemic cells) or an autoimmune cell population (e.g., CD45+ autoimmune lymphocytes, such as CD45+ T cells expressing T cell receptors that cross-react with self-antigens)). Insofar as the methods of the application are directed to preventing a disorder, it is understood that the term "prevention" does not require complete blocking of the disease state. Rather, the term prevention as used herein refers to the ability of one skilled in the art to identify a population predisposed to a disorder, such that a compound of the application can be administered prior to the onset of the disease. The term implies that complete avoidance of the disease state is not possible.

[0310] A patient in need of hematopoietic stem cell transplant as used herein includes patients exhibiting a deficiency or defect in one or more blood cell types as well as patients suffering from a stem cell disorder, an autoimmune disease, a cancer, or other condition described herein. Hematopoietic stem cells generally exhibit 1) multipotency and can therefore differentiate into multiple different blood lineages including, but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), thrombocytes (e.g., megakaryoblasts, megakaryocytes that produce platelets, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells), 2) self-renewal and can therefore generate daughter cells with equivalent potential to the parent cell, and 3) the ability to be re-introduced into a transplant recipient, whereupon they home to the hematopoietic stem cell niche and reestablish productive and sustained hematopoiesis. Thus, hematopoietic stem cells can be administered to a patient lacking or deficient in one or more cell types of the hematopoietic lineage to restore the in vivo deficient or defective cell population. For example, a patient can have a cancer and the deficiency can be caused by administration of a chemotherapeutic or other agent that selectively or non-specifically depletes the population of cancer cells. Additionally or alternatively, a patient can have a hemoglobinopathy (e.g., a non-malignant hemoglobinopathy), such as sickle cell anemia, thalassemia, Fanconi anemia, Diamond Blackfan anemia, and Wiskott-Aldrich syndrome. An individual can be an individual with adenosine deaminase severe combined immunodeficiency disease (ADA SCID), HIV / AIDS, metachromatic leukodystrophy, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome. An individual can have or be affected by a genetic blood disorder (e.g., sickle cell anemia) or an autoimmune disorder. Additionally or alternatively, an individual can have or be affected by a malignant disease (e.g., a neuroblastoma or a blood cancer). For example, an individual can have leukemia, lymphoma, or myeloma. In some embodiments, an individual has acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. In some embodiments, an individual has a myelodysplastic syndrome. In some embodiments, an individual has an autoimmune disease, such as scleroderma, multiple sclerosis, ulcerative colitis, Crohn's disease, type 1 diabetes, or another autoimmune condition described herein. In some embodiments, an individual is in need of chimeric antigen receptor T cell (CART) therapy. In some embodiments, an individual has or is otherwise affected by a metabolic storage disease.Individuals may suffer from or be otherwise affected by metabolic disorders selected from the following groups: glycogen storage diseases, mucopolysaccharidosis, Gaucher's disease, Hurlers disease, sphingolipid storage diseases, metachromatic leukodystrophy, or any other disease or condition that may benefit from the treatments and therapies described herein, including (but not limited to) severe combined immunodeficiency diseases, Wiscott-Aldrich syndrome, hyperimmune globulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteosclerosis, osteogenesis imperfecta, storage diseases, severe thalassemia, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and "Bone Marrow Transplantation for Non-Malignant Disease," ASH Education Book, 1:319-338. The diseases or conditions described in (2000), and the full text of that literature regarding conditions that can be treated by hematopoietic stem cell transplantation, are incorporated herein by reference. Alternatively or otherwise, a patient "requiring" a hematopoietic stem cell transplant may be a patient who has or does not have one of the aforementioned conditions, but still exhibits a reduced level (e.g., compared to other healthy individuals) of one or more endogenous cell types within the hematopoietic lineage, such as megakaryocytes, coagulatory cells, platelets, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes. Those familiar with this technique can easily determine, for example, by using flow cytometry and fluorescence activated cell sorting (FACS) methods, as well as other procedures known in this technique, whether the levels of one or more of the aforementioned cell types or other blood cell types in an individual are reduced relative to other healthy individuals.

[0311] The phrase "stem cell disorder" as used herein broadly refers to any disease, disorder, or condition that can be treated or cured by conditioning a target tissue of a subject, and / or by eliminating a population of endogenous stem cells in a target tissue (e.g., eliminating a population of endogenous hematopoietic stem or progenitor cells from a subject's bone marrow tissue), and / or by implanting or transplanting stem cells into a 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. Other 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. Other diseases that can be treated using the patient conditioning and / or hematopoietic stem cell transplantation methods described herein include genetic blood disorders (e.g., sickle cell anemia) and autoimmune disorders, such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease. Other diseases that can be treated using the conditioning and / or transplantation methods described herein include malignancies, such as neuroblastoma or a blood cancer, such as leukemia, lymphoma, and myeloma. For example, the cancer can be acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Other diseases that can be treated using the conditioning and / or transplantation methods described herein include myelodysplastic syndrome. In some embodiments, the subject has or is otherwise affected by a metabolic storage disorder. For example, the subject can have or otherwise be affected by a metabolic disorder selected from the group consisting of glycogen storage disease, mucopolysaccharidosis, Gaucher's disease, Hurler's disease, sphingolipidosis, metachromatic leukodystrophy, or any other disease or disorder that can benefit from the treatments and therapies disclosed herein, including but not limited to severe combined immunodeficiency disease, Wiskott-Aldrich syndrome, hyperimmunoglobulin M (IgM) syndrome, Chediak-Higashi disease, hereditary lymphohistiocytosis, osteosclerosis, osteogenesis imperfecta, storage disease, thalassemia major, sickle cell disease, systemic sclerosis, systemic lupus erythematosus, multiple sclerosis, juvenile rheumatoid arthritis, and those diseases or disorders described in "Bone Marrow Transplantation for Non-Malignant Disease," ASH Education Book, 1 :319-338 (2000), the entire disclosure of which regarding the diseases conditions that can be treated by administration of hematopoietic stem cell transplantation therapy is incorporated herein by reference.

[0312] The term "vector" as used herein includes nucleic acid vectors, such as plasmids, DNA vectors, plasmids, RNA vectors, viruses, or other suitable replicons. Expression vectors described herein can contain polynucleotide sequences as well as other sequence components, such as for expressing proteins and / or for integrating such polynucleotide sequences into the genome of a mammalian cell. Certain vectors useful for expressing antibodies and antibody fragments of the present application include plasmids containing regulatory sequences that direct transcription of the gene, such as promoter and enhancer regions. Other vectors useful for expressing antibodies and antibody fragments contain polynucleotide sequences that enhance the rate of translation of the gene or improve the stability or nuclear export of mRNA derived from transcription of the gene. Such sequence components can include, for example, 5' and 3' untranslated regions and polyadenylation signal sites to direct efficient transcription of genes carried on the expression vector. Expression vectors described herein can also contain polynucleotides encoding markers for selection of cells containing the vector. Examples of suitable markers include genes that encode antibiotic (e.g., ampicillin, chloramphenicol, kanamycin, and nourseothricin) resistance.

[0313] The term "conjugate" or "antibody drug conjugate" or "ADC" as used herein refers to an antibody linked to a cytotoxin or toxin, and are used interchangeably throughout. In one embodiment, an ADC is formed by the reaction of a reactive functional group of one molecule (e.g., an antibody or antigen-binding fragment thereof) with an appropriate reactive functional group of another molecule (e.g., a cytotoxin described herein). Non-limiting examples of cytotoxins that can be used in conjugates provided herein include small organic molecules (e.g., MW 1500 Da or less), biological molecules (e.g., proteins), drug-filled nanoparticles, or radionuclides. Conjugates can include a linker between two molecules that are conjugated to each other, such as between an antibody and a cytotoxin. Examples of linkers that can be used to form conjugates include peptide-containing linkers, such as those containing natural or unnatural amino acids (e.g., D-amino acids). Linkers can be prepared using a variety of strategies described herein and known in the art. Depending on the reactive components therein, linkers can be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under basic conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, e.g., Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0314] When used in the sense of at least two molecules being bound together, the terms "bind," "bind to," or "bind with" refer to a molecule (e.g., an antibody) being attached to or combined with a second molecule (e.g., a toxin). Anti-CD45 antibodies and fragments thereof may bind to other molecules, including toxins, labelers (e.g., fluorescein or biotin), and drug-loaded nanoparticles. The bound molecules may bind via covalent or non-covalent interactions. In some embodiments, anti-CD45 antibodies or fragments thereof bind to protein toxins to form protein fusions, such as scFv-toxin chimeras. In some embodiments, the bound molecules may be coupled via a non-covalent interaction between a first interacting portion (e.g., biotin) associated with the bound molecule and a second interacting portion (e.g., streptavidin).

[0315] As used herein, "drug-to-antibody ratio" or "DAR" refers to the amount of drug (e.g., phallotoxins) linked to the conjugate antibody. The DAR of an ADC can range from 1 to 8, but higher loadings are possible depending on the number of linking sites on the antibody. In some embodiments, the DAR of the conjugate is 1, 2, 3, 4, 5, 6, 7, or 8.

[0316] As used herein, the term "microtubule binder" refers to a compound that acts by disrupting the microtubule network essential for mitosis and interphase cell function. Examples of microtubule binders include (but are not limited to) maytansine, maytansine-like compounds and their derivatives (such as those described herein or known in the art), vinca alkaloids (such as vinblastine, vincristine sulfate, vindesine, and vinorelbine), taxanes (such as docetaxel and paclitaxel), macrolides (such as discodermolide, cochicine, and epothilone and their derivatives, such as epothilone B or its derivatives).

[0317] The term "phalloidin" as used herein refers to a member of the phalloidin family of peptides produced by Amanita phalloides mushrooms or derivatives thereof (e.g., variants or derivatives capable of inhibiting RNA polymerase II activity). Phalloidins that can be used in conjunction with the compositions and methods described herein include compounds described herein, such as a-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amatoxin, amatoxinamide, amanullin, amanullinol, or proamanullin. As described herein, phalloidins can be conjugated to antibodies or antigen-binding fragments thereof (thus forming an ADC), e.g., via a linker moiety (L). Exemplary phalloidin conjugation methods and linkers that can be used in such methods are set forth below. Exemplary linker-containing phalloidins that can be used in conjugation to antibodies or antigen-binding fragments according to the compositions and methods are also set forth herein.

[0318] The term "acyl" as used herein refers to -C(=0)R, wherein R is hydrogen ("aldehyde"), C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C7 carbocyclyl, C6-C20 aryl, 5-10 membered heteroaryl, or 5-10 membered heterocyclyl, as defined herein. Non-limiting examples include formyl, acetyl, propionyl, benzoyl, and propenoyl.

[0319] The term "C1-C12 alkyl" as used herein refers to a straight-chain or branched-chain saturated hydrocarbon having from 1 to 12 carbon atoms. Representative C1-C12 alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, and -n-hexyl; while branched C1-C12 alkyl groups include, but are not limited to, -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl. C1-C12 alkyl groups can be unsubstituted or substituted.

[0320] The term "alkenyl" as used herein refers to a C2-C12 hydrocarbon that contains normal, secondary, or tertiary carbon atoms and at least one site of unsaturation (i.e., a carbon-carbon sp2 bond). Examples include, but are not limited to: eth-1- enyl 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, propenyl, isopropenyl, butenyl, tert-butenyl, hexenyl, and the like. Alkenyl groups can be unsubstituted or substituted.

[0321] "Alkynyl" as used herein refers to a C2-C12 hydrocarbon that contains normal, secondary, or tertiary carbon atoms and at least one site of unsaturation (i.e., a carbon-carbon sp triple bond). Examples include, but are not limited to, alkynyl and propargyl, butynyl, pentynyl, hexynyl, and the like. Alkynyl groups can be unsubstituted or substituted.

[0322] "Aryl" as used herein refers to a C6-C20carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, and anthryl. The aryl group can be unsubstituted or substituted.

[0323] "Heteroaralkyl" as used herein refers to acyclic alkyl groups in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3carbon atom, is replaced by a heteroaryl group. Typical heteroaralkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furyl ethyl, and the like. Heteroaralkyl groups comprise 6 to 20 carbon atoms, e.g., the alkyl portion of the heteroaralkyl group (including alkanyl, alkenyl, or alkynyl) is 1 to 6 carbon atoms and the heteroaryl portion is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. The heteroaryl portion of the heteroaralkyl group can be a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), or a bicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), e.g., a bicyclo[4,5], [5,5], [5,6], or [6,6] system.

[0324] "Cycloalkyl" as used herein refers to saturated carbocyclic groups that can be monocyclic or bicyclic. Cycloalkyl groups include rings having 3 to 7 carbon atoms (as in monocyclic rings) or 7 to 12 carbon atoms (as in bicyclic rings). Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. The cycloalkyl group can be unsubstituted or substituted.

[0325] "Cycloalkenyl" as used herein refers to unsaturated carbocyclic groups that can be monocyclic or bicyclic. Cycloalkenyl groups include rings having 3 to 6 carbon atoms (as in monocyclic rings) or 7 to 12 carbon atoms (as in bicyclic rings). Examples of monocyclic cycloalkenyl groups include 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl. The cycloalkenyl group can be unsubstituted or substituted.

[0326] "Heteroaralkyl" as used herein refers to acyclic alkyl groups in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp3carbon atom, is replaced by a heteroaryl group. Typical heteroaralkyl groups include, but are not limited to, 2-benzimidazolylmethyl, 2-furyl ethyl, and the like. Heteroaralkyl groups comprise 6 to 20 carbon atoms, e.g., the alkyl portion of the heteroaralkyl group (including alkanyl, alkenyl, or alkynyl) is 1 to 6 carbon atoms and the heteroaryl portion is 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. The heteroaryl portion of the heteroaralkyl group can be a monocyclic ring having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), or a bicyclic ring having 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), e.g., a bicyclo[4,5], [5,5], [5,6], or [6,6] system.

[0327] "Heteroaryl" and "heterocycloalkyl" as used herein refer to aromatic or non-aromatic ring systems, respectively, in which one or more ring atoms are heteroatoms (e.g., nitrogen, oxygen, and sulfur). Heteroaryl or heterocycloalkyl groups comprise 2 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. Heteroaryl or heterocycloalkyl groups can be monocyclic with 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S) or bicyclic with 7 to 10 ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S), e.g., bicyclo[4,5], [5,5], [5,6], or [6,6] systems. Heteroaryl and heterocycloalkyl groups can be unsubstituted or substituted.

[0328] Heteroaryl and heterocycloalkyl groups are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (W. A. Benjamin, New York, 1968), in particular 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), in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc. (1960) 82:5566.

[0329] Examples of heteroaryl groups include, for example, but are not limited to, pyridyl, thiazolyl, tetrahydrothienyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthalenyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl, benzimidazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, lH-indazolyl, purinyl, 4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl, phenarsenazinyl, acridinyl, pyrimidinyl, phenoxazinyl, phenothiazinyl, furazanyl, phenazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, benzotriazolyl, benzisoxazolyl, and indatrinyl.

[0330] Examples of heterocycloalkyl groups include, for example, but are not limited to, dihydropyridinyl, tetrahydropyridinyl (hexahydropyridinyl), tetrahydrothienyl, hexahydropyridinyl, 4-hexahydropyridinonyl, pyrrolidinyl, 2-pyrrolidinonyl, tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, hexahydropyrazinyl, quinuclidinyl, and morpholinyl.

[0331] By way of example but not limitation, carbon-bound heteroaryl and heterocycloalkyl groups are bound to position 2, 3, 4, 5, or 6 of pyridine, position 3, 4, 5, or 6 of pyrazine, position 2, 4, 5, or 6 of pyrimidine, position 2, 3, 5, or 6 of pyrazine, position 2, 3, 4, or 5 of furan, tetrahydrofuran, thiophene, pyrrole, or tetrahydropyrrole, position 2, 4, or 5 of oxazole, imidazole, or thiazole, position 3, 4, or 5 of isoxazole, pyrazole, or isothiazole, position 2 or 3 of aziridine, position 2, 3, or 4 of azetidine, position 2, 3, 4, 5, 6, 7, or 8 of quinoline, or position 1, 3, 4, 5, 6, 7, or 8 of isoquinoline. More typically, carbon-bound heterocycles include 2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyrazinyl, 4-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

[0332] By way of example but not limitation, nitrogen-bound heteroaryl and heterocycloalkyl groups are bound to position 1 of aziridine, azetidine, pyrrole, pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, hexahydropyridine, hexahydropyrazine, indole, indoline, 1H-indazole, position 2 of isoindole or isoindoline, position 4 of morpholine, and position 9 of carbazole or β-carboline. More typically, nitrogen-bound heterocycles include 1-aziridinyl, 1-azetidinyl, 1-pyrrolyl, 1-imidazolyl, 1-pyrazolyl, and 1-hexahydropyridinyl.

[0333] “Substituted” as used herein and as applied to any of the above-mentioned alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclyl, and the like, means that one or more hydrogen atoms are each independently replaced with a substituent. Typical substituents include, but are not limited to, -X, -R, -OH, -OR, -SH, -SR, NH2, -NHR, -N(R)2, -N+(R)3, -CX3, -CN, -OCN, -SCN, -NCO, -NCS, -NO, -NO2, -N3, -NC(=O)H, -NC(=O)R, -C(=O)H, -C(=O)R, -C(=O)NH2, -C(=O)N(R)2, -SO3-, -SO3H, -S(=O)2R, -OS(=O)2OR, -S(=O)2NH2, -S(=O)2N(R)2, -S(=O)R, -OP(=O)(OH)2, -OP(=O)(OR)2, -P(=O)(OR)2, -PO3, -PO3H2, -C(=O)X, -C(=S)R, -CO2H, -CO2R, -CO2-, -C(=S)OR, -C(=O)SR, -C(=S)SR, -C(=O)NH2, -C(=O)N(R)2, -C(=S)NH2, -C(=S)N(R)2, -C(=NH)NH2, and -C(=NR)N(R)2; wherein each X is independently selected at each occurrence from F, Cl, Br, and I; and each R is independently selected at each occurrence from C1-C12 alkyl, C6-C20 aryl, C3-C14 heterocycloalkyl or heteroaryl, a protecting group, and a prodrug moiety. When a group is described as “optionally substituted,” that group can be substituted independently at each occurrence with one or more of the above-mentioned substituents.

[0334] It should be understood that certain group naming conventions can include either a mono- or di- group, depending on the context. For example, when a substituent requires two points of attachment to the remainder of the molecule, it should be understood that the substituent is a di- group. For example, substituents identified as alkyl groups requiring two points of attachment include di- groups such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, and the like. Other group naming conventions explicitly indicate that the group is a di- group, such as “extended alkyl,” “extended alkenyl,” “extended aryl,” “extended heterocycloalkyl,” and the like.

[0335] Unless otherwise indicated, when a substituent is depicted as a di- group (i.e., having two points of attachment to the remainder of the molecule), it should be understood that the substituent can be attached in either orientation configuration.

[0336] "Isomerism" means compounds having the same molecular formula but different sequences of bonding of their atoms or different spatial arrangements of their atoms. Isomers that differ in the spatial arrangement of their atoms are called "stereoisomers." Stereisomers that are not mirror images of one another are called "diastereomers" and those that are mirror images of one another are called "enantiomers" or sometimes "optical isomers."

[0337] A carbon atom bonded to four different substituents is called a "chiral center." "Chiral isomers" means compounds having at least one chiral center. Compounds having more than one chiral center can exist as individual diastereomers or as mixtures of diastereomers, called "diastereomeric mixtures." When there is one chiral center, the stereoisomers can be characterized by the absolute configuration (R or S) of the chiral center. Absolute configuration refers to the spatial arrangement of substituents attached to the chiral center. According to the sequence rules of Cahn, Ingold and Prelog (Cahn et al., Angew. Chem. Inter. Edit., 1966, 5, 385; errata 511; Cahn et al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951 (London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem. Educ. 1964, 41, 116), substituents attached to a chiral center are ranked. A mixture containing equal amounts of individual enantiomeric forms of opposite chirality is called a "racemic mixture."

[0338] The compounds disclosed in the specification and application patent range can contain one or more asymmetric centers and can exist in different enantiomeric and / or diastereomeric forms. The description or description of any compound herein is intended to include all isomers, enantiomeric and diastereomeric, as well as mixtures of any of the foregoing. In addition, unless otherwise specified, the description or description of any compound herein is intended to include individual enantiomers, as well as any mixtures of enantiomers, racemic or otherwise. When a structure is depicted as a particular enantiomer, it is to be understood that the disclosure of the application is not limited to that particular enantiomer. Thus, the mirror image isomer, optical isomer, and diastereomeric isomer of each structural formula of the present disclosure are encompassed herein. In the specification, for the sake of convenience, the structural formula of a compound is represented in some cases as a certain isomer, but the disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, and the like, it is understood that not all isomers can have the same level of activity. Compounds can exist in different tautomeric forms. Unless otherwise specified, the compounds of the present disclosure are intended to include all tautomeric forms. When a structure is depicted as a particular tautomer, it is to be understood that the disclosure of the application is not limited to that particular tautomer.

[0339] If applicable, a compound of any formula described herein includes the compound itself as well as salts and solvates thereof. For example, salts can form between an anion on a compound of the disclosure and a positively charged group (e.g., an amine group). Suitable anions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate). The term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Likewise, salts can also form between a cation on a compound of the disclosure and a negatively charged group (e.g., a carboxylate). Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion, and ammonium cations, such as tetramethylammonium ion. Examples of some suitable substituted ammonium ions are those derived from the following: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. Compounds of the disclosure also include those salts containing a quaternary nitrogen atom. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyloxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0340] In addition, the compounds of the present disclosure (e.g., salts of the compounds) can exist in hydrated or unhydrated (anhydrous) form or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, and the like. Non-limiting examples of solvates include ethanol solvates, acetone solvates, and the like. A "solvate" means a solvate addition form that contains either a stoichiometric or non-stoichiometric amount of solvent molecules. Some compounds have a tendency to trap a fixed or variable number of solvent molecules as solvate molecules within the crystalline solid state. If the solvent is water, the solvate is a hydrate; and if the solvent is alcohol, the solvate is an alcoholate. Hydrates are formed by the combination of water molecules with molecules of a substance in which water retains its molecular state as H2O. Hydrates are referred to, for example, monohydrates, dihydrates, trihydrates, and the like.

[0341] In addition, the compounds represented by the formulae disclosed herein, or salts thereof, can exist in crystal polymorphs. It is noted that any crystal form, mixture of crystal forms, or anhydride or hydrate thereof is included within the scope of the present disclosure.

[0342] Anti-CD45 antibodies

[0343] Encompassed herein are antibodies or antigen-binding fragments thereof capable of binding CD45 that can be used as a sole therapeutic agent or as a conjugate (ADC) for, e.g., (i) treating cancers characterized by CD45+ cells and autoimmune diseases, and (ii) facilitating engraftment of transplanted hematopoietic stem cells in patients in need of transplant therapy. Such therapeutic activity can arise, e.g., by binding of an isolated anti-CD45 antibody, antigen-binding fragment thereof, or ADC to CD45 expressed on the surface of a cell (e.g., a cancer cell, an autoimmune cell, or a hematopoietic stem cell), and subsequently inducing cell death. Depletion of endogenous hematopoietic stem cells can provide a niche for transplanted hematopoietic stem cells to home to and subsequently establish productive hematopoiesis. In this way, transplanted hematopoietic stem cells can successfully engraft in a patient, e.g., a human patient suffering from a stem cell disorder described herein. In addition, the combination of depleting white blood cells in a patient in need thereof with an HSC transplant can re-set the immune system of the patient, thereby curing a patient of, e.g., an autoimmune disease.

[0344] CD45, also known as leukocyte common antigen and receptor-type tyrosine-protein phosphatase C, is a hematopoietic cell-specific transmembrane protein tyrosine phosphatase that is required for T and B cell antigen receptor-mediated signaling. CD45 comprises a large extracellular domain and a cytoplasmic domain containing the phosphatase. CD45 can act as a positive and negative regulator, depending on the nature of the stimulus and the cell type involved. Although a large number of hypermutations can occur in the CD45 gene, only six isoforms are typically identified in humans. The isoforms are RA, RO, RB, RAB, RBC, and RABC (Hermiston et al., 2003 "CD45: a critical regulator of signaling thresholds in immune cells", Annu Rev Immunol. 2: 107-137). CD45RA is expressed on naive T cells, and CD45RO is expressed on activated and memory T cells, some B cell subsets, activated monocytes / macrophages, and granulocytes. CD45RB is expressed on peripheral B cells, naive T cells, thymocytes, macrophages (weakly), and dendritic cells. The amino acid sequence of CD45RABC is provided herein as SEQ ID NO: 112. The amino acid sequence of CD45RA is provided herein as SEQ ID NO: 107. The amino acid sequence of CD45RO is provided herein as SEQ ID NO: 108. The amino acid sequence of CD45RB is provided herein as SEQ ID NO: 109. The amino acid sequence of CD45RAB is provided herein as SEQ ID NO: 110. The amino acid sequence of CD45RBC is provided herein as SEQ ID NO: 111.

[0345] As described below, in certain embodiments, novel anti-human CD45 (hCD45) antibodies were identified by screening a yeast display library displaying fully human antibodies. Seven human antibodies were identified in the screen (designated Antibody 1 (Ab1), Antibody 2 (Ab2), Antibody 3 (Ab3), Antibody 4 (Ab4), Antibody 5 (Ab5), Antibody 6 (Ab6), and Antibody 7 (Ab7)) that bind to human CD45 (all isoforms) and cross-react with non-human primate CD45 (e.g., cynomolgus monkey CD45 and / or rhesus monkey CD45).

[0346] In other embodiments, humanized and affinity matured antibodies are prepared from three rat anti-CD45 antibodies. In this manner, three additional antibodies are identified (designated Antibody A (AbA), Antibody B (AbB), and Antibody C (AbC) that bind to human CD45 (all isoforms) and cross-react with non-human primate CD45 (e.g., cynomolgus monkey CD45 and / or rhesus monkey CD45). The identified antibodies have diagnostic and therapeutic characteristics, as described herein.

[0347] Accordingly, provided herein are antibodies, or antigen binding portions thereof, that specifically bind to human CD45 and cross-react with non-human primate CD45.

[0348] In one embodiment, the present disclosure provides antibodies, or antigen binding portions thereof, that bind to human CD45 (SEQ ID NO: 112) and cynomolgus monkey CD45 (SEQ ID NO: 145) and / or rhesus monkey CD45 (SEQ ID NO: 146). In some embodiments, the antibodies, or antigen binding portions thereof, can bind to human CD45 and K D about 100 nM or less, e.g., about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 10 nM or less, or about 0.1 nM or less, as determined by Bio-Layer Interferometry (BLI). In some embodiments, the antibodies, or antigen binding portions thereof, can bind to cynomolgus monkey CD45 and K D about 100 nM or less, e.g., about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 10 nM or less, or about 0.1 nM or less, as determined by Bio-Layer Interferometry (BLI). In some embodiments, the antibodies, or antigen binding portions thereof, can bind to cynomolgus monkey CD45 and K Dabout 100 nM or less, for example, about 100 nM or less, about 90 nM or less, about 80 nM or less, about 70 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 30 nM or less, about 20 nM or less, about 10 nM or less, about 10 nM or less, or about 0.1 nM or less, as determined by Biolayer Interferometry (BLI). In some embodiments, the antibody is a fully human antibody or antigen binding portion thereof. In other embodiments, the antibody is a humanized antibody or antigen binding portion thereof. In some embodiments, the antibody is a chimeric antibody or antigen binding portion thereof. In some embodiments, the antibody is a deimmunized antibody or antigen binding portion thereof.

[0349] The extracellular region of human CD45 includes a mucin-like domain and four fibronectin-like domains (d1, d2, d3, and d4). Without wishing to be bound by any theory, it is believed that antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, and Ab7 interact with residues of human CD45 located within the d3 and d4 fibronectin-like domains. In particular, the antibodies can interact with the human CD45 fragment set forth in SEQ ID NO: 115 and the human CD45 fragment set forth in SEQ ID NO: 117. Crosslinking studies described herein indicate that the antibodies can specifically interact with one or more CD45 amino acid residues that are conserved between human CD45, cynomolgus CD45, and rhesus CD45. Such residues include 405T, 407K, 419Y, 425K, and 505R (referring to the hCD45 fragment numbering set forth in SEQ ID NO: 113). In addition, the antibodies can interact with residues 481R and / or 509H in human CD45 (referring to the hCD45 fragment numbering set forth in SEQ ID NO: 113). Accordingly, in some embodiments, provided herein are antibodies or antigen-binding portions thereof that bind to human CD45 at an epitope located in the d3 and / or d4 fibronectin-like domain. In some embodiments, provided herein are antibodies or antigen-binding portions thereof that bind to CD45 at an epitope located within CD45 fragment 2 (SEQ ID NO: 115) and / or CD45 fragment 4 (SEQ ID NO: 117) of human CD45. In some embodiments, provided herein are antibodies or antigen-binding portions thereof that bind to CD45 at an epitope located within CD45 fragment 1 (SEQ ID NO: 114) and / or CD45 fragment 3 (SEQ ID NO: 116) of human CD45. In some embodiments, provided herein are antibodies or antigen-binding portions thereof that bind to CD45 at an epitope comprising at least one, at least two, at least three, at least four, or at least five amino acid residues that are conserved between human CD45, cynomolgus CD45, and / or rhesus CD45. For example, in some embodiments, the antibodies or antigen-binding portions thereof can bind to at least one, at least two, at least three, at least four, or all five of the following amino acid residues in human CD45: 405T, 407K, 419Y, 425K, and 505R (referring to the hCD45 fragment numbering set forth in SEQ ID NO: 113).In some embodiments, the antibody or antigen-binding portion thereof can bind to one or more, two or more, three or more, four or more, five or more, six or more, or seven of the following amino acid residues in human CD45: 405T, 407K, 419Y, 425K, 481R, and 505R, 509H (referring to hCD45 fragment numbering set forth in SEQ ID NO: 113). Also provided herein are antibodies or antigen-binding portions thereof that compete with Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, and / or Ab7 for binding to human CD45 (SEQ ID NO: 112). In some embodiments, the antibody or antigen-binding portion thereof can also compete with Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, and / or Ab7 for binding to cynomolgus monkey CD45 (SEQ ID NO: 145) and / or rhesus monkey CD45 (SEQ ID NO: 146). In some embodiments, the antibody is a fully human antibody or antigen-binding portion thereof. In other embodiments, the antibody is a humanized antibody or antigen-binding portion thereof. In some embodiments, the antibody is a chimeric antibody or antigen-binding portion thereof. In some embodiments, the antibody is a deimmunized antibody or antigen-binding portion thereof.

[0350] Without wishing to be bound by any theory, it is believed that the antibody AbA described herein also binds to the d4 fibronectin-like domain of CD45, but does not compete for binding to CD45 with any of Ab1-Ab7. Antigenic determinant mapping experiments described herein indicate that AbA binds to the d4 fibronectin-like domain on an opposite face of the molecule from Ab1-Ab7. In particular, it is believed that this antibody interacts with the human CD45 fragment shown in SEQ ID NO: 118. Crosslinking studies described herein indicate that AbA can specifically interact with one or more CD45 amino acid residues that are conserved between human CD45, cynomolgus CD45, and rhesus CD45. Such residues include 493Y and 502T (referring to the hCD45 fragment numbering shown in SEQ ID NO: 113). In addition, this antibody can interact with residue 486R in human CD45 (referring to the hCD45 fragment numbering shown in SEQ ID NO: 113). Accordingly, in some embodiments, provided herein are antibodies or antigen-binding portions thereof that bind to human CD45 at an antigenic determinant located in the d4 fibronectin-like domain. In some embodiments, provided herein are antibodies or antigen-binding portions thereof that bind to CD45 at an antigenic determinant located within CD45 fragment 5 (SEQ ID NO: 118) of human CD45. In some embodiments, provided herein are antibodies or antigen-binding portions thereof that bind to CD45 at an antigenic determinant comprising at least one or at least two amino acid residues that are conserved in human CD45, cynomolgus CD45, and / or rhesus CD45. For example, in some embodiments, the antibody or antigen-binding portion thereof can bind to one or both of the following amino acid residues in human CD45: 493Y and 502T (referring to the hCD45 fragment numbering shown in SEQ ID NO: 113). In some embodiments, the antibody or antigen-binding portion thereof can bind to one or more, two or more, or three of the following amino acid residues in human CD45: 486R, 493Y, and 502T (referring to the hCD45 fragment numbering shown in SEQ ID NO: 113). Also provided herein are antibodies or antigen-binding portions thereof that compete with AbA for binding to human CD45 (SEQ ID NO: 112). In some embodiments, the antibody or antigen-binding portion thereof can also compete with AbA for binding to cynomolgus CD45 (SEQ ID NO: 145) and / or rhesus CD45 (SEQ ID NO: 146). In some embodiments, the antibody is a fully human antibody or antigen-binding portion thereof. In other embodiments, the antibody is a humanized antibody or antigen-binding portion thereof. In some embodiments, the antibody is a chimeric antibody or antigen-binding portion thereof. In some embodiments, the antibody is a deimmunized antibody or antigen-binding portion thereof.

[0351] In other embodiments, provided herein are antibodies or antigen-binding portions thereof that bind to the same epitope of human CD45 as AbB. In some embodiments, the antibodies or antigen-binding portions thereof cross-react with cynomolgus CD45 and / or rhesus CD45. Also provided herein are antibodies or antigen-binding portions thereof that compete with AbB for binding to human CD45 (SEQ ID NO: 112). In some embodiments, the antibodies or antigen-binding portions thereof can also compete with AbB for binding to cynomolgus CD45 (SEQ ID NO: 145) and / or rhesus CD45 (SEQ ID NO: 146). In some embodiments, the antibodies are fully human antibodies or antigen-binding portions thereof. In other embodiments, the antibodies are humanized antibodies or antigen-binding portions thereof. In some embodiments, the antibodies are chimeric antibodies or antigen-binding portions thereof. In some embodiments, the antibodies are deimmunized antibodies or antigen-binding portions thereof.

[0352] In other embodiments, provided herein are antibodies or antigen-binding portions thereof that bind to the same epitope of human CD45 as AbC. In some embodiments, the antibodies or antigen-binding portions thereof cross-react with cynomolgus CD45 and / or rhesus CD45. Also provided herein are antibodies or antigen-binding portions thereof that compete with AbC for binding to human CD45 (SEQ ID NO: 112). In some embodiments, the antibodies or antigen-binding portions thereof can also compete with AbC for binding to cynomolgus CD45 (SEQ ID NO: 145) and / or rhesus CD45 (SEQ ID NO: 146). In some embodiments, the antibodies are fully human antibodies or antigen-binding portions thereof. In other embodiments, the antibodies are humanized antibodies or antigen-binding portions thereof. In some embodiments, the antibodies are chimeric antibodies or antigen-binding portions thereof. In some embodiments, the antibodies are deimmunized antibodies or antigen-binding portions thereof.

[0353] The amino acid sequences of each of the binding regions of anti-CD45 antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, and AbC are set forth in Table 27. In various aspects, the present application provides antibodies comprising the heavy and / or light chain CDR sequences of the antibodies described in Table 27. In some aspects, the present application provides antibodies comprising the heavy chain variable region and / or light chain variable region of the antibodies described in Table 27. In some aspects, the present application provides antibodies comprising the heavy and / or light chain of the antibodies described in Table 27. Additional features of the antibodies and antigen-binding portions thereof provided herein are set forth below.

[0354] Ab1

[0355] Antibody 1 (Ab1) cross-reacts with human CD45, cynomolgus CD45, and rhesus CD45, and can bind to multiple isoforms of human CD45.

[0356] The amino acid sequences of each of the binding regions of the anti-CD45 Ab1 are set forth in Table 27. The present disclosure includes, for example, anti-CD45 antibodies based on Ab1 comprising the CDRs as shown in Table 27.

[0357] In one embodiment, the present disclosure provides an anti-CD45 antibody or antigen-binding fragment thereof comprising an antigen-binding region, for example, CDRs and / or a variable region corresponding to those of Ab1. The heavy chain variable region (VH) amino acid sequence of Ab1 is shown in SEQ ID NO: 1 (see Table 27). The VH CDR domain amino acid sequences of Ab1 are shown in SEQ ID NO: 2 (VH CDR1); SEQ ID NO: 3 (VH CDR2); and SEQ ID NO: 4 (VH CDR3). The light chain variable region (VL) amino acid sequence of Ab1 is set forth in SEQ ID NO: 5 (see Table 27). The VL CDR domain amino acid sequences of Ab1 are shown in SEQ ID NO: 6 (VL CDR1); SEQ ID NO: 7 (VL CDR2); and SEQ ID NO: 8 (VL CDR3).

[0358] Thus, in some embodiments, provided herein is an anti-CD45 antibody or antigen-binding portion thereof, comprising a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 2, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 3, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 4; and / or a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 6, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 7; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 8. In certain embodiments, the anti-CD45 antibody comprises the CDRs described herein (SEQ ID Nos: 2-4 and 6-8), wherein the CDRs comprise conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., similar to the specificity of Ab1).

[0359] In some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 1, and / or a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 5. In certain embodiments, the antibody can comprise a modified heavy chain (HC) variable region comprising a HC variable domain comprising SEQ ID NO: 1 or a variant of SEQ ID NO: 1 that (i) differs from SEQ ID NO: 1 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 1 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 1 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of Ab1), or has an enhanced biological activity relative to the biological activity of another Ab1 heavy chain variable region. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising a LC variable domain comprising SEQ ID NO: 5 or a variant of SEQ ID NO: 5 that (i) differs from SEQ ID NO: 5 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 5 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 5 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 5, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of Ab1), or has an enhanced biological activity relative to the biological activity of another Ab1 light chain variable region.

[0360] Antibodies comprising CDR and / or variable region sequences of Ab1 can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multi-chain or single-chain antibodies, and / or binding fragments that specifically bind human CD45, including but not limited to Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibody (including surrogate light chain constructs), single domain antibodies, camelized antibodies, and the like. They can also have or be derived from any isotype, including, for example, IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), or IgM. In some embodiments, anti-45 antibodies are IgG (e.g., IgGl, IgG2, IgG3, or IgG4).

[0361] In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab1 can further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgGl constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some embodiments, the heavy chain constant region can be a modified constant region. Exemplary constant region substitutions and / or modifications are set forth herein and include, but are not limited to, substitutions at one or more of the following positions: 234, 235, 265, and 435 (according to the EU index of Kabat). In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab1 can further comprise an IgGl heavy chain constant region comprising one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the EU index of Kabat). In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab1 can further comprise a heavy chain constant region set forth in SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106. In some embodiments antibodies comprise a light chain constant region set forth in SEQ ID NO: 101.

[0362] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing the amino acid sequence shown in SEQ ID NO: 9. In some embodiments, the antibody comprises a modified heavy chain (HC) region containing an HC domain containing SEQ ID NO: 9 or a variant of SEQ ID NO: 9, which (i) differs from SEQ ID NO: 9 in that 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 9 in that up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 9 in that 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions; and / or (iv) contains a heavy chain containing the amino acid sequence shown in SEQ ID NO: 9. 9. An amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, wherein in any of (i)-(iv), the amino acid substitutions may be conserved or non-conserved; and wherein the modified heavy chain region retains the antibody’s CD45 specificity (i.e., Ab1-like specificity) or has enhanced biological activity relative to another Ab1 heavy chain region.

[0363] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a light chain containing an amino acid sequence as shown in SEQ ID NO: 10. In some embodiments, the antibody comprises a modified light chain (LC) region containing an LC domain containing SEQ ID NO: 10 or a variant of SEQ ID NO: 10, the variant being (i) different from SEQ ID NO: 10 in that 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) different from SEQ ID NO: 10 in that up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) different from SEQ ID NO: 10 in that 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions; and / or (iv) containing the amino acid sequence shown in SEQ ID NO: 10. 10 An amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, wherein in any of (i)-(iv), the amino acid substitutions may be conserved or non-conserved; and wherein the modified light chain region retains the antibody’s CD45 specificity (i.e., Ab1-like specificity) or has enhanced biological activity relative to another Ab1 light chain region.

[0364] In some embodiments, antibodies comprising the CDR regions and / or variable regions of Ab1 can be incorporated into antibody-drug conjugates, as described herein. In addition, antibodies comprising the CDR regions and / or variable regions of Ab1 can be used in the methods described herein, for example, for depleting CD45+ cells in a subject.

[0365] Ab2

[0366] Antibody 2 (Ab2) cross-reacts with human CD45, cynomolgus CD45, and rhesus CD45, and can bind to multiple isoforms of human CD45.

[0367] The amino acid sequences of each binding region of anti-CD45 Ab2 are set forth in Table 27. The present disclosure includes anti-CD45 antibodies based on Ab2, for example, comprising the CDRs as shown in Table 27.

[0368] In one embodiment, the present disclosure provides an anti-CD45 antibody or antigen-binding fragment thereof comprising an antigen binding region, for example, a CDR and / or variable region corresponding to those of Ab2. The heavy chain variable region (VH) amino acid sequence of Ab2 is shown in SEQ ID NO: 11 (see Table 27). The VH CDR domain amino acid sequences of Ab2 are shown in SEQ ID NO: 12 (VH CDR1); SEQ ID NO: 13 (VH CDR2); and SEQ ID NO: 14 (VH CDR3). The light chain variable region (VL) amino acid sequence of Ab2 is set forth in SEQ ID NO: 15 (see Table 27). The VL CDR domain amino acid sequences of Ab2 are shown in SEQ ID NO: 16 (VL CDR1); SEQ ID NO: 17 (VL CDR2); and SEQ ID NO: 18 (VL CDR3).

[0369] Accordingly, provided herein in some embodiments are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 12, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 13, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 14; and / or a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 16, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 17; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 18. In certain embodiments, the anti-CD45 antibodies comprise the CDRs described herein (SEQ ID NOs: 12-14 and 16-18), wherein the CDRs comprise conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., similar to the specificity of Ab2).

[0370] In some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 11, and / or a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 15. In certain embodiments, the antibody can comprise a modified heavy chain (HC) variable region comprising a HC variable domain comprising SEQ ID NO: 11 or a variant of SEQ ID NO: 11 that (i) differs from SEQ ID NO: 11 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 11 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 11 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of Ab2), or has an enhanced biological activity relative to the biological activity of another Ab2 heavy chain variable region. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising a LC variable domain comprising SEQ ID NO: 15 or a variant of SEQ ID NO: 15 that (i) differs from SEQ ID NO: 15 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 15 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 15 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of Ab2), or has an enhanced biological activity relative to the biological activity of another Ab2 light chain variable region.

[0371] Antibodies comprising CDRs and / or variable region sequences of Ab2 can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multi-chain or single-chain antibodies, and / or binding fragments that specifically bind human CD45, including, but not limited to, Fabs, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibody (including surrogate light chain constructs), single domain antibodies, camelized antibodies, and the like. They can also have or be derived from any isotype, including, for example, IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), or IgM. In some embodiments, anti-45 antibodies are IgG (e.g., IgGl, IgG2, IgG3, or IgG4).

[0372] In some embodiments, antibodies comprising CDRs and / or variable region sequences of Ab2 can further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgGl constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some embodiments, the heavy chain constant region can be a modified constant region. Exemplary constant region substitutions and / or modifications are set forth herein and include, but are not limited to, substitutions at one or more of the following positions: 234, 235, 265, and 435 (according to the EU index of Kabat). In some embodiments, antibodies comprising CDRs and / or variable region sequences of Ab2 can further comprise an IgGl heavy chain constant region comprising one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the EU index of Kabat). In some embodiments, antibodies comprising CDRs and / or variable region sequences of Ab2 can further comprise a heavy chain constant region set forth in SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106. In some embodiments antibodies comprise a light chain constant region set forth in SEQ ID NO: 101.

[0373] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 19. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 19 or a variant of SEQ ID NO: 19 that (i) differs from SEQ ID NO: 19 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 19 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 19 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 19, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of Ab2), or has an enhanced biological activity relative to the biological activity of another Ab2 heavy chain region.

[0374] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 20. In certain embodiments, the antibody comprises a modified light chain (LC) region comprising a LC domain comprising SEQ ID NO: 20 or a variant of SEQ ID NO: 20 that (i) differs from SEQ ID NO: 20 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 20 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 20 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 20, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of Ab2), or has an enhanced biological activity relative to the biological activity of another Ab2 light chain region.

[0375] In some embodiments, antibodies containing the CDR region and / or variable region of Ab2 may be incorporated into antibody-drug conjugates, as described herein. Additionally, antibodies containing the CDR region and / or variable region of Ab2 may be used in the methods described herein, for example, for depleting CD45+ cells in an individual.

[0376] Ab3

[0377] Antibody 3 (Ab3) cross-reacts with human CD45, cynomolgus monkey CD45 and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0378] The amino acid sequences of the various binding regions of anti-CD45 Ab3 are described in Table 27. This invention includes, for example, Ab3-based anti-CD45 antibodies comprising the CDRs shown in Table 27.

[0379] In one embodiment, the present invention provides an anti-CD45 antibody or an antigen-binding fragment thereof comprising an antigen-binding region, such as a CDR and / or variable region corresponding to the CDR and / or variable region of Ab3. The amino acid sequence of the heavy chain variable region (VH) of Ab3 is shown in SEQ ID NO: 21 (see Table 27). The amino acid sequence of the VH CDR domain of Ab3 is shown in SEQ ID NO: 22 (VH CDR1); SEQ ID NO: 23 (VH CDR2) and SEQ ID NO: 24 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of Ab3 is described in SEQ ID NO: 25 (see Table 27). The amino acid sequence of the VL CDR domain of Ab3 is shown in SEQ ID NO: 26 (VL CDR1); SEQ ID NO: 27 (VL CDR2) and SEQ ID NO: 28 (VL CDR3).

[0380] Accordingly, in some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 22, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 23, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 24; and / or a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 26, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 27; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 28. In certain embodiments, the anti-CD45 antibodies comprise the CDRs described herein (SEQ ID NOs: 22-24 and 26-28), wherein the CDRs comprise conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., similar to the specificity of Ab3).

[0381] In some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 21, and / or a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 25. In certain embodiments, the antibody can comprise a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO: 21 or a variant of SEQ ID NO: 21 that (i) differs from SEQ ID NO: 21 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 21 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 21 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain variable region retains the CD45 specificity of the antibody (i.e., similar to the specificity of Ab3), or has an enhanced biological activity relative to the biological activity of another Ab3 heavy chain variable region. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain comprising SEQ ID NO: 25 or a variant of SEQ ID NO: 25 that (i) differs from SEQ ID NO: 25 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 25 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 25 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain variable region retains the CD45 specificity of the antibody (i.e., similar to the specificity of Ab3), or has an enhanced biological activity relative to the biological activity of another Ab3 light chain variable region.

[0382] Antibodies comprising CDR and / or variable region sequences of Ab3 can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multi-chain or single-chain antibodies, and / or binding fragments that specifically bind human CD45, including but not limited to Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibody (including surrogate light chain constructs), single domain antibodies, camelized antibodies, and the like. They can also have or be derived from any isotype, including, for example, IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), or IgM. In some embodiments, anti-45 antibodies are IgG (e.g., IgGl, IgG2, IgG3, or IgG4).

[0383] In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab3 can further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgGl constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some embodiments, the heavy chain constant region can be a modified constant region. Exemplary constant region substitutions and / or modifications are set forth herein and include, but are not limited to, substitutions at one or more of the following positions: 234, 235, 265, and 435 (according to the EU index of Kabat). In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab3 can further comprise an IgGl heavy chain constant region comprising one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the EU index of Kabat). In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab3 can further comprise a heavy chain constant region set forth in SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106. In some embodiments antibodies comprise a light chain constant region set forth in SEQ ID NO: 101.

[0384] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 29. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 29 or a variant of SEQ ID NO: 29 that (i) differs from SEQ ID NO: 29 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 29 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 29 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., similar to the specificity of Ab3), or has an enhanced biological activity relative to the biological activity of another Ab3 heavy chain region.

[0385] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 29. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 29 or a variant of SEQ ID NO: 29 that (i) differs from SEQ ID NO: 29 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 29 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 29 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., similar to the specificity of Ab3), or has an enhanced biological activity relative to the biological activity of another Ab3 heavy chain region.

[0386] In some embodiments, antibodies comprising the CDR regions and / or variable regions of Ab3 can be incorporated into antibody-drug conjugates, as described herein. In addition, antibodies comprising the CDR regions and / or variable regions of Ab3 can be used in the methods described herein, for example, for depleting CD45+ cells in a subject.

[0387] Ab4

[0388] Antibody 4 (Ab4) cross-reacts with human CD45, cynomolgus CD45, and rhesus CD45, and can bind to multiple isoforms of human CD45.

[0389] The amino acid sequences of each binding region of anti-CD45 Ab4 are set forth in Table 27. The present disclosure includes anti-CD45 antibodies based on Ab4, e.g., comprising the CDRs as shown in Table 27.

[0390] In one embodiment, the present disclosure provides an anti-CD45 antibody or antigen-binding fragment thereof comprising an antigen-binding region, e.g., a CDR and / or variable region corresponding to those of Ab4. The heavy chain variable region (VH) amino acid sequence of Ab4 is set forth in SEQ ID NO: 31 (see Table 27). The VH CDR domain amino acid sequences of Ab4 are set forth in SEQ ID NO: 32 (VH CDR1); SEQ ID NO: 33 (VH CDR2); and SEQ ID NO: 34 (VH CDR3). The light chain variable region (VL) amino acid sequence of Ab4 is set forth in SEQ ID NO: 35 (see Table 27). The VL CDR domain amino acid sequences of Ab4 are set forth in SEQ ID NO: 36 (VL CDR1); SEQ ID NO: 37 (VL CDR2); and SEQ ID NO: 38 (VL CDR3).

[0391] Thus, in some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 32, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 33, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 34; and / or a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 36, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 37; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 38. In certain embodiments, the anti-CD45 antibodies comprise the CDRs described herein (SEQ ID NOs: 32-34 and 36-38) wherein the CDRs comprise conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., similar to the specificity of Ab4).

[0392] In some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 31, and / or a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 35. In certain embodiments, the antibody can comprise a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO: 31 or a variant of SEQ ID NO: 31 that (i) differs from SEQ ID NO: 31 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 31 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 31 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 31, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain variable region retains the CD45 specificity of the antibody (i.e., specificity similar to that of Ab4), or has an enhanced biological activity relative to the biological activity of another Ab4 heavy chain variable region. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain comprising SEQ ID NO: 35 or a variant of SEQ ID NO: 35 that (i) differs from SEQ ID NO: 35 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 35 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 35 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 35, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain variable region retains the CD45 specificity of the antibody (i.e., specificity similar to that of Ab4), or has an enhanced biological activity relative to the biological activity of another Ab4 light chain variable region.

[0393] Antibodies comprising CDR and / or variable region sequences of Ab4 can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multi-chain or single-chain antibodies, and / or binding fragments that specifically bind human CD45, including but not limited to Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibody (including surrogate light chain constructs), single domain antibodies, camelized antibodies, and the like. They can also have or be derived from any isotype, including, for example, IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), or IgM. In some embodiments, anti-45 antibodies are IgG (e.g., IgGl, IgG2, IgG3, or IgG4).

[0394] In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab4 can further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgGl constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some embodiments, the heavy chain constant region can be a modified constant region. Exemplary constant region substitutions and / or modifications are set forth herein and include, but are not limited to, substitutions at one or more of the following positions: 234, 235, 265, and 435 (according to the EU index of Kabat). In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab4 can further comprise an IgGl heavy chain constant region comprising one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the EU index of Kabat). In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab4 can further comprise a heavy chain constant region set forth in SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106. In some embodiments antibodies comprise a light chain constant region set forth in SEQ ID NO: 101.

[0395] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 39. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 39 or a variant of SEQ ID NO: 39 that (i) differs from SEQ ID NO: 39 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 39 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 39 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of Ab4), or has an enhanced biological activity relative to the biological activity of another Ab4 heavy chain region.

[0396] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 39. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 39 or a variant of SEQ ID NO: 39 that (i) differs from SEQ ID NO: 39 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 39 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 39 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 39, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of Ab4), or has an enhanced biological activity relative to the biological activity of another Ab4 heavy chain region.

[0397] In some embodiments, antibodies comprising the CDR regions and / or variable regions of Ab4 can be incorporated into antibody-drug conjugates, as described herein. In addition, antibodies comprising the CDR regions and / or variable regions of Ab4 can be used in the methods described herein, for example, for depleting CD45+ cells in a subject.

[0398] Ab5

[0399] Antibody 5 (Ab5) cross-reacts with human CD45, cynomolgus CD45, and rhesus CD45, and can bind to multiple isoforms of human CD45.

[0400] The amino acid sequences of each binding region of anti-CD45 Ab5 are set forth in Table 27. The present disclosure includes anti-CD45 antibodies based on Ab5, for example, comprising the CDRs as shown in Table 27.

[0401] In one embodiment, the present disclosure provides an anti-CD45 antibody or antigen-binding fragment thereof comprising an antigen-binding region, for example, a CDR and / or variable region corresponding to those of Ab5. The heavy chain variable region (VH) amino acid sequence of Ab5 is set forth in SEQ ID NO: 41 (see Table 27). The VH CDR domain amino acid sequences of Ab5 are set forth in SEQ ID NO: 42 (VH CDR1); SEQ ID NO: 43 (VH CDR2); and SEQ ID NO: 44 (VH CDR3). The light chain variable region (VL) amino acid sequence of Ab5 is set forth in SEQ ID NO: 45 (see Table 27). The VL CDR domain amino acid sequences of Ab5 are set forth in SEQ ID NO: 46 (VL CDR1); SEQ ID NO: 47 (VL CDR2); and SEQ ID NO: 48 (VL CDR3).

[0402] Thus, in some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 42, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 43, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 44; and / or a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 46, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 47; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 48. In certain embodiments, the anti-CD45 antibodies comprise the CDRs described herein (SEQ ID NOs: 42-44 and 46-48), wherein the CDRs comprise conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., specificity similar to that of Ab5).

[0403] In some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 41, and / or a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 45. In certain embodiments, the antibody can comprise a modified heavy chain (HC) variable region comprising a HC variable domain comprising SEQ ID NO: 41 or a variant of SEQ ID NO: 41 that (i) differs from SEQ ID NO: 41 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 41 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 41 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of Ab5), or has an enhanced biological activity relative to the biological activity of another Ab5 heavy chain variable region. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising a LC variable domain comprising SEQ ID NO: 45 or a variant of SEQ ID NO: 45 that (i) differs from SEQ ID NO: 45 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 45 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 45 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 45, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of Ab5), or has an enhanced biological activity relative to the biological activity of another Ab5 light chain variable region.

[0404] Antibodies comprising CDRs and / or variable region sequences of Ab5 can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multi-chain or single-chain antibodies, and / or binding fragments that specifically bind human CD45, including, but not limited to, Fabs, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including surrogate light chain constructs), single domain antibodies, camelized antibodies, and the like. They can also have or be derived from any isotype, including, for example, IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), or IgM. In some embodiments, anti-45 antibodies are IgG (e.g., IgGl, IgG2, IgG3, or IgG4).

[0405] In some embodiments, antibodies comprising CDRs and / or variable region sequences of Ab5 can further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgGl constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some embodiments, the heavy chain constant region can be a modified constant region. Exemplary constant region substitutions and / or modifications are set forth herein and include, but are not limited to, substitutions at one or more of the following positions: 234, 235, 265, and 435 (according to the EU index of Kabat). In some embodiments, antibodies comprising CDRs and / or variable region sequences of Ab5 can further comprise an IgGl heavy chain constant region comprising one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the EU index of Kabat). In some embodiments, antibodies comprising CDRs and / or variable region sequences of Ab5 can further comprise a heavy chain constant region set forth in SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106. In some embodiments antibodies comprise a light chain constant region set forth in SEQ ID NO: 101.

[0406] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 49. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 49 or a variant of SEQ ID NO: 9 that (i) differs from SEQ ID NO: 49 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 49 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 49 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 49, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of Ab5), or has an enhanced biological activity relative to the biological activity of another Ab5 heavy chain region.

[0407] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 49. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 49 or a variant of SEQ ID NO: 9 that (i) differs from SEQ ID NO: 49 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 49 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 49 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 49, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of Ab5), or has an enhanced biological activity relative to the biological activity of another Ab5 heavy chain region.

[0408] In some embodiments, antibodies comprising the CDR regions and / or variable regions of Ab5 can be incorporated into antibody-drug conjugates, as described herein. In addition, antibodies comprising the CDR regions and / or variable regions of Ab5 can be used in the methods described herein, for example, for depleting CD45+ cells in a subject.

[0409] Ab6

[0410] Antibody 6 (Ab6) cross-reacts with human CD45, cynomolgus CD45, and rhesus CD45, and can bind to multiple isoforms of human CD45.

[0411] The amino acid sequences of each binding region of anti-CD45 Ab6 are set forth in Table 27. The present disclosure includes anti-CD45 antibodies based on Ab6, e.g., comprising the CDRs as shown in Table 27.

[0412] In one embodiment, the present disclosure provides an anti-CD45 antibody or antigen-binding fragment thereof comprising an antigen-binding region, e.g., a CDR and / or variable region corresponding to those of Ab6. The heavy chain variable region (VH) amino acid sequence of Ab6 is shown in SEQ ID NO: 51 (see Table 27). The VH CDR domain amino acid sequences of Ab6 are shown in SEQ ID NO: 52 (VH CDR1); SEQ ID NO: 53 (VH CDR2); and SEQ ID NO: 54 (VH CDR3). The light chain variable region (VL) amino acid sequence of Ab6 is set forth in SEQ ID NO: 55 (see Table 27). The VL CDR domain amino acid sequences of Ab6 are shown in SEQ ID NO: 56 (VL CDR1); SEQ ID NO: 57 (VL CDR2); and SEQ ID NO: 58 (VL CDR3).

[0413] Accordingly, in some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 52, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 53, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 54; and / or a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 56, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 57; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 58. In certain embodiments, the anti-CD45 antibodies comprise the CDRs described herein (SEQ ID NOs: 52-54 and 56-58) wherein the CDRs comprise conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., specificity similar to that of Ab6).

[0414] In some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 51, and / or a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 55. In certain embodiments, the antibody can comprise a modified heavy chain (HC) variable region comprising a HC variable domain comprising SEQ ID NO: 51 or a variant of SEQ ID NO: 51 that (i) differs from SEQ ID NO: 51 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 51 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 51 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 51, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain variable region retains the CD45 specificity of the antibody (i.e., specificity similar to Ab6), or has an enhanced biological activity relative to the biological activity of another Ab6 heavy chain variable region. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising a LC variable domain comprising SEQ ID NO: 55 or a variant of SEQ ID NO: 55 that (i) differs from SEQ ID NO: 55 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 55 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 55 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 55, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain variable region retains the CD45 specificity of the antibody (i.e., specificity similar to Ab6), or has an enhanced biological activity relative to the biological activity of another Ab6 light chain variable region.

[0415] Antibodies comprising CDR and / or variable region sequences of Ab6 can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multi-chain or single-chain antibodies, and / or binding fragments that specifically bind human CD45, including, but not limited to, Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibody (including surrogate light chain constructs), single domain antibodies, camelized antibodies, and the like. They can also have or be derived from any isotype, including, for example, IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), or IgM. In some embodiments, anti-45 antibodies are IgG (e.g., IgGl, IgG2, IgG3, or IgG4).

[0416] In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab6 can further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgGl constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some embodiments, the heavy chain constant region can be a modified constant region. Exemplary constant region substitutions and / or modifications are set forth herein and include, but are not limited to, substitutions at one or more of the following positions: 234, 235, 265, and 435 (according to the EU index of Kabat). In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab6 can further comprise an IgGl heavy chain constant region comprising one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the EU index of Kabat). In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab6 can further comprise a heavy chain constant region set forth in SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106. In some embodiments the antibodies comprise a light chain constant region set forth in SEQ ID NO: 101.

[0417] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 59. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 59 or a variant of SEQ ID NO: 59 that (i) differs from SEQ ID NO: 59 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 59 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 59 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 59, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., specificity analogous to Ab6), or has an enhanced biological activity relative to the biological activity of another Ab6 heavy chain region.

[0418] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 60. In certain embodiments, the antibody comprises a modified light chain (LC) region comprising a LC domain comprising SEQ ID NO: 60 or a variant of SEQ ID NO: 60 that (i) differs from SEQ ID NO: 60 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 60 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 60 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 60, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain region retains the CD45 specificity of the antibody (i.e., specificity analogous to Ab6), or has an enhanced biological activity relative to the biological activity of another Ab6 light chain region.

[0419] In some embodiments, antibodies comprising the CDR regions and / or variable regions of Ab6 can be incorporated into antibody-drug conjugates, as described herein. In addition, antibodies comprising the CDR regions and / or variable regions of Ab6 can be used in the methods described herein, for example, for depleting CD45+ cells in a subject.

[0420] Ab7

[0421] Antibody 7 (Ab7) cross-reacts with human CD45, cynomolgus CD45, and rhesus CD45, and can bind to multiple isoforms of human CD45.

[0422] The amino acid sequences of each binding region of anti-CD45 Ab7 are set forth in Table 27. The present disclosure includes anti-CD45 antibodies based on Ab7, e.g., comprising the CDRs as shown in Table 27.

[0423] In one embodiment, the present disclosure provides an anti-CD45 antibody or antigen-binding fragment thereof comprising an antigen-binding region, e.g., a CDR and / or variable region corresponding to those of Ab7. The heavy chain variable region (VH) amino acid sequence of Ab7 is set forth in SEQ ID NO: 61 (see Table 27). The VH CDR domain amino acid sequences of Ab7 are set forth in SEQ ID NO: 62 (VH CDR1); SEQ ID NO: 63 (VH CDR2); and SEQ ID NO: 64 (VH CDR3). The light chain variable region (VL) amino acid sequence of Ab7 is set forth in SEQ ID NO: 65 (see Table 27). The VL CDR domain amino acid sequences of Ab7 are set forth in SEQ ID NO: 66 (VL CDR1); SEQ ID NO: 67 (VL CDR2); and SEQ ID NO: 68 (VL CDR3).

[0424] Accordingly, in some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 62, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 63, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 64; and / or a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 66, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 67; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 68. In certain embodiments, the anti-CD45 antibodies comprise the CDRs described herein (SEQ ID NOs: 62-64 and 66-68) wherein the CDRs comprise conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., specificity analogous to that of Ab7).

[0425] In some embodiments, this document provides an anti-CD45 antibody or its antigen-binding portion comprising a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO: 61, and / or a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 65. In some embodiments, the antibody may comprise a modified heavy chain (HC) variable region comprising an HC variable domain containing a variant of SEQ ID NO: 61 or SEQ ID NO: 61, which (i) differs from SEQ ID NO: 61 in that 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 61 in that up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 61 in that 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions; and / or (iv) comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO: 65. 61. An amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, wherein in any of (i)-(iv), the amino acid substitutions may be conserved or non-conserved; and wherein the modified heavy chain variable region retains the antibody’s CD45 specificity (i.e., Ab7-like specificity) or has enhanced biological activity relative to another Ab7 heavy chain variable region. In some embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain containing a variant of SEQ ID NO: 65 or SEQ ID NO: 65, the variant being (i) different from SEQ ID NO: 65 in that 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) different from SEQ ID NO: 65 in that up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) different from SEQ ID NO: 65 in that 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions; and / or (iv) containing a variant of SEQ ID NO: 65 has an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, wherein in any of (i)-(iv), the amino acid substitutions may be conserved or non-conserved; and wherein the modified light chain variable region retains the antibody’s CD45 specificity (i.e., Ab7-like specificity) or has enhanced biological activity relative to another Ab7 light chain variable region.

[0426] Antibodies comprising CDR and / or variable region sequences of Ab7 can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multi-chain or single-chain antibodies, and / or binding fragments that specifically bind human CD45, including, but not limited to, Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibody (including surrogate light chain constructs), single domain antibodies, camelized antibodies, and the like. They can also have or be derived from any isotype, including, for example, IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), or IgM. In some embodiments, anti-45 antibodies are IgG (e.g., IgGl, IgG2, IgG3, or IgG4).

[0427] In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab7 can further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgGl constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some embodiments, the heavy chain constant region can be a modified constant region. Exemplary constant region substitutions and / or modifications are set forth herein and include, but are not limited to, substitutions at one or more of the following positions: 234, 235, 265, and 435 (according to the EU index of Kabat). In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab7 can further comprise an IgGl heavy chain constant region comprising one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the EU index of Kabat). In some embodiments, antibodies comprising CDR and / or variable region sequences of Ab7 can further comprise a heavy chain constant region set forth in SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106. In some embodiments antibodies comprise a light chain constant region set forth in SEQ ID NO: 101.

[0428] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 69. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 69 or a variant of SEQ ID NO: 69 that (i) differs from SEQ ID NO: 9 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 69 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 69 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 69, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of Ab7), or has an enhanced biological activity relative to the biological activity of another Ab7 heavy chain region.

[0429] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 69. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 69 or a variant of SEQ ID NO: 69 that (i) differs from SEQ ID NO: 9 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 69 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 69 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 69, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of Ab7), or has an enhanced biological activity relative to the biological activity of another Ab7 heavy chain region.

[0430] In some embodiments, antibodies comprising the CDR regions and / or variable regions of Ab7 can be incorporated into antibody-drug conjugates, as described herein. In addition, antibodies comprising the CDR regions and / or variable regions of Ab7 can be used in the methods described herein, for example, for depleting CD45+ cells in a subject.

[0431] AbA

[0432] Antibody A (AbA) cross-reacts with human CD45, cynomolgus monkey CD45, and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0433] The amino acid sequences of each binding region of anti-CD45 AbA are set forth in Table 27. The present disclosure includes anti-CD45 antibodies based on AbA, e.g., comprising the CDRs as shown in Table 27.

[0434] In one embodiment, the present disclosure provides an anti-CD45 antibody or antigen-binding fragment thereof comprising an antigen-binding region, e.g., a CDR and / or variable region corresponding to those of AbA. The heavy chain variable region (VH) amino acid sequence of AbA is set forth in SEQ ID NO: 71 (see Table 27). The VH CDR domain amino acid sequences of AbA are set forth in SEQ ID NO: 72 (VH CDR1); SEQ ID NO: 73 (VH CDR2); and SEQ ID NO: 74 (VH CDR3). The light chain variable region (VL) amino acid sequence of AbA is set forth in SEQ ID NO: 75 (see Table 27). The VL CDR domain amino acid sequences of AbA are set forth in SEQ ID NO: 76 (VL CDR1); SEQ ID NO: 77 (VL CDR2); and SEQ ID NO: 78 (VL CDR3).

[0435] Accordingly, in some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 72, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 73, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 74; and / or a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 76, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 77; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 78. In certain embodiments, the anti-CD45 antibodies comprise the CDRs described herein (SEQ ID NOs: 72-74 and 76-78), wherein the CDRs comprise conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., specificity similar to that of AbA).

[0436] In some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 71, and / or a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 75. In certain embodiments, the antibody can comprise a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO: 71 or a variant of SEQ ID NO: 71 that (i) differs from SEQ ID NO: 71 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 71 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 71 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 71, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of AbA), or has an enhanced biological activity relative to the biological activity of another AbA heavy chain variable region. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain comprising SEQ ID NO: 75 or a variant of SEQ ID NO: 75 that (i) differs from SEQ ID NO: 75 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 75 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 75 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 75, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of AbA), or has an enhanced biological activity relative to the biological activity of another AbA light chain variable region.

[0437] Antibodies comprising CDRs and / or variable region sequences of AbA can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multi-chain or single-chain antibodies, and / or binding fragments that specifically bind human CD45, including, but not limited to, Fabs, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibody (including surrogate light chain constructs), single domain antibodies, camelized antibodies, and the like. They can also have or be derived from any isotype, including, for example, IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), or IgM. In some embodiments, anti-45 antibodies are IgG (e.g., IgGl, IgG2, IgG3, or IgG4).

[0438] In some embodiments, antibodies comprising CDRs and / or variable region sequences of AbA can further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgGl constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some embodiments, the heavy chain constant region can be a modified constant region. Exemplary constant region substitutions and / or modifications are set forth herein and include, but are not limited to, substitutions at one or more of the following positions: 234, 235, 265, and 435 (according to the EU index of Kabat). In some embodiments, antibodies comprising CDRs and / or variable region sequences of AbA can further comprise an IgGl heavy chain constant region comprising one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the EU index of Kabat). In some embodiments, antibodies comprising CDRs and / or variable region sequences of AbA can further comprise a heavy chain constant region set forth in SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106. In some embodiments antibodies comprise a light chain constant region set forth in SEQ ID NO: 101.

[0439] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 79. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 79 or a variant of SEQ ID NO: 79 that (i) differs from SEQ ID NO: 79 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 79 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 79 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 79, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of AbA), or has an enhanced biological activity relative to the biological activity of another AbA heavy chain region.

[0440] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 80. In certain embodiments, the antibody comprises a modified light chain (LC) region comprising a LC domain comprising SEQ ID NO: 80 or a variant of SEQ ID NO: 80 that (i) differs from SEQ ID NO: 80 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 80 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 80 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 80, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of AbA), or has an enhanced biological activity relative to the biological activity of another AbA light chain region.

[0441] In some embodiments, antibodies comprising the CDR regions and / or variable regions of AbA can be incorporated into antibody-drug conjugates, as described herein. In addition, antibodies comprising the CDR regions and / or variable regions of AbA can be used in the methods described herein, for example, for depleting CD45+ cells in a subject.

[0442] AbB

[0443] Antibody B (AbB) cross-reacts with human CD45, cynomolgus CD45, and rhesus CD45, and can bind to multiple isoforms of human CD45.

[0444] The amino acid sequences of each binding region of anti-CD45 AbB are set forth in Table 27. The present disclosure includes anti-CD45 antibodies based on AbB, for example, comprising the CDRs as shown in Table 27.

[0445] In one embodiment, the present disclosure provides an anti-CD45 antibody or antigen binding fragment thereof comprising an antigen binding region, for example, a CDR and / or variable region corresponding to those of AbB. The heavy chain variable region (VH) amino acid sequence of AbB is set forth in SEQ ID NO: 81 (see Table 27). The VH CDR domain amino acid sequences of AbB are set forth in SEQ ID NO: 82 (VH CDR1); SEQ ID NO: 83 (VH CDR2); and SEQ ID NO: 84 (VH CDR3). The light chain variable region (VL) amino acid sequence of AbB is set forth in SEQ ID NO: 85 (see Table 27). The VL CDR domain amino acid sequences of AbB are set forth in SEQ ID NO: 86 (VL CDR1); SEQ ID NO: 87 (VL CDR2); and SEQ ID NO: 88 (VL CDR3).

[0446] Thus, in some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 82, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 83, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 84; and / or a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 86, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 87; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 88. In certain embodiments, the anti-CD45 antibodies comprise the CDRs described herein (SEQ ID NOs: 82-84 and 86-88), wherein the CDRs comprise conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., similar to the specificity of AbB).

[0447] In some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 81, and / or a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 85. In certain embodiments, the antibody can comprise a modified heavy chain (HC) variable region comprising a HC variable domain comprising SEQ ID NO: 81 or a variant of SEQ ID NO: 81 that (i) differs from SEQ ID NO: 81 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 81 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 81 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 81, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of AbB), or has an enhanced biological activity relative to the biological activity of another AbB heavy chain variable region. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising a LC variable domain comprising SEQ ID NO: 85 or a variant of SEQ ID NO: 85 that (i) differs from SEQ ID NO: 85 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 85 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 85 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 85, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of AbB), or has an enhanced biological activity relative to the biological activity of another AbB light chain variable region.

[0448] Antibodies comprising CDRs and / or variable region sequences of AbB can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multi-chain or single-chain antibodies, and / or binding fragments that specifically bind human CD45, including, but not limited to, Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibody (including surrogate light chain constructs), single domain antibodies, camelized antibodies, and the like. They can also have or be derived from any isotype, including, for example, IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), or IgM. In some embodiments, anti-45 antibodies are IgG (e.g., IgGl, IgG2, IgG3, or IgG4).

[0449] In some embodiments, antibodies comprising CDRs and / or variable region sequences of AbB can further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgGl constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some embodiments, the heavy chain constant region can be a modified constant region. Exemplary constant region substitutions and / or modifications are set forth herein and include, but are not limited to, substitutions at one or more of the following positions: 234, 235, 265, and 435 (according to the EU index of Kabat). In some embodiments, antibodies comprising CDRs and / or variable region sequences of AbB can further comprise an IgGl heavy chain constant region comprising one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the EU index of Kabat). In some embodiments, antibodies comprising CDRs and / or variable region sequences of AbB can further comprise a heavy chain constant region set forth in SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106. In some embodiments antibodies comprise a light chain constant region set forth in SEQ ID NO: 101.

[0450] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 89. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 89 or a variant of SEQ ID NO: 89 that (i) differs from SEQ ID NO: 89 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 89 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 89 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 89, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of AbB), or has an enhanced biological activity relative to the biological activity of another AbB heavy chain region.

[0451] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 90. In certain embodiments, the antibody comprises a modified light chain (LC) region comprising a LC domain comprising SEQ ID NO: 90 or a variant of SEQ ID NO: 90 that (i) differs from SEQ ID NO: 90 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 90 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 90 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 90, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of AbB), or has an enhanced biological activity relative to the biological activity of another AbB light chain region.

[0452] In some embodiments, antibodies comprising the CDR regions and / or variable regions of AbB can be incorporated into antibody-drug conjugates, as described herein. In addition, antibodies comprising the CDR regions and / or variable regions of AbB can be used in the methods described herein, for example, for depleting CD45+ cells in a subject.

[0453] AbC

[0454] AbC is cross-reactive with human CD45, cynomolgus monkey CD45, and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0455] The amino acid sequences of each binding region of anti-CD45 AbC are set forth in Table 27. The present disclosure includes anti-CD45 antibodies based on AbC, for example, comprising the CDRs as shown in Table 27.

[0456] In one embodiment, the present disclosure provides an anti-CD45 antibody or antigen-binding fragment thereof comprising an antigen-binding region, for example, a CDR and / or variable region corresponding to those of AbC. The heavy chain variable region (VH) amino acid sequence of AbC is set forth in SEQ ID NO: 91 (see Table 27). The VH CDR domain amino acid sequences of AbC are set forth in SEQ ID NO: 92 (VH CDR1); SEQ ID NO: 93 (VH CDR2); and SEQ ID NO: 94 (VH CDR3). The light chain variable region (VL) amino acid sequence of AbC is set forth in SEQ ID NO: 95 (see Table 27). The VL CDR domain amino acid sequences of AbC are set forth in SEQ ID NO: 96 (VL CDR1); SEQ ID NO: 97 (VL CDR2); and SEQ ID NO: 98 (VL CDR3).

[0457] Accordingly, in some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 92, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 93, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 94; and / or a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 96, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 97; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 98. In certain embodiments, the anti-CD45 antibodies comprise the CDRs described herein (SEQ ID NOs: 92-94 and 96-98), wherein the CDRs comprise conservative amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., similar to the specificity of AbC).

[0458] In some embodiments, provided herein are anti-CD45 antibodies, or antigen binding portions thereof, comprising a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 991, and / or a light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO: 95. In certain embodiments, the antibody can comprise a modified heavy chain (HC) variable region comprising an HC variable domain comprising SEQ ID NO: 91 or a variant of SEQ ID NO: 91 that (i) differs from SEQ ID NO: 91 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 91 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 91 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 91, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of AbC), or has an enhanced biological activity relative to the biological activity of another AbC heavy chain variable region. In certain embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain comprising SEQ ID NO: 95 or a variant of SEQ ID NO: 95 that (i) differs from SEQ ID NO: 95 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 95 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 95 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 95, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain variable region retains the CD45 specificity of the antibody (i.e., the specificity of AbC), or has an enhanced biological activity relative to the biological activity of another AbC light chain variable region.

[0459] Antibodies comprising CDRs and / or variable region sequences of AbC can be in the form of full-length antibodies, bispecific antibodies, dual variable domain antibodies, multi-chain or single-chain antibodies, and / or binding fragments that specifically bind human CD45, including, but not limited to, Fabs, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including surrogate light chain constructs), single domain antibodies, camelized antibodies, and the like. They can also have or be derived from any isotype, including, for example, IgA (e.g., IgAl or IgA2), IgD, IgE, IgG (e.g., IgGl, IgG2, IgG3, or IgG4), or IgM. In some embodiments, anti-45 antibodies are IgG (e.g., IgGl, IgG2, IgG3, or IgG4).

[0460] In some embodiments, antibodies comprising CDRs and / or variable region sequences of AbC can further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgGl constant region, a human IgG2 constant region, a human IgG3 constant region, or a human IgG4 constant region. In some embodiments, the heavy chain constant region can be a modified constant region. Exemplary constant region substitutions and / or modifications are set forth herein and include, but are not limited to, substitutions at one or more of the following positions: 234, 235, 265, and 435 (according to the EU index of Kabat). In some embodiments, antibodies comprising CDRs and / or variable region sequences of AbC can further comprise an IgGl heavy chain constant region comprising one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the EU index of Kabat). In some embodiments, antibodies comprising CDRs and / or variable region sequences of AbC can further comprise a heavy chain constant region set forth in SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106. In some embodiments antibodies comprise a light chain constant region set forth in SEQ ID NO: 101.

[0461] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 99. In certain embodiments, the antibody comprises a modified heavy chain (HC) region comprising a HC domain comprising SEQ ID NO: 99 or a variant of SEQ ID NO: 99 that (i) differs from SEQ ID NO: 99 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 99 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 99 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 99, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of AbC), or has an enhanced biological activity relative to the biological activity of another AbC heavy chain region.

[0462] In certain embodiments, the anti-CD45 antibody, or antigen-binding portion thereof, comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 100. In certain embodiments, the antibody comprises a modified light chain (LC) region comprising a LC domain comprising SEQ ID NO: 100 or a variant of SEQ ID NO: 100 that (i) differs from SEQ ID NO: 100 by 1, 2, 3, 4, or 5 amino acid substitutions, additions, or deletions; (ii) differs from SEQ ID NO: 100 by up to 5, 4, 3, 2, or 1 amino acid substitutions, additions, or deletions; (iii) differs from SEQ ID NO: 100 by 1-5, 1-3, 1-2, 2-5, or 3-5 amino acid substitutions, additions, or deletions, and / or (iv) comprises an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 100, wherein in any of (i)-(iv), the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions; and wherein the modified light chain region retains the CD45 specificity of the antibody (i.e., specificity similar to that of AbC), or has an enhanced biological activity relative to the biological activity of another AbC light chain region.

[0463] In some embodiments, antibodies comprising the CDR regions and / or variable regions of AbC can be incorporated into antibody-drug conjugates, as described herein. In addition, antibodies comprising the CDR regions and / or variable regions of AbC can be used in the methods described herein, for example, for depleting CD45+ cells in a subject.

[0464] Consensus CDRs

[0465] A comparison of the CDR amino acid sequences of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, and Ab7 is provided below. Figure 32 These antibodies bind to the same epitope on human CD45 and share certain consensus residues in their CDR regions. The consensus heavy chain amino acid CDR sequences are presented in SEQ ID NO: 119, SEQ ID NO: 120, and SEQ ID NO: 121; and the consensus light chain amino acid CDR sequences are presented in SEQ ID NO: 122, SEQ ID NO: 123, and SEQ ID NO: 124.

[0466] Accordingly, in some embodiments, provided herein are isolated anti-CD45 antibodies, or antigen-binding portions thereof, comprising a heavy chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 119, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 120, and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 121; and a light chain variable region comprising a CDR1 domain comprising an amino acid sequence as set forth in SEQ ID NO: 122, a CDR2 domain comprising an amino acid sequence as set forth in SEQ ID NO: 123; and a CDR3 domain comprising an amino acid sequence as set forth in SEQ ID NO: 124. In some embodiments, the foregoing antibodies can further comprise a heavy chain constant region and / or a light chain constant region. For example, in some embodiments, the foregoing antibodies can further comprise a heavy chain constant region selected from the heavy chain constant region set forth in any one of SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, or SEQ ID NO: 106 and / or a light chain constant region set forth in SEQ ID NO: 101.

[0467] Fc-Modified Antibodies

[0468] Encompassed herein are antibodies or antigen-binding fragments thereof that are capable of binding CD45 and have Fc modifications that allow Fc silencing, wherein the antibodies or antigen-binding fragments thereof can be used as a standalone therapeutic or as an ADC to deplete CD45-expressing cells in a patient in need thereof. For example, in some embodiments, the antibodies or antigen-binding fragments thereof encompassed herein can be used to deplete certain cell types, including HSCs and leukocytes. Thus, in certain embodiments, the antibodies or antigen-binding fragments thereof encompassed herein can be used to condition a patient for HSC transplantation. In some embodiments, the antibodies or antigen-binding fragments thereof encompassed herein can be used to reconstitute a patient's immune system by, for example, depleting HSCs and leukocytes in the patient and administering an HSC graft to the patient. In some embodiments, the antibodies or antigen-binding fragments thereof encompassed herein can be used to treat diseases associated with CD45-positive cells, including but not limited to cancer and autoimmune diseases, by eliminating pathogenic CD45+ cells from a patient.

[0469] For example, encompassed herein are antibodies or antigen-binding fragments thereof that are capable of binding an antigen expressed by hematopoietic stem cells, such as CD45, and have Fc modifications that allow Fc silencing, wherein the antibodies or antigen-binding fragments thereof can be used as a standalone therapeutic or as an ADC to (i) treat cancers and autoimmune diseases characterized by CD45+ hematopoietic stem cells; and (ii) promote engraftment of transplanted hematopoietic stem cells in a patient in need of transplant therapy. Such therapeutic activities can arise, for example, by binding an anti-CD45 antibody or antigen-binding fragment thereof that binds to, for example, a cancer cell, an autoimmune cell, or a hematopoietic stem cell expressed by a hematopoietic cell, such as a hematopoietic stem cell or a mature immune cell, such as a T cell, and subsequently inducing cell death. Depletion of endogenous hematopoietic stem cells can provide a niche in which transplanted hematopoietic stem cells can home and subsequently establish productive hematopoiesis. In this way, transplanted hematopoietic stem cells can successfully engraft in a patient, such as a human patient suffering from a stem cell disorder described herein. The Fc-modified antibodies and ADCs encompassed herein not only allow selective depletion of endogenous hematopoietic stem cells, but also have reduced cytotoxic effects on exogenous hematopoietic stem cell grafts, thereby further promoting engraftment of hematopoietic stem cell grafts.

[0470] The antibodies or binding fragments described herein can also include modifications and / or mutations that alter the properties of the antibody and / or fragment, such as those that extend or shorten half-life or increase or decrease ADCC. In one embodiment, an antibody comprising one or more radiolabeled amino acids is provided. Radiolabeled antibodies can be used for diagnostic and therapeutic purposes (binding to a radiolabeled molecule is another possible feature). Non-limiting examples of labels for polypeptides include, but are not limited to,3H,14C,15N,35S,90Y,99Tc, and125I,131I, and186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, e.g., Junghans et al., Cancer Chemotherapy and Biotherapy 655-686 (2nd ed., Chafner and Longo, eds., Lippincott Raven (1996)) and U.S. Pat. No. 4,681,581, U.S. Pat. No. 4,735,210, U.S. Pat. No. 5,101,827, U.S. Pat. No. 5,102,990 (U.S. RE 35,500), U.S. Pat. No. 5,648,471, and U.S. Pat. No. 5,697,902. For example, a radioisotope can be bound by the chloramine-T method.

[0471] In one embodiment, the anti-CD45 antibody or binding fragment thereof comprises a modified Fc region, wherein the modified Fc region comprises at least one amino acid modification relative to a wild-type Fc region such that the molecule has altered FcyR (FcyR) affinity or binding to FcyR (FcyR). Certain amino acid positions within the Fc region are known via crystallographic studies to make direct contact with FcyR. In particular, amino acids 234-239 (hinge region), amino acids 265-269 (B / C loop), amino acids 297-299 (C' / E loop), and amino acids 327-332 (F / G) loop. (See Sondermann et al., 2000 Nature, 406:267-273). The antibodies described herein can comprise a variant Fc region comprising a modification of at least one residue that makes direct contact with FcyR 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), which is expressly incorporated herein by reference. "EU index as in Kabat" refers to the numbering of the human IgGl 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.

[0472] In one embodiment, the Fc region comprises mutations at amino acid positions D265, V205, H435, I253, and / or H310. For example, particular mutations at these positions include D265C, V205C, H435A, I253A, and / or H310A.

[0473] 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 another embodiment, the Fc region comprises L234A and L235A mutations. In another embodiment, the Fc region comprises D265C, L234A, and L235A mutations. In another embodiment, the Fc region comprises D265C, L234A, L235A, and H435A mutations. In another embodiment, the Fc region comprises D265C and H435A mutations.

[0474] In some embodiments, an anti-CD45 antibody herein comprises an Fc region comprising one or a combination of the following modifications: D265A, D265C, D265C / H435A, D265C / LALA, D265C / LALA / H435A, D265C / N297G, D265C / N297G / H435A, D265C (IgG2 ), D265C (IgG2) / H435A, D265C / N297Q / H435A, D265C / N297Q, EPLVLAdelG / H435A, N297A, N297G, or N297Q (according to the EU index of Kabat).

[0475] Binding or affinity between a modified Fc region and an Fc gamma receptor can be determined using a variety of techniques known in the art, such as, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al., Analytical Biochemistry, vol. 373:52-60, 2008; or radioimmunoassay (RIA)) or surface plasmon resonance assays or other kinetic-based assays (e.g., BIACORE.RTM. assays or Octet TM assays, competitive binding assays fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods can utilize labeling on one or more of the components being examined and / or employ a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. Detailed descriptions of binding affinity and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Edition, Lippincott-Raven, Philadelphia (1999), for antibody-immunogen interactions. One example of a competitive binding assay is a radioimmunoassay, which includes incubating labeled antigen with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and detecting antibody bound to the labeled antigen. The affinity and dissociation rate of the antibody of interest for a particular antigen can be determined from data analyzed by Scatchard plot. Competition with a second antibody can also be determined using a radioimmunoassay. In this case, the antigen is incubated with the antibody of interest bound to a labeled compound in the presence of increasing amounts of unlabeled second antibody.

[0476] In one embodiment, an antibody having an Fc modification described herein (e.g., D265C, L234A, L235A, and / or H435A) has at least 70% reduction, at least 80% reduction, at least 90% reduction, at least 95% reduction, at least 98% reduction, at least 99% reduction, or about 100% reduction in binding to an Fc gamma receptor relative to the binding of the same antibody comprising an unmodified Fc region (e.g., as assessed by biolayer interferometry (BLI)).

[0477] The binding interaction of the Fc region with Fc gamma receptors is required 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 aspects, antibodies comprising a modified Fc region (e.g., comprising a L234A, L235A, and / or D265C mutation) have substantially reduced or eliminated effector functions. Effector functions can be analyzed using a variety of methods known in the art, e.g., by measuring cellular responses (e.g., mast cell degranulation or cytokine release) in response to the antibody of interest. For example, using standard methods in the art, an Fc-modified antibody can be analyzed, e.g., for its ability to trigger mast cell degranulation or to trigger cytokine release by human peripheral blood mononuclear cells.

[0478] Thus, in one embodiment, the Fc region comprises a mutation that results in a shortened half-life (e.g., relative to an antibody having an unmodified Fc region). Antibodies having a short half-life can be advantageous in certain situations in which the antibody is expected to play a short-term therapeutic role (e.g., opsonization steps described herein), where the antibody is administered followed by HSCs. Typically, the antibody will be substantially cleared prior to delivery of the HSCs, which also typically express the target antigen (e.g., CD45), but are not the target of the anti-CD45 antibody, unlike endogenous stem cells. In one embodiment, the Fc region comprises a mutation at position 435 (EU index according to Kabat). In one embodiment, the mutation is a H435A mutation.

[0479] In one embodiment, the half-life of an anti-CD45 antibody described herein (e.g., in humans) is equal to or less than 24 hours, equal to or less than 23 hours, equal to or less than 22 hours, equal to or less than 21 hours, equal to or less than 20 hours, equal to or less than 19 hours, equal to or less than 18 hours, equal to or less than 17 hours, equal to or less than 16 hours, equal to or less than 15 hours, equal to or less than 14 hours, equal to or less than 13 hours, equal to or less than 12 hours, or equal to or less than 11 hours.

[0480] In one embodiment, the anti-CD45 antibody described herein has a half-life (e.g., in humans) of about 1-2 hours, about 1-3 hours, about 1-5 hours, about 1-10 hours, about 5-10 hours, about 5-15 hours, about 10-15 hours, about 10-20 hours, about 15-20 hours, about 15-25 hours, or about 20-25 hours.

[0481] In some aspects, the Fc region comprises two or more mutations that confer a shortened half-life and reduce the effector function of the antibody. In some embodiments, the Fc region comprises a mutation that causes a shortening of the half-life and a mutation of at least one residue that can make direct contact with an FcyR (e.g., as based on structural and crystallographic analysis). In one embodiment, the Fc region comprises a H435A mutation, a L234A mutation, and a L235A mutation. In one embodiment, the Fc region comprises a H435A mutation and a D265C mutation. In one embodiment, the Fc region comprises a H435A mutation, a L234A mutation, a L235A mutation, and a D265C mutation.

[0482] In some embodiments, the antibody or antigen-binding fragment thereof binds to a cytotoxin (e.g., a venome peptide) via a cysteine residue in the Fc domain of the antibody or antigen-binding fragment thereof. In some embodiments, the cysteine residue is introduced via a mutation of the Fc domain of the antibody or antigen-binding fragment thereof. For example, the cysteine residue can be selected from the group consisting of Cys118, Cys239, and Cys265. In 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. In one embodiment, the Fc region comprises a D265C mutation. In one embodiment, the Fc region comprises a D265C and a H435A mutation. In one embodiment, the Fc region comprises a D265C, a L234A, and a L235A mutation. In one embodiment, the Fc region comprises a D265C, a L234A, a L235A, and a H435A mutation.

[0483] It should be noted that Fc amino acid positions are referenced with respect to the EU numbering index, unless otherwise indicated.

[0484] The disclosure of each of the foregoing publications relating to anti-CD45 antibodies is incorporated herein by reference. Antibodies and antigen-binding fragments that can be used in conjunction with the compositions and methods described herein include the antibodies and antigen-binding fragments described above, as well as variants of the non-human antibodies and antigen-binding fragments described above, and antibodies or antigen-binding fragments that bind to the same epitope as the antibodies and antigen-binding fragments described above, as evaluated, for example, by a competitive antigen-binding assay.

[0485] Engineered antibodies to include any of the Fc modifications herein are well known in the art. Such methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis of prepared DNA molecules encoding the antibody or at least the constant region of the antibody, PCR mutagenesis, and cassette mutagenesis. Site-directed mutagenesis is well known in the art (see, e.g., Carter et al., Nucleic Acids Res., 13:4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA, 82:488 (1987)). PCR mutagenesis is also suitable for making amino acid sequence variants of starting polypeptides. See Higuchi, PCR Protocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17:723-733 (1989). Another method of making sequence variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene, 34:315-323 (1985).

[0486] Nucleic Acids, Vectors, and Host Cells

[0487] Also provided herein are nucleic acid molecules (e.g., DNA or mRNA) comprising a nucleic acid sequence encoding an anti-CD45 antibody or antigen-binding portion thereof described herein.

[0488] Thus, in some embodiments, provided herein are isolated nucleic acid molecules encoding a heavy chain variable region comprising the heavy chain CDR1, CDR2, and CDR3 of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In other embodiments, provided herein are isolated nucleic acid molecules encoding the heavy chain variable region of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In other embodiments, provided herein are isolated nucleic acid molecules encoding the heavy chain of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC.

[0489] In some embodiments, provided herein are isolated nucleic acid molecules encoding a light chain variable region comprising the light chain CDR1, CDR2, and CDR3 of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In other embodiments, provided herein are isolated nucleic acid molecules encoding the light chain variable region of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In other embodiments, provided herein are isolated nucleic acid molecules encoding the light chain of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC.

[0490] In some embodiments, provided herein are isolated nucleic acid molecules encoding a heavy chain variable region comprising the heavy chain CDR1, CDR2, and CDR3 of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC, and a light chain variable region comprising the light chain CDR1, CDR2, and CDR3 of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In other embodiments, provided herein are isolated nucleic acid molecules encoding the heavy chain variable region and the light chain variable region of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In other embodiments, provided herein are isolated nucleic acid molecules encoding the heavy chain and the light chain of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC.

[0491] A nucleic acid encoding an antibody heavy chain, or a portion thereof, can be present in the same nucleic acid molecule (e.g., expression vector) as a nucleic acid encoding an antibody light chain, or a portion thereof. Alternatively, the heavy chain and light chain sequences can be present on separate nucleic acid molecules (e.g., separate expression vectors).

[0492] In one embodiment, the present application provides an isolated nucleic acid molecule comprising:

[0493] (a) the nucleic acid sequence set forth in SEQ ID NO: 125;

[0494] (b) the nucleic acid sequence set forth in SEQ ID NO: 126;

[0495] (c) the nucleic acid sequence set forth in SEQ ID NO: 127;

[0496] (d) the nucleic acid sequence set forth in SEQ ID NO: 128;

[0497] (e) the nucleic acid sequence set forth in SEQ ID NO: 129;

[0498] (f) the nucleic acid sequence set forth in SEQ ID NO: 130;

[0499] (g) the nucleic acid sequence set forth in SEQ ID NO: 131 ;

[0500] (h) the nucleic acid sequence set forth in SEQ ID NO: 132;

[0501] (i) the nucleic acid sequence set forth in SEQ ID NO: 133;

[0502] (j) the nucleic acid sequence set forth in SEQ ID NO: 134;

[0503] (k) the nucleic acid sequence set forth in SEQ ID NO: 135;

[0504] (l) the nucleic acid sequence set forth in SEQ ID NO: 136;

[0505] (m) the nucleic acid sequence set forth in SEQ ID NO: 137;

[0506] (n) the nucleic acid sequence set forth in SEQ ID NO: 138;

[0507] (o) the nucleic acid sequence set forth in SEQ ID NO: 139;

[0508] (p) the nucleic acid sequence set forth in SEQ ID NO: 140;

[0509] (q) the nucleic acid sequence set forth in SEQ ID NO: 141 ;

[0510] (r) the nucleic acid sequence set forth in SEQ ID NO: 142;

[0511] (s) the nucleic acid sequence set forth in SEQ ID NO: 143; and / or

[0512] (t) the nucleic acid sequence set forth in SEQ ID NO: 144;

[0513] wherein the isolated nucleic acid encodes an anti-CD45 antibody or a portion thereof.

[0514] In another embodiment, the present application provides an isolated nucleic acid molecule comprising:

[0515] (a) the nucleic acid sequence set forth in SEQ ID NO: 150;

[0516] (b) the nucleic acid sequence set forth in SEQ ID NO: 151 ;

[0517] (c) the nucleic acid sequence set forth in SEQ ID NO: 152;

[0518] (d) the nucleic acid sequence set forth in SEQ ID NO: 153;

[0519] (e) the nucleic acid sequence set forth in SEQ ID NO: 154;

[0520] (f) the nucleic acid sequence set forth in SEQ ID NO: 155;

[0521] (g) the nucleic acid sequence set forth in SEQ ID NO: 156;

[0522] (h) the nucleic acid sequence set forth in SEQ ID NO: 157;

[0523] (i) the nucleic acid sequence set forth in SEQ ID NO: 158;

[0524] (j) the nucleic acid sequence set forth in SEQ ID NO: 159;

[0525] (k) the nucleic acid sequence set forth in SEQ ID NO: 160; and / or

[0526] (l) the nucleic acid sequence set forth in SEQ ID NO: 161;

[0527] wherein the isolated nucleic acid encodes an anti-CD45 antibody or a portion thereof.

[0528] Antibodies can be produced using, for example, recombinant methods and compositions as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-CD45 antibody described herein is provided. The nucleic acid can encode an amino acid sequence comprising a VL of the antibody (e.g., light chain and / or heavy chain of the antibody) and / or an amino acid sequence comprising a VH of the antibody. In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acid are provided. In another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of the antibody and an amino acid sequence comprising a VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising a VL of the antibody; and a second vector comprising a nucleic acid encoding an amino acid sequence comprising a VH of the antibody. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-CLL-1 antibody is provided, wherein the method comprises culturing a host cell comprising a 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).

[0529] To recombinantly produce an anti-CD45 antibody, nucleic acid encoding, e.g., an antibody as provided above, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. The nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are specific for the genes encoding the heavy and light chains of the antibody).

[0530] Host cells suitable for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, in particular when glycosylation and Fc effector functions are not needed. See, e.g., U.S. Pat. Nos. 5,648,237; 5,789,199; and 5,840,523, for expression of antibody fragments and polypeptides in bacteria. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell mass in the soluble fraction and can be further purified.

[0531] Vertebrate cells can also be used as hosts. For example, mammalian cell strains adapted to grow in suspension can be used. Other examples of useful mammalian host cell lines are the COS (ATCC CRL 1651) cell line, derived from the African green monkey kidney line CV1 (ATCC CRL 1587); 293 cells (ATCC CRL 1573); and human embryonic kidney line 293 cells (HEK 293, ATCC CRL 1573); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, Mather et al., Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CRL 1711); African green monkey kidney cells (VERO-76, ATCC CRL- 1573); human cervical carcinoma cells (HELA); canine kidney cells (MDCK, ATCC CRL 1972); buffalo rat liver cells (BRL 3CB); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

[0532] In one embodiment, the anti-CD45 antibody or antigen-binding fragment thereof comprises a variable region having an amino acid sequence that is at least 95%, 96%, 97%, or 99% identical to a SEQ ID No disclosed herein. Alternatively, the anti-CD45 antibody or antigen-binding fragment thereof comprises CDRs comprising a SEQ ID No disclosed herein, and a framework region of the variable region having an amino acid sequence that is at least 95%, 96%, 97%, or 99% identical to a SEQ ID No disclosed herein.

[0533] In one embodiment, the anti-CD45 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a heavy chain constant region having an amino acid sequence disclosed herein. In another embodiment, the anti-CD45 antibody or antigen-binding fragment thereof comprises a light chain variable region and a light chain constant region having an amino acid sequence disclosed herein. In another embodiment, the anti-CD45 antibody or antigen-binding fragment thereof comprises a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region having an amino acid sequence disclosed herein.

[0534] Methods of identifying antibodies

[0535] Provided herein are novel anti-CD45 antibodies that can be used, for example, to deplete CD45+ cells in a patient. The antibodies can be used, for example, in conditioning methods for stem cell transplantation. Other anti-CD45 antibodies can be identified in light of the disclosure provided herein.

[0536] Methods of high-throughput screening of libraries of antibodies or antibody fragments capable of binding CD45 expressed by hematopoietic stem cells can be used to identify anti-CD45 antibodies that can be used to treat cancer, autoimmune diseases, and to condition patients (e.g., human patients) in need of hematopoietic stem cell therapy as described herein. The methods can be used to identify improved versions of the anti-CD45 antibodies described herein. The methods include in vitro display technologies known in the art, including, for example, phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display.

[0537] The use of phage display to isolate antibodies or antigen-binding fragments that bind biologically relevant molecules is reviewed, for example, in Felici et al., Biotechnol. Annual Rev. 1 : 149-183, 1995; Katz, Annual Rev. Biophys. Biomol. Struct. 26: 27-45, 1997; and Hoogenboom et al., Immunotechnology 4: 1-20, 1998, the disclosure of each of which is incorporated herein by reference for its disclosure of in vitro display technology. Randomized combinatorial peptide libraries have been constructed to select polypeptides that bind cell surface antigens, as described in Kay, Perspect. Drug Discovery Des. 2: 251-268, 1995 and Kay et al., Mol. Divers. 1 : 139-140, 1996, the disclosure of each of which is incorporated herein by reference for its disclosure of finding antigen-binding molecules. Proteins (e.g., multimeric proteins) have been successfully phage displayed as functional molecules (see, e.g., EP 0349578; EP 4527839; and EP 0589877, and Chiswell and McCafferty, Trends Biotechnol. 10: 80-84 1992, the disclosure of each of which is incorporated herein by reference for its disclosure of finding antigen-binding molecules using in vitro display technology. In addition, functional antibody fragments, such as Fab and scFv fragments, have been expressed in in vitro display formats (see, e.g., McCafferty et al., Nature 348: 552-554, 1990; Barbas et al., Proc. Natl. Acad. Sci. USA 88: 7978-7982, 1991; and Clackson et al., Nature 352: 624-628, 1991, the disclosure of each of which is incorporated herein by reference for its disclosure of in vitro display platforms for finding antigen-binding molecules). Human anti-CD45 antibodies can also be generated in, e.g., HuMAb-Mouse® or XenoMouse™. These technologies are particularly useful for identifying and improving the affinity of antibodies, antibodies or fragments that are capable of binding CD45 expressed by hematopoietic stem cells, which in turn are used to deplete endogenous hematopoietic stem cells in a patient (e.g., a human patient) in need of hematopoietic stem cell transplant therapy.

[0538] In addition to in vitro display techniques, computational modeling techniques can be used to design and identify antibodies that are capable of binding to an antigen expressed by hematopoietic stem cells (e.g., CD45). For example, using computational modeling techniques, one skilled in the art can screen libraries of antibodies or antibody fragments to find molecules that are capable of binding to a particular epitope (e.g., an extracellular epitope of an antigen) on an antigen expressed by hematopoietic stem cells (e.g., CD45).

[0539] Other techniques can be used to identify antibodies or antibody fragments that are capable of binding to CD45 expressed by hematopoietic stem cells and internalized by cells, e.g., by receptor-mediated endocytosis. For example, the in vitro display techniques described above can be adapted to screen for antibodies or antibody fragments that bind to CD45 and are subsequently internalized. Phage display represents one technique that can be used in conjunction with this screening paradigm. To identify anti-CD45 antibodies or antibody fragments that are internalized by hematopoietic stem cells, one skilled in the art can use the phage display techniques described in Williams et al., Leukemia 19: 1432-1438, 2005, the disclosure of which is incorporated herein by reference in its entirety. For example, one can generate a recombinant phage library using mutagenesis methods known in the art that encode antibodies, antibody fragments (e.g., scFv fragments, Fab fragments, diabodies, triabodies, and 10 Fn3 domains), or ligands containing randomized amino acid boxes (e.g., in one or more or all CDRs or equivalent regions thereof or antibodies or antibody fragments). The framework regions, hinge, Fc domain, and other regions of the antibodies or antibody fragments can be designed to be non-immunogenic in humans, e.g., by having human germline antibody sequences or sequences that exhibit only minimal changes relative to human germline antibody sequences.

[0540] Using phage display technology as described herein or known in the art, a phage library containing randomized antibodies or antibody fragments covalently bound to phage particles can be incubated with CD45, for example by first incubating the phage library with blocking agents (e.g., milk proteins, bovine serum albumin, and / or IgG) to remove phage encoding antibodies or antibody fragments that exhibit non-specific protein binding and phage encoding antibodies or fragments thereof that bind Fc domains, and then incubating the phage library with a population of cells expressing CD45 (e.g., hematopoietic stem cells). The phage library can be incubated with the hematopoietic stem cells for a time sufficient to allow anti-CD45 antibodies or antibody fragments to bind to the cognate cell surface antigen and subsequently be internalized by the hematopoietic stem cells (e.g., for 30 minutes to 6 hours at 4°C, e.g., for 1 hour at 4°C). Phage containing antibodies or antibody fragments that do not exhibit sufficient affinity for CD45 to permit binding to and internalization by the hematopoietic stem cells can then be removed by, e.g., washing the cells with cold (4°C) 0.1 M glycine buffer at pH 2.8. Phage bound to antibodies or antibody fragments that have been internalized by the hematopoietic stem cells can be identified, e.g., by lysing the cells and recovering the internalized phage from the cell culture media. The phage can then be amplified in bacterial cells, e.g., by incubating the bacterial cells with the recovered phage in 2x YT media using methods known in the art. The encoded antibodies or antibody fragments can then be prepared de novo, e.g., by chemical synthesis (e.g., of antibody fragments, e.g., scFv fragments) or by recombinant expression (e.g., of full-length antibodies).

[0541] The internalization capacity of the prepared antibodies or antibody fragments can be evaluated, e.g., using radionuclide internalization assays known in the art. For example, the antibodies or antibody fragments can be labeled with 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 Bior 225 Ac) will functionalize the anti-CD45 antibody or antibody fragment identified using in vitro display techniques described herein or known in this art. For example, beads (e.g., polystyrene beads containing electrophilic halogen reagents, such as iodinated beads, Thermo Fisher Scientific, Inc., Cambridge, MA) can be used to functionalize radioactive halogens (e.g., 18 F, 75 Br、 77 Br、 122 I, 123 I, 124 I, 125 I, 129 I, 131 I, 211 The ADC (antibody-associated antibody) is incorporated into the antibody or antibody fragment. The radiolabeled antibody, its fragment, or ADC can be cultured with hematopoietic stem cells for a time sufficient for internalization (e.g., 30 minutes to 6 hours at 4°C, or 1 hour at 4°C). The cells are then washed to remove uninternalized antibodies or fragments (e.g., using cold (4°C) 0.1 M glycine buffer at pH 2.8). The internalized antibody or antibody fragment can be identified by comparing the radiation emitted by the hematopoietic stem cells (e.g., gamma radiation) with the radiation emitted by the recovered wash buffer. The aforementioned internalization analysis can also be used to characterize the ADC.

[0542] Antibodies can be produced using, for example, recombinant methods and compositions as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-CD45 antibody described herein is provided. The nucleic acid can encode an amino acid sequence comprising an antibody (e.g., light chain and / or heavy chain) VL and / or an amino acid sequence comprising an antibody VH. In another embodiment, one or more vectors (e.g., expression vectors) comprising the nucleic acid are provided. In another embodiment, a host cell comprising the nucleic acid is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL and an amino acid sequence comprising an antibody VH, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VL; and a second vector comprising a nucleic acid encoding an amino acid sequence comprising an antibody VH. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of making an anti-CLL-1 antibody is provided, wherein the method comprises culturing a host cell comprising a 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).

[0543] To recombinantly produce an anti-CD45 antibody, a nucleic acid encoding, for example, an antibody described above is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. The nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0544] Host cells suitable for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, in particular when glycosylation and Fc effector functions are not needed. See, e.g., U.S. Patent Nos. 5,648,237; 5,789,199; and 5,840,523, for expression of antibody fragments and polypeptides in bacteria. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody can be isolated from the bacterial cell mass in the soluble fraction and can be further purified.

[0545] Vertebrate cells can also be used as hosts. For example, mammalian cell strains such as the COS-7 line of monkey kidney cells, derived from the African green monkey kidney line CV1 transformed by SV40 virus (J. Exp. Med. 1984, 159, 757-768); 293 or 293 cells, for example, as described in Graham et al., J. Gen Virol. 36:59 (1977); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells, as described, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); hamster lung cells (CH1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells can be used as hosts. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0 and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003). In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp20 cell).

[0546] Antibody drug conjugates (ADCs)

[0547] The anti-CD45 antibodies or antigen-binding fragments thereof described herein can be conjugated (linked) to a cytotoxin via a linker. In some embodiments, a cytotoxic molecule is conjugated to an intracellularly internalizing antibody or antigen-binding fragment thereof as disclosed herein, such that upon cellular uptake of the antibody or fragment thereof, the cytotoxin can enter its intracellular target and mediate ...

Claims

1. An isolated anti-CD45 antibody or its antigen-binding moiety, comprising: (a) A heavy chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:2, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:3, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:4; and a light chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:6, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:7, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:8; (b) A heavy chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:12, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:13, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:14; and a light chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:16, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:17, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:18; (c) A heavy chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:22, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:23, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:24; and a light chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:26, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:27, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:28; (d) A heavy chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:32, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:33, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:34; and a light chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:36, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:37, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:38; (e) A heavy chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:42, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:43, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:44; and a light chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:46, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:47, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:48; (f) A heavy chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:52, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:53, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:54; and a light chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:56, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:57, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:58; (g) A heavy chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:62, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:63, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:64; and a light chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:66, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:67, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:68; (h) A heavy chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:72, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:73, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:74; and a light chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:76, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:77, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:78; (i) a heavy chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:82, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:83, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:84; and a light chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:86, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:87, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:88; or (j) A heavy chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:92, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:93, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:94; and a light chain variable region comprising a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:96, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:97, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:

98.

2. An isolated anti-CD45 antibody or its antigen-binding moiety, comprising... The heavy chain variable region includes a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:119, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:120, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:121; and the light chain variable region includes a CDR1 domain containing the amino acid sequence shown in SEQ ID NO:122, a CDR2 domain containing the amino acid sequence shown in SEQ ID NO:123, and a CDR3 domain containing the amino acid sequence shown in SEQ ID NO:

124.

3. An isolated anti-CD45 antibody or its antigen-binding moiety, comprising three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR); and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR), wherein the isolated anti-CD45 antibody or its antigen-binding moiety comprises: (a) A heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:1, and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:5; (b) A heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:11, and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:15; (c) A heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:21, and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:25; (d) A heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:31, and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:35; (e) A heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:41, and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:45; (f) A heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:51, and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:55; (g) A heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:61, and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:65; (h) A heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:71, and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:75; (i) A heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:81, and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:85; (j) A heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO:91, and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO:

95.

4. The isolated anti-CD45 antibody or its antigen-binding portion as claimed in any one of claims 1 to 3, wherein the antibody or its antigen-binding portion comprises an Fc region.

5. The isolated anti-CD45 antibody or its antigen-binding portion as claimed in claim 4, wherein the Fc region is a human IgG1 Fc region or a human IgG4 Fc region.

6. The isolated anti-CD45 antibody or its antigen-binding portion as claimed in any one of claims 1 to 5, wherein the antibody is a monoclonal antibody.

7. The isolated anti-CD45 antibody according to any one of claims 1 to 6, wherein the antibody is a complete antibody containing a constant region.

8. The isolated anti-CD45 antibody according to any one of claims 1 to 7, wherein the antibody is IgG.

9. The isolated anti-CD45 antibody of claim 8, wherein the IgG is IgG1 or IgG4.

10. The isolated anti-CD45 antibody according to any one of claims 1 to 9, wherein the antibody comprises a constant region, and wherein the constant region comprises at least one, at least two, at least three, at least four, or at least five amino acid substitutions selected from the group consisting of: L234A, L235A, D265C, H310A, and H435A (according to EU index number).

11. The isolated anti-CD45 antibody of claim 10, wherein the constant region comprises amino acid substitutions L234A, L235A, and D265C (according to EU index number).

12. The isolated anti-CD45 antibody according to any one of claims 1 to 11, comprising a constant region, wherein the constant region comprises (a) The heavy chain amino acid sequence as shown in SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105 or SEQ ID NO:106; and (b) The light chain amino acid sequence as shown in SEQ ID NO:

101.

13. An isolated anti-CD45 antibody comprising... (a) The heavy chain amino acid sequence shown in SEQ ID NO:9 and the light chain amino acid sequence shown in SEQ ID NO:10; (b) The heavy chain amino acid sequence shown in SEQ ID NO:19 and the light chain amino acid sequence shown in SEQ ID NO:20; (c) The heavy chain amino acid sequence shown in SEQ ID NO:29 and the light chain amino acid sequence shown in SEQ ID NO:30; (d) The heavy chain amino acid sequence as shown in SEQ ID NO:39 and the light chain amino acid sequence as shown in SEQ ID NO:40; (e) The heavy chain amino acid sequence shown in SEQ ID NO:49 and the light chain amino acid sequence shown in SEQ ID NO:50; (f) The heavy chain amino acid sequence as shown in SEQ ID NO:59 and the light chain amino acid sequence as shown in SEQ ID NO:60; (g) The heavy chain amino acid sequence shown in SEQ ID NO:69 and the light chain amino acid sequence shown in SEQ ID NO:70; (h) The heavy chain amino acid sequence as shown in SEQ ID NO:79 and the light chain amino acid sequence as shown in SEQ ID NO:80; (i) The amino acid sequence of the heavy chain as shown in SEQ ID NO:89, and the amino acid sequence of the light chain as shown in SEQ ID NO:90; or (j) The heavy chain amino acid sequence as shown in SEQ ID NO:99 and the light chain amino acid sequence as shown in SEQ ID NO:

100.

14. The isolated anti-CD45 antibody or its antigen-binding moiety as claimed in any one of claims 1 to 13, wherein the antibody or its antigen-binding moiety binds to human CD45 and the dissociation rate (K0) is... 解离 ) is 1×10 -2 Up to 1×10 -3 1×10 -3 Up to 1×10 -4 1×10 -4 Up to 1×10 -5 1×10 -5 Up to 1×10 -6 1×10 -6 Up to 1×10 -7 Or 1×10 -7 Up to 1×10 -8 Such as measurements obtained by biological layer interference (BLI).

15. The isolated anti-CD45 antibody or its antigen-binding portion as claimed in any one of claims 1 to 14, wherein the antibody or its antigen-binding portion binds to human CD45 and K D The values ​​are approximately 100 nM or less, approximately 90 nM or less, approximately 80 nM or less, approximately 70 nM or less, approximately 60 nM or less, approximately 50 nM or less, approximately 40 nM or less, approximately 30 nM or less, approximately 20 nM or less, approximately 10 nM or less, approximately 10 nM or less, or approximately 0.1 nM or less, as determined by biolayer interference (BLI).

16. A pharmaceutical composition comprising an antibody or antigen-binding portion thereof as claimed in any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

17. An isolated nucleic acid comprising a nucleic acid sequence encoding an antibody or an antigen-binding portion thereof as claimed in any one of claims 1 to 13.

18. A vector comprising the isolated nucleic acid as claimed in claim 17.

19. A host cell comprising the isolated nucleic acid of claim 17 or the vector of claim 18.

20. The host cell of claim 19, wherein the cell is a Chinese hamster ovary (CHO) cell or a HEK293T cell.

21. A pharmaceutical composition comprising an antibody or an antigen-binding portion thereof as claimed in any one of claims 1 to 15, for depleting a population of CD45+ cells in a human patient.

22. Use of an antibody or its antigen-binding portion for the preparation of a medicament for depleting a population of CD45+ cells in a human patient, comprising administering to the human patient an antibody or its antigen-binding portion as claimed in any one of claims 1 to 15.

23. The use as claimed in claim 22, wherein the CD45+ cells are CD3+, CD19+, CD33+, CD34+, or CD45+ / B2M+.

24. The use as claimed in claim 22, wherein the CD45+ cells are hematopoietic stem cells (HSCs), T cells, B cells, and / or bone marrow cells.

25. The use as claimed in claim 22, wherein the CD45+ cells are cancer cells.

26. The use as claimed in any one of claims 22 to 25, wherein the CD45+ cells are depleted from the patient’s bone marrow and / or from the patient’s peripheral blood.

27. The use as claimed in any one of claims 22 to 26, wherein the patient requires a hematopoietic stem cell transplant.

28. The use as claimed in any one of claims 22 to 27, further comprising administering a graft containing hematopoietic stem cells to the patient.

29. The use as claimed in claim 27 or 28, wherein the graft is an allogeneic graft.

30. The use as claimed in claim 27 or 28, wherein the graft is autologous.

31. An antibody-drug conjugate (ADC) comprising an anti-CD45 antibody or its antigen-binding portion bound to a cytotoxic agent via a linker, wherein the anti-CD45 antibody or its antigen-binding portion is an antibody or its antigen-binding portion as claimed in any one of claims 1 to 15.

32. The ADC of claim 31, wherein the antibody binds to the cytotoxin via a cysteine ​​residue in a non-constant domain of the antibody.

33. The ADC of claim 32, wherein the cysteine ​​residue is introduced by means of an amino acid substitution in the constant region of the antibody.

34. The ADC of claim 33, wherein the amino acid substitution is D265C and / or V205C (EU number).

35. The ADC of any one of claims 31 to 34, wherein the ADC has a drug-to-antibody ratio (DAR) of 1, 2, 3, 4, 5, 6, 7 or 8.

36. The ADC of any one of claims 31 to 35, wherein the cytotoxic agent is an RNA polymerase inhibitor, a DNA intercalating agent, a DNA alkylating agent, a DNA cross-linking agent, an agent that disrupts protein synthesis, an agent that disrupts microtubule dynamics, or an agent that disrupts the mitotic spindle.

37. The ADC of any one of claims 31 to 36, wherein the cytotoxic agent is selected from the group consisting of: amatoxin, anthracycline, auristatin, calicheamicin, deBouganin, diphtheria toxin, duocarmycin, indolinobenzodiazepine (IGN), indolinobenzodiazepine dimer, irinotecan, maytansine, maytansinoid, Pseudomonas exotoxin A, pyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine dimer, saporin, and SN-38.

38. The ADC of claim 36, wherein the cytotoxin is an RNA polymerase inhibitor.

39. The ADC of claim 38, wherein the RNA polymerase inhibitor is amatoxins.

40. The ADC of claim 39, wherein the ADC is represented by the formula Ab-ZL-Am, wherein Ab is the antibody or its antigen-binding portion, L is the linker, Z is the chemical portion, and Am is amatoxins.

41. The ADC of claim 40, wherein the phallotoxin-linker conjugate is represented by formula (I): Where R1 is H, OH, OR A OR C ; R2 represents H, OH, or OR. B OR C ; R A and R B (If present) it combines with the oxygen atom it is bonded to to form, as appropriate, a substituted 5-membered heterocyclic alkyl group; R3 is H, R C or R D ; R4, R5, R6, and R7 are each independently H, OH, and OR. C OR D R C or R D ; R8 represents OH, NH2, and OR. C OR D NHR C or NR C R D ; R9 represents H, OH, and OR. C OR D ; X is -S-, -S(O)-, or -SO2-; R C Yes, it's LZ; R D It may be a C1-C6 alkyl group, a C1-C6 heteroalkyl group, a C2-C6 alkenyl group, a C2-C6 heteroalkenyl group, a C2-C6 alkynyl group, a C2-C6 heteroalkynyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, or a heteroaryl group, depending on the situation. L is, as appropriate, a C1-C6 alkyl group, a C1-C6 heteroalkyl group, a C2-C6 alkenyl group, a C2-C6 heteroalkyl group, a C2-C6 alkyne group, a C2-C6 heteroalkynyl group, a cycloalkyl group, a heterocycloalkyl group, an aryl group, a heteroaryl group, a peptide, a dipeptide, -(C=O)-, a disulfide, a hydrazone, or a combination thereof; and Z is the chemical moiety formed by the coupling reaction between the reactive substituent present on L and the reactive substituent present in the antibody or its antigen-binding fragment. Where Am contains exactly one R C Substituents.

42. The ADC of claim 41, wherein LZ is or 。 43. The ADC of claim 39 or 40, wherein the ADC is represented by one of the following: Where X is -S-, -S(O)-, or -SO2-.

44. The ADC of claim 39 or 40, wherein the ADC has the following formula ， , , or Ab represents the binding site of the anti-CD45 antibody.

45. The ADC of claim 37, wherein the cytotoxin is pyrrolobenzodiazepine (PBD).

46. ​​The ADC of claim 45, wherein the cytotoxin is a PBD dimer.

47. The ADC of claim 46, wherein the PBD dimer is represented by the following formula: The waveform line indicates the connection point with the connector of the ADC.

48. The ADC of any one of claims 45 to 47, wherein the linker comprises one or more of the following: peptide, oligosaccharide, -(CH2). p -、-(CH2CH2O) q -、-(C=O)(CH2) r -、-(C=O)(CH2CH2O) t -、-(NHCH2CH2) u -, -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, where each of p, q, r, t and u is an integer from 1 to 12, and is independently chosen each time it appears.

49. The ADC of any one of claims 45 to 47, wherein the connector has the following structure R1 is CH3 (Ala) or (CH2)3NH(CO)NH2 (Cit).

50. The ADC of any one of claims 45 to 47, wherein the linker has the following structure before being bound to the antibody and, together with the reactive substituent Z', in an L-Z' form: R1 is CH3 (Ala) or (CH2)3NH(CO)NH2 (Cit).

51. The ADC of claim 50, wherein R1 is CH3.

52. The ADC of claim 35 or 36, wherein the cytotoxin-linker conjugate is tesirine having the following structure before being bound to the antibody and, together with the reactive substituent Z', in the Cy-L-Z' form: 。 53. The ADC of claim 35 or 36, having the following structure: ， Where Ab is the anti-CD45 antibody or its antigen-binding portion, and S represents a sulfur atom present in or introduced into the antibody or its antigen-binding portion.

54. The ADC of claim 37, wherein the cytotoxin is indolinobenzodiazepine (IGN).

55. The ADC of claim 54, wherein the cytotoxin is an IGN dimer or an IGN pseudodimer.

56. The ADC of claim 54 or 55, wherein the cytotoxin is an IGN pseudodimer represented by the following formula: ， The waveform line indicates the covalent connection point with the connector of the ADC.

57. The ADC of any one of claims 54 to 56, wherein the linker comprises a dipeptide, a disulfide, a C1-C12 alkyl group, a C=O group, or a combination thereof.

58. The ADC of any one of claims 54 to 56, wherein the connector comprises 。 59. The ADC of claim 27 or 28, wherein the cytotoxic-linker conjugate has the following structure before binding to the antibody or its antigen-binding moiety and forming a Cy-L-Z' form including the reactive substituent Z': 。 60. A pharmaceutical composition comprising an ADC as claimed in any one of claims 31 to 59 and a pharmaceutically acceptable carrier.

61. Use of an ADC in the preparation of a medicament for depleting a population of CD45+ cells in a human patient, comprising administering to the patient an effective amount of the ADC as claimed in any one of claims 31 to 59.

62. The use as claimed in claim 61, wherein the CD45+ cells are CD3+, CD19+, CD33+, CD34+, or CD45+ / B2M+.

63. The use as claimed in claim 61, wherein the CD45+ cells are hematopoietic stem cells (HSCs), T cells, B cells, and / or bone marrow cells.

64. The use of any one of claims 61 to 63, wherein the CD45+ cells are depleted from the patient’s bone marrow and / or from the patient’s peripheral blood.

65. The use as claimed in any one of claims 61 to 64, wherein the patient requires a hematopoietic stem cell transplant.

66. The use as claimed in any one of claims 61 to 65, further comprising administering to the patient a graft containing hematopoietic stem cells.

67. Use of an ADC in the preparation of a medicament for depleting a population of CD45+ cells in a human patient requiring a hematopoietic stem cell (HSC) graft, including administering the ADC as claimed in any one of claims 31 to 59 to the patient prior to receiving a graft containing hematopoietic stem cells.

68. Use of ADC in the preparation of a drug, said drug being used in a method comprising: (a) Administering to a human patient an amount sufficient to deplete the CD45+ cell population in the patient the ADC of any one of claims 31 to 59; and (b) The patient was then given a graft containing hematopoietic stem cells.

69. The use as claimed in any one of claims 65 to 68, wherein the graft is an allogeneic graft.

70. The use of any one of claims 65 to 69, wherein the hematopoietic stem cell graft is administered to the patient after the concentration of the ADC has been substantially removed from the patient's blood.

71. The use as claimed in any one of claims 65 to 70, wherein the hematopoietic stem cells or their progeny are capable of localizing to hematopoietic tissue and / or reconstructing hematopoiesis after the hematopoietic stem cells are transplanted into the patient.

72. The use as claimed in any one of claims 65 to 71, wherein, upon transplantation into the patient, the hematopoietic stem cells induce a recovery of a cell population selected from the following groups: megakaryocytes, coagulating cells, platelets, erythrocytes, mast cells, myeloid blasts, basophils, neutrophils, eosinophils, microglia, granulocytes, monocytes, osteoclasts, antigen-presenting cells, macrophages, dendritic cells, natural killer cells, T lymphocytes, and B lymphocytes.

73. The use as claimed in any one of claims 61-72, wherein the patient suffers from a blood disorder, metabolic disorder, cancer, or autoimmune disease or severe combined immunodeficiency disease (SCID).

74. The use as claimed in claim 73, wherein the cancer is a blood cancer.

75. The use as claimed in claim 74, wherein the blood cancer is acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, or multiple myeloma.

76. The method of claim 73, wherein the autoimmune disease is multiple sclerosis, type 1 diabetes, or scleroderma.

77. The method of claim 73, wherein the autoimmune disease is selected from the group consisting of: multiple sclerosis, human lupus, rheumatoid arthritis, inflammatory bowel disease, psoriasis, type 1 diabetes, acute diffuse encephalomyelitis, Addison's disease, alopecia universalis, adhesive capsulitis, antiphospholipid syndrome, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, autoimmune lymphoproliferative syndrome, autoimmune oophoritis, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Chagas' disease, chronic fatigue immune dysfunction syndrome, chronic inflammatory demyelination polyneuropathy, Crohn's disease. Diseases including cicatricial pemphigoid, celiac-herpetic dermatitis, cold agglutinin disease, CREST syndrome, Degos disease, discoid lupus, autonomic dysfunction, endometriosis, idiopathic mixed cryoglobulinemia, fibromyalgia-fibromyositis, Goodpasture's syndrome, Grave's disease, Guillain-Barré syndrome, 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, and Meniere's disease. Diseases, mixed connective tissue diseases, myasthenia gravis, neurogenic myotonia, oculoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus vulgaris, pernicious anemia, polychondritis, polymyositis and dermatomyositis, primary biliary cirrhosis, polyarteritis nodosa, polyglandular syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjögren's syndrome, stiff body syndrome, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis, leukoplakia, vulvar dysphagia, and Wegener's granulomatosis.

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