Antibodies targeting CD45

Abstract

The present disclosure relates to antibodies and antibody fragments specific for CD45. The antibodies are improved humanized versions of murine antibodies. In addition to complex humanization activities, the antibodies are engineered to remove multiple detrimental motifs within the CDR region without losing any beneficial properties. The antibodies are useful for the treatment of diseases associated with CD45.

Description

Technical Field

[0001] The present disclosure relates to antibodies and antibody fragments specific for CD45. The antibodies are improved humanized versions of murine antibodies. In addition to complex humanization, the antibodies are engineered to remove multiple deleterious motifs within the CDR regions without losing any beneficial properties. The antibodies are useful for treating diseases associated with CD45.

[0002] Statement on Funding

[0003] The project leading to this application has received funding from the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (grant scheme number 818806). Background Art

[0004] CD45, also known as protein tyrosine phosphatase receptor type C (PTPRC), is an enzyme encoded by the PTPRC gene (Kaplan et al., PNAS 87:7000-7004 (1990)). CD45 is a member of the protein tyrosine phosphatase (PTP) family, which includes signaling molecules that regulate a variety of cellular processes, including cell growth, differentiation, mitotic cycle, and oncogenic transformation. CD45 contains an extracellular domain, a single transmembrane segment, and two tandem intracytoplasmic catalytic domains, and therefore belongs to the receptor type PTP family. CD45 is a type I transmembrane protein that is present in differentiated hematopoietic cells (except, for example, erythrocytes) in a variety of subtypes (Holmes, Immunology 7:145-55 (2006)). CD45 has been shown to be a regulator of T cell and B cell antigen receptor signaling. It functions by directly interacting with components of the antigen receptor complex through its extracellular domain or by activating various Src family kinases (SFKs) (such as Lck) required for antigen receptor signaling via its cytoplasmic domain. CD45 also inhibits JAK kinases and thus functions as a negative regulator of cytokine receptor signaling.

[0005] CD45 is present on the surface of hematopoietic cells (including HSCs, leukocytes, and osteoclasts), all of which are of hematopoietic origin (Shivtiel et al., J Exp Med 205:2381 (2008)). Deletion mutations within CD45 in humans are associated with severe immunodeficiency. This is primarily due to the lack of CD45 on T cells, which is normally abundant and required to regulate SFK activity during antigenic responses. The bone marrow of CD45-deficient (CD45- / -) mice contains a normal number of hematopoietic cells, but the number of the most primitive HSCs is reduced, and their mobilization in response to G-CSF is impaired. In part, this defect is inherent to HSCs; without CD45-mediated downregulation of SFK activity, integrin-mediated adhesion would be higher and HSCs would be more likely to remain in the stem cell niche. CD45- / - HSCs also lack G-CSF-stimulated mobilization and homing to the chemokine CXCL12 / SDF-1, which can negatively impact cell engraftment after transplantation. These defects can be restored by supplementing SFK inhibitors, indicating that this role is usually performed by CD45. Similarly, CD45- / - recipients also showed defective implantation and subsequent mobilization of normal HSC, indicating the role of CD45 in stem cell niche and HSC (Shivtiel et al., J Exp Med 205:2381 (2008)). For example, because CD45 is expressed on HSC and leukocytes, it becomes a therapeutic target including conditioning therapy, immune resetting and disease treatment.

[0006] A variety of anti-CD45 moieties are known in the art, some of which are currently under development. BC8 is a mouse hybridoma antibody commercially available from IchorBio (#ICH1155). The BC8 antibody is the basis for anti-CD45 antibody-radioconjugates developed by Actinium Pharmaceuticals (WO2017155937, WO2019084258, WO2020159656). Other anti-CD45 antibodies and antibody-based moieties are disclosed in WO2016016442, WO2019115791, WO2020058495, WO2017009473, WO2019129178, WO2020018580, WO2020170254, and WO2020219959. These and other anti-CD45 moieties can be used in the context of the present disclosure. Summary of the Invention

[0007] The present disclosure relates to humanized antibodies or antibody fragments specific for human CD45, wherein the antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises at least one of the following mutations:

[0008] a) glycine in the LCDR1 region (SEQ ID NO: 7) was replaced by alanine,

[0009] b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by lysine,

[0010] c) the arginine in the LCDR2 region (SEQ ID NO: 8) is replaced by leucine, or

[0011] d) The phenylalanine in the LCDR2 region (SEQ ID NO: 8) was replaced by alanine.

[0012] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises at least one of the following mutations:

[0013] a) glycine in the LCDR1 region (SEQ ID NO: 7) was replaced by alanine,

[0014] b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by lysine, or

[0015] c) Arginine in the LCDR2 region (SEQ ID NO: 8) was replaced by leucine.

[0016] The present disclosure also relates to a humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises:

[0017] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 75, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9,

[0018] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9,

[0019] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9,

[0020] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 110, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9,

[0021] e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9,

[0022] f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9, or

[0023] g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9.

[0024] The present disclosure also relates to the aforementioned humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment further comprises a mutation of the second glycine of the triple glycine motif in the framework region 4 of the variable light chain to glutamine.

[0025] The present disclosure also relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises:

[0026] a) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 98,

[0027] b) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 99,

[0028] c) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 101,

[0029] d) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 102,

[0030] e) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 103,

[0031] f) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 104, or

[0032] g) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 105.

[0033] The present disclosure also relates to the aforementioned humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a mutation:

[0034] a) glycine in the LCDR1 region (SEQ ID NO: 7) was replaced by alanine,

[0035] b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by lysine, and

[0036] c) Arginine in the LCDR2 region (SEQ ID NO: 8) was replaced by leucine.

[0037] The present disclosure also relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104.

[0038] The present disclosure also relates to the aforementioned humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody comprises a modification in the Fc region, preferably wherein the Fc modification is a silent mutation, more preferably wherein the mutation is a PA-LALA, PG-LALA or AEASS mutation.

[0039] The present disclosure also relates to the aforementioned humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment is a monoclonal antibody or antibody fragment.

[0040] The present disclosure also relates to an antibody drug conjugate comprising the aforementioned humanized antibody or antibody fragment specific for human CD45 and a cytotoxic drug. Preferably, the cytotoxic moiety is a pyrrolobenzodiazepine (PBD). More preferably, the pyrrolobenzodiazepine It's tesirine.

[0041] The present disclosure also relates to the aforementioned humanized antibodies, antibody fragments, and antibody drug conjugates comprising the humanized antibodies or antibody fragments, for use in medicine.

[0042] The present disclosure also relates to the aforementioned humanized antibodies, antibody fragments, and antibody drug conjugates comprising the humanized antibodies or antibody fragments for use in treating hematological cancers, such as leukemia, myeloma, or lymphoma.

[0043] The present disclosure also relates to the aforementioned humanized antibodies, antibody fragments, and antibody drug conjugates comprising the humanized antibodies or antibody fragments for use in treating inflammatory or autoimmune diseases, such as multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn's disease, type 1 diabetes, rheumatoid arthritis, or idiopathic arthritis.

[0044] The present disclosure also relates to nucleic acid compositions comprising one or more nucleic acid sequences encoding the aforementioned humanized antibodies and antibody fragments. The present disclosure also relates to vectors comprising the nucleic acid compositions. The present disclosure also relates to host cells comprising the vectors or nucleic acid compositions.

[0045] The present disclosure also relates to a pharmaceutical composition comprising the aforementioned humanized antibody, antibody fragment, or antibody drug conjugate comprising the humanized antibody or antibody fragment and a pharmaceutically acceptable carrier or excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Five humanized variants are shown with Alignment of the original murine VH sequences of SEQ ID NO: 1. VHO is the murine sequence and VH1-5 are humanized variants. Key residues important for the VH / VL interface and canonical loop structure have been retained as much as possible.

[0047] Figure 2 Five humanized variants and antibodies are shown Alignment of the original murine VL sequences of SEQ ID NO: 1. VL0 is the murine sequence and VL1-6 are humanized variants. Key residues important for the VH / VL interface and canonical loop structure have been retained as much as possible.

[0048] Figure 3 showed that compared with the PBS control group, all human CD45+ cells and LT-HSCs were completely depleted 3 weeks after a single dose of 0.3 mg / kg of Kiebitz-Tescillin, whereas BC8-Tescillin treatment only led to partial depletion.

[0049] Figure 4 Blockade with naked antibodies mimics cellular shielding and prevents the decline in cell viability via the respective ADCs in both cell lines tested. BC08-tetracillin exhibits lower potency than Kiebitz-tetracillin. Panel A: MV4-11; Panel B: Molm-13.

[0050] Figure 5 We showed that, also in HSPCs, blocking with naked Kiebitz antibodies shielded the cells and was able to prevent the reduction of cell viability via the respective ADCs.

[0051] Figure 6 The IC50 of Kiebitz-Tecillin in Jurkat CD45 knockout cells was shown to be significantly higher than that in Jurkat wild-type cells (0.46 μg / ml vs. 0.032 μg / ml).

[0052] Figure 7 Display antibodies Amandine and Ballerina bound equally well to endogenously expressed CD45 on Jurkat cells.

[0053] definition

[0054] The present disclosure relates to antibodies that specifically bind CD45, and uses of such antibodies, particularly therapeutic uses.

[0055] The term "CD45" refers to a protein also known as PTPRC, leukocyte common antigen (L-CA), or T200. Human CD45 has the following amino acid sequence (UniProt P08575-3, defined as the canonical sequence):

[0056] MTMYLWLKLLAFGFAFLDTEVFVTGQSPTPSPTGLTTAKMPSVPLSSDPLPT

[0057] HTTAFSPASTFERENDFSETTTSLSPDNTSTQVSPDSLDNASAFNTTGVSSV

[0058] QTPHLPTHADSQTPSAGTDTQTFSGSAANAKLNPTPGSNAISDVPGERSTAS

[0059] TFPTDPVSPLTTTLSLAHHSSAALPARTSNTTITANTSDAYLNASETTTLSP

[0060] SGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVEC

[0061] GNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCT

[0062] QVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHE

[0063] YKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQ

[0064] RSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQ

[0065] RNGSAAMCHFTTKSAPPSQVWNMTVSTMTSDNSMHVKCRPPRDRNGPHERYHL

[0066] EVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHST

[0067] VISIONSCALEIAFLAFLIIVTSIALLVVLYKIYDLHKKRSCNLDQQELVERDDE

[0068] KQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIPRVFSKFPIKEARKPF

[0069] NQNKNRYVDILPYDYNRVELSEINGDAGSNYINASYIDGFKEPRKYIAAQGP

[0070] RDETVDDFWRMIWEQKATVIVMVTRCEEGNNRNKCAEYWPSMEEGTRAFGDVV

[0071] VKINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDHGVPEDPHLLLL

[0072] KLRRRVNAFSNFFSGPIVVHCSAGVGRTGTYIGIDAMLEGLEAENKVDVYGY

[0073] VVKLRRQRCLMVQVEAQYILIHQALVEYNQFGETEVNLSELHPYLHNMKKRD

[0074] PPSEPSPLEAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLK

[0075] HELEMSKESEHDSDESSDDDSDSEEPSKYINASFIMSYWKPEVMIAAQGPLK

[0076] ETIGDFWQMIFQRKVKVIVMLTELKHGDQEICAQYWGEGKQTYGDIEVDLKD

[0077] TDKSSTYTLRVFELRHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQVV

[0078] KQKLPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEVVD

[0079] IFQVVKALRKARPGMVSTFEQYQFLYDVIASTYPAQNGQVKKNNHQEDKIEF

[0080] DNEVDKVKQDANCVNPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGPASP

[0081] ALNQGS (SEQ ID No. 1)

[0082] The term "antibody" as used herein refers to a protein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, which interact with an antigen. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), and more conserved regions interspersed therein, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of an antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., Igd, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both light and heavy chains are divided into regions of structural and functional homology.

[0083] As used herein, the term "antibody fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, or stabilizing spatial distribution). Examples of binding fragments include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; an Fd fragment consisting of the VH and CH1 domains; an Fv fragment consisting of the VL and VH domains of a single antibody arm; a dAb fragment (Ward et al., (1989) Nature 341:544-546), consisting of the VH domain; and isolated complementarity determining regions (CDRs). In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, using recombinant methods, they can be joined together by synthetic linkers, enabling them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the utility of the fragments is screened in the same manner as for intact antibodies. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs and bis-scFv (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23: 1 126-1 136). Antibody fragments can be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monomers). Antibody fragments can be incorporated into single chain molecules comprising a pair of tandem Fv fragments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen binding sites (Zapata et al., (1995) Protein Eng. 8: 1057-1062; and U.S. Patent No. 5,641,870).

[0084] The structure and position of immunoglobulin variable domains (e.g., CDRs) can be defined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (see, e.g., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services (1991), Kabat et al., ed.; Lazikani et al., (1997) J. Mol. Biol. 273:927-948); Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1993) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1993) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services. al., (1997) J. Mol. Biol. 273:927-948; Annals of the New York Academy of Sciences, 764, 47-49 (1995); Nucleic Acids Research, 25, 206-211 (1997)).

[0085] "Human antibody" or "human antibody fragment" as used herein refers to antibodies and antibody fragments with variable regions, wherein the framework regions and CDR regions are all derived from sequences of human origin. Human antibodies can also be separated from synthetic libraries or transgenic mice (e.g., Xenomouse, OmniMouse, Harbour mice, ATX-Gx mice, Trianni mice), provided that each system produces antibodies with variable regions, wherein the framework regions and CDR regions are all derived from sequences of human origin. In addition, if the antibody contains a constant region, the constant region is also derived from such sequences. Human origin includes, for example, human germline sequences, or mutant versions of human germline sequences, or antibodies containing consensus framework sequences derived from human framework sequence analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296:57-86).

[0086] "Humanized antibody" or "humanized antibody fragment" is defined herein as an antibody molecule having a constant antibody region derived from a sequence of human origin, and a variable antibody region or portion thereof or only the CDRs derived from another species. For example, a humanized antibody can be CDR-grafted, wherein the CDRs of the variable domains are from a non-human source, while one or more frameworks of the variable domains are of human origin, and the constant domains (if any) are of human origin.

[0087] The term "chimeric antibody" or "chimeric antibody fragment" is defined herein as an antibody molecule having constant antibody regions derived from or corresponding to sequences found in one species and variable antibody regions derived from another species. Preferably, the constant antibody regions are derived from or correspond to sequences found in humans, and the variable antibody regions (e.g., VH, VL, CDR or FR regions) are derived from sequences found in non-human animals (e.g., mouse, rat, rabbit or hamster).

[0088] The term "isolated antibody" or "isolated antibody fragment" refers to an antibody or antibody fragment that is substantially free of other antibodies or antibody fragments having different antigenic specificities. In addition, an isolated antibody or antibody fragment may be substantially free of other cellular material and / or chemicals. Thus, in some aspects, the antibody provided is an isolated antibody that has been separated from antibodies having different specificities. The isolated antibody may be a monoclonal antibody. The isolated antibody may be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds to an epitope, subtype, or variant of a target may have cross-reactivity with other related antigens, such as antigens from other species (e.g., species homologs).

[0089] As used herein, the term "recombinant antibody" or "recombinant antibody fragment" includes all antibodies or antibody fragments that are prepared, expressed, created or isolated by a method not found in nature. For example, antibodies isolated from host cells transformed to express antibodies, antibodies selected and isolated from recombinant, combinatorial human antibody libraries, and antibodies prepared, expressed, created or isolated by any other method involving splicing all or part of a human immunoglobulin gene sequence to other DNA sequences, or antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal to human immunoglobulin genes or hybridomas prepared therefrom. Preferably, such recombinant antibodies have variable regions in which the framework regions and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when using transgenic animals for human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies, although derived from and related to human germline VH and VL sequences, may not naturally exist in the human antibody germline library in vivo. The recombinant antibody may be a monoclonal antibody.

[0090] The term "monoclonal" as used herein has the meaning commonly given to it in the art, ie, an antibody or antibody fragment (or its corresponding functional fragment) derived from a single clone of antibody-producing cells that recognizes a single epitope on the bound antigen.

[0091] As used herein, an antibody "specifically binds," "binds specifically," "is specific for," or "specifically recognizes" an antigen (e.g., human CD45) if it can distinguish between the antigen and one or more reference antigens, as binding specificity is not absolute but a relative property. For example, a standard ELISA assay or a standard flow cytometry assay can be performed. Scoring can be performed by standard color development (e.g., secondary antibody with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide) or by binding of a secondary antibody labeled with PE or other dyes or markers. Reactions in certain wells are scored by optical density (OD) (e.g., at 450 nm) or by mean or median fluorescence intensity (MFI) in flow cytometry. A typical background (=negative reaction) might be 0.1 OD; a typical positive reaction might be 1 OD. Background and positive reaction MFIs are highly dependent on instrument settings. Positive / negative differences can exceed 10-fold. Typically, binding specificity is determined not using a single reference antigen, but rather using a panel of approximately three to five unrelated antigens, such as milk powder, BSA, transferrin, etc. For flow cytometry, various antigen-negative cells can be used. However, antibodies that specifically bind to an antigen may have cross-reactivity to corresponding orthologous antigens from other species (e.g., species homologs). In certain embodiments, such cross-reactivity to orthologous antigens is even preferred.

[0092] As used herein, the term "affinity" refers to the strength of interaction between a polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts with the target at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0093] The term "epitope" includes any protein region that is specifically recognized by an antibody or antibody fragment thereof or that otherwise interacts with a molecule. Typically, an epitope is a chemically active surface group of a molecule, such as an amino acid or a carbohydrate or sugar side chain, and typically can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Those skilled in the art will appreciate that virtually anything to which an antibody can specifically bind can be an epitope.

[0094] The term "domain" or "protein domain" refers to a region of a protein polypeptide chain that forms a functional unit and / or independently forms a three-dimensional structure.

[0095] The "compositions" of the present disclosure can be used for therapeutic or prophylactic applications. Thus, the present disclosure includes a pharmaceutical composition comprising an antibody or antibody fragment disclosed herein and a pharmaceutically acceptable carrier or excipient. In a related aspect, the present disclosure provides a method for treating inflammatory diseases, autoimmune diseases, hematological malignancies, and potentially other diseases. Such methods comprise the step of administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising an antibody or antibody fragment described herein.

[0096] The present disclosure provides methods of treatment comprising administering to a subject in need of such treatment a therapeutically effective amount of an antibody or antibody fragment disclosed herein. As used herein, "therapeutically effective amount" or "effective amount" refers to the amount of an anti-CD45 antibody necessary to elicit a desired biological response. According to the present disclosure, a therapeutically effective amount is the amount of an anti-CD45 antibody necessary to treat and / or prevent a disease.

[0097] "Administered" or "administration" includes, but is not limited to, delivering a drug by an injectable form (such as, for example, intravenous, intramuscular, intradermal or subcutaneous routes) or a mucosal route (e.g., as a nasal spray or an aerosol for inhalation) or as an ingestible solution, capsule or tablet. Preferably, administration is by an injectable form.

[0098] As used herein, "treatment," "treat," or "treating" refers to a clinical intervention that attempts to alter the natural course of a disease in a subject being treated, and can be used for prevention or during clinical pathology. The desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies or antibody fragments according to the present disclosure are used to delay the development of a disease or slow the progression of a disease.

[0099] "Preventing" or "prevention" refers to reducing the risk of contracting or developing a disease (i.e., preventing the development of at least one clinical symptom of a disease in a subject who may be exposed to a disease-causing agent or who is susceptible to the disease before the onset of the disease). "Prevention" also refers to an approach intended to prevent the onset of a disease or its symptoms or to delay the onset of a disease or its symptoms.

[0100] As used herein, "subject" or "species" refers to any mammal, including rodents such as mice or rats, and primates such as crab monkeys (Macaca fascicularis), marmosets (Marmoset fascicularis), rhesus monkeys (Macaca mulatta), or humans (Homo sapiens). Preferably, the subject is a primate, most preferably a human.

[0101] The term "effector function" refers to those biological activities attributed to the Fc region of an antibody, which vary with the antibody isotype. Non-limiting examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and direct cell activation or direct cell inhibition.

[0102] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bind to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. NK cells, the primary cells used to mediate ADCC, express only FcγRIII, while monocytes / macrophages express FcγRI, FcγRII, and FcγRIII.

[0103] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (CIq) to an antibody (of the appropriate subclass) of the present disclosure bound to its cognate antigen.

[0104] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells are eliminated by internalization by phagocytes, such as macrophages or dendritic cells.

[0105] As used herein, the term "antibody-drug conjugate" or "ADC" refers to an antibody or antibody fragment chemically linked to a second chemical moiety, such as a therapeutic or cytotoxic agent.

[0106] As used herein, the term "cytotoxic drug" is art-recognized and includes, but is not limited to, daunorubicin, mitoxantrone, doxorubicin, cucurbitacin, chaetocin, globotomycin, chlamydomycin, calicheamicin, maytansine, nemorubicin, cryptophyscin, mensacarcin, ansamitocin, mitomycin C, geldanamycin, mechercharmycin, rebeccamycin, safracin, okilactomycin, oligomycin, actinomycin, yamamyocin, saframy ... ampicillin, hypothemycin, polyketomycin, hydroxyellipticine, thiocolchicine, methotrexate, triptolide, taltobulin, lactacystin, dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), temostatin, tubastatin A, combretatin, maytansinoids, MMAD, MMAF, DM1, DM4, DTT, 16-GMB-APA-GA, 17-DMAP-GA, JW 55, pyrrolobenzodiazepine SN-38, Ro 5-3335, puwainaphycin, duocarmycin, bafilomycin, taxane, tubulysin, ferulic acid, lusiol A, fumagillin, hygrolidin, glucocorticoidin, amanitin, antrienin, emberinoxin, carboxydihydroxyphalloidin, phalloidin, phytosphongosine, piericide, poronetin, podophyllotoxin, gramicidin A, sanguinarine, sinefungin, herboxidiene, microcolin B, microcystin, muscotoxin A, tolytoxin, triptorelin A, myostrobin, mytoxin B, nocuolin A, psuedolaric acid B, pseurotin A, cyclodopamine dopamine), curvulin, colchicine, aphidicolin, englerin, cordycepin, apoptolide, epothilone A, limaquinone, isotropolone, isofistularin, quinaldopeptin, ixabepilone, aeroplysinin, arruginosin, agrochelin, or epothilone. Exemplary pyrrolobenzodiazepines It's Tessilin.

[0107] Throughout this specification, unless the context requires otherwise, the words “comprise,” “have,” and “include,” and variations thereof such as “comprises,” “comprising,” “has,” “having,” “includes,” and “including,” will be understood to imply the inclusion of stated elements or integers or groups of elements or integers but not the exclusion of any other elements or integers or groups of elements or integers.

[0108] As used herein, the terms "engineered" or "modified" include manipulation of nucleic acids or polypeptides by synthetic methods (e.g., by recombinant techniques, in vitro peptide synthesis, by enzymatic or chemical conjugation of peptides, or some combination of these techniques). Preferably, the antibodies or antibody fragments according to the present disclosure are engineered or modified to improve one or more properties, such as antigen binding, stability, half-life, effector function, immunogenicity, safety, etc.

[0109] As used herein, "variant" refers to a polypeptide that differs from a reference polypeptide by one or more modifications, such as amino acid substitutions, insertions, or deletions. A variant polypeptide typically retains most of the properties of the reference polypeptide, such as binding to a target antigen, but introduces new, additional features or properties, such as a variant polypeptide having a higher affinity for the target antigen than the reference polypeptide, or a variant polypeptide that is a humanized version of a reference polypeptide.

[0110] The term "amino acid mutation" as used herein is intended to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be performed, as long as the final construct has the desired properties, such as reduced binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions of the N-terminus and / or C-terminus of an amino acid residue. A special amino acid mutation is an amino acid substitution. Amino acid substitutions include replacements by non-natural amino acids or by naturally occurring amino acid derivatives of twenty standard amino acids. Amino acid mutations can be produced using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is envisioned that methods for changing the side chain groups of amino acid residues by methods other than genetic engineering (e.g., chemical modification) may also be useful. Various names can be used herein to indicate the same amino acid mutation. For example, the glycine at position 237 of the antibody Fc region is substituted with alanine, which can be indicated as 237A, G237, G237A, or Gly237Ala.

[0111] As used herein, the term "EC50" refers to the concentration of an antibody or antibody fragment that induces a response halfway between baseline and maximum in an assay. Thus, it represents the concentration of antibody or ligand at which 50% of the maximal effect is observed.

[0112] As used herein, the term "Ka" refers to the on-rate of a particular antibody-antigen interaction.

[0113] The term "Kd" as used herein refers to the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using methods established in the art.

[0114] As used herein, the term "KD" refers to the dissociation constant of a specific antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka), and is expressed as a molar concentration. A preferred method for determining the Kd of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore system, or biolayer interferometry using an Octet BLI instrument.

[0115] The terms "inhibition" or "inhibit" or "reduction" or "reduce" or "neutralization" or "neutralize" refer to a reduction or cessation of any phenotypic characteristic (e.g., binding or biological activity or function), or a reduction or cessation of the incidence, extent, or likelihood of that characteristic. "Inhibition," "reduction," or "neutralization" need not be complete, as long as it is detectable using an appropriate assay. In some embodiments, "reduction" or "inhibit" or "neutralize" refers to the ability to cause a reduction of 20% or greater. In another embodiment, "reduction" or "inhibit" or "neutralize" refers to the ability to cause a reduction of 50% or greater. In yet another embodiment, "reduction" or "inhibit" or "neutralize" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or greater.

[0116] As used herein, the term "antagonist" antibody refers to an antibody or antibody fragment that interacts with an antigen and partially or completely inhibits or neutralizes the biological activity or function or any other phenotypic characteristic of the target antigen.

[0117] A "wild-type" protein is a version or variant of a protein found in nature. The amino acid sequence of a wild-type protein (e.g., the Fc region of a human IgG1 antibody) is the amino acid sequence of that protein as it occurs in nature. Due to allotypic differences, a wild-type protein may have more than one amino acid sequence. For example, there are several naturally occurring allotypes of the human IgG1 heavy chain constant region (see, e.g., Jeffries et al. (2009) mAbs 1:1).

[0118] "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain. The Fc region of an immunoglobulin typically contains two constant domains, a CH2 domain and a CH3 domain. Although the boundaries of the IgG heavy chain Fc region may vary slightly, the human IgG heavy chain Fc region is typically defined as extending from Cys226 or from Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may or may not be present. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Various Fc modifications are commonly used. For a review, see, for example, Antibodies (2020) 9: 64. Silent functions include (numbered according to the EU index) LALA (L234A / L235A), PA-LALA (L234A / L235A / P329A) and PG-LALA (L234A / L235A / P329G) mutations, as well as AEASS mutations (L234A / L235E / G237A / A330S / P331S). The preferred FC modification is PA-LALA. The mutation may also be one that results in reduced binding to FcRn, thereby reducing the in vivo half-life of the antibody. Such mutations include I253A, H310A, H435A, and H435Q. Alternatively, the mutation may be one that results in increased binding to FcRn, thereby increasing the in vivo half-life of the antibody. Such mutations include T250Q / M428L, M252Y / S254T / T256E(YTE), H433K / N434F, and M252Y / S254T / T256E / H433K / N434F. DETAILED DESCRIPTION

[0119] peptides

[0120] In certain embodiments, the present disclosure relates to antibodies or antibody fragments specific for CD45. In certain embodiments, the present disclosure relates to antibodies or antibody fragments specific for human CD45. In certain embodiments, the present disclosure relates to humanized antibodies or antibody fragments specific for CD45. In certain embodiments, the present disclosure relates to humanized antibodies or antibody fragments specific for human CD45.

[0121] In certain embodiments, the present disclosure relates to humanized antibodies or antibody fragments that are specific for a polypeptide consisting of the amino acid sequence of SEQ ID No. 1. In certain embodiments, the present disclosure relates to humanized antibodies or antibody fragments that are specific for a polypeptide comprising the amino acid sequence of SEQ ID No. 1.

[0122] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0123] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein a glycine in the LCDR1 region (SEQ ID NO: 7) of the humanized antibody or antibody fragment is replaced by an alanine.

[0124] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the second asparagine in the LCDR1 region (SEQ ID NO: 7) of the humanized antibody or antibody fragment is replaced by lysine.

[0125] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the arginine in the LCDR2 region (SEQ ID NO: 8) of the humanized antibody or antibody fragment is replaced by leucine.

[0126] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein a glycine in the LCDR1 region (SEQ ID NO: 7) of the humanized antibody or antibody fragment is replaced with an alanine, wherein the second asparagine in the LCDR1 region (SEQ ID NO: 7) of the humanized antibody or antibody fragment is replaced with a lysine, and wherein an arginine in the LCDR2 region (SEQ ID NO: 8) of the humanized antibody or antibody fragment is replaced with a leucine.

[0127] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises a mutation from the second glycine of the triple glycine motif in framework region 4 of the variable light chain to glutamine.

[0128] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the glycine in the LCDR1 region (SEQ ID NO: 7) of the humanized antibody or antibody fragment is replaced by an alanine, wherein the second asparagine in the LCDR1 region (SEQ ID NO: 7) of the humanized antibody or antibody fragment is replaced by a lysine, wherein the arginine in the LCDR2 region (SEQ ID NO: 8) of the humanized antibody or antibody fragment is replaced by a leucine, and wherein the humanized antibody or antibody fragment comprises a mutation from the second glycine of the triple glycine motif of framework region 4 of the variable light chain to a glutamine.

[0129] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 63, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0130] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 65, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0131] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0132] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 69, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0133] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 71, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0134] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 73, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0135] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 75, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0136] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 77, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0137] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0138] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0139] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 83, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0140] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 85, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0141] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 87, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0142] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 89, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0143] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 91, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0144] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9.

[0145] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9.

[0146] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 95, and a LCDR3 region of SEQ ID NO: 9.

[0147] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9.

[0148] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 97, and a LCDR3 region of SEQ ID NO: 9.

[0149] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9.

[0150] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9.

[0151] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 69, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9.

[0152] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 75, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9.

[0153] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0154] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0155] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9.

[0156] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 110, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9.

[0157] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9.

[0158] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9.

[0159] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9.

[0160] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 34.

[0161] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 34.

[0162] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 23 and a variable light chain of SEQ ID NO: 34.

[0163] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 38.

[0164] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 39.

[0165] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 40.

[0166] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 41.

[0167] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 42.

[0168] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 43.

[0169] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 44.

[0170] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 45.

[0171] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 46.

[0172] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 47.

[0173] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 48.

[0174] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 49.

[0175] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 50.

[0176] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 51.

[0177] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 52.

[0178] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 53.

[0179] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 54.

[0180] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 55.

[0181] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 56.

[0182] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 57.

[0183] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 58.

[0184] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 60 and a variable light chain of SEQ ID NO: 58.

[0185] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 60 and a variable light chain of SEQ ID NO: 59.

[0186] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 98.

[0187] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 99.

[0188] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 100.

[0189] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 101.

[0190] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 102.

[0191] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 103.

[0192] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104.

[0193] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45, wherein the humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 105.

[0194] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises 6 (six) CDRs of one of the antibodies disclosed in Table 14.

[0195] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises 6 (six) CDRs as defined by Kabat of one of the antibodies disclosed in Table 14.

[0196] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises 6 (six) CDRs as defined by IMGT of one of the antibodies disclosed in Table 14.

[0197] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises the variable heavy chain and variable light chain of one of the antibodies disclosed in Table 14.

[0198] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is a monoclonal antibody or antibody fragment.

[0199] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is a recombinant antibody or antibody fragment.

[0200] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is of the IgG isotype.

[0201] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is of the IgG1 class.

[0202] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is of human IgG1 class.

[0203] In certain embodiments, the antigen binding region of the antibodies disclosed herein is used as scFv. Such antibodies are particularly useful for certain applications, such as in CAR or on LNP. Not all antigen binding regions are equally suitable for use as scFv, mainly because of low expression levels. Antibodies that can be well expressed as scFv include Jelly (VH2 / VL5.22), LadyRosetta (VH2 / VL5.38), Levinata (VH2 / VL5.39), and Osira (VH2 / VL5.40).

[0204] Thus, in certain embodiments, the present disclosure relates to a scFv comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9.

[0205] In certain embodiments, the present disclosure relates to a scFv comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 120, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9.

[0206] In certain embodiments, the present disclosure relates to a scFv comprising the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 58.

[0207] In certain embodiments, the present disclosure relates to a scFv comprising a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 121.

[0208] In certain embodiments, the present disclosure relates to a scFv comprising the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 122.

[0209] In certain embodiments, the present disclosure relates to a scFv comprising the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 123.

[0210] The antibody or antibody fragment according to the separation of the present disclosure may or may not be fused with one or more other amino acid residues, polypeptide or part. Such fusion proteins can be prepared in any suitable manner, including genetic methods or chemical methods. The connected part can contain a secretory sequence or a leader sequence, a sequence that helps to detect, express, separate or purify, or a sequence that gives increased protein stability (e.g., during recombinant production). The non-limiting examples of potential parts include beta-galactosidase, glutathione-S-transferase, luciferase, T7 polymerase fragment, secretion signal peptide, antibody or antibody fragment, toxin, cytokine, chemokine, reporter enzyme, a part that can bind metal ions (such as a polyhistidine tag), a label suitable for detection and / or purification, a homologous or heterologous associated domain, a part that increases protein solubility, or a part that comprises an enzymatic cleavage site.

[0211] Thus, an isolated antibody or antibody fragment according to the present disclosure may optionally contain one or more moieties for binding to other targets or target proteins of interest. It should be clear that such further moieties may or may not provide further functionality to the antibody and may or may not alter properties.

[0212] In certain embodiments, the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45 disclosed herein, and (b) a cytotoxic drug. In certain embodiments, the cytotoxic drug is selected from daunorubicin, mitoxantrone, doxorubicin, cucurbitacin, chaetocin, globochaetocin, chlamydomycin, calicheamicin, maytansine, nemorubicin, moniliformin, mensacacin, ansamicin, mitomycin C, geldanamycin, mechercharmycin, rabekamycin, saframycin, oxiramycin, oligomycin, actinomycin, shan Aminomycin, paramycin, polyketide-containing mycin, hydroxyellipticine, thiocolchicine, methotrexate, triptolide, tatoprine, lactacystin, dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), temostatin, tibacterin A, combretatin, maytansinoids, MMAD, MMAF, DM1, DM4, DTT, 16-GMB-APA-GA, 17-DMAP-GA, JW 55, pyrrolobenzodiazepine , SN-38, Ro 5-3335, puranafilin, duocarmycin, bafilomycin, taxane, tubulysin, ferulicidin, lusiol A, fumagillin, hygromycin, glucocidin, amanitin, antrienol, emberizin, carboxydihydroxyphalloidin, phalloidin, phytosphingosine, piericide, poronetin, podophyllotoxin, gramicidin A, sanguinarine, sinefungin, herbicide, microcystin B, microcystin, muscotoxin A, monocystin, triptorelin A, myoblastin, mytoxin B, nocuolin A, ceramide B, pseudoneurin A, cyclodopamine, curvulin, colchicine, aphidicolin, engellin, cordycepin, apoptin, epothilone A, limaquinone, isotropolone, isofistularin, quinaldol, ixabepilone, aeroplysinin, arruginosin, agrochelin and epothilone. In certain embodiments, the cytotoxic drug is a pyrrolobenzodiazepine In other embodiments, the pyrrolobenzodiazepine It's Tessilin.

[0213] The antibody-drug conjugate comprises (a) a humanized antibody or antibody fragment specific for human CD45 disclosed herein, and (b) a cytotoxic drug.

[0214] In certain embodiments, the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein the humanized antibody or antibody fragment specific for human CD45 comprises:

[0215] i. A variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 63, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0216] ii. a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 65, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0217] iii. a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 67, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0218] iv. a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 69, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0219] v. a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 71, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0220] vi. A variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 73, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0221] vii. A variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 75, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0222] viii. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 77, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0223] ix. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 79, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0224] x. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 81, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0225] xi. a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 83, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0226] xii. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 85, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0227] xiii. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 87, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0228] xiv. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 89, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0229] xv. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 91, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0230] xvi. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 93, and the LCDR3 region of SEQ ID NO: 9;

[0231] xvii. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 94, and the LCDR3 region of SEQ ID NO: 9;

[0232] xviii. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 95, and the LCDR3 region of SEQ ID NO: 9;

[0233] xix. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 7, the LCDR2 region of SEQ ID NO: 96, and the LCDR3 region of SEQ ID NO: 9;

[0234] xx. A variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 97, and a LCDR3 region of SEQ ID NO: 9;

[0235] xxi. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 67, the LCDR2 region of SEQ ID NO: 94, and the LCDR3 region of SEQ ID NO: 9;

[0236] xxii. a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9;

[0237] xxiii. a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 69, the LCDR2 region of SEQ ID NO: 94, and the LCDR3 region of SEQ ID NO: 9;

[0238] xxiv. a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 75, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0239] xxv. a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0240] xxvi. a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 108, the LCDR2 region of SEQ ID NO: 8, and the LCDR3 region of SEQ ID NO: 9;

[0241] xxvii. A variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0242] xxviii. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 110, the LCDR2 region of SEQ ID NO: 96, and the LCDR3 region of SEQ ID NO: 9;

[0243] xxix. A variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 96, and the LCDR3 region of SEQ ID NO: 9;

[0244] xxx. a variable heavy chain comprising the HCDR1 region of SEQ ID NO: 4, the HCDR2 region of SEQ ID NO: 5, the HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising the LCDR1 region of SEQ ID NO: 106, the LCDR2 region of SEQ ID NO: 93, and the LCDR3 region of SEQ ID NO: 9; or

[0245] xxxi. A variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9.

[0246] In a preferred embodiment, the cytotoxic drug is a pyrrolobenzodiazepine In other preferred embodiments, the pyrrolobenzodiazepine For Tessilin.

[0247] In certain embodiments, the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein the humanized antibody or antibody fragment specific for human CD45 comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9.

[0248] In certain embodiments, the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein the humanized antibody or antibody fragment specific for human CD45 comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9, and wherein the cytotoxic drug is a pyrrolobenzodiazepine .

[0249] In certain embodiments, the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein the humanized antibody or antibody fragment specific for human CD45 comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9, and wherein the cytotoxic drug is tecillin.

[0250] In certain embodiments, the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein the humanized antibody or antibody fragment specific for human CD45 comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104.

[0251] In certain embodiments, the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein the humanized antibody or antibody fragment specific for human CD45 comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104, and wherein the cytotoxic drug is a pyrrolobenzodiazepine .

[0252] In certain embodiments, the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45, and (b) a cytotoxic drug, wherein the humanized antibody or antibody fragment specific for human CD45 comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104, and wherein the cytotoxic drug is tecillin.

[0253] Nucleic Acids

[0254] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding the isolated antibody or antibody fragment specific for human CD45 of the present disclosure.

[0255] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises:

[0256] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 63, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0257] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 65, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0258] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0259] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 69, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0260] e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 71, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0261] f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 73, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0262] g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 75, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0263] h) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 77, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0264] i) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0265] j) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0266] k) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 83, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0267] 1) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 85, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0268] m) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 87, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0269] n) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 89, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0270] o) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 91, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0271] p) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9;

[0272] q) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9;

[0273] r) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 95, and a LCDR3 region of SEQ ID NO: 9;

[0274] s) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0275] t) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 97, and a LCDR3 region of SEQ ID NO: 9;

[0276] u) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9;

[0277] v) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9;

[0278] w) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 69, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9;

[0279] x) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 75, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0280] y) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0281] z) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0282] aa) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0283] bb) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 110, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0284] cc) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0285] dd) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9; or

[0286] ee) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9.

[0287] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises:

[0288] a) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 34;

[0289] b) the variable heavy chain of SEQ ID NO: 22 and the variable light chain of SEQ ID NO: 34;

[0290] c) the variable heavy chain of SEQ ID NO: 23 and the variable light chain of SEQ ID NO: 34;

[0291] d) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 38;

[0292] e) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 39;

[0293] f) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 40;

[0294] g) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 41;

[0295] h) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 42;

[0296] i) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 43;

[0297] j) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 44;

[0298] k) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 45;

[0299] 1) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 46;

[0300] m) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 47;

[0301] n) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 48;

[0302] o) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 49;

[0303] p) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 50;

[0304] q) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 51;

[0305] r) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 52;

[0306] s) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 53;

[0307] t) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 54;

[0308] u) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 55;

[0309] v) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 56;

[0310] w) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 57;

[0311] x) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 58;

[0312] y) a variable heavy chain of SEQ ID NO: 60 and a variable light chain of SEQ ID NO: 58;

[0313] z) a variable heavy chain of SEQ ID NO: 60 and a variable light chain of SEQ ID NO: 59;

[0314] aa) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 98;

[0315] bb) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 99;

[0316] cc) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 100;

[0317] dd) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 101;

[0318] ee) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 102;

[0319] ff) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 103;

[0320] gg) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 104; or

[0321] hh) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 105.

[0322] In one embodiment, the present disclosure relates to a nucleic acid composition comprising one or more nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises six (six) CDRs as defined by Kabat of one of the antibodies disclosed in Table 14.

[0323] In one embodiment, the present disclosure relates to a nucleic acid composition comprising one or more nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises 6 (six) CDRs as defined by IMGT of one of the antibodies disclosed in Table 14.

[0324] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding an isolated antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises the variable heavy chain and variable light chain of one of the antibodies disclosed in Table 13.

[0325] In one embodiment, the nucleic acid composition and / or the nucleic acid sequence and / or the plurality of nucleic acid sequences are isolated.

[0326] carrier

[0327] In one embodiment, the present disclosure provides a vector composition comprising one or more vectors, wherein the one or more vectors comprise a nucleic acid composition, wherein the nucleic acid composition comprises one or more nucleic acid sequences, wherein the one or more nucleic acid sequences encode a humanized antibody or antibody fragment specific for human CD45 according to the present disclosure.

[0328] In one embodiment, the present disclosure provides a vector composition comprising one or more vectors, wherein the one or more vectors comprise a nucleic acid composition, wherein the nucleic acid composition comprises one or more nucleic acid sequences, wherein the one or more nucleic acid sequences encode any one of the humanized antibodies or antibody fragments specific for human CD45 disclosed in Table 14.

[0329] host cells

[0330] In one embodiment, the present disclosure provides a host cell comprising a vector composition comprising one or more vectors, wherein the one or more vectors comprise a nucleic acid composition, wherein the nucleic acid composition comprises one or more nucleic acid sequences, wherein the one or more nucleic acid sequences encode a humanized antibody or antibody fragment specific for human CD45 according to the present disclosure.

[0331] In one embodiment, the present disclosure relates to a host cell comprising a vector composition comprising one or more vectors, or one or more vectors comprising a nucleic acid composition comprising one or more nucleic acid sequences encoding any one of the humanized antibodies or antibody fragments specific for human CD45 disclosed in Table 14.

[0332] In one embodiment, the host cell according to the present disclosure is capable of expressing a humanized antibody or antibody fragment specific for human CD45 encoded by the vector composition or nucleic acid composition.

[0333] In a further embodiment, the host cell is an isolated host cell. In a further embodiment, the host cell is a mammalian cell. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a CHO cell. In one embodiment, the cell is a HEK cell. In another embodiment, the cell is a PERC.6 cell. In one embodiment, the cell is a HKB11 cell.

[0334] Those skilled in the art will recognize that the nucleic acid sequence or sequences encoding the heavy and / or light chains of the antibodies or antibody fragments of the present disclosure can be cloned into different vectors or into the same vector.

[0335] The vector can be introduced into an appropriate host cell, such as a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a yeast or mammalian cell) by methods well known in the art (see, for example, "Current Protocol in Molecular Biology," Ausubel et al. (eds.), Greene Publishing Assoc and John Wiley Interscience, New York, 1989 and 1992). A variety of cloning vectors are known to those skilled in the art, and selecting an appropriate cloning vector is a matter of choice. The gene can be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and an optional operator (collectively referred to herein as "control" elements) so that the nucleic acid sequence encoding the desired protein is transcribed into RNA in a host cell transformed with a vector containing the expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. After expression in the host cell, the antibody or antibody fragment of the present disclosure is obtained. As is known to those skilled in the art, these steps can be achieved in different ways. In general, such steps typically include transforming or transfecting a suitable host cell with a nucleic acid composition or vector composition or infectious particles encoding the antibody or antibody fragment. Further, such steps are generally included in culturing the host cell under conditions suitable for the host cell proliferation (breeding, growth), and a culturing step under conditions suitable for producing (expressing, synthesizing) the encoded antibody or antibody fragment. Cultivating host cells under conditions suitable for proliferation or expression is generally accomplished in the presence of a culture medium comprising components suitable for cell growth or induced expression. In a particular embodiment, the method for producing an antibody or antibody fragment of the present invention further comprises the step of separating and purifying the antibody or antibody fragment produced from the host cell or culture medium. If the expression system secretes the protein into the growth medium, the protein can be directly purified from the culture medium. If the protein is not secreted, it is separated from the cell lysate or recovered from the cell membrane fraction. Selecting suitable growth conditions and recovery methods is within the capabilities of those skilled in the art. Then, the antibody or antibody fragment of the present invention can be purified by various techniques well known to those skilled in the art.

[0336] In one embodiment, the present disclosure relates to a method for producing a humanized antibody or antibody fragment specific for human CD45 of any of the antibodies disclosed in Table 14. In one embodiment, a method for producing an isolated antibody or antibody fragment according to the present disclosure is provided, wherein the method comprises culturing a host cell under conditions suitable for expression of the antibody or antibody fragment, the host cell comprising a vector composition comprising one or more vectors, the one or more vectors comprising a nucleic acid composition comprising one or more nucleic acid sequences encoding an antibody or antibody fragment according to the present disclosure, and isolating the antibody or antibody fragment from the host cell or host cell culture medium. The isolated antibody or antibody fragment as described herein can be purified by techniques known in the art, such as high performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The conditions used to purify a particular antibody or antibody fragment will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen to which the antibody or antibody fragment can bind can be used. For example, for affinity chromatography purification of antibodies or antibody fragments according to the present disclosure, a matrix with protein A or protein G can be used. The purity of the antibody or antibody fragment can be determined by a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.

[0337] Specificity

[0338] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45. In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human CD45 disclosed in Table 14. In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for a polypeptide encoded by the amino acid sequence of SEQ ID NO: 1. In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0339] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment is conjugated to an antibody Cross-competition for binding to CD45. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment cross-competes for binding to CD45 with an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0340] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment is conjugated to an antibody Binds to the same epitope on CD45. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment binds to the same epitope on CD45 as an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0341] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment retains the In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment retains the binding specificity of an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0342] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment is expressed in a manner similar to that of the antibody In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment binds to CD45 with about the same affinity as an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0343] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment is expressed in a manner comparable to that of the antibody. Higher affinity binding to CD45. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment disclosed herein that is specific for CD45, wherein the antibody or antibody fragment binds to CD45 with a higher affinity than an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0344] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment binds to CD45 with an affinity that is at least two-fold greater than that of an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, and wherein the humanized antibody or antibody fragment comprises at least one of the following mutations:

[0345] a) the first asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by arginine, or

[0346] b) Arginine in the LCDR2 region (SEQ ID NO: 8) was replaced by leucine.

[0347] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment has a higher affinity than the antibody. Fewer critical deamidation sequence motifs. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment has a fewer number of critical deamidation sequence motifs than an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0348] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment has a higher affinity than the antibody. In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment has a lower number of key sequence motifs for T cell epitopes than an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0349] Biological function

[0350] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment is functionally equivalent to the antibody In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment is functionally equivalent to an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0351] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment inhibits the biological function of CD45 to at least the same degree as the antibody In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment inhibits the biological function of CD45 to at least the same extent as an antibody or antibody fragment comprising a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9.

[0352] In another embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45 as disclosed herein, wherein the antibody or antibody fragment is conjugated to a toxin and thereby inhibits the proliferation of CD45-positive cells.

[0353] Effector function

[0354] The Fc region of an immunoglobulin generally confers favorable pharmacokinetic properties to antibodies, such as a prolonged half-life in serum and the ability to induce effector functions by binding to Fc receptors expressed on cells. On the other hand, binding to Fc receptors may also lead to undesirable activation of certain cell surface receptors, resulting in unwanted cytokine release and severe side effects upon systemic administration.

[0355] Therefore, for certain treatment situations, it is necessary to reduce or eliminate the normal binding of the wild-type Fc region of the antibody (such as wild-type IgG Fc region) to one or more or all Fc receptors and / or to complement components (such as C1q) to reduce or eliminate the ability of the antibody to induce effector functions. For example, it may be necessary to reduce or eliminate the binding of the Fc region of the antibody to one or more or all Fc receptors (such as: FcγRI, FcγRIla, FcγRIIb, FcγRIIIa). Effector functions may include, but are not limited to, one or more of the following: complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen-presenting cell uptake of antigens, binding to NK cells, binding to macrophages, binding to monocytes, binding to polymorphonuclear cells, direct signaling to induce apoptosis, cross-linking of target-bound antibodies, dendritic cell maturation, or T cell priming.

[0356] Reduction or elimination of Fc region binding to Fc receptors and / or C1q is typically achieved by mutating a wild-type Fc region (such as an IgG1 Fc region, more particularly a human IgG1 Fc region), thereby generating a variant or engineered Fc region of the wild-type Fc region, e.g., a variant human IgG1 Fc region. Substitutions that result in reduced binding may be useful. To reduce or eliminate Fc region binding to Fc receptors, non-conservative amino acid substitutions are preferred, i.e., replacement of one amino acid with another having different structural and / or chemical properties.

[0357] Thus, in one embodiment, an isolated antibody or antibody fragment specific for human CD45 according to the present disclosure comprises a variant Fc region that has reduced or eliminated binding to an Fc receptor and / or C1q compared to a wild-type Fc region. In one such embodiment, an isolated antibody or antibody fragment according to the present disclosure comprises a variant Fc region that reduces or eliminates the ability of the antibody to induce effector function. In a further embodiment, an isolated antibody or antibody fragment according to the present disclosure does not substantially induce effector function.

[0358] In certain embodiments, the effector function is one or more selected from CDC, ADCC, and ADCP. In one embodiment, the effector function is ADCC. In one embodiment, the effector function is CDC. In one embodiment, the effector function is ADCP. In one embodiment, the antibody or antibody fragment separated according to the present disclosure does not substantially induce ADCC and / or CDC and / or ADCP. In one embodiment, the antibody or antibody fragment separated according to the present disclosure does not induce ADCC or ADCP in vitro.

[0359] In one embodiment, the variant Fc region of the isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions, which, when compared to the wild-type Fc region, reduce or eliminate the binding of the variant Fc region to one or more Fc receptors and / or C1 q. In one embodiment, the variant Fc region of the isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions, which, when compared to the wild-type Fc region, reduce or eliminate the ability of the antibody to induce effector functions. In a particular embodiment, when compared to the wild-type Fc region, the one or more amino acid substitutions can reduce the binding affinity of the variant Fc region to one or more Fc receptors and / or C1 q by at least 2 times, at least 5 times, at least 10 times, at least 20 times or even at least 50 times. In an alternative embodiment, when compared to the wild-type Fc region, the one or more amino acid substitutions can reduce the ability of the isolated antibody or antibody fragment according to the present disclosure to induce effector functions by at least 2 times, at least 5 times, at least 10 times, at least 20 times or even at least 50 times.

[0360] In one embodiment, the variant Fc region of the isolated antibody or antibody fragment according to the present disclosure does not substantially bind to one or more Fc receptors and / or C1q. In one embodiment, the variant Fc region of the antibody according to the present disclosure does substantially eliminate the ability of the antibody to induce effector functions. In one embodiment, the antibody or antibody fragment according to the present disclosure does not substantially induce effector functions. In one embodiment, the effector function is ADCC and / or ADCP and / or CDC. In one embodiment, the antibody or antibody fragment according to the present disclosure does not substantially induce effector function, meaning that the level of induced effector function is not significantly higher than the background measured in the absence of the antibody.

[0361] In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is human FcγRIIIa, FcγRI, FcγRIla and / or FcγRIlb.

[0362] In one embodiment, an isolated antibody or antibody fragment according to the present disclosure comprises a variant human IgG1 Fc region comprising one or more amino acid substitutions compared to a wild-type human IgG1 Fc region. In one embodiment, the one or more amino acid substitutions reduce or eliminate binding of the variant Fc region to an Fc receptor and / or C1q and / or reduce the ability of the antibody to induce effector function when compared to a wild-type Fc region.

[0363] Various Fc modifications are commonly used. For a review, see, for example, Antibodies (2020) 9:64. Functional silencing includes (numbered according to the EU index) LALA (L234A / L235A), PA-LALA (L234A / L235A / P329A), and PG-LALA (L234A / L235A / P329G) mutations, as well as AEASS mutations (L234A / L235E / G237A / A330S / P331S).

[0364] In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibodies have a silent Fc region. In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibodies or antibody fragments are in AEASS format.

[0365] In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibody comprises an Fc region in which at least the following modifications are present: L234A, L235E, G237A, A330S, P331S (numbering according to the EU index). In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibody comprises an Fc region in which the following modifications are present: L234A, L235E, G237A, A330S, P331S (numbering according to the EU index).

[0366] In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibody or antibody fragment is in the form of PA-LALA. In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibody comprises an Fc region in which at least the following modifications are present: L234A, L235A, P329A (numbering according to the EU index). In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibody comprises an Fc region in which at least the following modifications are present: L234A, L235A, P329A (numbering according to the EU index).

[0367] In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibodies or antibody fragments are in the PG-LALA format. In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibodies comprise an Fc region in which at least the following modifications are present: L234A, L235A, P329G (numbering according to the EU index). In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibodies comprise an Fc region in which at least the following modifications are present: L234A, L235A, P329G (numbering according to the EU index).

[0368] In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibody or antibody fragment has a mutation that results in reduced binding to FcRn, thereby reducing the in vivo half-life of the antibody. In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibody comprises an Fc region having an I253A mutation (numbered according to the EU index). In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibody comprises an Fc region having an H310A mutation (numbered according to the EU index). In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibody comprises an Fc region having an H435A mutation (numbered according to the EU index). In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibody comprises an Fc region having an H435Q mutation (numbered according to the EU index).

[0369] In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibodies or antibody fragments have mutations that result in increased binding to FcRn, thereby increasing the in vivo half-life of the antibodies. In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibodies comprise T250Q / M428L mutations in the Fc region (numbered according to the EU index). In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibodies comprise M252Y / S254T / T256E(YTE) mutations in the Fc region (numbered according to the EU index). In certain embodiments, the present disclosure relates to humanized antibodies specific for CD45, wherein the humanized antibodies comprise H433K / N434F mutations in the Fc region (numbered according to the EU index). In certain embodiments, the disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises M252Y / S254T / T256E / H433K / N434F mutations in the Fc region (numbering according to the EU index).

[0370] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody comprises:

[0371] a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 63, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0372] b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 65, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0373] c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0374] d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 69, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0375] e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 71, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0376] f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 73, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0377] g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 75, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0378] h) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 77, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0379] i) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 79, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0380] j) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 81, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0381] k) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 83, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0382] 1) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 85, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0383] m) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 87, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0384] n) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 89, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0385] o) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 91, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0386] p) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9;

[0387] q) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9;

[0388] r) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 95, and a LCDR3 region of SEQ ID NO: 9;

[0389] s) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0390] t) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 97, and a LCDR3 region of SEQ ID NO: 9;

[0391] u) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9;

[0392] v) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 67, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9;

[0393] w) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 69, a LCDR2 region of SEQ ID NO: 94, and a LCDR3 region of SEQ ID NO: 9;

[0394] x) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 75, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0395] y) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0396] z) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9;

[0397] aa) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0398] bb) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 110, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0399] cc) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9;

[0400] dd) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9; or

[0401] ee) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9.

[0402] The Fc region of the humanized antibody comprises modifications L234A, L235A and P329A (numbering according to the EU index).

[0403] Treatment

[0404] The antibodies and antibody fragments disclosed herein or pharmaceutical compositions incorporating the antibodies and antibody fragments can be used to treat various conditions. For example, such antibodies can be used to ablate endogenous hematopoietic stem and progenitor cells (HSPCs) in subjects in need. The ablation of endogenous HSPCs is an initial step in stem cell replacement therapy. Stem cell replacement therapy typically involves reducing or eliminating endogenous HSPCs that are defective in some respects and replacing them with alternative HSPCs. Alternative HSPCs can be autologous, allogeneic, or xenogeneic. Endogenous HSPCs may be due to genetic mutations that impair function or expression (e.g., sickle cell anemia or thalassemia), due to hematologic cancers, or due to defects caused by damage caused by chemotherapy used in the treatment of cancer. Endogenous HSPCs may also be replaced together with organ transplantation because endogenous HSPCs can cause immune attacks on the transplant.

[0405] Antibodies against CD45 can also be used in the treatment of cancers that express CD45. Such cancers include blood cancers such as leukemia, myeloma, or lymphoma.

[0406] Antibodies against CD45 can also be used in the treatment of inflammatory or autoimmune diseases such as multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn's disease, type 1 diabetes, rheumatoid arthritis, or idiopathic arthritis.

[0407] In certain embodiments, the present disclosure relates to the use of a humanized antibody or antibody fragment of the present disclosure for use in medicine.

[0408] In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of cancer. In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of hematological cancers. In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of leukemia, myeloma, or lymphoma.

[0409] In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of inflammatory or autoimmune diseases. In other embodiments, the present disclosure relates to the use of the humanized antibodies or antibody fragments of the present disclosure for use in the treatment of multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn's disease, type 1 diabetes, rheumatoid arthritis, or idiopathic arthritis.

[0410] In certain embodiments, the present disclosure relates to the use of a humanized antibody or antibody fragment of the present disclosure for use in the treatment of a disease associated with the undesirable presence of CD45. In certain embodiments, the present disclosure relates to the use of a humanized antibody or antibody fragment of the present disclosure for use in the treatment of a disease associated with the undesirable presence of CD45-positive cells.

[0411] In one embodiment, the present disclosure provides a method for treating a disease.

[0412] In one embodiment, the present disclosure provides a method for treating a disease, comprising administering to a patient an antibody or antibody fragment of the present disclosure.

[0413] In one embodiment, the present disclosure provides a method for treating a disease, comprising administering an antibody or antibody fragment of the present disclosure to a subject in need thereof.

[0414] In one embodiment, the present disclosure provides a method for preventing a disease.

[0415] In one embodiment, the present disclosure provides a method for preventing a disease, comprising administering the antibody or antibody fragment of the present disclosure to a subject.

[0416] In one embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use in treating a disease. In one embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use in the treatment of a disease. In one embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for use in the treatment of a disease in a subject in need thereof.

[0417] In one embodiment, the present disclosure provides an antibody or antibody fragment according to the separation of the present disclosure for use in the manufacture of a medicament. In one embodiment, the present disclosure provides an antibody or antibody fragment according to the separation of the present disclosure for use as a medicament. In one embodiment, the present disclosure provides an antibody or antibody fragment according to the separation of the present disclosure for use in medicine. In one embodiment, the present disclosure provides an antibody or antibody fragment according to the separation of the present disclosure for use as a medicament for treating a subject in need.

[0418] In one embodiment, the present disclosure provides an isolated antibody or antibody fragment specific for human CD45 according to the present disclosure for use in a method of treating a subject having a disease, the method comprising administering to the subject a therapeutically effective amount of the antibody or antibody fragment according to the present disclosure.

[0419] In one embodiment, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent. The subject in need of treatment is typically a mammal, more particularly a human. For use in therapeutic methods, the isolated antibodies or antibody fragments according to the present disclosure will be formulated, dosed, and administered in a manner consistent with good medical practice.

[0420] Pharmaceutical composition

[0421] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or antibody fragment according to the present disclosure and a pharmaceutically acceptable carrier or excipient.

[0422] The antibody is administered at an effective regimen, which refers to a dose, route of administration, and frequency of administration that achieves the intended purpose, such as reduction of endogenous HSPCs, tissue-resident or circulating immune cells, or CD45-expressing cancer cells. In some cases, efficacy can be observed in an individual patient relative to historical controls or prior experience in the same patient. In other cases, efficacy can be demonstrated in a preclinical or clinical trial population of treated patients relative to a control population of untreated patients.

[0423] The pharmaceutical composition may further comprise at least one other pharmaceutically active compound. The pharmaceutical composition according to the present disclosure may be used in the diagnosis, prevention and / or treatment of diseases associated with the undesirable presence of CD45 (particularly human CD45). The pharmaceutical composition according to the present disclosure may be used in the diagnosis, prevention and / or treatment of diseases associated with the undesirable presence of CD45-positive cells (particularly CD45-positive human cells). In particular, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure, which is suitable for prophylactic, therapeutic and / or diagnostic use in mammals (more particularly humans).

[0424] In general, the antibodies or antibody fragments according to the present disclosure can be formulated into pharmaceutical compositions comprising at least one antibody or antibody fragment according to the present disclosure and at least one pharmaceutically acceptable carrier or excipient and optionally one or more further pharmaceutically active compounds. Such formulations can be suitable for oral, parenteral, topical administration or administration by inhalation. Therefore, the pharmaceutical composition comprising at least one antibody or antibody fragment according to the present disclosure can be administered parenterally, such as intravenously, intramuscularly or subcutaneously. Alternatively, the antibodies of the present invention can be administered via non-parenteral routes, such as orally or topically. In a preferred embodiment, the pharmaceutical composition comprising the antibody or antibody fragment according to the present disclosure is administered intravenously or subcutaneously.

[0425] In particular, the antibodies or antibody fragments according to the present disclosure can be used in combination with one or more pharmaceutically active compounds that are used or can be used to prevent and / or treat diseases involving the target antigen of interest, with the result that a synergistic effect may or may not be achieved. Examples of such compounds, as well as routes, methods, and pharmaceutical formulations or compositions for administering them, will be clear to the clinician.

[0426] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure, for use in the prevention and / or treatment of diseases associated with the unwanted presence of CD45 (particularly human CD45). In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure, for use in the prevention and / or treatment of diseases associated with the unwanted presence of CD45-positive cells (particularly CD45-positive human cells). In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure, for use as a medicament. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure, for use in the prevention and / or treatment of autoimmune diseases and / or inflammatory diseases and / or cancer.

[0427] In one embodiment, the present disclosure provides a method of treating autoimmune diseases and / or inflammatory diseases and / or cancer in a subject in need thereof using a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure.

[0428] Further provided is a method for producing an antibody or antibody fragment according to the present disclosure in a form suitable for in vivo administration, the method comprising (a) obtaining an antibody or antibody fragment by a method according to the present disclosure, and (b) formulating the antibody or antibody fragment with at least one pharmaceutically acceptable carrier or excipient, thereby formulating a formulation of the antibody or antibody fragment for in vivo administration. The pharmaceutical composition according to the present disclosure comprises a therapeutically effective amount of one or more antibodies or antibody fragments according to the present disclosure dissolved in a pharmaceutically acceptable carrier or excipient.

[0429] Diagnostic uses

[0430] In one embodiment, the present disclosure provides the use of an antibody or antibody fragment specific for human CD45 according to the present disclosure for diagnosing a disease. In one embodiment, the present disclosure provides the use of an antibody or antibody fragment according to the present disclosure for detecting CD45 (particularly human CD45). In one embodiment, the present disclosure provides a method for detecting CD145 in a subject or sample, comprising the step of contacting the subject or sample with an antibody or antibody fragment specific for human CD45 according to the present disclosure. In one embodiment, the present disclosure provides a method for diagnosing a disease in a subject, comprising the step of contacting the subject or sample with an antibody or antibody fragment isolated according to the present disclosure. The antibody can also be used to determine the level of CD45 expression in patient cells. CD45 expression levels can be used as therapeutic biomarkers, for example, for patient stratification.

[0431] Example

[0432] Example 1: Anti-CD45 antibodies of the present invention

[0433] Antibody Is a mouse anti-CD45 antibody. The sequences of the variable chain and CDRs are shown in the following table:

[0434] Table 1:

[0435]

[0436]

[0437] By a complex method, the inventors of the present application have successfully transformed the antibody Humanization was achieved while simultaneously removing unwanted post-translational modifications and fragmentation sites. Surprisingly, the resulting antibodies did not compromise the affinity and activity of the antibodies. This was achieved through a stepwise approach, as further described in the following examples.

[0438] Example 2: Murine Antibodies Humanization

[0439] The humanization process relies on combining CDR grafting technology with the latest research on antibody structure and the latest databases of mature human IgG sequences. A large number of human framework sequences have been identified and used as "acceptor" frameworks for the target CDR sequences. All acceptor sequences are derived from mature human IgG. As a result, the humanized sequences are expected to be non-immunogenic and retain the canonical structure of the CDR loops.

[0440] Example 2.1: Design of humanized variants of the variable heavy chain

[0441] The closest human germline gene V-region identified in Homo sapiens is IGHV3-23:

[0442]

[0443] A BLAST search algorithm was used to search the human IgG sequence database for comparison with the murine VH domains, and candidate human variable domains were selected from the top 200 BLAST results. These were reduced to four candidates based on a combination of framework homology, maintenance of key framework residues, and canonical loop structure.

[0444] The four acceptor frameworks are:

[0445] >QDF60926

[0446]

[0447] >ABF83355

[0448]

[0449] >QAV55037

[0450]

[0451] >AMK70661

[0452]

[0453] The closest human germline IGHV3-23 (SEQ ID NO. 15) was selected as the fifth receptor sequence.

[0454] Will The CDRs of the mouse VH of α-glucan were transplanted into the aforementioned acceptor framework, and the following humanized variants were selected and synthesized:

[0455] >VH1

[0456]

[0457] >VH2

[0458]

[0459] >VH3*

[0460]

[0461] >VH4*

[0462]

[0463] >VH5*

[0464]

[0465] Figure 1 The humanized variants are shown to be different from the original mouse Sequence alignment. The table below shows the homology of the humanized variants to the original murine VH sequence.

[0466] Table 2:

[0467] The same amino acids consensus amino acids VH1 89.1％ 94.1％ VH2 87.4％ 93.3％ VH3 87.4％ 93.3％ VH4 91.6％ 95.8％ VH5 90.8％ 95.8％

[0468] Example 2.2: Design of humanized variants of the variable light chain

[0469] The closest human germline gene V-region identified in Homo sapiens is IGKV2-30:

[0470]

[0471] A BLAST search algorithm was used to search the human IgK sequence database for comparison with the murine VL domain, and candidate human variable domains were selected from the top 200 BLAST results. These were reduced to four candidates based on a combination of framework homology, maintenance of key framework residues, and canonical loop structure.

[0472] The four acceptor frameworks are:

[0473] >QEP26792

[0474]

[0475] >BAH04700

[0476]

[0477] >ABA26062

[0478]

[0479] >BAH04694

[0480]

[0481] The closest human germline IGKV2-30 (SEQ ID NO. 25) was selected as the fifth receptor sequence.

[0482] Will The CDRs of the mouse VL of α-glucanase were transplanted into the aforementioned acceptor framework, and the following humanized variants were selected and synthesized:

[0483] >VL1

[0484]

[0485] >VL2*

[0486]

[0487] >VL3*

[0488]

[0489] >VL4*

[0490]

[0491] >VL5*

[0492]

[0493] *VL2-VL5 mutations have been introduced to make the antibody more similar to the parent antibody while maintaining humanized nomenclature.

[0494] Figure 2 The humanized variants are shown to be different from the original mouse Sequence alignment. The table below shows the homology of the humanized variants to the original murine VL sequence.

[0495] Table 3:

[0496] The same amino acids consensus amino acids VL1 87.6％ 93.8％ VL2 89.4％ 96.5％ VL3 88.5％ 96.5％ VL4 90.3％ 97.3％ VL5 92.0％ 97.3％

[0497] Example 2.3: Expression and purification of humanized variants

[0498] DNA encoding the amino acid sequence of each antibody was synthesized and cloned into the mammalian transient expression plasmid pETE V2 (Fusion Antibodies, Northern Ireland). Antibodies were expressed using a CHO-based transient expression system, and the resulting cell culture supernatant containing the antibody was clarified by centrifugation and filtered. Antibodies were purified from the cell culture supernatant by affinity chromatography using state-of-the-art chromatography equipment. The purified antibodies were buffer-exchanged into phosphate-buffered saline solution. The purity of these antibodies was determined to be >95% as judged by reducing and denaturing sodium dodecyl sulfate polyacrylamide gels. Antibody concentration was determined by measuring absorbance at 280 nm.

[0499] Combinations of five humanized variable heavy chains and five humanized variable light chains were tested, resulting in a total of twenty-five (25) combinations. Of these 25 combinations, six were successfully produced. These six combinations all included the humanized light chain VL5, regardless of the humanized heavy chain used. In addition, combinations of VH2 and VL3 were successfully produced. These six combinations can be produced in sufficient quantities for further characterization.

[0500] Example 2.4: Affinity determination of humanized variants

[0501] IgG antibodies were immobilized on the biosensor using a suitable capture surface and the binding of soluble antigen to the immobilized antibody was monitored by BLI (Octet). The resulting sensorgrams were analyzed using the supplied software (Fortebio). Human recombinant CD45 from R&D Systems was used as the antigen (Catalog No. 1430-CD-050; Batch No. NUZ142001). As a preliminary experiment, a series of experiments were performed to optimize the assay parameters. The following parameters were selected:

[0502] Table 4:

[0503] Antibody loading concentration 0.15ug / ml Antigen stability @25℃, oscillation @1000rpm >4h Running buffer HBS-P+ with 10uM DTT Time of Contract 1200 seconds Dissociation time 1800 seconds Required kinetic screening protocol Modified Antigen screening range (seven concentrations, 3-fold dilution) 10-0.1563nM Interaction / fitting models 1:1

[0504] In the main experiment, kinetic measurements were first performed by capturing IgG using an anti-human Fc biosensor. The mAb-captured biosensors were then immersed in wells containing different concentrations of antigen (association phase), followed by a dissociation step in running buffer. To allow for dual reference correction, the IgG-captured sensors were immersed in wells containing buffer alone, and blank sensors were immersed in wells containing antigen. This reference provided a means of compensating for the natural dissociation of captured IgG and nonspecific binding of antigen to the sensor surface. The steps were performed at 25°C with constant shaking at 1000 rpm. A new sensor was used for each sample. The dissociation rate constant (KD) was calculated using ForteBio data analysis software. All consumables used were recommended by ForteBio.

[0505] All samples were diluted in freshly prepared running buffer. Antibody variants were immobilized on the surface of a series of biosensors using the capture method described. Antigen was passed over the surface to generate a binding response. Binding data for IgG:antigen interactions were collected on the biosensors at 25°C. An antigen dilution series was used in the binding step in order to globally fit the results and obtain optimal values for ka, kd, and KD. The binding response data for antigen to surface-immobilized IgG were fit to a 1:1 binding model. The kinetic parameters are summarized in the table below. HC0 LC0 refers to mouse

[0506] Table 5:

[0507]

[0508] NF: Not fitted to a 1:1 binding model

[0509] Five antibodies showed Among these five antibodies, four had KD better than That is, the combinations VH1 / VL5, VH2 / VL5, VH3 / VL5, and VH4 / VL5. The combination VH2 / VL3 did not fit a 1:1 binding model and was therefore not explored further.

[0510] Example 2.5: Biophysical Characterization of Three Preselected Antibodies

[0511] It is generally believed that protein aggregation along non-native pathways is caused by protein conformational instability or partial unfolding of the protein. Protein unfolding exposes hydrophobic core residues, which then form dimers with other partially unfolded monomers and ultimately act as nucleation sites for mass aggregation. Therefore, conformational stability indicates the propensity of a protein to non-natively aggregate. Thermal stability is considered a feasible method to monitor protein conformational stability by determining the melting point (Tm) of the protein.

[0512] Differential Scanning Fluorescence (DSF) allows for the monitoring of conformational stability. Multiple studies have found highly correlated data between Differential Scanning Calorimetry (DSC) and DSF, suggesting that DSF can successfully monitor the thermal stability of proteins and ultimately their non-native aggregation propensity. DSF uses a fluorescent dye that is quenched in an aqueous environment but fluoresces under hydrophobic conditions. Therefore, upon thermal exposure, the antibody begins to lose conformational stability and unfold, exposing hydrophobic core residues, which can be measured by an increase in the fluorescence signal. A temperature versus fluorescence (melting curve) plot can then be used to determine the melting temperature of the protein of interest (dF / dT curve).

[0513] Many antibody molecules exhibit a two-phase melting profile. Published studies have shown that mAbs display unique unfolding profiles due to the melting transitions of their various domains (CH2, CH3, and Fab). The thermal stability of IgG molecules is affected by all of these domains. Reported antibody DSF curves show two clearly distinguishable transitions: a lower temperature representing the unfolding of the CH2 domain (Tm1), and a higher temperature representing the melting of the CH3-Fab domain (Tm2). The Fab melting transition is usually well-defined, with a peak that is up to 2-3 times larger than the peaks of either CH2 or CH3.

[0514] For DSF analysis, 5 μl of a solution of Sypro Orange (diluted 1 / 200 in water; Sigma) and 45 μl of 0.3 mg / ml of the test antibody were added to a test tube (Bio-Rad; TLS0831). The tube was sealed with an optical flat cap (Bio-Rad; TCS0803) and heated from 20 to 95°C in 0.5°C increments in an i-CycleriQ5 Real-Time PCR Detection System (Bio-Rad). Fluorescence changes in the wells were monitored simultaneously using a charge-coupled device (CCD) camera. The excitation and emission wavelengths were 485 and 575 nm, respectively. The temperature midpoint, Tm, of the protein unfolding transition was calculated using Bio-Rad iQ5 software.

[0515] For size exclusion chromatography (SEC), samples were diluted to a final concentration of 0.1 mg / ml using phosphate-buffered saline (PBS). Highly purified antibody samples were individually loaded onto a Superdex 200 Increase 10 / 300 GL gel filtration column. 50 μl of sample was injected, and the column flow rate was maintained at 0.75 ml / min. Separation and equilibration steps were performed at 19°C in phosphate-buffered saline. Protein peaks were monitored using absorbance at 214 nm, and spectra were analyzed using the Unicorn evaluation software package (Cytiva).

[0516] Melting curves were successfully generated for all samples. All antibodies tested showed a single phase transition melting profile. VH2 / VL5 exhibited the highest TM1 melting temperature, indicating that this IgG was the most conformationally stable of the tested molecules. Tm values are summarized in the table below.

[0517] All samples were successfully analyzed by SEC. SEC peak quality was good for all samples. All antibodies eluted as a single major peak. Based on column calibration, the major peak corresponded to monomeric IgG.

[0518] Table 6:

[0519] Antibody Tm1[℃] Tm2[℃] %monomer Commentary VH0 / VL0 67.0 N / A >97 Good peak quality VH2 / VL5 76.0 N / A >97 Good peak quality VH3 / VL5 70.5 N / A >98 Good peak quality VH4 / VL5 66.5 N / A >97 Good peak quality

[0520] Example 2.6: Summary and selection of final candidates for further engineering

[0521] All combinations containing VL5 were successfully expressed and purified. In addition, the combination VH2 / VL3 was also successfully expressed and purified. From SDS-PAGE analysis, all antibodies showed sufficient levels of purity. Under reducing conditions, the heavy and light chains of the antibodies were visible and observed at the expected molecular weights of ~50 and 25 kDa, respectively. Under non-reducing conditions, a single major band and multiple minor bands were observed. Additional bands (impurities) may be the result of non-glycosylated IgG and IgG degradation products (e.g., a single [partial] light chain, a combination of two heavy chains and one light chain, two heavy chains, and two heavy chains and one light chain).

[0522] In kinetic (Octet) analysis, all combinations with VL5 (VH1 / VL5, VH2 / VL5, VH3 / VL5, VH4 / VL5, VH5 / VL5) exhibited binding properties similar to the control antibody VH0 / VL0, showing dissociation constants within 2-fold of that of the control antibody.

[0523] The biophysical properties of all antibodies were similar to those of the original Antibodies are just as good.

[0524] For further engineering, we selected antibody VH2 / VL5, which has an affinity of 162 pM, which is higher than the original The IgG antibody (354 pM) was more than 2-fold higher and exhibited the highest melting temperature (TM1), indicating that this IgG was the most conformationally stable of the tested molecules.

[0525] Example 3: Removal of key sites prone to PTM, immunogenicity, increased flexibility or hydrophobicity

[0526] Example 3.1: Design of engineered, humanized variants

[0527] In the next engineering step, the antibody was screened for sites prone to Fv glycosylation, deamidation, isomerization, fragmentation, other types of post-translational modification (PTM) sequence hotspots, increased flexibility or hydrophobicity. Several such sites were identified in the CDRs of the antibody. See the table below.

[0528] Table 7:

[0529]

[0530]

[0531] To remove these deleterious amino acid motifs, variants of the antibody VH2 / VL5 were generated. The approach to each change was rationalized by analysis as described below.

[0532] Suitable variants were identified by computer-calculated sequence- and structure-based descriptors that maintain the structural integrity and biological activity of the antibody while enhancing developability, binding affinity, and solubility. For this purpose, a therapeutic antibody dataset retrieved from the TABS database (https: / / tabs.craic.com / ) and a second dataset consisting of a paired antibody library from the Observed Antibody Space database (http: / / opig.stats.ox.ac.uk / webapps / oas / ) were evaluated for physicochemical descriptors. After alignment of the variable domain sequences with an isotype-specific hidden Markov model (HMM), sequence alignments, antibody numbering, and CDR canonical structures were annotated, as described in Nat Protoc (2014) 9:2771-83. Based on Nucleic Acid Res (2017) 45: W17-W23 and Bioinformatics (2014) 30: the method described in 2733-40, using the template-based method for framework, the canonical structure modeling for CDR and the random forest machine learning modeling method for heavy chain ring H3, a three-dimensional model was constructed. For VH / VL packaging, the template-based method based on the overall similarity and identity of residue position L44 was used, as described in FEBS J. (2011) 278: 2858-66. In the sequence descriptor, the antibody solubility curve is calculated as a linear combination of different physicochemical properties, such as hydrophobicity, static charge at neutral pH, α-helix and β-chain tendency of each amino acid (Nat Struct Biol (1996) 3: 842-8, J Mol Biol (1994) 238: 693-708). To account for the effects of neighboring amino acids, we used a sliding window approach in which each residue solubility descriptor was averaged over a window of seven consecutive amino acids and corrected for the occurrence of polar / non-polar aggregation-prone patterns (J Mol Biol (2000) 296:961-8; Methods Mol Biol (2022) 2313:57-113). Although we did not impose specific target values for local and global solubility during the engineering step, we excluded variants that would lead to the formation of covalent aggregates (cysteines) and solvent-exposed hydrophobic patches, as assessed by the relative solvent accessibility of each residue calculated on a 3D model of a paired VH / VL domain (F1000 Res (2016) 5:189). In addition, variants that would affect the CDR conformation and, therefore, the antibody binding mode were also excluded.This was done by assessing the contribution of the engineered sites to any established canonical structure (Nat Protocol (2014) 9:2771-83) and by evaluating the expected binding modes of native and engineered molecules using per-residue probability scores (Bioinformatics (2013) 29:2285-91). In addition, we excluded / prioritized variants that would result in increased / decreased immunogenicity risk, as assessed by comparing the in silico immunogenicity profiles with those of a large dataset of therapeutic antibodies of natural human repertoire and known immunogenicity (ADA frequency). To this end, we employed a repurposed NetMHCIIpan neural network algorithm to screen native and variant antibody sequences for the presence of class II restricted HLA ligands and putative T cell epitopes (Nucl Acids Res (2020) 48:W449-W454). We calculated global and local immunogenicity profiles, corrected for the presence of HLA ligands shared by the human germline that are likely to be tolerogenic or tolerogenic. Higher priority was given to variants that did not carry neoepitopes (new HLA binders versus native ones) or cross-reactive epitopes (ligands recognized by multiple HLA alleles), and lower priority was given to variants that showed high epitope content in antibody regions that showed low immunogenicity scores in the reference antibody dataset.

[0533] The sequences of the new VH and VL chains are shown below.

[0534] >VL5.2

[0535]

[0536] >VL5.3

[0537]

[0538] >VL5.4

[0539]

[0540] >VL5.5

[0541]

[0542] >VL5.6

[0543]

[0544] >VL5.7

[0545]

[0546] >VL5.8

[0547]

[0548] >VL5.9

[0549]

[0550] >VL5.10

[0551]

[0552] >VL5.11

[0553]

[0554] >VL5.12

[0555]

[0556] >VL5.13

[0557]

[0558] >VL5.14

[0559]

[0560] >VL5.15

[0561]

[0562] >VL5.16

[0563]

[0564] >VL5.17

[0565]

[0566] >VL5.18

[0567]

[0568] >VL5.19

[0569]

[0570] >VL5.20

[0571]

[0572] >VL5.21

[0573]

[0574] >VL5.22

[0575]

[0576] >VL5.23

[0577]

[0578] >VH2.2

[0579]

[0580] The following table shows the SEQ ID No. of the variable light chain including their CDRs.

[0581] Table 9:

[0582]

[0583]

[0584] SEQ ID No.61:RYDVWYYFDV

[0585] SEQ ID No.62: ARRYDVWYYFDV

[0586] SEQ ID No.63: RSSQSIVHSRGNTYLE

[0587] SEQ ID No.64: QSIVHSRGNTY

[0588] SEQ ID No.65: RSSQSIVHSKGNTYLESEQ ID No.66: QSIVHSKGNTY

[0589] SEQ ID No.67: RSSQSIVHSQGNTYLESEQ ID No.68: QSIVHSQGNTY

[0590] SEQ ID No.69: RSSQSIVHSSGNTYLESEQ ID No.70: QSIVHSSGNTY

[0591] SEQ ID No.71:RSSQSIVHSNANTYLE

[0592] SEQ ID No.72: QSIVHSNANTY

[0593] SEQ ID No.73: RSSQSIVHSNGQTYLE

[0594] SEQ ID No.74: QSIVHSNGQTY

[0595] SEQ ID No.75:RSSQSIVHSNGKTYLE

[0596] SEQ ID No.76:QSIVHSNGKTY

[0597] SEQ ID No.77:RSSQSIVHSNGETYLE

[0598] SEQ ID No.78:QSIVHSNGETY

[0599] SEQ ID No.79:RSSQSIVHSNGHTYLE

[0600] SEQ ID No.80:QSIVHSNGHTY

[0601] SEQ ID No.81:RSSQSIVHSNGNKYLE

[0602] SEQ ID No.82:QSIVHSNGNKY

[0603] SEQ ID No.83:RSSQSIVHSQGQTYLE

[0604] SEQ ID No.84:QSIVHSQGQTY

[0605] SEQ ID No.85:RSSQSIVHSQGKTYLE

[0606] SEQ ID No.86:QSIVHSQGKTY

[0607] SEQ ID No.87:RSSQSIVHSSGQTYLE

[0608] SEQ ID No.88:QSIVHSSGQTY

[0609] SEQ ID No.89:RSSQSIVHSKGQTYLE

[0610] SEQ ID No.90:QSIVHSKGQTY

[0611] SEQ ID No.91:RSSQSIVHSNANKYLE

[0612] SEQ ID No.92:QSIVHSNANKY

[0613] SEQ ID No.93:KVSNLFS

[0614] SEQ ID No.94: KVSNRAS

[0615] SEQ ID No.95: KVSNRES

[0616] SEQ ID No.96: KVSNLAS

[0617] SEQ ID No.97: KVSNLES

[0618] Example 3.2: Testing of Engineered, Humanized Variants

[0619] The engineered variants were subjected to the same type of analysis and characterization as the initial humanized variants. For the experimental setup, similar conditions to those in Example 2 were used, with the following differences. Binding affinity was determined using an Octet system Red96e or R8 at 25°C and 1000 rpm using 1x kinetic buffer (Sartorius, PN: 18-1105). The antibody was captured on an anti-human capture biosensor (Sartorius, PN_18-5060) at 0.5-1 ug / mL for 300-600 s. The antigen was titrated at different concentrations ranging from 200 nM to 0.5 nM, and association / interpretation was monitored for 300-600 s and 1000-2000 s. Reference subtraction was performed for wells containing only buffer. The AHC tip was regenerated using 10 mM Gly-HCl pH 1.7. Data were analyzed using Octet data analysis software HT 12.0. The data were fit to a 1:1 binding model. Kinetic rates ka and kd were globally fitted.

[0620] As in Example 2, conformational stability was assessed by DSF using Sypro Orange (Sigma) and RT-PCR machine (C1000 thermal cycler, Biorad). Samples were measured in duplicate at 0.25-1.0 mg / mL in PBS buffer, and Sypro Orange was added at a final concentration of 5x. The gradient identical to that used in Example 2 was used.

[0621] All variants can be expressed and purified. The dissociation constants KD of most antibodies are similar to those of the original The monomer content of both variants was less than 85% (VH2 / VL5.22 and VH2.2 / VL5.22). The following table summarizes the results.

[0622] Table 10:

[0623]

[0624]

[0625] Example 3.3: Summary and selection of candidates for further engineering

[0626] In summary, the parent antibody Unwanted motifs were removed from the sequence of the α-terminal peptide (α-terminal peptide) without affecting the binding behavior of the binder. This was particularly surprising because the sequence motifs were located within the CDR regions. Despite this, the new variants retained their ability to bind approximately the same target antigen, and some even showed increased affinity. At the same time, the biophysical properties of the binder remained within acceptable ranges.

[0627] For further engineering, we selected six antibodies (VH2 / VL5.2, VH2 / VL5.6, VH2 / VL5.7, VH2 / VL5.8, VH2 / VL5.17, and VH2 / VL5.20) that overall showed the most promising properties for further derivatization.

[0628] Example 4: Removal of multiple sites prone to PTM or fragmentation

[0629] Example 4.1: Design of further engineered, humanized variants

[0630] Success from antibodies Encouraged by the removal of deleterious sequence motifs from the CDRs of β-actin-binding proteins, the inventors next investigated whether it would even be possible to remove multiple such motifs without losing affinity or function of the binder.

[0631] The following variants were generated.

[0632] Table 11 ("---" indicates that the site has not changed):

[0633] Antibody NG in LCDR1 NT LCDR1 RF in LCDR2 GGG in VL VH2 / VL5.24 NG KT LA GQG VH2 / VL5.25 NA KT --- GQG VH2 / VL5.26 RG QT --- GQG VH2 / VL5.27 RG QT LA GQG VH2 / VL5.28 RG KT LA GQG VH2 / VL5.29 NA KT LA GQG VH2 / VL5.30 NA KT LF GQG VH2 / VL5.31 RG QT LF GQG

[0634] The sequence of the new VL chain is shown below.

[0635] >VL5.24

[0636]

[0637] >VL5.25

[0638]

[0639] >VL5.26

[0640]

[0641] >VL5.27

[0642]

[0643] >VL5.28

[0644]

[0645] >VL5.29

[0646]

[0647] >VL5.30

[0648]

[0649] >VL5.31

[0650]

[0651] The following table shows the SEQ ID No. of the variable light chain including their CDRs.

[0652] Table 12:

[0653]

[0654]

[0655] SEQ ID No.106: RSSQSIVHSNAKTYLE

[0656] SEQ ID No. 107: QSIVHSNAKTY

[0657] SEQ ID No.108: RSSQSIVHSRGQTYLE

[0658] SEQ ID No. 109: QSIVHSRGQTY

[0659] SEQ ID No.110: RSSQSIVHSRGKTYLE

[0660] SEQ ID No.111: QSIVHSRGKTY

[0661] Example 4.2: Testing of further engineered, humanized variants

[0662] The engineered variants were subjected to the same type of analysis and characterization as the original humanized variants. The experimental setup is shown in Example 2.

[0663] All variants could be expressed and purified in sufficient yields. All antibodies showed Dissociation constant K in the antibody range D The results are summarized in the table below.

[0664] Table 13:

[0665]

[0666]

[0667] Example 4.3: Summary and selection of candidates for further engineering

[0668] In short, even from Multiple unnecessary motifs were removed from the sequences of the humanized variants. At the same time, the affinity of some variants was even higher than that of the original antibodies, and the biophysical properties of the binder are maintained at within the range.

[0669] Example 5: Summary of the sequences of antibodies disclosed in this patent

[0670] The following table summarizes the key antibodies produced and tested in this disclosure. The VH and VL sequences of the antibodies are indicated. The corresponding VH sequences, VL sequences and CDR sequences can be found in the aforementioned examples.

[0671] Table 14:

[0672]

[0673]

[0674] Example 6: Specificity analysis of humanized antibodies

[0675] The specificity of the humanized antibodies produced in the above examples for the target antigen was tested. For this experiment, a membrane proteome array (MPA) (Integral Molecular) was used. MPA is a protein library consisting of 6,000 different human membrane protein clones, each of which is overexpressed in living HEK-293T cells by expression plasmids. Each clone was individually transfected into a separate well of a 384-well plate and then incubated for 24 hours (Proc Natl Acad Sci USA (2018) 115: eE4990-9). Cells expressing each individual MPA protein clone were arranged in duplicate in a matrix format for high-throughput screening. Before screening on MPA, the optimal concentration of the antibody to be tested (antibody Kiebitz) was determined on cells expressing positive (membrane-bound protein A) and negative (mock-transfected) binding controls, and then detected by flow cytometry using a fluorescent-labeled secondary antibody (Jackson ImmunoResearch, Cat#109-606-008).

[0676] The optimal screening concentration of antibody Kiebitz was determined to be 20 μg / ml. Kiebitz was added to MPA at a concentration of 20 μg / mL, and the binding of the entire protein library was measured on Intellicyt iQue using a fluorescently labeled secondary antibody (see above). Each array plate contained positive (Fc binding) and negative (empty vector) controls to ensure reproducibility across the plate. Interactions of antibody Kiebitz above background were observed for five targets.

[0677] Binding to these five targets (NOX5, CPOX, FRFL2, PHKG1, and LYSMD4) was evaluated in more detail in a second flow cytometry experiment using a serial dilution of the Kiebitz antibody (0.3, 1.3, 5, and 20 μg / mL). Binding to the five antigens identified in the large-scale MPA assay could not be confirmed. At the two highest concentrations tested, binding was less than 2-fold above background levels. The Kiebitz antibody is highly specific for CD45.

[0678] Example 7: Depletion of CD45+ cells and LT-HSCs in humanized mice

[0679] The Jackson Laboratory humanized NSG mice by transplanting human umbilical cord blood-derived CD34+ HSPCs. Eighteen weeks after HSPC injection, humanized mice received a single intravenous injection of 0.3 mg / kg of Kiebitz-tetracillin or BC8-tetracillin conjugates. Control animals were treated with phosphate-buffered saline (PBS). Twenty-one days after treatment, mice were euthanized, and blood, spleen, and bone marrow were analyzed by FACS.

[0680] The table below shows the VH, VL and CDR sequences of antibody BC8 according to Kabat.

[0681] Table 15:

[0682]

[0683] Figure 3 Results are shown for the depletion of human CD45+ cells (gated as live / hCD45+) and long-term (LT)-HSCs in the bone marrow. LT-HSCs were gated as live / hCD45+ / CD34+ / CD38- / CD90+ / CD45RA-. Results show that compared to PBS controls, all human CD45+ cells and LT-HSCs were completely depleted 3 weeks after a single dose of 0.3 mg / kg of the ADC Kiebitz-Tescillin, while BC8-Tescillin treatment resulted in only partial depletion.

[0684] Example 8: Blockade with naked antibody mimics shielding of cells and can be abrogated by subsequent treatment with ADC Prevents loss of cell viability in cell lines

[0685] In this experiment, various human cell lines were treated with tecillin conjugates of selected naked anti-CD45 antibodies. Some samples were pre-incubated with the same anti-CD45 antibodies in naked (unconjugated) form.

[0686] To this end, MV4-11 cells were seeded at a concentration of 75,000 cells / mL in 135 μl of culture medium (IMDM supplemented with GlutaMAX + 10% heat-inactivated FBS) in a 96-well plate. Some wells were pretreated with 50 μg / mL naked antibody (Kiebitz or Ref043) or PBS as a control for 1 h, and then various concentrations of Kiebitz-tecillin or BC08-tecillin were added (for both cell lines, the starting concentration of naked antibody pretreated wells was 10 μg / ml, and the starting concentration of PBS pretreated wells was 1 μg / ml) for 72 h. The cells were then incubated with 50 μl of CTG2.0 ( 2.0 Cell Viability Assay #G9241, Promega), the plates were incubated on a shaker at room temperature for 10 minutes and luminescence was read (EnVision, PerkinElmer) to measure cell viability.

[0687] In addition, Molm-13 cells were analyzed according to the same protocol described. Cells were seeded in 135ul of culture medium (RPMI1640 supplemented with GlutaMAX+20% heat-inactivated FBS) in 96-well plates at a concentration of 75,000 cells / mL. The starting concentration of naked antibody pretreatment was 3ug / ml.

[0688] The results are as follows Figure 4 As shown in Figure 3, naked antibody blocking mimics cellular shielding and prevents the decline in cell viability via the respective ADCs in both cell lines tested. BC08-Tescillin is less potent than Kiebitz-Tescillin.

[0689] Example 9: Blockade with naked antibodies can also be observed with HSPCs

[0690] The experiment of embodiment 8 was repeated with HSPC. From peripheral blood, CD34-positive HSPC (CliniMACS Prodigy, Miltenyi Biotech) was isolated. Cells were seeded in 135ul low or high cytokine culture medium in 96-well plates at a concentration of 75000 cells / ml. Low cytokine culture medium consists of StemPro culture medium (Gibco), StemPro nutrients (Gibco), 50ng / ml LDL (STEMcell Technologies), 1% penicillin / streptomycin (Thermo Fisher), 1% glutamine (Thermo Fisher), 20ng / ml Flt3 (Miltenyi Biotec), 50ng / ml TPO (Milteny Biotec) and 100ng / ml SCF (Milteny Biotec). High cytokine medium consisted of the same ingredients plus 50 ng / ml IL6 (Milteny Biotec), 10 ng / ml IL3 (Milteny Biotec), 10 ng / ml IL2 (Milteny Biotec), 20 ng / ml IL7 (Milteny Biotec), 50 ng / ml IL11 (Milteny Biotec), 3 ng / ml EPO (STEMcell Technologies) and 20 ng / ml GM-CSF (Milteny Biotec). Wells were pretreated with 50 ug / mL naked antibody (Kiebitz) or PBS as a control for 1 h, followed by the addition of various concentrations of Kiebitz-Tescillin (for both cell lines, the starting concentration for naked antibody pretreated wells was 10 ug / ml and the starting concentration for PBS pretreated wells was 1 ug / ml) for 72 h. Cells were then incubated with 50 ul of CTG2.0 ( 2.0 Cell Viability Assay #G9241, Promega), the plate was incubated on a shaker at room temperature for 10 minutes and luminescence was read (EnVision, Perkin Elmer) to measure cell viability.

[0691] The results are as follows Figure 5 As shown in Figure 3 . Similarly, in HSPCs, blocking with naked Kiebitz antibodies shielded the cells and prevented the reduction in cell viability via the respective ADCs. The IC 50 increased 58-fold in low cytokine medium (0.0029 μg / ml vs. 0.17 μg / ml) and 38-fold in high cytokine medium (0.0059 μg / ml vs. 0.23 μg / ml).

[0692] Example 10: Blockade is seen in wild-type cells but not in CD45 knockout cells

[0693] Similar experiments were performed using wild-type and CD45 knockout Jurkat cells. Jurkat cells or Jurkat cells engineered for CD45KO were seeded at a concentration of 75,000 cells / mL in 135ul of culture medium (RPMI1640 (ATCC modified) + 10% heat-inactivated FBS) in a 96-well plate. Various concentrations of Kiebitz-Tecillin were added. The starting concentration was 100ug / ml. The cells were incubated for 72h. 50ul of CTG2.0 ( 2.0 Cell Viability Assay #G9241, Promega), the plates were incubated on a shaker at room temperature for 10 minutes and luminescence was read (EnVision, Perkin Elmer) to measure cell viability.

[0694] The results are as follows Figure 6 The IC50 of Kiebitz-Tecillin in Jurkat CD45 knockout cells was significantly higher than that in Jurkat wild-type cells (0.46 μg / ml vs. 0.032 μg / ml).

[0695] Example 11: Antibody Binding to Endogenously Expressed CD45 on Cells

[0696] Use different concentrations of antibodies KASUMI-1 cells (ATCC No. CRL-2724) were stained with IgG goat anti-human AF488 secondary antibody or the humanized derivatives Amandine or Ballerina. Cells stained with human IgG1 isotype control were used to determine background staining.

[0697] The results are as follows Figure 7 All three antibodies tested bound equally well to CD45 endogenously expressed on Jurkat cells.

[0698] In similar experiments, Jurkat cells or Jurkat CD45 knockout (KO) cells were stained with different dilutions of the antibody Kiebitz-AF647. Unstained Jurkat cells and anti-human Ig-AF647 were used to determine background staining levels. AF647-labeled Kiebitz recognized endogenously expressed CD45 on Jurkat cells in a concentration-dependent manner. Kiebitz did not bind to CD45 KO Jurkat cells.

[0699] Example 12: Antibody expression as scFv-Fc

[0700] Not all antibodies can be expressed well in the scFv-Fc format. One antibody that can be expressed well in this format is the antibody Jelly (VH2 / VL5.22). Such antibodies are particularly useful for certain purposes, such as for use in CARs or LNPs. Certain additional antibodies were constructed that all expressed well as scFv-Fc. Table 16 shows these antibodies.

[0701] Table 16:

[0702]

[0703]

[0704]

Claims

1. A humanized antibody or antibody fragment specific for human CD45, wherein: The antibody or antibody fragment comprises: a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, and a HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising a LCDR1 region of SEQ ID NO: 7, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises at least one of the following mutations: a) glycine in the LCDR1 region (SEQ ID NO: 7) is replaced by alanine, b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by lysine, c) the arginine in the LCDR2 region (SEQ ID NO: 8) is replaced by leucine, or d) The phenylalanine in the LCDR2 region (SEQ ID NO: 8) is replaced by alanine.

2. The humanized antibody or antibody fragment according to claim 1, wherein The humanized antibody or antibody fragment comprises: a) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 75, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9, b) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 8, and a LCDR3 region of SEQ ID NO: 9, c) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9, d) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 110, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9, e) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 96, and a LCDR3 region of SEQ ID NO: 9, f) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 106, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO: 9, or g) a variable heavy chain comprising a HCDR1 region of SEQ ID NO: 4, a HCDR2 region of SEQ ID NO: 5, a HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising a LCDR1 region of SEQ ID NO: 108, a LCDR2 region of SEQ ID NO: 93, and a LCDR3 region of SEQ ID NO:

9.

3. The humanized antibody or antibody fragment according to claim 1 or 2, wherein The humanized antibody or antibody fragment comprises a mutation of the second glycine of the triple glycine motif in framework region 4 of the variable light chain to glutamine.

4. The humanized antibody or antibody fragment according to any one of the preceding claims, wherein The humanized antibody or antibody fragment comprises: a) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 98, b) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 99, c) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 101, d) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 102, e) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 103, f) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO: 104, or g) the variable heavy chain of SEQ ID NO: 21 and the variable light chain of SEQ ID NO:

105.

5. The humanized antibody or antibody fragment according to any one of the preceding claims, wherein The humanized antibody or antibody fragment comprises the following mutations: a) glycine in the LCDR1 region (SEQ ID NO: 7) is replaced by alanine, b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by lysine, and c) Arginine in the LCDR2 region (SEQ ID NO: 8) was replaced by leucine.

6. The humanized antibody or antibody fragment according to claim 5, wherein The humanized antibody or antibody fragment comprises a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO:

104.

7. A humanized antibody according to any one of the preceding claims, wherein The humanized antibody comprises modifications in the FC region, preferably wherein, The Fc modifications are silent mutations selected from PA-LALA (L234A / L235A / P329A), PG-LALA (L234A / L235A / P329G) and AEASS (L234A / L235E / G237A / A330S / P331S) (numbered according to the EU index).

8. The humanized antibody or antibody fragment according to any one of the preceding claims, wherein The humanized antibody or antibody fragment is a monoclonal antibody or antibody fragment.

9. An antibody drug conjugate comprising the humanized antibody or antibody fragment according to any one of the preceding claims and a cytotoxic drug.

10. The antibody drug conjugate according to claim 9, wherein The cytotoxic drug is a pyrrolobenzodiazepine (PBD), preferably tecillin.

11. The humanized antibody or antibody fragment according to any one of claims 1 to 8 or the antibody drug conjugate according to claim 9 or 10, for use in medicine, preferably wherein the use in medicine is the treatment of blood cancers, such as leukemia, myeloma or lymphoma, or the treatment of inflammatory or autoimmune diseases, such as multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn's disease, type 1 diabetes, rheumatoid arthritis or idiopathic arthritis.

12. A nucleic acid composition comprising one or more nucleic acid sequences, wherein the one or more nucleic acid sequences encode the humanized antibody or antibody fragment according to any one of claims 1 to 8 or the antibody drug conjugate according to claim 9 or 10.

13. A vector comprising the nucleic acid composition of claim 12.

14. A host cell comprising the vector of claim 13 or the nucleic acid composition of claim 12.

15. A pharmaceutical composition comprising the humanized antibody or antibody fragment according to any one of claims 1 to 8 or the antibody drug conjugate according to claim 9 or 10 and a pharmaceutically acceptable carrier or excipient.

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