Anti-CD45 antibodies and their conjugates

Abstract

This article discloses anti-CD45 antibodies that specifically bind to human CD45, their antigen-binding fragments, and antibody-drug conjugates (ADCs). These antibodies and ADCs can be used in therapeutic applications, including methods that deplete a patient's CD45+ cells. The compositions and methods described herein can be used, for example, to directly treat conditions by depleting CD45+ cancer cells or autologous immune cell populations. The compositions and methods described herein can also be used to prepare patients for hematopoietic stem cell transplantation and to improve the implantation of hematopoietic stem cell grafts by selectively depleting endogenous CD45+ cells prior to the transplantation procedure.

Description

[0001] Related applications

[0002] This application claims 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; and U.S. Provisional Application No. 62 / 929,283, filed November 1, 2019. Priority claims to U.S. Provisional Application No. 62 / 929,347; 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 entire contents of each of the foregoing priority applications are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy was created on October 29, 2020, named 2020-10-29_M103034 1525WO_SL_ST25.txt, and is 233 kilobytes in size. Technical Field

[0005] This article describes anti-CD45 antibodies, their antigen-binding fragments, and antibody-drug conjugates. It also describes the treatment of patients with various conditions, particularly blood disorders, metabolic disorders, cancer, and autoimmune diseases, by administering anti-CD45 antibodies, their antigen-binding fragments, or their antibody-drug conjugates (ADCs), in which the antibodies, their antigen-binding fragments, or ADCs can bind to CD45 on target cells (such as hematopoietic stem cells, immune cells, or other cell types). Background Technology

[0006] CD45, also known as the C-type protein tyrosine phosphatase receptor (PTPRC), is an enzyme encoded by the PTPRC gene in humans (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 multiple cellular processes, including cell growth, differentiation, the mitotic cycle, and oncogenic transformation. CD45 contains an extracellular domain, a single transmembrane domain, and two tandem intracellular catalytic domains, and therefore belongs to the receptor-type PTP family. CD45 is a type I transmembrane protein present in several isoforms on differentiated hematopoietic cells (excluding erythrocytes 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 the form of co-stimulation) or activates various Src family kinases required for antigen receptor signaling via its cytoplasmic domain. CD45 also inhibits JAK kinase and thus acts as a negative regulator of intercellular receptor signaling.

[0007] 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 in CD45 in humans are associated with severe immunodeficiency. This is primarily attributed to the absence of CD45 on T cells, where CD45 is normally abundant and essential for regulating SFK activity during antigen responses. CD45 deficiency (CD45...) - / - Mouse bone marrow contains a normal number of hematopoietic cells, but the number of primitive hematopoietic stem cells (HSCs) is reduced, and their mobilization in response to G-CSF is impaired. This deficiency is partly inherent to HSCs; in the absence of 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 chemokines CXCL12 / SDF-1, which negatively impacts cell engraftment after transplantation. These deficiencies can be reversed by supplementing with SFK inhibitors, indicating that this function is normally mediated by CD45. Similarly, CD45... - / - Recipients also showed defective engraftment and subsequent mobilization of normal HSCs, indicating the role of CD45 in stem cell niches and in HSCs (Shivtiel et al., J Exp Med 205:2381 (2008)).

[0008] Despite advances in the medical field, there remains a need to treat conditions of the hematopoietic system, particularly diseases of specific blood cells, metabolic disorders, cancer, and autoimmune disorders. While hematopoietic stem cells (HSCs) hold significant therapeutic potential, a limiting factor hindering their clinical application is the difficulty in ensuring HSC graft implantation in the host. Specifically, hematopoietic stem cell therapies involving antibodies targeting cell surface antigens on endogenous HSCs can trigger unwanted immune stimulation and effector functions, thereby preventing the implantation of exogenous HSC grafts. Because CD45 is expressed on, for example, HSCs and leukocytes, it presents a target for therapies including opsonization, immune reactivation, and disease treatment. Summary of the Invention

[0009] Given the crucial role of CD45 in cell biology, there is a need for anti-CD45 antibodies and their fragments. This article describes anti-CD45 antibodies, their antigen-binding fragments, and their antibody-drug conjugates (ADCs). Anti-CD45 antibodies, their antigen-binding fragments, and their antibody-drug conjugates (ADCs) can bind to hematopoietic stem cells (HSCs) and can be used as conditioning agents, for example, in HSC transplantation. Specifically, the anti-CD45 antibodies, their antigen-binding fragments, and their ADCs described herein can be used to specifically deplete, for example, host HSCs, immune cells (e.g., leukocytes), or pathogenic cells. Furthermore, the anti-CD45 antibodies, their antigen-binding fragments, and their ADCs described herein can be used to treat patients with leukemia or lymphoma, or patients with autoimmune diseases (e.g., multiple sclerosis and scleroderma). The anti-CD45 antibodies, their antigen-binding fragments, and ADCs described herein meet the need for compositions and methods for promoting the implantation of exogenous hematopoietic stem cell grafts, thereby preserving the pluripotency and hematopoietic function of these cells after transplantation.

[0010] In a first state, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising: (a) a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:2, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:3, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:4; and a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:6, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:7, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:8; (b) a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:12, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:13, and a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:8; and a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:12, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:13, and a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:4; and a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:5; and a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:6; and a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:7; and a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:8; and a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:12; and a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:13 ... (c) A heavy chain variable region comprising a CDR1 domain of the amino acid sequence shown in SEQ ID NO:16, a CDR2 domain of the amino acid sequence shown in SEQ ID NO:17, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO:18; (c) a heavy chain variable region comprising a CDR1 domain of the amino acid sequence shown in SEQ ID NO:22, a CDR2 domain of the amino acid sequence shown in SEQ ID NO:23, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO:24; and a light chain variable region comprising a CDR1 domain of the amino acid sequence shown in SEQ ID NO:26, a CDR2 domain of the amino acid sequence shown in SEQ ID NO:27, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO:18; (d) A heavy chain variable region comprising a CDR1 domain of an amino acid sequence as shown in SEQ ID NO:32, a CDR2 domain of an amino acid sequence as shown in SEQ ID NO:33, and a CDR3 domain of an amino acid sequence as shown in SEQ ID NO:34; and a light chain variable region comprising a CDR1 domain of an amino acid sequence as shown in SEQ ID NO:36, a CDR2 domain of an amino acid sequence as shown in SEQ ID NO:37, and a CDR3 domain of an amino acid sequence as shown in SEQ ID NO:38.(e) A heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:42, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:43, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:44; and a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:46, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:47, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:48; (f) a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:52, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:53, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:54; and a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:42, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:53, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:54; and a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:42, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:53, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:44; (g) A heavy chain variable region comprising a CDR1 domain of the amino acid sequence shown in SEQ ID NO:56, a CDR2 domain of the amino acid sequence shown in SEQ ID NO:57, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO:58; and a light chain variable region comprising a CDR1 domain of the amino acid sequence shown in SEQ ID NO:62, a CDR2 domain of the amino acid sequence shown in SEQ ID NO:63, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO:64; and a light chain variable region comprising a CDR1 domain of the amino acid sequence shown in SEQ ID NO:66, a CDR2 domain of the amino acid sequence shown in SEQ ID NO:67, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO:68; and a heavy chain variable region comprising a CDR1 domain of the amino acid sequence shown in SEQ ID NO:72, a CDR2 domain of the amino acid sequence shown in SEQ ID NO:67, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO:68; and a light chain variable region comprising a CDR1 domain of the amino acid sequence shown in SEQ ID NO:72, a CDR2 domain of the amino acid sequence shown in SEQ ID NO:67, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO:68; and a heavy chain variable region comprising a CDR1 domain of the amino acid sequence shown in SEQ ID NO:72, a CDR2 domain of the amino acid sequence shown in SEQ ID NO:67, and a CDR3 domain of the amino acid sequence shown in SEQ ID NO:68; The CDR2 domain of the amino acid sequence shown in NO:73 and the CDR3 domain of the amino acid sequence shown in SEQ ID NO:74; and the light chain variable region, which includes the CDR1 domain of the amino acid sequence shown in SEQ ID NO:76, the CDR2 domain of the amino acid sequence shown in SEQ ID NO:77, and the CDR3 domain of the amino acid sequence shown in SEQ ID NO:78; (i) the heavy chain variable region, which includes the CDR1 domain of the amino acid sequence shown in SEQ ID NO:82, the CDR2 domain of the amino acid sequence shown in SEQ ID NO:83, and the CDR3 domain of the amino acid sequence shown in SEQ ID NO:84;and (j) a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:86, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:87, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:88; or (j) a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:92, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:93, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:94; and a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:96, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:97, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:98.

[0011] In another embodiment, this document provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising: a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:119, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:120, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:121; and a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:122, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:123, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:124.

[0012] In another embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising: (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:15. (f) The amino acid sequence shown in NO:45 contains a light chain variable region; (g) The amino acid sequence shown in SEQ ID NO:51 contains a heavy chain variable region and the amino acid sequence shown in SEQ ID NO:55 contains a light chain variable region; (g) The amino acid sequence shown in SEQ ID NO:61 contains a heavy chain variable region and the amino acid sequence shown in SEQ ID NO:65 contains a light chain variable region; (h) The amino acid sequence shown in SEQ ID NO:71 contains a heavy chain variable region and the amino acid sequence shown in SEQ ID NO:75 contains a light chain variable region; (i) The amino acid sequence shown in SEQ ID NO:81 contains a heavy chain variable region and the amino acid sequence shown in SEQ ID NO:85 contains a light chain variable region; or (j) The amino acid sequence shown in SEQ ID NO:91 contains a heavy chain variable region and the amino acid sequence shown in SEQ ID NO:95 contains a light chain variable region.

[0013] In some embodiments of the aforementioned samples, the isolated anti-CD45 antibody or its antigen-binding portion contains an Fc region. In some embodiments, the Fc region is a human IgG1 Fc region or a human IgG4 Fc region.

[0014] In some embodiments, anti-system monoclonal antibodies.

[0015] In some embodiments, the anti-system comprises an intact antibody with a constant region.

[0016] In some embodiments, the anti-IgG system is used. In a particular embodiment, the IgG is IgG1 or IgG4.

[0017] In some embodiments of the aforementioned samples, 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 (according to EU index numbers). In specific embodiments, the constant region comprises amino acid substitutions of L234A, L235A, and D265C (according to EU index numbers).

[0018] In some embodiments, the isolated anti-CD45 antibody of this disclosure includes a constant region, wherein the constant region includes (a) a 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) a light chain amino acid sequence as shown in SEQ ID NO:101.

[0019] In another embodiment, the present invention provides an isolated anti-CD45 antibody comprising: (a) a heavy chain amino acid sequence as shown in SEQ ID NO:9 and a light chain amino acid sequence as shown in SEQ ID NO:10; (b) a heavy chain amino acid sequence as shown in SEQ ID NO:19 and a light chain amino acid sequence as shown in SEQ ID NO:20; (c) a heavy chain amino acid sequence as shown in SEQ ID NO:29 and a light chain amino acid sequence as shown in SEQ ID NO:30; (d) a heavy chain amino acid sequence as shown in SEQ ID NO:39 and a light chain amino acid sequence as shown in SEQ ID NO:40; (e) a heavy chain amino acid sequence as shown in SEQ ID NO:49 and a light chain amino acid sequence as shown in SEQ ID NO:50; (f) a heavy chain amino acid sequence as shown in SEQ ID NO:59 and a light chain amino acid sequence as shown in SEQ ID NO:60; (g) as shown in SEQ ID NO:9; The heavy chain amino acid sequence shown in NO:69 and the light chain amino acid sequence shown in SEQ ID NO:70; (h) the heavy chain amino acid sequence shown in SEQ ID NO:79 and the light chain amino acid sequence shown in SEQ ID NO:80; (i) the heavy chain amino acid sequence shown in SEQ ID NO:89 and the light chain amino acid sequence shown in SEQ ID NO:90; or (j) the heavy chain amino acid sequence shown in SEQ ID NO:99 and the light chain amino acid sequence shown in SEQ ID NO:100.

[0020] In another embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, which specifically binds to human CD45 at antigenic determinants 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).

[0021] In some embodiments of the above-described forms, the isolated anti-CD45 antibody or its antigen-binding portion specifically binds to (a) the antigenic determinant of human CD45 located in fragment 2 of CD45 and the antigenic determinant of human CD45 located in fragment 4; (b) the antigenic determinant of human CD45 located in fragment 1 of CD45 and the antigenic determinant of human CD45 located in fragment 3 of CD45; or (c) the antigenic determinant of human CD45 located in fragment 5 of CD45.

[0022] In some embodiments, the isolated anti-CD45 antibody or its antigen-binding portion specifically binds to (a) one or more residues selected from the group consisting of 405T, 407K, 419Y, 425K, 481R, 505R and 509H of human CD45 (see SEQ ID NO: 113); or (b) one or more residues selected from the group consisting of 486R, 493Y and 502T of human CD45 (see SEQ ID NO: 113).

[0023] In some embodiments, the isolated anti-CD45 antibody or antigen-binding moiety of this disclosure specifically binds to human CD45 and cross-reacts with cynomolgus monkey CD45.

[0024] In some embodiments, the isolated anti-CD45 antibody or antigen-binding moiety of this disclosure binds to human CD45 and the dissociation rate (K) 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).

[0025] In some embodiments, the isolated anti-CD45 antibody or antigen-binding moiety of this disclosure 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).

[0026] In another embodiment, this document provides a pharmaceutical composition comprising an antibody of the present invention or an antigen-binding portion thereof and a pharmaceutically acceptable carrier.

[0027] In another instance, this document provides isolated nucleic acids containing nucleic acid sequences encoding the antibody of the present invention or its antigen-binding portion.

[0028] In another instance, this document provides a vector containing the isolated nucleic acid of the present invention.

[0029] In another embodiment, this document provides a host cell containing the isolated nucleic acid of the present invention or the vector of the present invention. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell.

[0030] In another respect, this article provides a pharmaceutical composition comprising an antibody or its antigen-binding portion of any of the states mentioned above, for use in depleted populations of CD45+ cells in human patients.

[0031] In another embodiment, this document provides a method for depleting a population of CD45+ cells in a human patient by administering an antibody or its antigen-binding portion of any of the embodiments mentioned above to the human patient. In some embodiments, the CD45+ cell lines are CD3+, CD19+, CD33+, CD34+, or CD45+ / B2M+. In other embodiments, the CD45+ cell line is a hematopoietic stem cell (HSC). In other embodiments, the CD45+ cell lines are HSCs, T cells, B cells, and / or bone marrow cells.

[0032] In some of the above-described embodiments, the CD45+ cell line was depleted from the patient's bone marrow and / or from the patient's peripheral blood.

[0033] In some embodiments of the above-described embodiments, the patient requires a hematopoietic stem cell graft. In some embodiments of the above-described embodiments, the method further includes delivering a graft containing hematopoietic stem cells to the patient. In some embodiments, the graft is allogeneic. In alternative embodiments, the graft is autologous.

[0034] In another embodiment, the present invention provides 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 of any of the embodiments mentioned above.

[0035] In some embodiments of the above-described forms, the antibody binds to the cytotoxin via cysteine ​​residues in the antibody constant domain. In some embodiments, the cysteine ​​residues are introduced via amino acid substitution in the antibody constant region. In specific embodiments, the amino acid substitution is D265C and / or V205C (EU designation).

[0036] In some of the above embodiments, the drug-to-antibody ratio (DAR) of the ADC is 1, 2, 3, 4, 5, 6, 7 or 8.

[0037] In some embodiments of the above-described forms, the cytotoxin is an RNA polymerase inhibitor, DNA intercalating agent, DNA alkylating agent, DNA cross-linking agent, agent that disrupts protein synthesis, agent that disrupts microtubule dynamics, or agent that disrupts the mitotic spindle.

[0038] In some embodiments of the above-described embodiments, the cytotoxin 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. In some embodiments, the cytotoxin is an RNA polymerase inhibitor. In some embodiments, the RNA polymerase inhibitor is amatoxin.

[0039] In some embodiments, 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 the phallotoxin. In some embodiments, Am-LZ is represented by formula (I):

[0040]

[0041] Among them, R1 series H, OH, OR A OR C ;

[0042] R2 series H, OH, OR B OR C ;

[0043] 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;

[0044] R3 series H, R C or R D ;

[0045] R4, R5, R6, and R7 each independently generate H, OH, and OR. C OR D R C or R D ;

[0046] R8 series OH, NH2, OR C OR D NHR C or NR C R D ;

[0047] R9 series H, OH, OR C OR D ;

[0048] X series -S-, -S(O)- or -SO2-;

[0049] R C LZ;

[0050] 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.

[0051] L-series substituted C1-C6 alkyl groups, substituted C1-C6 heteroalkyl groups, substituted C2-C6 alkenyl groups, substituted C2-C6 heteroalkyl groups, substituted C2-C6 alkynyl groups, substituted C2-C6 heterokynyl groups, substituted cycloalkyl groups, substituted heterocycloalkyl groups, substituted aryl groups, substituted heteroaryl groups, peptides, dipeptides, -(C=O)-, disulfides, hydrazones, or combinations thereof; and

[0052] Z is a chemical moiety formed by the coupling reaction between reactive substituents present on L and reactive substituents present in the antibody or its antigen-binding fragment.

[0053] Where Am contains exactly one R C Substituents.

[0054] In some embodiments, the LZ system

[0055]

[0056] In some embodiments, ADC is represented by one of the following:

[0057]

[0058] Among them, the X series are -S-, -S(O)- or -SO2-.

[0059] In some embodiments, the ADC has the following formula:

[0060]

[0061] Ab represents the binding site of the anti-CD45 antibody.

[0062] In other embodiments, the ADC has the following formula:

[0063]

[0064] Ab represents the binding site of the anti-CD45 antibody.

[0065] In an alternative embodiment, the ADC has the following formula:

[0066]

[0067] Ab represents the binding site of the anti-CD45 antibody.

[0068] In other embodiments, the ADC has the following formula:

[0069]

[0070] Ab represents the binding site of the anti-CD45 antibody.

[0071] In other embodiments, the ADC has the following formula:

[0072]

[0073] Ab represents the binding site of the anti-CD45 antibody.

[0074] In some embodiments of the ADCs disclosed herein, the cytotoxin is pyrrolobenzodiazepine (PBD). In some embodiments, the cytotoxin is a PBD dimer. In a particular embodiment, the PBD dimer system is represented by the following formula.

[0075]

[0076] The waveform line indicates the connection point with the ADC connector.

[0077] In some embodiments of the ADC disclosed herein, 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.

[0078] In some embodiments of the ADC disclosed herein, the connector has the following structure:

[0079]

[0080] Among them, R1 series CH3(Ala) or (CH2)3NH(CO)NH2(Cit).

[0081] In some embodiments of the ADC disclosed herein, the linker has the following structure prior to binding to the anti-CD45 antibody and including the reactive substituent Z' (together in L-Z' form):

[0082]

[0083] In some embodiments, R1 is CH3.

[0084] In some embodiments of the ADCs disclosed herein, the cytotoxin-linker conjugate is tesirine having the following structure prior to binding to the anti-CD45 antibody and including the reactive substituent Z' (together in the Cy-L-Z' form):

[0085]

[0086] In some embodiments, the ADC disclosed herein has a structure

[0087]

[0088] Ab represents the anti-CD45 antibody or its antigen-binding portion, and S represents a sulfur atom present in or introduced into the anti-CD45 antibody or its antigen-binding portion.

[0089] In some embodiments of the ADCs disclosed herein, the cytotoxin is indolinobenzodiazepine (IGN). In some embodiments, the cytotoxin is an IGN dimer or an IGN pseudodimer.

[0090] In some embodiments, the cytotoxin is an IGN pseudodimer represented by the following formula:

[0091]

[0092] The waveform line indicates the covalent connection point with the ADC connector.

[0093] In some embodiments, the linker comprises a dipeptide, a disulfide, a C1-C12 alkyl group, a C=O group, or a combination thereof.

[0094] In some embodiments, the connector includes

[0095]

[0096] In some embodiments, the cytotoxin-linker conjugate has the following structure prior to binding to an anti-CD45 antibody or its antigen-binding portion and including the reactive substituent Z' (together in the Cy-L-Z' form):

[0097]

[0098] In another embodiment, the present invention provides a pharmaceutical composition comprising the ADC described above and a pharmaceutically acceptable carrier.

[0099] In another instance, this paper provides a method for depleting the CD45+ cell population in a human patient by administering an effective dose of the anti-CD45 ADC described above.

[0100] In some embodiments of the above-described configurations, CD45+ cell lines are CD3+, CD19+, CD33+, CD34+, or CD45+ / B2M+. In some embodiments, CD45+ cell lines are hematopoietic stem cells (HSCs). In other embodiments, CD45+ cell lines are HSCs, T cells, B cells, and / or bone marrow cells.

[0101] In some of the above-described embodiments, the CD45+ cell line was depleted from the patient's bone marrow and / or from the patient's peripheral blood.

[0102] In some of the above-described embodiments, the patient requires a hematopoietic stem cell transplant.

[0103] In some embodiments of the above-described manner, the method further includes delivering a graft containing hematopoietic stem cells to the patient.

[0104] In another embodiment, the present invention provides a method for depleting the CD45+ cell population in a human patient by administering the aforementioned ADC to the patient prior to receiving a graft containing hematopoietic stem cells in a human patient requiring hematopoietic stem cell (HSC) transplantation.

[0105] In another embodiment, the present invention provides a method comprising: (a) administering to a human patient an amount of the ADC described above sufficient to deplete the CD45+ cell population in the patient; and (b) subsequently administering to the patient a graft comprising hematopoietic stem cells.

[0106] In some embodiments, the graft is an allogeneic graft. In other embodiments, the graft is autologous.

[0107] In some embodiments, the graft containing hematopoietic stem cells is administered to the patient after the concentration of ADC has been substantially cleared from the patient's blood.

[0108] In some embodiments, hematopoietic stem cells or their progeny can be localized to hematopoietic tissue and / or reconstitute hematopoiesis after transplantation of hematopoietic stem cells into a patient.

[0109] In some embodiments, after transplantation into a patient, hematopoietic stem cells induce the recovery of a cell population selected from the following groups: megakaryocytes, coagulation 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.

[0110] In some embodiments, the patient has a blood disorder, metabolic disorder, cancer, or autoimmune disease or severe combined immunodeficiency disease (SCID). In some embodiments, the patient has cancer. In a particular embodiment, the cancer is a blood cancer. In a specific embodiment, the blood cancer is acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, or multiple myeloma.

[0111] In some embodiments, the patient has an autoimmune disease. In specific embodiments, the autoimmune disease is multiple sclerosis, type 1 diabetes, or scleroderma. In other embodiments, autoimmune diseases are 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, and 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, polytrichosis syndrome, polymyalgia rheumatica, primary agammaglobulinemia, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, sarcoidosis, scleroderma, Scheregler's syndrome ( Stiff body syndrome, Takayasu's arteritis, temporal arteritis, ulcerative colitis, uveitis, vasculitis, leukoplakia, vulvar dystrophy, and Wegener's granulomatosis.

[0112] In addition, the present invention also includes the following embodiments:

[0113] In one embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:2, an amino acid sequence CDR2 as shown in SEQ ID NO:3, and an amino acid sequence CDR3 as shown in SEQ ID NO:4; and comprising a light chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:6, an amino acid sequence CDR2 as shown in SEQ ID NO:7, and an amino acid sequence CDR3 as shown in SEQ ID NO:8.

[0114] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:1.

[0115] In other embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:5.

[0116] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:1; and a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:5.

[0117] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:9.

[0118] 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.

[0119] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:9; and a light chain containing an amino acid sequence as shown in SEQ ID NO:10.

[0120] In one embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:12, an amino acid sequence CDR2 as shown in SEQ ID NO:13, and an amino acid sequence CDR3 as shown in SEQ ID NO:14; and comprising a light chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:16, an amino acid sequence CDR2 as shown in SEQ ID NO:17, and an amino acid sequence CDR3 as shown in SEQ ID NO:18.

[0121] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:11.

[0122] In other embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:15.

[0123] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:11; and

[0124] The light chain variable region contains an amino acid sequence as shown in SEQ ID NO:15.

[0125] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:19.

[0126] 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:20.

[0127] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:19; and a light chain containing an amino acid sequence as shown in SEQ ID NO:20.

[0128] In one embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:22, an amino acid sequence CDR2 as shown in SEQ ID NO:23, and an amino acid sequence CDR3 as shown in SEQ ID NO:24; and comprising a light chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:26, an amino acid sequence CDR2 as shown in SEQ ID NO:27, and an amino acid sequence CDR3 as shown in SEQ ID NO:28.

[0129] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:21.

[0130] In other embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:25.

[0131] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:21; and

[0132] The light chain variable region contains an amino acid sequence as shown in SEQ ID NO:25.

[0133] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:29.

[0134] 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:30.

[0135] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:29; and a light chain containing an amino acid sequence as shown in SEQ ID NO:30.

[0136] In one embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:32, an amino acid sequence CDR2 as shown in SEQ ID NO:33, and an amino acid sequence CDR3 as shown in SEQ ID NO:34; and comprising a light chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:36, an amino acid sequence CDR2 as shown in SEQ ID NO:37, and an amino acid sequence CDR3 as shown in SEQ ID NO:38.

[0137] In some embodiments, the anti-CD45 antibody or its antigen-binding portion includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:31.

[0138] In other embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:35.

[0139] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:31; and

[0140] The light chain variable region contains an amino acid sequence as shown in SEQ ID NO:35.

[0141] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:39.

[0142] 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:40.

[0143] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:39; and a light chain containing an amino acid sequence as shown in SEQ ID NO:40.

[0144] In one embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:42, an amino acid sequence CDR2 as shown in SEQ ID NO:43, and an amino acid sequence CDR3 as shown in SEQ ID NO:44; and comprising a light chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:46, an amino acid sequence CDR2 as shown in SEQ ID NO:47, and an amino acid sequence CDR3 as shown in SEQ ID NO:48.

[0145] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:41.

[0146] In other embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:45.

[0147] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:41; and

[0148] The light chain variable region contains an amino acid sequence as shown in SEQ ID NO:45.

[0149] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:49.

[0150] 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:50.

[0151] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:49; and a light chain containing an amino acid sequence as shown in SEQ ID NO:50.

[0152] In one embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:52, an amino acid sequence CDR2 as shown in SEQ ID NO:53, and an amino acid sequence CDR3 as shown in SEQ ID NO:54; and comprising a light chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:56, an amino acid sequence CDR2 as shown in SEQ ID NO:57, and an amino acid sequence CDR3 as shown in SEQ ID NO:58.

[0153] In some embodiments, the anti-CD45 antibody or its antigen-binding portion includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:51.

[0154] In other embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:55.

[0155] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:51; and

[0156] The light chain variable region contains an amino acid sequence as shown in SEQ ID NO:55.

[0157] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:59.

[0158] 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:60.

[0159] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:59; and a light chain containing an amino acid sequence as shown in SEQ ID NO:60.

[0160] In one embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:62, an amino acid sequence CDR2 as shown in SEQ ID NO:63, and an amino acid sequence CDR3 as shown in SEQ ID NO:64; and comprising a light chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:66, an amino acid sequence CDR2 as shown in SEQ ID NO:67, and an amino acid sequence CDR3 as shown in SEQ ID NO:68.

[0161] In some embodiments, the anti-CD45 antibody or its antigen-binding portion includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:61.

[0162] In other embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:65.

[0163] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:61; and

[0164] The light chain variable region contains an amino acid sequence as shown in SEQ ID NO:65.

[0165] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:69.

[0166] 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:70.

[0167] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:69; and a light chain containing an amino acid sequence as shown in SEQ ID NO:70.

[0168] In one embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:72, an amino acid sequence CDR2 as shown in SEQ ID NO:73, and an amino acid sequence CDR3 as shown in SEQ ID NO:74; and comprising a light chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:76, an amino acid sequence CDR2 as shown in SEQ ID NO:77, and an amino acid sequence CDR3 as shown in SEQ ID NO:78.

[0169] In some embodiments, the anti-CD45 antibody or its antigen-binding portion includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:71.

[0170] In other embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:75.

[0171] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:71; and

[0172] The light chain variable region contains an amino acid sequence as shown in SEQ ID NO:75.

[0173] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:79.

[0174] 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:80.

[0175] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:79; and a light chain containing an amino acid sequence as shown in SEQ ID NO:80.

[0176] In one embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:82, an amino acid sequence CDR2 as shown in SEQ ID NO:83, and an amino acid sequence CDR3 as shown in SEQ ID NO:84; and comprising a light chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:86, an amino acid sequence CDR2 as shown in SEQ ID NO:87, and an amino acid sequence CDR3 as shown in SEQ ID NO:88.

[0177] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:81.

[0178] In other embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:85.

[0179] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:81; and

[0180] The light chain variable region contains an amino acid sequence as shown in SEQ ID NO:85.

[0181] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:89.

[0182] 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:90.

[0183] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:89; and a light chain containing an amino acid sequence as shown in SEQ ID NO:90.

[0184] In one embodiment, the present invention provides an isolated anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:92, an amino acid sequence CDR2 as shown in SEQ ID NO:93, and an amino acid sequence CDR3 as shown in SEQ ID NO:94; and comprising a light chain having a variable region having an amino acid sequence CDR1 as shown in SEQ ID NO:96, an amino acid sequence CDR2 as shown in SEQ ID NO:97, and an amino acid sequence CDR3 as shown in SEQ ID NO:98.

[0185] In some embodiments, the anti-CD45 antibody or its antigen-binding portion includes a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:91.

[0186] In other embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:95.

[0187] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain variable region containing an amino acid sequence as shown in SEQ ID NO:91; and

[0188] The light chain variable region contains an amino acid sequence as shown in SEQ ID NO:95.

[0189] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:99.

[0190] 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:100.

[0191] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:99; and a light chain containing an amino acid sequence as shown in SEQ ID NO:100.

[0192] In one embodiment, the anti-CD45 system described herein is complete.

[0193] In some embodiments, the anti-CD45 antibody fragment is selected from the group consisting of Fab, F(ab')2 and scFv.

[0194] In other embodiments, the anti-CD45 antibody or its antigen-binding portion is a human antibody or its binding fragment.

[0195] 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).

[0196] In some embodiments, the anti-CD45 antibody or its antigen-binding portion is IgG, such as IgG1 or IgG4.

[0197] 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.

[0198] 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.

[0199] In one embodiment, the anti-CD45 ADC comprises a cytotoxin, which is a phallotoxin represented by formula (IA).

[0200]

[0201] Among them, R1 series H, OH, OR A OR C ;

[0202] R2 series H, OH, OR B OR C ;

[0203] 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;

[0204] R3 series H, R C or R D ;

[0205] R4, R5, R6, and R7 each independently generate H, OH, and OR. C OR D R C or R D ;

[0206] R8 series OH, NH2, OR C OR D NHR C or NR C R D ;

[0207] R9 series H, OH, OR C OR D ;

[0208] X series -S-, -S(O)- or -SO2-;

[0209] R C LZ;

[0210] 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.

[0211] L is a substituted C1-C6 alkyl group, a substituted C1-C6 heteroalkyl group, a substituted C2-C6 alkenyl group, a substituted C2-C6 heteroalkyl group, a substituted C2-C6 alkyne group, a substituted C2-C6 heterokyne group, a substituted cycloalkyl group, a substituted heterocycloalkyl group, a substituted aryl group, or a substituted heteroaryl group; and Z is a chemical moiety formed by the coupling reaction between a reactive substituent present on L and a reactive substituent present in the antibody or its antigen-binding fragment.

[0212] Where Am contains exactly one R C Substituents.

[0213] In one embodiment, the anti-CD45 ADC has the following formula:

[0214]

[0215] Ab represents the binding site of the anti-CD45 antibody.

[0216] In one embodiment, the anti-CD45 ADC has the following formula:

[0217]

[0218] Ab represents the binding site of the anti-CD45 antibody.

[0219] In one embodiment, the anti-CD45 ADC comprises cytotoxins such as amanitin (e.g., α-amanitin, β-amanitin, γ-amanitin, ε-amanitin), amanin, amanitin amide, amanullin, amanullinic acid, and proto-amanitin.

[0220] In one embodiment, the anti-CD45 ADC comprises a cytotoxic agent selected from the group consisting of: Pseudomonas exotoxin A, debendazole, diphtheria toxin, saposhnikovia toxin, maytansin, maytansin-like substances, orrisstatin (e.g., MMAE or MMAF), anthracycline, cazidromycin, irinotecan, SN-38, pyrrolobenzodiazepine, pyrrolobenzodiazepine dimer, indolinobenzodiazepine, and indolinobenzodiazepine dimer.

[0221] In one embodiment, the anti-CD45 ADC comprises a cytotoxic agent containing a benzodiazepine moiety. In some embodiments, the anti-CD45 ADC comprises pyrrolobenzodiazepine (“PBD”). In some embodiments, the anti-CD45 ADC comprises indolinobenzodiazepine (“IGN”).

[0222] In one embodiment, the anti-CD45 ADC comprises an anti-CD45 antibody that binds to the toxin via a cysteine ​​residue in the antibody's Fc domain. In one embodiment, the cysteine ​​residue is introduced via amino acid substitution in the antibody's Fc domain. In one embodiment, the amino acid substitution is D265C and / or V205C (EU number).

[0223] In some embodiments, the drug-to-antibody ratio (DAR) of the anti-CD45 ADC is 1, 2, 3, 4, 5, 6, 7, or 8.

[0224] The present invention also includes pharmaceutical compositions comprising the anti-CD45 antibody or ADC described herein and a pharmaceutically acceptable carrier.

[0225] The anti-CD45 antibodies, fragments, and ADCs described in this article can be used in the treatment of human patients, including (but not limited to) conditioning therapy prior to allogeneic or autologous transplantation.

[0226] In one embodiment, this document discloses a method for 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 as described herein. In some embodiments, the method further comprises administering to the patient a graft containing hematopoietic stem cells.

[0227] In other embodiments, this document discloses a method of administering a graft (allogeneic or autologous) comprising hematopoietic stem cells to a human patient, wherein the patient has previously been administered an amount of the anti-CD45 antibody, fragment, or ADC described herein sufficient to deplete the patient's hematopoietic stem cell population. In some embodiments, the hematopoietic stem cells are CD45+ cells.

[0228] In other embodiments, the anti-CD45 antibodies, fragments, or ADCs described herein are used to treat human patients with blood disorders, metabolic disorders, cancer, or autoimmune diseases or severe combined immunodeficiency diseases (SCID).

[0229] In one embodiment, the anti-CD45 antibody, fragment, or ADC described herein is administered to a human patient to treat leukemia in a human patient.

[0230] In other embodiments, this document discloses a method of administering a graft comprising hematopoietic stem cells to a human patient, wherein the patient has previously been administered an amount of the anti-CD45 antibody, fragment, or ADC described herein sufficient to deplete the patient's immune cell population. In one embodiment, the immune cell line is CD137+, CD2+, or CD5+ cells. In other embodiments, the immune cell line is T cells. Attached Figure Description

[0231] Figure 1 The results of the in vitro binding assays are presented graphically, evaluating the binding affinity of mature anti-CD45 antibodies and their corresponding parental antibodies (parental 1-4) to human CD45 or cynomolgus monkey CD45. Parental 1 corresponds to Ab1 as described herein. Binding was measured using biolayer interference (BLI) of purified IgG (sensory-related) incubated with purified human CD45 or cynomolgus monkey CD45 extracellular domains. Figure 2 The results of in vitro cell binding assays evaluating the binding of indicated anti-CD45 antibodies (Ab5 and Ab7) to REH cells, human PBMCs, and cynomolgus monkey PBMCs are presented graphically. The Fc variants of Ab5 and Ab7, Ab5_D265C_LALA_H435A and A7_D265C_LALA_H435A, were used in this study. Non-targeted isotype IgGs with the Fc-modified region (D265C LALA H435A) were evaluated as controls.

[0232] Figure 3The results of a study on the localization of the CD45 antigenic determinant using the anti-CD45 antibody Ab5 are depicted. A schematic diagram of the Ab5 interaction sites on CD45 is shown, with the amino acid sequences surrounding the Ab5 antigenic determinant (SEQ ID NO: 115 and SEQ ID NO: 117) marked. Contact residues are highlighted as 405T, 407K, 419Y, 425K, 481R, 505R, and 509H, with residue numbering referring to the human CD45 fragment represented by SEQ ID NO: 113.

[0233] Figure 4 This paper presents the results of an in vitro internalization analysis evaluating the internalization of an Ab5-constructed anti-CD45 antibody-drug conjugate (ADC) in human CD34+ bone marrow cells. The Fc variant of Ab5, Ab5D265C.LALA.H435A, binds to amatoxins (Amatoxin 1 (AM1)) to form Ab5-AM1D265C.LALA.H435A. The anti-CD45 ADC binds to a water-soluble, bright, photosensitizing, and pH-responsive pHAb dye. After internalization, the bound antibody migrates to acidic endosomes / lysosomes, where the pHAb dye emits at 563 nM and can be detected by flow cytometry. Human bone marrow CD34+ cells were cultured on ice for 2 hours, followed by incubation with saturated concentrations of ADC for 0, 2, 24, 48, or 72 hours. The left graph plots the pHAb levels over time. The binding of surface hIgG1 to fluorescently labeled antiIgG molecules was evaluated by flow cytometry, and the percentage of surface IgG over time was calculated, as depicted in the figure on the right.

[0234] Figure 5A and 5B The results of the in vitro cell line killing analysis are presented in a graphical format, showing that the anti-CD45 ADC 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)). The Fc variants of Ab4 and Ab5, Ab4 D265C.LALA.H435A and Ab5 D265C.LALA.H435A, bind to phallotoxins (phallotoxins 1 (AM1) or phallotoxins 2 (AM2)) to form Ab4-AM2 D265C.LALA.H435A ("Ab4-AM2") and Ab5-AM1 D265C.LALA.H435A ("Ab5-AM1"). Figure 5AIn this study, SKNO1, Jurkat, REH (CD45+), or REH (CD45- / -) cell lines were cultured for 7 days in the presence of Ab5-AM1 or a control non-targeted isotype-matched ADC ("isotype-AM1"). Cell viability was measured by changes in antibody concentration (x-axis) as observed by Celltiter Glo luminescence (y-axis). Figure 5B In this study, SKNO1, Jurkat, or REH (CD45+) cell lines were cultured for 7 days in the presence of Ab4-AM2 or a control non-targeted isotype-matched ADC ("isotype-AM2"). Cell viability was measured based on changes in antibody concentration (x-axis) using Celltiter Glo luminescence (y-axis).

[0235] Figures 6A-6C The results of in vitro primary cell killing analysis are presented graphically, 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). The Fc variants of Ab2, Ab4, and Ab5, Ab2D265C.LALA.H435A, Ab4D265C.LALA.H435A, and Ab5D265C.LALA.H435A, bind to phallotoxins (phallotoxins 1 (AM1) or phallotoxins 2 (AM2)) to form Ab2-AM2D265C.LALA.H435A ("Ab2-AM2"), Ab4-AM2D265C.LALA.H435A ("Ab4-AM2"), and Ab5-AM1D265C.LALA.H435A ("Ab5-AM1"). Figure 6A In Figure 76B, human PBMCs were cultured for 7 days on 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 Celltiter Glo. 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).

[0236] Figure 7The 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.

[0237] 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).

[0238] Figure 9 The results of serum stability analysis evaluating the stability of the anti-CD45 ADC constructed from Ab5 in human serum (top plot) or cynomolgus monkey ("cynomolgus monkey") serum (bottom plot) are presented graphically. The Fc variant of Ab5, Ab5D265C.LALA.H435A, binds to phallotoxin 1 (AM1) to form Ab5-AM1D265C.LALA.H435A ("Ab5-AM1"). To determine the stability of this ADC in serum, the ADC was pre-incubated in human or cynomolgus monkey ("cynomolgus monkey") serum at 37°C for 0 or 72 hours, and then the cytotoxic effect of the ADC on REH cells was evaluated. Cell viability % (y-axis) of REH cells as a function of serum-treated ADC concentration (x-axis) was evaluated using Cell-titer Glo.

[0239] Figures 10A-10EThe results of the in vivo cell depletion analysis are presented graphically, showing the selective depletion of human HSCs and immune cells in humanized NSG mice with short half-life anti-CD45 ADCs constructed from Ab2, Ab3, Ab5, and Ab7. Ab2D265C.LALA.H435A, Ab3D265C.LALA.H435A, Ab5D265C.LALA.H435A, and Ab7D265C.LALA.H435A (Fc variants of Ab2, Ab3, Ab5, and Ab7) bind to phallotoxin 1 (AM1) or phallotoxin 2 (AM2) to form Ab2D265C.LALA.H435A-AM2 (“Ab2-AM2”), Ab3D265C.LALA.H435A-AM2 (“Ab3-AM2”), Ab5D265C.LALA.H435A-AM1 (“Ab5-AM1”), and Ab7D265C.LALA.H435A-AM1 (“Ab7-AM1”). Mice treated with PBS served as controls. Figure 10A Shows the percentage of human CD3+ T cells, CD19+ B cells, and CD33+ bone marrow cells in mice 14 days after administration of anti-CD45-ADC (Ab2-AM2, Ab3-AM2, AbA-AM2) relative to baseline. Figure 10B This shows the percentage of human CD45+ cells in peripheral blood at 0, 7, and 14 days after administration of anti-CD45-ADC (Ab2-AM2 or Ab3-AM2). Figure 10C The absolute number of human CD45 cells (left) and human HSCs (right) in the bone marrow of humanized NSG mice 14 days after administration of anti-CD45-ADC (Ab2-AM2 or Ab3-AM2). Figure 10D The 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).

[0240] Figure 11A and 11BThe 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-CD45 ADC (1 mg / kg Ab4-AM1 or 0.5 or 2 mg / kg Ab5-AM1) were plotted in a graphical format.

[0241] Figure 12 The pharmacokinetic analysis results of the short half-life anti-CD45 ADCs constructed from Ab4 in cynomolgus monkeys are presented graphically. The Fc variant of Ab4, Ab4 D265C.LALA.H435A, binds to phallotoxin 1 (AM1) or phallotoxin 2 (AM2) to form Ab4 D265C.LALA.H435A-AM1 ("Ab4-AM1") and Ab4 D265C.LALA.H435A-AM2 ("Ab4-AM2"). The mean plasma drug concentration (y-axis) of each ADC was monitored over time (x-axis).

[0242] Figure 13 The results of multispecific reagent (PSR) analysis evaluating the binding of humanized pure lines A, B and C derived from parental pure lines in Example 15 to a mixture of membrane and cytoplasmic proteins are presented graphically.

[0243] Figure 14 The results of in vitro cell binding assays used to evaluate the binding of anti-CD45 antibodies AbA, AbB, and AbC (identified in Example 15) to human PBMCs and cynomolgus monkey ("cynomolgus monkey") PBMCs are presented graphically.

[0244] Figure 15The results of the CD45 antigenic determinant localization study of the anti-CD45 antibody AbA identified in Example 15 are depicted. A schematic diagram of the AbA interaction site on CD45 is shown, in which the amino acid sequence surrounding the AbA antigenic determinant (SEQ ID NO: 117) is indicated. Contact residues are highlighted as 486R, 493Y, and 502T, where the residue numbering refers to the human CD45 fragment represented by SEQ ID NO: 113.

[0245] Figure 16 This paper presents the results of an in vitro internalization analysis evaluating the internalization of an anti-CD45 antibody-drug conjugate (ADC) constructed from AbA in human CD34+ bone marrow cells. The Fc variant of AbA, AbA_D265C_LALA_H435A, binds to phallotoxin (phallotoxin 1) to form the anti-CD45 ADC AbA_D265C_LALA_H435A-AM1. The anti-CD45 ADC binds to a water-soluble, bright, photosensitizing, and pH-responsive pHAb dye. After internalization, the bound antibody migrates to acidic endosomes / lysosomes, 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, followed by incubation with saturated concentrations of ADC for 0, 2, 24, 48, or 72 hours. The left graph plots the pHAb levels over time. The binding of surface hIgG1 to fluorescently labeled antiIgG molecules was evaluated by flow cytometry, and the percentage of surface IgG over time was calculated, as depicted in the figure on the right.

[0246] Figure 17A and 17B The results of in vitro primary cell killing analysis are presented graphically, showing that the anti-CD45 ADC constructed from AbA can effectively kill in vitro primary human or cynomolgus monkey peripheral blood mononuclear cells (PBMCs). The Fc variant of AbA, AbA_D265C_LALA_H435A, binds to one of two amatoxins, namely amatoxin 1 (AM1) or amatoxin 2 (AM2), to form AbA_D265C_LALA_H435A-AM1 or AbA_D265C_LALA_H435A-AM2. The indicated CD45-AM conjugate (AbA_D265C_LALA-H435A-AM1)... Figure 17A ) or AbA_D265C_LALA-H435A-AM2( Figure 17B Human PBMCs were cultured for 7 days in the presence of a non-targeted isotype-matched ADC ("isotype-AM1" or "isotype-AM2") and cell viability (y-axis) was measured as a function of antibody concentration (x-axis) using Celltiter Glo.

[0247] Figure 18 The results of in vitro primary cell killing assays are presented graphically, showing that anti-CD45 ADCs constructed from AbA can effectively kill in vivo human hematopoietic stem cells (HSCs). The Fc variants of AbA, AbA_D265C_LALA_H435A and AbA_D265C_LALA_IHH, bind to phallotoxins 1 (AM1), 2 (AM2), or PBD to form AbA_D265C_LALA_H435A-AM1, AbA_D265C_LALA_H435A-AM2, or AbA_D265C_LALA_IHH-PBD. Primary human CD45 cells were killed in the presence of the indicated ADCs (AbA_D265C_LALA_H435A-AM1 and AbA_D265C_LALA_IHH-PBD) or isotype controls. + Bone marrow cells were cultured for 5 days. The active CD34+CD90+ HSC count (y-axis) was measured by flow cytometry as a function of antibody concentration (x-axis).

[0248] Figure 19 The results of the in vitro killing assay are presented graphically, showing that the anti-CD45 ADC constructed from AbA can effectively kill in vivo macrophages. The Fc variant of AbA, AbA_D265C_LALA_H435A, binds to phallotoxin 1 (AM1) to form the CD45 ADC AbA_D265C_LALA_H435A-AM1. Macrophages were cultured for 6 days in the presence of the ADC (AbA_D265C_LALA_H435A-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).

[0249] Figure 20 The results of an in vivo depletion analysis in humanized NSG mice treated with an anti-CD45 antibody-drug conjugate (AbA-PBD) are presented graphically, evaluating the depletion of human cells in peripheral blood. hNSG mice were administered a single dose of the indicated mediator (PBS), isotype control-PBD ("Iso-PBD"), or CD45-PBD (AbA-PBD). Peripheral blood was collected at indicated time points, and the total human hematopoietic cell count (hβ2M) was assessed. + ), bone marrow cell content (CD33) + ), B cell content (CD19) + ) and T cell content (CD3) + The result is presented as the depletion percentage normalized to baseline.

[0250] Figure 21The results of an in vivo depletion analysis in humanized NSG mice treated with anti-CD45-ADC (AbA-PBD) are presented graphically, evaluating the depletion of human cells in the bone marrow. hNSG mice were administered a single dose of the indicated mediator (PBS), isoform-PBD, or CD45-PBD (AbA-PBD). Bone marrow samples were collected on day 14 post-treatment, and the human progenitor cell / HSC content was assessed. Results are presented as the percentage and absolute number of human cells per femur.

[0251] Figure 22 The results of in vivo depletion analysis in humanized NSG mice treated with anti-CD45-ADC (AbA-PBD) are presented graphically, including the evaluation of human CD45+ cells, double-positive (DP) thymocytes, and mature CD4+ cells at 14 days post-treatment. + Single positive (SP) thymocytes or mature CD8 + Depletion of single-positive (SP) thymocytes. hNSG mice were administered a single dose of the indicated mediator (PBS), isotype-PBD, or CD45-PBD (AbA-PBD).

[0252] Figure 23 The results of an in vivo depletion analysis in humanized NSG mice treated with an anti-CD45 antibody-drug conjugate (AbA-IGN) are presented graphically, evaluating the depletion of human cells in peripheral blood. hNSG mice were administered a single dose of the indicated mediator (PBS), isotype control-IGN ("Iso-IGN"), or CD45-IGN (AbA-IGN). Peripheral blood was collected at indicated time points, and the total human hematopoietic cell count (hβ2M) was assessed. + ), bone marrow cell content (CD33) + ), B cell content (CD19) + ) and T cell content (CD3) + The result is presented as the depletion percentage normalized to baseline.

[0253] Figure 24 Results of an in vivo depletion analysis in humanized NSG mice treated with CD45-IGN are presented graphically, evaluating the depletion of human cells in the bone marrow. hNSG mice were administered a single dose of the indicated mediator (PBS), isoform-IGN, or CD45-IGN. Bone marrow samples were collected on day 14 post-treatment, and the human progenitor cell / HSC content was assessed. Results are presented as the percentage and absolute number of human cells per femur.

[0254] Figure 25 The results of in vivo depletion analysis in humanized NSG mice treated with AbA-IGN are presented graphically, evaluating human CD45+ cells, double-positive (DP) thymocytes, and mature CD4+ cells.+ Single positive (SP) thymocytes or mature CD8 + Depletion of single-positive (SP) thymocytes. hNSG mice were administered a single dose of the indicated mediator (PBS), isotype-IGN, or AbA-IGN.

[0255] Figure 26 The results of the in vivo cell depletion analysis are presented graphically, showing that the anti-CD45-phalloidin ADC constructed from AbA effectively depletes HSCs and immune cells in non-human primates in vivo. The Fc variant of AbA (introducing Fc region modifications to shorten the half-life), AbA_D265C_LALA_H435A, binds to phallotin 1 (AM1) or phallotin 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 bone marrow cells) in the bone marrow of cynomolgus monkeys were measured at indicated time points (days 6, 14, 20, and 28) following a single 1 mg / kg injection of the indicated ADC.

[0256] Figure 27 The pharmacokinetic analysis results of anti-CD45 ADCs constructed from AbA in cynomolgus monkeys are presented graphically. The Fc variant of AbA (with modifications to the Fc region to shorten the half-life), AbA_D265C_LALA_H435A, binds to either amatoxin 1 (AM1) or amatoxin 2 (AM2) to form AbA_D265C_LALA_H435A-AM1 ("CD45 ADC-AM1") or AbA_D265C_LALA_H435A-AM2 ("CD45 ADC-AM2"). The mean plasma drug concentration (y-axis) of each ADC was monitored over time (x-axis).

[0257] 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.

[0258] 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 graphically. Figure 29B The radiation levels of REH-luciferase in the indicated treatment groups during the study period are plotted graphically (mean ± SEM). Figure 29C These are representative bioluminescent signal false-color images captured on days 22-23 post-implantation in all treatment groups.

[0259] 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 delayed tumor growth. Figure 30A The survival rate of mice in the indicated treatment group as of days post-implantation is plotted graphically. Figure 30BThe percentage of tumor burden (hCD45+) in peripheral blood of mice in each indicated treatment group as of days post-implantation is plotted graphically.

[0260] Figures 31A-31D The results of in vivo mouse studies are illustrated graphically, showing that short-half-life CD45-DC (AbA-AM) effectively depletes human leukemia cells in two patient-derived AML models. Figure 31A and 31B The PDX model AML#1 mice in the indicated treatment groups are depicted graphically. Figure 31A ) or PDX model AML#2 mice ( Figure 31B Survival rate as of post-implantation days. Figure 31A and 31B The illustrations for each model depict flow cytometry analysis of each AML PDX model to evaluate the expression of CD117 and CD45 cells on the spleen cells of diseased mice. Figure 31C and 31D The PDX model AML#1 mice in each indicated treatment group are depicted graphically. Figure 31C ) and PDX model AML#2 mice ( Figure 31D The percentage of tumor burden (hCD45+) in peripheral blood as a function of the number of days after implantation.

[0261] Figure 32 Multiple sequence alignments of the heavy chain variable regions and light chain variable regions of anti-CD45 antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, and Ab7 were depicted. The CDRs of each variable region are indicated in bold. Detailed Implementation

[0262] This article discloses novel anti-CD45 antibodies, their antigen-binding fragments, and conjugates (e.g., antibody-drug conjugates; ADCs), which are useful because, for example, they exhibit cross-reactivity between human CD45 and non-human primate CD45. Furthermore, the anti-CD45 antibodies and fragments described herein can be used as therapeutic agents, such as for the treatment of leukemia, or as transplant conditioning agents. Therefore, this article includes anti-hematopoietic cell antibodies (anti-CD45 antibodies) that can be used in hematopoietic stem cell therapy. For example, the antibodies or ADCs described herein can be used in conditioning procedures in which patients are prepared to receive grafts comprising hematopoietic stem cells. These procedures facilitate the implantation of hematopoietic stem cell grafts. According to the methods described herein, patients can be conditioning for hematopoietic stem cell transplantation therapy by administering anti-CD45 ADCs, antibodies, or antigen-binding fragments thereof that bind to CD45 (e.g., CD45 expressed by hematopoietic cells, such as hematopoietic stem cells or mature immune cells, such as T cells). As described herein, anti-CD45 antibodies can covalently bind to cytotoxins to form antibody-drug conjugates (ADCs). Administering CD45-binding ADCs to patients requiring hematopoietic stem cell transplantation can promote the implantation of hematopoietic stem cell grafts, for example, by selectively depleting endogenous hematopoietic stem cells, thereby creating vacancies to be filled by exogenous hematopoietic stem cell grafts.

[0263] The following sections provide novel anti-CD45 antibodies and fragments thereof with unique properties, such as cross-reactivity with human and non-human primate CD45. The following sections also provide descriptions of anti-CD45 antibodies or conjugates thereof that can be administered to patients (e.g., patients with cancer or autoimmune diseases or patients requiring hematopoietic stem cell transplantation therapy) to promote hematopoietic stem cell graft implantation, and methods for administering such therapeutic agents to patients (e.g., prior to hematopoietic stem cell transplantation).

[0264] definition

[0265] As used herein, the term "about" refers to a value within 5% above or below the stated value. For example, the term "about 100 nM" indicates a range of 95-105 nM.

[0266] As used in this article, the term "allogeneic" is used in the context of transplantation to define a graft (e.g., a cell, tissue, or organ graft) that is transplanted from a donor to a recipient, where the recipient is a different individual of the same species as the donor.

[0267] As used in this article, the term "autologous" in the context of transplantation refers to a graft in which the donor and recipient are the same individual, or the same subject.

[0268] As used in this article, the term "heterogeneous" in the context of transplantation refers to a graft in which the donor and recipient are of different species.

[0269] As used herein, the term "immune cell" is intended to include (but is not limited to) cells of hematopoietic origin that play a role in the immune response. Immune cells include (but are not limited to) T cells and natural killer (NK) cells. Natural killer cells are well known in the art. In 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 may be allogeneic or autologous.

[0270] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with a specific antigen. Antibodies include (but are not limited to) monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0271] Typically, antibodies consist of a heavy chain and a light chain containing antigen-binding regions. Each heavy chain contains a heavy chain variable region (abbreviated as HCVR or VH in this text) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated as LCVR or VL in this text) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) and more conserved regions (called framework regions (FRs)), which are interspersed. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors (including various cells of the immune system, such as effector cells, and the first component (Clq) of the classical complement system).

[0272] As used herein, the term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more portions of the antibody that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of the full-length antibody. Antibody fragments can be, for example, Fab, F(ab')2, scFv, bivalent antibodies, trivalent antibodies, affinity antibodies, nanoantibodies, aptamers, or domain antibodies. Examples of binding fragments covered by the term "antigen-binding fragment" of an antibody include (but are not limited to): (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, which are bivalent fragments containing two Fab fragments connected by disulfide bridges in the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of an antibody arm; (v) dAb fragments including VH and VL domains; (vi) dAb fragments consisting of VH domains (e.g., see Ward et al., Nature 341:544-546, 1989); (vii) dAb fragments consisting of VH or VL domains; (viii) separated complementarity-determining regions (CDRs); and (ix) combinations of two or more (e.g., two, three, four, five, or six) separated CDRs that may be linked by synthetic linkers, as appropriate. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be linked together using recombinant methods via a linker, allowing the formation of a single protein chain of monovalent molecules with the VL and VH regions paired therein (referred to as single-chain Fv (scFv); see, for example, Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and their efficacy can be screened in the same manner as for intact antibodies. Antigen-binding fragments can be generated using recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or, in some cases, chemical peptide synthesis procedures known in this art.

[0273] As used herein, "intact" or "full-length" antibody systems refer to antibodies having two heavy chain (H) polypeptides and two light chain (L) polypeptides linked by disulfide bonds. In some embodiments, toxins may bind to intact anti-CD45 antibodies having the heavy chain and / or light chain amino acid sequences described herein.

[0274] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a single pure line (including any eukaryotic, prokaryotic, or phage pure line) by any method available or known in this art, and is not limited to antibodies produced via hybridoma technology. Monoclonal antibodies used in this disclosure can be prepared using a variety of techniques known in this art, including hybridoma, recombinant, and phage display technologies, or combinations thereof.

[0275] As used herein, the terms "Fc region," "Fc domain," and "IgG Fc domain" refer to the portion of an immunoglobulin (e.g., the IgG molecule) associated with the crystallizable fragment obtained by papain digestion of the IgG molecule. The Fc region contains the C-terminal half of the two heavy chains of the IgG molecule linked by disulfide bonds. It is not antigen-binding active but contains a carbohydrate portion and binding sites for complement and Fc receptors (including FcRn receptors) (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).

[0276] Fc can refer to this region alone, or to this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphism has been observed at multiple positions in the Fc domain (including but not limited to EU positions 270, 272, 312, 315, 356, and 358), and therefore slight differences may exist between the sequence presented in this application and sequences known in this art. Therefore, "wild-type IgG Fc domain" or "WT IgG Fc domain" refers to any naturally occurring IgG Fc region (i.e., any paired gene). Heavy chain sequences of human IgG1, IgG2, IgG3, and IgG4 can be found in multiple 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).

[0277] As used herein, the terms "modified Fc region" or "variant Fc region" refer to an IgG Fc domain containing one or more amino acid substitutions, deletions, insertions, or modifications introduced at any position within the Fc domain. In some states, the variant IgG Fc domain contains one or more amino acid substitutions that reduce or eliminate binding affinity for FcγR and / or C1q compared to the wild-type Fc domain which does not contain one or more amino acid substitutions. Furthermore, Fc binding interactions are essential for a variety of effector functions and downstream signaling events, including but not limited to antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore, in certain states, antibodies containing variant Fc domains (e.g., antibodies, fusion proteins, or conjugates) may exhibit altered binding affinity to at least one or more Fc ligands (e.g., FcγR) relative to corresponding antibodies that originally have the same amino acid sequence but do not contain one or more amino acid substitutions, deletions, insertions, or modifications (e.g., unmodified Fc regions containing native amino acid residues at corresponding positions in the Fc region).

[0278] The variant Fc domain is defined according to the amino acid modifications that constitute it. For all amino acid substitutions discussed in this paper concerning the Fc region, the numbering is always based on 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 replaced by cysteine ​​(C) relative to the parental Fc domain. It should be noted that the order of substitutions provided is arbitrary.

[0279] As used herein, the term "Fcγ receptor" or "FcγR" refers to any member of the protein family that binds to the Fc region of IgG antibodies and is encoded by the FcγR gene. In humans, this family includes (but is not limited to) FcγRI (CD64), including isotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), including isotypes FcγRIIa (including isotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), including isotypes FcγRIIIa (including isotypes V158 and F158) and FcγRIIIb (including isotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), as well as any undiscovered human FcγR or FcγR isotypes or isotypes. FcγRs can be derived from any organism, including (but not limited to) humans, mice, rats, rabbits, and monkeys. Mouse FcγRs include (but are not limited to) FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγR or FcγR isoforms or variants.

[0280] As used herein, "effective function" refers to a biochemical event arising from the interaction between the Fc domain and the Fc receptor. Effector functions include (but are not limited to) ADCC, ADCP, and CDC. As used herein, "effective cell" refers to a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include (but are not limited to) monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and γ-δ T cells, and can be derived from any organism, including (but not limited to) humans, mice, rats, rabbits, and monkeys.

[0281] As used herein, the terms "silent," "silenced," or "silent" refer to an antibody having the modified Fc region described herein, which exhibits reduced binding to the Fcγ receptor (FcγR) compared to the binding of the same antibody containing the unmodified Fc region (e.g., a reduction in FcγR binding by at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, as measured by, for example, BLI). In some embodiments, the Fc-silencing antibody does not have detectable FcγR binding. The binding of antibodies with modified Fc regions to FcγR can be determined using a variety of techniques known in this field, such as (but not limited to) equilibration methods (e.g., enzyme-linked immunosorbent assay (ELISA); Kin Ex A, Rathanaswami et al., Analytical Biochemistry, Vol. 373: 52-60, 2008; or radioimmunoassay (RIA)) or surface plasma resonance analysis or other kinetic-based analyses (e.g., BIACORE RTM analysis or Octet). 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), are employed. These and other methods utilize labeling on one or more tested components and / or employ various detection methods, including (but not limited to) chromogenic, fluorescent, luminescent, or isotopic labeling. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th ed., Lippincott-Raven, Philadelphia (1999), which addresses antibody-immunogen interactions. An example of competitive binding assays is radioimmunoassay, which involves co-culturing a labeled antigen and an antibody of interest in the presence of incrementally added unlabeled antigen, and detecting the antibody bound to the labeled antigen. The affinity of the antibody of interest for a specific antigen and the binding-dissociation rate can be determined from data analyzed using a scatchard plot. Competition with a secondary antibody can also be determined using radioimmunoassay. In this case, the antigen is cultured together with the antibody of interest bound to the labeled compound in the presence of incrementally added unlabeled second antibody.

[0282] As used herein, "identical antibody containing an unmodified Fc region" refers to an antibody that lacks the listed amino acid substitutions (e.g., D265C, H435A) but originally has the same amino acid sequence as the Fc-modified antibody to which it is compared.

[0283] The term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which a polypeptide (e.g., an antibody) containing an Fc domain binds to an Fc receptor (FcR) present on certain cytotoxic cells (e.g., primarily NK cells / neutrophils and macrophages), enabling these cytotoxic effector cells to specifically bind to "target cells" carrying antigens and subsequently kill the target cells with cytotoxins. (Hogarth et al., Nature review DrugDiscovery 2012, 11:313) In addition to antibodies and their fragments, it encompasses other polypeptides containing an Fc domain (e.g., Fc fusion proteins and Fc binding proteins) that have the ability to specifically bind to target cells carrying antigens, thus enabling cell-mediated cytotoxicity.

[0284] For the sake of brevity, cell-mediated cytotoxicity derived from the activity of peptides containing Fc domains is also referred to herein as ADCC activity. The ability of any specific peptide of this disclosure to mediate ADCC lysis of target cells can be analyzed. To evaluate ADCC activity, a combination of the peptide of interest (e.g., an antibody) and immune effector cells is added to target cells, inducing cell lysis. Cell lysis is typically detected by the release of a marker (e.g., a radioactive receptor, a fluorescent dye, or a native intracellular protein) from the lysed cells. Effector cells that can be used for these analyses include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Specific examples of in vitro ADCC analysis 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 otherwise, the ADCC activity of the antibody of interest can be evaluated in vivo in animal models, for example, as disclosed by Clynes et al., Proc. Natl. Acad. Sci. USA 95:652 (1998).

[0285] As used herein, the terms "conditioning" and "conditioning" refer to the process of preparing a patient for the reception of a graft (e.g., a graft containing hematopoietic stem cells). These procedures promote the implantation of the hematopoietic stem cell graft (e.g., inferred from the consistent increase in the amount of viable hematopoietic stem cells in blood samples isolated from the patient after a conditioning procedure and subsequent hematopoietic stem cell transplantation). According to the methods described herein, patients can be conditioned for hematopoietic stem cell transplantation therapy by administering an ADC, antibody, or antigen-binding fragment thereof that binds to CD45 expressed by hematopoietic stem cells. As described herein, antibodies can covalently bind to cytotoxins to form drug-antibody conjugates. Administering antibodies, antigen-binding fragments, or ADCs that bind to the aforementioned antigens to patients requiring hematopoietic stem cell transplantation therapy can promote the implantation of the hematopoietic stem cell graft, for example, by selectively depleting endogenous hematopoietic stem cells, thereby creating vacancies to be filled by exogenous hematopoietic stem cell grafts.

[0286] As used herein, the term "effective amount" or "therapeutic effective amount" refers to an amount of a therapeutic agent (e.g., an anti-CD45 ADC) sufficient to achieve the desired outcome in the context of treating, preventing, improving, or alleviating symptoms of a patient's disease or condition. For example, in some embodiments, a therapeutically effective amount of an anti-CD45 antibody or ADC is sufficient to reduce or deplete the CD45+ cell population in a patient. In other embodiments, a therapeutically effective amount of an anti-CD45 antibody or ADC is sufficient to condition a patient to receive a hematopoietic stem cell graft. In these embodiments, a therapeutically effective amount may be, for example, sufficient to selectively deplete a patient's endogenous hematopoietic stem cells, and / or sufficient to promote the engraftment of a hematopoietic stem cell graft into the patient. In other embodiments, a therapeutically effective amount of an anti-CD45 antibody or ADC is sufficient to be effective against autoimmune diseases or cancer in human patients.

[0287] As used in this article, "half-life" refers to the time it takes for the plasma concentration of an antibody drug in an individual (e.g., a human individual) to decrease by half or 50%. This 50% decrease in serum concentration reflects the amount of drug circulating.

[0288] As used herein, the phrase "substantially cleared from the blood" refers to the point in time at which, after administration of a therapeutic agent (e.g., an anti-CD45 antibody or its antigen-binding fragment), the concentration of the therapeutic agent in a blood sample separated from the patient becomes undetectable by conventional methods (e.g., undetectable at noise levels above the noise threshold of the device or analysis used to detect the therapeutic agent). Various techniques known in this art can be used to detect antibodies or antibody fragments, such as ELISA-based detection analyses known in this art or described herein. Other analyses that can be used to detect antibodies or antibody fragments include, in particular, immunoprecipitation techniques and immunodotting analyses known in this art.

[0289] As used herein, "specific binding" or "specifically binding" refers to the ability of an antibody to recognize and bind to a specific protein structure (antigenic determinant) rather than a regular protein. If an antibody specifically targets antigenic determinant "A," then in a reaction containing labeled "A" and an antibody, molecules containing antigenic determinant A (or free, unlabeled A) will reduce the amount of labeled A bound to the antibody. For example, if an antibody, when labeled, can compete with a corresponding unlabeled antibody to move away from its target, then the antibody "specifically binds" to the target. In one embodiment, if the antibody targets the K... D For 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, as used herein, the terms "specific binding to CD45" or "specifically binds to CD45" refer to binding to CD45 with a dissociation constant (K). D The value, as 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.

[0290] As used herein, the term "human antibody" is intended to include antibodies having variable regions derived from human germline immunoglobulin sequences. In embodiments where the human antibody contains a constant region, the constant region may also be derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis during in vitro or gene rearrangement processes, or by in vivo somatic mutations). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) has been grafted onto a human framework sequence. Human antibodies may be produced in human cells (e.g., through recombinant expression) or by non-human animal or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and / or light chain) genes. When human antibodies are single-chain antibodies, they may include linker peptides not found in native human antibodies. For example, Fv may contain a linker peptide that connects the heavy chain variable region and the light chain variable region, such as two to eight glycine or other amino acid residues. Human antibodies can be prepared using antibody libraries derived from human immunoglobulin sequences by various methods known in this technology, including phage display or yeast display methods. Human antibodies can also be produced using genetically modified mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes (see, for example, PCT Publications WO 1998 / 24893; WO 1992 / 01047; WO 1996 / 34096; WO 1996 / 33735; U.S. Patents 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).

[0291] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a rat antibody and the constant region sequence is derived from a human antibody.

[0292] A “humanized” form of a non-human (e.g., mouse or rat) antibody is an immunoglobulin containing a minimal sequence derived from a non-human immunoglobulin. Generally, a humanized antibody will contain substantially all at least one, and usually two, variable domains, wherein all or substantially all CDR regions correspond to their respective CDR regions of the non-human immunoglobulin and all or substantially all FR regions correspond to their respective FR regions of the human immunoglobulin sequence. Humanized antibodies may also contain all or part of the immunoglobulin constant region (Fc) of a sequence common to human immunoglobulins. Methods for antibody humanization are known in this art. For example, see Riechmann et al., 1988, Nature 332:323-7; U.S. Patents 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370 to Queen et al.; EP239400; PCT Publication WO 91 / 09967; U.S. Patent No. 5,225,539; EP592106; EP519596; Padlan, 1991, Mol. Immunol., 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and U.S. Patent No. 5,565,332.

[0293] Conserved sequence modifications of the sequence shown in SEQ ID NO described herein are also provided. Conserved sequence modifications include nucleotide and amino acid sequence modifications that do not eliminate the binding of the antibody or its antigen-binding portion containing an amino acid sequence encoded by a nucleotide sequence provided herein to its homologous antigen (e.g., CD45). These conserved sequence modifications include conserved nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into SEQ ID NO described herein using standard techniques known in this art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conserved sequence modifications include conserved amino acid substitutions, wherein an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in this art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-polar side chains (e.g., glycine, asparagine, glutamic acid, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the predicted non-essential amino acid residues in anti-CD73 antibodies are preferably substituted with another amino acid residue from the same side chain family. Methods for identifying nucleotides and amino acid substitutions that do not eliminate antigen binding are well known in this technique (see, for example, 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)).

[0294] As used herein, the term "engrafting potential" refers to the ability of hematopoietic stem cells and progenitor cells to rebuild a population in a tissue, whether these cell lines circulate naturally or are provided through transplantation. This term encompasses all events surrounding or resulting in engraftment, such as tissue homing of cells and colonization of cells within the tissue of interest. Engrafting efficiency or engraftment rate can be assessed or quantified using any clinically acceptable parameters known to those skilled in this art, and may include, for example, evaluating competitive population rebuilding units (CRUs); the inclusion or expression of markers in tissues where stem cells have been homegrown, colonized, or engrafted; or assessing individual progression via disease progression, survival of hematopoietic stem cells and progenitor cells, or survival of the recipient. Engrafting can also be determined by measuring the white blood cell count in peripheral blood at post-transplantation time. Engrafting can also be evaluated by measuring the recovery of bone marrow cells from donor cells in bone marrow aspirate samples.

[0295] As used herein, "hematopoietic stem cells" ("HSCs") refer to immature blood cells capable of self-renewal and differentiation into mature blood cells comprising different lineages, including (but not limited to) granulocytes (e.g., premyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, red blood cells), coagulation cells (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). These cells may include CD34. + Cells. CD34 +Cell lines are immature cells exhibiting the CD34 cell surface marker. In humans, CD34+ cells are considered to comprise a subset of cells with stem cell characteristics as defined above, while in mice, HSCs are CD34-. Additionally, HSCs also refer to long-term population reconstructive HSCs (LT-HSCs) and short-term population reconstructive HSCs (ST-HSCs). LT-HSCs and ST-HSCs differentiate based on functional potential and cell surface marker expression. For example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for maturity lineage markers including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, and CD235A). In mice, the bone marrow LT-HSC lineage was CD34-, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, CD48-, and lin- (negative for maturity lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL7ra), while the ST-HSC lineage was also CD34+, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, and lin- (negative for maturity lineage markers including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL7ra). Furthermore, under homeostatic conditions, ST-HSCs were less quiescent and more proliferative than LT-HSCs. However, LT-HSCs have a greater self-renewal potential (i.e., they survive throughout adulthood and can be transplanted continuously from successive recipients), while ST-HSCs have limited self-renewal potential (i.e., they survive only for a limited period of time and do not have the potential for continuous transplantation). Either of these HSCs can be used in the methods described herein. ST-HSCs are particularly useful because they are highly proliferating and therefore produce differentiated offspring more quickly.

[0296] As used herein, the term "functional potential of hematopoietic stem cells" refers to the functional properties of hematopoietic stem cells, including: 1) pluripotency (which refers to the ability to differentiate into multiple different blood lineages, including (but not limited to) granulocytes (e.g., premyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), coagulation cells (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, T cells, and B cells); 2) self-renewal (which refers to the ability of hematopoietic stem cells to produce progeny cells with potential equivalent to the parent cells, and this ability can be repeated throughout the individual's life without depletion); and 3) the ability of hematopoietic stem cells or their progeny to be reintroduced into graft recipients, subsequently home to the hematopoietic stem cell niche, and to re-establish productive and continuous hematopoiesis.

[0297] As used herein, the terms "individual" and "patient" refer to an organism, such as a human, that receives treatment for a particular disease or ailment as described herein. In some embodiments, the individual or patient mentioned in the methods provided herein refers to a human individual.

[0298] As used herein, "recipient" refers to a patient who receives a graft (e.g., a graft containing a population of hematopoietic stem cells). The transplanted cells given to the recipient may be, for example, autologous, syngeneic, or allogeneic cells.

[0299] As used herein, "treatment" or "treatment" means any improvement in disease outcome, such as prolonged survival, reduced morbidity, and / or mitigation of the side effects of alternative treatments; while complete eradication of the disease is preferred, but not required, as is readily understood in this art. For example, treatment may refer to reducing the severity and / or frequency of disease symptoms, eliminating disease symptoms and / or their underlying causes, reducing the frequency or likelihood of disease symptoms and / or their underlying causes, and improving or remedying damage directly or indirectly caused by the disease. Beneficial or expected clinical outcomes include (but are not limited to) promoting the engraftment of exogenous hematopoietic cells in patients following antibody conditioning therapy and subsequent hematopoietic stem cell transplantation as described herein. Other beneficial outcomes include an increase in the cell count or relative concentration of hematopoietic stem cells in patients requiring hematopoietic stem cell transplants following conditioning therapy and subsequent administration of exogenous hematopoietic stem cell transplants. Beneficial outcomes of the therapy described herein may also include an increase in the cell count or relative concentration of one or more hematopoietic lineage cells (e.g., megakaryocytes, coagulatory 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, or B lymphocytes) following conditioning therapy and subsequent hematopoietic stem cell transplantation. Other beneficial outcomes may include a reduction in the amount of pathogenic cell populations (e.g., cancer cell populations (e.g., CD45+ leukemia cells) or autoimmune cell populations (e.g., CD45+ autoimmune lymphocytes, such as CD45+ T cells expressing T cell receptors that cross-react with self-antigens)). In the context of the method of this invention relating to disease prevention, the term "prevention" should be understood as not requiring complete cessation of the disease state. Conversely, as used herein, the term "prevention" refers to the ability of those skilled in this art to identify susceptible populations, enabling the administration of the compounds of this invention before the onset of disease. This term implies that disease states cannot be completely avoided.

[0300] Patients who "need" hematopoietic stem cell grafts, as used in this article, include those exhibiting the absence or deficiency of one or more blood cell types, as well as those with stem cell disorders, autoimmune diseases, cancer, or other conditions described herein. Hematopoietic stem cells typically exhibit 1) pluripotency and can therefore differentiate into a variety of different blood lineages, including (but not limited to) granulocytes (e.g., premyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), coagulating cells (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells), 2) self-renewal and can therefore produce progeny cells with the potential to be equivalent to the parent cells, and 3) the ability to be reintroduced into the graft recipient, subsequently home to the hematopoietic stem cell niche, and restore productive and sustainable hematopoiesis. Therefore, hematopoietic stem cells can be administered to patients who lack or are deficient in one or more cell types of the hematopoietic lineage to restore the in vivo population of missing or deficient cells. For example, the patient may have cancer, and the deficiency may be caused by administration of chemotherapy agents or other agents that selectively or non-specifically deplete the cancer cell population. Alternatively, the patient may have heme disorders (e.g., non-malignant heme disorders), such as sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome. Individuals may be those with adenosine deaminase severe immunodeficiency disease (ADA SCID), HIV / AIDS, metachromatic leukotrophic disorder, Diamond-Blackfan anemia, and Schwachman-Diamond syndrome. Individuals may have or be affected by inherited blood disorders (e.g., sickle cell anemia) or autoimmune diseases. Alternatively or additionally, an individual may have a malignant disease (such as neuroblastoma or blood cancer) or be affected by such a disease. For example, an individual may have leukemia, lymphoma, or myeloma. In some embodiments, an individual may have acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphoblastic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. In some embodiments, an individual may have myelodyplasia syndrome. In some embodiments, an individual may have 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 may require chimeric antigen receptor T-cell (CAR-T) therapy. In some embodiments, an individual may have a metabolic storage disorder or be otherwise affected by such a 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. The diseases or conditions described in Book 1:319-338 (2000), and the full text of that document regarding conditions treatable by hematopoietic stem cell transplantation, are incorporated herein by reference. Alternatively, 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.

[0301] As used herein, the term "stem cell disease" broadly refers to any disease, condition, or ailment that can be treated or cured by conditioning an individual's target tissues, and / or by eliminating endogenous stem cell populations in the target tissues (e.g., eliminating endogenous hematopoietic stem cell or progenitor cell populations from an individual's bone marrow), and / or by implanting or transplanting stem cells into an individual's target tissues. For example, type 1 diabetes has been shown to be cured by hematopoietic stem cell grafts and can benefit from conditioning according to the compositions and methods described herein. Other conditions that can be treated with the compositions and methods described herein include (but are not limited to) sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, Weill II syndrome, ADASCID, HIV / AIDS, metachromatic leukotrophic disorder, Dionysius-Budd-Chiari anemia, and Shudlow-Die syndrome. Other diseases that can be treated with the patient conditioning and / or hematopoietic stem cell transplantation methods described herein include hereditary blood disorders (e.g., sickle cell anemia) and autoimmune diseases such as scleroderma, multiple sclerosis, ulcerative colitis, and Crohn's disease. Other diseases that can be treated with the conditioning and / or transplantation methods described herein include malignancies such as neuroblastoma or blood cancers such as leukemia, lymphoma, and myeloma. For example, cancers may be acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, or non-Hodgkin's lymphoma. Other diseases that can be treated with the conditioning and / or transplantation methods described herein include myelodyplasia syndrome. In some embodiments, an individual has a metabolic storage disorder or is otherwise affected by it. For example, an individual may suffer from or be otherwise affected by a metabolic disorder selected from the following groups: glycogen storage disease, mucopolysaccharidosis, Gaucher's disease, Huller's disease, sphingolipid storage disease, metachromatic leukodystrophy, or any other disease or condition that may benefit from the treatments and therapies disclosed herein, including (but not limited to) severe combined immunodeficiency disease, Werner-Oxley syndrome, hyperimmune globulin M (IgM) syndrome, Cheshire's 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. The diseases or conditions described in Book, 1:319-338 (2000), and the full text of the disclosure of the conditions that can be treated by hematopoietic stem cell transplantation, are incorporated herein by reference.

[0302] As used herein, the term "vector" includes nucleic acid vectors, such as plastids, DNA vectors, RNA vectors, viruses, or other suitable replicons. Expression vectors described herein may contain polynucleotide sequences and other sequence components, such as those for expressing proteins and / or integrating such polynucleotide sequences into the gene body of mammalian cells. Some vectors that can be used to express antibodies and antibody fragments of the present invention include plastids containing regulatory sequences (e.g., promoter and enhancer regions) that guide gene transcription. Other vectors that can be used to express antibodies and antibody fragments contain polynucleotide sequences that enhance the translation rate of such genes or improve the stability of mRNA derived from gene transcription or nuclear export. These sequence components may include, for example, 5' and 3' untranslated regions and polyadenylation signaling sites to guide efficient transcription of the gene carried on the expression vector. Expression vectors described herein may also contain polynucleotides encoding markers for selecting cells containing the vector. Examples of suitable markers include genes encoding antibiotic resistance (such as ampicillin, chloramphenicol, kanamycin, and nourseothricin).

[0303] As used herein, the terms "conjugate," "antibody-drug conjugate," or "ADC" refer to an antibody linked to a cytotoxin or toxin and are used interchangeably throughout. In one embodiment, an ADC is formed by chemically bonding a reactive functional group of one molecule (e.g., an antibody or its antigen-binding fragment) to a suitable 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 in some embodiments include small organic molecules (e.g., MW1500 Da or smaller), biomolecules (e.g., proteins), drug-filled nanoparticles, or radionuclides. Conjugates may include linkers between two molecules that are bound together, such as an antibody and a cytotoxin. Examples of linkers that can be used to form conjugates include peptide-containing linkers, such as linkers containing natural or non-natural 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, the linker can be cleaved, for example, by enzymatic hydrolysis, photolysis, hydrolysis under acidic conditions, hydrolysis under alkaline conditions, oxidation, disulfide reduction, nucleophilic cleavage, or organometallic cleavage (see, for example, Leriche et al., Bioorg. Med. Chem., 20:571-582, 2012).

[0304] 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).

[0305] 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.

[0306] 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).

[0307] As used herein, the term "phalloidin" refers to a member of the amatoxins family of peptides produced by *Amanita phalloides* or its derivatives (e.g., variants or derivatives capable of inhibiting RNA polymerase II activity). Amatoxins that can be used in conjunction with the compositions and methods described herein include compounds such as α-glucosinolates, β-glucosinolates, γ-glucosinolates, ε-glucosinolates, amatoxins, amatoxin amides, amatoxin cyclic peptides, monohydroxyamatoxin carboxylic acid, or protoamatoxin cyclic peptides. As described herein, amatoxins can, for example, bind to an antibody or its antigen-binding fragment via a linker portion (L) (thus forming an ADC). Exemplary methods for binding amatoxins and linkers that can be used in such methods are described below. Amatoxins containing exemplary linkers that can be used to bind to antibodies or antigen-binding fragments according to the compositions and methods are also described herein.

[0308] As used herein, the term "acyl" refers to -C(=O)R, where R is hydrogen ("aldehyde"), C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, C3-C7 carbocyclic, C6-C20 aryl, 5-10 member heteroaryl, or 5-10 member heterocyclic, as defined herein. Non-limiting examples include formyl, acetyl, propionyl, benzoyl, and acryloyl.

[0309] As used herein, the term "C1-C12 alkyl" refers to a straight-chain or branched saturated hydrocarbon having 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, -dibutyl, -isobutyl, -tert-butyl, -isopentyl, and 2-methylbutyl. C1-C12 alkyl groups may be unsubstituted or substituted.

[0310] As used herein, the term "alkenyl" refers to a C2-C12 hydrocarbon containing a normal, secondary, or tertiary carbon atom and at least one unsaturated site (i.e., a carbon-carbon sp2 double bond). Examples include (but are not limited to): ethyl or vinyl, -allyl, -1-butenyl, -2-butenyl, -isobutenyl, -1-pentenyl, -2-pentenyl, -3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl, propenyl, isopropenyl, butenyl, terbutenyl, hexenyl, and the like. Alkenyl groups can be unsubstituted or substituted.

[0311] As used herein, "alkynyl" refers to a C2-C12 hydrocarbon containing a normal, secondary, or tertiary carbon atom and at least one unsaturated site (i.e., a carbon-carbon sp triple bond). Examples include (but are not limited to) alkynyl and alkynylpropynyl, butynyl, pentynyl, hexynyl, and the like. The alkynyl group can be unsubstituted or substituted.

[0312] As used herein, "aryl" refers to a C6-C20 carbocyclic aromatic group. Examples of aryl groups include (but are not limited to) phenyl, naphthyl, and anthracene. Aryl groups can be unsubstituted or substituted.

[0313] As used herein, "arylalkyl" refers to an acyclic alkyl group in which a hydrogen atom bonded to a carbon atom, usually terminal or sp3 carbon atom, is replaced by an aryl group. Typical arylalkyl groups include (but are not limited to) benzyl, 2-phenylethyl-1-yl, 2-phenylvinyl-1-yl, naphthylmethyl, 2-naphthylethyl-1-yl, 2-naphthylvinyl-1-yl, naphthobenzyl, 2-naphthophenylethyl-1-yl, and the like. Arylalkyl groups contain 6 to 20 carbon atoms; for example, the alkyl portion (including aliphatic, alkenyl, or ynyl) of an arylalkyl group has 1 to 6 carbon atoms and the aryl portion has 5 to 14 carbon atoms. The alkylaryl group can be unsubstituted or substituted.

[0314] As used herein, "cycloalkyl" refers to a saturated carbocyclic group that can be monocyclic or bicyclic. Cycloalkyl groups include rings having 3 to 7 carbon atoms (e.g., monocyclic) or 7 to 12 carbon atoms (e.g., bicyclic). Examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Cycloalkyl groups can be unsubstituted or substituted.

[0315] As used herein, "cycloalkenyl" refers to an unsaturated carbocyclic group that can be monocyclic or bicyclic. Cycloalkenyl groups include rings having 3 to 6 carbon atoms (e.g., monocyclic) or 7 to 12 carbon atoms (e.g., bicyclic). Examples of monocyclic cycloalkenyl groups include 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, and 1-cyclohex-3-enyl. Cycloalkenyl groups can be unsubstituted or substituted.

[0316] As used herein, "heteroarylalkyl" refers to an acyclic alkyl group in which a hydrogen atom bonded to a carbon atom, usually terminal or sp3 carbon atom, is replaced by a heteroaryl group. Typical heteroarylalkyl groups include (but are not limited to) 2-benzimidazolylmethyl, 2-furanylethyl, and the like. Heteroarylalkyl groups contain 6 to 20 carbon atoms, for example, the alkyl portion (including aliphatic, alkenyl, or alkynyl) of a heteroarylalkyl group has 1 to 6 carbon atoms and the heteroaryl portion has 5 to 14 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. The heteroaryl moiety of a heteroarylalkyl 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), for example: bicyclic [4,5], [5,5], [5,6] or [6,6] systems.

[0317] As used herein, "heteroaryl" and "heterocyclic alkyl" refer to aromatic or non-aromatic ring systems in which one or more ring atoms are heteroatoms (e.g., nitrogen, oxygen, and sulfur). Heteroaryl or heterocyclic alkyl systems comprise 2 to 20 carbon atoms and 1 to 3 heteroatoms selected from N, O, P, and S. Heteroaryl or heterocyclic alkyl systems 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), for example, bicyclic [4,5], [5,5], [5,6], or [6,6] systems. Heteroaryl and heterocyclic alkyl systems can be unsubstituted or substituted.

[0318] Heteroaryl and heterocyclic alkyl groups are described in Paquette, Leo A.; "Principles of Modern Heterocyclic Chemistry" (WA Benjamin, New York, 1968), specifically Chapters 1, 3, 4, 6, 7 and 9; "The Chemistry of Heterocyclic Compounds, A series of Monographs" (John Wiley & Sons, New York, 1950 to present), specifically Volumes 13, 14, 16, 19 and 28; and J. Am. Chem. Soc. (1960) 82:5566.

[0319] Examples of heteroaryl groups include, for example (but not limited to), pyridyl, thiazolyl, tetrahydrothiophene, pyrimidinyl, furanyl, thiophene, pyrrole, pyrazolyl, imidazolyl, tetrazolyl, benzofuranyl, thianaphthyl, indole, pseudoindole, quinolinyl, isoquinolinyl, benzimidazolyl, isoxazolyl, pyrazinyl, darazinyl, indazinyl, isoindoleyl, 3H-indoleyl, 1H-indazolyl, purinyl, 4H-quinazinyl, phthalazinyl, naphthidyl, quinolinyl, quinazolinyl, and quinazolinyl. Linolyl, pteridinyl, 4aH-carbazolyl, carbazolyl, phenidinyl, acridinel, pyrimidinyl, phenazinyl, phenothiazinyl, furazinyl, phenotoxazinyl, isocyaninyl, chrominyl, imidazodinyl, imidazolinyl, pyrazolinyl, pyrazolinyl, benzotriazolyl, benzoisoxazolyl, and indigo acyl.

[0320] Examples of heterocyclic alkyl groups include, for example (but not limited to), dihydropyridyl, tetrahydropyridyl (hexahydropyridyl), tetrahydrothiophenyl, hexahydropyridyl, 4-hexahydropyridinone, pyrrolidyl, 2-pyrrolidone, tetrahydrofuranyl, tetrahydropyranyl, bis-tetrahydrofuranyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl, hexahydropyrazinyl, quininecycloyl, and morpholinyl.

[0321] For example, but not limited to, carbon-bonded heteroaryl and heterocycloalkyl groups bonded to positions 2, 3, 4, 5, or 6 of pyridine, positions 3, 4, 5, or 6 of darazine, positions 2, 4, 5, or 6 of pyrimidine, positions 2, 3, 5, or 6 of pyrazine, positions 2, 3, 5, or 6 of furan, tetrahydrofuran, thiofuran, thiophene, pyrrole, or tetrahydropyrrole, positions 2, 3, 4, or 5 of oxazole, imidazole, or thiazole, positions 3, 4, or 5 of isoxazole, pyrazole, or isothiazole, positions 2 or 3 of aziridine, positions 2, 3, or 4 of aziridine, positions 2, 3, or 4 of aziridine, positions 2, 3, 4, 5, 6, 7, or 8 of quinoline, or positions 1, 3, 4, 5, 6, 7, or 8 of isoquinoline. More typically, the heterocycles of the carbon bonds 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-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

[0322] For example, but not limited to, nitrogen-bonded heteroaryl and heterocyclic alkyl groups are bonded to position 1 of aziridinium, aziridine, pyrrole, pyrrolidine, 2-pyrrololine, 3-pyrroleline, imidazole, imidazoline, 2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline, hexahydropyridine, hexahydropyrazine, indole, indoline, and 1H-indazole; position 2 of isoindole or isoindoline; position 4 of morpholine; and position 9 of carbazole or β-carbline. More typically, nitrogen-bonded heterocycles include 1-aziridinyl, 1-aziridinebutyl (1-azetedyl), 1-pyrroleyl, 1-imidazolyl, 1-pyrazolyl, and 1-hexahydropyridinyl.

[0323] As used herein and as applied to the above-mentioned alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, heteroaryl, heterocyclic and any of the like, "substituted" means that one or more hydrogen atoms are each independently substituted by 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 from F, Cl, Br and I each time it appears; and each R is independently selected from C1-C12 alkyl, C6-C20 aryl, C3-C14 heterocyclic alkyl or heteroaryl, protecting group and prodrug moiety each time it appears. When a group is described as "substituted as appropriate", the group may be independently substituted by one or more of the above substituents each time it appears.

[0324] It should be understood that, depending on the context, certain group naming conventions may include mono- or di-group diagrams. For example, when a substituent requires two connection points to the remainder of the molecule, it should be understood that the substituent is a di-group. For instance, substituents identified as alkyl groups requiring two connection points 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 "alkyl," "alkenyl," "aryl," "heterocyclic alkyl," and the like.

[0325] Unless otherwise specified, when a substituent is described as a digroup (i.e., having two connection points with the remainder of the molecule), it should be understood that the substituent can be connected in any directional configuration.

[0326] "Isomerism" refers to compounds with the same molecular formula but different sequences of atomic bonds or different spatial arrangements of atoms. Isomers with different spatial arrangements of atoms are called "stereoisomers". Stereoisomers that are not mirror images of each other are called "non-mirror image isomers", and stereoisomers that are not superimposed mirror images of each other are called "mirror image isomers" or sometimes "optical isomers".

[0327] A carbon atom bonded to four different substituents is called a "chiral center." A "chiral isomer" refers to a compound having at least one chiral center. Compounds having more than one chiral center can exist individually as non-mirror image isomers or as mixtures of non-mirror image isomers (called "non-mirror image isomer mixtures"). When a chiral center is present, the stereoisomer is characterized by the absolute configuration (R or S) of that chiral center. The absolute configuration refers to the spatial arrangement of the substituents attached to the chiral center. According to the sequence rules of Cahn, Ingold, and Prelog (Cahn et al., Angew. Chem. Inter. ed., 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), the substituents considered to be attached to the chiral center are classified. A mixture containing equal amounts of individual mirror-image isomers with opposite chirality is called a "racemic mixture".

[0328] The compounds disclosed in this specification and the claims may contain one or more asymmetric centers and may have different non-mirror image isomers and / or mirror image isomers for each compound. Unless otherwise stated, the description of any compound in this specification and the claims is intended to include all mirror image isomers, non-mirror image isomers, and mixtures thereof. Furthermore, unless otherwise stated, the description of any compound in this specification and the claims is intended to include individual mirror image isomers and any mixtures, racemic or other forms of mirror image isomers. When the structure of a compound is described as a particular mirror image isomer, it should be understood that the disclosure of this application is not limited to that particular mirror image isomer. Therefore, this document covers mirror image isomers, optical isomers, and non-mirror image isomers of each structural formula disclosed herein. In this specification, for convenience, in some cases the structural formula of a compound represents a particular isomer; however, this disclosure includes all isomers, such as geometric isomers, optical isomers based on asymmetric carbon, stereoisomers, tautomers, and the like. It should be understood that not all isomers may have the same level of activity. Compounds may exist in different tautomer forms. Unless otherwise stated, the compounds in this disclosure are intended to include all tautomer forms. When the structure of a compound is described as a specific tautomer, it should be understood that the disclosure of this application is not limited to that specific tautomer.

[0329] Where applicable, any compound of any formula described herein includes the compound itself, its salts, and its solvates. For example, salts can be formed between an anion on the compound of this disclosure and a positively charged group (e.g., an amino group). Suitable anions include chloride, bromide, iodide, sulfate, hydrogen sulfate, aminosulfonate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate, tartrate, toluenesulfonate, salicylate, lactate, naphthalenesulfonate, and acetate (e.g., trifluoroacetate). The term "pharmaceutically acceptable anion" refers to an anion suitable for forming a pharmaceutically acceptable salt. Similarly, salts can also be formed between a cation on the compound of this disclosure and a negatively charged group (e.g., a carboxyl group). Suitable cations include sodium, potassium, magnesium, calcium, and ammonium cations, such as tetramethylammonium ions. Examples of suitable substituted ammonium ions are derived from the following ammonium ions: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, hexahydropyrazine, benzylamine, phenylbenzylamine, choline, meglumine, and glycerol, as well as amino acids such as lysine and arginine. The compounds disclosed herein also include salts containing a quaternary nitrogen atom. Examples of suitable inorganic anions include (but are not limited to) anions derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid. Examples of suitable organic anions include (but are not limited to) anions derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, ethylenediaminetetraacetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheponic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthoic acid, hydroxyethylsulfonic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, mesylic acid, mucilage, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, p-aminobenzenesulfonic acid, tartaric acid, toluenesulfonic acid, and valeric acid. Examples of suitable polymerizable organic anions include (but are not limited to) anions derived from the following polymeric acids: tannic acid and carboxymethyl cellulose.

[0330] Furthermore, the compounds disclosed herein (e.g., salts of compounds) may exist in hydrated or non-hydrated (anhydrous) forms or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc. "Solvate" means a solvation form containing stoichiometric or non-stoichiometric amounts of solvent. Some compounds have a tendency to trap solvent molecules in a fixed molar ratio in a crystalline solid state, thus forming solvates. If the solvent is water, the formed solvate is a hydrate; and if the solvent is an alcohol, the formed solvate is an alcohol. Hydrates are formed by combining one or more molecules of water with a molecule of a substance in which the water retains its molecular state as H2O. Hydrates refer to, for example, monohydrates, dihydrates, trihydrates, etc.

[0331] Furthermore, compounds or their salts represented by the formulas disclosed herein may exhibit crystal polymorphism. It should be noted that any crystalline form, mixture of crystalline forms, or their anhydrides or hydrates are included within the scope of this disclosure.

[0332] Anti-CD45 antibody

[0333] This article covers antibodies or antigen-binding fragments thereof capable of binding to CD45, which can be used as a single therapeutic agent or as an adjuvant (ADC) for, for example, (i) treating cancers and autoimmune diseases characterized by CD45+ cells, and (ii) promoting the engraftment of transplanted hematopoietic stem cells into patients requiring transplant therapy. Such therapeutic activity can be induced, for example, by binding to isolated anti-CD45 antibodies, their antigen-binding fragments, or ADCs, and subsequently inducing cell death, whereby the isolated anti-CD45 antibodies, their antigen-binding fragments, or ADCs bind to CD45 expressed on the surface of cells (e.g., cancer cells, autoimmune cells, or hematopoietic stem cells). Depletion of endogenous hematopoietic stem cells provides a niche for transplanted hematopoietic stem cells to home and subsequently establish productive hematopoiesis. In this way, transplanted hematopoietic stem cells can be successfully engrafted into patients, such as human patients with stem cell diseases described herein. Additionally, the combination of depleted white blood cells in patients in need with HSC grafts can reset the patient's immune system, thereby curing patients with, for example, autoimmune diseases.

[0334] CD45, also known as leukocyte-shared antigen and receptor-type tyrosine phosphatase C, is a hematopoietic cell-specific transmembrane protein tyrosine phosphatase essential for T and B cell antigen receptor-mediated signal transduction. CD45 comprises a large extracellular domain and a phosphatase-containing cytoplasmic domain. CD45 can function as both a positive and negative regulator, depending on the nature of the stimulus and the cell type involved. Although numerous hypermutations may exist in the CD45 gene, only six isotypes are typically identified in humans. These isotypes 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, while 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 (weak expression), 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 SEQ ID NO:RBC is provided herein as SEQ ID NO:111.

[0335] As described below, in some embodiments, the novel anti-human CD45 (hCD45) anti-system is identified by screening a yeast display library displaying all human antibodies. Seven human antibodies (named Antibody 1 (Ab1), Antibody 2 (Ab2), Antibody 3 (Ab3), Antibody 4 (Ab4), Antibody 5 (Ab5), Antibody 6 (Ab6), and Antibody 7 (Ab7)) were identified during the screening. These antibodies bind to human CD45 (all isotypes) and cross-react with non-human primate CD45 (e.g., cynomolgus monkey CD45 and / or rhesus monkey CD45).

[0336] In other embodiments, humanized and affinity-matured antibody systems were prepared from three rat anti-CD45 antibodies. In this manner, three additional antibodies (named antibody A (AbA), antibody B (AbB), and antibody C (AbC)) were identified that bind to human CD45 (all isotypes) and cross-react with CD45 in non-human primates (e.g., cynomolgus monkey CD45 and / or rhesus monkey CD45). The identified antibodies possess diagnostic and therapeutic characteristics, as described herein.

[0337] Therefore, this article provides antibodies or their antigen-binding portions that specifically bind to human CD45 and cross-react with CD45 in non-human primates.

[0338] In one embodiment, the present invention provides an antibody or its antigen-binding portion thereof that binds to human CD45 (SEQ ID NO: 112), cynomolgus monkey CD45 (SEQ ID NO: 145), and / or rhesus monkey CD45 (SEQ ID NO: 146). In some embodiments, the antibody or its antigen-binding portion may bind to human CD45 and K D The value is approximately 100 nM or less, such as 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). In some embodiments, the antibody or its antigen-binding portion may bind to cynomolgus monkey CD45 and K D The value is approximately 100 nM or less, such as 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). In some embodiments, the antibody or its antigen-binding portion may bind to rhesus monkey CD45 and K DThe value is about 100 nM or less, such as 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 biological layer interference (BLI). In some embodiments, the anti-system fully human antibody or its antigen-binding portion thereof. In other embodiments, the anti-system humanized antibody or its antigen-binding portion thereof. In some embodiments, the anti-system chimeric antibody or its antigen-binding portion thereof. In some embodiments, the anti-system deimmunized antibody or its antigen-binding portion thereof.

[0339] The extracellular region of human CD45 includes a mucin-like domain and four fibronectin-like domains (d1, d2, d3, and d4). Without being bound by any theory, antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, and Ab7 are considered to interact with residues within the d3 and d4 fibronectin-like domains of human CD45. Specifically, these antibodies can interact with the human CD45 fragments shown in SEQ ID NO:115 and SEQ ID NO:117. The crosslinking studies described herein demonstrate that the antibodies can specifically interact with one or more conserved CD45 amino acid residues among human CD45, cynomolgus monkey CD45, and rhesus monkey CD45. These residues include 405T, 407K, 419Y, 425K, and 505R (refer to the hCD45 fragment numbering shown in SEQ ID NO:113). Additionally, these antibodies can interact with residues 481R and / or 509H of human CD45 (refer to the hCD45 fragment number shown in SEQ ID NO: 113). Therefore, in some embodiments, this document provides an antibody or antigen-binding portion thereof that binds to human CD45 at antigenic determinants located in the d3 and / or d4 fibronectin-like domains. In some embodiments, this document provides an antibody or antigen-binding portion thereof that binds to CD45 at antigenic determinants located within CD45 fragment 2 (SEQ ID NO: 115) and / or CD45 fragment 4 (SEQ ID NO: 117) of human CD45. In some embodiments, this document provides an antibody or antigen-binding portion thereof that binds to CD45 at antigenic determinants located within CD45 fragment 1 (SEQ ID NO: 114) and / or CD45 fragment 3 (SEQ ID NO: 116) of human CD45. In some embodiments, this document provides an antibody or antigen-binding portion thereof that binds to CD45 at an antigenic determinant comprising at least one, at least two, at least three, at least four, or at least five conserved amino acid residues in human CD45, cynomolgus monkey CD45, and / or rhesus monkey CD45. For example, in some embodiments, the antibody or antigen-binding portion thereof may 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 (refer to the hCD45 fragment number shown in SEQ ID NO: 113).In some embodiments, the antibody or its antigen-binding portion may 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 (refer to the hCD45 fragment number shown in SEQ ID NO: 113). An antibody or its antigen-binding portion is also provided herein that competitively binds to human CD45 with Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, and / or Ab7 (SEQ ID NO: 112). In some embodiments, the antibody or its antigen-binding portion may also competitively bind to cynomolgus monkey CD45 (SEQ ID NO: 145) and / or rhesus monkey CD45 (SEQ ID NO: 146) with Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, and / or Ab7. In some embodiments, the antibody is a whole-human antibody or its antigen-binding portion. In other embodiments, the anti-system humanized antibody or its antigen-binding portion thereof. In some embodiments, the anti-system chimeric antibody or its antigen-binding portion thereof. In some embodiments, the anti-system deimmunizing antibody or its antigen-binding portion thereof.

[0340] To avoid being limited by any particular theory, it is believed that the antibody AbA described herein also binds to the d4 fibronectin-like domain of CD45, but does not compete with any of Ab1-Ab7 for binding to CD45. The antigenic determinant localization experiments described herein show that AbA binds to the d4 fibronectin-like domain on the side of the molecule opposite to Ab1-Ab7. Specifically, this antibody is believed to interact with the human CD45 fragment shown in SEQ ID NO:118. The crosslinking studies described herein show that AbA can specifically interact with one or more conserved CD45 amino acid residues among human CD45, cynomolgus monkey CD45, and rhesus monkey CD45. These residues include 493Y and 502T (refer to the hCD45 fragment number shown in SEQ ID NO:113). Additionally, this antibody can interact with residue 486R in human CD45 (refer to the hCD45 fragment number shown in SEQ ID NO:113). Therefore, in some embodiments, this document provides an antibody or antigen-binding portion thereof that binds to human CD45 at an antigenic determinant located within the d4 fibronectin-like domain. In some embodiments, this document provides an antibody or antigen-binding portion thereof that binds to CD45 at an antigenic determinant located within CD45 fragment 5 (SEQ ID NO: 118) of human CD45. In some embodiments, this document provides an antibody or antigen-binding portion thereof that binds to CD45 at an antigenic determinant comprising at least one or at least two conserved amino acid residues in human CD45, cynomolgus monkey CD45, and / or rhesus monkey CD45. For example, in some embodiments, the antibody or antigen-binding portion thereof may bind to one or both of the following amino acid residues in human CD45: 493Y and 502T (refer to the hCD45 fragment number shown in SEQ ID NO: 113). In some embodiments, the antibody or its antigen-binding portion may bind to one or more, two or more, or three of the following amino acid residues in human CD45: 486R, 493Y, and 502T (refer to the hCD45 fragment number shown in SEQ ID NO: 113). An antibody or its antigen-binding portion is also provided herein that competitively binds to human CD45 (SEQ ID NO: 112) in conjunction with AbA. In some embodiments, the antibody or its antigen-binding portion may also competitively bind to cynomolgus monkey CD45 (SEQ ID NO: 145) and / or rhesus monkey CD45 (SEQ ID NO: 146) in conjunction with AbA. In some embodiments, the anti-system is a fully human antibody or its antigen-binding portion. In other embodiments, the anti-system is a humanized antibody or its antigen-binding portion. In some embodiments, the anti-system is a chimeric antibody or its antigen-binding portion. In some embodiments, the anti-system is a deimmunized antibody or its antigen-binding portion.

[0341] In other embodiments, this document provides an antibody or its antigen-binding portion thereof that binds to the same antigenic determinant as AbB on human CD45. In some embodiments, the antibody or its antigen-binding portion thereof cross-reacts with cynomolgus monkey CD45 and / or rhesus monkey CD45. This document also provides an antibody or its antigen-binding portion thereof that competitively binds to human CD45 (SEQ ID NO: 112) against AbB. In some embodiments, the antibody or its antigen-binding portion thereof may also competitively bind to cynomolgus monkey CD45 (SEQ ID NO: 145) and / or rhesus monkey CD45 (SEQ ID NO: 146) against AbB. In some embodiments, an anti-system fully human antibody or its antigen-binding portion thereof. In other embodiments, an anti-system humanized antibody or its antigen-binding portion thereof. In some embodiments, an anti-system chimeric antibody or its antigen-binding portion thereof. In some embodiments, an anti-system deimmunized antibody or its antigen-binding portion thereof.

[0342] In other embodiments, this document provides an antibody or its antigen-binding portion thereof that binds to the same antigenic determinant as AbC on human CD45. In some embodiments, the antibody or its antigen-binding portion thereof cross-reacts with cynomolgus monkey CD45 and / or rhesus monkey CD45. This document also provides an antibody or its antigen-binding portion thereof that competitively binds to human CD45 (SEQ ID NO: 112) against AbC. In some embodiments, the antibody or its antigen-binding portion thereof may also competitively bind to cynomolgus monkey CD45 (SEQ ID NO: 145) and / or rhesus monkey CD45 (SEQ ID NO: 146) against AbC. In some embodiments, an anti-system fully human antibody or its antigen-binding portion thereof. In other embodiments, an anti-system humanized antibody or its antigen-binding portion thereof. In some embodiments, an anti-system chimeric antibody or its antigen-binding portion thereof. In some embodiments, an anti-system deimmunized antibody or its antigen-binding portion thereof.

[0343] The amino acid sequences of the binding regions of the anti-CD45 antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, and AbC are described in Table 27. In several samples, the present invention provides antibodies comprising the heavy chain and / or light chain CDR sequences of the antibodies described in Table 27. In some samples, the present invention provides antibodies comprising the heavy chain variable region and / or light chain variable region of the antibodies described in Table 27. In some samples, the present invention provides antibodies comprising the heavy chain and / or light chain of the antibodies described in Table 27. Further characteristics of the antibodies and their antigen-binding regions provided herein are described below.

[0344] Ab1

[0345] Antibody 1 (Ab1) cross-reacts with human CD45, cynomolgus monkey CD45 and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0346] The amino acid sequences of the various binding regions of anti-CD45 Ab1 are described in Table 27. This invention includes, for example, Ab1-based anti-CD45 antibodies comprising CDRs as shown in Table 27.

[0347] 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 Ab1. The amino acid sequence of the heavy chain variable region (VH) of Ab1 is shown in SEQ ID NO:1 (see Table 27). The amino acid sequence of the VH CDR domain of Ab1 is shown in SEQ ID NO:2 (VHCDR1); SEQ ID NO:3 (VH CDR2) and SEQ ID NO:4 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of Ab1 is described in SEQ ID NO:5 (see Table 27). The amino acid sequence of the VL CDR domain of Ab1 is shown in SEQ ID NO:6 (VL CDR1); SEQ ID NO:7 (VL CDR2) and SEQ ID NO:8 (VL CDR3).

[0348] Therefore, in some embodiments, this document provides an anti-CD45 antibody or its antigen-binding portion comprising a heavy chain variable region containing a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:2, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:3, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:4; and / or a light chain variable region containing a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:6, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:7, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:8. In some embodiments, the anti-CD45 antibody comprises the CDRs described herein (SEQ ID Nos:2 to 4 and 6 to 8), wherein the CDRs contain conserved amino acid substitutions (or 2, 3, 4, or 5 amino acid substitutions) while retaining the CD45 specificity of the antibody (i.e., similar to Ab1 specificity).

[0349] 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:1, and / or a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:5. In some embodiments, the antibody may comprise a modified heavy chain (HC) variable region comprising an HC variable domain containing SEQ ID NO:1 or a variant of SEQ ID NO:1, which (i) differs from SEQ ID NO:1 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:1 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:1 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:5. NO:1 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain variable region retains the antibody’s CD45 specificity (i.e., similar to Ab1 specificity) or has enhanced biological activity relative to another Ab1 heavy chain variable region. In some embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain containing SEQ ID NO:5 or a variant of SEQ ID NO:5, wherein the variant (i) differs from SEQ ID NO:5 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:5 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:5 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) comprises a variant of SEQ ID NO:5. NO:5 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain variable region retains the antibody’s CD45 specificity (i.e., similar to Ab1 specificity) or has enhanced biological activity relative to another Ab1 light chain variable region.

[0350] Antibodies containing the CDR and / or variable region sequence 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 to human CD45, including (but not limited to) Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including substitute light chain constructs), single-domain antibodies, camelified antibodies, and the like. They may also have or be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM. In some embodiments, the anti-45 anti-system is IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0351] In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab1 may further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgG1 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 may 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 Kabat EU index). In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab1 may further comprise an IgG1 heavy chain constant region containing one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the Kabat EU index). In some embodiments, the antibody containing the CDR and / or variable region sequence of Ab1 may further contain the heavy chain constant region shown 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 antibody contains the light chain constant region shown in SEQ ID NO:101.

[0352] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as 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, the variant being (i) different from SEQ ID NO:9 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:9 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:9 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a heavy chain containing an amino acid sequence as shown in SEQ ID NO:9. IDNO:9 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 heavy chain region retains the antibody’s CD45 specificity (i.e., similar to Ab1 specificity) or has enhanced biological activity relative to another Ab1 heavy chain region.

[0353] 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 one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:10 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:10 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a light chain containing an amino acid sequence as shown in SEQ ID NO:10. NO:10 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain region retains the antibody’s CD45 specificity (i.e., similar to Ab1 specificity) or has enhanced biological activity relative to another Ab1 light chain region.

[0354] In some embodiments, antibodies containing the CDR region and / or variable region of Ab1 may be incorporated into antibody-drug conjugates, as described herein. Additionally, antibodies containing the CDR region and / or variable region of Ab1 may be used in the methods described herein, for example, for depleting CD45+ cells in an individual.

[0355] Ab2

[0356] Antibody 2 (Ab2) cross-reacts with human CD45, cynomolgus monkey CD45 and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0357] The amino acid sequences of the various binding regions of anti-CD45 Ab2 are described in Table 27. This invention includes, for example, Ab2-based anti-CD45 antibodies comprising CDRs as shown in Table 27.

[0358] 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 Ab2. The amino acid sequence of the heavy chain variable region (VH) of Ab2 is shown in SEQ ID NO:11 (see Table 27). The amino acid sequence of the VH CDR domain of Ab2 is shown in SEQ ID NO:12 (VHCDR1); SEQ ID NO:13 (VH CDR2) and SEQ ID NO:14 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of Ab2 is illustrated in SEQ ID NO:15 (see Table 27). The amino acid sequence of the VL CDR domain of Ab2 is shown in SEQ ID NO:16 (VL CDR1); SEQ ID NO:17 (VL CDR2) and SEQ ID NO:18 (VL CDR3).

[0359] Therefore, in some embodiments, this document provides an anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:12, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:13, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:14; and / or a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:16, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:17, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:18. In some embodiments, the anti-CD45 antibody comprises the CDR (SEQ ID NO: 12 to 14 and 16 to 18) described herein, wherein the CDR comprises a conserved amino acid substitution (or two, three, four or five amino acid substitutions) while retaining the antibody’s CD45 specificity (i.e., similar to Ab2 specificity).

[0360] 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:11, and / or a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:15. In some embodiments, the antibody may comprise a modified heavy chain (HC) variable region comprising an HC variable domain containing SEQ ID NO:11 or a variant of SEQ ID NO:11, the variant being (i) different from SEQ ID NO:11 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:11 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:11 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) containing a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:15. NO:11 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain variable region retains the antibody’s CD45 specificity (i.e., similar to Ab2 specificity) or has enhanced biological activity relative to another Ab2 heavy chain variable region. In some embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain containing SEQ ID NO:15 or a variant of SEQ ID NO:15, the variant being (i) different from SEQ ID NO:15 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:15 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:15 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a variable domain containing SEQ ID NO:15. NO:15 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain variable region retains the antibody’s CD45 specificity (i.e., similar to Ab2 specificity) or has enhanced biological activity relative to another Ab2 light chain variable region.

[0361] Antibodies containing the CDR and / or variable region sequence 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 to human CD45, including (but not limited to) Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including substitute light chain constructs), single-domain antibodies, camelified antibodies, and the like. They may also have or be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM. In some embodiments, the anti-45 antibody system is IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0362] In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab2 may further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgG1 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 may 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 Kabat EU index). In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab2 may further comprise an IgG1 heavy chain constant region containing one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the Kabat EU index). In some embodiments, the antibody containing the CDR and / or variable region sequence of Ab2 may further contain the heavy chain constant region shown 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 antibody contains the light chain constant region shown in SEQ ID NO:101.

[0363] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:19. In some embodiments, the antibody comprises a modified heavy chain (HC) region containing an HC domain containing SEQ ID NO:19 or a variant of SEQ ID NO:19, which (i) differs from SEQ ID NO:19 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:19 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:19 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a heavy chain containing an amino acid sequence as shown in SEQ ID NO:19. NO:19 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain region retains the antibody’s CD45 specificity (i.e., Ab2-like specificity) or has enhanced biological activity relative to another Ab2 heavy chain region.

[0364] 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:20. In some embodiments, the antibody comprises a modified light chain (LC) region containing an LC domain containing SEQ ID NO:20 or a variant of SEQ ID NO:20, the variant being (i) different from SEQ ID NO:20 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:20 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:20 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a light chain containing an amino acid sequence as shown in SEQ ID NO:20. NO:20 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain region retains the CD45 specificity of the antibody (i.e., similar to Ab2 specificity) or has enhanced biological activity relative to another Ab2 light chain region.

[0365] 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.

[0366] Ab3

[0367] Antibody 3 (Ab3) cross-reacts with human CD45, cynomolgus monkey CD45 and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0368] 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.

[0369] 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 sequences of the VH CDR domain of Ab3 are shown in SEQ ID NO:22 (VHCDR1); 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 illustrated in SEQ ID NO:25 (see Table 27). The amino acid sequences of the VL CDR domain of Ab3 are shown in SEQ ID NO:26 (VL CDR1); SEQ ID NO:27 (VL CDR2) and SEQ ID NO:28 (VL CDR3).

[0370] Therefore, in some embodiments, this document provides an anti-CD45 antibody or its antigen-binding portion comprising a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:22, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:23, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:24; and / or a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:26, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:27, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:28. In some embodiments, the anti-CD45 antibody comprises the CDR (SEQ ID NO: 22 to 24 and 26 to 28) described herein, wherein the CDR comprises a conserved amino acid substitution (or two, three, four or five amino acid substitutions) while retaining the antibody’s CD45 specificity (i.e., similar to Ab3 specificity).

[0371] 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:21, and / or a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:25. In some embodiments, the antibody may comprise a modified heavy chain (HC) variable region comprising an HC variable domain containing SEQ ID NO:21 or a variant of SEQ ID NO:21, the variant being (i) different from SEQ ID NO:21 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:21 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:21 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) containing a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:25. NO:21 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain variable region retains the antibody’s CD45 specificity (i.e., similar to Ab3 specificity) or has enhanced biological activity relative to another Ab3 heavy chain variable region. In some embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain containing SEQ ID NO:25 or a variant of SEQ ID NO:25, wherein the variant (i) differs from SEQ ID NO:25 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:25 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:25 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) comprises a variant of SEQ ID NO:25. NO:25 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain variable region retains the antibody’s CD45 specificity (i.e., similar to Ab3 specificity) or has enhanced biological activity relative to another Ab3 light chain variable region.

[0372] Antibodies containing the CDR and / or variable region sequence 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 to human CD45, including (but not limited to) Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including substitute light chain constructs), single-domain antibodies, camelified antibodies, and the like. They may also have or be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM. In some embodiments, the anti-45 anti-system is IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0373] In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab3 may further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgG1 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 may 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 Kabat EU index). In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab3 may further comprise an IgG1 heavy chain constant region containing one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the Kabat EU index). In some embodiments, the antibody containing the CDR and / or variable region sequence of Ab3 may further contain the heavy chain constant region shown 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 antibody contains the light chain constant region shown in SEQ ID NO:101.

[0374] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:29. In some embodiments, the antibody comprises a modified heavy chain (HC) region containing an HC domain containing SEQ ID NO:29 or a variant of SEQ ID NO:29, the variant being (i) different from SEQ ID NO:29 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:29 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:29 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a heavy chain containing an amino acid sequence as shown in SEQ ID NO:29. NO:29 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain region retains the antibody’s CD45 specificity (i.e., similar to Ab3 specificity) or has enhanced biological activity relative to another Ab3 heavy chain region.

[0375] 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:30. In some embodiments, the antibody comprises a modified light chain (LC) region containing an LC domain containing SEQ ID NO:30 or a variant of SEQ ID NO:30, the variant being (i) different from SEQ ID NO:30 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:30 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:30 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a light chain containing an amino acid sequence as shown in SEQ ID NO:30. NO:30 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain region retains the antibody’s CD45 specificity (i.e., similar to Ab3 specificity) or has enhanced biological activity relative to another Ab3 light chain region.

[0376] In some embodiments, antibodies containing the CDR region and / or variable region of Ab3 may be incorporated into antibody-drug conjugates, as described herein. Additionally, antibodies containing the CDR region and / or variable region of Ab3 may be used in the methods described herein, for example, for depleting CD45+ cells in an individual.

[0377] Ab4

[0378] Antibody 4 (Ab4) cross-reacts with human CD45, cynomolgus monkey CD45 and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0379] The amino acid sequences of the various binding regions of anti-CD45 Ab4 are described in Table 27. This invention includes, for example, Ab4-based anti-CD45 antibodies comprising the CDRs shown in Table 27.

[0380] 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 Ab4. The amino acid sequence of the heavy chain variable region (VH) of Ab4 is shown in SEQ ID NO:31 (see Table 27). The amino acid sequence of the VH CDR domain of Ab4 is shown in SEQ ID NO:32 (VHCDR1); SEQ ID NO:33 (VH CDR2) and SEQ ID NO:34 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of Ab4 is illustrated in SEQ ID NO:35 (see Table 27). The amino acid sequence of the VL CDR domain of Ab4 is shown in SEQ ID NO:36 (VL CDR1); SEQ ID NO:37 (VL CDR2) and SEQ ID NO:38 (VL CDR3).

[0381] Therefore, in some embodiments, this document provides an anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:32, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:33, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:34; and / or a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:36, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:37, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:38. In some embodiments, the anti-CD45 antibody comprises the CDR (SEQ ID NO: 32 to 34 and 36 to 38) described herein, wherein the CDR comprises a conserved amino acid substitution (or two, three, four or five amino acid substitutions) while retaining the antibody’s CD45 specificity (i.e., similar to Ab4 specificity).

[0382] 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:31, and / or a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:35. In some embodiments, the antibody may comprise a modified heavy chain (HC) variable region comprising an HC variable domain containing SEQ ID NO:31 or a variant of SEQ ID NO:31, the variant being (i) different from SEQ ID NO:31 in that 1, 2, 3, 4, or 5 amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:31 in that up to 5, 4, 3, 2, or 1 amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:31 in that 1-5, 1-3, 1-2, 2-5, or 3-5 amino acids are substituted, added, or deleted; and / or (iv) comprising a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:35. NO:31 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain variable region retains the antibody’s CD45 specificity (i.e., Ab4-like specificity) or has enhanced biological activity relative to another Ab4 heavy chain variable region. In some embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain containing SEQ ID NO:35 or a variant of SEQ ID NO:35, wherein the variant (i) differs from SEQ ID NO:35 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:35 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:35 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) comprises a variant of SEQ ID NO:35. NO:35 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain variable region retains the antibody’s CD45 specificity (i.e., Ab4-like specificity) or has enhanced biological activity relative to another Ab4 light chain variable region.

[0383] Antibodies containing the CDR and / or variable region sequence 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 to human CD45, including (but not limited to) Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including substitute light chain constructs), single-domain antibodies, camelified antibodies, and the like. They may also have or be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM. In some embodiments, the anti-45 antibody system is IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0384] In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab4 may further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgG1 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 may 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 Kabat EU index). In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab4 may further comprise an IgG1 heavy chain constant region containing one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the Kabat EU index). In some embodiments, the antibody containing the CDR and / or variable region sequence of Ab4 may further contain the heavy chain constant region shown 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 antibody contains the light chain constant region shown in SEQ ID NO:101.

[0385] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:39. In some embodiments, the antibody comprises a modified heavy chain (HC) region containing an HC domain containing SEQ ID NO:39 or a variant of SEQ ID NO:39, the variant being (i) different from SEQ ID NO:39 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:39 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:39 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a heavy chain containing an amino acid sequence as shown in SEQ ID NO:39. NO:39 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain region retains the antibody’s CD45 specificity (i.e., Ab4-like specificity) or has enhanced biological activity relative to another Ab4 heavy chain region.

[0386] 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:40. In some embodiments, the antibody comprises a modified light chain (LC) region containing an LC domain containing SEQ ID NO:40 or a variant of SEQ ID NO:40, the variant being (i) different from SEQ ID NO:40 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:40 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:40 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a light chain containing an amino acid sequence as shown in SEQ ID NO:40. NO:40 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain region retains the CD45 specificity of the antibody (i.e., similar to Ab4 specificity) or has enhanced biological activity relative to another Ab4 light chain region.

[0387] In some embodiments, antibodies containing the CDR region and / or variable region of Ab4 may be incorporated into antibody-drug conjugates, as described herein. Additionally, antibodies containing the CDR region and / or variable region of Ab4 may be used in the methods described herein, for example, for depleting CD45+ cells in an individual.

[0388] Ab5

[0389] Antibody 5 (Ab5) cross-reacts with human CD45, cynomolgus monkey CD45 and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0390] The amino acid sequences of the various binding regions of anti-CD45 Ab5 are described in Table 27. This invention includes, for example, Ab5-based anti-CD45 antibodies comprising CDRs as shown in Table 27.

[0391] 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 Ab5. The amino acid sequence of the heavy chain variable region (VH) of Ab5 is shown in SEQ ID NO:41 (see Table 27). The amino acid sequences of the VH CDR domain of Ab5 are shown in SEQ ID NO:42 (VHCDR1); SEQ ID NO:43 (VH CDR2) and SEQ ID NO:44 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of Ab5 is illustrated in SEQ ID NO:45 (see Table 27). The amino acid sequences of the VL CDR domain of Ab5 are shown in SEQ ID NO:46 (VL CDR1); SEQ ID NO:47 (VL CDR2) and SEQ ID NO:48 (VL CDR3).

[0392] Therefore, in some embodiments, this document provides an anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:42, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:43, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:44; and / or a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:46, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:47, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:48. In some embodiments, the anti-CD45 antibody comprises the CDR (SEQ ID NO: 42 to 44 and 46 to 48) described herein, wherein the CDR comprises a conserved amino acid substitution (or two, three, four or five amino acid substitutions) while retaining the antibody’s CD45 specificity (i.e., similar to Ab5 specificity).

[0393] 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:41, and / or a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:45. In some embodiments, the antibody may comprise a modified heavy chain (HC) variable region comprising an HC variable domain containing SEQ ID NO:41 or a variant of SEQ ID NO:41, the variant being (i) different from SEQ ID NO:41 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:41 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:41 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) containing a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:45. NO:41 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain variable region retains the antibody’s CD45 specificity (i.e., Ab5-like specificity) or has enhanced biological activity relative to another Ab5 heavy chain variable region. In some embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain containing SEQ ID NO:45 or a variant of SEQ ID NO:45, wherein the variant (i) differs from SEQ ID NO:45 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:45 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:45 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) comprises a variant of SEQ ID NO:45. NO:45 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain variable region retains the CD45 specificity of the antibody (i.e., similar to Ab5 specificity) or has enhanced biological activity relative to another Ab5 light chain variable region.

[0394] Antibodies containing the CDR and / or variable region sequence 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 to human CD45, including (but not limited to) Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including substitute light chain constructs), single-domain antibodies, camelified antibodies, and the like. They may also have or be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM. In some embodiments, the anti-45 anti-system is IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0395] In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab5 may further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgG1 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 may 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 Kabat EU index). In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab5 may further comprise an IgG1 heavy chain constant region containing one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the Kabat EU index). In some embodiments, the antibody containing the CDR and / or variable region sequence of Ab5 may further contain the heavy chain constant region shown 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 antibody contains the light chain constant region shown in SEQ ID NO:101.

[0396] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:49. In some embodiments, the antibody comprises a modified heavy chain (HC) region containing an HC domain containing a variant of SEQ ID NO:49 or SEQ ID NO:9, the variant being (i) different from SEQ ID NO:49 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:49 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:49 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a heavy chain containing an amino acid sequence as shown in SEQ ID NO:49. NO:49 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain region retains the antibody’s CD45 specificity (i.e., Ab5-like specificity) or has enhanced biological activity relative to another Ab5 heavy chain region.

[0397] 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:50. In some embodiments, the antibody comprises a modified light chain (LC) region containing an LC domain containing SEQ ID NO:50 or a variant of SEQ ID NO:50, the variant being (i) different from SEQ ID NO:50 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:50 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:50 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a light chain containing an amino acid sequence as shown in SEQ ID NO:50. NO:50 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain region retains the CD45 specificity of the antibody (i.e., similar to Ab5 specificity) or has enhanced biological activity relative to another Ab5 light chain region.

[0398] In some embodiments, antibodies containing the CDR region and / or variable region of Ab5 may be incorporated into antibody-drug conjugates, as described herein. Additionally, antibodies containing the CDR region and / or variable region of Ab5 may be used in the methods described herein, for example, for depleting CD45+ cells in an individual.

[0399] Ab6

[0400] Antibody 6 (Ab6) cross-reacts with human CD45, cynomolgus monkey CD45 and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0401] The amino acid sequences of the various binding regions of anti-CD45 Ab6 are described in Table 27. This invention includes, for example, Ab6-based anti-CD45 antibodies comprising the CDRs shown in Table 27.

[0402] 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 Ab6. The amino acid sequence of the heavy chain variable region (VH) of Ab6 is shown in SEQ ID NO:51 (see Table 27). The amino acid sequences of the VH CDR domain of Ab6 are shown in SEQ ID NO:52 (VHCDR1); SEQ ID NO:53 (VH CDR2) and SEQ ID NO:54 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of Ab6 is illustrated in SEQ ID NO:55 (see Table 27). The amino acid sequences of the VL CDR domain of Ab6 are shown in SEQ ID NO:56 (VL CDR1); SEQ ID NO:57 (VL CDR2) and SEQ ID NO:58 (VL CDR3).

[0403] Therefore, in some embodiments, this document provides an anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:52, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:53, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:54; and / or a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:56, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:57, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:58. In some embodiments, the anti-CD45 antibody comprises the CDR (SEQ ID NO: 52 to 54 and 56 to 58) described herein, wherein the CDR comprises a conserved amino acid substitution (or two, three, four or five amino acid substitutions) while retaining the antibody’s CD45 specificity (i.e., similar to Ab6 specificity).

[0404] 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:51, and / or a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:55. In some embodiments, the antibody may comprise a modified heavy chain (HC) variable region comprising an HC variable domain containing SEQ ID NO:51 or a variant of SEQ ID NO:51, the variant being (i) different from SEQ ID NO:51 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:51 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:51 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) containing a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:55. NO:51 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain variable region retains the antibody’s CD45 specificity (i.e., similar to Ab6 specificity) or has enhanced biological activity relative to another Ab6 heavy chain variable region. In some embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain containing SEQ ID NO:55 or a variant of SEQ ID NO:55, wherein the variant (i) differs from SEQ ID NO:55 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:55 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:55 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) comprises a variant of SEQ ID NO:55. NO:55 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain variable region retains the antibody’s CD45 specificity (i.e., similar to Ab6 specificity) or has enhanced biological activity relative to another Ab6 light chain variable region.

[0405] Antibodies containing the CDR and / or variable region sequence 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 to human CD45, including (but not limited to) Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including substitute light chain constructs), single-domain antibodies, camelified antibodies, and the like. They may also have or be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM. In some embodiments, the anti-45 antibody system is IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0406] In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab6 may further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgG1 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 may 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 Kabat EU index). In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab6 may further comprise an IgG1 heavy chain constant region containing one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the Kabat EU index). In some embodiments, the antibody containing the CDR and / or variable region sequence of Ab6 may further contain the heavy chain constant region shown 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 antibody contains the light chain constant region shown in SEQ ID NO:101.

[0407] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:59. In some embodiments, the antibody comprises a modified heavy chain (HC) region containing an HC domain containing SEQ ID NO:59 or a variant of SEQ ID NO:59, the variant being (i) different from SEQ ID NO:59 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:59 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:59 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a heavy chain containing an amino acid sequence as shown in SEQ ID NO:59. NO:59 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain region retains the CD45 specificity of the antibody (i.e., similar to Ab6 specificity) or has enhanced biological activity relative to another Ab6 heavy chain region.

[0408] 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:60. In some embodiments, the antibody comprises a modified light chain (LC) region containing an LC domain containing SEQ ID NO:60 or a variant of SEQ ID NO:60, the variant being (i) different from SEQ ID NO:60 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:60 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:60 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a light chain containing an amino acid sequence as shown in SEQ ID NO:60. NO:60 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain region retains the antibody’s CD45 specificity (i.e., similar to Ab6 specificity) or has enhanced biological activity relative to another Ab6 light chain region.

[0409] In some embodiments, antibodies containing the CDR region and / or variable region of Ab6 may be incorporated into antibody-drug conjugates, as described herein. Additionally, antibodies containing the CDR region and / or variable region of Ab6 may be used in the methods described herein, for example, for depleting CD45+ cells in an individual.

[0410] Ab7

[0411] Antibody 7 (Ab7) cross-reacts with human CD45, cynomolgus monkey CD45 and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0412] The amino acid sequences of the various binding regions of anti-CD45 Ab7 are described in Table 27. This invention includes, for example, Ab7-based anti-CD45 antibodies comprising the CDRs shown in Table 27.

[0413] 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 Ab7. The amino acid sequence of the heavy chain variable region (VH) of Ab7 is shown in SEQ ID NO:61 (see Table 27). The amino acid sequences of the VH CDR domain of Ab7 are shown in SEQ ID NO:62 (VHCDR1); SEQ ID NO:63 (VH CDR2) and SEQ ID NO:64 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of Ab7 is illustrated in SEQ ID NO:65 (see Table 27). The amino acid sequences of the VL CDR domain of Ab7 are shown in SEQ ID NO:66 (VL CDR1); SEQ ID NO:67 (VL CDR2) and SEQ ID NO:68 (VL CDR3).

[0414] Therefore, in some embodiments, this document provides an anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:62, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:63, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:64; and / or a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:66, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:67, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:68. In some embodiments, the anti-CD45 antibody comprises the CDR (SEQ ID NO: 62 to 64 and 66 to 68) described herein, wherein the CDR comprises a conserved amino acid substitution (or two, three, four or five amino acid substitutions) while retaining the antibody’s CD45 specificity (i.e., similar to Ab7 specificity).

[0415] 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 SEQ ID NO:61 or a variant of SEQ ID NO:61, the variant being (i) different from SEQ ID NO:61 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:61 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:61 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) containing a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:65. NO:61 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; 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 SEQ ID NO:65 or a variant of SEQ ID NO:65, wherein the variant (i) differs from SEQ ID NO:65 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:65 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:65 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) comprises a variant of SEQ ID NO:65. 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; 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.

[0416] Antibodies containing the CDR and / or variable region sequence 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 to human CD45, including (but not limited to) Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including substitute light chain constructs), single-domain antibodies, camelified antibodies, and the like. They may also have or be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM. In some embodiments, the anti-45 anti-system is IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0417] In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab7 may further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgG1 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 may 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 Kabat EU index). In some embodiments, antibodies comprising the CDR and / or variable region sequence of Ab7 may further comprise an IgG1 heavy chain constant region containing one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the Kabat EU index). In some embodiments, the antibody containing the CDR and / or variable region sequence of Ab7 may further contain the heavy chain constant region shown 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 antibody contains the light chain constant region shown in SEQ ID NO:101.

[0418] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:69. In some embodiments, the antibody comprises a modified heavy chain (HC) region containing an HC domain containing SEQ ID NO:69 or a variant of SEQ ID NO:69, which (i) differs from SEQ ID NO:9 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:69 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:69 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a heavy chain containing an amino acid sequence as shown in SEQ ID NO:69. NO:69 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain region retains the antibody’s CD45 specificity (i.e., Ab7-like specificity) or has enhanced biological activity relative to another Ab7 heavy chain region.

[0419] 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:70. In some embodiments, the antibody comprises a modified light chain (LC) region containing an LC domain containing SEQ ID NO:70 or a variant of SEQ ID NO:70, the variant being (i) different from SEQ ID NO:70 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:70 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:70 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a light chain containing an amino acid sequence as shown in SEQ ID NO:70. NO:70 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain region retains the antibody’s CD45 specificity (i.e., Ab7-like specificity) or has enhanced biological activity relative to another Ab7 light chain region.

[0420] In some embodiments, antibodies containing the CDR region and / or variable region of Ab7 may be incorporated into antibody-drug conjugates, as described herein. Additionally, antibodies containing the CDR region and / or variable region of Ab7 may be used in the methods described herein, for example, for depleting CD45+ cells in an individual.

[0421] AbA

[0422] Antibody A (AbA) cross-reacts with human CD45, cynomolgus monkey CD45 and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0423] The amino acid sequences of the various binding regions of anti-CD45 AbA are described in Table 27. This invention includes, for example, AbA-based anti-CD45 antibodies comprising CDRs as shown in Table 27.

[0424] 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 AbA. The amino acid sequence of the heavy chain variable region (VH) of AbA is shown in SEQ ID NO:71 (see Table 27). The amino acid sequences of the VH CDR domain of AbA are shown in SEQ ID NO:72 (VHCDR1); SEQ ID NO:73 (VH CDR2) and SEQ ID NO:74 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of AbA is described in SEQ ID NO:75 (see Table 27). The amino acid sequences of the VL CDR domain of AbA are shown in SEQ ID NO:76 (VL CDR1); SEQ ID NO:77 (VL CDR2) and SEQ ID NO:78 (VL CDR3).

[0425] Therefore, in some embodiments, this document provides an anti-CD45 antibody or its antigen-binding portion comprising a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:72, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:73, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:74; and / or a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:76, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:77, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:78. In some embodiments, the anti-CD45 antibody comprises the CDR (SEQ ID NO: 72 to 74 and 76 to 78) described herein, wherein the CDR comprises a conserved amino acid substitution (or two, three, four or five amino acid substitutions) while retaining the antibody’s CD45 specificity (i.e., similar to AbA specificity).

[0426] 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:71, and / or a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:75. In some embodiments, the antibody may comprise a modified heavy chain (HC) variable region comprising an HC variable domain containing SEQ ID NO:71 or a variant of SEQ ID NO:71, the variant being (i) different from SEQ ID NO:71 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:71 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:71 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) comprising a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:75. NO:71 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain variable region retains the antibody’s CD45 specificity (i.e., similar to AbA specificity) or has enhanced biological activity relative to the biological activity of another AbA heavy chain variable region. In some embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain containing SEQ ID NO:75 or a variant of SEQ ID NO:75, the variant being (i) different from SEQ ID NO:75 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:75 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:75 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a domain containing the same SEQ ID NO:75 as SEQ ID NO:75. NO:75 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain variable region retains the antibody’s CD45 specificity (i.e., similar to AbA specificity) or has enhanced biological activity relative to another AbA light chain variable region.

[0427] Antibodies containing the CDR and / or variable region sequence 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 to human CD45, including (but not limited to) Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including substitute light chain constructs), single-domain antibodies, camelified antibodies, and the like. They may also have or be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM. In some embodiments, the anti-45 antibody system is IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0428] In some embodiments, antibodies comprising the CDR and / or variable region sequence of AbA may further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgG1 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 may 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 Kabat EU index). In some embodiments, antibodies comprising the CDR and / or variable region sequence of AbA may further comprise an IgG1 heavy chain constant region containing one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the Kabat EU index). In some embodiments, the antibody containing the CDR and / or variable region sequence of AbA may further contain the heavy chain constant region shown 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 antibody contains the light chain constant region shown in SEQ ID NO:101.

[0429] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:79. In some embodiments, the antibody comprises a modified heavy chain (HC) region containing an HC domain containing SEQ ID NO:79 or a variant of SEQ ID NO:79, the variant being (i) different from SEQ ID NO:79 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:79 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:79 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a heavy chain containing an amino acid sequence as shown in SEQ ID NO:79. NO:79 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain region retains the antibody’s CD45 specificity (i.e., similar to AbA specificity) or has enhanced biological activity relative to another AbA heavy chain region.

[0430] 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:80. In some embodiments, the antibody comprises a modified light chain (LC) region containing an LC domain containing SEQ ID NO:80 or a variant of SEQ ID NO:80, the variant being (i) different from SEQ ID NO:80 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:80 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:80 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a light chain containing an amino acid sequence as shown in SEQ ID NO:80. NO:80 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain region retains the CD45 specificity of the antibody (i.e., similar to the specificity of AbA), or has enhanced biological activity relative to the light chain region of another AbA.

[0431] In some embodiments, antibodies containing the CDR region and / or variable region of AbA may be incorporated into antibody-drug conjugates, as described herein. Additionally, antibodies containing the CDR region and / or variable region of AbA may be used in the methods described herein, for example, for depleting CD45+ cells in an individual.

[0432] AbB

[0433] Antibody B (AbB) cross-reacts with human CD45, cynomolgus monkey CD45 and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0434] The amino acid sequences of the various binding regions of anti-CD45 AbB are described in Table 27. This invention includes, for example, AbB-based anti-CD45 antibodies comprising CDRs as shown in Table 27.

[0435] 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 AbB. The amino acid sequence of the heavy chain variable region (VH) of AbB is shown in SEQ ID NO:81 (see Table 27). The amino acid sequences of the VH CDR domain of AbB are shown in SEQ ID NO:82 (VHCDR1); SEQ ID NO:83 (VH CDR2) and SEQ ID NO:84 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of AbB is described in SEQ ID NO:85 (see Table 27). The amino acid sequences of the VL CDR domain of AbB are shown in SEQ ID NO:86 (VL CDR1); SEQ ID NO:87 (VL CDR2) and SEQ ID NO:88 (VL CDR3).

[0436] Therefore, in some embodiments, this document provides an anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:82, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:83, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:84; and / or a light chain variable region comprising a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:86, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:87, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:88. In some embodiments, the anti-CD45 antibody comprises the CDR (SEQ ID NO: 82 to 84 and 86 to 88) described herein, wherein the CDR comprises a conserved amino acid substitution (or two, three, four or five amino acid substitutions) while retaining the antibody’s CD45 specificity (i.e., similar to AbB specificity).

[0437] 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:81, and / or a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:85. In some embodiments, the antibody may comprise a modified heavy chain (HC) variable region comprising an HC variable domain containing SEQ ID NO:81 or a variant of SEQ ID NO:81, the variant being (i) different from SEQ ID NO:81 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:81 in that up to five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:81 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) containing a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:85. NO:81 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain variable region retains the antibody’s CD45 specificity (i.e., similar to AbB specificity) or has enhanced biological activity relative to the biological activity of another AbB heavy chain variable region. In some embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain containing SEQ ID NO:85 or a variant of SEQ ID NO:85, wherein the variant (i) differs from SEQ ID NO:85 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:85 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:85 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) comprises a variant of SEQ ID NO:85. NO:85 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain variable region retains the antibody’s CD45 specificity (i.e., similar to AbB specificity) or has enhanced biological activity relative to the biological activity of another AbB light chain variable region.

[0438] Antibodies containing the CDR and / or variable region sequence 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 to human CD45, including (but not limited to) Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including substitute light chain constructs), single-domain antibodies, camelified antibodies, and the like. They may also have or be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM. In some embodiments, the anti-45 antibody system is IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0439] In some embodiments, antibodies comprising the CDR and / or variable region sequence of AbB may further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgG1 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 may 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 Kabat EU index). In some embodiments, antibodies comprising the CDR and / or variable region sequence of AbB may further comprise an IgG1 heavy chain constant region containing one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the Kabat EU index). In some embodiments, the antibody containing the CDR and / or variable region sequence of AbB may further contain the heavy chain constant region shown 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 antibody contains the light chain constant region shown in SEQ ID NO:101.

[0440] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:89. In some embodiments, the antibody comprises a modified heavy chain (HC) region containing an HC domain containing SEQ ID NO:89 or a variant of SEQ ID NO:89, the variant being (i) different from SEQ ID NO:89 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:89 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:89 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a heavy chain containing an amino acid sequence as shown in SEQ ID NO:89. NO:89 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain region retains the antibody’s CD45 specificity (i.e., similar to AbB specificity) or has enhanced biological activity relative to the biological activity of another AbB heavy chain region.

[0441] 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:90. In some embodiments, the antibody comprises a modified light chain (LC) region containing an LC domain containing SEQ ID NO:90 or a variant of SEQ ID NO:90, the variant being (i) different from SEQ ID NO:90 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:90 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:90 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a light chain containing an amino acid sequence as shown in SEQ ID NO:90. NO:90 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain region retains the CD45 specificity of the antibody (i.e., similar to the specificity of AbB), or has enhanced biological activity relative to the light chain region of another AbB.

[0442] In some embodiments, antibodies containing the CDR region and / or variable region of AbB may be incorporated into antibody-drug conjugates, as described herein. Additionally, antibodies containing the CDR region and / or variable region of AbB may be used in the methods described herein, for example, for depleting CD45+ cells in an individual.

[0443] AbC

[0444] Antibody C (AbC) cross-reacts with human CD45, cynomolgus monkey CD45 and rhesus monkey CD45, and can bind to multiple isoforms of human CD45.

[0445] The amino acid sequences of the various binding regions of anti-CD45 AbC are described in Table 27. This invention includes, for example, AbC-based anti-CD45 antibodies comprising CDRs as shown in Table 27.

[0446] 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 AbC. The amino acid sequence of the heavy chain variable region (VH) of AbC is shown in SEQ ID NO:91 (see Table 27). The amino acid sequence of the VH CDR domain of AbC is shown in SEQ ID NO:92 (VHCDR1); SEQ ID NO:93 (VH CDR2) and SEQ ID NO:94 (VH CDR3). The amino acid sequence of the light chain variable region (VL) of AbC is illustrated in SEQ ID NO:95 (see Table 27). The amino acid sequence of the VL CDR domain of AbC is shown in SEQ ID NO:96 (VL CDR1); SEQ ID NO:97 (VL CDR2) and SEQ ID NO:98 (VL CDR3).

[0447] Therefore, in some embodiments, this document provides an anti-CD45 antibody or its antigen-binding portion thereof, comprising a heavy chain variable region including a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:92, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:93, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:94; and / or a light chain variable region including a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:96, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:97, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:98. In some embodiments, the anti-CD45 antibody comprises the CDR (SEQ ID NO: 92 to 94 and 96 to 98) described herein, wherein the CDR comprises a conserved amino acid substitution (or two, three, four or five amino acid substitutions) while retaining the antibody’s CD45 specificity (i.e., similar to AbC specificity).

[0448] 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:991, and / or a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:95. In some embodiments, the antibody may comprise a modified heavy chain (HC) variable region comprising an HC variable domain containing SEQ ID NO:91 or a variant of SEQ ID NO:91, which (i) differs from SEQ ID NO:91 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:91 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:91 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) comprises a light chain variable region containing an amino acid sequence as shown in SEQ ID NO:95. NO:91 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain variable region retains the antibody’s CD45 specificity (i.e., similar to AbC specificity) or has enhanced biological activity relative to the biological activity of another AbC heavy chain variable region. In some embodiments, the antibody comprises a modified light chain (LC) variable region comprising an LC variable domain containing SEQ ID NO:95 or a variant of SEQ ID NO:95, wherein the variant (i) differs from SEQ ID NO:95 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:95 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:95 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) comprises a variant of SEQ ID NO:95. NO:95 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain variable region retains the antibody’s CD45 specificity (i.e., similar to AbC specificity) or has enhanced biological activity relative to the biological activity of another AbC light chain variable region.

[0449] Antibodies containing the CDR and / or variable region sequence 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 to human CD45, including (but not limited to) Fab, Fab', (Fab')2, Fv, scFv (single-chain Fv), alternative antibodies (including substitute light chain constructs), single-domain antibodies, camelified antibodies, and the like. They may also have or be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4), or IgM. In some embodiments, the anti-45 antibody system is IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0450] In some embodiments, antibodies comprising the CDR and / or variable region sequence of AbC may further comprise a heavy chain constant region and / or a light chain constant region. In some embodiments, the constant region is a human IgG1 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 may 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 Kabat EU index). In some embodiments, antibodies comprising the CDR and / or variable region sequence of AbC may further comprise an IgG1 heavy chain constant region containing one or more of the following substitutions: L234A, L235A, D265C, and H435A (according to the Kabat EU index). In some embodiments, the antibody containing the CDR and / or variable region sequence of AbC may further contain the heavy chain constant region shown 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 antibody contains the light chain constant region shown in SEQ ID NO:101.

[0451] In some embodiments, the anti-CD45 antibody or its antigen-binding portion comprises a heavy chain containing an amino acid sequence as shown in SEQ ID NO:99. In some embodiments, the antibody comprises a modified heavy chain (HC) region containing an HC domain containing SEQ ID NO:99 or a variant of SEQ ID NO:99, which (i) differs from SEQ ID NO:99 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) differs from SEQ ID NO:99 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) differs from SEQ ID NO:99 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a heavy chain containing an amino acid sequence as shown in SEQ ID NO:99. NO:99 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified heavy chain region retains the antibody’s CD45 specificity (i.e., similar to AbC specificity) or has enhanced biological activity relative to another AbC heavy chain region.

[0452] 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:100. In some embodiments, the antibody comprises a modified light chain (LC) region containing an LC domain containing SEQ ID NO:100 or a variant of SEQ ID NO:100, the variant being (i) different from SEQ ID NO:100 in that one, two, three, four, or five amino acids are substituted, added, or deleted; (ii) different from SEQ ID NO:100 in that at most five, four, three, two, or one amino acid is substituted, added, or deleted; (iii) different from SEQ ID NO:100 in that one to five, one to three, one to two, two to five, or three to five amino acids are substituted, added, or deleted; and / or (iv) contains a light chain containing an amino acid sequence as shown in SEQ ID NO:100. NO:100 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 substitution may be a conserved amino acid substitution or a non-conserved amino acid substitution; and wherein the modified light chain region retains the antibody’s CD45 specificity (i.e., similar to AbC specificity) or has enhanced biological activity relative to the light chain region of another AbC.

[0453] In some embodiments, antibodies containing the CDR region and / or variable region of AbC may be incorporated into antibody-drug conjugates, as described herein. Additionally, antibodies containing the CDR region and / or variable region of AbC may be used in the methods described herein, for example, for depleting CD45+ cells in an individual.

[0454] There are a total of CDRs

[0455] A comparison of the CDR amino acid sequences of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, and Ab7 is as follows: Figure 32 Provided. These antibodies bind to the same antigenic determinant on human CD45 and share certain common residues in their CDR regions. The common heavy chain amino acid CDR sequences are shown in SEQ ID NO:119, SEQ ID NO:120, and SEQ ID NO:121; and the common light chain amino acid CDR sequences are shown in SEQ ID NO:122, SEQ ID NO:123, and SEQ ID NO:124.

[0456] Therefore, in some embodiments, this document provides an isolated anti-CD45 antibody or its antigen-binding portion comprising a heavy chain variable region containing a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:119, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:120, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:121; and a light chain variable region containing a CDR1 domain containing an amino acid sequence as shown in SEQ ID NO:122, a CDR2 domain containing an amino acid sequence as shown in SEQ ID NO:123, and a CDR3 domain containing an amino acid sequence as shown in SEQ ID NO:124. In some embodiments, the aforementioned antibody may further comprise a heavy chain constant region and / or a light chain constant region. For example, in some embodiments, the aforementioned antibody may further comprise a heavy chain constant region selected from 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 selected from SEQ ID NO:101.

[0457] Fc-modified antibodies

[0458] This document covers antibodies or antigen-binding fragments thereof capable of binding to CD45 and having Fc modifications that allow Fc silencing, wherein such antibodies or antigen-binding fragments thereof can be used as a single therapeutic agent or as an ADC to deplete CD45-expressing cells in a patient in need. For example, in some embodiments, such antibodies or antigen-binding fragments thereof covered herein can be used to deplete certain cell types, including HSCs and leukocytes. Thus, in some embodiments, the antibodies or antigen-binding fragments thereof covered herein can be used to condition a patient for HSC transplantation. In some embodiments, the antibodies or antigen-binding fragments thereof covered herein can be used to reset a patient's immune system by, for example, depleting HSCs and leukocytes in a patient and delivering an HSC graft to the patient. In some embodiments, the antibodies or antigen-binding fragments thereof covered 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.

[0459] For example, this article covers antibodies or antigen-binding fragments thereof capable of binding to antigens expressed by hematopoietic stem cells (e.g., CD45) and having Fc modifications that allow Fc silencing, wherein such antibodies or antigen-binding fragments thereof can be used as a single therapeutic agent or as an ADC for (i) treating cancers and autoimmune diseases characterized by CD45+ hematopoietic stem cells; and (ii) promoting the engraftment of transplanted hematopoietic stem cells into patients requiring transplant therapy. Such therapeutic activity can be induced, for example, by binding to an anti-CD45 antibody or antigen-binding fragment thereof and subsequently inducing cell death, the anti-CD45 antibody or antigen-binding fragment thereof binding to antigens expressed by hematopoietic cells (e.g., hematopoietic stem cells or mature immune cells (e.g., T cells)), such as cancer cells, autoimmune cells, or hematopoietic stem cells. Depletion of endogenous hematopoietic stem cells provides a niche for transplanted hematopoietic stem cells to home and subsequently establish productive hematopoiesis. In this way, transplanted hematopoietic stem cells can be successfully engrafted into patients, such as human patients with stem cell diseases described herein. The Fc-modified antibodies and ADCs covered in this article not only allow for the selective depletion of endogenous hematopoietic stem cells, but also have reduced cytotoxic effects on exogenous hematopoietic stem cell grafts, thereby further promoting the implantation of hematopoietic stem cell grafts.

[0460] The antibodies or binding fragments described herein may also include modifications and / or mutations that alter the properties of the antibody and / or fragment, such as extending or shortening the half-life or increasing or decreasing 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 characteristic). Non-limiting examples of labeling for peptides include (but are not limited to) 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in this art (see, for example, Junghans et al., Cancer Chemotherapy and Biotherapy 655-686 (2nd edition, edited by Chafner and Longo, Lippincott Raven (1996)) and U.S. Patents 4,681,581, 4,735,210, 5,101,827, 5,102,990 (USRE35,500), 5,648,471, and 5,697,902). For instance, radioisotopes can be bound using the chloramine-T method.

[0461] In one embodiment, an anti-CD45 antibody or its binding fragment comprises a modified Fc region, wherein the modified Fc region contains at least one amino acid modification relative to the wild-type Fc region, such that the molecule has an altered FcγR affinity or binding to FcγR. Certain amino acid positions within the Fc region are known through crystallographic studies to create direct contact with FcγR. Specifically, amino acids 234-239 (hinge region), 265-269 (B / C ring), 297-299 (C' / E ring), and 327-332 (F / G ring). (See Sondermann et al., 2000 Nature, 406:267-273). The antibodies described herein may comprise a variant Fc region containing modifications based on structural and crystallographic analysis to create direct contact with FcγR. In one embodiment, the Fc region of the anti-CD45 antibody (or a fragment thereof) contains an amino acid substitution at amino acid 265 according to the EU index as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, NH1, MD (1991), which is expressly incorporated herein by reference. "EU index as described in Kabat" refers to the number of the human IgG1 EU antibody. In one embodiment, the Fc region contains a D265A mutation. In one embodiment, the Fc region contains a D265C mutation. In some embodiments, the Fc region of the antibody (or a fragment thereof) contains an amino acid substitution at amino acid 234 according to the EU index as described in Kabat.

[0462] In one embodiment, the Fc region contains mutations at the amino acid positions of D265, V205, H435, I253, and / or H310. For example, specific mutations at these positions include D265C, V205C, H435A, I253A, and / or H310A.

[0463] In one embodiment, the Fc region contains an L234A mutation. In some embodiments, the Fc region of the anti-CD45 antibody (or a fragment thereof) contains an amino acid substitution at amino acid 235 according to the EU index as described in Kabat. In one embodiment, the Fc region contains an L235A mutation. In another embodiment, the Fc region contains both L234A and L235A mutations. In another embodiment, the Fc region contains D265C, L234A, and L235A mutations. In another embodiment, the Fc region contains D265C, L234A, L235A, and H435A mutations. In yet another embodiment, the Fc region contains both D265C and H435A mutations.

[0464] In some embodiments, the anti-CD45 antibody described herein comprises an Fc region containing 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 Kabat's EU index).

[0465] The binding or affinity between the modified Fc region and the Fcγ receptor can be determined using a variety of techniques known in this field, 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 plasma resonance analysis or other kinetic-based analyses (e.g., BIACORE.RTM. analysis or Octet. TM Analysis (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), are employed. These and other methods utilize labeling on one or more tested components and / or employ various detection methods, including (but not limited to) chromogenic, fluorescent, luminescent, or isotopic labeling. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th ed., Lippincott-Raven, Philadelphia (1999), which addresses antibody-immunogen interactions. An example of competitive binding assays is radioimmunoassay, which involves co-culturing a labeled antigen and an antibody of interest in the presence of incrementally added unlabeled antigen, and detecting the antibody bound to the labeled antigen. The affinity of the antibody of interest for a specific antigen and the binding-dissociation rate can be determined using data analyzed by Scarjad plots. Competition with a secondary antibody can also be determined using radioimmunoassay. In this case, the antigen is cultured together with the antibody of interest bound to the labeled compound in the presence of incrementally added unlabeled second antibody.

[0466] In one embodiment, compared to the binding of a homologous antibody containing an unmodified Fc region to the Fcγ receptor (e.g., as evaluated by biological layer interference (BLI)), the binding of an antibody with the Fc modifications described herein (e.g., D265C, L234A, L235A and / or H435A) to the Fcγ receptor is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or about 100%.

[0467] The binding interaction between the Fc region and the Fcγ receptor is essential for a variety of effector functions and downstream signaling events, including (but not limited to) antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore, in certain morphologies, antibodies containing modified Fc regions (e.g., containing L234A, L235A, and / or D265C mutations) exhibit substantially reduced or eliminated effector functions. Effector functions can be analyzed using a variety of methods known in this technique, such as by measuring cellular responses to the antibody of interest (e.g., mast cell degranulation or intercytokine release). For example, using standard methods in this technique, the ability of Fc-modified antibodies to trigger mast cell degranulation or intercytokine release, for example, by human peripheral blood mononuclear cells, can be analyzed.

[0468] Therefore, in one embodiment, the Fc region contains a mutation that causes a shortened half-life (e.g., relative to an antibody with an unmodified Fc region). Antibodies with short half-lives may be advantageous in certain cases where the antibody is expected to have a short-term therapeutic effect (e.g., the conditioning steps described herein), where HSCs are performed after antibody administration. Typically, the antibody will be substantially cleared prior to HSC delivery, and these HSCs typically represent target antigens (e.g., CD45), but are not the target of anti-CD45 antibodies, unlike endogenous stem cells. In one embodiment, the Fc region contains a mutation at position 435 (according to Kabat's EU index). In one embodiment, the mutation is the H435A mutation.

[0469] In one embodiment, the half-life (e.g. in humans) of the anti-CD45 antibody described herein 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.

[0470] In one embodiment, the half-life (e.g. in humans) of the anti-CD45 antibody described herein is 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.

[0471] In some embodiments, the Fc region contains two or more mutations that confer a shortened half-life and reduce the antibody's effector function. In some embodiments, the Fc region contains a mutation that causes a shortened half-life and a mutation that creates at least one residue that makes a direct contact with FcγR (e.g., based on structural and crystallographic analysis). In one embodiment, the Fc region contains an H435A mutation, an L234A mutation, and an L235A mutation. In one embodiment, the Fc region contains an H435A mutation and a D265C mutation. In another embodiment, the Fc region contains an H435A mutation, an L234A mutation, an L235A mutation, and a D265C mutation.

[0472] In some embodiments, an antibody or its antigen-binding fragment binds to a cytotoxin (e.g., amatoxins) via a cysteine ​​residue in the Fc domain of the antibody or its antigen-binding fragment. In some embodiments, a cysteine ​​residue is introduced by mutation of the Fc domain of the antibody or its antigen-binding fragment. For example, the cysteine ​​residue may be selected from the group consisting of Cys118, Cys239, and Cys265. In one embodiment, the Fc region of an anti-CD45 antibody (or a fragment thereof) contains an amino acid substitution at amino acid 265 according to the EU index in Kabat. In one embodiment, the Fc region contains a D265C mutation. In one embodiment, the Fc region contains D265C and H435A mutations. In one embodiment, the Fc region contains D265C, L234A, and L235A mutations. In one embodiment, the Fc region contains D265C, L234A, L235A, and H435A mutations.

[0473] It should be noted that, unless otherwise specified, the position of the Fc amino acid is referenced to the EU number index.

[0474] The disclosures of each of the aforementioned publications regarding anti-CD45 antibodies are incorporated herein by reference. Antibodies and antigen-binding fragments that can be used in conjunction with the compositions and methods described herein include the aforementioned antibodies and their antigen-binding fragments, variants of the aforementioned non-human antibodies and antigen-binding fragments, and antibodies or antigen-binding fragments that bind to the same antigenic determinant as the aforementioned antibodies and their antigen-binding fragments, as evaluated, for example, by means of competitive antigen binding assays.

[0475] Engineered antibodies are well known in the art through methods including any of the Fc modifications described herein. These methods include (but are not limited to) site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of DNA molecules encoding the antibody or at least a constant region of the antibody. Site-directed mutagenesis is well known in the art (see, for example, Carter et al., Nucleic Acids Res., 13:4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA, 82:488 (1987)). PCR mutagenesis is also suitable for producing amino acid sequence variants of the starting polypeptide. See Higuchi, PCR Protocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17:723-733 (1989). Another method for preparing sequence variants and cassette mutagenesis is based on the technique described by Wells et al., Gene, 34:315-323 (1985).

[0476] Nucleic acids, vectors and host cells

[0477] This article also provides nucleic acid molecules (such as DNA or mRNA) containing nucleic acid sequences encoding the anti-CD45 antibody described herein or its antigen-binding portion.

[0478] Therefore, in some embodiments, this document provides isolated nucleic acid molecules that encode heavy chain variable regions comprising heavy chain CDR1, CDR2, and CDR3 of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In other embodiments, this document provides isolated nucleic acid molecules that encode heavy chain variable regions of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In still other embodiments, this document provides isolated nucleic acid molecules that encode heavy chains of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC.

[0479] In some embodiments, this document provides isolated nucleic acid molecules encoding light chain variable regions comprising CDR1, CDR2, and CDR3 of light chains Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In other embodiments, this document provides isolated nucleic acid molecules encoding light chain variable regions of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In other embodiments, this document provides isolated nucleic acid molecules encoding light chains of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC.

[0480] In some embodiments, this document provides isolated nucleic acid molecules encoding heavy chain variable regions comprising heavy chain CDR1, CDR2, and CDR3 of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC, and light chain variable regions comprising light chain CDR1, CDR2, and CDR3 of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In other embodiments, this document provides isolated nucleic acid molecules encoding both heavy and light chain variable regions of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC. In still other embodiments, this document provides isolated nucleic acid molecules encoding both heavy and light chains of Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, AbA, AbB, or AbC.

[0481] The nucleic acid encoding the antibody heavy chain, or a portion thereof, may be present in a nucleic acid molecule (e.g., an expression vector) that is identical to the nucleic acid encoding the antibody light chain, or a portion thereof. Alternatively, the heavy and light chain sequences may be present on separate nucleic acid molecules (e.g., separate expression vectors).

[0482] In one embodiment, the present invention provides an isolated nucleic acid molecule comprising:

[0483] (a) The nucleic acid sequence shown in SEQ ID NO:125;

[0484] (b) The nucleic acid sequence shown in SEQ ID NO:126;

[0485] (c) The nucleic acid sequence shown in SEQ ID NO:127;

[0486] (d) The nucleic acid sequence shown in SEQ ID NO:128;

[0487] (e) The nucleic acid sequence shown in SEQ ID NO:129;

[0488] (f) The nucleic acid sequence shown in SEQ ID NO:130;

[0489] (g) The nucleic acid sequence shown in SEQ ID NO:131;

[0490] (h) The nucleic acid sequence shown in SEQ ID NO:132;

[0491] (i) The nucleic acid sequence shown in SEQ ID NO:133;

[0492] (j) The nucleic acid sequence shown in SEQ ID NO:134;

[0493] (k) The nucleic acid sequence shown in SEQ ID NO:135;

[0494] (l) The nucleic acid sequence shown in SEQ ID NO:136;

[0495] (m)The nucleic acid sequence shown in SEQ ID NO:137;

[0496] (n) The nucleic acid sequence shown in SEQ ID NO:138;

[0497] (o) The nucleic acid sequence shown in SEQ ID NO:139;

[0498] (p) The nucleic acid sequence shown in SEQ ID NO:140;

[0499] (q)The nucleic acid sequence shown in SEQ ID NO:141;

[0500] (r)The nucleic acid sequence shown in SEQ ID NO:142;

[0501] (s) the nucleic acid sequence shown in SEQ ID NO:143; and / or

[0502] The nucleic acid sequence shown in (t)SEQ ID NO:144;

[0503] The isolated nucleic acid encodes an anti-CD45 antibody or a portion thereof.

[0504] In another embodiment, the present invention provides an isolated nucleic acid molecule comprising:

[0505] (a) The nucleic acid sequence shown in SEQ ID NO:150;

[0506] (b) The nucleic acid sequence shown in SEQ ID NO:151;

[0507] (c) The nucleic acid sequence shown in SEQ ID NO:152;

[0508] (d) The nucleic acid sequence shown in SEQ ID NO:153;

[0509] (e) The nucleic acid sequence shown in SEQ ID NO:154;

[0510] (f) The nucleic acid sequence shown in SEQ ID NO:155;

[0511] (g) The nucleic acid sequence shown in SEQ ID NO:156;

[0512] (h) The nucleic acid sequence shown in SEQ ID NO:157;

[0513] (i) The nucleic acid sequence shown in SEQ ID NO:158;

[0514] (j) The nucleic acid sequence shown in SEQ ID NO:159;

[0515] (k) The nucleic acid sequence shown in SEQ ID NO:160; and / or

[0516] (l) The nucleic acid sequence shown in SEQ ID NO:161;

[0517] The isolated nucleic acid encodes an anti-CD45 antibody or a portion thereof.

[0518] Antibodies can be generated 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 the anti-CD45 antibody described herein is provided. The nucleic acid may encode an amino acid sequence comprising an antibody (e.g., the light chain and / or heavy chain of the antibody) 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 embodiment, the host cell comprises (e.g., having been converted to): (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 Chinese hamster ovary (CHO) cells or lymphoid cells (e.g., Y0, NSO, Sp20 cells). In one embodiment, a method for manufacturing an anti-CLL-1 antibody is provided, wherein the method includes culturing a host cell containing a nucleic acid encoding an antibody as provided above under conditions suitable for antibody expression, and, where appropriate, recovering the antibody from the host cell (or host cell culture medium).

[0519] To generate anti-CD45 antibodies through recombinant synthesis, a nucleic acid encoding an antibody, such as that described above, is isolated and inserted into one or more vectors for further selection and / or expression in host cells. This nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the antibody heavy and light chains).

[0520] Suitable host cells for the selection or expression of vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, specifically without glycosylation and Fc effector function. For information on the expression of antibody fragments and peptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, HumanaPress, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in *Escherichia coli*.) After expression, the antibody can be isolated from the bacterial cell mass in the soluble fraction and can be further purified.

[0521] Vertebrate cells can also be used as a host. For example, mammalian cell lines suitable for suspension growth can be used. Other examples of useful mammalian host cell lines include monkey kidney CV1 (COS-7) transformed from SV40; human embryonic kidney lines (e.g., 293 or 293 cells as described in Graham et al., J. Gen Virol. 36:59 (1977)); young hamster kidney cells (BHK); mouse cetelli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); TRI cells as described in Mather et al., Annals NYAcad. 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, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (edited by BKCLo, Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0522] In one embodiment, the anti-CD45 antibody or its antigen-binding fragment comprises a variable region having an amino acid sequence that is at least 95%, 96%, 97%, or 99% identical to the SEQ ID No disclosed herein. Alternatively, the anti-CD45 antibody or its antigen-binding fragment comprises a CDR containing the SEQ ID No disclosed herein, and a frame region of the variable region described herein having an amino acid sequence that is at least 95%, 96%, 97%, or 99% identical to the SEQ ID No disclosed herein.

[0523] In one embodiment, the anti-CD45 antibody or its antigen-binding fragment includes a heavy chain variable region and a heavy chain constant region having the amino acid sequences disclosed herein. In another embodiment, the anti-CD45 antibody or its antigen-binding fragment includes a light chain variable region and a light chain constant region having the amino acid sequences disclosed herein. In yet another embodiment, the anti-CD45 antibody or its antigen-binding fragment includes a heavy chain variable region, a light chain variable region, a heavy chain constant region, and a light chain constant region having the amino acid sequences disclosed herein.

[0524] Methods for identifying antibodies

[0525] This article provides novel anti-CD45 antibodies that can be used, for example, in patients with depleted CD45+ cells. These antibodies can be used, for example, in conditioning methods for stem cell transplantation. Other anti-CD45 antibodies can be identified based on the disclosures provided herein.

[0526] High-throughput screening of antibodies or antibody fragment libraries that bind to CD45 expressed in hematopoietic stem cells can be used to identify anti-CD45 antibodies suitable for treating cancer, autoimmune diseases, and conditioning patients requiring hematopoietic stem cell therapy as described herein (e.g., human patients). These methods can also be used to identify modified forms of the anti-CD45 antibodies described herein. These methods include in vitro display techniques known in this field, particularly phage display, bacterial display, yeast display, mammalian cell display, ribosome display, mRNA display, and cDNA display.

[0527] A review of the use of phage display to isolate antibodies or antigen-binding fragments that bind to biologically relevant molecules is available in references such as 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. All disclosures regarding in vitro display techniques in these publications are incorporated herein by reference. Randomized combinatorial peptide libraries have been constructed to select peptides that bind to cell surface antigens, as described in Kay, Perspect. Drug Discovery Des. 2:251-268, 1995 and Kay et al., Mol. Divers. 1:139-140, 1996. All disclosures regarding the discovery of antigen-binding molecules in these publications are incorporated herein by reference. Proteins (e.g., multimeric proteins) have been successfully displayed as functional molecules in bacteriophages (see, for example, EP 0349578; EP 4527839; and EP0589877, and Chiswell and McCafferty, Trends Biotechnol. 10:80-84 1992, each of which discloses information on the use of in vitro display techniques for the discovery of antigen-binding molecules, all of which are incorporated herein by reference). In addition, functional antibody fragments, such as Fab and scFv fragments, have been displayed in in vitro formats (see, for example, 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 details of each of these documents concerning in vitro display platforms for the discovery of antigen-binding molecules are incorporated herein by reference). Also available, for example, HuMAb- Or XenoMouse TM Human anti-CD45 antibodies are generated. These technologies can be used in particular to identify and improve the affinity of antibodies, antibodies, or fragments that can bind to CD45 expressed by hematopoietic stem cells, and these antibodies, antibodies, or fragments can then be used to deplete endogenous hematopoietic stem cells in patients (e.g., human patients) who require hematopoietic stem cell transplantation therapy.

[0528] In addition to in vitro display techniques, computational modeling techniques can be used to design and identify antibodies that can bind to antigens expressed by hematopoietic stem cells (such as CD45). For example, using computational modeling techniques, those skilled in this technique can screen libraries of antibodies or antibody fragments to find molecules that can bind to specific antigenic determinants (such as extracellular antigenic determinants) on antigens expressed by hematopoietic stem cells (such as CD45).

[0529] Other techniques can be used to identify antibodies or antibody fragments that can bind to CD45 expressed by hematopoietic stem cells and are internalized by cells, for example, through receptor-mediated endocytosis. For example, the above-described in vitro display technique can be used to screen for antibodies or antibody fragments that bind to CD45 and are subsequently internalized. Phage display represents a technique that can be used in conjunction with this screening paradigm. To identify anti-CD45 antibodies or antibody fragments that can be internalized by hematopoietic stem cells, those skilled in this technique can use the phage display technique described in Williams et al., Leukemia 19:1432-1438, 2005, the full text of which is incorporated herein by reference. For example, recombinant phage libraries encoding antibodies, antibody fragments (especially, for example, scFv fragments, Fab fragments, bivalent antibodies, trivalent antibodies, and...) can be generated using mutagenesis methods known in this technique. 10 The Fn3 domain or ligands containing randomized amino acid cassettes (e.g., in one or more or all CDRs or their equivalent regions or antibodies or antibody fragments). The frame region, hinge, Fc domain and other regions of the antibody or antibody fragment can be designed to be non-immunogenic in humans, for example by using sequences containing human germline antibody sequences or sequences that show only minimal changes relative to human germline antibodies.

[0530] Using phage display techniques described herein or known in this art, a phage library containing randomized antibodies or antibody fragments covalently bound to phage particles can be co-cultured with CD45, for example by first co-culturing the phage library with a blocking agent (e.g., milk protein, bovine serum albumin, and / or IgG) to remove phages encoding antibodies or antibody fragments exhibiting nonspecific protein binding and phages encoding antibodies or fragments of antibodies binding to the Fc domain, and then co-culturing the phage library with a population of CD45-expressing cells (e.g., hematopoietic stem cells). The phage library can be co-cultured with hematopoietic stem cells for a time sufficient to allow anti-CD45 antibodies or antibody fragments to bind to homologous cell surface antigens and subsequently be internalized by the hematopoietic stem cells (e.g., incubation at 4°C for 30 minutes to 6 hours, or for example, incubation at 4°C for 1 hour). Cells can then be washed, for example, with a cold (4°C) 0.1M glycine buffer at pH 2.8 to remove phages containing antibodies or antibody fragments that do not exhibit sufficient CD45 affinity to bind to and be internalized by hematopoietic stem cells. Phages that have bound to antibodies or antibody fragments already internalized by hematopoietic stem cells can be identified, for example, by lysing cells and recovering the internalized phages from the cell culture medium. The phages can then be amplified in the bacterial cells by co-culturing the bacterial cells and the recovered phages in 2×YT medium, for example, using methods known in this technique. The phages recovered from this culture medium can then be characterized, for example, by determining the nucleic acid sequence of the gene encoding the antibody or antibody fragment inserted into the phage genome. The encoded antibody or antibody fragment can then be reconstituted by chemical synthesis (e.g., of antibody fragments, such as scFv fragments) or by recombinant expression (e.g., of full-length antibodies).

[0531] The internalization capability of the prepared antibody or antibody fragment can be evaluated, for example, using radioactive nucleus internalization analysis known in this technique. For instance, it can be evaluated by incorporating a radioactive isotope (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) 0.1M 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.

[0532] Antibodies can be generated 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 the anti-CD45 antibody described herein is provided. The nucleic acid may encode an amino acid sequence comprising an antibody (e.g., the light chain and / or heavy chain of the antibody) 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 embodiment, the host cell comprises (e.g., having been converted to): (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 ...

Claims

1. An isolated anti-CD45 antibody or its antigen-binding portion, comprising: a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR1 domain as shown in SEQ ID NO:42, a CDR2 domain as shown in SEQ ID NO:43, and a CDR3 domain as shown in SEQ ID NO:44; and the light chain variable region comprises a CDR1 domain as shown in SEQ ID NO:46, a CDR2 domain as shown in SEQ ID NO:47, and a CDR3 domain as shown in SEQ ID NO:

48.

2. An isolated anti-CD45 antibody or its antigen-binding portion, comprising: a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:41, and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

45.

3. The isolated anti-CD45 antibody or its antigen-binding portion as claimed in claim 1 or 2, wherein the antibody or its antigen-binding portion comprises an Fc region.

4. The isolated anti-CD45 antibody or its antigen-binding portion as claimed in claim 3, wherein the Fc region is a human IgG1 Fc region or a human IgG4 Fc region.

5. The isolated anti-CD45 antibody or its antigen-binding portion as claimed in any one of claims 1 to 4, wherein the antibody is a monoclonal antibody.

6. The isolated anti-CD45 antibody as claimed in claim 1 or 2, wherein the antibody is a complete antibody containing a constant region.

7. The isolated anti-CD45 antibody as claimed in claim 1 or 2, wherein the antibody is IgG.

8. The isolated anti-CD45 antibody of claim 7, wherein the IgG is IgG1 or IgG4.

9. The isolated anti-CD45 antibody of claim 1 or 2, wherein the antibody comprises a constant region, and wherein the constant region comprises at least one amino acid substitution selected from the group consisting of: L234A, L235A, D265C, H310A and H435A, wherein the substitution is based on the EU index number.

10. The isolated anti-CD45 antibody of claim 1 or 2, wherein the antibody comprises a constant region, and wherein the constant region comprises at least two amino acid substitutions selected from the group consisting of: L234A, L235A, D265C, H310A and H435A, wherein the substitutions are based on EU index numbers.

11. The isolated anti-CD45 antibody of claim 1 or 2, wherein the antibody comprises a constant region, and wherein the constant region comprises at least three amino acid substitutions selected from the group consisting of: L234A, L235A, D265C, H310A and H435A, wherein the substitutions are based on EU index numbers.

12. The isolated anti-CD45 antibody of claim 1 or 2, wherein the antibody comprises a constant region, and wherein the constant region comprises at least four amino acid substitutions selected from the group consisting of: L234A, L235A, D265C, H310A and H435A, wherein the substitutions are based on EU index numbers.

13. The isolated anti-CD45 antibody of claim 1 or 2, wherein the antibody comprises a constant region, and wherein the constant region comprises five amino acid substitutions selected from the group consisting of: L234A, L235A, D265C, H310A and H435A, wherein the substitutions are based on EU index numbers.

14. The isolated anti-CD45 antibody of claim 9, wherein the constant region comprises amino acid substitutions L234A, L235A and D265C, wherein according to EU index number.

15. The isolated anti-CD45 antibody of claim 1 or 2, comprising a constant region, wherein the constant region comprises The heavy chain amino acid sequence shown in SEQ ID NO:106; and The light chain amino acid sequence is shown in SEQ ID NO:

101.

16. An isolated anti-CD45 antibody comprising 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.

17. The isolated anti-CD45 antibody or its antigen-binding portion as claimed in claim 1 or 2, which specifically binds to human CD45 and cross-reacts with cynomolgus monkey CD45.

18. The isolated anti-CD45 antibody or its antigen-binding moiety as claimed in claim 1 or 2, 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).

19. The isolated anti-CD45 antibody or its antigen-binding portion as claimed in claim 1 or 2, wherein the antibody or its antigen-binding portion binds to human CD45 and K D It is 10 nM or less, as determined by biolayer interference (BLI).

20. The isolated anti-CD45 antibody or its antigen-binding portion as claimed in claim 1 or 2, wherein the antibody or its antigen-binding portion binds to human CD45 and K D The values ​​range from 0.1 nM to 100 nM, as determined by biolayer interference (BLI).

21. A pharmaceutical composition comprising an antibody or antigen-binding portion thereof as claimed in any one of claims 1 to 20 and a pharmaceutically acceptable carrier.

22. 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 16.

23. A vector comprising the isolated nucleic acid as claimed in claim 22.

24. A host cell comprising the isolated nucleic acid of claim 22 or the vector of claim 23.

25. The host cell of claim 24, wherein the cell is a Chinese hamster ovary (CHO) cell or a HEK293T cell.

26. A pharmaceutical composition comprising an antibody or an antigen-binding portion thereof as claimed in any one of claims 1 to 20, for depleting a population of CD45+ cells in a human patient.

27. 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 20 and is IgG.

28. The ADC of claim 27, wherein the antibody binds to the cytotoxin via a cysteine ​​residue in a constant domain of the antibody.

29. The ADC of claim 28, wherein the cysteine ​​residue is introduced by substitution of an amino acid in the constant region of the antibody.

30. The ADC of claim 29, wherein the amino acid substitution is D265C and / or V205C, wherein according to the EU index number.

31. The ADC of claim 27, wherein the ADC has a drug-to-antibody ratio (DAR) of 1, 2, 3, 4, 5, 6, 7 or 8.

32. The ADC of claim 27, 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.

33. The ADC of claim 27, wherein the cytotoxin is selected from the group consisting of: amatoxin, anthracycline, auristatin, calicheamicin, deBouganin, diphtheria toxin, duocarmycin, and indolinobenzodiazepine. (IGN), indolinobenzodiazepine Dimer, irinotecan, maytansine, maytansinoid, Pseudomonas exotoxin A, pyrrolobenzodiazepine (PBD), pyrrolobenzodiazepine Dimer, saporin, and SN-38.

34. The ADC of claim 32, wherein the cytotoxin is an RNA polymerase inhibitor.

35. The ADC of claim 34, wherein the RNA polymerase inhibitor is phallotoxin.

36. The ADC of claim 35, wherein the ADC is represented by the formula Ab-ZL-Am, wherein Ab is an antibody or antigen-binding portion thereof as claimed in any one of claims 1 to 20, L is a linker, Z is a chemical portion, and Am is a phallotoxin.

37. The ADC of claim 36, 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 ; If it exists, R A and R B Together with the oxygen atom it is bonded to, it forms an optionally 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 can be a substituted C1-C6 alkyl, a substituted C1-C6 heteroalkyl, a substituted C2-C6 alkenyl, a substituted C2-C6 heteroalkenyl, a substituted C2-C6 ynyl, a substituted C2-C6 heteroynyl, a substituted cycloalkyl, a substituted heterocycloalkyl, a substituted aryl, or a substituted heteroaryl. L is an optionally substituted C1-C6 alkylene group, an optionally substituted C1-C6 heteroalkylene group, an optionally substituted C2-C6 alkenyl group, an optionally substituted C2-C6 heteroalkenyl group, an optionally substituted C2-C6 alkyne group, an optionally substituted C2-C6 heterokyne group, an optionally substituted cycloalkylene group, an optionally substituted heterocycloalkylene group, an optionally substituted arylene group, an optionally substituted heteroarylene 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.

38. The ADC of claim 37, wherein LZ is 39. The ADC of claim 35 or 36, wherein the ADC is represented by one of the following: Where X is -S-, -S(O)-, or -SO2-.

40. The ADC of claim 35 or 36, wherein the ADC has the following formula: Ab represents the binding site of the anti-CD45 antibody.

41. The ADC of claim 35 or 36, wherein the ADC has the following formula: Where Ab represents the linker site of the anti-CD45 antibody.

42. The ADC of claim 35 or 36, wherein the ADC has the following formula: Where Ab represents the linker site of the anti-CD45 antibody.

43. The ADC of claim 35 or 36, wherein the ADC has the following formula: Where Ab represents the linker site of the anti-CD45 antibody.

44. The ADC of claim 35 or 36, wherein the ADC has the following formula: Where Ab represents the linker site of the anti-CD45 antibody.

45. The ADC of claim 33, 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 claim 45, 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 claim 45, wherein the connector has a structure R1 is CH3(Ala) or (CH2)3NH(CO)NH2(Cit).

50. The ADC of claim 45, 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 50, wherein the cytotoxic-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 45, 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 33, 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 cytotoxic agent 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 claim 54, 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 claim 54, wherein the connector comprises 59. The ADC of claim 54, wherein the cytotoxic-linker conjugate has the following structure prior to 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 27 to 59 and a pharmaceutically acceptable carrier.

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