Antibodies targeting CD45

Abstract

The present disclosure relates to antibodies and antibody fragments specific to CD45. The antibodies are improved humanized versions of mouse antibodies. In addition to elaborate humanization campaigns, the antibodies have also been engineered to remove some harmful motifs in the CDR region without losing their beneficial properties. The antibodies are useful for treating diseases related to CD45.

Description

[Technical Field]

[0001] The present disclosure relates to antibodies and antibody fragments specific to CD45. The antibodies are improved humanized versions of mouse antibodies. In addition to elaborate humanization campaigns, the antibodies have also been engineered to remove some harmful motifs in the CDR region without losing their beneficial properties. The antibodies are useful for treating diseases related to CD45.

[0002] Financial support statement 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 agreement No 818806). [Background technology]

[0003] 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 various cellular processes, including cell growth, differentiation, the 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 present in various isoforms on differentiated hematopoietic cells (e.g., excluding erythrocytes) (Holmes, Immunology 7:145-55 (2006)). CD45 has been shown to be a regulator of T and B cell antigen receptor signaling. It functions by direct interaction 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 through its cytoplasmic domain. CD45 also inhibits JAK kinases and thus functions as a negative regulator of cytokine receptor signaling.

[0004] CD45 is present on the surface of hematopoietic cells, including hematopoietic stem cells (HSCs), leukocytes, and osteoclasts (Shivtiel et al., J Exp Med 205:2381 (2008)). Deletion mutations within CD45 in humans are associated with severe immunodeficiency. This is primarily due to the absence of CD45 on T cells, which are typically abundant and required for regulating SFK activity during antigen responses. The bone marrow of CD45-deficient (CD45- / -) mice contains normal numbers 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 specific to HSCs; without CD45-mediated downregulation of SFK activity, integrin-mediated adhesion is enhanced, and HSCs are more likely to remain in the stem cell niche. CD45- / - HSCs also exhibit defects in G-CSF-stimulated mobilization and homing to the chemokine CXCL12 / SDF-1, which adversely impacts cell engraftment after transplantation. These defects can be restored by supplementing with SFK inhibitors, indicating that this role is normally performed by CD45. Similarly, CD45- / - recipients also exhibit defective engraftment and subsequent mobilization of normal HSCs, demonstrating the role of CD45 in the stem cell niche and HSCs (Shivtiel et al., J Exp Med 205:2381 (2008)). Because CD45 is expressed on HSCs and leukocytes, for example, it presents a target for therapeutic applications, including conditioning therapy, immune resetting, and disease treatment.

[0005] Several 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 the anti-CD45 antibody-radioconjugates developed by Actinium Pharmaceuticals (WO2017155937, WO2019084258, WO2020159656). Other anti-CD45 antibodies and antibody-based moieties are disclosed in WO 2016016442, WO 2019115791 and WO 2020058495, WO 2017009473, WO 2019129178, WO 2020018580, WO 2020170254 and WO 2020219959. These and other anti-CD45 moieties may be used in the context of the present disclosure. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2017155937 [Patent Document 2] International Publication No. 2019084258 [Patent Document 3] International Publication No. 2020159656 [Patent Document 4] International Publication No. 2016016442 [Patent Document 5] International Publication No. 2019115791 [Patent Document 6] International Publication No. 2020058495 [Patent Document 7] International Publication No. 2017009473 [Patent Document 8] International Publication No. 2019129178 [Patent Document 9] International Publication No. 2020018580 [Patent Document 10] International Publication No. 2020170254 [Patent Document 11] International Publication No. 2020219959 [Non-patent literature]

[0007] [Non-Patent Document 1] Kaplan et al., PNAS 87:7000-7004(1990) [Non-patent document 2] Holmes, Immunology 7:145-55(2006) [Non-patent document 3] Shivtiel et al., J Exp Med 205:2381(2008) Summary of the Invention

[0008] The present invention provides a humanized antibody or antibody fragment specific for human CD45, the antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment comprises: a) a glycine in the LCDR1 region (SEQ ID NO: 7) is replaced with an alanine; b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced with a lysine; c) an arginine in the LCDR2 region (SEQ ID NO: 8) is replaced by a leucine, or d) A humanized antibody or antibody fragment comprising at least one mutation in the LCDR2 region (SEQ ID NO: 8) in which phenylalanine is replaced by alanine.

[0009] In a specific embodiment, the present invention provides a humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, wherein the antibody humanization or antibody fragment comprises one of the following: a) a glycine in the LCDR1 region (SEQ ID NO: 7) is replaced with an alanine; b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced by a lysine, or c) A humanized antibody or antibody fragment comprising at least one mutation in the LCDR2 region (SEQ ID NO: 8) in which arginine is replaced by leucine.

[0010] The present disclosure also provides a humanized antibody or antibody fragment specific for human CD45, the antibody or antibody fragment comprising: a) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 75, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; b) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; c) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; d) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 110, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; e) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; f) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9, or g) A humanized antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9.

[0011] 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 framework region 4 of the variable light chain to glutamine.

[0012] The present disclosure also provides a humanized antibody or antibody fragment specific for human CD45, the humanized antibody or antibody fragment comprising: a) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 98; b) a variable heavy chain of SEQ ID NO: 21 and a 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) a variable heavy chain of SEQ ID NO: 21 and a 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) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104, or g) A humanized antibody or antibody fragment comprising a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 105.

[0013] The present disclosure also relates to a humanized antibody or antibody fragment specific to human CD45 as described above, wherein the humanized antibody or antibody fragment comprises: a) a glycine in the LCDR1 region (SEQ ID NO: 7) is replaced with an alanine; b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced with a lysine, and c) A humanized antibody or antibody fragment comprising the following mutation in the LCDR2 region (SEQ ID NO: 8): arginine is replaced by leucine.

[0014] 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.

[0015] The present disclosure also relates to the aforementioned humanized antibodies or antibody fragments specific for human CD45, wherein the humanized antibodies comprise a modification in the Fc region, preferably, the Fc modification is a silencing mutation, more preferably, the mutation is a PA-LALA, PG-LALA, or AEASS mutation.

[0016] 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.

[0017] 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 is tesirine.

[0018] The present disclosure also relates to the aforementioned humanized antibodies, antibody fragments and antibody drug conjugates comprising said humanized antibodies or antibody fragments for use in medicine.

[0019] 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.

[0020] 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.

[0021] The present disclosure also relates to nucleic acid compositions comprising a nucleic acid sequence or 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.

[0022] 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 explanation of the drawings]

[0023] [Figure 1] Figure 1 shows the alignment of the five humanized variants to the original murine VH sequence of Atti. VH0 is the murine sequence, and VH1-5 are humanized variants. Key residues important for the VH / VL interface and canonical loop structure have been maintained as much as possible. [Figure 2] Figure 2 shows the alignment of the five humanized variants to the original murine VL sequence of antibody Atti. VL is the murine sequence, and VL1-6 are humanized variants. Key residues important for the VH / VL interface and canonical loop structure have been maintained where possible. [Figure 3]Figure 3 shows complete depletion of all human CD45+ cells and LT-HSCs after 3 weeks of a single dose of 0.3 mg / kg Kiebitz-tetherin compared to the PBS control group, whereas BC8-tetherin treatment resulted in only partial depletion. [Figure 4] Figure 4 shows that naked antibody blocking mimics cell shielding and prevents the loss of cell viability mediated by the respective ADCs in both cell lines tested. BC08-Tesilin shows lower efficacy than Kiebitz-Tesilin. Panel A: MV4-11; Panel B: Molm-13. [Figure 5] Figure 5 shows that even in HSPCs, blocking with naked Kiebitz antibodies can block the cells and prevent cell viability from decreasing with the respective ADC. [Figure 6] FIG. 6 shows that Jurkat CD45 knockout cells have a significantly higher IC50 for Kiebitz-Teslin than that of Jurkat wild-type cells (0.46 μg / ml vs. 0.032 μg / ml). [Figure 7] FIG. 7 shows that the antibodies Atti, Amandine and Ballerina bind equally well to CD45 expressed endogenously on Jurkat cells. DETAILED DESCRIPTION OF THE INVENTION

[0024] definition The present disclosure relates to antibodies that specifically bind to CD45 and to uses, particularly therapeutic uses, of such antibodies.

[0025] 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):

[0026] As used herein, the term "antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds that interact with an antigen. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (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, lgG2, lgG3, lgG4, lgA1, and lgA2), or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

[0027] As used herein, the term "antibody fragment" refers to one or more parts of an antibody that retains the ability to specifically interact with antigen (for example, by binding, steric hindrance, stabilizing spatial distribution).Examples of binding fragments include, but are not limited to, Fab fragments, VL, VH, CL and CH1 domains; F(ab)2 fragments, bivalent fragments that comprise two Fab fragments linked by disulfide bridges at hinge regions; Fd fragments that comprise VH domains and CH1 domains; Fv fragments that comprise the VL domain and VH domain of a single arm of antibody; dAb fragments that comprise VH domains (Ward et al., (1989) Nature 341:544-546); and monovalent fragments that comprise isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., 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 by the term "antibody fragment." These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1 126-1 136). Antibody fragments can be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).Antibody fragments can be assembled into single-chain molecules comprising a pair of tandem Fv segments (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).

[0028] The structure and location 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 (e.g., Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services (1991), eds. Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948; Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 USDapartment of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:877-883; and See Al-Lazikani et 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).

[0029] As used herein, "human antibodies" or "human antibody fragments" refer to antibodies and antibody fragments having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Human antibodies can also be isolated from synthetic libraries or transgenic mice (e.g., Xenomouse, OmniMouse, Harbour Mouse, ATX-Gx Mouse, Trianni Mouse), providing a high yield of antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such sequences. Examples of human origin include human germline sequences or mutated versions of antibodies containing consensus framework sequences derived from human framework sequence analysis, e.g., as described in Knappik et al. (2000) J Mol Biol 296:57-86).

[0030] A "humanized antibody" or "humanized antibody fragment" is defined herein as an antibody molecule having constant antibody regions derived from sequences of human origin, with only the variable antibody regions or portions thereof, or CDRs, derived from another species. For example, a humanized antibody can be CDR-grafted, in which the CDRs of the variable domain are of non-human origin, while one or more frameworks of the variable domain are of human origin, and the constant domains, if any, are of human origin.

[0031] 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 a non-human animal, such as a mouse, rat, rabbit, or hamster.

[0032] 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 with different antigen specificities. Furthermore, an isolated antibody or antibody fragment may be substantially free of other cellular material and / or chemicals. Thus, in some embodiments, the provided antibody is an isolated antibody that is separated from antibodies with different specificities. An isolated antibody may be a monoclonal antibody. An isolated antibody may be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds to a target epitope, isoform, or variant may have cross-reactivity with other related antigens, such as antigens from other species (e.g., species homologs).

[0033] As used herein, the term "recombinant antibody" or "recombinant antibody fragment" includes all antibodies or antibody fragments prepared, expressed, created, or isolated by means that do not occur in nature. For example, antibodies isolated from host cells transformed to express the antibody, antibodies selected and isolated from recombinant combinatorial human antibody libraries, and antibodies prepared, expressed, created, or isolated by any other means, including splicing of 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 for human immunoglobulin genes or hybridomas prepared therefrom. Preferably, such recombinant antibodies have variable regions in which the framework 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 animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo. The recombinant antibody can be a monoclonal antibody.

[0034] As used herein, the term "monoclonal" has the meaning typically ascribed to it in the art, i.e., an antibody or antibody fragment (or corresponding functional fragment thereof) arising from a single clone of antibody-producing cells that recognizes a single epitope on the bound antigen.

[0035] As used herein, binding specificity is a relative, not absolute, property; therefore, an antibody "binds specifically to," "specifically binds to," "specific for," or "specifically recognizes" an antigen, such as human CD45, if it can distinguish between that antigen and one or more reference antigen(s). 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 antibodies containing tetramethylbenzidine with horseradish peroxide and hydrogen peroxide) or by binding of a secondary antibody labeled with PE or another dye or marker. Reactions in a particular well are scored, for example, by optical density (OD) at 450 nm or by mean or median fluorescence intensity (MFI) in flow cytometry. A typical background (=negative reaction) can be 0.1 OD; a typical positive reaction can be 1 OD. The background and positive reaction MFIs are highly dependent on instrument settings. The difference between positive and negative reactions can be more than 10-fold. Typically, binding specificity is determined not using a single reference antigen, but using a set of approximately 3-5 unrelated antigens, such as milk powder, BSA, or transferrin. A variety of antigen-negative cells can be used for flow cytometry. However, antibodies that specifically bind to an antigen may have cross-reactivity with their respective orthologous antigens from other species (e.g., species homologs). In certain embodiments, such cross-reactivity with orthologous antigens is even preferable.

[0036] 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 its target at multiple sites through weak non-covalent forces. The more interactions, the stronger the affinity.

[0037] The term "epitope" includes any proteinaceous region that is specifically recognized by an antibody or antibody fragment thereof or that otherwise interacts with a molecule. Generally, epitopes are chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and generally may have specific three-dimensional structural characteristics, as well as specific charge characteristics. As will be appreciated by those skilled in the art, virtually anything to which an antibody can specifically bind can be an epitope.

[0038] The term "domain" or "protein domain" refers to a region of a polypeptide chain of a protein that forms a functional unit and / or forms an independent three-dimensional structure.

[0039] The "composition" or compositions of the present disclosure can be used for therapeutic or prophylactic purposes. Accordingly, the present disclosure includes pharmaceutical compositions containing the antibodies or antibody fragments disclosed herein and a pharmaceutically acceptable carrier or excipient therefor. In a related aspect, the present disclosure provides methods of treating inflammatory diseases, autoimmune diseases, hematological malignancies, and potentially other diseases. Such methods include administering to a subject in need thereof an effective amount of a pharmaceutical composition containing an antibody or antibody fragment as described herein.

[0040] The present disclosure provides a method of treatment comprising administering a therapeutically effective amount of an antibody or antibody fragment disclosed herein to a subject in need of such treatment. As used herein, a "therapeutically effective amount" or "effective amount" refers to the amount of an anti-CD45 antibody required to induce a desired biological response. According to the disclosed subject matter, a therapeutically effective amount is the amount of an anti-CD45 antibody required to treat and / or prevent a disease.

[0041] "Administering" or "administration" includes, but is not limited to, delivery of a drug by, for example, intravenous, intramuscular, intradermal or subcutaneous routes, or mucosal routes, in an injectable form, for example, as a nasal spray or aerosol for inhalation, or as an ingestible solution, capsule or tablet. Preferably, administration is in an injectable form.

[0042] As used herein, "treatment," "treat," or "treating," etc., refer to a clinical intervention that seeks to alter the natural history of a disease in the subject being treated and can be performed prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies or antibody fragments of the present disclosure are used to delay the onset of disease or to slow the progression of the disease.

[0043] "Preventing" or "prevention" refers to a reduction in the risk of acquiring or developing a disease (i.e., not developing 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 prior to disease onset). "Prevention" also refers to methods aimed at preventing the onset of a disease or its symptoms, or delaying the onset of a disease or its symptoms.

[0044] "Subject" or "species," as used in this context, refers to any mammal, including rodents such as mice or rats, and primates such as cynomolgus monkeys (Macaca fascicularis), marmoset monkeys (Callithrix jacchus), rhesus monkeys (Macaca mulatta), or humans (Homo sapiens). Preferably, the subject is a primate, most preferably a human.

[0045] The term "effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies depending on 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.

[0046] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) allow these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. The primary cells for mediating ADCC, NK cells, express only FcyRIII, whereas monocytes / macrophages express FcyRI, FcyRII, and FcyRIII.

[0047] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) of the present disclosure that is bound to its cognate antigen.

[0048] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism for elimination of antibody-coated target cells by internalization by phagocytic cells such as macrophages or dendritic cells.

[0049] 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.

[0050] As used herein, the term "cytotoxic drug" is art-recognized and includes, but is not limited to, daunorubicin, mitoxantrone, doxorubicin, cucurbitacin, chaetocin, chaetoglobosin, chlamydocin, calicheamicin, mertansine, nemorubicin, cryptophyscin, mensacarcin, ansamitocin, mitomycin C, thiamin monophosphate ... C), geldanamycin, mechercharmycin, rebeccamycin, safracin, okilactomycin, oligomycin, actinomycin, sandramycin, hypothemycin, polyketomycin, hydroxyellipticine, thiocolchicine, methotrexate, triptolide, taltobulin, lactacystin, dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAE), monomethyl auristatin F) (MMAF), telomestatin, tubastatin A, combretastatin, maytansinoid, MMAD, MMAF, DM1, DM4, DTT, 16-GMB-APA-GA, 17-DMAP-GA, JW55, pyrrolobenzodiazepine, SN-38, Ro 5-3335, puwainaphycin, duocarmycin, bafilomycin, taxoid, tubulysin, ferulenol, lusiol A A), fumagillin, hygrolidin, glucopyricidin, amanitin, ansatrienin, cinerubin, phallacidin, phalloidin, phytosphongosine, piericidin, poronetin, phosphophyllotoxin, gramicidin A, sanguinarine, sinefungin, herboxidiene, microcolin B, microcystin, muscotoxin A, tolytoxin, tripolin A A), myoseverin, myotoxin B, nocuolin A, pseudolaric acid B, pseurotin A, cy dopamine, curvulin, colchicine, aphidicolin, englerin, cordycepin, apoptolidin, epothilone AA), limaquinone, isotropolone, isofistularin, quinaldopeptin, ixabepilone, aeropressinin, arruginosin, agrochelin, or epothilone. An exemplary pyrrolobenzodiazepine is tesirine.

[0051] Throughout this specification, unless the context requires otherwise, the words "comprise," "have," and "include," and their respective variations such as "comprises," "comprising," "has," "having," "includes," and "including," are understood to mean the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.

[0052] As used herein, the terms "engineered" or "modified" include the manipulation of nucleic acids or polypeptides by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, or some combination of these techniques). Preferably, 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.

[0053] 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, e.g., the variant polypeptide has a higher affinity for the target antigen compared to the reference polypeptide, or the variant polypeptide is a humanized version of the reference polypeptide.

[0054] As used herein, the term "amino acid mutation" is intended to encompass amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions, and modifications can be made as long as the final construct possesses the desired characteristics, such as reduced binding to Fc receptors. Deletions and insertions in the amino acid sequence include deletions and insertions at the N- and / or C-terminus of amino acid residues. A specific amino acid mutation is an amino acid substitution. Amino acid substitutions include substitutions with unnatural amino acids or with natural amino acid derivatives of the 20 standard amino acids. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods for modifying the side chain groups of amino acid residues by methods other than genetic engineering, such as chemical modification, may also be useful. Various names may be used herein to refer to the same amino acid mutation. For example, a substitution of glycine with alanine at position 237 of an antibody Fc region can be designated as 237A, G237, G237A, or Gly237Ala.

[0055] As used herein, the term "EC50" refers to the concentration of an antibody or antibody fragment that induces a response in an assay halfway between baseline and maximum, and thus represents the antibody or ligand concentration at which 50% of the maximal effect is observed.

[0056] As used herein, the term "Ka" refers to the association rate of a particular antibody-antigen interaction.

[0057] As used herein, the term "Kd" refers to the dissociation rate of a particular antibody-antigen interaction. The Kd value of an antibody can be determined using methods well established in the art.

[0058] As used herein, the term "KD" refers to the dissociation constant of a particular antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and 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 by using biolayer interferometry with an Octet BLI instrument.

[0059] The terms "inhibition" or "inhibiting" or "reduction" or "reducing" or "neutralization" or "neutralizing" refer to a decrease or cessation of any phenotypic characteristic (such as binding or biological activity or function), or a decrease or cessation of the incidence, degree, 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, "reduce" or "inhibit" or "neutralizing" refers to the ability to cause a 20% or greater reduction. In other embodiments, "reduce" or "inhibit" or "neutralizing" refers to the ability to cause a 50% or greater reduction. In yet other embodiments, "reduce" or "inhibiting" or "neutralizing" refers to the ability to cause a 75%, 85%, 90%, 95% or greater overall reduction.

[0060] 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 property of the target antigen.

[0061] A "wild-type" protein is a version or variant of a protein found in nature. The amino acid sequence of a wild-type protein, such as the Fc region of a human IgG1 antibody, is the amino acid sequence of a naturally occurring protein. Due to allotypic differences, a wild-type protein may have more than one amino acid sequence. For example, there are several allotypes of the naturally occurring human IgG1 heavy chain constant region (see, e.g., Jeffries et al. (2009) mAbs 1:1).

[0062] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region of an immunoglobulin generally contains two constant domains, a CH2 domain and a CH3 domain. While the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region follows the EU numbering system, also known as the EU index, as described by 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. Silencing functions include 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) (numbering according to the EU index). A preferred FcR modification is PA-LALA. Mutations may also result in reduced binding to FcRn, thereby reducing the in vivo half-life of the antibody. Such mutations include I253A, H310A, H435A, and H435Q. Alternatively, mutations may also result 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.

[0063] Embodiments of the present invention Polypeptides 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.

[0064] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1. In certain embodiments, 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.

[0065] In a specific embodiment, the present invention relates to a humanized antibody or antibody fragment specific for human CD45, wherein the antibody or antibody fragment comprises a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0066] In a specific embodiment, the present invention relates to a humanized antibody or antibody fragment specific for human CD45, the antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, wherein glycine in the LCDR1 region (SEQ ID NO: 7) of the humanized antibody or antibody fragment has been replaced by alanine.

[0067] In a specific embodiment, the present invention relates to a humanized antibody or antibody fragment specific for human CD45, the antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an 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 has been replaced with a lysine.

[0068] In a specific embodiment, the present invention relates to a humanized antibody or antibody fragment specific for human CD45, the antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, wherein arginine in the LCDR2 region (SEQ ID NO: 8) of the humanized antibody or antibody fragment has been replaced with leucine.

[0069] In a specific embodiment, the present invention relates to a humanized antibody or antibody fragment specific for human CD45, the antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an 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 has been replaced with an alanine, a second asparagine in the LCDR1 region (SEQ ID NO: 7) of the humanized antibody or antibody fragment has been replaced with a lysine, and an arginine in the LCDR2 region (SEQ ID NO: 8) of the humanized antibody or antibody fragment has been replaced with a leucine.

[0070] In a specific embodiment, the invention relates to a humanized antibody or antibody fragment specific for human CD45, the antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, 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 a glutamine.

[0071] In a specific embodiment, the invention relates to a humanized antibody or antibody fragment specific for human CD45, the antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an 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, a second asparagine in the LCDR1 region (SEQ ID NO: 7) of the humanized antibody or antibody fragment is replaced by a lysine, and an arginine in the LCDR2 region (SEQ ID NO: 8) of the humanized antibody or antibody fragment is replaced by a leucine, and 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 a glutamine.

[0072] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 63, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0073] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 65, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0074] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0075] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 69, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0076] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 71, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0077] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 73, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0078] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 75, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0079] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 77, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0080] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0081] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 81, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0082] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 83, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0083] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 85, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0084] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 87, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0085] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 89, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0086] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 91, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0087] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9.

[0088] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9.

[0089] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 95, and an LCDR3 region of SEQ ID NO: 9.

[0090] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9.

[0091] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 97, and an LCDR3 region of SEQ ID NO: 9.

[0092] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9.

[0093] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9.

[0094] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 69, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9.

[0095] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 75, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9.

[0096] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0097] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0098] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9.

[0099] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 110, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9.

[0100] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9.

[0101] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9.

[0102] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[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 of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 98.

[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 of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 99.

[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 of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 100.

[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 of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 101.

[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 of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 102.

[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 of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 103.

[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 of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104.

[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 of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 105.

[0137] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises six CDRs of one of the antibodies disclosed in Table 14.

[0138] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises the six CDRs as defined by Kabat of one of the antibodies disclosed in Table 14.

[0139] In certain embodiments, the present disclosure provides a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment comprises six CDRs as defined by the IMGT of one of the antibodies disclosed in Table 14.

[0140] 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 any of the antibodies disclosed in Table 14.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is of the lgG1 class.

[0145] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment, wherein the humanized antibody or antibody fragment is of the human IgG1 class.

[0146] In certain embodiments, the antigen-binding region of the antibody disclosed herein is used as scFv. Such antibodies are particularly useful for certain purposes, such as CAR or LNP. Not all antigen-binding regions are equally suitable for use as scFv, mainly due to low expression levels. Antibodies that can be fully expressed as scFv include Jelly (VH2 / VL5.22), Lady Rosetta (VH2 / VL5.38), Levinata (VH2 / VL5.39) and Osira (VH2 / VL5.40).

[0147] Thus, in certain embodiments, the present disclosure relates to an scFv comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9.

[0148] In certain embodiments, the present disclosure relates to an scFv comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 120, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9.

[0149] In certain embodiments, the present disclosure relates to an scFv comprising a variable heavy chain comprising the variable heavy chain of SEQ ID NO:21 and the variable light chain of SEQ ID NO:58.

[0150] In certain embodiments, the present disclosure relates to an scFv comprising a variable heavy chain comprising the variable heavy chain of SEQ ID NO:21 and the variable light chain of SEQ ID NO:121.

[0151] In certain embodiments, the present disclosure relates to an scFv comprising a variable heavy chain comprising the variable heavy chain of SEQ ID NO:21 and the variable light chain of SEQ ID NO:122.

[0152] In certain embodiments, the present disclosure relates to an scFv comprising a variable heavy chain comprising the variable heavy chain of SEQ ID NO:21 and the variable light chain of SEQ ID NO:123.

[0153] Isolated antibodies or antibody fragments according to the present disclosure may or may not be fused to one or more other amino acid residues, polypeptides, or moieties. Such fusion proteins can be prepared by any suitable method, including genetic or chemical approaches. The linked moieties may include secretory or leader sequences, sequences that aid in detection, expression, separation, or purification, or sequences that provide increased protein stability, for example, during recombinant production. Non-limiting examples of potential moieties include β-galactosidase, glutathione-S-transferase, luciferase, a T7 polymerase fragment, a secretory signal peptide, an antibody or antibody fragment, a toxin, a cytokine, a chemokine, a reporter enzyme, a moiety capable of binding to a metal ion such as a polyhistidine tag, a tag suitable for detection and / or purification, a homo- or hetero-association domain, a moiety that increases protein solubility, or a moiety containing an enzymatic cleavage site.

[0154] Thus, an isolated antibody or antibody fragment according to the present disclosure may optionally include one or more moieties for binding to other targets or target proteins of interest. It should be clear that such additional moieties may or may not provide additional functionality to the antibody and may or may not alter its properties.

[0155] In certain embodiments, the present disclosure relates to an antibody-drug conjugate comprising (a) a humanized antibody or antibody fragment specific for human CD45 as disclosed herein, and (b) a cytotoxic agent, such as daunorubicin, mitoxantrone, doxorubicin, cucurbitacin, chaetocin, chaetoglobosin, chlamydocin, calicheamicin, mertansine, nemorubicin, cryptophyscin, mensacarcin, ansamitocin, or mitomycin C. C), geldanamycin, mechercharmycin, rebeccamycin, safracin, okilactomycin, oligomycin, actinomycin, sandramycin, hypothemycin, polyketomycin, hydroxyellipticine, thiocolchicine, methotrexate, triptolide, taltobulin, lactacystin, dolastatin, auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAE), monomethyl auristatin F) (MMAF), telomestatin, tubastatin AA), combretastatin, maytansinoid, MMAD, MMAF, DM1, DM4, DTT, 16-GMB-APA-GA, 17-DMAP-GA, JW 55, pyrrolobenzodiazepine, SN-38, Ro 5-3335, puwainaphycin, duocarmycin, bafilomycin, taxoid, tubulysin, ferulenol, lusiol A A), fumagillin, hygrolidin, glucopyricidin, amanitin, ansatrienin, cinerubin, phallacidin, phalloidin, phytosphongosine, piericidin, poronetin, phosphophyllotoxin, gramicidin A, sanguinarine, sinefungin, herboxidiene, microcolin B, microcystin, muscotoxin A, tolytoxin, tripolin A A), myoseverin, myotoxin B, nocuolin A, pseudolaric acid B, pseurotin A, cy dopamine, curvulin, colchicine, aphidicolin, englerin, cordycepin, apoptolidin, epothilone AA), limaquinone, isotropolone, isofistularin, quinaldopeptin, ixabepilone, aeropressinin, arruginosin, agrochelin, and epothilone. In certain embodiments, the cytotoxic agent is a pyrrolobenzodiazepine. In other embodiments, the pyrrolobenzodiazepine is tesirine.

[0156] An antibody-drug conjugate comprising: (a) a humanized antibody or antibody fragment specific for human CD45 as disclosed herein; and (b) a cytotoxic drug. In certain embodiments, the present disclosure provides 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 CD45 is i. a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 63, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; ii. a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 65, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; iii. a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; iv. a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 69, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; v. a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 71, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; vi. a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 73, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; vii. a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 75, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; viii. a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 77, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; ix. a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; x. a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 81, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; xi. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 83, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; xii. a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 85, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; xiii. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 87, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; xiv. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 89, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; xv. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 91, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; xvi. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9; xvii. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; xviii. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 95, and an LCDR3 region of SEQ ID NO: 9; xix. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; xx. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 97, and an LCDR3 region of SEQ ID NO: 9; xxi. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; xxii. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; xxiii. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 69, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; xxiv. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 75, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; xxv. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; xxvi. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; xxvii. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; xxviii. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 110, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; xxix. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; xxx. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9; or xxxi. A variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9.

[0157] In a preferred embodiment, the cytotoxic agent is a pyrrolobenzodiazepine. In another preferred embodiment, the pyrrolobenzodiazepine is tesirine.

[0158] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9.

[0159] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9; and the cytotoxic drug is a pyrrolobenzodiazepine.

[0160] 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 an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9; and the cytotoxic drug is tesirin.

[0161] 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.

[0162] 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 the cytotoxic drug is a pyrrolobenzodiazepine.

[0163] 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 the cytotoxic drug is tesirin.

[0164] nucleic acid In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or sequences encoding an isolated antibody or antibody fragment specific for human CD45 of the present disclosure.

[0165] 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, the antibody or antibody fragment comprising: a) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 63, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; b) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 65, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; c) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; d) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 69, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; e) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 71, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; f) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 73, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; g) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 75, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; h) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 77, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; i) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; j) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 81, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; k) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 83, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; l) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 85, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; m) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 87, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; n) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 89, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; o) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 91, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; p) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9; q) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; r) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 95, and an LCDR3 region of SEQ ID NO: 9; s) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; t) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 97, and an LCDR3 region of SEQ ID NO: 9; u) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; v) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; w) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 69, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; x) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 75, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; y) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; z) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; aa) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; bb) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 110, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; cc) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; dd) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9; or ee) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9.

[0166] 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, the antibody or antibody fragment comprising: a) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 34; b) a variable heavy chain of SEQ ID NO: 22 and a variable light chain of SEQ ID NO: 34; c) a variable heavy chain of SEQ ID NO: 23 and a variable light chain of SEQ ID NO: 34; d) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 38; e) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 39; f) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 40; g) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 41; h) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 42; i) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 43; j) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 44; k) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 45; l) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 46; m) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 47; n) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 48; o) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 49; p) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 50; q) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 51; r) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 52; s) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 53; t) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 54; u) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 55; v) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 56; w) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 57; x) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 58; y) a variable heavy chain of SEQ ID NO: 60 and a variable light chain of SEQ ID NO: 58; z) a variable heavy chain of SEQ ID NO: 60 and a variable light chain of SEQ ID NO: 59; aa) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 98; bb) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 99; cc) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 100; dd) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 101; ee) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 102; ff) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 103; gg) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104, or hh) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 105.

[0167] 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 six CDRs as defined by Kabat of one of the antibodies disclosed in Table 14.

[0168] 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 six CDRs as defined by the IMGT of one of the antibodies disclosed in Table 14.

[0169] 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 any of the antibodies disclosed in Table 13.

[0170] In one embodiment, the nucleic acid composition and / or the nucleic acid sequence and / or the plurality of nucleic acid sequences is isolated.

[0171] vector In one embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a humanized antibody or antibody fragment specific for human CD45 according to the present disclosure.

[0172] In one embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the humanized antibodies or antibody fragments specific for human CD45 disclosed in Table 14.

[0173] host cell In one embodiment, the present disclosure provides a host cell comprising a vector or a vector composition comprising a plurality of vectors, comprising a nucleic acid sequence or a nucleic acid composition comprising a plurality of nucleic acid sequences encoding a humanized antibody or antibody fragment specific for human CD45 according to the present disclosure.

[0174] In one embodiment, the present disclosure refers to a host cell comprising a vector or a vector composition comprising a plurality of vectors that includes a nucleic acid sequence or a nucleic acid composition that includes a plurality of nucleic acid sequences encoding any one of the humanized antibodies or antibody fragments specific for human CD45 disclosed in Table 14.

[0175] In one embodiment, a 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.

[0176] In further embodiments, the host cell is an isolated host cell. In further embodiments, 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 an HKB11 cell.

[0177] One skilled in the art will understand that the nucleic acid sequence or sequences encoding the heavy and / or light chains of an antibody or antibody fragment of the present disclosure can be cloned into different vectors or into the same vector.

[0178] The vector can be introduced into an appropriate host cell, such as a prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or mammalian) cell, by methods well known in the art (e.g., "Current Protocols in Molecular Biology," Ausubel et al. (eds.), Greene Publishing Assoc and John Wiley Interscience, New York, 1989 and 1992). Numerous cloning vectors are known to those skilled in the art, and the selection of 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 optionally, an 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 this expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. Upon expression in a host cell, the antibody or antibody fragment of the present disclosure is obtained. These steps can be achieved in different ways, as known to those skilled in the art. In general, such processes typically involve transforming or transfecting suitable host cells with a nucleic acid composition, vector composition, or infectious particle encoding the antibody or antibody fragment. Furthermore, such processes typically involve culturing the host cells under conditions suitable for their proliferation (expansion, growth) and for their production (expression, synthesis) of the encoded antibody or antibody fragment. Culturing host cells under conditions suitable for proliferation or expression is typically accomplished in the presence of a medium containing components suitable for inducing cell growth or expression. In particular, in embodiments, the disclosed methods for producing antibodies or antibody fragments further include isolating and purifying the produced antibody or antibody fragment from the host cells or medium. If the expression system secretes the protein into the growth medium, the protein can be purified directly from the medium. If the protein is not secreted, it is isolated from a cell lysate or recovered from a cell membrane fraction. The selection of appropriate growth conditions and recovery methods is within the skill of one of ordinary skill in the art.The antibodies or antibody fragments of the present disclosure can then be purified by several techniques known to those of skill in the art.

[0179] In one embodiment, the present disclosure provides a method for producing a humanized antibody or antibody fragment specific to 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, comprising culturing a host cell containing a vector or vector composition comprising a plurality of vectors, the vector comprising a nucleic acid sequence or nucleic acid sequence encoding an antibody or antibody fragment according to the present disclosure, under conditions suitable for expression of the antibody or antibody fragment, and isolating the antibody or antibody fragment from the host cell or host cell culture medium. Antibodies or antibody fragments isolated as described herein can be purified using techniques known in the art, such as high-performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The conditions used to purify a particular antibody or antibody fragment will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., 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 binds can be used. For example, a matrix with Protein A or Protein G can be used for affinity chromatography purification of antibodies or antibody fragments according to the present disclosure. The purity of the antibody or antibody fragment can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.

[0180] specificity In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for human CD45. In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for human CD45 disclosed in Table 14. In one embodiment, the 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 disclosure relates to a humanized antibody or antibody fragment specific for a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0181] In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, wherein the antibody or antibody fragment cross-competes with the antibody Atti for binding to CD45. In another embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, wherein the antibody or antibody fragment cross-competes with an antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9 for binding to CD45.

[0182] In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which binds to the same epitope on CD45 as antibody Atti. In another embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which binds to the same epitope on CD45 as an antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0183] In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which antibody or antibody fragment retains the binding specificity of antibody Atti. In another embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which antibody or antibody fragment retains the binding specificity of an antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0184] In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which antibody or antibody fragment binds to CD45 with approximately the same affinity as antibody Atti. In another embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which antibody or antibody fragment binds to CD45 with approximately the same affinity as an antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0185] In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which binds to CD45 with greater affinity than antibody Atti. In another embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which binds to CD45 with greater affinity than an antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0186] In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, wherein the antibody or antibody fragment binds to CD45 with at least two-fold greater affinity than an antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9, wherein the humanized antibody or antibody fragment has the following affinity: a) the first asparagine of the LCDR1 region (SEQ ID NO: 7) is replaced with an arginine, or b) contains at least one mutation in which arginine in the LCDR2 region (SEQ ID NO: 8) is replaced with leucine.

[0187] In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which has a fewer number of critical deamidation sequence motifs than antibody Atti. In another embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which has a fewer number of critical deamidation sequence motifs than an antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0188] In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which has key sequence motifs for a fewer number of T-cell epitopes than antibody Atti. In another embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which has key sequence motifs for a fewer number of T-cell epitopes than an antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0189] biological function In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which is functionally equivalent to the antibody Atti. In another embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which is functionally equivalent to an antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0190] In one embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which inhibits a biological function of CD45 at least to the same extent as antibody Atti. In another embodiment, the disclosure relates to a humanized antibody or antibody fragment specific for CD45 disclosed herein, which inhibits a biological function of CD45 at least to the same extent as an antibody or antibody fragment comprising a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, an HCDR3 region of SEQ ID NO: 6, and a variable heavy chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9.

[0191] 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 coupled to a toxin, thereby inhibiting the proliferation of CD45-positive cells.

[0192] Effector function The Fc region of an immunoglobulin generally confers favorable pharmacokinetic properties to antibodies, such as extended serum half-life and the ability to induce effector functions via binding to Fc receptors expressed on cells. However, Fc receptor binding can also result in the undesired activation of certain cell surface receptors, potentially resulting in undesired cytokine release and severe side effects upon systemic administration.

[0193] Thus, in certain therapeutic situations, it may be desirable to reduce or eliminate the normal binding of a wild-type Fc region of an antibody, such as a wild-type IgG Fc region, to one or more or all of the Fc receptors and / or binding to complement components, such as C1q, to ​​reduce or eliminate the ability of the antibody to induce effector functions. For example, it may be desirable to reduce or eliminate binding of the Fc region of an antibody to one or more or all of the Fcy receptors, such as FcyRI, FcyRI1a, FcyRIIb, and FcyRIIIa. Effector functions may include, but are not limited to, one or more of complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, binding to NK cells, binding to macrophages, binding to monocytes, binding to polymorphonuclear cells, direct signaling to induce apoptosis, crosslinking of target-bound antibodies, dendritic cell maturation, or T cell priming.

[0194] Reducing or eliminating the binding of an Fc region to an Fc receptor and / or C1q is typically achieved by mutating a wild-type Fc region, such as an IgG1 Fc region, more specifically a human IgG1 Fc region, to produce a mutant or engineered Fc region of the wild-type Fc region, such as a mutant human IgG1 Fc region. Substitutions that result in reduced binding can be useful. To reduce or eliminate the binding properties of an Fc region to an Fc receptor, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid with different structural and / or chemical properties, are preferred.

[0195] 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 when compared to the wild-type Fc region. In one such embodiment, the 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, the isolated antibody or antibody fragment according to the present disclosure does not substantially induce effector function.

[0196] In certain embodiments, the effector function is one or more selected from the group consisting of 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, an isolated antibody or antibody fragment according to the present disclosure does not substantially induce ADCC and / or CDC and / or ADCP. In one embodiment, an isolated antibody or antibody fragment according to the present disclosure does not induce ADCC or ADCP in vitro.

[0197] In one embodiment, the variant Fc region of an isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions that reduce or eliminate binding of the variant Fc region to one or more Fc receptors and / or C1q when compared to the wild-type Fc region. In one embodiment, the variant Fc region of an isolated antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions that reduce or eliminate the ability of the antibody to induce effector function when compared to the wild-type Fc region. In certain embodiments, the one or more amino acid substitutions may reduce the binding affinity of the variant Fc region to one or more Fc receptors and / or C1q by at least two-fold, at least five-fold, at least ten-fold, at least twenty-fold, or even at least fifty-fold when compared to the wild-type Fc region. In alternative embodiments, the one or more amino acid substitutions may reduce the ability of the isolated antibody or antibody fragment according to the present disclosure to induce effector function by at least two-fold, at least five-fold, at least ten-fold, at least twenty-fold, or even at least fifty-fold when compared to the wild-type Fc region.

[0198] In one embodiment, the variant Fc region of an 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 an antibody according to the present disclosure substantially eliminates the ability of the antibody to induce effector function. In one embodiment, an antibody or antibody fragment according to the present disclosure does not substantially induce effector function. In one embodiment, the effector function is ADCC and / or ADCP and / or CDC. In one embodiment, an 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 above background measured in the absence of the antibody.

[0199] In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an Fcy receptor. In one embodiment, the Fc receptor is human FcyRIIIa, FcyRI, FcyRIla and / or FcyRIlb.

[0200] 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 relative to the 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 the wild-type Fc region.

[0201] Various Fc modifications are commonly used. For a review, see, for example, Antibodies (2020) 9:64. Silencing functions include LALA (L234A / L235A), PA-LALA (L234A / L235A / P329A) and PG-LALA (L234A / L235A / P329G) mutations, and AEASS mutations (L234A / L235E / G237A / A330S / P331S) (numbering according to the EU index).

[0202] In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody has a silenced Fc region. In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody or antibody fragment is in the AEASS format.

[0203] In certain embodiments, the disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises an Fc region having at least the following modifications: L234A, L235E, G237A, A330S, P331S (numbering according to the EU index). In certain embodiments, the disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises an Fc region having the following modifications: L234A, L235E, G237A, A330S, P331S (numbering according to the EU index).

[0204] In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody or antibody fragment is in the PA-LALA format. In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises an Fc region having at least the following modifications: L234A, L235A, P329A (numbering according to the EU index). In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises an Fc region having the following modifications: L234A, L235A, P329A (numbering according to the EU index).

[0205] In certain embodiments, the present disclosure relates to a humanized antibody or antibody fragment specific for CD45, wherein the humanized antibody or antibody fragment is in the PG-LALA format. In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises an Fc region having at least the following modifications: L234A, L235A, P329G (numbering according to the EU index). In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises an Fc region having the following modifications: L234A, L235A, P329G (numbering according to the EU index).

[0206] In certain embodiments, the present disclosure relates to a humanized antibody 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 a humanized antibody specific for CD45, wherein the humanized antibody comprises an Fc region with a I253A mutation (numbering according to the EU index). In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises an Fc region with a H310A mutation (numbering according to the EU index). In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises an Fc region with a H435A mutation (numbering according to the EU index). In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises an Fc region having an H435Q mutation (numbering according to the EU index).

[0207] In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody or antibody fragment has mutations that result in increased binding to FcRn, thereby increasing the in vivo half-life of the antibody. In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises T250Q / M428L mutations in the Fc region (numbering according to the EU index). In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises M252Y / S254T / T256E (YTE) mutations in the Fc region (numbering according to the EU index). In certain embodiments, the present disclosure relates to a humanized antibody specific for CD45, wherein the humanized antibody comprises H433K / N434F mutations in the Fc region (numbering according to the EU index). In certain embodiments, the present 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).

[0208] In certain embodiments, the present disclosure relates to a humanized antibody, wherein the humanized antibody comprises: a) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 63, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; b) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 65, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; c) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; d) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 69, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; e) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 71, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; f) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 73, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; g) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 75, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; h) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 77, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; i) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 79, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; j) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 81, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; k) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 83, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; l) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 85, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; m) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 87, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; n) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 89, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; o) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 91, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; p) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9; q) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; r) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 95, and an LCDR3 region of SEQ ID NO: 9; s) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; t) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 7, an LCDR2 region of SEQ ID NO: 97, and an LCDR3 region of SEQ ID NO: 9; u) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; v) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 67, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; w) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 61, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 69, an LCDR2 region of SEQ ID NO: 94, and an LCDR3 region of SEQ ID NO: 9; x) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 75, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; y) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; z) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 8, and an LCDR3 region of SEQ ID NO: 9; aa) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; bb) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 110, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; cc) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 96, and an LCDR3 region of SEQ ID NO: 9; dd) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 106, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9; or ee) a variable heavy chain comprising an HCDR1 region of SEQ ID NO: 4, an HCDR2 region of SEQ ID NO: 5, and an HCDR3 region of SEQ ID NO: 6, and a variable light chain comprising an LCDR1 region of SEQ ID NO: 108, an LCDR2 region of SEQ ID NO: 93, and an LCDR3 region of SEQ ID NO: 9; The Fc region of the humanized antibody contains the modifications L234A, L235A and P329A (numbering according to the EU index).

[0209] Treatment method The antibodies and antibody fragments of the present disclosure, or pharmaceutical compositions incorporating them, can be used to treat a variety of conditions. For example, such antibodies can be used to remove endogenous hematopoietic stem and progenitor cells (HSPCs) in subjects in need of such removal. Ablation of endogenous HSPCs is the first step in stem cell replacement therapy, which generally involves reducing or eliminating endogenous HSPCs that are defective in some way and replacing them with replacement HSPCs. Replacement HSPCs can be autologous, allogeneic, or xenogeneic. Endogenous HSPCs can be lost as a result of inherited mutations that impair function or expression (e.g., sickle cell anemia or thalassemia), as a result of blood cancer, or as a result of damage from chemotherapy used to treat cancer. Endogenous HSPCs can also be replaced in conjunction with organ transplants, as endogenous HSPCs pose an immune attack on the transplant.

[0210] Antibodies to CD45 can also be used to treat cancers that express CD45, including blood cancers such as leukemia, myeloma, or lymphoma.

[0211] Antibodies against CD45 can also be used to treat inflammatory or autoimmune diseases such as multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn's disease, type 1 diabetes, rheumatoid arthritis or idiopathic arthritis.

[0212] 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.

[0213] In other embodiments, the present disclosure relates to the use of a humanized antibody or antibody fragment of the present disclosure for use in treating cancer. In other embodiments, the present disclosure relates to the use of a humanized antibody or antibody fragment of the present disclosure for use in treating a hematological cancer. In other embodiments, the present disclosure relates to the use of a humanized antibody or antibody fragment of the present disclosure for use in treating a leukemia, myeloma, or lymphoma.

[0214] In other embodiments, the present disclosure relates to the use of a humanized antibody or antibody fragment of the present disclosure for use in treating an inflammatory disease or an autoimmune disease, hi other embodiments, the present disclosure relates to the use of a humanized antibody or antibody fragment of the present disclosure for use in treating multiple sclerosis, systemic sclerosis, systemic lupus erythematosus, Crohn's disease, type 1 diabetes, rheumatoid arthritis, or idiopathic arthritis.

[0215] In certain embodiments, the present disclosure relates to the use of a humanized antibody or antibody fragment of the present disclosure for use in treating a disease associated with the unwanted 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 treating a disease associated with the unwanted presence of CD45-positive cells.

[0216] In one embodiment, the present disclosure provides a method of treating a disease.

[0217] In one embodiment, the present disclosure provides a method of treating a disease comprising administering to a patient an antibody or antibody fragment of the present disclosure.

[0218] In one embodiment, the present disclosure provides a method of treating a disease comprising administering to a subject in need thereof an antibody or antibody fragment of the present disclosure.

[0219] In one embodiment, the present disclosure provides a method for preventing a disease.

[0220] In one embodiment, the present disclosure provides a method of preventing disease comprising administering to a subject an antibody or antibody fragment of the present disclosure.

[0221] In one embodiment, the present disclosure provides an isolated antibody or antibody fragment according to the present disclosure for treating a disease. 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 treating a disease in a subject in need thereof.

[0222] In one embodiment, the present disclosure provides for the use of an isolated antibody or antibody fragment according to the present disclosure for the manufacture of a medicament. In one embodiment, the present disclosure provides for an isolated antibody or antibody fragment according to the present disclosure for use as a medicament. In one embodiment, the present disclosure provides for an isolated antibody or antibody fragment according to the present disclosure for use in medicine. In one embodiment, the present disclosure provides for an isolated antibody or antibody fragment according to the present disclosure for use as a medicament for the treatment of a subject in need thereof.

[0223] 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, comprising administering to the subject a therapeutically effective amount of an antibody or antibody fragment according to the present disclosure.

[0224] 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 a method of treatment, an isolated antibody or antibody fragment according to the present disclosure will be formulated, dosed, and administered in a manner consistent with good medical practice.

[0225] Pharmaceutical Composition 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.

[0226] The antibody is administered in an effective regimen, meaning a dosage, route of administration, and frequency of administration that achieves the intended purpose, such as reducing endogenous HSPCs, tissue-resident or circulating immune cells, or cancer cells expressing CD45. In some cases, efficacy can be observed in an individual patient compared to historical controls or previous experience in the same patient. In other instances, efficacy can be demonstrated in preclinical or clinical trials in a population of treated patients compared to a control population of untreated patients.

[0227] The pharmaceutical composition may further comprise at least one other pharmaceutically active compound. The pharmaceutical composition according to the present disclosure can be used for the diagnosis, prevention, and / or treatment of diseases associated with the undesired presence of CD45, particularly human CD45. The pharmaceutical composition according to the present disclosure can be used for the diagnosis, prevention, and / or treatment of diseases associated with the undesired presence of CD45-positive cells, particularly CD45-positive human cells. In particular, the present disclosure provides pharmaceutical compositions comprising an antibody or antibody fragment according to the present disclosure suitable for prophylactic, therapeutic, and / or diagnostic use in mammals, more particularly humans.

[0228] Generally, antibodies or antibody fragments according to the present disclosure can be formulated as pharmaceutical compositions comprising at least one antibody or antibody fragment according to the present disclosure, at least one pharmaceutically acceptable carrier or excipient, and, optionally, one or more additional pharmaceutically active compounds. Such formulations can be suitable for oral, parenteral, topical, or inhalation administration. Thus, pharmaceutical compositions comprising at least one antibody or antibody fragment according to the present disclosure can be administered parenterally, for example, intravenously, intramuscularly, or subcutaneously. Alternatively, antibodies of the present invention can be administered parenterally, for example, orally or topically. In a preferred embodiment, pharmaceutical compositions comprising antibodies or antibody fragments according to the present disclosure are administered intravenously or subcutaneously.

[0229] In particular, antibodies or antibody fragments according to the present disclosure are or can be used for the prevention and / or treatment of diseases involving the target antigen of interest, and can be used in combination with one or more pharmaceutically active compounds, which may or may not result in a synergistic effect. Examples of such compounds, as well as routes, methods and pharmaceutical formulations or compositions for administering them, will be apparent to the clinician.

[0230] 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 a disease associated with the undesired 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 a disease associated with the undesired 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 an autoimmune disease and / or an inflammatory disease and / or cancer.

[0231] In one embodiment, the present disclosure provides a method for treating an autoimmune disease and / or inflammatory disease and / or cancer in a subject in need thereof using a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure.

[0232] Further provided is a method of producing an antibody or antibody fragment according to the present disclosure in a form suitable for in vivo administration, comprising: (a) obtaining the 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, whereby the antibody or antibody fragment preparation is formulated for in vivo administration. Pharmaceutical compositions according to the present disclosure comprise 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.

[0233] Diagnostic Use In one embodiment, the present disclosure provides the use of an isolated antibody or antibody fragment specific to human CD45 according to the present disclosure for the diagnosis of disease. In one embodiment, the present disclosure provides the use of an antibody or antibody fragment according to the present disclosure for the detection of CD45, particularly human CD45. In one embodiment, the present disclosure provides a method for detecting CD145 in a subject or sample, comprising contacting the subject or sample with an isolated antibody or antibody fragment specific to human CD45 according to the present disclosure. In one embodiment, the present disclosure provides a method for diagnosing disease in a subject, comprising contacting the subject or sample with an isolated antibody or antibody fragment according to the present disclosure. The antibody can also be used to determine CD45 expression levels in cells derived from a patient. CD45 expression levels may serve as a therapeutic biomarker, for example, for patient stratification. [Example]

[0234] Example 1: Anti-CD45 antibodies of the present invention Antibody Atti is a murine anti-CD45 antibody. The sequences of the variable chains and CDRs of antibody Atti are shown in the table below: (Table 1) JPEG2026500540000001.jpg91128

[0235] Using an elegant approach, the inventors of the present application successfully humanized the antibody Atti while simultaneously removing undesired post-translational modifications and fragmentation sites. Surprisingly, the antibody produced did not lose its affinity and activity. This was achieved through a stepwise approach, as further detailed in the Examples below.

[0236] Example 2: Humanization of the murine antibody Atti The humanization process relied on a combination of CDR-grafting techniques coupled with the latest research on antibody structure and an up-to-date database of mature human IgG sequences. Several human framework sequences were identified that were used as "acceptor" frameworks for the target CDR sequences. All acceptor sequences were 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.

[0237] Example 2.1: Design of humanized variants of the variable heavy chain The closest human germline gene V region identified in humans was IGHV3-23: EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVIYSGGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (SEQ ID NO: 15).

[0238] A database of human IgG sequences was searched for comparison with the mouse VH domain using the BLAST search algorithm, 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, preserving critical framework residues and canonical loop structure.

[0239] The four acceptor frameworks are: >QDF60926 EVQLVESGGGLVQPGGSLRLSCAASGFTLSNYDMNWVRQAPGKRLEWVSYISSSGSTTYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGHNNWFLYFDSWGQGTLVTVSS (SEQ ID NO: 16) >ABF83355 QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQAPGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDGSWYFDLWGRGTLVTVSS (SEQ ID NO: 17) >QAV55037 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYEMSWVRQAPGKGLEWVSYISSSANTIYYADSVKGRFTISRDNAKKSLFLQMNSLRAEDTAVYYCARVVLGYWYFDLWGRGTLVTVSS (SEQ ID NO: 18) >AMK70661 EVQLVESGGDLVQPGGSLRLSCAASGFSFSSYSMNWVRQAPEKGLEWVSYISSSSRTIYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARGNGDERAVYYYYGMDVWGQGTTVTVSS (SEQ ID NO: 19)

[0240] As the fifth acceptor sequence, the closest human germline IGHV3-23 (SEQ ID NO: 15) was selected.

[0241] Using the CDRs of the mouse VH of Atti grafted onto the acceptor framework described above, the following humanized variants were selected and synthesized: >VH1 EVQLVESGGGLVQPGGSLRLSCAASGFAFSNYDMSWVRQAPGKRLEWVSYISSGGVSTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRYDVWWYFDVWGQGTLVTVSS (SEQ ID NO: 20) >VH2 QVQLVESGGGLVKPGGSLRLSCAASGFAFSNYDMSWIRQAPGKGLEWVSYISSGGVSTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRYDVWWYFDVWGRGTLVTVSS (SEQ ID NO: 21) >VH3* EVQLVESGGGLVQPGGSLRLSCAASGFAFSNYDMSWVRQAPGKGLEWVSYISSGGVSTYYPDTVKGRFTISRDNAKKSLFLQMNSLRAEDTAVYYCARRYDVWWYFDVWGAGTLVTVSS (SEQ ID NO: 22) >VH4* EVQLVESGGDLVQPGGSLRLSCAASGFAFSNYDMSWVRQAPEKGLEWVSYISSGGVSTYYPDTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARRYDVWWYFDVWGAGTTVTVSS (SEQ ID NO: 23) >VH5* EVQLLESGGGLVQPGGSLRLSCAASGFAFSNYDMSWVRQAPEKGLEWVAYISSGGVSTYYPDTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRYDVWWYFDVWGAGTTVTVSS (SEQ ID NO: 24)

[0242] The alignment of the humanized variants to the original mouse Atti sequence is shown in Figure 1. The homology of the humanized variants to the original mouse VH sequence is shown in the table below. (Table 2) JPEG2026500540000002.jpg39133

[0243] Example 2.2: Design of humanized variants of the variable light chain The closest human germline gene V region identified in humans was IGKV2-30: DVVMTQSPLSLPVTLGQPASISCRSSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWP (SEQ ID NO: 25)

[0244] A database of human IgK sequences was searched for comparison with the mouse VL domain using the BLAST search algorithm, 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, preserving key framework residues and canonical loop structure.

[0245] The four acceptor frameworks are: >QEP26792 DIVMTQTPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLQQRPGQPPRLLIYKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCMQATHFPPYTFGQGTKLEIK (SEQ ID NO: 26) >BAH04700 EIVLTQSPLSLPVTLGQPASISCRPSQSLVHSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPWTFGQGTKVEIK (SEQ ID NO: 27) >ABA26062 DIVMTQTPLSLPVTLGQPASISCRSSQSLLYSDGNTYLNWFHQRPGQSPRRLIYRVSNRDSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPPTTFGQGTRLEIK (SEQ ID NO: 28) >BAH04694 DVVMTQSPLSLPVTLGQPASISCRSSQSLVHTDGNTYLNWFLQRPGQSPRRLIYKVSDRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQNTHWPLTFGQGTKVEIK (SEQ ID NO: 29)

[0246] As the fifth acceptor sequence, the closest human germline IGKV2-30 (SEQ ID NO: 25) was selected.

[0247] Using the CDRs of the mouse VL of Atti grafted onto the acceptor framework described above, the following humanized variants were selected and synthesized: >VL1 DIVMTQTPLSSPVTLGQPASISCRSSQSIVHSNGNTYLEWLQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 30) >VL2* DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKVEIK (SEQ ID NO: 31) >VL3* DIVMTQTPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFHQRPGQSPRRLIYKVSNRFSGIPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTRLEIK (SEQ ID NO: 32) >VL4* DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFLQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKVEIK (SEQ ID NO: 33) >VL5* DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKLEIK (SEQ ID NO: 34)

[0248] *VL2-VL5 have mutations introduced to make the antibodies more similar to the parent antibody while maintaining the humanized nomenclature.

[0249] The alignment of the humanized variants to the original mouse Atti sequence is shown in Figure 2. The homology of the humanized variants to the original mouse VL sequence is shown in the table below. (Table 3) JPEG2026500540000003.jpg41133

[0250] Example 2.3: Expression and purification of humanized variants 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 antibody-containing cell culture supernatant was clarified by centrifugation and filtration. Antibodies were purified from the cell culture supernatant via affinity chromatography using state-of-the-art chromatography equipment. The purified antibodies were buffer-exchanged into phosphate-buffered saline. The purity of these antibodies was determined to be greater than 95%, as judged by reducing and denaturing sodium dodecyl sulfate polyacrylamide gels. Antibody concentrations were determined by measuring absorbance at 280 nm.

[0251] Five humanized variable heavy chains were tested in combination with five humanized variable light chains, resulting in a total of 25 combinations. Of these 25 combinations, six combinations could be produced. All six of these combinations, independent of the humanized heavy chain used, contain the humanized light chain VL5. Additionally, VH2 combined with VL3 could be successfully produced. These six combinations can be produced in sufficient quantities for further characterization.

[0252] Example 2.4: Affinity determination of humanized variants IgG antibodies were immobilized on a biosensor using an appropriate capture surface, and the binding of soluble antigens to the immobilized antibodies was monitored by BLI (Octet). The resulting sensorgrams were analyzed using the provided software (Fortebio). Human recombinant CD45 from R&D Systems (catalog number 1430-CD-050; lot number NUZ142001) was used as the antigen. As a preliminary experiment, a series of experiments was performed to optimize the assay parameters. The following parameters were selected: (Table 4) JPEG2026500540000004.jpg48128

[0253] In the main experiment, a kinetic assay was performed by first capturing IgG using an anti-human Fc biosensor. The mAb-captured biosensor was then immersed in wells containing different concentrations of antigen (association step), followed by a dissociation step in running buffer. To allow for double referencing, the IgG-captured sensor was immersed in a well containing buffer only, and a blank sensor was also immersed in a well containing antigen. This referencing provided a means of compensating for both spontaneous dissociation of the captured IgG and nonspecific binding of antigen to the sensor surface. The process was 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 those recommended by ForteBio.

[0254] All samples were diluted with freshly prepared running buffer. Antibody variants were immobilized on a series of biosensor surfaces using the described capture method. 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. A dilution series of antigen was used in the association step to globally fit the results and obtain the best values ​​for ka, kd, and KD. Response data for antigen binding to immobilized IgG were fit to a 1:1 binding model. Kinetic parameters are summarized in the table below. HC0 LC0 refers to mouse Atti. (Table 5) JPEG2026500540000005.jpg120128NF: Does not fit the 1:1 combination model

[0255] Five antibodies exhibited dissociation constants (KD) within the range of the original antibody Atti (HC0 LC0). Of these five antibodies, four had better KDs than Atti: VH1 / VL5, VH2 / VL5, VH3 / VL5, and VH4 / VL5. The VH2 / VL3 combination did not fit the 1:1 binding model and was therefore not investigated further.

[0256] Example 2.5: Biophysical characterization of three preselected antibodies Protein aggregation along non-native pathways is recognized to be caused by conformational instability or partial unfolding of the protein. Protein unfolding exposes hydrophobic core residues, which then combine with other partially unfolded monomers to form dimers, ultimately acting as nucleation points for mass aggregation. Therefore, conformational stability indicates a protein's propensity for non-native aggregation. Thermal stability, through the determination of its melting temperature (Tm), has been accepted as a viable method for monitoring the conformational stability of a protein.

[0257] Differential scanning fluorimetry (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 a protein's thermal stability and, ultimately, its tendency toward non-native aggregation. DSF uses a fluorescent dye that is quenched in an aqueous environment but fluoresces under hydrophobic conditions. Thus, upon thermal exposure, the antibody loses conformational stability and begins to unfold, exposing hydrophobic core residues, which can be measured as an increase in fluorescent signal. A temperature vs. fluorescence (melting profile) plot can then be used to determine the melting temperature (dF / dT curve) of the protein of interest.

[0258] Many antibody molecules exhibit biphasic melting profiles. Published studies have shown that mAbs exhibit characteristic unfolding profiles as a result of the melting transitions of their individual domains, CH2, CH3, and Fab. The thermal stability of IgG molecules is affected by all of these domains. DSF curves reported for antibodies show two clearly distinguishable transitions: a lower temperature representing unfolding of the CH2 domain (Tm1), and a higher temperature representing melting of the CH3-Fab domain (Tm2). The Fab melting transition is generally well-defined, with a peak up to 2–3 times larger than the CH2 or CH3 peaks.

[0259] For DSF analysis, 5 μl of Sypro Orange (diluted 1 / 200 in water; Sigma) and 45 μl of a 0.3 mg / ml solution of the antibody to be tested were added to a tube (Bio-Rad; TLS0831). The tube was sealed with an optical flat cap (Bio-Rad; TCS0803) and heated in 0.5°C increments from 20°C to 95°C in an i-Cycler iQ5 real-time PCR detection system (Bio-Rad). Fluorescence changes in the plate wells were simultaneously monitored with a charge-coupled device (CCD) camera. The excitation and emission wavelengths were 485 nm and 575 nm, respectively. The temperature midpoint (Tm) of the protein unfolding transition was calculated using Bio-Rad iQ5 software.

[0260] For size exclusion chromatography (SEC), samples were diluted to a final concentration of 0.1 mg / ml with phosphate-buffered saline (PBS). Highly purified antibody samples were independently loaded onto a Superdex 200 Increase 10 / 300GL 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 in phosphate-buffered saline at 19°C. Protein peaks were monitored using absorbance at 214 nm, and spectra were analyzed using the Unicorn evaluation software package (Cytiva).

[0261] Melting curves were successfully generated for all samples. All antibodies tested exhibit a single phase transition melting profile. VH2 / VL5 exhibited the highest TM1 melting temperature, indicating that this IgG has the highest conformational stability of the molecules tested. The Tm values ​​are summarized in the table below.

[0262] SEC analysis was also successfully performed on all samples. Peak quality in SEC analysis was good for all samples. All antibodies eluted as one major peak. From column calibration, the main peak corresponded to monomeric IgG. (Table 6) JPEG2026500540000006.jpg25128

[0263] Example 2.6: Summary and selection of finalists for further engineering All combinations containing VL5 were successfully expressed and purified. Furthermore, VH2 / VL3 combinations were also successfully expressed and purified. SDS-PAGE analysis demonstrated sufficient levels of purity for all antibodies. Under reducing conditions, both the heavy and light chains of the antibodies were visible, with expected molecular weights of approximately 50 kDa and 25 kDa, respectively. Under non-reducing conditions, a single major band and several minor bands were observed. The additional bands (impurities) likely represent non-glycosylated IgG and IgG degradation products (e.g., a single [partial] light chain, two heavy chains and one light chain, two heavy chains, or two heavy chains and one light chain combination).

[0264] In kinetic (Octet) analysis, all combinations with VL5 (VH1 / VL5, VH2 / VL5, VH3 / VL5, VH4 / VL5, VH5 / VL5) showed binding properties similar to those of the control antibody VH0 / VL0, with dissociation constants within 2-fold of the control antibody.

[0265] The biophysical properties of all antibodies were as good as the original Atti antibody.

[0266] Antibody VH2 / VL5 was selected for further manipulation, as it displayed an affinity of 162 pM, more than two-fold higher than that of the original Atti antibody (354 pM), and exhibited the highest melting temperature (TM1), indicating that this IgG was the most conformationally stable of the molecules tested.

[0267] Example 3: Removal of critical sites prone to increased PTM, immunogenicity, flexibility, or hydrophobicity Example 3.1: Design of engineered humanized variants In the next engineering step, the antibodies were screened for sites susceptible to Fv glycosylation, deamidation, isomerization, fragmentation, other types of post-translational modifications (PTMs), sequence hotspots, increased flexibility, or hydrophobicity. Several such sites were identified in the CDRs of the antibodies. See the table below. (Table 7) JPEG2026500540000007.jpg29128

[0268] To remove these deleterious amino acid motifs, antibody VH2 / VL5 mutants were generated, and the approach to each change was rationalized by the analysis described below.

[0269] Sequence- and structure-based descriptors were calculated in silico to identify suitable variants that preserve the structural integrity and biological activity of antibodies while enhancing their developability, binding affinity, and solubility. To this end, physicochemical descriptors were evaluated against a dataset of therapeutic antibodies retrieved from the TABS database (https: / / tabs.craic.com / ) and a second dataset consisting of paired antibody repertoires from the Observational Antibody Space Database (http: / / opig.stats.ox.ac.uk / webapps / oas / ). Sequence alignments, antibody numbering, and CDR canonical structures were annotated upon alignment of variable domain sequences to isotype-specific hidden Markov models (HMMs) constructed as described in Nat Protoc (2014) 9:2771-83. Based on the methods described in Nucleic Acid Res (2017) 45:W17-W23 and Bioinformatics (2014) 30:2733-40, a three-dimensional model was constructed using a template-based approach for the framework, canonical structure modeling for the CDRs, and a random forest machine learning modeling approach for the heavy chain loop H3. For VH / VL packing, a template-based approach based on overall similarity and identity at residue position L44 was used, as described in FEBS J. (2011) 278:2858-66. Among the sequence descriptors, the antibody solubility profile was calculated as a linear combination of different physicochemical properties, such as the hydrophobicity of each amino acid, electrostatic charge at neutral pH, and α-helical and β-strand propensity ( Nat Struct Biol (1996) 3:842-8, J Mol Biol (1994) 238:693-708). To account for the influence of neighboring amino acids, we use a sliding window approach, in which per-residue solubility descriptors are averaged over a window of seven consecutive amino acids and corrected for the occurrence of polar / non-polar aggregation propensity patterns (J Mol Biol (2000) 296:961-8; Methods Mol Biol (2022) 2313:57-113).During the engineering process, we did not impose specific targets for local and global solubility. However, we excluded mutants that would result in the formation of covalent aggregates (cysteines) and solvent-exposed hydrophobic patches, as assessed by the relative solvent accessibility per residue calculated in a 3D model of the paired VH / VL domains (F1000Res(2016)5:189). Furthermore, we also excluded mutants that would affect the conformation of the CDRs and thus the antibody binding mode. This was done by assessing the contribution of the engineered sites to one of the established canonical structures (Nat Protocol(2014)9:2771-83) and by evaluating the predicted binding modes of the native and engineered molecules using per-residue probability scores (Bioinformatics(2013)29:2285-91). Furthermore, we eliminated / prioritized variants that would likely increase / decrease immunogenicity risk, as assessed by comparing the in silico calculated immunogenicity profile with that of the natural human repertoire and a large dataset of known immunogenic therapeutic antibodies (ADA frequency). To this end, we used a re-adaptation of the NetMHCIIpan neural network algorithm to screen natural 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 shared HLA ligands to the human germline that are likely to be tolerated or tolerogenic. Higher priority was given to variants that did not harbor neoepitopes (new HLA binders vs. native) or cross-reactive epitopes (ligands recognized by multiple HLA alleles), and lower priority was given to variants that showed high epitope content in regions of antibodies that showed low immunogenicity scores in the reference antibody dataset.

[0270] The sequences of the new VH and VL chains are shown below. >VL5.2 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSRGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKLEIK (SEQ ID NO: 38) >VL5.3 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSKGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 39) >VL5.4 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSQGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKLEIK (SEQ ID NO: 40) >VL5.5 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSSGNTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 41) >VL5.6 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNANTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKLEIK (SEQ ID NO: 42) >VL5.7 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGQTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 43) >VL5.8 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGKTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 44) >VL5.9 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGETYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKLEIK (SEQ ID NO: 45) >VL5.10 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGHTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKLEIK (SEQ ID NO: 46) >VL5.11 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNKYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 47) >VL5.12 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSQGQTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKLEIK (SEQ ID NO: 48) >VL5.13 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSQGKTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 49) >VL5.14 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSSGQTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 50) >VL5.15 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSKGQTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKLEIK (SEQ ID NO: 51) >VL5.16 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNANKYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 52) >VL5.17 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNLFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKLEIK (SEQ ID NO: 53) >VL5.18 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 54) >VL5.19 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKLEIK (SEQ ID NO: 55) >VL5.20 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 56) >VL5.21 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYQQRPGQSPRLLIYKVSNLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGGGTKLEIK (SEQ ID NO: 57) >VL5.22 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSQGNTYLEWYQQRPGQSPRLLIYKVSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 58) >VL5.23 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSSGNTYLEWYQQRPGQSPRLLIYKVSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 59) >VH2.2 QVQLVESGGGLVKPGGSLRLSCAASGFAFSNYDMSWIRQAPGKGLEWVSYISSGGVSTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARRYDVWYYFDVWGRGTLVTVSS (SEQ ID NO: 60)

[0271] The SEQ ID NOs of the variable light chains containing the CDRs are shown in the table below. (Table 9) JPEG2026500540000008.jpg130128 Sequence number 61:RYDVWYYFDV SEQ ID NO: 62: ARRYDVWYYFDV SEQ ID NO: 63: RSSQSIVHSRGNTYLE SEQ ID NO: 64: QSIVHSRGNTY SEQ ID NO: 65: RSSQSIVHSKGNTYLE SEQ ID NO: 66: QSIVHSKGNTY SEQ ID NO: 67: RSSQSIVHSQGNTYLE SEQ ID NO: 68: QSIVHSQGNTY SEQ ID NO: 69: RSSQSIVHSSGNTYLE SEQ ID NO: 70: QSIVHSSGNTY SEQ ID NO: 71: RSSQSIVHSNANTYLE SEQ ID NO: 72: QSIVHSNANTY SEQ ID NO: 73: RSSQSIVHSNGQTYLE SEQ ID NO: 74: QSIVHSNGQTY SEQ ID NO: 75: RSSQSIVHSNGKTYLE SEQ ID NO: 76: QSIVHSNGKTY SEQ ID NO: 77: RSSQSIVHSNGETYLE SEQ ID NO: 78: QSIVHSNGETY SEQ ID NO: 79: RSSQSIVHSNGHTYLE SEQ ID NO: 80: QSIVHSNGHTY SEQ ID NO: 81: RSSQSIVHSNGNKYLE SEQ ID NO: 82: QSIVHSNGNKY SEQ ID NO: 83: RSSQSIVHSQGQTYLE SEQ ID NO: 84: QSIVHSQGQTY SEQ ID NO: 85: RSSQSIVHSQGKTYLE SEQ ID NO: 86: QSIVHSQGKTY SEQ ID NO: 87: RSSQSIVHSSGQTYLE SEQ ID NO: 88: QSIVHSSGQTY SEQ ID NO: 89: RSSQSIVHSKGQTYLE SEQ ID NO: 90: QSIVHSKGQTY SEQ ID NO: 91: RSSQSIVHSNANKYLE SEQ ID NO: 92: QSIVHSNANKY SEQ ID NO: 93: KVSNLFS SEQ ID NO: 94: KVSNRAS SEQ ID NO: 95: KVSNRES SEQ ID NO: 96: KVSNLAS SEQ ID NO: 97: KVSNLES

[0272] Example 3.2: Testing of engineered humanized variants The engineered variants were subjected to the same types of analysis and characterization as the initially humanized variants. For the experimental setup, conditions similar to those in Example 2 were used, with the following differences: Binding affinity was determined using the Octet Red96e or R8 system at 25°C and 1000 rpm with 1x dynamic buffer (Sartorius, PN:18-1105). Antibodies were captured on anti-human capture biosensors (Sartorius, PN_18-5060) at 0.5-1 μg / mL for 300-600 seconds. Antigen was titrated at different concentrations ranging from 200 nM to 0.5 nM, and association / dissociation was monitored over 300-600 seconds and 1000-2000 seconds. Reference subtraction was performed against buffer-only wells. The AHC chip was regenerated using 10 mM Gly-HCl pH 1.7. Data were analyzed using Octet Data Analysis software HT 12.0. Data were fitted to a 1:1 binding model. The kinetic rates ka and kd were fitted globally.

[0273] Conformational stability was assessed by DSF as in Example 2 using Sypro Orange (Sigma) and an RT-PCR machine (C1000 Thermal Cycler, Biorad). Samples were run in duplicate at 0.25-1.0 mg / mL in PBS buffer, and Sypro Orange was added at 5x the final concentration. The same gradient as in Example 2 was used.

[0274] All mutants could be expressed and purified. The dissociation constants (KD) of most antibodies were comparable to that of the original Atti. Two mutants had a monomer content of less than 85% (VH2 / VL5.22 and VH2.2 / VL5.22). The results are summarized in the table below. (Table 10) JPEG2026500540000009.jpg125128

[0275] Example 3.3: Summary and Selection of Candidates for Further Engineering In summary, it was possible to remove undesirable motifs from the sequence of the parent antibody Atti without affecting the binding behavior of the binder. This was particularly surprising because the sequence motifs were located within the CDR regions. Nevertheless, the new variants retained their ability to bind to the target antigen almost identically, and some variants even increased affinity. At the same time, the biophysical properties of the binder remained within acceptable limits.

[0276] Six antibodies were selected for further manipulation (VH2 / VL5.2, VH2 / VL5.6, VH2 / VL5.7, VH2 / VL5.8, VH2 / VL5.17, and VH2 / VL5.20), which collectively exhibited the most promising properties for further derivatization.

[0277] Example 4: Removal of PTMs or Multiple Fragmentation-Prone Sites Example 4.1: Design of further engineered humanized variants Encouraged by the successful removal of deleterious sequence motifs from the CDRs of antibody Atti, we next investigated whether it was even possible to remove multiple such motifs without losing the affinity or functionality of the binder.

[0278] The following mutants were generated: (Table 11) ("---" indicates no change in site): JPEG2026500540000010.jpg54129

[0279] The sequence of the new VL chain is shown below. >VL5.24 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGKTYLEWYQQRPGQSPRLLIYKVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 98) >VL5.25 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNAKTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 99) >VL5.26 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSRGQTYLEWYQQRPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 100) >VL5.27 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSRGQTYLEWYQQRPGQSPRLLIYKVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 101) >VL5.28 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSRGKTYLEWYQQRPGQSPRLLIYKVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 102) >VL5.29 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNAKTYLEWYQQRPGQSPRLLIYKVSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 103) >VL5.30 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNAKTYLEWYQQRPGQSPRLLIYKVSNLFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 104) >VL5.31 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSRGQTYLEWYQQRPGQSPRLLIYKVSNLFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPMYTFGQGTKLEIK (SEQ ID NO: 105)

[0280] The SEQ ID NOs of the variable light chains containing the CDRs are shown in the table below. (Table 12) JPEG2026500540000011.jpg51128 Sequence number 106:RSSQSIVHSNAKTYLE SEQ ID NO: 107: QSIVHSNAKTY SEQ ID NO: 108: RSSQSIVHSRGQTYLE SEQ ID NO: 109: QSIVHSRGQTY SEQ ID NO: 110: RSSQSIVHSRGKTYLE SEQ ID NO: 111: QSIVHSRGKTY

[0281] Example 4.2: Testing of further engineered humanized variants The engineered variants were subjected to the same types of analysis and characterization as the original humanized variants (see Example 2 for experimental setup).

[0282] All variants could be expressed and purified in sufficient yields. All antibodies had dissociation constants K within the range of the original Atti antibody. D The results are summarized in the table below. (Table 13) JPEG2026500540000012.jpg60128

[0283] Example 4.3: Summary and Selection of Candidates for Further Engineering In summary, it was even possible to remove some undesirable motifs from the sequences of the humanized variants of Atti, while the affinity of some variants was even higher than that of the original Atti antibody, and the biophysical properties of the binders remained within the range of Atti.

[0284] Example 5: Summary of antibody sequences disclosed in this patent The following table summarizes the important antibodies that were produced and tested in this disclosure. The VH and VL sequences of the antibodies are shown. The respective VH, VL, and CDR sequences can be found in the previous examples. (Table 14) JPEG2026500540000013.jpg202113

[0285] Example 6: Specificity profiling of humanized antibodies The humanized antibodies generated in the previous examples were tested for specificity against their target antigens. For this experiment, a membrane proteome array (MPA) was used (Integral Molecular). MPA is a protein library composed of 6,000 different human membrane protein clones, each overexpressed in live HEK-293T cells from an expression plasmid. Each clone was individually transfected into a separate well of a 384-well plate, followed by 24 hours of incubation (Proc Natl Acad Sci USA (2018) 115: eE4990-9). Cells expressing each individual MPA protein clone were arrayed in duplicate in a matrix format for high-throughput screening. Prior to screening with MPA, the optimal concentration of the antibody to be tested for screening (antibody Kiebitz) was determined in cells expressing positive (membrane-associated protein A) and negative (mock-transfected) binding controls, followed by flow cytometric detection using a fluorescently labeled secondary antibody (Jackson ImmunoResearch, catalog no. 109-606-008).

[0286] The optimal screening concentration of antibody Kiebitz was determined to be 20 μg / ml. Kiebitz was added to MPA at 20 μg / mL, and binding of the entire protein library was measured on an Intellicyt iQue using a fluorescently labeled secondary antibody (see above). Each array plate includes both positive (Fc binding) and negative (empty vector) controls to ensure plate-to-plate reproducibility. Antibody Kiebitz interactions above background were observed for five targets.

[0287] Binding to these five targets (NOX5, CPOX, FRFL2, PHKG1, and LYSMD4) was evaluated in more detail in a second flow cytometry experiment using serial dilutions 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 was not confirmed. Binding was less than twice background levels at the two highest concentrations tested. The Kiebitz antibody is highly specific for CD45.

[0288] Example 7: Depletion of CD45+ cells and LT-HSCs in humanized mice NSG mice were humanized by transplantation of human umbilical cord blood-derived CD34+ HSPCs from The Jackson Laboratory. Eighteen weeks after HSPC injection, humanized mice were intravenously injected with Kiebitz-tetherin or BC8-tetherin conjugates at a single dose of 0.3 mg / kg. Control animals were treated with phosphate-buffered saline (PBS) solution. After 21 days of treatment, mice were euthanized, and blood, spleen, and bone marrow were analyzed by FACS.

[0289] The VH, VL and CDR sequences of antibody BC8 according to Kabat are shown in the table below. (Table 15) JPEG2026500540000014.jpg65128

[0290] Results for depletion of human CD45+ cells (gated as live / hCD45+) and long-term (LT)-HSCs in bone marrow are shown in Figure 3. LT-HSCs were gated as live / hCD45+ / CD34+ / CD38- / CD90+ / CD45RA-. Results show complete depletion of all human CD45+ cells and LT-HSCs after 3 weeks of a single dose of 0.3 mg / kg with the ADC Kiebitz-Tetherin compared to the PBS control group, whereas BC8-Tetherin treatment resulted in only partial depletion.

[0291] Example 8: Naked antibody blocking can mimic cell shielding and prevent loss of cell viability in cell lines upon subsequent treatment with ADC In this experiment, various human cell lines were treated with tetherin-conjugated versions of selected naked anti-CD45 antibodies. Certain samples were preincubated with the same anti-CD45 antibodies in naked (unconjugated) form.

[0292] To do so, MV4-11 cells were seeded at a concentration of 75,000 cells / mL in 135 μl of medium (IMDM supplemented with GlutaMAX + 10% heat-inactivated FBS) in 96-well plates. Some wells were pretreated with 50 μg / mL of naked antibody (Kiebitz or Ref043) or PBS as a control for 1 hour, followed by the addition of various concentrations of Kiebitz-Teslin or BC08-Teslin (the starting concentration for naked antibody-pretreated wells was 10 μg / mL, and the starting concentration for PBS-pretreated wells was 1 μg / mL for both cell lines) for 72 hours. Cell viability measurements were performed by adding 50 μl CTG2.0 (CellTiter-Glo® 2.0 Cell Viability Assay #G9241, Promega) per well, incubating the plate on a shaker at room temperature for 10 minutes, and reading luminescence (EnVision, Perkin Elmer).

[0293] Molm-13 cells were also analyzed following the same protocol described above. Cells were seeded in 96-well plates at a concentration of 75,000 cells / mL in 135 μl of medium (RPMI 1640 supplemented with GlutaMAX + 20% heat-inactivated FBS). The starting concentration of naked antibody pretreatment was 3 μg / mL.

[0294] The results are shown in Figure 4. It can be seen that in both cell lines tested, naked antibody blocking mimics cell shielding and prevents the loss of cell viability mediated by the respective ADCs. BC08-Tesilin exhibits lower efficacy than Kiebitz-Tesilin.

[0295] Example 9: Naked antibody blocking can also be observed in HSPCs The experiment in Example 8 was repeated using HSPCs. CD34-positive HSPCs were isolated from peripheral blood (CliniMACS Prodigy, Miltenyi Biotech). Cells were seeded at a concentration of 75,000 cells / ml in 135 μl of either low-cytokine or high-cytokine medium in 96-well plates. Low-cytokine medium consisted of StemPro medium (Gibco), StemPro nutrients (Gibco), 50 ng / ml LDL (STEMcell Technologies), 1% penicillin / streptomycin (Thermo Fisher), 1% glutamine (Thermo Fisher), 20 ng / ml Flt3 (Miltenyi Biotech), 50 ng / ml TPO (Milteny Biotech), and 100 ng / ml SCF (Milteny Biotech). High cytokine medium consisted of the same components 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 μg / ml naked antibody (Kiebitz) or PBS as a control for 1 hour, followed by the addition of various concentrations of Kiebitz-Teslin (starting concentration was 10 μg / ml for naked antibody-pretreated wells and 1 μg / ml for PBS-pretreated wells for both cell lines) for 72 hours. Cell viability measurements were performed by adding 50ul CTG2.0 (CellTiter-Glo® 2.0 Cell Viability Assay #G9241, Promega) / well, incubating the plate on a shaker at room temperature for 10 minutes, and reading luminescence (EnVision, Perkin Elmer).

[0296] The results are shown in Figure 5. Even in HSPCs, blocking with naked Kiebitz antibodies shields the cells and prevents cell viability from decreasing with each ADC. The IC50 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).

[0297] Example 10: Blocking is seen in wild-type cells but not in CD45 knockout cells Similar experiments were performed using wild-type and CD45 knockout Jurkat cells. CD45 KO-engineered Jurkat or Jurkat cells were seeded in 96-well plates at a concentration of 75,000 cells / mL in 135 μl of medium (RPMI 1640 (ATCC modified) + 10% heat-inactivated FBS). Various concentrations of Kiebitz-Teslin were added. The starting concentration was 100 μg / mL. Cells were incubated for 72 hours. Cell viability measurements were performed by adding 50 μl CTG2.0 (CellTiter-Glo® 2.0 Cell Viability Assay #G9241, Promega) per well, incubating the plate on a shaker at room temperature for 10 minutes, and reading the luminescence (EnVision, Perkin Elmer).

[0298] The results are shown in Figure 6. Jurkat CD45 knockout cells have an IC50 for Kiebitz-Teslin that is significantly higher than that of Jurkat wild type cells (0.46 μg / ml vs. 0.032 μg / ml).

[0299] Example 11: Antibodies bind to CD45 endogenously expressed on cells KASUMI-1 cells (ATCC No. CRL-2724) were stained with different concentrations of antibody Atti or its humanized derivatives, Amandine or Ballerina, using an IgG goat anti-human AF488 secondary antibody. Background staining was determined using cells stained with a human IgG1 isotype control.

[0300] The results are shown in Figure 7. All three antibodies tested bind equally well to CD45 endogenously expressed on Jurkat cells.

[0301] In a similar experiment, 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 recognizes endogenously expressed CD45 on Jurkat cells in a concentration-dependent manner. Kiebitz does not bind to CD45 KO Jurkat cells.

[0302] Example 12: Antibody expression as scFv-Fc Not all antibodies can be fully expressed in the scFv-Fc format. One antibody that can be fully expressed 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 have been generated that can all be fully expressed as scFv-Fc. These antibodies are listed in Table 16. (Table 16) JPEG2026500540000015.jpg182128

Claims

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

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

3. 3. The humanized antibody or antibody fragment of claim 1, 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 a) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 98; b) a variable heavy chain of SEQ ID NO: 21 and a 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) a variable heavy chain of SEQ ID NO: 21 and a 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) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 104, or g) a variable heavy chain of SEQ ID NO: 21 and a variable light chain of SEQ ID NO: 105 The humanized antibody or antibody fragment of any one of claims 1 to 3, comprising:

5. The humanized antibody or antibody fragment is a) the glycine in the LCDR1 region (SEQ ID NO: 7) is replaced with an alanine; b) the second asparagine in the LCDR1 region (SEQ ID NO: 7) is replaced with a lysine; and c) the arginine in the LCDR2 region (SEQ ID NO: 8) is replaced with a leucine; The humanized antibody or antibody fragment of any one of claims 1 to 4, comprising the following mutation:

6. 6. The humanized antibody or antibody fragment of 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. 7. The humanized antibody of any one of claims 1 to 6, wherein the humanized antibody comprises a modification in the Fc region, preferably the Fc modification is a silencing mutation selected from (numbering according to the EU index): PA-LALA (L234A / L235A / P329A), PG-LALA (L234A / L235A / P329G) and AEASS (L234A / L235E / G237A / A330S / P331S).

8. The humanized antibody or antibody fragment of any one of claims 1 to 7, 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 of any one of claims 1 to 8 and a cytotoxic drug.

10. 10. The antibody drug conjugate of claim 9, wherein the cytotoxic drug is a pyrrolobenzodiazepine (PBD), preferably tesirine.

11. 11. The humanized antibody or antibody fragment of any one of claims 1 to 8 or the antibody drug conjugate of claim 9 or 10 for use in medicine, preferably wherein said use in medicine is the treatment of a blood cancer such as leukemia, myeloma or lymphoma, or the treatment of an inflammatory or autoimmune disease 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 a nucleic acid sequence or a plurality of nucleic acid sequences encoding the humanized antibody or antibody fragment of any one of claims 1 to 8 or the antibody drug conjugate of 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 of any one of claims 1 to 8, or the antibody-drug conjugate of claim 9 or 10, and a pharmaceutically acceptable carrier or excipient.

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