Antibodies

Abstract

This invention specifically provides antibodies or antigen-binding fragments thereof that are specific to CD45. The antibodies include monospecific antibodies to CD45 and antibodies with dual complementary sites to CD45. The antibodies are typically capable of binding to both human and cynomolgus monkey CD45. The antibodies of this invention have various uses, including targeting, exhausting, and killing cells expressing CD45, particularly for the treatment of cancers or autoimmune diseases mediated by CD45-positive cells, or prior to cell transplantation.

Description

Technical Field

[0001] This invention relates to antibodies specific to CD45. The antibodies include those with single or dual complementary sites specific to CD45. These antibodies can be used, for example, to kill or deplete CD45-positive target cells, particularly for treating cancers or autoimmune diseases mediated by CD45-positive cells, or prior to cell transplantation. Background Technology

[0002] CD45 (a first and prototype receptor-like protein tyrosine phosphatase) is expressed on nucleated hematopoietic cells and plays a central role in the regulation of cellular responses. CD45 is also known as PTPRC, T200, Ly5, leukocyte common antigen (LCA), and B220. CD45 is the most abundant cell surface protein expressed on the surface of both T and B cells. It is essential for B and T cell development and activation. Studies in CD45 mutant cell lines, CD45-deficient mice, and CD45-deficient humans initially demonstrated the essential role of CD45 in T and B cell antigen receptor signaling and lymphocyte development. It is now known that CD45 also modulates signals from integrin and cytokine receptors. Contrary to its positive role in antigen receptor signaling, CD45 acts as a negative regulator of integrin-mediated signaling, such as in macrophages. CD45 also plays a role in regulating hematopoiesis and interferon-dependent antiviral responses. CD45 also functions in cell survival.

[0003] CD45 comprises a highly and variablely glycosylated extracellular domain of approximately 400 to 550 amino acids, followed by a single transmembrane domain and a long intracellular domain of 705 amino acids containing two tandemly repeated phosphatase domains. Regulation of CD45 expression and the expression of various alternative splicing isoforms (which selectively splice exons 4, 5, and 6 from the CD45 gene and are designated A, B, and C) critically regulate phosphatase activity and differential signal transduction. CD45 influences cellular responses by controlling the relative sensitivity threshold to external stimuli. Disruption of this function may contribute to autoimmunity, immunodeficiency, and malignancy.

[0004] All CD45 isoforms exhibit tyrosine phosphatase activity, mediated by a cytoplasmic domain of the molecule containing two tandemly repeated phosphatase domains, D1 and D2, each of which contains a highly conserved H+ phosphatase. C(X)5R motif. All tyrosine phosphatase activities of CD45 are thought to originate from the D1 domain, with the D2 domain potentially involved in regulation. One of the main targets of CD45 tyrosine phosphatases is the Src-family kinases, reflecting the role of CD45 in cellular signaling. Depending on where CD45 phosphatase activity is located, it can activate or downregulate the activity of these Src-family kinases.

[0005] CD45 is an attractive target for treating cancer and other therapeutic scenarios. Given the importance of CD45, there is a continued need for agents that can target CD45 and be used to treat the aforementioned diseases. Summary of the Invention

[0006] This invention provides antibodies specific to CD45. The provided antibodies include monospecific and dual complementary antibodies specific to CD45. Both can be used to kill and / or deplete cells expressing CD45.

[0007] In particular, the present invention provides an antibody or antigen-binding fragment thereof comprising at least one variable domain specific to CD45, the variable domain comprising the following light and heavy chain variable regions:

[0008] (a) A light chain variable region comprising: CDR1 comprising the sequence of SEQ ID NO: 33, CDR2 comprising the sequence of SEQ ID NO: 34, and CDR3 comprising a sequence selected from any one of SEQ ID NO: 35 and 39 to 42; and

[0009] (b) A heavy chain variable region comprising: CDR1 comprising a sequence selected from any one of SEQ ID NO: 46, 52, 53 and 54, CDR2 comprising a sequence selected from any one of SEQ ID NO: 47, 55, 56 and 57, and CDR3 comprising a sequence of SEQ ID NO: 48.

[0010] The present invention also provides an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one variable domain specific to CD45, said variable domain comprising the following light chain and heavy chain variable regions:

[0011] (a') A light chain variable region comprising: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and

[0012] (b') Heavy chain variable region comprising: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence selected from any one of SEQ ID NO: 102 to 105.

[0013] The variable regions of the light and heavy chains may be humanized. The antibody or antigen-binding fragment may be bicomponent, comprising a first variable domain specific to CD45 from the first group of antibodies described above (i.e., antibodies having (a) and (b) chains) and a second variable domain specific to CD45 from the second group of antibodies described above (i.e., antibodies having (a') and (b') chains). The constant region of the antibody or its antigen-binding fragment may contain one or more modifications to reduce or eliminate binding to the Fc receptor. The antibody or antigen-binding fragment may specifically bind to both human CD45 and cynomolgus monkey CD45.

[0014] The present invention also provides pharmaceutical compositions comprising the antibodies set forth herein and pharmaceutically acceptable loads or diluents. These pharmaceutical compositions can be used to kill or deplete CD45-expressing cells in subjects, for example in the treatment of hematologic malignancies such as leukemia, lymphoma, or multiple myeloma. Attached Figure Description

[0015] Figure 1 The ability of monospecific (monoclonal) antibodies 17415, 17552, and 4133 to kill CD45-expressing human PBMCs (peripheral blood mononuclear cells) was compared. A VR5604 isotype control antibody that does not bind to CD45 was also included.

[0016] Figure 2 Sequence alignments are provided for various sequences involved in the humanization of the variable region of the light chain of the original rabbit 17415 antibody. The CDR of the rabbit antibody was grafted onto either the human IGKV1-9 acceptor framework (top set of alignments) or the IGKV4-1 acceptor framework (bottom set of alignments). Numerous donor framework residues were also transferred, and several CDR modifications were included. The CDRs are shown in bold and underlined. Amino acid residues deviating from the original CDR or the human donor framework are shown in bold and italicized with shading. The resulting grafted variants are shown below the acceptor sequence.

[0017] Figure 3Sequence alignments are provided for the various sequences involved in the humanization of the variable region of the heavy chain of the original rabbit 17415 antibody. The CDR of the rabbit antibody was grafted onto the human IGHV3-72 acceptor framework. Numerous donor framework residues were also transferred, and several CDR modifications were included. The CDRs are shown in bold and underlined. Amino acid residues deviating from the original CDR or the human donor framework are shown in bold and italicized with shading. The resulting grafted variants are shown below the acceptor sequence.

[0018] Figure 4 Sequence alignments are provided for the various sequences involved in the humanization of the original rabbit 17552 antibody, with the top set of alignments concerning the humanization of the light chain into the human IGKV1-8 acceptor framework, and the bottom set concerning the humanization of the heavy chain into the human IGHV4-4 acceptor framework. Numerous donor framework residues were also transferred, and some CDR modifications were included. CDRs are shown in bold and underlined. Amino acid residues deviating from the original CDR or the human donor framework are shown in bold and italics with shading. The resulting grafted variants are shown below the acceptor sequence.

[0019] Figure 5 shows the results of the human lymphocyte depletion assay, which compares: (A) 17415 IgG LALA heavy chain grafted variant H1 paired with 17415 light chain grafted variants L1 to L5, and (B) 17415 IgG LALA heavy chain grafted variants H1 to H4 paired with 17415 light chain grafted variant L1.

[0020] Figure 6 shows the results of human lymphocyte assays for the following items: (A) 17415 light chain grafted variants L1 to L5 paired with chimeric 17415 heavy chains containing the variable region of the original rabbit 17415 heavy chain, compared with chimeric 17415 light and heavy chains containing the variable regions of the original rabbit 17415 light and heavy chains; and (B) chimeric 17415 light chains paired with 17415 heavy chain grafted variants H1 to H4 containing the variable region of the original rabbit 17415 light chain, compared with chimeric 17415 light and heavy chains containing the variable regions of the original rabbit 17415 light and heavy chains.

[0021] Figure 7 shows the results of the human T-cell depletion assay for the following items: (A) 17415 heavy chain grafted variant H5 paired with 17415 light chain grafted variants L6, L7, L13, L14, L15 and L16, and (B) 17415 heavy chain grafted variant H6 paired with the same light chain grafted variants.

[0022] Figure 8 shows the results of human Jurkat cell killing for the following antibodies in the IgG1 LALA pattern: (A) 17415 light chain variable region grafted variants L1 to L5 paired with 17415 heavy chain grafted variant H1, (B) 17415 light chain variable region grafted variant L1 paired with 17415 heavy chain grafted variants H1 to H4, (C) 17415 chimeric light and heavy chain antibodies containing the original variable region of the original rabbit 17415 antibody compared with antibodies having chimeric light chains paired with each of the 17415 heavy chain grafted variants H1 to H4, and (D) 17415 chimeric light and heavy chain antibodies containing the original variable region of the original rabbit 17415 antibody compared with antibodies having 17415 light chain variants L1 to L5 paired with chimeric heavy chains.

[0023] Figure 9 Results for the human T-cell depletion assay are shown for the following antibodies: IgG1 LALA antibodies containing 17415 light chain graft variant 7, 15 or 16 and 17415 heavy chain graft variant H6; and IgG1 LALA antibodies containing 17552 light chain graft variant 1 and 17552 heavy chain graft variant 1 or 4.

[0024] Figure 10 Results for the human Jurkat cell depletion assay are shown for the following antibodies: IgG1 LALA antibodies containing 17415 light chain grafted variants 7, 15 or 16 and 17415 heavy chain grafted variant H6.

[0025] Figure 11 Results are shown regarding the binding of humanized 17415 IgG1 LALA grafts (which contain 17415 light chain graft variants 7, 15, and 16 paired with 17415 heavy chain graft variant 6) to human CD45.

[0026] Figure 12 shows the results of binding to human CD45 with various dual complementary site 17415 / 17552, 17415 / 5604 and 5604 / 17552 antibody variants and monospecific 5604 antibody, for the various specified antibodies, including those with 17415 light chain grafted variants 7 (A), 15 (B) and 16 (C).

[0027] Figure 13 shows the results of binding of various dual complementary site 17415 / 17552, 17415 / 5604 and 5604 / 17552 antibody variants and monospecific 5604 antibody to cynomolgus monkey CD45, for the various specified antibodies, including those with 17415 light chain grafted variants 7 (A), 15 (B) and 16 (C).

[0028] Figure 14 shows the results of a human T-cell depletion assay for various dual complementary site 17415 / 17552, 17415 / 5604, and 5604 / 17552 antibody variants containing light chain grafted variants 7 (A), 15 (B), and 16 (C).

[0029] Figure 15 shows the results of the human Jurkat cell depletion assay for various dual complementary site 17415 / 17552, 17415 / 5604 and 5604 / 17552 antibody variants containing light chain grafted variants 7 (A), 15 (B) and 16 (C).

[0030] Figure 16 The results of the cynomolgus monkey T-cell exhaustion assay for the 17415 and 17552 monospecific (monoclonal) IgG1 antibodies are shown, compared with the 17415 / 17552 IgG1 antibody and the 4133 / 6294 IgG1 antibody with double complementary sites.

[0031] Figure 17 This study compares the ability of the bicomplementary 17415-17552 IgG1 and 4133-6294 IgG1 antibodies to kill CD45-expressing human PBMCs (peripheral blood mononuclear cells). A VR5604 isotype control antibody (which does not bind to CD45) is also included.

[0032] Figure 18 This study compares the ability of the bicomplementary 17415-17552 IgG1 and YTH24.5-YTH54.12 IgG1 antibodies to reduce T cell numbers in a PBMC population. Results for the isotype (5604 IgG1) control are also included.

[0033] Figure 19 Results regarding human Jurkat cell killing were shown for antibodies against the dual complementary sites 4133-6294 IgG1 and 17415-17552 IgG1. The VR5604 isotype control antibody (which does not bind to CD45) was also included.

[0034] Figure 20 shows the induction levels of the following cytokines in whole blood by double complementary site 17415gL15gH6-17552gL1gH4 IgG1 LALA antibody or monospecific (monoclonal) 17415gL15gH6 IgG1 LALA antibody: (A) IFNγ, (B) IL-6 and (C) TNFα, compared with unrelated specificity controls (5604 IgG1 LALA), Camppath or PBS.

[0035] Figure 21This study compares the ability of the dual complementary site 17415-17552 IgG1 and YTH24.5-YTH54.12 IgG1 antibodies to kill Jurkat cells. A VR5604 isotype control antibody (which does not bind to CD45) is also included.

[0036] Figure 22 The percentage reduction in T-cells in T-cell lines (A) Peers, (B) SUPT11, and (C) SUDHL1 is shown for the use of the dual complementary site 17415gL15gH6-17552gL1gH4 IgG1 LALA antibody and the monospecific (monoclonal) 17415gL15gH6 IgG1 LALA antibody.

[0037] Figure 23 The percentage reduction in B-cells in B-cell lines (A) Ramos and (B) DOHH2 is shown for the bicomplementary site 17415gL15gH6-17552gL1gH4 antibody and the monospecific (monoclonal) 17415gL15gH6 IgG1 LALA antibody.

[0038] Figure 24 The percentage reduction in T cells in PBMCs derived from (A) healthy volunteers 336BB+330CD and (B) T-cell leukemia patients 4368POS is shown compared to the control, using either the double complementary site 17415gL15gH6-17552gL1gH4 IgG1 LALA antibody or the monospecific (monoclonal) 17415gL15gH6 IgG1 LALA antibody.

[0039] Figure 25 The percentage reduction in B-cells in PBMCs derived from (A) healthy volunteers 336BB+330CD and (B) B-cell leukemia patients 4650ADG is shown compared to the control, using either the double complementary site 17415gL15gH6-17552gL1gH4 IgG1 LALA antibody or the monospecific (monoclonal) 17415gL15gH6 IgG1 LALA antibody.

[0040] Figure 26 and 27 The experimental results for some of the preferred monospecific and dual complementary antibodies of the present invention are summarized respectively.

[0041] Figure 28 , 29The amino acid sequences of full-length human CD45 (SEQ ID NO: 127), extracellular domains D1 to D4 of human CD45 (SEQ ID NO: 128), and full-length cynomolgus monkey CD45 (SEQ ID NO: 129) are provided in SEQ ID NO: 30, respectively.

[0042] Figure 31 The full heavy chain (top) and light chain (bottom) sequences of the VR17415gL15gH6 IgG1 LALA antibody are provided, with the constant region sequences of each shown in italics. The heavy chain constant region can be further subdivided from the N-terminus to the C-terminus into the CH1 region (underlined), the hinge region (ununderlined), the CH2 region (underlined), and the CH3 region (ununderlined).

[0043] Figure 32 The full heavy and light chain sequences of the VR17415gL15gH6 x VR17552gL1gH4 IgG1 LALA dual complementary site antibody are shown. The heavy and light chain sequences for VR17415gL15gH6 are the topmost and second-to-last sequences shown. The heavy and light chain sequences specific to VR17552gL1gH4 are the second-to-last and last sequences shown. For all sequences, constant regions are shown in italics. The heavy chain constant region can be subdivided from the N-terminus to the C-terminus into the CH1 region (underlined), hinge region (ununderlined), CH2 region (underlined), and CH3 region (ununderlined). Both heavy chains have "knobs-into-holes" modifications, with mutations related to this shown in shaded areas.

[0044] Figure 33 Examples of preferred heavy and light chain constant region sequences for use in the antibodies of this invention are provided, wherein the constant region is a modified IgG1 LALA constant region. The table provides amino acid sequences for the heavy chain with and without the "mortar" modification. In the case of dual complementary antibody, the "mortar" modification can be employed to promote heterodimer formation, and thus promote the formation of dual complementary antibody, rather than monospecific antibody. Detailed Implementation

[0045] This invention particularly provides antibodies specific to CD45, especially monospecific antibodies to CD45 and antibodies with dual complementary sites to CD45. These antibodies are useful for targeting CD45-expressing cells, particularly for exhausting and / or killing CD45-expressing cells. In a preferred embodiment, the provided antibody is capable of specifically binding to both human and monkey CD45. In a particularly preferred embodiment, the antibody is capable of specifically binding to both human CD45 and cynomolgus monkey CD45. Such antibodies are particularly useful because they can be studied in preclinical trials using cynomolgus monkeys. In one embodiment, the antibody of the present invention is monospecific to CD45, i.e., has single-species specificity for CD45. In a particularly preferred embodiment, the provided antibody is a dual complementary site antibody to CD45, wherein two of the monospecific binding specificities for CD45 listed herein are combined in a single molecule. In a further preferred embodiment, a dual complementary site antibody is provided that binds to and kills both human and cynomolgus monkey cells. In a particularly preferred embodiment, an antibody with dual complementary sites to CD45 is provided that binds to and kills both human and cynomolgus monkey cells.

[0046] Further details about the antibody and its uses are provided below.

[0047] CD45 molecules

[0048] The antibody of this invention is specific to CD45. As explained above, CD45 is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known signaling molecules that regulate a wide variety of 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 is therefore a receptor-type PTP. Various isoforms of CD45 exist: CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, and CD45R (ABC). CD45 splice variant isoforms A, B, and C are differentially expressed in many leukocyte subsets. Despite the existence of different CD45 isoforms, they share a common sequence, meaning that all isoforms can be targeted by a single antibody.

[0049] The intracellular (COOH-terminal) region of CD45 contains two PTP catalytic domains, and the extracellular region is highly variable due to alternative splicing of exons 4, 5, and 6 (named A, B, and C, respectively), coupled with inconsistent glycosylation levels. The CD45 isoforms detected are cell type, maturity, and activation state specific. Typically, the long form of the protein (A, B, or C) is expressed on naive or resting B cells, while the mature or truncated form of CD45 (RO) is expressed on activated or mature / memory B cells.

[0050] The human sequence of CD45 is available in UniProt entry number P08575 and is provided herein in SEQ ID NO: 127, or in amino acids 24-1304 of SEQ ID NO: 127, lacking the signal peptide. The amino acid sequence of the extracellular domain of human CD45 domains 1-4 is provided in SEQ ID NO: 128.

[0051] The mouse version of CD45 is available in UniProt entry P06800.

[0052] The cynomolgus monkey version of CD45 is provided in this article as SEQ ID NO: 129 ( Figure 21 ).

[0053] In one embodiment, the CD45 bound by the antibody of the present invention is mammalian CD45. In a particularly preferred embodiment, CD45 refers to human CD45 and its natural variants and isoforms. In a preferred embodiment, the antibody of the present invention is capable of binding to all isoforms of CD45 expressed by a given species; for example, the antibody can bind to all human isoforms of CD45. Preferably, the antibody of the present invention can bind to both human and cynomolgus monkey CD45, and more preferably, it binds to all isoforms of human and cynomolgus monkey CD45.

[0054] Antibodies - Overview

[0055] The antibodies of this invention have at least one specificity for CD45. The “specificity” of an antibody refers to the target to which the antibody binds. The portion of the antibody that binds to its target may be called an antigen-binding site, or in some cases, a complementary site of the antibody. The portion of the antigen bound by the antibody may be called an epitope. The specificity of an antibody can be described based on the antigen bound or on which epitope of the antigen it binds. Bis-complementary antibodies are a subset of bispecific antibodies because bispecific antibodies can recognize two different epitopes on different antigens or two different epitopes on the same antigen. In the latter case, they are bi-complementary antibodies. The number of binding sites an antibody has may be called its valency, where each valency represents one antigen-binding site of the antibody.

[0056] The antibodies provided by this invention are specific for CD45. Therefore, the antibodies of this invention contain at least one antigen-binding site specific for CD45, i.e., a complementary site. An antibody recognizing a single epitope of CD45 can be called a monospecific antibody for CD45. An antibody recognizing two different epitopes of CD45 can be called a dual complementary antibody for CD45. This invention provides both monospecific antibodies for CD45 and dual complementary antibodies for CD45. The CD45-specific dual complementary antibody of this invention can be monovalent for each epitope or polyvalent for each epitope.

[0057] In a preferred embodiment, the antibody of the present invention specifically binds to CD45 but does not significantly bind to non-CD45 proteins. In one embodiment, such specificity relates only to the antigen-binding site of the antibody that recognizes CD45, but the antibody may have other antigen-binding sites with other specificities. For example, in one embodiment, the antibody of the present invention has at least one antigen-binding site specific to molecules other than CD45, and an antigen-binding site specific to CD45. In one embodiment, the further specificity is against serum albumin. In an alternative embodiment, all specificity of the antibody of the present invention is against CD45.

[0058] In one embodiment, the antibody specifically binds to CD45 from at least one species, but not necessarily to CD45 from all species. Preferably, the antibody of the present invention specifically binds to human CD45. In a more preferred embodiment, the antibody of the present invention specifically binds to human CD45, and also to CD45 from at least one or more other species. Preferably, the antibody specifically binds to CD45 from the species used in animal studies, which will help in developing the antibody into a therapeutic agent. In a particularly preferred embodiment, the antibody of the present invention specifically binds to both human and monkey CD45. In a particularly preferred embodiment, the antibody of the present invention specifically binds to both human and cynomolgus monkey CD45. In a further particularly preferred embodiment, it kills and / or depletes both human cells expressing CD45 and cynomolgus monkey cells expressing CD45.

[0059] In one embodiment, the antibody of the present invention exhibits trans binding, meaning it binds more than one molecule of CD45 simultaneously. Such trans binding typically leads to cross-linking of CD45 and thus represents a preferred embodiment of the invention. In one embodiment, the antibody of the present invention exhibits cis binding of CD45, thereby specifically binding only one molecule of CD45 with its binding site.

[0060] In a particularly preferred embodiment, the antibody of the present invention specifically binds to CD45 and induces CD45 polymerization. In one embodiment, it may be able to polymerize CD45 on the surface of target cells. The CD45 polymers are particularly higher-order structures having more than one CD45. In one embodiment, the antibody of the present invention specifically binds to the extracellular portion of CD45 and induces CD45 polymerization on the surface of target cells. In a particularly preferred embodiment, the CD45 polymer comprises at least three CD45 molecules. In one embodiment, the CD45 polymer may comprise at least three, four, five, six, seven, or more CD45 molecules linked together by the antibody of the present invention. Techniques such as mass spectrometry can be used to identify CD45 polymers conjugated with the antibody of the present invention and thus measure the ability of the antibody of the present invention to generate CD45 polymers.

[0061] The degree of specificity (or specificity) for the target molecule (specifically, CD45) used herein can refer to the situation where the interacting partner or its associated portion recognizes only each other or has a significantly higher affinity for each other than for non-partners, for example, at least 10-fold, at least 100-fold, at least 1000-fold, at least 10,000-fold, at least 100,000-fold, or at least 1,000,000-fold higher affinity than, for example, background binding or binding to another unrelated protein (e.g., egg white lysozyme). In one embodiment, such a degree of specificity is directed against CD45. In another embodiment, such specificity is directed not only against CD45, but also against a specific epitope of CD45 bound by the antigen-binding site (specifically, the complementary site) of the antibody, compared to other epitopes of CD45.

[0062] In one implementation, through the dissociation constant (K) D The antibody affinity measured is approximately 100 nM or less, for example, approximately 50 nM or less, 20 nM or less, 10 nM or less, 1 nM or less, 500 pM or less, 250 pM or less, 200 pM or less, or 100 pM or less. In one embodiment, the K... D The concentration is 50 pM or less. In one embodiment, at least one complementary site of the antibody has such affinity for CD45. In another embodiment, the antibody has two complementary sites, each with a different specificity for CD45, wherein all complementary sites individually have such affinity for CD45. In one embodiment, that is the total affinity of the antibody for CD45. In one embodiment, the K of the complementary site for CD45... D It can be less than 1 μM, less than 750 nM, less than 500 nM, less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM, less than 75 nM, less than 50 nM, less than 10 nM, less than 1 nM, less than 0.1 nM, less than 10 pM, less than 1 pM, or less than 0.1 pM. In some embodiments, the K... D The affinity is approximately 0.1 pM to approximately 1 μM. In one embodiment, the antibody of the present invention generally has that level of affinity for CD45. Affinity is typically measured using surface plasmon resonance assays such as the Biacore assay.

[0063] Antibodies derived from 17415

[0064] (a) Overview of antibodies and variable regions derived from 17415

[0065] The preferred antibody of the present invention is an antibody comprising at least one antigen-binding site derived from the original rabbit 17415 antibody described herein, particularly a pair of humanized light and heavy chain variable regions derived from the original rabbit 17415 antibody described herein. The original rabbit 17415 antibody has a light chain variable region of SEQ ID NO: 1 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 15. The light chain variable region CDR of the original rabbit 17415 antibody is LCDR1, LCDR2, and LCDR3 having the sequences of SEQ ID NO: 33, 34, and 35, respectively. The heavy chain variable region CDR of the original rabbit 17415 is HCDR1, HCDR2, and HCDR3 having the sequences of SEQ ID NO: 46, 47, and 48, respectively.

[0066] The term "17415-derived antibody" includes any of the specific 17415-derived sequences listed herein, as well as variants of such antibodies. References to such "17415-derived" sequences include... Figure 2 and 3 The humanized light and heavy chain variable regions shown in the figures, as well as the variable regions with CDR sets of CDR1, CDR2, and CDR3 shown in those figures. Figure 2 and 3 The specific grafting variants shown are particularly preferred. Pairs of light-chain and heavy-chain variable-region grafting variants generated in the embodiments of this application are also preferred.

[0067] The resulting original rabbit 17415 antibody and antibodies containing the grafted variants described herein for the 17415 antibody are particularly effective in killing or depleting cells expressing CD45. They also have the further advantage of specifically binding to both human CD45 and cynomolgus monkey CD45, making them particularly suitable for development as therapeutics. Preferably, they are capable of killing or depleting both human and cynomolgus monkey cells expressing CD45.

[0068] In one embodiment, the antibody comprises one or more 17415-derived antigen-binding sites specific to CD45. In a particularly preferred embodiment, it comprises one or more antigen-binding sites formed from a pair of humanized 17415-derived light and heavy chain variable regions.

[0069] In one embodiment, the antibody is monospecific for CD45, such that the 17415-derived antigen-binding site present in the antibody is the only CD45-specific antigen-binding site contained in the antibody. In another embodiment, the antibody of the present invention has a plurality of complementary sites that bind to CD45, wherein one of those CD45-specific complementary sites is provided by the 17415-derived antigen-binding site. In a particularly preferred embodiment, the antibody of the present invention is dual-complementary for CD45, wherein one of the complementary sites is a 17415-derived antigen-binding site. A particularly preferred antibody of the present invention with dual CD45 complementarity is an antibody comprising: one complementary site is 17415-derived, and the other complementary site is a CD45-specific 17552-derived complementary site.

[0070] Therefore, in a particularly preferred embodiment, the present invention provides an antibody or antigen-binding fragment thereof comprising at least one variable domain specific to CD45, the variable domain comprising the following light and heavy chain variable regions:

[0071] (a) A light chain variable region comprising: CDR1 comprising the sequence of SEQ ID NO: 33, CDR2 comprising the sequence of SEQ ID NO: 34, and CDR3 comprising a sequence selected from any one of SEQ ID NO: 35 and 39 to 42; and

[0072] (b) A heavy chain variable region comprising: CDR1 comprising a sequence selected from any one of SEQ ID NO: 46, 52, 53 and 54, CDR2 comprising a sequence selected from any one of SEQ ID NO: 47, 55, 56 and 57, and CDR3 comprising a sequence of SEQ ID NO: 48.

[0073] In a preferred embodiment, the light chain variable regions comprise LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 35, respectively. In another embodiment, they comprise sequences of SEQ ID NO: 33, 34, and 39. In another embodiment, they comprise sequences of SEQ ID NO: 33, 34, and 40. In another embodiment, they comprise sequences of SEQ ID NO: 33, 34, and 41. In yet another embodiment, they comprise sequences of SEQ ID NO: 33, 34, and 42.

[0074] In a preferred embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 46, 47, and 48, respectively. In another embodiment, they comprise sequences of SEQ ID NO: 52, 55, and 48. In yet another embodiment, they comprise sequences of SEQ ID NO: 53, 56, and 48. In still another embodiment, they comprise sequences of SEQ ID NO: 54, 57, and 48.

[0075] The humanization of the original rabbit 17415 antibody is described herein. In a preferred embodiment, the 17415-derived antigen-binding site is a humanized antigen-binding site. In a particularly preferred embodiment, a 17415-derived complementary site is generated by transferring a CDR from the variable region (donor sequence) of the original rabbit 17415 into the framework region from a second antibody (recipient sequence). The humanization may also require the transfer of some framework residues from the donor sequence as well as the CDR.

[0076] Preferred light chain variable regions used as acceptor sequences for the architecture include IGKV1-9 and IGKV4-1. Particularly preferred acceptor sequences are those derived from IGKV4-1. For the heavy chain, a particularly preferred acceptor sequence for the CDR is the IGHV3-72 acceptor architecture. In a preferred embodiment, the complementary site derived from 17415 comprises a light chain variable region based on the IGKV4-1 acceptor architecture and a heavy chain variable region based on the IGHV3-72 acceptor architecture.

[0077] Table 1 below summarizes the original 17415 light and heavy chain variable regions, the scaffold acceptor sequences used, and the specific grafting variants derived from the original 17415 antibody. Figure 2 and 3 It also provides comparisons of various original donor and acceptor sequences, as well as the specific grafted variants generated. Shaded and italicized individual amino acids indicate residues that are not simply those directly transferred from the original 17415 antibody CDR sequence to the human acceptor framework, but rather those that are donor framework residues that were already transferred with the CDR sequence, or those that are different from those in the original 17415 CDR.

[0078] Table 1 - 17415 antibody and humanized graft variants

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Below are various examples of the preferred 17415-derived sequences:

[0087] (b) A preferred 17415 light chain grafted variant containing an IGKV4-1 / IGKJ4-derived framework.

[0088] In a preferred embodiment, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the rabbit 17415 antibody. In an alternative preferred embodiment, LCDR1, LCDR2, and LCDR3 are those of the rabbit 17415 antibody, except that LCDR3 includes the mutant C90S.

[0089] In a preferred embodiment, the humanized 17415 light chain variable region grafted variant comprises a set of LCDR1, LCDR2, and LCDR3 derived from the rabbit 17415 antibody, having an IGKV4-1 / IGKJ4-derived framework. In a preferred embodiment, the light chain variable region comprises: CDR1 containing the sequence of SEQ ID NO: 33, CDR2 containing the sequence of SEQ ID NO: 34, and CDR3 containing a sequence selected from any one of SEQ ID NO: 35 and 39 to 42, wherein the framework region is an IGKV4-1 / IGKJ4-derived framework.

[0090] In a preferred embodiment, in addition to the 17415 light chain LCDR1, LCDR2, and LCDR3, one or more donor residues from the 17415 rabbit antibody light chain framework are retained at one or more positions selected from the group consisting of: residues 2 (valine, V2), 4 (leucine, L4), 12 (serine, S12), 19 (valine, V19), 60 (serine, S60), 63 (lysine, K63), 70 (glutamic acid, E70), 83 (alanine, A83), 85 (threonine, T85), 106 (glutamic acid, E106), and 108 (valine, V108).

[0091] In one embodiment, donor residues 2 (valine, V2) and 4 (leucine, L4) are retained in the light chain variable region FWR1 of the rabbit 17415 antibody. In another embodiment, donor residues (valine, V2), 4 (leucine, L4), 12 (serine, S12), and 19 (valine, V19) from 17415 are retained in FWR1. In an alternative embodiment, FWR1 corresponds to the FWR1 acceptor sequence from IGKV4-1 / IGKJ4.

[0092] In a preferred embodiment, FWR2 corresponds to the FWR2 acceptor sequence from IGKV4-1 / IGKJ4.

[0093] In one embodiment, donor residues 60 (serine, S60), 63 (lysine, K63), 83 (alanine, A83), and 85 (threonine, T85) (and optionally, (glutamic acid, E70)) from the rabbit 17415 antibody are retained in the light chain variable region FWR3. In one embodiment, donor residues 60 (serine, S60), 63 (lysine, K63), and 85 (threonine, T85) from 17415 are retained in FWR3.

[0094] In one embodiment, donor residues 106 (glutamic acid, E106) and 108 (valine, V108) from the rabbit 17415 antibody are retained in the light chain variable region FWR4. In an alternative embodiment, FWR4 corresponds to the FWR4 acceptor sequence from IGKV4-1 / IGKJ4.

[0095] Examples of preferred light chain architectures based on the IGKV4-1 acceptor sequence include those having FWR1, FWR2, FWR3, and FWR4 with SEQ ID NO: 77, 74, 79, and 81, respectively. In another preferred embodiment, they have sequences with SEQ ID NO: 78, 74, 80, and 76.

[0096] Examples of particularly preferred 17415-derived light chain variable region grafted variants that can be used include those of 17415gL13 (SEQ ID NO: 11), 17415gL14 (SEQ ID NO: 12), 17415gL15 (SEQ ID NO: 13), and 17415gL16 (SEQ ID NO: 14).

[0097] (c) A preferred 17415 light chain grafted variant comprising an IGKV1-9 / IGKJ4-derived framework

[0098] In a preferred embodiment, the light chain variable region derived from the original rabbit 17415 antibody comprises: CDR1 containing the sequence of SEQ ID NO: 33, CDR2 containing the sequence of SEQ ID NO: 34, and CDR3 containing the sequence selected from any one of SEQ ID NO: 35 and 39 to 42, wherein the framework region is derived from the IGKV1-9 acceptor framework.

[0099] In one such preferred embodiment, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the rabbit 17415 antibody. In an alternative preferred embodiment, LCDR1, LCDR2, and LCDR3 are those of the rabbit 17415 antibody, except that LCDR3 includes the mutant C90S. Alternatively, C90 may be mutated to A (alanine), V (valine), or Q (glutamine).

[0100] In one embodiment, donor residues from the rabbit 17415 antibody light chain framework are retained at one or more of positions 2 (valine, V2), 3 (valine, V3), and 63 (lysine, K63) in the light chain variable region. In another embodiment, all of those residues are retained.

[0101] In another embodiment, donor residues from the rabbit 17415 antibody light chain framework are retained at one or more positions selected from 2 (valine, V2), 3 (valine, V3), 10 (serine, S10), 42 (glutamine, Q42), 63 (lysine, K63), 83 (alanine, A83), 106 (glutamate, E106), and 108 (valine, V108). In a preferred embodiment, all of those donor framework residues are retained.

[0102] In one embodiment, donor residues from the framework are retained at positions 2 (valine, V2) and 3 (valine, V3) in FWR1. In a particularly preferred embodiment, donor residues from the framework are retained at positions 2 (valine, V2), 3 (valine, V3), and 10 (serine, S10) in FWR1.

[0103] In one embodiment, a donor residue is retained at position 42 (glutamine, Q42) in FWR2. In an alternative embodiment, FWR2 is identical to the acceptor sequence, and no donor residue is retained in FWR2.

[0104] In one embodiment, donor residues are retained at positions 63 (lysine, K63) and 83 (alanine, A83) in FWR3.

[0105] In one embodiment, donor residues are retained at positions 106 (glutamic acid, E106) and 108 (valine, V108) in FWR4. In another embodiment, the sequence of FWR4 is identical to that of the acceptor sequence.

[0106] Examples of particularly preferred donor framework regions to be retained are those at all positions 2 (valine, V2), 3 (valine, V3), 10 (serine, S10), 42 (glutamine, Q42), 63 (lysine, K63), 83 (alanine, A83), 106 (glutamate, E106), and 108 (valine, V108). In a further preferred embodiment, the LCDR3 comprises the C90S mutation. In another embodiment, it is not so.

[0107] In a preferred embodiment, the receiver architecture comprises FWR1, FWR2, FWR3, and FWR4 of SEQ ID NOs: 58, 59, 60, and 61, respectively. In another embodiment, the FWRs comprise sequences of SEQ ID NOs: 66, 63, 70, and 65, respectively. In yet another embodiment, they comprise sequences of SEQ ID NOs: 67, 69, 71, and 72, respectively. In yet another embodiment, they comprise sequences of SEQ ID NOs: 68, 69, 71, and 72, respectively.

[0108] Examples of preferred light chain variable region grafted variants include those from 17415gL1 to 17415gL7 (SEQ ID NO: 3 to 9, respectively). A particularly preferred example of a grafted variant light chain is that of SEQ ID NO: 8 (17415gL6). A further particularly preferred example of a grafted variant light chain is that of SEQ ID NO: 9 (17415gL6). Those from SEQ ID NO: 3-7 (17415gL1 to 17415gL5) are also provided.

[0109] (d) A preferred 17415 heavy chain grafted variant containing an IGV3-72-derived framework.

[0110] In a further preferred embodiment, the IGHV3-72 / IGHJ4 J-region acceptor architecture is used to generate the 17415-derived heavy chain graft variant. Therefore, in a preferred embodiment, the heavy chain variable region comprises: CDR1 containing a sequence selected from any one of SEQ ID NO: 46, 52, 53, and 54; CDR2 containing a sequence selected from any one of SEQ ID NO: 47, 55, 56, and 57; and CDR3 containing a sequence of SEQ ID NO: 48, wherein the acceptor architecture is derived from IGHV3-72 / IGHJ4.

[0111] In one embodiment, the 17415-derived heavy chain variable region contains HCDR1 of the rabbit 17415 antibody. In another embodiment, the last residue of said HCDR1 is changed from C (cysteine) to S (serine), A (alanine), or V (valine).

[0112] In one embodiment, the 17415-derived heavy chain variable region contains HCDR2 of the rabbit 17415 antibody. In another embodiment, the first residue of said HCDR2 is changed from C (cysteine) to S (serine), A (alanine), or V (valine).

[0113] In one implementation, the derived heavy chain variable region contains the original HCDR3 of the rabbit 17415 antibody.

[0114] The preferred CDR combination is shown in Figure 3 In the 17415-derived heavy chain variable region variants, any combination of the three heavy chain CDRs shown for each individual variant can be used. Specifically, in one embodiment, the 17415-derived heavy chain variant has a CDR of 17415gH1 (SEQ ID NO: 17). In another embodiment, it has those of 17415gH2 (SEQ ID NO: 18). In a further embodiment, it has those of 17415gH3 (SEQ ID NO: 19). In a further embodiment, it has those of 17415gH4 (SEQ ID NO: 20). In another embodiment, it has those of 17415gH5 (SEQ ID NO: 21). In yet another embodiment, it has a CDR of 17415gH6 (SEQ ID NO: 22).

[0115] In a preferred embodiment, the 17415-derived heavy chain variant has FWR1 of the IGHV3-72 acceptor, but retains donor residues from the rabbit 17415 antibody heavy chain framework at position 23 (threonine, T23).

[0116] In a preferred embodiment, the 17415-derived heavy chain variant has FWR2 of the IGHV3-72 acceptor, but retains the donor residue at position 49 (isoleucine, I49).

[0117] In a preferred embodiment, the 17415-derived heavy chain variant has FWR3 of the IGHV3-72 acceptor, but retains donor residues at positions 74 (lysine, K74), 76 (serine, S76), 79 (threonine, T79), 81 (valine, V81), 99 (glutamic acid, E99), and 100 (leucine, L100).

[0118] In a preferred embodiment, the 17415-derived heavy chain variant has FWR4 of the IGHV3-72 / IGHJ4 acceptor, but without further sequence changes.

[0119] The preferred architecture is shown Figure 3 In the heavy chain variable region derived from 17415, information about... Figure 3 Any combination of the individual variants FWR1, FWR2, FWR3 and FWR4 shown in the figure.

[0120] Therefore, in a preferred embodiment, the acceptor architecture for the heavy chain variable region derived from 17415 has FWR1, FWR2, FWR3, and FWR4 comprising sequences of SEQ ID NO: 90, 91, 92, and 89, respectively. In another embodiment, they comprise sequences of SEQ ID NO: 90, 91, 93, and 89, respectively.

[0121] The particularly preferred 17415-derived heavy-chain grafted variant shows Figure 3 In a preferred embodiment, the heavy chain variable region of 17415gH1 (SEQ ID NO: 17) can be used. In another embodiment, the type of 17415gH2 (SEQ ID NO: 18) is used. In another embodiment, the type of 17415gH3 (SEQ ID NO: 19) is used. In a further embodiment, the type of 17415gH4 (SEQ ID NO: 20) is used. In another embodiment, the type of 17415gH5 (SEQ ID NO: 21) can be used. In another embodiment, the type of 17415gH6 (SEQ ID NO: 22) can be used.

[0122] (e) Further preferred 17415-derived light and heavy chain pairings

[0123] In one embodiment, the antibody of the present invention comprises a 17415-derived light chain variable region and a 17415-derived heavy chain variable region. In one embodiment, the light chain variable region is... Figure 2 Any of those shown in the figure, and the heavy chain variable region is in Figure 3Any of those shown in the examples. Particularly preferred are the light chain and heavy chain variable region pairs as those in the embodiments of this application. Also preferred are the CDR sets of six CDRs (LCDR1, LCDR2, and LCDR3, and HCDR1, HCDR2, and HCDR3) of a particular light chain and heavy chain variable region pair used in the embodiments of this application.

[0124] In a particularly preferred embodiment, the light chain variable region is a grafted variant of 17415gL6 (SEQ ID NO: 8), 17415gL7 (SEQ ID NO: 9), or 17415gL13 to 17415gL16 (SEQ ID NO: 11 to 14, respectively). In a particularly preferred embodiment, the heavy chain variable region is a grafted variant of 17415gH1 to H6 (SEQ ID NO: 17 to 22, respectively).

[0125] In one embodiment, 17415gL6 (SEQ ID NO: 8) is paired with any one of 17415gH1 to H6 (SEQ ID NO: 17 to 22, respectively). In another embodiment, 17415gL7 (SEQ ID NO: 9) is paired with any one of 17415gH1 to H6 (SEQ ID NO: 17 to 22, respectively). In another embodiment, 17415gL13 (SEQ ID NO: 11) is paired with any one of 17415gH1 to H6 (SEQ ID NO: 17 to 22, respectively). In another embodiment, 17415gL14 (SEQ ID NO: 12) is paired with any one of 17415gH1 to H6. In another embodiment, 17415gL15 (SEQ ID NO: 13) is paired with any one of 17415gH1 to H6. In another embodiment, 17415gL16 (SEQ ID NO: 14) is paired with any one of 17415gH1 to H6 (SEQ ID NO: 17 to 22, respectively).

[0126] In one embodiment, 17415gH1 (SEQ ID NO: 17) is paired with any one of 17415gL6 (SEQ ID NO: 8), 17415gL7 (SEQ ID NO: 9), or 17415gL13 to 17415gL16 (SEQ ID NO: 11 to 14, respectively). In one embodiment, 17415gH2 (SEQ ID NO: 18) is paired with any one of 17415gL6 (SEQ ID NO: 8), 17415gL7 (SEQ ID NO: 9), or 17415gL13 to 17415gL16 (SEQ ID NO: 11 to 14, respectively). In one embodiment, 17415gH3 (SEQ ID NO: 19) is paired with any one of 17415gL6, 17415gL7, or 17415gL13 to 17415gL16. In one embodiment, 17415gH4 (SEQ ID NO: 20) is paired with any one of 17415gL6 (SEQ ID NO: 8), 17415gL7 (SEQ ID NO: 9), or 17415gL13 to 17415gL16 (SEQ ID NO: 11 to 14, respectively). In one embodiment, 17415gH5 (SEQ ID NO: 21) is paired with any one of 17415gL6 (SEQ ID NO: 8), 17415gL7 (SEQ ID NO: 9), or 17415gL13 to 17415gL16 (SEQ ID NO: 11 to 14, respectively). In one embodiment, 17415gH6 (SEQ ID NO: 22) is paired with any one of 17415gL6 (SEQ ID NO: 8), 17415gL7 (SEQ ID NO: 9), or 17415gL13 to 17415gL16 (SEQ ID NO: 11 to 14, respectively).

[0127] In one embodiment, the 17415-derived antigen-binding site comprises a pair of light and heavy chain variable regions of 17415gL7 (SEQ ID NO: 9) and 17415gH6 (SEQ ID NO: 22). In another embodiment, the 17415-derived antigen-binding site comprises a pair of light and heavy chain variable regions of 17415gL15 (SEQ ID NO: 13) and 17415gH6 (SEQ ID NO: 22).

[0128] In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions, namely 17415gL16 (SEQ ID NO: 14) and 17415gH6 (SEQ ID NO: 22).

[0129] In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL1 (SEQ ID NO: 3) and 17415gH1 (SEQ ID NO: 17). In another embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL1 (SEQ ID NO: 3) and 17415gH4 (SEQ ID NO: 20).

[0130] In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL2 (SEQ ID NO: 4) and 17415gH1 (SEQ ID NO: 17). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL3 (SEQ ID NO: 5) and 17415gH1 (SEQ ID NO: 17). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL4 (SEQ ID NO: 6) and 17415gH1 (SEQ ID NO: 17). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL5 (SEQ ID NO: 7) and 17415gH1 (SEQ ID NO: 17).

[0131] In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL1 (SEQ ID NO: 3) and 17415gH2 (SEQ ID NO: 18). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL1 (SEQ ID NO: 3) and 17415gH3 (SEQ ID NO: 19). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL1 (SEQ ID NO: 3) and 17415gH4 (SEQ ID NO: 20).

[0132] In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL1 (SEQ ID NO: 3) and 17415gH5 (SEQ ID NO: 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL6 (SEQ ID NO: 8) and 17415gH5 (SEQ ID NO: 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL7 (SEQ ID NO: 9) and 17415gH5 (SEQ ID NO: 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL13 (SEQ ID NO: 11) and 17415gH5 (SEQ ID NO: 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL14 (SEQ ID NO: 12) and 17415gH5 (SEQ ID NO: 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL15 (SEQ ID NO: 13) and 17415gH5 (SEQ ID NO: 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL16 (SEQ ID NO: 14) and 17415gH5 (SEQ ID NO: 21).

[0133] In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL7 (SEQ ID NO: 9) and 17415gH6 (SEQ ID NO: 22). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL15 (SEQ ID NO: 13) and 17415gH6 (SEQ ID NO: 22). In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL16 (SEQ ID NO: 14) and 17415gH6.

[0134] (f) Variants and Derivatives

[0135] In addition to the specific 17415-derived sequences listed herein, variants and derivatives of the specific sequences described elsewhere herein are provided. Such variants will retain the ability to specifically bind to CD45. Preferably, they will retain the ability to kill or deplete cells expressing CD45.

[0136] Antibodies derived from 17552

[0137] (a) Overview of antibodies and variable regions derived from 17552

[0138] A further preferred antibody of the present invention is an antibody comprising at least one antigen-binding site derived from the rabbit 17552 antibody described herein, particularly a pair of humanized light and heavy chain variable regions derived from the rabbit 17552 antibody described herein. The original light and heavy chain variable region sequences of the rabbit 17552 antibody are provided as SEQ ID NO: 23 and 27, respectively. The light chain variable regions LCDR1, LCDR2, and LCDR3 of the original rabbit antibody are provided as SEQ ID NO: 94, 56, and 96, respectively. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 of the original rabbit antibody are provided as SEQ ID NO: 100, 101, and 102, respectively.

[0139] In a preferred embodiment, the 17552-derived antibody has a light chain and heavy chain CDR set of one of the antibodies in the embodiments of this application, including... Figure 17 The specificity of 17552-derived antibodies among the dual complementary site antibodies listed herein. In another preferred embodiment, it contains... Figure 17 The specific light and heavy chain variable regions derived from 17552 are shown in the figure.

[0140] The resulting original rabbit 17552 antibody and the antibody containing the grafted variant described herein have the advantage that they specifically bind to both human CD45 and cynomolgus monkey CD45, thus helping to make them particularly suitable for development as therapeutic agents. The 17552-derived antigen-binding site also has a further advantage: when used in conjunction with a second CD45 specificity that induces CD45 killing or elimination, the 17552 specificity acts as an “auxiliary” specificity, potentially enhancing the efficacy of the other specificity. Therefore, the 17552-derived specificity is particularly effective in antibodies having more than one complementary site or specificity for CD45. In a more preferred embodiment, the 17552-derived specificity is used as part of an antibody with two complementary sites for CD45. In a particularly preferred embodiment, the present invention provides a double-complementary antibody comprising 17415 and 17552-derived specificities, wherein the 17415 specificity acts as a “killing” specificity and the 17552 specificity acts as an “auxiliary” specificity.

[0141] The humanization of the original rabbit 17552 antibody is described herein. In a preferred embodiment, the 17552-derived complementary site present in the antibody of the present invention is a humanized antigen-binding site. In a particularly preferred embodiment, the 17552-derived complementary site is generated by transferring a CDR from the 17552 variable region (donor sequence) into a framework region from a second antibody (recipient sequence). The humanization may also require the transfer of some framework residues from the donor sequence as well as the CDR.

[0142] Therefore, in a particularly preferred embodiment, the present invention provides an antibody or antigen-binding fragment thereof comprising at least one variable domain specific to CD45, the variable domain comprising the following light and heavy chain variable regions:

[0143] (a) A light chain variable region comprising: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and

[0144] (b) A heavy chain variable region comprising: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence selected from any one of SEQ ID NO: 102 to 105.

[0145] Preferred light chain variable regions for serving as acceptor sequences with respect to the architecture include IGKV1-8 / IGKJ4. For the heavy chain, a particularly preferred acceptor sequence with respect to the CDR is the IGHV4-4 / IGHJ4 acceptor architecture. In a preferred embodiment, the complementary site derived from 17552 comprises a light chain variable region based on the IGKV1-8 acceptor architecture and a heavy chain variable region based on the IGHV4-4 acceptor architecture.

[0146] Table 2 below summarizes the original 17552 light and heavy chain variable regions, the scaffold acceptor sequences used, and the specific grafting variants derived from the original 17552 antibody. Figure 4 It also provides comparisons of various original donor and acceptor sequences, as well as the specific grafted variants generated. Shaded and italicized individual amino acids indicate residues that are not simply those directly transferred from the original 17552 antibody CDR sequence to the human acceptor framework, but rather those that are donor framework residues that were already transferred with the CDR sequence, or those that are different from the original 17552 CDR.

[0147] Table 2 - 17552 antibody and humanized graft variants

[0148]

[0149]

[0150]

[0151]

[0152] (b) A preferred 17552 light chain grafted variant comprising an IGKV11-8 derived framework.

[0153] In a particularly preferred embodiment, the 17552-derived light chain variable region comprises the original CDR of the 17552 light chain variable region. Specifically, it comprises a light chain variable region containing: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96. Such a set of CDRs can be combined with any of the framework regions listed below.

[0154] In a preferred embodiment, the 17552-derived light chain variable region has an IGKV1-8 / IGKJ4 framework region, except that one or more residues selected from the group consisting of leucine (L2), valine (V3), and glutamic acid (E63) are retained. In one embodiment, leucine (L2) and valine (V3) are retained. In another embodiment, leucine (L2), valine (V3), and glutamic acid (E63) are retained.

[0155] In one embodiment, the free cysteine ​​residue (C77) at position 77 in FWR3 is mutated to serine (C77S). Therefore, in one embodiment, leucine (L2) and valine (V3) are retained from the donor sequence, and the (C77S) modification is also present. In another embodiment, leucine (L2), valine (V3), and glutamic acid (E63) are retained from the donor sequence, and the (C77S) modification is also present.

[0156] In a preferred embodiment, the FWR1, FWR2, FWR3 and FWR4 framework sequences of the light chain variable region respectively comprise SEQ ID NO: 114, 111, 115 and 113.

[0157] In a further preferred embodiment, the FWR1, FWR2, FWR3 and FWR4 framework sequences of the light chain variable region comprise SEQ ID NO: 114, 111, 112 and 113, respectively.

[0158] A particularly preferred 17552-derived light chain grafted variant is 17552gL1 (SEQ ID NO: 25). A further particularly preferred 17552-derived light chain grafted variant is 17552gL2 (SEQ ID NO: 26).

[0159] (b) A preferred 17552 heavy chain grafted variant containing an IGHV4-4 derived framework

[0160] In a particularly preferred embodiment, the 17552-derived heavy chain variable region comprises the original HCDR1 and HCDR2 of the 17552 heavy chain variable region. In a preferred embodiment, it also comprises the original 17552 HCDR3. Alternatively, in a further preferred embodiment, the HCDR3 has a sequence change from aspartic acid (D) to glutamic acid (E) at the fourth amino acid of the HCDR3. In a further preferred embodiment, it has a sequence change from glycine (G) to serine (S) or alanine (A) at the fifth amino acid of the CDR.

[0161] In a preferred embodiment, the heavy chain variable region derived from 17552 comprises: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence selected from any one of SEQ ID NO: 102 to 105.

[0162] In one embodiment, the framework region of the 17552-derived heavy chain variable region has a framework region in which FWR1 corresponds to that of the IGHV4-4 acceptor, except that the first amino acid glutamine (Q1) has been replaced with glutamic acid (E1), and threonine (T23) has been retained from the donor sequence.

[0163] In one embodiment, the FWR2 corresponds to the acceptor sequence except that tyrosine (Y47) is retained from the donor sequence.

[0164] In one embodiment, the FWR3 corresponds to the acceptor sequence except that it retains phenylalanine (F67), lysine (K71), serine (S73), valine (V78), and threonine (T96).

[0165] In one implementation, the FWR4 corresponds to the IGHV4-4 / IGHJ4 acceptor sequence.

[0166] In one embodiment, one or more of the following residues are retained from the donor 17552 sequence: threonine (T23), 47-tyrosine (Y47), phenylalanine (F67), lysine (K71), serine (S73), valine (V78), and threonine (T96). In a preferred embodiment, all of those residues are retained. In a further preferred embodiment, all of those residues are retained, and glutamine (Q1) has been replaced with glutamic acid (E1).

[0167] In one particular embodiment, the FWR1, FWR2, FWR3 and FWR4 regions have amino acid sequences of SEQ ID NO:123, 124, 125 and 126, respectively.

[0168] A particularly preferred 17552-derived heavy chain variable region is that of 17552gH1 (SEQ ID NO: 29). Another preferred variant is 17552gH2 (SEQ ID NO: 30). A further preferred variant is 17552gH3 (SEQ ID NO: 31). A further preferred variant is 17552gH4 (SEQ ID NO: 32).

[0169] (c) Preferred pairing of light and heavy chains derived from 17552

[0170] In a preferred embodiment, the antibody of the present invention comprises 17552-derived light and heavy chain variable regions, such as any pair of 17552-derived light and heavy chain variable regions listed above.

[0171] Therefore, in one embodiment, the antibody or antigen-binding fragment of the present invention comprises at least one variable domain specific to CD45, said variable domain comprising the following light chain and heavy chain variable regions:

[0172] (a) A light chain variable region comprising: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and

[0173] (b) A heavy chain variable region comprising: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence selected from any one of SEQ ID NO: 102 to 105.

[0174] Preferred pairs include the 17552gL1 light chain variable region (SEQ ID NO: 25) and the 17552gH1 (SEQ ID NO: 29) heavy chain variable region. A further preferred pair is 17552gL1 (SEQ ID NO: 25) and 17552gH2 (SEQ ID NO: 30). Another preferred pair is 17552gL1 (SEQ ID NO: 25) and 17552gH3 (SEQ ID NO: 31). Another example of a preferred pair is 17552gL1 (SEQ ID NO: 25) and 17552gH4 (SEQ ID NO: 32).

[0175] Further preferred pairings include the light chain variable region of 17552gL2 (SEQ ID NO: 26) and the heavy chain variable region of 17552gH1 (SEQ ID NO: 29). A further preferred pair is 17552gL2 (SEQ ID NO: 26) and 17552gH2 (SEQ ID NO: 30). Another preferred pair is 17552gL2 (SEQ ID NO: 26) and 17552gH3 (SEQ ID NO: 31). Another example of a preferred pair is 17552gL2 (SEQ ID NO: 26) and 17552gH4 (SEQ ID NO: 32).

[0176] (d) Variants and Derivatives

[0177] In addition to the specific 17552-derived sequences listed herein, variants and derivatives of the specific sequences described elsewhere herein are provided. Such variants will retain the ability to specifically bind to CD45. Preferably, they will retain the ability to specifically bind to both human and cynomolgus monkey CD45.

[0178] Illustrative antibody patterns

[0179] The term "antibody" is not limited to a conventional quadruple-chain IgG antibody having two identical light chains and two identical heavy chains. It includes any pattern having at least one antigen-binding site formed by a CDR, particularly by a set of six CDRs. The antibodies of the present invention can be complete antibodies or fragments thereof having full-length heavy and light chains. Unless otherwise stated, any reference to antibodies herein also covers antigen-binding fragments of said antibodies that may be used alternatively. Examples of types of antibodies and antibody fragments include, for example, Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single-domain antibodies (e.g., VH or VL or VHH), scFv, bivalent, trivalent or quadruple antibodies, Bis-scFv, double-chain antibodies, triple-chain antibodies, quadruple-chain antibodies, or epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech.23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3)). , 209-217). In one embodiment, the antibody of the present invention is not a single-domain antibody. Methods for creating and preparing antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments used in the present invention include the Fab and Fab' fragments described in international patent applications WO2005 / 003169, WO 2005 / 003170 and WO 2005 / 003171. Multivalent antibodies may contain multiple specificities, such as bispecific, or may be monospecific (see, for example, WO 92 / 22853, WO05 / 113605, WO 2009 / 040562 and WO 2010 / 035012). In a particularly preferred embodiment, the antibody of the present invention is a monospecific antibody for CD45. In a further particularly preferred embodiment, the antibody of the present invention is a double complementary site for CD45. The term "antibody" includes monospecific, bispecific, and multispecific antibodies. Thus, for example, the term "antibody" specifically includes patterns such as Fab-X / Fab-Y, BYbe, and TrYbe. The term "antibody" also includes antibody fragments, preferably those mentioned herein. Unless the specific context otherwise requires, wherever "antibody" is mentioned herein, antigen-binding antibody fragments may also be used.

[0180] Examples of possible antibody patterns are known in the art, for example, as disclosed in the following reviews: “The coming of Age of Engineered Multivalent Antibodies, Nunez-Prado et al., DrugDiscovery Today Vol 20 Number 5 Mar 2015, pp. 588-594; D. Holmes, Nature RevDrug Disc Nov 2011:10, 798; Chan and Carter, Nature Reviews Immunology vol. 10, May 2010.” 301, which is incorporated herein by reference. In one embodiment, the antibody of the present invention may comprise, consist substantially of, or consist of any of the patterns listed below. Antibodies based on sequences derived from the rabbit 17415 and 17552 antibodies described herein are particularly preferred. Thus, for any of the patterns listed below, in a preferred embodiment, the antibody will comprise at least one antigen-binding site comprising humanized light and heavy chain variable regions derived from the rabbit 17415 antibody. In another preferred embodiment, for any of the patterns listed below, the antibody will comprise at least one antigen-binding site comprising humanized light and heavy chain variable regions derived from the rabbit 17552 antibody. In a particularly preferred embodiment, for those patterns having at least two antigen-binding sites, they will comprise at least one antigen-binding site comprising humanized light and heavy chain variable regions derived from the rabbit 17415 antibody and at least one antigen-binding site comprising humanized light and heavy chain variable regions derived from the rabbit 17552 antibody.

[0181] A particularly preferred antibody pattern is an IgG pattern antibody.

[0182] As used herein, "binding fragment" refers to a fragment capable of binding to a target peptide or antigen with sufficient affinity to characterize the fragment as specific for said peptide or antigen. As used herein, the term "Fab fragment" refers to an antibody fragment comprising: a V... L (Variable light) structural domains and constant structural domains (C L The light chain fragments and the V of the heavy chain H (Variable-weight) domains and a first constant domain (CH1). The term "Fv" refers to two variable domains, such as cooperative variable domains, or variable domains with maturing affinity pairs, i.e., V. H and V LYes. In one embodiment, such fragments are used as the antibody molecule of the present invention. The cooperative variable domains used herein are complementary to each other and / or both contribute to antigen binding such that the Fv(V) H / V L (Yes) Variable domains specific to the antigens in question.

[0183] As used herein, "antigen binding site" refers to a binding region, typically a polypeptide, capable of binding a target antigen, for example, with sufficient affinity to characterize the site as specific for the antigen. In one embodiment, the binding site comprises at least one variable domain or a derivative thereof, such as a pair of variable domains or their derivatives, or an associated pair of variable domains or their derivatives. Typically, this is a VH / VL pair. Variable regions (also referred to herein as variable domains) typically comprise three CDRs and a suitable framework. In one embodiment, the antigen binding site comprises two variable regions (a light chain variable region and a heavy chain variable region), and these elements together contribute to the specificity of the binding interaction of the antibody or binding fragment with respect to CD45, particularly regarding the specificity of where the binding site binds to CD45. In a particularly preferred embodiment, the VH / VL pair is humanized. The six CDRs provided by the pairing of light and heavy chain variable regions can be referred to as a "CDR set".

[0184] Residues in the antibody variable domain are routinely numbered according to a system devised by Kabat et al., described in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter “Kabat et al. (ibid.)”). Kabat residue nomenclature does not always directly correspond to linear amino acid residue numbering. The actual linear amino acid sequence may contain fewer or additional amino acids than in a strict Kabat numbering, corresponding to shortening or insertion of structural components of the basic variable domain structure, whether the framework or the complementarity-determining region (CDR). The correct Kabat residue number for a given antibody can be determined by comparing homologous residues in the antibody sequence with a “standard” Kabat-numbered sequence. According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31-35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3). However, according to Chothia (Chothia, C. and Lesk, AMJMol. Biol., 196, 901-917 (1987)), the ring equivalent to CDR-H1 extends from residue 26 to residue 32. Therefore, unless otherwise stated, “CDR-H1” as used herein is intended to refer to residues 26 to 35, as described by the combination defined by the Kabat numbering system and the Chothia topological ring. According to the Kabat numbering system, the CDRs of the variable domains of the light chain are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2), and residues 89-97 (CDR-L3).

[0185] In this application, linear amino acid numbering, rather than Kabat numbering, is used when discussing humanization. Therefore, the discussion of amino acid positions in grafted variants adopts linear amino acid numbering.

[0186] Derivatives, modifications and humanization

[0187] In addition to the specific antibodies listed herein, variants and derivatives thereof are provided. Such variants and derivatives will at least retain the ability to bind specifically to CD45. In a preferred embodiment, they will retain their biological functions described herein, such as the ability to kill or deplete target cells expressing CD45. Any of the specific assays and target cells used herein can be used, for example, to confirm such activity. In a particularly preferred embodiment, the variants or derivatives will retain the ability to bind specifically to both human and cynomolgus monkey CD45. Such ability can be confirmed using assays such as Biacore or cell-based assays. Again, the ability to specifically bind to human and cynomolgus monkey CD45 can be confirmed using any of the specific methods used in the embodiments of this application.

[0188] The “variants” or “derivatives” used herein may have, for example, one, two, three, four, five, or more amino acid sequence variations relative to the specific sequences listed herein. In one embodiment, the complementary site or antigen-binding site may be contained in one of the sets of the six specific CDRs listed herein, in addition to a total of up to seven amino acid sequence variations in all six CDRs relative to the specific sequence. In one embodiment, up to six amino acid sequence variations are present. In another embodiment, up to five amino acid sequence variations are present. In yet another embodiment, up to four amino acid sequence variations are present. In a preferred embodiment, up to three amino acid sequence variations are present. In a more preferred embodiment, up to two amino acid sequence variations are present. In a particularly preferred embodiment, only one amino acid sequence variation is present relative to the specific six CDRs listed. Any such variant will retain the ability to specifically bind to human CD45. Preferably, the variant will retain the ability to specifically bind to both human and cynomolgus monkey CD45. In another embodiment, such a number of sequence variations may be in the overall variable region with respect to the complementary site, compared to those of the specific antibodies listed herein.

[0189] Modifications in the CDR can include, for example, replacing one or more cysteine ​​residues with, for example, serine residues. Asn can be a substrate for deamination, and this tendency can be reduced by replacing Asn and / or adjacent amino acids with alternative amino acids (e.g., conserved substitution). The amino acid Asp in the CDR can undergo isomerization. The latter can be minimized by replacing Asp and / or adjacent amino acids with alternative amino acids (e.g., conserved substitution). The amino acid sequence can be used to eliminate or reduce undesirable properties, but characteristic features are preserved. Examples of modifications include those used to remove glycosylation sites, GPI anchors, or solvent-exposed lysines. These modifications can be achieved by replacing the relevant amino acid residues with conserved amino acid substitutions.

[0190] Antibody constant region and Fc region function

[0191] In a preferred embodiment, the antibody of the present invention does not contain an Fc domain. In an alternative preferred embodiment, the antibody of the present invention contains a modified Fc domain as described herein. In a preferred embodiment, the antibody of the present invention contains an Fc domain, but the sequence of said Fc domain has been altered to remove one or more Fc effector functions. In another embodiment, the Fc region of the antibody of the present invention has been modified to optimize specific properties of said antibody, such as any of those discussed herein.

[0192] In one embodiment, the antibody of the present invention comprises a "silenced" Fc region. For example, in one embodiment, the antibody of the present invention does not exhibit one or more effector functions associated with a normal Fc region.

[0193] Unless the context clearly indicates otherwise, the Fc domain as used in this paper generally refers to -(CH2CH3)2.

[0194] In one embodiment, the antibody of the present invention does not contain the -CH2CH3 fragment.

[0195] In one embodiment, the antibody of the present invention does not contain a CH2 domain.

[0196] In one embodiment, the antibody of the present invention does not contain a CH3 domain.

[0197] In one embodiment, the antibody of the present invention does not bind to the Fc receptor.

[0198] In one embodiment, the antibody of the present invention does not bind complement. In a preferred embodiment, the antibody of the present invention does not bind a first complement factor, C1q or C1. In one embodiment, the antibody of the present invention does not bind those factors because, for example, it lacks the Fc region. In another embodiment, the antibody of the present invention does not bind those factors because it has modifications in the constant region that prevent it from doing so. In an alternative embodiment, the antibody of the present invention does not bind FcγR, but binds complement. For example, in one embodiment, the antibody of the present invention does not bind FcγR, but binds C1q and / or C1.

[0199] In one embodiment, the antibody of the present invention does not contain an active Fc region because the antibody does not trigger the release of one or more cytokines that would normally be released by a normal Fc region. For example, the Fc region of the antibody of the present invention may not trigger the release of cytokines when it binds to an Fc receptor, or may not do so significantly.

[0200] In one embodiment, the antibody of the present invention may typically contain a modification that alters the serum half-life of the antibody. Therefore, in another embodiment, the antibody of the present invention has an Fc region modification that alters the half-life of the antibody. Such modifications may also be present in addition to those that alter Fc function. In one embodiment, the antibody of the present invention has a modification that increases or decreases the serum half-life of the antibody compared to an antibody lacking such a modification. In another embodiment, the antibody of the present invention contains a modification that collectively silences the Fc region and increases or decreases the serum half-life of the antibody compared to an antibody lacking such a modification.

[0201] The antibody constant region domain (if present) of the antibody of the present invention can be selected taking into account the proposed functions of the antibody molecule, particularly the effector functions that may be required. In a preferred embodiment, the antibody is an antibody lacking Fc or lacking one or more effector functions (preferably all effector functions) of the Fc region. In other embodiments of the invention, the effector function of the Fc region of the antibody may still be present. In one embodiment, the antibody of the present invention may comprise a human constant region, such as an IgA, IgD, IgE, IgG, or IgM constant region domain. In particular, when the antibody molecule is intended for therapeutic use in which antibody effector function is required, a human IgG constant region domain, especially those of the IgG1 and IgG3 isotypes, may be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector function is not required, IgG2 and IgG4 isotypes may be used. Particularly preferred IgG isotypes are IgG2 and IgG4. In a preferred embodiment, the constant region may have been modified so that the antibody does not have effector function. Therefore, it will be appreciated that sequence variants of these constant region domains may also be used. For example, an IgG4 molecule in which the serine at position 241 has been replaced with proline can be used, as described in Angal et al., 1993, Molecular Immunology, 1993, 30:105-108. Therefore, in this embodiment, when the antibody is an IgG4 antibody, the antibody may include a mutant S241P. In another embodiment, the antibody of the present invention may lack the Fc region.

[0202] In one embodiment, the antibody of the present invention may have a silenced Fc region. As used herein, the terms “silent,” “silenced,” or “silent” refer to an antibody having the modified Fc region described herein, which has reduced binding to the Fcγ receptor (FcγR) relative to the binding of the same antibody containing an unmodified Fc region to FcγR (e.g., a reduction in binding to FcγR of at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, as measured by, for example, BLI, relative to the binding of the same antibody containing an unmodified Fc region to FcγR). In some embodiments, the Fc-silenced antibody has no detectable binding to FcγR. The binding of antibodies with modified Fc regions to FcγR can be determined using a wide variety of techniques known in the art, such as, but not limited to, equilibration methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et al., Analytical Biochemistry, Vol. 373: 52-60, 2008; or radioimmunoassay (RIA)), or by surface plasmon resonance assays or other kinetic-based assays (e.g., BIACORE). TM Analysis or Octet TM Analysis (forteBIO), and other methods, such as indirect binding assays, competitive binding assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). In another embodiment, the antibody of the present invention may have been modified to reduce or eliminate binding to FcγR, but still allow complement activation. In another embodiment, the antibody of the present invention may have a modified Fc region such that it does not activate cytokine release, but is still able to activate complement.

[0203] In one embodiment, the antibody heavy chain comprises a CH1 domain, and the antibody light chain comprises a CL domain (κ or λ). In another embodiment, the antibody heavy chain comprises a CH1 domain, a CH2 domain, and a CH3 domain, and the antibody light chain comprises a CL domain (κ or λ). The κ light chain is preferred.

[0204] The four human IgG isotypes bind to activating Fcγ receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa), inhibitory FcγRIIb receptors, and complement component 1 (C1q) with different affinities, thus producing very different effector functions (Bruhns P. et al., 2009. Specificity and affinity of human FcγRs and their polymorphic variants for human IgG subclasses. Blood. 113(16):3716-25); see also Jeffrey B. Stavenhagen et al., Cancer Research 2007 Sep 15, 67(18):8882-90). In one embodiment, the antibody of the present invention does not bind to the Fc receptor. In another embodiment of the present invention, the antibody binds to one or more types of Fc receptors.

[0205] The binding of IgG to FcγR or C1q depends on residues located in the hinge region and the CH2 domain. Both regions of the CH2 domain are crucial for FcγR and C1q binding and have unique sequences in IgG2 and IgG4. Substitution of human IgG1 with IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330, and 331 has been shown to significantly reduce ADCC and CDC (Armour KL). .et al., 1999. Recombinant human IgG molecules lacking Fc gamma receptor I binding and monocyte triggering activities. Eur J Immunol. 29(8):2613-24; and Shields RL. et al., 2001, High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma RJ Biol Chem. 276(9):6591-604). Furthermore, Idusogie et al. demonstrated that alanine substitution at different locations (including K322) significantly reduced complement activation (Idusogie EE. et al., 2000. Mapping of the C1qbinding site on rituxan, a chimeric antibody with a human IgG1 Fc. J Immunol.164(8):4178-84). Similarly, mutations in the CH2 domain of mouse IgG2A have shown reduced binding to FcγRI and C1q (Steurer W. et al., 1995). Ex vivo coating of islet cell allografts withmurine CTLA4 / Fc promotes graft tolerance. J Immunol. 155(3):1165-74).

[0206] In one embodiment, the Fc region used is mutated, particularly the mutations described herein. In one embodiment, the mutation is intended to remove binding and / or effector function. In a preferred embodiment, the antibody of the present invention has been mutated so that it does not bind to the Fc receptor. In another preferred embodiment, the antibody of the present invention does not contain an Fc region and therefore does not exhibit Fc effector activity for that reason. In one embodiment, the Fc mutation is selected from the group consisting of: mutations for removing or enhancing the binding of the Fc region to the Fc receptor, mutations for increasing or removing effector function, mutations for increasing or decreasing the half-life of the antibody, and combinations thereof. In a preferred embodiment, the modification eliminates or reduces binding to the Fc receptor. In another preferred embodiment, the modification eliminates or reduces Fc effector function. In another preferred embodiment, the modification increases or decreases serum half-life. In another preferred embodiment, the constant region of the antibody contains one or more modifications that reduce or eliminate Fc receptor binding and Fc effector function and increase or decrease serum half-life. In one embodiment, where the effect of the modification is mentioned, it can be demonstrated by comparison with an equivalent antibody that lacks the modification.

[0207] In another embodiment of the invention, the antibody may have heavy chain modifications that alter its ability to bind to protein A, specifically eliminating protein A binding. Such methods can be preferably used to facilitate the purification of bispecific antibodies, as discussed herein. However, in other embodiments, if any antibody of the invention has an Fc region, it can be modified to alter protein A binding. For example, both heavy chains may include modifications. Alternatively, both heavy chains may be unmodified. However, in a preferred embodiment, one heavy chain is modified while the other is unmodified.

[0208] Some antibodies that selectively bind FcRn at pH 6.0 rather than pH 7.4 exhibit longer half-lives in a wide variety of animal models. Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q / M428L (Hinton PR. et al., 2004. Engineered human IgG antibodies with longer serum half-lives in primates. J Biol Chem. 279(8):6213-6) and M252Y / S254T / T256E +H433K / N434F (Vaccaro C. et al., 2005. Engineering the Fc region of immunoglobulin G to modulate...), have shown longer half-lives in various animal models. in vivo antibody levels. Nat Biotechnol. 23(10):1283-8) and M428L / N434S (Zalevsky et al., 2010, Enhanced antibody half-life improves in vivo (Activity. Nature Biotech 28:157-159) has been shown to increase binding affinity to FcRn and the in vivo half-life of IgG1. Therefore, modifications altering the serum half-life can be present at M252 / S254 / T256 + H44 / N434, particularly M252Y / S254T / T256E + H433K / N434F. However, a direct relationship is not always present between increased FcRn binding and increased half-life (Datta-Mannan A. et al., 2007. Humanized IgG1 Variants with Differential Binding Properties to the Neonatal Fc Receptor: Relationship to Pharmacokinetics in Mice and Primates. Drug Metab. Dispos.35: 86-94). In one embodiment, increasing the half-life is desirable. In another embodiment, it may actually be desirable to reduce the serum half-life of the antibody, and therefore modifications reducing the serum half-life may be present.

[0209] The IgG4 subclass exhibits reduced Fc receptor (FcγRIIIa) binding. Antibodies against other IgG subclasses typically show strong binding. Reduced receptor binding in these other IgG subclasses can be influenced by altering, for example, by substituting one or more amino acids selected from the group consisting of: Pro238, Asp265, Asp270, Asn270 (loss of Fc carbohydrates), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435. In one embodiment, the molecule according to the invention has an Fc of an IgG subclass (e.g., IgG1, IgG2, or IgG3), wherein the Fc is mutated at one, two, or all of the following positions: S228, L234, and / or D265. In one embodiment, the mutation in the Fc region is independently selected from S228P, L234A, L235A, L235A, L235E and combinations thereof.

[0210] In one embodiment, the antibody of the present invention may contain modifications that affect whether the antibody induces cytokine release. In particular, L234F and K274Q modifications exhibit the ability to reduce antibody-induced cytokine release. Therefore, in one embodiment, the antibody of the present invention may contain modifications that alter cytokine release at L234 and / or K274, particularly L234F and K274Q modifications. Further, the L234 residue may have an effect on platelet activation, and that residue may be modified additionally or alternatively. In one embodiment of the invention, for example, an L234 modification that alters platelet binding, particularly L234F modification, may be introduced. P331 also exhibits a role in C1q binding, therefore in one embodiment, P331 may be unmodified to preserve complement activation. In another embodiment, it may be modified to reduce or eliminate complement activation; for example, the heavy chain may contain a P331S modification. In another embodiment, a P329 modification, particularly P329A modification, is present to reduce or eliminate complement binding. In another embodiment, the antibody may include one or more modifications at positions P329, P331, K332, and / or D265. In a preferred embodiment, the antibody may include modifications at P329A, P331S, K332A, and D265A to affect complement binding, particularly reducing C1q binding.

[0211] It may be desirable to reduce or increase the effector function of the Fc region. In a preferred embodiment, it is desirable to reduce such effector function. In another embodiment, it is desirable to optimize it. For antibodies targeting cell surface molecules (especially those on immune cells), it is typically necessary to eliminate effector function. In other cases, particularly where the purpose is to deplete cells, it may be desirable that Fc effector function has been eliminated or reduced to the lowest possible level. For example, in a particularly preferred embodiment, the antibody of the present invention is capable of inducing cell death in target cells expressing CD45, but does not exhibit Fc effector function. Thus, in a preferred embodiment, the antibody of the present invention lacks an active Fc region. For example, the antibody may be physically absent from the Fc region, or the antibody may contain modifications that inactivate the Fc region. The latter may be referred to, for example, as Fc silencing. In one embodiment, Fc silencing may mean that the antibody of the present invention is less able to induce or does not induce the release of one or more cytokines that an antibody with an unmodified Fc region would normally trigger. In a preferred embodiment, the antibody of the present invention is capable of stimulating cell death, but does not exhibit Fc function. Further examples of Fc function include stimulating mast cell degranulation, and again that function may be reduced or absent in the antibodies of the present invention. The degree of reduction in Fc function may be, for example, at least 65%, and for example, at least 75%. In one embodiment, the reduction is at least 80%. In another embodiment, the reduction is at least 90%. The reduction may be, for example, at least 95%. In a preferred embodiment, the reduction is at least 99%. In another embodiment, the reduction may be 100%, meaning that in such cases, Fc function is completely eliminated.

[0212] Numerous mutations have been made in the CH2 domain of human IgG1, and their effects on ADCC and CDC have been tested in vitro (Idusogie EE. et al., 2001. Engineered antibodies with increased activity to recruit complement. J Immunol. 166(4):2571-5). Notably, an alanine substitution at position 333 has been reported to increase both ADCC and CDC. Therefore, in one embodiment, modifications at position 333 can be present, and in particular modifications that alter the ability to recruit complement. Lazar et al. described a triple mutant (S239D / I332E / A330L) with high affinity for FcγRIIIa and low affinity for FcγRIIb, resulting in enhanced ADCC (Lazar GA. et al., 2006). Therefore, modifications at S239 / I332 / A330 can exist, particularly those that alter the affinity for the Fc receptor, especially S239D / I332E / A330L. (Engineered antibody Fc variants with enhanced effector function. PNAS 103(11): 4005-4010). The same mutations can be used to generate antibodies with increased ADCC (Ryan MC. et al., 2007. Antibody targeting of B-cell maturation antigen on malignant plasma cells. Mol. CancerTher., 6: 3009-3018). Richards et al. investigated a slightly different triple mutant (S239D / I332E / G236A) with improved FcγRIIIa affinity and FcγRIIa / FcγRIIb ratio, which mediates enhanced macrophage phagocytosis of target cells (Richards JO et al., (2008) Optimization of antibody binding to Fcgamma RIIa enhances macrophage phagocytosis of tumor cells. MolCancer Ther. 7(8):2517-27). Therefore, in one embodiment, the S239D / I332E / G236A modification may be present.

[0213] Because they lack effector function, IgG4 antibodies represent a suitable subclass of IgG for receptor blocking. IgG4 molecules can exchange half-molecules in a dynamic process called Fab-arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous IgG4. In a preferred embodiment, the antibodies of the present invention have a modification at S228, specifically S228P. The S228P mutation has been shown to prevent this recombination process, thereby allowing the design of less predictable therapeutic IgG4 antibodies (Labrijn AF. et al., 2009. Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4). in vivo (Nat Biotechnol. 27(8):767-71). This technique can be used to create bispecific antibody molecules. In a preferred embodiment, the modifications listed herein can be used in the context of IgG4.

[0214] WO 2008 / 145142 discloses examples of modifications, particularly modifications for IgG4 isotype antibodies that can be used in this invention.

[0215] In one embodiment, the heavy chain of the antibody of the present invention may comprise a human IgG4 constant region having substitutions of an Arg residue at position 409, a Phe residue at position 405, and / or a Lys residue at position 370. For example, in a preferred embodiment, the heavy chain of the antibody includes a modification at position 409, particularly a modification selected from those introducing Lys, Ala, Thr, Met, or Leu residues at that position. In one embodiment, the modification is the introduction of a Lys, Thr, Met, or Leu residue at position 409. In another embodiment, the modification may be the introduction of a Lys, Met, or Leu residue at position 409. In one embodiment, the antibody does not contain Cys-Pro-Pro-Cys in the hinge region. In one embodiment, the antibody exhibits a reduced ability to induce Fab arm exchange in vivo. In one embodiment, the hinge region of the antibody comprises a CXPC or CPXC sequence, where X is any amino acid other than proline. In one embodiment, the antibody of the present invention may exhibit the ability to display specific properties using a specific antibody class, antibody allotype, or antibody allotype. Such natural diversity can be used to confer specific properties. For example, IgG1 has R409, while IgG4 has K409 at position 409 on the heavy chain, which may naturally affect the capabilities of said antibodies. A review of various naturally occurring sequence variations is provided in Jefferis et al., (2009) mAbs, 1(4): 332-338, which is incorporated herein by reference in its entirety, particularly concerning the sequence variations discussed herein.

[0216] Those skilled in the art will also understand that antibodies can undergo a wide variety of post-translational modifications. The type and extent of these modifications often depend on the host cell line used to express the antibody and the culture conditions. Such modifications can include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. A common modification is the loss of a basic C-terminal residue (e.g., lysine or arginine) due to the action of carboxypeptidase (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Therefore, the C-terminal lysine of the antibody heavy chain may be absent.

[0217] In one embodiment, the antibody of the present invention may be a glycosylated IgG, for example, to induce a reduced Fc function, particularly a nearly Fc-ineffective phenotype. In one embodiment, the antibody of the present invention has a modification at N297, particularly N297A. In one embodiment, the antibody of the present invention has a modification at F243 and / or F244, particularly meaning that the antibody is a glycosylated IgG. In one embodiment, the antibody of the present invention may contain a heavy chain modification at F243A and / or F244A. In another embodiment, one or more of F241, F243, V262, and V264 may be modified, particularly to affect amino acids involved in glycosylation. In one embodiment, the antibody of the present invention may have modifications at F241A, F243A, V262E, and V264E. This list of modifications is discussed in Yu et al. (2013) 135(26): 9723-9732, which is incorporated herein by reference in its entirety, particularly with respect to the modifications discussed herein. Such modifications provide a means of modulating, for example, Fc receptor binding. Modifications affecting the glycosylation of the antibody may be present. Further, as a further method for glycoengineering, the antibody of the present invention can be generated in a cell type that affects glycosylation. In one embodiment, the fucosylation, sialylation, galactosylation, and / or mannosylation of the antibody of the present invention can be altered by sequence modification and / or via the type of cell used to generate the antibody.

[0218] In one embodiment, the antibody of the present invention has modifications at positions 297 and / or 299. For example, in one embodiment, the antibody of the present invention includes an N297A modification, preferably N297Q, in its heavy chain, or a mutation of Ser or Thr at position 299 into other residues. In one embodiment, it has both modifications.

[0219] Particularly preferred examples of constant region modifications are Leu234Ala and Leu235Ala modifications (according to EU designations), also known as LALA modifications. In particular, IgG1 antibody patterns with LALA modifications are preferred. In a further preferred embodiment, the antibody of the present invention is an IgG4 antibody with FALA modification.

[0220] In another embodiment, the antibody of the present invention may have a modified hinge region and / or CH1 region. Alternatively, the isotype used may be selected because it has a specific hinge region. As in White et al., (2015) Cancer CellAs described in 27(1): 138-148, the IgG2 CH1 and hinge region impart specific properties, particularly concerning the disulfide bridge between the heavy and light chains. For example, modifications to promote or reduce flexibility in the hinge region can also be used in the antibodies of this invention. Methods for altering the flexibility of the hinge region are disclosed in Liu et al., (2019). Nature Communications 10:4206. White et al. (2015) and Liu et al. (2019) are incorporated herein by reference in their entirety, particularly with respect to the modifications discussed. In one embodiment, the heavy chain of the antibody of the present invention has IgG2 CH1 and / or hinge regions, and in another embodiment, both heavy chains have them. In one embodiment, the antibody used is an IgG1 antibody. In a particularly preferred embodiment, the antibody used may be an IgG2 or IgG4 antibody with hinge or CH1 modifications, particularly those with modified hinge regions, for example, those modified to alter disulfide bond formation. In another embodiment, IgG2 or IgG4 isotype antibodies are used because those isotypes exhibit less flexibility in their hinge regions than IgG3 isotype antibodies. In one embodiment, the IgG4 isotype antibody is used in a form that may be able to initiate CD32 cross-linking.

[0221] In another embodiment, the antibody exhibits optimal ability to spatially initiate cross-linking of CD45 molecules.

[0222] Particularly preferred examples of heavy chain constant region sequences containing LALA modification are provided as SEQ ID NO: 144, 146 and 148, respectively.

[0223] The heavy chain constant region of SEQ ID NO: 148 has a so-called "knob" modification, T336W. The heavy chain constant region of SEQ ID NO: 146 has a so-called "hole" modification, T366S, L366S, and Y407. The "hole" modification promotes heterodimer formation, and therefore the formation of bicomponent antibodies, preferentially over monospecific antibodies. Thus, in a particularly preferred embodiment, such heavy chain constant regions are used for the bicomponent antibodies of the present invention.

[0224] Examples of particularly preferred light chain constant region sequences are provided as SEQ ID NO: 145.

[0225] In a particularly preferred embodiment, the heavy and light chain constant regions in the monospecific antibody of the present invention are those of SEQ ID NO: 144 and 145. In one embodiment, those constant regions are used together with the VR17415gL15gH6 variable region.

[0226] In another particularly preferred embodiment, the constant regions of the heavy and light chains in the bicomponent antibody of the present invention are those of SEQ ID NO: 146 and 148 (for the heavy chain) and SEQ ID NO: 145 (for the light chain). In one embodiment, the constant regions of the VR17552gL1gH4 portion of the bicomponent antibody are those of SEQ ID NO: 148 and 145. In one embodiment, the constant regions of the VR17552gL1gH4 portion of the bicomponent antibody are those of SEQ ID NO: 146 and 145.

[0227] In one embodiment, the constant region of the VR17415gL15gH6 portion of the dual complementary site antibody is those of SEQ ID NO: 148 and 145. In one embodiment, the constant region of the VR17415gL15gH6 portion of the dual complementary site antibody is those of SEQ ID NO: 146 and 145.

[0228] In one embodiment, the constant regions of the VR17552gL1gH4 portion of the dual complementary site antibody are those of SEQ ID NO: 148 and 145, and the constant regions of the VR17415gL15gH6 portion of the dual complementary site antibody are those of SEQ ID NO: 148 and 145. In another embodiment, the constant regions of the VR17552gL1gH4 portion of the dual complementary site antibody are those of SEQ ID NO: 146 and 145, and the constant regions of the VR17415gL15gH6 portion of the dual complementary site antibody are those of SEQ ID NO: 146 and 145.

[0229] Bispecific and dual complementary site antibodies

[0230] In a preferred embodiment, the antibody of the present invention is bispecific. In a preferred embodiment, it is a dual complementary antibody concerning CD45, i.e., having two specificities for CD45. A wide variety of bispecific antibody profiles can be used to facilitate the formation or purification of bispecific antibodies, preferentially over monospecific antibodies, when different heavy and light chains with respect to said specificity are expressed together, and these can be used in the present invention.

[0231] In one embodiment, the antibody of the present invention may have modifications that promote the formation of the antibody of the present invention, preferred over unwanted ones. Such modifications are particularly preferred when the antibody of the present invention comprises at least two different antigen-binding sites that recognize different epitopes. In particular, such modifications are particularly preferred for bispecific and bicomplementary antibodies. For example, in one embodiment, the generation of the antibody of the present invention may involve two different antigen-binding sites, particularly two different complementary sites, which are on different polypeptide chains and associated. Therefore, it is desirable to form a heterodimer including both specificities, preferred over a homodimer including only one of the two specificities. An example of a method to promote heterodimer formation is to employ heavy chain modifications that promote the association of two different heavy chains rather than two identical heavy chains. In one embodiment, one (or at least one) of the binding partners cannot form a homodimer, for example, by mutating the amino acid sequence of the binding partner to eliminate or minimize homodimer formation. Examples of such modifications include so-called "mortar and pestle" modifications. Possible mortar and pestle modifications are listed, for example, in Merchant et al., (1998). Nature Biotechnology 16(7):677-681 and Carter et al., (2001) J Immunol Methods In references to 248(1-2): 7-15, all of which are combined by reference, particularly concerning the mortar and pestle modifications discussed herein. Charge modifications may alternatively or additionally be used to promote the formation of heterodimers rather than homodimers; for example, such modifications may be present in the heavy chain. In another embodiment, charge modifications are used to initiate pairing of a particular light chain with a particular heavy chain.

[0232] In one embodiment, such methods for promoting heterodimer formation are used in combination with common light chain methods. In another embodiment, it is possible that, instead of prioritizing homodimer formation, there is a modification that means the heterodimer can be more easily separated from the homodimer, for example, by chromatography. Again, in some embodiments, such methods can be used in conjunction with common light chain methods. In another embodiment, the portion of an antibody carrying a specific complementary site against CD45 is only able to associate with portions of antibodies containing different complementary sites of said antibody.

[0233] As used herein, “cannot form homodimers” means a low or zero tendency to form homodimers. “Low” as used herein means 20%, 10%, 5% or less, such as 4%, 3%, 2%, 1%, 0.5% or less of aggregates.

[0234] Heavy chain modifications can also be used to give one heavy chain a different affinity for the binding agent compared to another. For example, the two different heavy chains can have different affinities for protein A. In one embodiment, one heavy chain has a modification that eliminates protein A binding or is a homotype that does not bind protein A, while the other heavy chain still binds protein A. Although such methods do not change the proportion of heterodimers formed, they do allow the purification of heterodimer antibodies from either homodimer antibody based on protein A affinity. The antibodies of the present invention can have modifications at positions 95 and 96 of one of the heavy chains that affect protein A binding. Examples of such modifications that can be used include the use of H95R modification (for one heavy chain) or H95R and Y96F modification (for both) in the IMGT exon numbering system. Those modifications are H435R modification and H435R and Y436F modification in the EU numbering system. In one embodiment, the antibody of the present invention may also have modifications at D16, L18, N44, K52, V57, and V82. In one embodiment, such modifications are present in the heavy chain, as well as one or more of the modifications D16E, L18M, N44S, K52N, V57M, and V82I in the IMGT numbering system. In one embodiment, such modifications are used when the IgG is IgG1, IgG2, or IgG4. In a particularly preferred embodiment, they are used on one of the two heavy chains, wherein both heavy chains are IgG4 isotypes. Methods for influencing such modifications to affect protein A binding are described, for example, in US 2010 / 0331527 A1, which is incorporated herein by reference in its entirety, particularly with respect to the modifications disclosed therein involving protein A binding.

[0235] In a further embodiment, the isotypes of the heavy chains used can be selected based on their ability to bind to protein A. For example, in humans, IgG1, IgG2, and IgG4 in their wild-type forms all bind to protein A, while wild-type human IgG3 does not. In a particularly preferred embodiment, both heavy chains are IgG4, but one has modifications to reduce or eliminate protein A binding. That means that, based on protein A affinity, the heterodimeric form of the antibody will be more easily separated from the unwanted homodimeric form.

[0236] In one embodiment, the modification for promoting heterodimer formation can be combined with those modifications that allow for the purification of the heterodimer. In one embodiment, the modification can be at positions F405 and K409. For example, an example of a pair of modifications that can be introduced into the two heavy chains to promote heterodimer formation is F405L and K409R. Those modifications can be used alone or in combination with heavy chain modifications that allow for preferential purification of the heterodimer. In one embodiment, one heavy chain has modifications at positions 405, 409, 435, and 436, and the other heavy chain has a modification at position 409. In one embodiment, one heavy chain has the F405L modification, while the other has the K409R, H435R, and Y436F modifications. In another embodiment, one heavy chain has the F405L, H435R, and Y436F modifications, and the other heavy chain has the K409R modification. Examples of such methods are described in Steinhardt et al., (2020). Pharmaceutics In references to 12, 3, this document is incorporated herein by reference in its entirety, particularly concerning the described bispecific antibody patterns and heavy chain modifications. In another embodiment, methods relating to the light chain may be used, particularly in addition to the methods discussed above for the heavy chain. In one embodiment, the Roche Cross-Mab method is applied. In another embodiment, a common light chain may be used, thereby enabling the use of the same light chain for both specificities. Various bispecific antibody patterns are described in Spiess et al., (2015). Molecular Immunology The above is summarized in 67: 95-106 and can be used in this invention.

[0237] In a particularly preferred embodiment, the dual complementary site antibody of the present invention is an IgG-like antibody. In a preferred embodiment, it is an IgG1 or IgG4 antibody. In a particularly preferred embodiment, its constant region comprises an IgG1-like antibody modified with LALA. In a particularly preferred embodiment, its constant region comprises an IgG4-like antibody modified with FALA.

[0238] 17415 x 17552 derived double complementary site antibody

[0239] In a preferred embodiment, the antibody of the present invention is bicomponent with respect to CD45. In one embodiment, the antibody may also contain at least one specificity for molecules other than CD45, in addition to the two specificities with respect to CD45. For example, the antibody may contain further specificity for blood proteins or cell-specific surface proteins. A particularly preferred specificity is specificity for serum albumin. In an alternative embodiment, the antibody of the present invention is bicomponent with respect to CD45 and does not contain any further specificity.

[0240] In a particularly preferred embodiment, the antibody of the present invention is double-complementary to CD45 because it contains CD45 specificity derived from 17415 and CD45 specificity derived from 17552. It is possible that the 17552-derived specificity can be considered "helper" specificity, and the 17415-derived specificity can be considered "killer" specificity.

[0241] Any pairing of grafting variants derived from 17415 and 17552 can be used. For example, grafting variants derived from... Figures 2 to 4 Any pair. In one embodiment, the 17415 specificity is from Table 1, and the 17552 specificity is from Table 2. Particularly preferred combinations are those used in the embodiments and figures of this application.

[0242] In one embodiment, a preferred bicomplementary molecule is a 17415gL7gH6 x 17552gL1gH1 bicomplementary molecule. In another embodiment, a preferred bicomplementary molecule is a 17415gL7gH6 x 17552gL1gH4 bicomplementary molecule. In one embodiment, the bicomplementary molecule comprises those combined CDRs. In another embodiment, it will comprise those combined light and heavy chain variable regions.

[0243] In one embodiment, a preferred bicomplementary molecule is a 17415gL15gH6 x 17552gL1gH1 bicomplementary molecule. In another embodiment, a preferred bicomplementary molecule is a 17415gL15gH6 x 17552gL1gH4 bicomplementary molecule. In one embodiment, the bicomplementary molecule comprises those combined CDRs. In another embodiment, it will comprise those combined light and heavy chain variable regions.

[0244] In one embodiment, a preferred bicomplementary molecule is a 17415gL16gH6 x 17552gL1gH1 bicomplementary molecule. In another embodiment, a preferred bicomplementary molecule is a 17415gL16gH6 x 17552gL1gH4 bicomplementary molecule. In one embodiment, the bicomplementary molecule comprises those combined CDRs. In another embodiment, it will comprise those combined light and heavy chain variable regions.

[0245] In one embodiment, a preferred bicomplementary molecule is a 17415gL7gH6 x 17552gL1gH1 bicomplementary molecule. In another embodiment, a preferred bicomplementary molecule is a 17415gL7gH6 x 17552gL1gH4 bicomplementary molecule. In one embodiment, the bicomplementary molecule comprises those combined CDRs. In another embodiment, it will comprise those combined light and heavy chain variable regions.

[0246] In one embodiment, a preferred bicomplementary molecule is a 17415gL15gH6 x 17552gL1gH1 bicomplementary molecule. In another embodiment, a preferred bicomplementary molecule is a 17415gL15gH6 x 17552gL1gH4 bicomplementary molecule. In one embodiment, the bicomplementary molecule comprises those combined CDRs. In another embodiment, it will comprise those combined light and heavy chain variable regions.

[0247] In one embodiment, a preferred bicomplementary molecule is a 17415gL16gH6 x 17552gL1gH1 bicomplementary molecule. In another embodiment, a preferred bicomplementary molecule is a 17415gL16gH6 x 17552gL1gH4 bicomplementary molecule. In one embodiment, the bicomplementary molecule comprises those combined CDRs. In another embodiment, it will comprise those combined light and heavy chain variable regions.

[0248] In a particularly preferred embodiment, the bicomplementary antibody of the present invention is an IgG-like antibody. In a preferred embodiment, it is an IgG1 or IgG4 antibody. In a particularly preferred embodiment, it is an IgG1-like antibody whose constant region comprises LALA modification. In a particularly preferred embodiment, it is an IgG4-like antibody whose constant region comprises FALA modification. Thus, those styles can be used for any of the 17415 and 17552-derived antibodies listed herein, which contain humanized variable regions, particularly any of the combinations of light chain and heavy chain variable regions listed above.

[0249] The SEQ ID NOs of the light and heavy chain variable region sequences of the aforementioned dual complementary site antibodies can be found by referring to Tables 1 and 2, and particularly by referring to the SEQ ID NOs of the grafted variants listed in those tables. Dual complementary site antibodies comprising CDR sets or variable region pairs of specific dual complementary site antibodies described in the embodiments of this application are also preferred.

[0250] A particularly preferred dual complementary antibody of the present invention is one having in Figure 17 The CDR set of the double complementary antibody shown in the figure. A further particularly preferred double complementary antibody of the invention is one having in Figure 17 The variable regions of the light and heavy chains of the dual complementary antibody shown in the figure.

[0251] In a particularly preferred embodiment, while providing VR17415-derived specificity, a VR17415gL15gH6 IgG1 LALA antibody is provided, with the sequences of its light and heavy chains described in [the original text]. Figure 31 Therefore, in a particularly preferred embodiment, the antibody will comprise the heavy chain sequence of SEQ ID NO: 140 and the light chain sequence of SEQ ID NO: 141. In one embodiment, the antibody is a monospecific VR17415gL15gH6 IgG1 LALA antibody comprising two such heavy chains and two such light chains. In one embodiment, where the antibody is a dual complementary site molecule, one specificity of the antibody will comprise the heavy and light chain pairs of SEQ ID NO: 140 and 141. Variations of such specific antibodies discussed herein are also provided.

[0252] In a particularly preferred embodiment, when using VR17552-derived specificity, a VR17552gL1gH4 IgG1 LALA antibody is used. Therefore, in a particularly preferred embodiment, the antibody will comprise the heavy chain sequence of SEQ ID NO: 142 and the light chain sequence of SEQ ID NO: 143. In one embodiment, when the antibody is a monospecific VR17552gL1gH4 IgG1 LALA antibody, the antibody will comprise two such heavy chains and two such light chains. In one embodiment, when the antibody is a bicomponent molecule, one specificity of the antibody will comprise the heavy and light chain pairs of SEQ ID NO: 142 and 143. Variations of such specific bicomponent antibodies discussed herein are also provided.

[0253] In a particularly preferred embodiment, a dual complementary antibody is provided, which has in Figure 32The light and heavy chain sequences are given in the provided text, therefore VR17415gL15gH6 is provided by the heavy and light chain sequences of SEQ ID NO: 147 and 141, respectively, while the specificity of VR17552gL1gH4 is provided by the heavy and light chain sequences of SEQ ID NO: 142 and 143, respectively. Variants of this type of specific dual complementary site antibody discussed herein are also provided.

[0254] Antibody generation and screening

[0255] This section describes various methods that can be used to generate variants. In the case of antibodies with dual complementary sites for CD45, while antibodies containing both 17415-derived and 17552-derived CD45 specificities are preferred, the methods described herein can also be used to identify further CD45 specificity to pair with either the 17415-derived or 17552-derived specificity.

[0256] In one embodiment, the antibody or antibody / fragment component of the present invention has enhanced potency, optionally wherein the antibody or antibody / fragment component of the present invention also has enhanced potency compared to other known antibodies against CD45. "Enhanced potency" includes the meaning that, compared to prior art antibodies against CD45, the antibody of the present invention can achieve the same level of cell killing at a lower concentration / titer. In one embodiment, reference to "enhanced potency" means that, compared to prior art antibodies against CD45, the antibody of the present invention can achieve a higher maximum effect.

[0257] In one embodiment, the antibody of the present invention or an antibody / fragment component thereof is processed to provide improved affinity for one or more target antigens, particularly for CD45. Such variants can be obtained through a number of affinity maturation protocols, including CDR mutation (Yang et al., J. Mol. Biol., 254, 392-403, 1995), strand tampering (Marks et al., Bio / Technology, 10, 779-783, 1992), and the use of *E. coli* (…). E. coliThe methods used include proliferating strains (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996), and sexual PCR (Crameri et al., Nature, 391, 288-291, 1998). Vaughan et al. (ibid.) discuss these affinity maturation methods. The binding domains used in this invention can be generated by any suitable method known in the art; for example, the CDR can be derived from nonhuman antibodies, including commercially available antibodies, and grafted into a human framework, or alternatively, chimeric antibodies can be prepared using nonhuman variable regions and human constant regions, etc.

[0258] Examples of CD45 antibodies are known in the art, and complementary sites from such antibodies can be used in the antibodies of the present invention, having more than one specificity for CD45, or screened for suitability using the methods described herein, and subsequently modified, for example, humanized, if desired, using the methods described herein. Therapeutic anti-CD45 antibodies have been described in the art, such as the anti-CD45 antibody disclosed in US2011 / 0076270.

[0259] Those skilled in the art can generate antibodies for use in the antibodies of this invention using any suitable method known in the art. Antigenic peptides for use in antibody generation, such as for immunizing a host or for use in panning (e.g., in phage display), can be prepared from genetically modified host cells containing an expression system using processes well known in the art, or they can be recovered from natural biological sources. In this application, the term "peptide" includes peptide, polypeptide, and protein. Unless otherwise specified, these are used interchangeably. In some cases, the antigenic peptide may be part of a larger protein (e.g., a fusion protein, such as fused to an affinity tag or the like). In one embodiment, the host can be immunized using cells transfected with CD45 (e.g., cells expressing CD45 on their surface).

[0260] Antibodies against the antigenic peptide can be obtained, where animal immunization is necessary, by administering the peptide to animals, preferably non-human animals, using well-known and conventional procedures, see, for example, Handbook of Experimental Immunology, DM Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986. Many warm-blooded animals, such as rabbits, mice, rats, sheep, cattle, camels, or pigs, can be immunized. However, mice, rabbits, pigs, and rats are generally the most suitable. Monoclonal antibodies can be prepared by any method known in the art, such as hybridoma technology (Kohler & Milstein, 1975, Nature, 256:495-497), trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immunology Today, 4:72), and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

[0261] Antibodies can also be generated using a single-lymphocyte antibody method by cloning and expressing immunoglobulin variable region cDNA generated from a single lymphocyte selected for producing a specific antibody, via methods described, for example, by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15): 7843-7848l; WO 92 / 02551; WO 2004 / 051268; and WO 2004 / 106377. Antibodies used in this invention can also be generated using various phage display methods known in the art. In a preferred embodiment, the antibodies of the present invention have at least two distinct complementary sites specific to CD45, and it is possible that an antibody recognizing one complementary site of CD45 is first generated, and then, for example, two of those antibodies are used to generate antibodies of the present invention capable of specifically binding to at least two distinct complementary sites of CD45. For example, it is possible to generate multiple antibodies against CD45 using the methods discussed herein, and then screen them for desired properties (e.g., binding affinity). The best candidates can then be used to generate the antibodies of the present invention.

[0262] In one instance, the antigen-binding site (particularly the variable region) of the antibody according to the invention is humanized. As used herein, “humanized” (including CDR-grafted antibodies) refers to a molecule having one or more complementarity-determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule. It will be appreciated that only the specific determination residues of the CDR need to be transferred, rather than the entire CDR (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). However, in a preferred embodiment, the entire CDR is transplanted. The humanized antibody may optionally further comprise one or more framework residues derived from the non-human species from which the CDR originates. As used herein, the term “humanized antibody molecule” refers to an antibody molecule in which the heavy chain and / or light chain comprises one or more CDRs (and, if desired, one or more modified CDRs) from a donor antibody (e.g., a murine monoclonal antibody) grafted into the heavy chain and / or light chain variable region framework of a recipient antibody (e.g., a human antibody). For the review, see Vaughan et al., Nature Biotechnology, 16, 535-539, 1998. In one embodiment, instead of transferring the entire CDR, only one or more specificity-determining residues from any of the CDRs described above herein are transferred to the human antibody framework (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). In another embodiment, only one or more specificity-determining residues from the CDRs described above herein are transferred to the human antibody framework. In yet another embodiment, only specificity-determining residues from each of the CDRs described above herein are transferred to the human antibody framework.

[0263] When grafting CDRs or specificity-determining residues, any suitable acceptor variable region framework sequence, including mouse, primate, and human framework regions, can be used, taking into account the class / type of the donor antibody from which the CDR is derived. Suitably, humanized antibodies according to the invention have a variable region comprising a human acceptor framework region and one or more of the CDRs provided herein. Examples of human frameworks that can be used in the invention are KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al., ibid.). For example, KOL and NEWM can be used for the heavy chain, REI for the light chain, and EU, LAY, and POM for both the heavy and light chains. Alternatively, human germline sequences can be used; these are available at http: / / www2.mrc-lmb.cam.ac.uk / vbase / list2.php.

[0264] In the humanized antibody molecules of this invention, the recipient heavy and light chains do not necessarily need to originate from the same antibody, and may, if desired, comprise a complex chain having a framework region derived from a different chain. The framework region does not need to have the exact same sequence as those of the recipient antibody. For example, uncommon residues may be replaced with residues more frequently occurring for that particular recipient chain class or type. Alternatively, selected residues in the recipient framework region may be modified such that they correspond to residues found at the same positions in the donor antibody (see, Reichmann et al., 1998, Nature, 332, 323-324). Such modifications should be kept to the minimum necessary to restore affinity to the donor antibody. Procedures for selecting residues in the recipient framework region that may need to be modified are described in WO91 / 09967. Derivatives of the framework may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids replaced by alternative amino acids (e.g., donor residues). Donor residues are residues derived from the donor antibody (i.e., the antibody from which the CDR is originally derived), specifically residues from the donor sequence at the appropriate positions. Donor residues may be replaced by suitable residues derived from the human recipient framework (recipient residues).

[0265] In one embodiment, the invention extends to the antibody sequences disclosed herein, and in particular the humanized sequences disclosed herein.

[0266] In one instance, the binding domain is humanized.

[0267] In one instance, one or more CDRs provided herein may be modified to remove unwanted residues or sites, such as cysteine ​​residues or aspartic acid (D) isomerization sites or asparagine (N) deamidation sites. In one instance, asparagine deamidation sites may be removed from one or more CDRs by mutating an asparagine residue (N) and / or adjacent residues to any other suitable amino acid.

[0268] Technicians can test variants or humanized sequences of CDRs in any suitable assay (e.g., those described herein) to confirm that activity is maintained.

[0269] Any suitable assay can be used to test for specific binding to the antigen, including, for example, ELISA or surface plasmon resonance methods such as BIAcore, where binding to the antigen (CD45) can be measured. Such assays can use isolated natural or recombinant CD45 or suitable fusion proteins / peptides. In one example, recombinant CD45 (SEQ ID NO: 127 or amino acids 23-1304 of SEQ ID NO: 127) is used, and binding is measured by, for example, surface plasmon resonance such as BIAcore. Alternatively, the protein can be expressed in cells such as HEK cells, and affinity can be measured using a flow cytometry-based affinity assay. In one embodiment, where it is necessary to individually determine the characteristics of an antigen-binding site (particularly the complementary site), an antibody is generated using only that complementary site. For example, antibodies of the same pattern as the antibodies of the present invention are generated with two different specificities, but only one specific for CD45 is present. In one embodiment, an antibody against each of the complementary sites of CD45 can be generated from the antibody of the present invention having at least two complementary sites, for example to allow determination of the affinity for each complementary site or to determine whether the complementary sites exhibit cross-blocking against each other. In one embodiment, the ability to specifically bind to the extracellular region of CD45 is measured, for example using the protein of SEQ ID NO: 113. In one embodiment, a monovalent antibody, such as ScFv, is generated for comparison.

[0270] The degree of identity and similarity can be readily calculated (Computational Molecular Biology, Lesk, AM, eds., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, eds., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; BLAST data available from NCBI). TM Software (Altschul, SF et al., 1990, J. Mol. Biol. 215:403-410; Gish, W. & States, DJ 1993, Nature Genet. 3:266-272; Madden, TL et al., 1996, Meth. Enzymol. 266:131-141; Altschul, SF et al., 1997, Nucleic Acids Res. 25:3389-3402; Zhang, J. & Madden, TL 1997, Genome Res. 7:649-656).

[0271] This invention also extends to the novel polypeptide sequences disclosed herein and sequences that are at least 80% similar to or identical with them (e.g., 85% or greater, such as 90% or greater, particularly 95%, 96%, 97%, 98%, or 99% or greater similarity or identity). In one embodiment, the sequence may have at least 99% sequence identity with at least one of the specific sequences provided herein. As used herein, “identity” means that at any particular position in the compared sequences, the amino acid residues between the sequences are the same. As used herein, “similarity” means that at any particular position in the compared sequences, the amino acid residues between the sequences belong to a similar type. For example, leucine may be substituted with isoleucine or valine. Other amino acids that can often be substituted for each other include, but are not limited to:

[0272] - Phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains);

[0273] - Lysine, arginine, and histidine (amino acids with basic side chains);

[0274] - Aspartic acid and glutamic acid (amino acids with acidic side chains);

[0275] - Asparagine and glutamine (amino acids with amide side chains); and

[0276] - Cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0277] It will be appreciated that this aspect of the invention also extends to variants of these anti-CD45 antibodies, including those in which the amino acids in the CDR have been mutated to remove one or more of the isomerization, deamidation, glycosylation sites or cysteine ​​residues described above herein.

[0278] Preferred antibodies with FALA, LALA, and mortar modifications

[0279] Please note that the positions of amino acid residues given in this section are indicated using EU numbers rather than Kabat numbers.

[0280] In a particularly preferred embodiment of the invention, the antibody used comprises a heavy chain modified with FALA or LALA. Specifically, FALA and LALA modifications alter Fc receptor binding.

[0281] In a further preferred embodiment, the antibody includes modifications in the hinge region of the antibody, particularly at position 228, preferably 228P. In one embodiment, the antibody has a heavy chain containing modifications at positions 228, 234, and 235. In a particularly preferred embodiment, the heavy chain of the antibody of the present invention will contain S228P, F234A, and L235A FALA modifications. In a particularly preferred embodiment of the present invention, the provided antibody will be an IgG4(P) isotype antibody and will contain such modifications.

[0282] In another particularly preferred embodiment, the antibody of the present invention will contain a so-called "mortar and pestle" modification. In one embodiment, one heavy chain of the antibody contains a modification at T366, and another heavy chain contains modifications at T366, L368, and Y407, specifically creating complementary shapes in the two different heavy chains, meaning they preferentially pair rather than two identical heavy chains pairing. Specifically, the heavy chain of one specificity may have a T366W "mortar and pestle" modification, while the heavy chain of another specificity may have a T366S, L368A, and Y407V "mortar and pestle" modification. In a particularly preferred embodiment, the antibody of the present invention is an IgG4 isotype antibody and has such modifications.

[0283] In another particularly preferred embodiment of the invention, the FALA, hinge, and "mortar" modifications are combined. In a preferred embodiment, they are combined in the context of an IgG4 isotype antibody. In one embodiment, one heavy chain of the antibody has modifications at positions 228, 234, 235, and 355. In another embodiment, one heavy chain includes modifications at positions 228, 234, 235, 366, 368, and 407. For example, in one embodiment, one heavy chain has modifications of S228P, F234A, L235A, and T366W (therefore, both FALA and "mortar" modifications are present), and preferably another heavy chain has modifications of S228P, F234A, L235A, T366S, L368A, and Y407V (therefore, both FALA and "mortar" modifications are present).

[0284] In a particularly preferred embodiment, the antibody of the present invention is a FALA IgG4(P) antibody. In a further particularly preferred embodiment, it is a FALA club-and-mouth IgG4(P) antibody. In another embodiment, it is a LALA club-and-mouth IgG4(P) antibody.

[0285] In another embodiment, the above-described patterns can be combined with other patterns / modifications discussed herein. For example, they may also include modifications discussed herein to remove protein A binding at positions 95 and 96. In a further embodiment, they may include a common light chain and may also include protein A binding modifications.

[0286] Further preferred antibody patterns include BYbe and TrYbe.

[0287] In one aspect, an antibody molecule is provided that comprises or is composed of the following polypeptide chains:

[0288] a) The polypeptide chain of formula (VII):

[0289] V H -CH1-W-(V1) p ;

[0290] b) Polypeptide chain of formula (VIII):

[0291] V L -C L -Z-(V2) q ;

[0292] in:

[0293] V H Represents a heavy-chain variable structural domain;

[0294] CH1 represents the structural domain of the heavy chain constant region, such as its structural domain 1;

[0295] W represents a bond or linker, such as an amino acid linker, unless p or q is zero, in which case they will also be zero;

[0296] Z represents a bond or linker, such as an amino acid linker;

[0297] V1 represents dab, scFv, dsscFv, or dsFv;

[0298] V L This represents a variable structural domain, such as a variable structural domain in a light chain.

[0299] C L This indicates a domain originating from a constant region, such as a light chain constant region domain, for example, Cκ;

[0300] V2 represents dab, scFv, dsscFv, or dsFv;

[0301] p is 0 or 1;

[0302] q is 0 or 1; and

[0303] When p is 1, q is 0 or 1, and when q is 1, p is 0 or 1, meaning p and q do not both represent 0.

[0304] The antibody has at least two antigen-binding sites that are different complementary sites against CD45, each recognizing a different epitope against CD45, wherein at least one of VH and VL is a pair of 17415-derived variable regions or a pair of 17552-derived variable regions.

[0305] In one instance, the CD45-specific binding domain is selected from at least two of V1, V2, or VH / VL.

[0306] In one implementation, q is 0 and p is 1.

[0307] In one implementation, q is 1 and p is 1.

[0308] In one implementation, V1 is dab and V2 is dab, and together they form a single binding domain of a cooperative pair of variable regions (e.g., associated VH / VL pairs), which is optionally connected by disulfide bonds.

[0309] In one implementation scheme, V H and V L It is specific to CD45.

[0310] In one implementation, V1 is specific to CD45.

[0311] In one implementation, V2 is specific to CD45.

[0312] In one implementation, V1 and V2 together (e.g., as binding domains) are specific to CD45, and V H and V L It is specific to CD45.

[0313] In one implementation, V1 is specific to CD45.

[0314] In one implementation, V2 is specific to CD45.

[0315] In one implementation, V1 and V2 together (e.g., as a binding domain) are specific to CD45, and V H and V L It is specific to CD45.

[0316] In one implementation, V1 is specific to CD45, V2 is specific to CD45, and V...H and V L It is specific to CD45.

[0317] V1, V2, V in the above constructs H and V L Each can represent a combined structural field and can be included in any of the sequences provided in this paper.

[0318] W and Z can represent any suitable connector, for example, W and Z can be independently SGGGGSGGGGS (SEQ ID NO: 67) or SGGGGTGGGGS (SEQ ID NO: 114).

[0319] In one implementation, when V1 and / or V2 are dab, dsFv, or dsscFv, the variable structural domain V of V1 and / or V2... H and V L The disulfide bond between them is at position V H 44 and V L Formed between 100.

[0320] In a preferred embodiment of the invention, the antibody of the invention is in the BYbe antibody pattern. BYbe-style antibodies comprise a Fab linked to only one scFv or dsscFv, as in, for example, WO 2013 / 068571 and Dave et al., (2016). Mabs As described in , 8(7): 1319-1335. Therefore, for example, in a preferred embodiment, in the formula given above, one of (V1)p and (V2)q will be ScFv or dsscFv, and the other will be empty, thus making the BYbe-style antibody contain Fab and only one scFv or dsscFv. For either (V1)p or (V2)q being zero, the corresponding W or Z will also be empty, and the other will be a bond or linker. Preferably, the BYbe-style antibody contains Fab and dsscFv. In such BYbe-style antibodies, the two antigen-binding sites can preferably both be CD45-specific, wherein the two antigen-binding sites correspond to complementary sites of the two different epitopes of CD45.

[0321] In a further, particularly preferred embodiment of the invention, the antibody is in the TrYbe pattern. The TrYbe pattern comprises a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding to the same or different targets (e.g., one scFv or dsscFv binds to a therapeutic target, and another scFv or dsscFv increases half-life by binding to, for example, albumin). Such antibody fragments are described in WO 2015 / 197772. In the formulas given above, for a TrYbe antibody, p and q will both be one, where V1 and V2 are independently selected from scFvs and dsscFvs. In a preferred embodiment, V1 and V2 will both be scFvs. In another embodiment, V1 and V2 will both be dsscFvs. In yet another embodiment, one of V1 and V2 will be a scFv, and the other will be a dsscFv. At least two of the antigen-binding sites of TrYbe will be CD45-specific, wherein the antibody contains two distinct complementary sites, each specific to a different epitope of CD45. In a particularly preferred embodiment, a third antigen-binding site will be albumin-specific, specifically one of V1 and V2. For example, VH / VL may be CD45-specific (e.g., for a first epitope of CD45), one of V1 and V2 may be CD45-specific (e.g., for a second epitope of CD45), and the other of V1 and V2 may be albumin-specific.

[0322] In a preferred embodiment, the antibody of the present invention will contain at least one complementary site specific to albumin. In one embodiment, the antibody will be a TrYbe-style antibody containing two complementary sites specific to different epitopes of CD45 and a third complementary site specific to albumin. Examples of albumin-binding antibody sequences that can be used to specifically bind albumin include those disclosed in WO 2017 / 191062, the entire document of which is incorporated herein by reference, particularly relating to albumin-binding sequences. Therefore, the antibody of the present invention may contain a complementary site from one of the albumin-specific antibodies in WO 2017 / 191062.

[0323] In an alternative embodiment, the antibody discussed above has only one specificity for CD45, rather than at least two different specificities. For example, one of the antigen-binding sites of the antibody may be specific for CD45. In another embodiment, two of the antigen-binding sites are specific for CD45 but have the same specificity. In a further embodiment, all three antigen-binding sites of the antibody listed above have the same specificity for CD45. In another embodiment, two of the antigen-binding sites have the same specificity for CD45, and the third is specific for serum albumin.

[0324] In a particularly preferred embodiment, at least one of the variable regions in the molecule will be a 17415 or 17552 variable region. In one embodiment, at least one antigen-binding domain comprising a 17415-derived light and heavy chain variable region will be present. In one embodiment, at least one antigen-binding domain comprising a 17552-derived light and heavy chain variable region will be present. In one embodiment, both a 17415-derived antigen-binding site and a 17552-derived antigen-binding site will be present.

[0325] Disulfide bridge

[0326] In the case where one or more pairs of variable regions in the antibody of the present invention contain a disulfide bond between VH and VL, this can be at any suitable location, for example, between two of the residues listed below (Kabat numbers are used in the list below unless the context otherwise indicates). Wherever Kabat numbers are mentioned, the relevant reference is Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA.

[0327] In one implementation, when V1 and / or V2 are dsFv or dsscFv in the formulas discussed above, the disulfide bond between the variable domains VH and VL of VI and / or V2 is between two of the residues listed below (Kabat numbering is used in the list below unless the context otherwise requires). Wherever Kabat numbering is mentioned, the relevant reference is Kabat et al., 1987, Sequences of Proteins of Immunological Interest, USA Department of Health and Human Services, NIH, USA.

[0328] In one embodiment, the disulfide bond is located at a position selected from the group consisting of:

[0329] V H 37 + V L 95C, see, for example, Protein Science 6, 781-788, Zhu et al. (1997);

[0330] V H 44 + V L 100, see, for example, Biochemistry 33 5451-5459, Reiter et al. (1994); or Journal of Biological Chemistry Vol. 269 No. 28 pp.18327-18331, Reiter et al. (1994); or Protein Engineering, vol.10 no.12 pp.1453-1459, Rajagopal et al. (1997);

[0331] V H 44 + V L 105, see, for example, J Biochem. 118, 825-831, Luo et al. (1995);

[0332] V H 45+V L 87, see, for example, Protein Science 6, 781-788, Zhu et al. (1997);

[0333] V H55 + V L 101, see, for example, FEBS Letters 377 135-139, Young et al. (1995);

[0334] V H 100+V L 50, see, for example, Biochemistry 29 1362-1367, Glockshuber et al. (1990);

[0335] V H 100b + V L 49;

[0336] V H 98 + V L 46. ​​See, for example, Protein Science 6, 781-788, Zhu et al. (1997);

[0337] V H 101 + V L 46;

[0338] V H 105+V L 43, see, for example, Proc. Natl. Acad. Sci. USA Vol. 90 pp.7538-7542, Brinkmann et al. (1993); or Proteins 19, 35-47, Jung et al. (1994); and

[0339] V H 106 + V L 57. See, for example, FEBS Letters 377 135-139, Young et al. (1995).

[0340] and their corresponding positions in the variable regions located in the molecule.

[0341] In one embodiment, the disulfide bond is at position V H 44 and V L Formed between 100.

[0342] The amino acid pairs listed above are positioned at sites favorable for cysteine ​​substitution, thereby enabling the formation of disulfide bonds. Cysteine ​​can be modified to these desired positions using known techniques. In one embodiment, a modified cysteine ​​according to this disclosure refers to a case where a naturally occurring residue at a given amino acid position has been replaced with a cysteine ​​residue.

[0343] The introduction of modified cysteine ​​can be performed using any method known in the art. These methods include, but are not limited to, PCR extension overlap mutagenesis, site-directed mutagenesis, or cassette mutagenesis. Cassette mutagenesis can be performed based on Wells et al., 1985, Gene, 34:315-323. Alternatively, mutants can be prepared by whole-genome synthesis, annealing, ligation, and PCR amplification, as well as cloning of overlapping oligonucleotides.

[0344] WO 2015 / 197772 elaborates in detail on the preferred positions of disulfide bridges for BYbe and TrYbe style antibodies.

[0345] As discussed herein, the ability to alter residues in the hinge region of an antibody is a potential way to influence binding to CD45 and can be used in this invention.

[0346] tethered antibody

[0347] In one embodiment, the antibody of the present invention may comprise two parts linked together by a heterodimeric tether. For example, the antibody of the present invention may comprise two parts, each comprising a different antibody fragment having different complementary sites to CD45, and a tethering region that allows it to form a total antibody molecule with the other half of the antibody. In one embodiment, the antibody of the present invention is a Fab-X / Fab-Y antibody pattern (also known as a Fab-Kd-Fab pattern), as described, for example, in WO 2017 / 093402, see, for example. Figure 3 The Fab-X / Fab-Y antibody patterns are particularly useful for screening because they allow for rapid screening of different arrangements of complementary sites for CD45.

[0348] Therefore, in one embodiment, the antibody molecule according to the invention is an antibody comprising at least two different complementary sites specific to different epitopes of CD45, having the formula AX:YB, wherein:

[0349] AX is the first fusion protein;

[0350] YB is the second fusion protein;

[0351] X:Y represents heterodimer chains;

[0352] : This refers to the binding interaction between X and Y;

[0353] A is the first protein component of the antibody, which is selected from Fab or Fab' fragment;

[0354] B is the second protein component of the antibody, which is selected from Fab or Fab'.

[0355] X is the first binding partner of the binding pair, which is independently selected from the antigen or antibody or their binding fragments; and

[0356] Y is the second binding partner of the binding pair, which is independently selected from the antigen or antibody or their binding fragment;

[0357] The conditions are that when X is an antigen, Y is an antibody or its binding fragment that is specific to the antigen represented by X, and when Y is an antigen, X is an antibody or its binding fragment that is specific to the antigen represented by Y.

[0358] Illustrative examples of albumin antibodies and sequences

[0359] The antibodies used in this invention that have antigen-binding sites specific to albumin may have the following CDR sequence:

[0360] SEQ ID NO: 130 - CDRH1 GIDLSNYAIN

[0361] SEQ ID NO: 131 - CDRH2 IIWASGTTTFYATWAKG

[0362] SEQ ID NO: 132 - CDRH3 TVPGYSTAPYFDL

[0363] SEQ ID NO: 133 - CDRL1 QSSPSVWSNFLS

[0364] SEQ ID NO: 134 - CDRL2 EASKLTS

[0365] SEQ ID NO: 135 - CDRL3 GGGYSSISDTT.

[0366] Examples of albumin-binding specificity that may be adapted for use include those in WO 05 / 117984 and WO 2017 / 191062, which are combined by reference in their entirety and in relation to albumin-binding antibody specificity.

[0367] Effect molecules

[0368] The antibodies of the present invention can be conjugated to effector molecules. Thus, if desired, particularly antibodies used in the present invention, can be conjugated to one or more effector molecules. It will be appreciated that the effector molecule may comprise a single effector molecule, or two or more such molecules linked together to form a single portion that can be attached to the antibody. In cases where it is desired to obtain an antibody according to the invention conjugated to an effector molecule, this can be prepared by standard chemical or recombinant DNA procedures, wherein the antibody is conjugated to the effector molecule directly or via a conjugating agent. Techniques for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58; and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemical manipulation procedures include, for example, those described in WO 93 / 06231, WO 92 / 22583, WO 89 / 00195, WO 89 / 01476, and WO 03 / 031581. Alternatively, when the effector molecule is a protein or polypeptide, the ligation can be achieved using recombinant DNA manipulation procedures, such as those described in WO 86 / 01533 and EP0392745. In one embodiment, the antibody of the present invention may comprise an effector molecule. The term "effector molecule" as used herein includes, for example, antitumor agents, drugs, toxins, biologically active proteins such as enzymes, antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof such as DNA, RNA and fragments thereof, radionuclides, particularly radioiodides, radioisotopes, chelated metals, nanoparticles, and reporter groups, such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy.

[0369] Examples of effector molecules can include cytotoxic agents, including any agent that is harmful to cells (e.g., kills cells). Examples include compressoritine, dolalastatin, epomomycin, asteroidin, maytansine compounds, spongiformin, lisodium, levofloxacin, bacitracin, hammetrine, paclitaxel, pinocembrin B, bacitracin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthraxetine dione, mitoxantrone, styromycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as their analogues or homologues. Effector molecules also include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, aminoimide), alkylating agents (e.g., nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and cis-dichlorodiamine platinum(II) (DDP) (cisplatin)), anthracyclines (e.g., daunorubicin (formerly doxorubicin) and doxorubicin), antibiotics (e.g., dermatomycin (formerly actinomycin), bleomycin, photomycin, aztracin (AMC), galactamycin or pyromycin), and antimitotic agents (e.g., vinblastine and vinblastine).

[0370] Other effector molecules may include chelated radionuclides, such as 111 In and 90 Y, Lu 177 ,bismuth 213 ,californium 252 ,iridium 192 and tungsten 188 / rhenium 188Or drugs, such as, but not limited to, alkylphosphocholine, topoisomerase I inhibitors, taxanes, and suramin. Other effector molecules include proteins, peptides, and enzymes. Target enzymes include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, and transferases. Target proteins, polypeptides, and peptides include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin, proteins such as insulin, tumor necrosis factor (TNF), α-interferon, β-interferon, nerve growth factor, platelet-derived growth factor, or tissue plasminogen activator, thrombotic agents or anti-angiogenic agents such as styramine or endostatin, or biological response regulators such as lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), nerve growth factor (NGF), or other growth factors and immunoglobulins.

[0371] Other effector molecules may include, for example, detectable substances useful in diagnostics. Examples of detectable substances include various enzymes, cofactors, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron-emitting metals (used in positron emission tomography) and non-radioactive paramagnetic metal ions. For information on metal ions that can be conjugated to antibodies used as diagnostic agents, see U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; suitable cofactors include streptomycin, avidin, and biotin; suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazineamine luciferin, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and jellyfish luminescent protein; and suitable radionuclides include… 125 I, 131 I, 111 In and 99 Tc.

[0372] In another embodiment, the effector molecule may increase or decrease the in vivo half-life of the antibody, and / or decrease immunogenicity, and / or enhance delivery across the epithelial barrier to the immune system. Suitable examples of this type of effector molecule include polymers, albumins, albumin-binding proteins, or albumin-binding compounds, such as those described in WO 05 / 117984. In the case of a polymer, it can typically be a synthetic or naturally occurring polymer, such as optionally substituted linear or branched polyalkylene, polyolefin, or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homopolysaccharides or heteropolysaccharides. Specific optional substituents that may be present on the synthetic polymers mentioned above include one or more hydroxyl, methyl, or methoxy groups. Specific examples of synthetic polymers include optionally substituted linear or branched polyethylene glycol, polypropylene glycol, polyvinyl alcohol, or derivatives thereof, particularly optionally substituted polyethylene glycols such as methoxy polyethylene glycol or derivatives thereof.

[0373] The antibodies of the present invention can be conjugated to molecules that modulate or alter serum half-life. The antibodies of the present invention can specifically bind to albumin, for example, to modulate serum half-life. In one embodiment, the antibody of the present invention will further include a complementary site specific to albumin. In another embodiment, the antibody of the present invention may include a peptide linker, which is an albumin-binding peptide. Examples of albumin-binding peptides are included in WO2015 / 197772 and WO2007 / 106120, the entirety of which are incorporated herein by reference.

[0374] In another embodiment, the antibody of the present invention is not conjugated to an effector molecule. In one embodiment, the antibody of the present invention is not an antibody-drug conjugate. In one embodiment, the antibody of the present invention is not conjugated to a toxin, for example, via a linker. In another embodiment, the antibody of the present invention is not conjugated to a radioisotope. In another embodiment, the antibody of the present invention is not conjugated to a reagent used for imaging.

[0375] In a preferred embodiment, cell death is triggered by the ability of the antibody of the present invention to specifically bind to CD45, rather than by the ability of the conjugated effector molecule.

[0376] Cell death and killing

[0377] In a particularly preferred embodiment, the antibody of the present invention is capable of inducing cell death in target cells expressing CD45. Types of cell death that can be induced to kill target cells include intrinsic apoptosis, extrinsic apoptosis, necroptosis driven by mitochondrial permeability transition (MPT), necroptosis, ferroptosis, pyroptosis, parthanatos death, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic breakdown. In one embodiment, the antibody of the present invention is used to kill target cells.

[0378] In one embodiment, the target cells will be cells expressing CD45, particularly on the surface of said cells. In a preferred embodiment, the antibody of the present invention can induce cell death at least in T cells. In another preferred embodiment, the antibody of the present invention can induce cell death at least in B cells. In another preferred embodiment, the antibody of the present invention may be able to induce cell death in both B and T cells. In a preferred embodiment, the antibody of the present invention is able to induce cell death in hematopoietic stem cells. In one embodiment, the antibody of the present invention does not induce cell death in all immune cells, for example, it does not induce cell death in granulocytes, macrophages, and monocytes. In one embodiment, the antibody of the present invention induces cell death in all immune cells except granulocytes, macrophages, and monocytes. In one embodiment, the effect of inducing cell death in hematopoietic stem cells is practically to replace all hematopoietic cells. In one embodiment, the antibody of the present invention is used to kill the aforementioned target cells by inducing cell death.

[0379] In one embodiment, the antibody of the present invention may have different selectivity for cells expressing different CD45. In a preferred embodiment, the antibody of the present invention may be able to induce T cell death more effectively than inducing B cell cell death.

[0380] In another particularly preferred embodiment, the antibody of the present invention induces cell death but does not trigger significant cytokine release. In yet another preferred embodiment, the antibody of the present invention induces cell death but does not exhibit Fc effector function, for example, because the antibody lacks an Fc region or has an Fc region with silencing modifications.

[0381] Cytokines

[0382] In a particularly preferred embodiment, the antibody of the present invention does not induce significant cytokine release. In a particularly preferred embodiment, the antibody of the present invention is capable of inducing cell death in target cells without inducing significant cytokine release. Reduced or absent cytokine release may mean that the subject is not suffering from unwanted cytokine-driven inflammation. For example, the treatment of the present invention can kill target cells in the subject without triggering inflammation, particularly without the so-called "cytokine storm" associated with some treatments.

[0383] In one embodiment, the antibody of the present invention does not significantly induce the release of one or more of interferon-γ, IL-6, TNF-α, IL-1β, MCP1, and IL-8. In a preferred embodiment, the antibody of the present invention does not induce a significant release of any of those cytokines. In another embodiment, the antibody does not significantly induce the release of one or more of CCL2, IL-1RA, IL-6, and IL-8. In another preferred embodiment, it does not significantly induce the release of any of those cytokines. In one embodiment, such levels would be for one or more of interferon-γ, IL-6, TNF-α, IL-1β, MCP1, and IL-8; in another embodiment, such levels would be for one or more of CCL2, IL-1RA, IL-6, and IL-8; in yet another embodiment, such levels would be seen for at least one of CCL2, IL-1RA, IL-6, IL-8, IL-10, and IL-11. In yet another embodiment, such levels would be for at least one of CCL2, IL-1RA, IL-6, and IL-8.

[0384] Cytokine release can be measured using any suitable assay. For example, the ability of the antibody of the present invention to induce cytokine release can be determined by culturing cells with the antibody in vitro and measuring the cytokine release. In one embodiment, whole blood is incubated with the antibody, and then the cytokine level, such as any of the cytokines mentioned above, is measured. In another embodiment, leukocytes isolated from a whole blood sample can be incubated with the antibody of the present invention, and the cytokine level can be measured. Alternatively, it is possible to measure the cytokine level in a sample from a subject who has been administered the antibody of the present invention, particularly in a serum sample from the subject.

[0385] In one embodiment, "not significantly inducing" cytokine release means that the antibody of the present invention does not induce cytokine release to a degree exceeding five, four, three, or two times that seen with a negative control, for example, compared to a negative control treated in vitro with PBS alone. In some embodiments, the level of cytokine release will be compared to a positive control (e.g., in vitro treatment with Campath). In one embodiment, the antibody of the present invention will trigger no more than 50%, 40%, 30%, 20%, 10%, or less compared to that seen with Campath treatment. In one embodiment, the level of cytokine release seen with the antibody of the present invention will be less than one-tenth of that seen with Campath. In one embodiment, the level of cytokine release after incubation with Campath will be at least two, three, four, five, ten, or more times that seen after incubation with the antibody of the present invention. In one embodiment, the levels seen with Campath after 24 hours of incubation in whole blood will be those levels compared to the antibody of the present invention.

[0386] In another embodiment, the comparative antibody used to define the non-significantly induced antibody would be another antibody. For example, if the antibody of the present invention contains a modification designed to reduce cytokine release, the comparative antibody would be an equivalent antibody without such modification. In another embodiment, if the antibody has an Fc region modification intended to reduce cytokine release or has no Fc region, an equivalent antibody lacking such modification or having an Fc region would be used for comparison.

[0387] In another embodiment, comparisons regarding the non-significant release of cytokines will be performed in vivo. For example, when the antibody of the present invention is administered to a subject, it will show any of the levels of cytokine release discussed above, compared to the comparatives discussed above. In another embodiment, non-significant induction of cytokine release may be in terms of the levels of one or more cytokines compared to the levels before administration of the antibody of the present invention. For example, it is possible that the cytokine levels increase by no more than tenfold, fivefold, or less after administration of the antibody of the present invention. The measurement may be performed, for example, immediately before or simultaneously with the administration of the antibody, and, for example, one day, one week, or two weeks or more after administration. In one embodiment, the measurement is performed one day to one week after administration. In another embodiment, the antibody of the present invention does not significantly induce cytokine release because the treated subject does not experience adverse effects associated with unwanted cytokine release, such as fever, hypotension, or irregular or rapid heartbeats.

[0388] Functional testing

[0389] In one embodiment, functional assays can be used to determine whether the antibody of the present invention possesses a specific property, such as any of those mentioned herein. Therefore, functional assays can be used to evaluate the antibodies of the present invention. As used herein, "functional assay" refers to an assay that can be used to determine one or more desired properties or activities of the antibody or molecule of the present invention.

[0390] In a particularly preferred embodiment, the functional assay measures the ability to bind to CD45. In a preferred embodiment, the ability to bind to human CD45 can be measured. In another embodiment, the ability to bind to cynomolgus monkey CD45 can be measured. In a preferred embodiment, the ability to bind to both human CD45 and cynomolgus monkey CD45 can be measured. Such binding can be measured, for example, on the CD45 protein. Alternatively, it can be measured with respect to CD45 expressed on the surface of target cells expressing CD45. Preferred measurement techniques include those described in the embodiments of this application, such as SPR or flow cytometry. Further preferred methods that can be used include those listed herein with respect to cross-blocking, but without cross-blocking antibodies. Such assays can be with respect to the binding of a monospecific monovalent antibody. Alternatively, they can be with respect to any of the total antibody patterns and specific antibodies listed herein.

[0391] Further suitable functional assays may include binding assays, cell death (e.g., apoptosis) assays, antibody-dependent cytotoxicity (ADCC) assays, complement-dependent cytotoxicity (CDC) assays, cell growth or proliferation inhibition (inhibition of cell effects) assays, cell killing (cytotoxic effects) assays, cell signaling assays, cytokine production assays, antibody production and allotype conversion, and cell differentiation assays. In one embodiment, the assay may measure the degree of cell exhaustion, for example, for a specific cell type, using the antibody of the present invention. In a preferred embodiment, the assay may measure the ability of the antibody of the present invention to induce cell death in target cells expressing CD45. In a further preferred embodiment, the functional assay may measure the ability of the antibody of the present invention to induce cytokine release. In a preferred embodiment, the functional assay may be used to determine whether the antibody of the present invention can be used to kill cells without significantly inducing cytokines.

[0392] Preferred functional assays include those listed in the embodiments. For example, the PBMC cell killing assay described in the embodiments can be used. The Jurkat cell killing assay listed in the embodiments of this application can be used. In one preferred embodiment, the target cells are human cells expressing CD45. In another embodiment, the target cells are cynomolgus monkey cells expressing CD45.

[0393] The functional assay can be repeated multiple times as needed to enhance the reliability of the results. Various statistical tests known to those skilled in the art can be used to identify statistically significant results and thus identify antibodies with biological functions. In one embodiment, multiple antibodies are tested in parallel or substantially simultaneously. As used herein, "simultaneously" means in the case of analysis of a sample / molecule / complex in the same analysis, e.g., in the same "run". In one embodiment, "simultaneously" refers to a concomitant analysis in which the signal output is analyzed by the instrument at substantially the same time. This signal may need to be deconvolved to interpret the obtained results. Advantageously, testing multiple dual-complementary-site protein complexes allows for more efficient screening of a large number of antibodies and identification of novel and interesting relationships. Clearly, different variable regions of the target antigen CD45 can reveal subtle nuances in biological function.

[0394] In one embodiment, where the antibody of the present invention contains more than one specificity for CD45, a functional assay can be used to compare the properties of that antibody with, for example, an antibody having the same valence but only one of the specificities of the antibody of the present invention. In one embodiment, such an assay can be used to show that the antibody of the present invention having at least two different specificities for CD45 is superior to a comparative antibody. Therefore, in a preferred embodiment, the efficacy of the antibody of the present invention (particularly such an antibody according to the present invention) containing at least two different specificities for CD45 can be compared with a single “comparative” antibody (particularly a “comparative” antibody containing only one specificity for CD45 from the antibody of the present invention). For example, when performing the assay to study the cross-linking or cross-linking effect of CD45, an antibody having the same valence but only one specificity can be used as a comparative. In one embodiment, the antibody of the present invention can be compared with an antibody containing one of the same complementary sites from the antibody of the present invention at all antigen-binding sites. In one embodiment, the antibody of the present invention can be compared with an antibody having the same valence and pattern as the antibody of the present invention (but wherein one of the same complementary sites from the antibody of the present invention is present at all antigen-binding sites). In one embodiment, a bivalent antibody containing two different complementary sites specific to different epitopes of CD45 can be compared with each of the two possible bivalent antibodies containing only one of those complementary sites. In one embodiment, such comparisons are performed with respect to each different specificity (particularly each different complementary site) of the antibody of the present invention specific to CD45, using a comparative antibody. In one embodiment, the antibody of the present invention will show better results than against one such comparative antibody. In another embodiment, it will show better results than all comparative antibodies with respect to each specificity (particularly complementary site) of the antibody specific to CD45.

[0395] In another embodiment, where the antibody of the present invention contains at least two different specificities for CD45, a monospecific antibody is first evaluated, and then the selected candidate is used to generate the antibody of the present invention having at least two different specificities for CD45. In one embodiment, multiple antibodies are tested by using the multiplex system defined above and subjecting it to one or more functional assays.

[0396] As used herein, the term "biological function" refers to activity inherent to or intended for the biological entity being tested, such as the natural activity of a cell, protein, or analogue. Ideally, the presence of said function can be tested using in vitro functional assays, including assays using living mammalian cells. Natural functions as used herein include aberrant functions, such as those associated with cancer.

[0397] In one embodiment, the antibody of the present invention will be able to crosslink CD45 to a greater extent than comparative antibodies, particularly those discussed above. For example, the ability of the antibody of the present invention to form CD45 multimers of antibody:CD45 ECD can be studied when both are mixed (e.g., in equal amounts). The multimer can be a structure having at least two antibody:CD45 ECD units. A suitable technique is mass spectrometry, in which the antibody is mixed with an equal concentration of CD45 ECD, such as that of SEQ ID NO: 128, and mass spectrometry is performed on the test sample. Controls with separate antibodies and CD45 ECDs can be performed. The antibody of the present invention can produce more multimers than comparative antibodies. The antibody of the present invention can produce a greater number of multimers than the comparatives, having two, three, four, or more antibody:CD45 ECD units. This can be done for all possible comparatives regarding each of the specificities (particularly complementary sites) specific to CD45. A further suitable technique for such comparisons is analytical ultracentrifugation (AUC). Furthermore, the comparisons can also be made between an antibody mixture and each individual type of antibody in the mixture, compared separately with it.

[0398] In another embodiment, the comparison may be in terms of the ability of the antibody of the present invention to induce cell death. For example, the antibody of the present invention may induce cell killing in target cells expressing more CD45 than a comparison antibody, such as a comparison antibody. When measured using T cells, it may induce a higher amount of cell killing. For example, T cells isolated from PBMCs may be used. Any antibody of the present invention may induce a higher level of cell killing in CD4+ T cells. It may do so in CD8+ T cells. It may do so in CD4+ memory T cells. It may do so in CD4+ immature T cells. In another embodiment, the total cell count in whole blood may be measured after incubation with the antibody of the present invention and compared with the result seen for a comparison antibody. In one embodiment, the total cell count may be measured and compared with a control antibody of the present invention.

[0399] In one embodiment, in vivo assays, such as animal models including mouse tumor models, autoimmune disease models, rodent or primate models of viral or bacterial infections, can be used to test the antibodies of the present invention. In another embodiment, the degree of exhaustion of a specific cell type can be measured (e.g., in vivo). In one embodiment, in animal models of a disease, and in a preferred embodiment, in animal models of cancer, the antibodies of the present invention will induce a greater level of exhaustion than comparatives.

[0400] In one embodiment, the antibody molecule according to the invention has a novel or synergistic function. As used herein, the term "synergistic function" refers to a biological activity that is not observed or is higher than that observed when a comparative antibody is used instead. Therefore, "synergy" includes novel biological functions. In one embodiment, the antibody of the invention comprising at least two specificities for CD45 is synergistic because it is more effective than an antibody individually comprising any one of the specificities for CD45, such as the comparative antibodies discussed above. In a preferred embodiment, such synergy is manifested in terms of CD45 cross-linking. In one embodiment, the antibody mixture exhibits synergistic effects compared to any one of the individual antibodies constituting the mixture alone.

[0401] In one embodiment, “new biological function” as used herein refers to a function that is not apparent or present until the two or more synergistic entities [protein A and protein B] are brought together, or a function that has not been previously identified. “Higher” as used herein refers to an increase in activity, including an increase from zero, i.e., some activity in the antibody or molecule when the comparison compound is inactive in the relevant functional assay, which is also referred to herein as new activity or new biological function. “Higher” as used herein also includes a function greater than additive in the antibody in the relevant functional assay relative to a single complementary site, such as an increase of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300% or more in the relevant activity.

[0402] In one implementation, the new synergistic function is enhanced inhibitory activity.

[0403] In a particularly preferred embodiment of the invention, the synergistic effect is associated with cell exhaustion in target cell types expressing CD45. In one embodiment, the synergistic effect is associated with cell killing.

[0404] Suitable binding domains for use in this invention can also be identified by testing one or more pairs of binding domains in a functional assay. For example, antibodies, such as those containing at least one binding site specific to the antigen CD45, can be tested in one or more functional assays.

[0405] In one embodiment, the ability of an antibody to kill a CD45-expressing cancer cell line can be determined. The methods used in the embodiments of this application for assessing the ability of an antibody to induce the killing of such cell lines can be used to study the ability of a given antibody to kill cells. In one embodiment, the variant antibodies of the present invention will have the same or greater ability to kill cancer cells in such an assay as one of the specific antibodies listed herein. In one embodiment, they will have at least 50%, 75%, 80%, 90%, 100% or more of the activity of one of the specific antibodies listed herein in killing one of the aforementioned cancer cell lines in such an assay. In one embodiment, the antibodies of the present invention will kill at least 25%, 40%, 50%, 60% or 75% of cancer cells in such an assay. In another embodiment, the antibodies of the present invention will kill 100% of cancer cells in such an assay.

[0406] In one embodiment, the antibody of the present invention will have the same properties as for its function in... Figure 17 and 18 The results showed that those antibodies had similar activities.

[0407] For example, in one embodiment, the bicomponent antibody of the present invention will have an EC50 value of 0.01 to 0.30 nM in a human T lymphocyte depletion assay. In one embodiment, the EC50 value will be 0.02 to 0.200 nM. In one embodiment, it will have an Emax% value of 50 to 100%. In a preferred embodiment, it will have an Emax% value of 60 to 90%. In one embodiment, the bicomponent molecule may have a KD value of 5 to 20 nM, for example 5 to 15 nM, relative to human CD45, as measured by SPR Biocore. In one embodiment, the bicomponent molecule may have a KD value of 0.10 to 0.50 nM, for cynomolgus monkey CD45, as measured by SPR Biocore. In one embodiment, the bicomponent molecule of the present invention may have an EC50 value of 0.5 to 10 nM, for example 0.5 to 5 nM, relative to binding to human CD45 on cells. In one embodiment, the bicomplementary molecule of the present invention may have an EC50 value of 0.5 nM to 10 nM, for example 1 to 10 nM, regarding its binding to cynomolgus monkey CD45 on cells. In a preferred embodiment, the techniques used in the examples are used to perform such measurements. In a more preferred embodiment, the techniques used to generate in Figure 18 The technique shown in the results.

[0408] Pathological condition, medical use and cell exhaustion

[0409] This invention provides antibodies of the invention for use in methods of treating humans or animals. The antibodies of the invention can be used in any situation where targeting CD45 may have a therapeutic benefit, particularly in any scenario where killing such cells may have a therapeutic benefit. The antibodies of the invention can also be used in the diagnosis or detection of CD45. The invention further provides pharmaceutical compositions of the invention for such uses. The invention also provides nucleic acid molecules and vectors of the invention for such uses.

[0410] Therefore, the antibodies of the present invention can be used therapeutically. In one embodiment, instead of administering the antibodies of the present invention, the nucleic acid molecules or carriers of the present invention can be administered to induce the expression of the antibodies in target cells. In another embodiment, the pharmaceutical compositions of the present invention are preferred therapeutic agents. Although antibodies are listed below as preferred therapeutic agents, the pharmaceutical compositions, nucleic acid molecules, and carriers of the present invention can also be used in any of the listed embodiments. However, in a preferred embodiment, the antibody or the pharmaceutical composition comprising it is the preferred therapeutic agent.

[0411] In a particularly preferred embodiment, the present invention can be used to deplete target cells expressing CD45. In a particularly preferred embodiment, the present invention is used to deplete disease-causing cell types expressing CD45. In particular, the present invention can be used to deplete target cells expressing CD45 on their cell surface. In a particularly preferred embodiment, the antibody used is an antibody having at least two different specificities for CD45, i.e., an antibody with dual complementary sites for CD45.

[0412] In a preferred embodiment, when using the antibody of the present invention, inducing cell death in target cells via the antibody of the present invention means that the antibody of the present invention does not necessarily exhibit one or more Fc region effector functions that antibodies would normally exhibit. In a particularly preferred embodiment, the antibody of the present invention is therefore able to induce cell death in target cells but does not have an active Fc region. In a particularly preferred embodiment, the antibody induces cell death but does not induce significant cytokine release.

[0413] In a particularly preferred embodiment, cells or tissue are transferred to the subject after cell depletion by the invention. In a further particularly preferred embodiment, the transferred cells or tissue replace those already depleted by the invention. Thus, the treatments discussed herein include, rather than targeting the actual mechanism of the symptom, wholly or partially replacing the cell types involved in the symptom or whose killing (especially replacement) can simply have a therapeutic benefit. In one embodiment, the invention thus provides a method of depleting cells, which includes using the invention. In another embodiment, the method of the invention may include both cell depletion and subsequent transfer of cells or tissue. Cell depletion can be used in many therapeutic scenarios to effectively kill target cells.

[0414] In a preferred embodiment, the antibody of the present invention is used to kill immune cells. As used herein, the term "immune cells" is intended to include, but is not limited to, cells of hematopoietic origin and those that play a role in the immune response. In one embodiment, the present invention is used to deplete T cells in a subject. In one embodiment, the present invention is used to deplete B cells in a subject. In another embodiment, the present invention is used to deplete both. In another embodiment, the present invention is used to deplete T cells but not macrophages. In another embodiment, the present invention is used to deplete B cells but not macrophages. In another embodiment, the present invention is used to deplete both B and T cells but does not result in the depletion of macrophages. In one embodiment, the present invention is used to deplete hematopoietic stem cells (HSCs). In another embodiment, the present invention is used to deplete hematopoietic stem cells. In a preferred embodiment, HSCs are depleted in the subject via the present invention prior to transfer of HSCs to restore and rebuild the subject's immune system. In another embodiment, the present invention depletes a specific cell type but does not deplete hematopoietic stem cells. In another embodiment, the present invention is used to kill the cell types mentioned above. Therefore, in any of the embodiments mentioned herein with respect to cell depletion, the present invention can be used to kill the stated cells.

[0415] In one embodiment, the subject treated by the present invention is a subject with an autoimmune disease, blood disorder, metabolic disorder, cancer, or immunodeficiency. The ability to treat the condition by first depleting cells and then replacing them means that the antibodies of the present invention are particularly useful in treating cancer. In a particularly preferred embodiment, the condition to be treated is therefore cancer. In one embodiment, the present invention is therefore used to deplete cancer cells, such as cancer cells derived from immune system cells. In a preferred embodiment, the present invention provides a method of treating cancer comprising administering the present invention to deplete cancer cells expressing CD45. The method may further comprise transplanting cells into the subject. In one embodiment, the transferred cells replace the depleted cells. In one embodiment, the transferred cells are hematopoietic stem cells.

[0416] In a particularly preferred embodiment, the condition to be treated is a blood cancer. In another preferred embodiment, the cancer is a cancer involving the bone marrow, particularly a cancer involving cells of the hematopoietic system.

[0417] In a preferred embodiment, the cancer may be leukemia. In one embodiment, the cancer may be T-cell leukemia. In one embodiment, the cancer may be B-cell leukemia.

[0418] In one embodiment of the invention, the blood cancer to be treated may be lymphoma.

[0419] In one implementation scheme, the blood cell cancer to be treated is myeloma.

[0420] In another implementation, the subject to be treated has an autoimmune disease.

[0421] In one embodiment, the condition to be treated is a condition known to involve abnormal CD45 expression. In a particularly preferred embodiment, the treatment depletes the CD45-expressing cell types that play a role in the condition in the subject.

[0422] In one embodiment, the present invention is used to deplete cells prior to cell transplantation; therefore, in some embodiments, the method of the present invention may include a depletion step using the therapeutic agent (particularly an antibody) of the present invention, followed by a step of transferring the cells to the subject, for example, to help replace the depleted cells. In one embodiment, such transfer may be a transfer of allogeneic cells. In another embodiment, such transfer may be a transfer of autologous cells. In one embodiment, the transferred cells may be cells expressing a chimeric antigen receptor (CAR). In some embodiments, the subject requires chimeric antigen receptor T-cell (CAR) therapy. For example, such therapy may form part of the method of the present invention.

[0423] In another preferred embodiment, the present invention provides a method for promoting the influx of a cell population into a subject, wherein the method further includes depleting the cells using the antibody of the present invention prior to the influx of the cell population. Thus, the present invention provides a method for promoting the influx of transferred cells, comprising depleting CD45-expressing cells in a subject by administering the antibody of the present invention, and then transferring the target cells. In one embodiment, the present invention provides a method for promoting the influx of stem cells (particularly hematopoietic stem cells). In one embodiment, hematopoietic stem cells are administered to a subject who is deficient or lacking in one or more cell types of a hematopoietic lineage to reconstruct or partially reconstruct the deficient or lacking cell population in vivo. In one embodiment, the present invention is used to treat stem cell deficiency, for example, wherein the present invention is used to deplete target cells and replace them with transplanted cells, wherein the transplanted cells address the stem cell deficiency. In one embodiment, the reintroduced cells have been genetically modified. In one embodiment, cells from the subject have been removed and genetically modified, and then returned to the subject after the present invention has been used to kill target cells (e.g., unmodified cells of the type still present in the subject). In a preferred embodiment, the transferred genetically modified cells are hematopoietic stem cells.

[0424] In a preferred embodiment, the depleted cells and the transferred cells are of the same cell type, or contain the same cell type. In a preferred embodiment, the depleted cells are hematopoietic cells, particularly hematopoietic stem cells. In one embodiment, the invention is used to deplete cells prior to bone marrow transplantation. In another embodiment, the invention is used instead of radiation to deplete cells. In yet another embodiment, it is used in addition to radiation to deplete cells.

[0425] In another embodiment, the invention provides a method to help reduce the chance of rejection of transplanted cells, the method comprising administering a therapeutic agent of the invention to deplete the cells prior to transfer. In another embodiment, the invention can be used to promote the acceptance of transplanted immune cells in a subject by depleting target cells expressing CD45 prior to transfer of the immune cells. The target cells can be any of those discussed herein. In one embodiment, the cells transplanted or transferred to the subject are stem cells.

[0426] Any of the methods discussed herein for eliminating CD45-expressing cells can be used in cell exhaustion or killing. However, in a particularly preferred embodiment of the invention, the invention can be used to induce cell death in CD45-expressing cells, and thus exhaust such cells.

[0427] In one embodiment, bone marrow may be administered to the subject as part of cell transfer, serving as a means of cell transfer. In another embodiment, the subject may have been given umbilical cord blood or cells isolated from umbilical cord blood, serving as a means of cell transfer. In yet another embodiment, the transplanted cells may be derived from differentiated stem cells, for example, stem cells that have already differentiated in vitro and then been transplanted.

[0428] In one embodiment, when the antibody of the present invention is used to deplete or kill cells, a further cell-depleting or killing agent may also be used. In a preferred embodiment, the antibody of the present invention is the only cell-depleting agent administered to the subject. In one embodiment, the level of target cell depletion is sufficiently effective, for example, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the target cells. For example, in one embodiment, at least 50% of the target cells are depleted. In another embodiment, at least 75% of the target cells are depleted. In another embodiment, at least about 90% of the target cells are depleted. In another embodiment, at least about 95% of the target cells are depleted.

[0429] As discussed above, in a particularly preferred embodiment, the antibody of the present invention can be used to induce cell killing in cells expressing CD45. Any suitable method can be used to assess cell viability and thus cell killing.

[0430] In another embodiment, the present invention can be used in the context of graft-versus-host disease (GVHD), particularly in the treatment of cell populations, tissues, or organs with antibodies to deplete cells.

[0431] In another embodiment, the invention provides a treatment method comprising performing such ex vivo treatment first, followed by transplantation. In another embodiment, the invention is used to deplete or kill cells in a subject prior to transplantation, thereby reducing the number of host cells capable of attacking the transplanted material, as a way of reducing the chance of GVHD. Therefore, the invention also provides a method for treating or preventing GVHD, comprising administering the antibody of the invention to deplete cells in said cell population, tissue, or organ prior to transplantation of said cell population, tissue, or organ. The method may further include the transplantation itself. The depleted cells and the transplanted cells, tissue, or organ can be any of those mentioned herein. In a preferred embodiment, the transplanted cells are hematopoietic stem cells. In a preferred embodiment, the depleted cells are T cells. In another preferred embodiment, the ability of the invention to treat or prevent GVHD is used in heart, lung, kidney, or liver transplantation.

[0432] In another embodiment, the present invention provides a method for exhausting and / or killing cells in a cell population, tissue, or organ in vitro prior to transplantation by applying the antibody of the present invention, rather than treating the recipient. Therefore, the present invention also provides a method for removing target cells from a cell population, tissue, or organ in vitro prior to transplantation, comprising treating the cell population, tissue, or organ prior to transplantation, followed by transplantation.

[0433] In one embodiment, the invention can be used to deplete immune cells in organs or tissues, particularly where conventional therapies are not readily accessible or would lead to excessive inflammation as part of their inherent mechanisms. In one embodiment, the invention is used to deplete cells in closed organs, such as those in the brain, spinal cord, eye, or testis. In one embodiment, the invention can be used to deplete CD45 in immune-exempt organs. +Cells. The ability of the antibody of the present invention to deplete CD45+ cells without using Fc-mediated function can help avoid unwanted side effects and damage. In one embodiment, the present invention can be used to deplete cells in an immune-silencing manner and without requiring antibody effector mechanisms. This can have the advantage of minimizing or at least reducing unwanted damage, for example, because the closed organ may contain fragile and often undividing tissue cells that can be destroyed by infiltrating leukocytes. When the present invention is applied directly to organs such as the brain, spinal cord, eye, or testis, it can lead to the elimination of CD45-positive cells without inducing further tissue damage or inflammation or with reduced further damage. In one embodiment, the target cells in said closed organ are selected from lymphocytes, B cells, and T cells. In one embodiment, the target cells in said closed organ are or comprise CD4+ T cells. In another embodiment, said target cells are or comprise CD8+ T cells.

[0434] In a further preferred embodiment, the present invention is applied to a condition characterized by infiltrating CD8+ T cells.

[0435] Pharmaceutical Composition

[0436] In one aspect, a pharmaceutical composition is provided comprising: (a) an antibody, one or more nucleic acid molecules, or one or more carriers of the present invention; and (b) a pharmaceutically acceptable carrier or diluent. In a particularly preferred embodiment, it comprises one or more antibodies of the present invention. The composition may contain a variety of different components, including pharmaceutically acceptable carriers, excipients, and / or diluents. The composition may optionally contain further molecules capable of altering the characteristics of the molecules of the present invention, thereby, for example, reducing, stabilizing, delaying, modulating, and / or activating the function of the molecules. The composition may be in solid or liquid form, and in particular in powder, tablet, solution, or aerosol form.

[0437] The present invention also provides pharmaceutical or diagnostic compositions comprising the molecules of the present invention and one or more pharmaceutically acceptable excipients, diluents, or carriers in combination therewith. Thus, the use of the antibodies of the present invention in treating pathological conditions or diseases, and in the preparation of medicaments for treating pathological conditions or diseases, is provided. In one embodiment, in the case of administering the therapeutic agent of the present invention to a subject who is also given a second therapeutic agent, the two may be administered, for example, simultaneously, sequentially, or separately. In one embodiment, the two are administered in the same pharmaceutical composition. In another embodiment, the two are administered in separate pharmaceutical compositions. In one embodiment, the present invention provides an antibody of the present invention for use in a method in which said subject is also being treated with a second therapeutic agent. In another embodiment, the present invention provides a second therapeutic agent for use in a method in which said subject is being treated with the antibody of the present invention. The nucleic acid molecules and carriers of the present invention may also be administered in such combinations.

[0438] The compositions of the present invention are generally provided as sterile pharmaceutical compositions. The pharmaceutical compositions of the present invention may additionally contain pharmaceutically acceptable adjuvants. In another embodiment, such adjuvants are not present in the compositions of the present invention. The present invention also provides a method for preparing pharmaceutical or diagnostic compositions, comprising adding the antibody of the present invention and mixing it with one or more pharmaceutically acceptable excipients, diluents, or carriers.

[0439] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a pharmaceutically acceptable formulation carrier, solution, or additive to enhance the desired characteristics of the compositions of the present invention. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions may be encapsulated in liposomes or biodegradable microspheres. Using aseptic preparation methods, the formulations will generally be provided in a substantially sterile form.

[0440] This may include: generating a buffered solvent solution for the formulation and sterilizing it by filtration, aseptically suspending the antibody in the sterile buffered solvent solution, and dispensing the formulation into a sterile container by methods familiar to those skilled in the art.

[0441] The pharmaceutically acceptable carrier should not itself induce antibodies harmful to the individual receiving the composition, and should not be toxic. Suitable carriers can be large, slowly metabolized macromolecules, such as proteins, peptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive viral particles.

[0442] Pharmaceutically acceptable salts, such as inorganic acid salts like hydrochlorides, hydrobromides, phosphates, and sulfates, or organic acid salts like acetates, propionates, malonates, and benzoates, can be used. Pharmaceutically acceptable carriers in therapeutic compositions may additionally comprise liquids such as water, saline, glycerol, and ethanol. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, serums, and suspensions for patient ingestion.

[0443] A detailed discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ 1991).

[0444] As used herein, the term "therapeutic effective amount" refers to the amount of a therapeutic agent required to treat, improve, or prevent a targeted disease or condition, or to demonstrate a detectable therapeutic or preventative effect. For any antibody, the therapeutic effective amount can be initially estimated in a cell culture assay or in an animal model (typically rodents, rabbits, dogs, pigs, or primates). The animal model can also be used to determine appropriate concentration ranges and routes of administration. This information can then be used to determine a useful dose and route of administration for human use.

[0445] The composition can be administered to the patient individually or in combination with other agents, drugs, or hormones (e.g., simultaneously, sequentially, or separately). The dosage at which the invention is administered depends on the nature of the condition to be treated, the degree of inflammation present, and whether the antibody is used prophylactically or to treat an existing condition.

[0446] The present invention also provides a method for preparing a pharmaceutical or diagnostic composition, comprising adding an antibody and mixing it with one or more pharmaceutically acceptable excipients, diluents or carriers.

[0447] The antibody, nucleic acid molecule, or carrier may be the sole active ingredient in the pharmaceutical or diagnostic composition, or it may be accompanied by other active ingredients, including antibody or non-antibody components, such as steroids or other drug molecules.

[0448] The pharmaceutical composition suitably comprises a therapeutically effective amount of the antibody of the present invention. As used herein, the term "therapeutically effective amount" refers to the amount of therapeutic agent required to treat, improve, or prevent a targeted disease or condition, or to exhibit a detectable therapeutic or preventative effect. "Therapeutically effective amount" can also be the amount required to induce a desired level of cell exhaustion. For any antibody, the therapeutically effective amount can be initially estimated in a cell culture assay or in an animal model (typically rodents, rabbits, dogs, pigs, or primates). The animal model can also be used to determine suitable concentration ranges and routes of administration. Such information can then be used to determine a useful dose and route of administration for human use.

[0449] The precise therapeutically effective dose for human subjects will depend on the severity of the disease state, the subject's overall health, age, weight and sex, diet, timing and frequency of administration, drug combination, sensitivity to response, and tolerability / response to the therapy. Typically, the therapeutically effective dose will be from 0.01 mg / kg to 50 mg / kg daily, for example, from 0.1 mg / kg to 20 mg / kg. In one implementation, the amount in a given dose is at least sufficient to elicit the specific function.

[0450] The pharmaceutical composition can be conveniently available in unit dose form, which contains a predetermined amount of the active reagent / dose of the invention. The pharmaceutical composition of the invention can be provided in a container that provides an instrument for administration to a subject. The pharmaceutical composition of the invention can be provided in a pre-filled syringe. Therefore, the invention provides such loaded syringes. The invention also provides an auto-injector loaded with the pharmaceutical composition of the invention.

[0451] The composition can be administered to the patient individually or in combination with other agents, drugs or hormones (e.g., simultaneously, sequentially or separately).

[0452] As used herein, “pharmaceutical” refers to an entity that has a physiological effect when administered. As used herein, “medicine” refers to a chemical entity that has an appropriate physiological effect at therapeutic doses.

[0453] Once prepared, the composition of the present invention can be administered directly to the subject. The subject to be treated can be an animal. However, in one or more embodiments, the composition is adapted for administration to a human subject.

[0454] In one embodiment, the antibody of the present invention can be used to functionally alter the activity of one or more target antigens, particularly modulating CD45. For example, the present invention can directly or indirectly neutralize, antagonize, or activate the activity of said one or more antigens.

[0455] The present invention also extends to kits comprising the antibodies of the present invention. In one embodiment, a kit is provided comprising any of the antibodies of the present invention, optionally accompanied by instructions for use.

[0456] In another embodiment, the kit further comprises one or more reagents for performing one or more functional assays.

[0457] In one embodiment, the molecules of the present invention are provided, including the antibodies of the present invention, for use as laboratory reagents.

[0458] Further aspects

[0459] In a further aspect, one or more nucleotide sequences are provided, such as one or more DNA sequences encoding the antibody molecules of the present invention as described herein. In one embodiment, the nucleotide sequences are co-present on more than one polynucleotide, but together they collectively encode the antibody of the present invention.

[0460] This invention also extends to vectors containing the nucleotide sequences defined above. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One example of a vector is a "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be linked. Another type of vector is a viral vector, in which the additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). Other vectors (e.g., non-attached mammalian vectors) can be integrated into the genome of a host cell, where they subsequently replicate along with the host genome. In this specification, the terms "plasmid" and "vector" are used interchangeably, as plasmids are the most commonly used form of vector. General methods for constructing vectors, transfection methods, and culture methods are well known to those skilled in the art. For further information, see “Current Protocols in Molecular Biology”, 1999, FM Ausubel (ed.), Wiley Interscience, New York, and Maniatis Manual, published by Cold Spring Harbor Publishing.

[0461] The term "vector" as used herein also includes, for example, particles containing said vector, such as LNP (lipid nanoparticle) particles, particularly LNP-mRNA particles. It also includes viral particles for transferring the vectors of the present invention.

[0462] As used herein, the term "selective marker" refers to a protein whose expression allows for the identification of cells transformed or transfected with a vector containing a marker gene. A wide variety of selective markers are known in the art. For example, the selective marker gene typically confers resistance to drugs such as G418, hygromycin, or methotrexate to host cells in which the vector has been introduced. Selective markers can also be visually recognizable markers, such as fluorescent markers. Examples of fluorescent markers include rhodamine, FITC, TRITC, Alexa Fluors, and their various conjugates.

[0463] In one embodiment, the present invention provides a vector encoding an antibody of the present invention. In another embodiment, the present invention provides a vector that commonly encodes an antibody of the present invention.

[0464] Host cells comprising one or more cloning or expression vectors containing one or more DNA sequences encoding the antibodies of the present invention are also provided. Any suitable host cell / vector system can be used to express the DNA sequence encoding the antibody molecule of the present invention. Bacterial (e.g., *Escherichia coli*) and other microbial systems can be used, or eukaryotic (e.g., mammalian) host cell expression systems can also be used. Suitable mammalian host cells include CHO, myeloma, or hybridoma cells. Host cells comprising the nucleic acid molecule or vector of the present invention are also provided.

[0465] The present invention also provides a method for producing a molecule or a component thereof according to the invention, comprising culturing a host cell containing a vector of the invention under conditions suitable for causing the expression of a protein from DNA encoding a molecule of the invention, and isolating said molecule.

[0466] A method for generating an antibody comprising heterodimeric tethers may further include mixing the two portions of the antibody and allowing the binding partners of the heterodimeric tethers to associate. The method may further include purification, for example, to remove any species other than the desired heterodimer.

[0467] The antibodies of the present invention can be used in diagnostic / testing kits. In one embodiment, the antibodies of the present invention are immobilized on a solid surface. The solid surface can be, for example, a chip or an ELISA plate.

[0468] The antibodies of the present invention can, for example, be conjugated to fluorescent labels that drive the detection of the bound antibody-antigen complex. They can be used for immunofluorescence microscopy. Alternatively, the antibodies can also be used for Western blotting or ELISA.

[0469] In one embodiment, a method for purifying an antibody or a component thereof according to the present invention is provided. In one embodiment, a method for purifying an antibody or a component thereof according to the present invention is provided, comprising the steps of: performing anion exchange chromatography in a non-binding mode, such that impurities are retained on the column and the antibody remains in an unbound fraction. This step may be performed, for example, at a pH of approximately 6-8. The method may further include an initial capture step using cation exchange chromatography, which is performed, for example, at a pH of approximately 4-5. The method may further include additional chromatographic steps to ensure that the product and process-related impurities are properly resolved from the product stream. The purification method may also include one or more ultrafiltration steps, such as concentration and percolation steps.

[0470] The term "purified form" as used above refers to a purity of at least 90%, such as 91, 92, 93, 94, 95, 96, 97, 98, 99% w / w or higher.

[0471] In the context of this specification, "comprising" is interpreted as "including". The aspects of the invention that include certain elements are also intended to be extended to alternative embodiments that are "composed of related elements" or "substantially composed of related elements". For example, a CDR described as "comprising" a sequence of a specific SEQ ID NO may be "composed" of that sequence, i.e., containing only that sequence and excluding any further sequences.

[0472] In this paper, a clearly described implementation plan can be used as the basis for the abandonment.

[0473] Where the singular is mentioned in this text, the plural is also included, unless otherwise stated or clearly indicated. In particular, the singular forms “a,” “an,” “the,” etc., include plural indicators unless the context clearly specifies otherwise.

[0474] All references mentioned in this article are specifically combined by reference.

[0475] The subheadings in this document are used to help structure the instructions and are not intended to constitute the meaning of the technical terms used in this document.

[0476] The sequence of the present invention is provided below.

[0477] In the context of this specification, "including" is interpreted as "including".

[0478] The aspects of the invention that include certain elements are also intended to be extended to alternative embodiments that are “composed of related elements” or “substantially composed of related elements”.

[0479] In this paper, a clearly described implementation plan can also be used as the basis for abandonment.

[0480] All references mentioned in this article are specifically grouped together by reference. They are further grouped together in relation to the specific context in which they are cited.

[0481] Example

[0482] Example 1: Evaluation of antibodies binding to human CD45 via Biacore

[0483] This embodiment illustrates a method for measuring binding affinity via Biacore in other embodiments.

[0484] The kinetics of binding between humanized IgG grafts or dual complementary IgG molecules and human CD45 were evaluated by surface plasmon resonance on a Biacore T200 or 8K+ instrument (Cytiva).

[0485] Goat anti-human IgG Fc-specific F(ab')2 (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip (Cytiva) via amine coupling chemistry to a level typically between 5000 and 7000 RU. Each analytical cycle consisted of the following: capture of anti-CD45 IgG molecules onto the anti-Fc surface, followed by injection of human CD45 (internally prepared) at 25 °C and a flow rate of 30 or 50 μl / min. At the end of each cycle, the surface was regenerated using the following method at a flow rate of 10 μL / min: 60 s injection of 50 mM HCl, followed by 30 s injection of 5 mM NaOH, and a final 60 s injection of 50 mM HCl. Human CD45 was injected in HBS-EP+ run buffer (Cytiva) onto the captured sample and reference flow cell at concentrations ranging from 800 nM to 3.3 nM (5×3 serial dilutions) or 800 to 3.13 nM (4×4 serial dilutions) in HBS-EP+ run buffer (Cytiva). The binding response of the reference flow cell was subtracted from the binding response of the active flow cell, and buffer blank injections were included to subtract equipment noise and drift.

[0486] Use Biacore T200 Evaluation software (version 3.0) or Biacore Insight Evaluation (version 4.0.8.20368) to determine the dynamic parameters using a 1:1 dynamic combination model.

[0487] Example 2: Generation and characterization of CD45 antibody

[0488] introduction:

[0489] This example describes the initial generation and characterization of an antibody specific to CD45.

[0490] Immunization:

[0491] Rabbits were immunized with a mixture of rabbit fibroblasts expressing human CD45RO (a truncated form of CD45, SEQ ID NO: 136) or the equivalent cynomolgus monkey CD45RO (SEQ ID NO: 137). After 3 to 5 immunizations, the animals were sacrificed and PBMCs, spleen, bone marrow, and lymph nodes were harvested. Serum binding to human and cynomolgus monkey CD45RO proteins was monitored by ELISA.

[0492] Antibody discovery:

[0493] B cell cultures were prepared using a method similar to that described by Zubler RH, Erard F., Lees RK et al., Mutant EL-4 ThymomaCells Polyclonally Activate Murine and Human B Cells via Direct Cell Interaction. J. Immunol. 1985, 134, (6), 3662-3668. In summary, spleen or PBMC-derived B cells from immunized rabbits were cultured at a density of approximately 2000-5000 cells / well in barcoded 96-well tissue culture plates at 37°C in a 5% CO2 atmosphere for seven days. The tissue culture plates contained 200 μL / well supplemented with 10% FCS (PAA laboratories ltd), 2% HEPES (Sigma Aldrich), 1% L-glutamine (Gibco BRL), 1% penicillin / streptomycin solution (Gibco BRL), 0.1% β-mercaptoethanol (Gibco BRL), 3% activated spleen cell culture supernatant, and γ-irradiated mutant EL4 murine thymoma cells (5 × 10⁻⁶ cells / well). 4 RPMI 1640 medium (Gibco BRL) per well.

[0494] Initial screening:

[0495] B-cell cultures were established, and 34 supernatants were first screened for their binding ability to human and cynomolgus monkey CD45RO using a bead-based Mirrorball FMAT assay. This is a homogeneous assay using biotinylated human and cynomolgus monkey CD45RO and goat anti-rabbit Fcγ fragment-specific FITC conjugates coated on streptoacidin beads (as revealing reagents). After initial screening, positive supernatants were pooled onto 96-well barcoded master plates while B-cells in the cell culture plates were frozen at -80°C. To determine cell binding and species cross-reactivity, the master plate supernatants were then screened for binding to HEK293 cells transfected with human and cynomolgus monkey CD45RO.

[0496] Fluorescence focusing method:

[0497] To allow for the recovery of antibody variable region genes from antigen-specific B cells in wells containing heterogeneous B cell populations, a deconvolution step is performed. This is achieved using a fluorescence focusing method (Clargo et al., 2014. Mabs 2014 Jan. 1: 6(1) 143-159; EP1570267B1). Briefly, immunoglobulin-secreting B cells from positive wells are mixed with streptavidin beads coated with biotinylated human CD45RO (New England Biolabs) and a goat anti-rabbit Fcγ fragment-specific FITC conjugate (Jackson) at a final dilution of 1:1200. After incubation at 37°C for 1 hour, antigen-specific B cells can be identified due to the presence of a fluorescent halo around that B cell. A number of these individual B cell clones are then picked and placed into PCR tubes. The fluorescence focusing method has also been used to identify antigen-specific B cells from heterogeneous B cell populations directly from the bone marrow of immunized rabbits.

[0498] Antibody V-region discovery:

[0499] The antibody variable region gene was recovered from a single cell by reverse transcription (RT)-PCR. cDNA was synthesized from a single B cell using SuperScript IV VILO Master Mix (Life Technologies) in the presence of 0.5% Nonidet P-40 (Roche). Two rounds of PCR were performed using heavy and light chain variable region-specific primers, with a nested second PCR incorporating restriction sites at the 3′ and 5′ ends, allowing the variable region to be cloned into Fab X and Fab Y mammalian expression vectors to allow Fab-X / Fab-Y expression (e.g., as described in WO 2015 / 181282 and WO 2017 / 093402).

[0500] Using Expifectamine (Life Technologies), Fab X and Fab Y constructs for the expression vector were co-transfected into Expi293 cells, and the recombinant antibody was expressed in 30 ml volumes in tissue culture flasks. After 5–7 days of expression, the supernatant was harvested. To confirm the specificity of the recombinant antibody, the supernatant was tested on HEK293 cells transfected with human and cynomolgus monkey CD45RO using a homogeneous fluorescence-based binding assay.

[0501] Assessment of antibody binding and Jurkat cell killing

[0502] The methods described in other embodiments are used to evaluate the ability of antibodies to bind to CD45 and kill Jurkat cells.

[0503] result:

[0504] Numerous clones were screened using KD values ​​for binding to human and cynomolgus monkey CD45RO measured by surface plasmon resonance (SPR), and two clones were identified as monospecific antibodies for Jurkat cell killing for further research and development. Antibodies 17415 and 17552 bind to both human and cynomolgus monkey CD45. The rabbit 17415 antibody achieved potent killing of Jurkat cells expressing CD45. Subsequently, a bi-complementary antibody was developed, based on using a 17415-derived sequence as the "killing arm" of the bi-complementary antibody, while a 17552-derived sequence was developed as the "non-killing" or "helper" arm of the bi-complementary antibody. Both the 17415 and 17552 sequences were humanized, as further described below.

[0505] Example 3: Comparison of the ability of antibodies 17415 and 4133 to kill cells expressing human CD45

[0506] introduction:

[0507] The ability of the 17415 antibody to kill CD45-expressing target cells was compared with that of the 4133 antibody described in international application PCT / EP2021 / 078516 (published as WO 2022 / 079199 A1).

[0508] PBMC Depletion Determination Method

[0509] Human PBMCs derived from blood apheresis cones (NHSBT Oxford) were used as frozen aliquots for library construction. Before the assay, one vial of frozen cells (each containing 5 × 10⁶ cells in 1 ml) was prepared. 7 Cells were thawed in a 37°C water bath and then added to 50 ml of complete medium (RPMI 1640 + 2 mM GlutaMAX + 1% Pen / Strep, all supplied by Invitrogen, + 10% fetal bovine serum (FBS), Sigma-Aldrich). Cells were centrifuged (300 g, 5 min, at RT) and resuspended in 50 ml of complete medium for washing, and centrifuged again. Cells were resuspended in 10 ml of complete medium and then counted using a ChemoMetec NucleoCounter NC-3000. Cells were then counted in 50 μl at a concentration of 1×10⁻⁶ cells. 5 One cell / well was added to each well of a CorningCostar 96-well microplate with a U-shaped bottom and pre-treated cell culture (catalog number 07-200-95). PBMCs from UCB-Cone 1014 were used in this assay.

[0510] Stockpile was prepared in Greiner 96-well unbound microplates containing 400 nM purified VR17415, VR4133, and 17552 IgG1 antibodies and isotype control IgG1 (5604) in complete medium. The reagents were serially diluted seven times (1:5) in complete medium (twice for the isotype control) to generate an 8-point dose profile. 50 μL of each dilution (final well concentration 200–0.0026 nM) was added to cells and incubated at 37°C, 5% CO2 for 4 hours. After incubation, plates were centrifuged at 300 g for 5 minutes at room temperature. Buffer was aspirated using a BioTek ELx405 microplate washer, and cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2mM EDTA, Sigma Aldrich) for washing. Centrifugation was then performed again, and buffer was aspirated, leaving cells in 20 μl of residual culture medium. Cell viability was analyzed using Intellicyt iQue 3. Lymphocytes were gated using FSC vs. SSC plotting, and live cell counts were extracted as a metric and graphically represented using Graphpad Prism version 8.1 (Graphpad). Asymmetric (4-parameter) curve fitting was applied to derive EC50 and Emax values.

[0511] result:

[0512] The percentage reduction in lymphocytes in the PBMC population treated with CD45 IgG1 antibody was shown in... Figure 1 In the study, VR17415 had an EC50 of 0.71 and VR4133 had an EC50 of 44.13, showing a >60-fold difference in the EC50 of the antibodies. VR17552 IgG1 did not deplete lymphocytes.

[0513] Example 4: Humanization of Antibody 17415

[0514] Rabbit antibody 17415, obtained and evaluated using the method described in Example 2, was humanized by grafting a CDR from the rabbit V-region onto the human antibody V-region framework. To restore the activity of the antibody, numerous framework residues from the rabbit V-region were also retained in the humanized sequence. These residues were selected using the procedures outlined by Adair et al. (1991) (Humanised Antibodies - WO91 / 09967, which is combined by reference for its overall and particularly concerning procedures for selecting residues to be retained during humanization). Alignment of the rabbit antibody (donor) V-region sequence with the human (recipient) V-region sequence, along with the designed humanized sequence, is shown in the figure. Figure 2 and 3 The CDR grafted from the donor sequence to the recipient sequence is as defined by Kabat (Kabat et al., ibid.), except for CDR-H1, which uses the combined Chothia / Kabat definition (see Adair et al.). , 1991 Humanisedantibodies, WO91 / 09967).

[0515] Initially, human V-region IGKV1-9 and IGKJ4-1 J-region (IMGT, http: / / www.imgt.org / ) were selected as recipients for the CDRs of the antibody 17415 light chain. Donor residues from the rabbit antibody light chain framework were retained at positions 2 (valine, V2), 3 (valine, V3), and 63 (lysine, K63). Figure 2The CDRL3 of antibody 17415 contains an unpaired / free cysteine ​​residue at position 90 (C90). Free cysteine ​​residues can undergo post-translational modifications, such as cysteylation, and can contribute to covalent aggregation and poor stability. Cysteine ​​amino acid side chains possess both polar and hydrophobic characteristics; therefore, in humanized graft variants gL2 to gL5, the C90 residue is removed by mutating to amino acids with polar side chains (serine (C90S), glutamine (Q90V)) or amino acids with hydrophobic side chains (alanine (C90A), valine (C90V)). Figure 2 ).

[0516] Human V-region IGHV3-72 and IGHJ4-1 J-region (IMGT, http: / / www.imgt.org / ) were selected as acceptors for the heavy chain CDR of antibody 17415. Like many rabbit antibodies, the 17415 rabbit VH region's 3-structure lacks a residue (78, see SEQ ID NO: 17 gH1) in the loop between D and E of the β-sheet chain: in the original humanized graft variant, this gap was filled with the corresponding residue (lysine 78, K78) from the selected human acceptor sequence. Figure 3 Donor residues from the rabbit antibody heavy chain framework are retained at positions 23 (threonine T23), 49 (isoleucine, I49), 74 (lysine, K74), 76 (serine, S76), 79 (threonine, T79), 81 (valine, V81), 99 (glutamate, E99), and 100 (leucine, L100). In some humanized grafting variants (gH2 to gH4), the disulfide bond formed between cysteine ​​residues at positions 36 (C36) and 51 (C51) is removed by mutating these two residues to serine (S36 and S51), alanine (A36 and A51), or valine (V36 and V51). Figure 3 ).

[0517] Genes encoding the humanized V-region sequences of the modified heavy and light chains were designed and constructed by ATUM (Newark, CA) using an automated synthesis method. For transient expression in mammalian cells, the humanized light chain V-region gene was cloned into the UCB light chain expression vector pMhCK, which contains DNA encoding the human κ chain constant region (Km3 allotype). The humanized heavy chain V-region gene was cloned into the UCB human γ-1 heavy chain expression vector pMhg1 L234A L235A, which contains DNA encoding the human γ-1 heavy chain constant region (G1m17,1 allotype) with L234A and L235A mutations to reduce binding to the Fc γ receptor. The rabbit V-region gene of antibody 17415 was also cloned into a human antibody expression vector: the light chain V-region was cloned into a modified version of the human κ vector containing the S171C mutation to recreate the additional disulfide bonds found in the rabbit VK light chain. The resulting heavy and light chain vectors were co-transfected into CHOS-XE suspension cells to produce expression of humanized and chimeric recombinant antibodies in the human IgG1 LALA pattern. The antibodies were evaluated for activity and binding properties in an in vitro T lymphocyte depletion assay. All humanized antibodies exhibited reduced potency (EC50) and efficacy (Emax) compared to the chimeric parent antibody 17415 (Table 3 below).

[0518] Table 3

[0519]

[0520] To clarify whether the humanized heavy chain or the light chain was responsible for the loss of functional activity, the humanized light chain and heavy chain grafts gL1 and gH1 were expressed in combination with their respective chimeric antibody chains (cL and cH), and the antibodies were screened for activity in a T-cell exhaustion assay and for their binding affinity to human CD45 by SPR using the methods described in the following examples. The results are presented in Table 4 below. The humanized heavy chain graft paired with the chimeric light chain (cLgH1) exhibited reduced potency and efficacy in T-cell exhaustion, as well as reduced binding affinity to CD45. Conversely, the combination with the humanized light chain paired with the chimeric antibody heavy chain (gL1cH) retained the binding affinity to CD45 and the potency in the T-cell exhaustion assay, but with reduced efficacy compared to the parent antibody 17415.

[0521] Combining humanized heavy chain variants gH2 to gH4 with chimeric light chains revealed that removing the disulfide bond between C36 and C51 by mutating cysteine ​​residues to serine (cLgH2) resulted in a surprisingly increased binding affinity for CD45 compared to cLgH1, as well as increased potency and efficacy in T-cell exhaustion assays. Conversely, substituting cysteine ​​residues with valine (cLgH4) led to a further loss of binding affinity and function (in T-cell exhaustion assays) (compared to cLgH1), while substituting said residues with alanine (cLgH3) had little effect on binding or T-cell exhaustion activity (Table B).

[0522] Combining humanized light chain graft variants gL2 to gL5 with chimeric heavy chains demonstrated that removing free cysteine ​​residues (C90S, gL2cH) by mutation to serine maintained binding affinity, but resulted in some loss of functional activity in T lymphocyte exhaustion assays, exhibiting reduced potency and efficacy compared to gL1cH. Mutating C90 to alanine (gL3cH), valine (gL4cH), or glutamine (gL5cH) resulted in loss of binding affinity for CD45 and reduced T cell exhaustion activity (Table 4).

[0523] Table 4

[0524]

[0525] To attempt to restore functional activity, the humanized heavy and light chains were further modified as described herein. In heavy chain grafts gH5 and gH6, residue 78 (lysine, K78) was deleted to restore the cleavage in structure 3 to its original state and recreate the rabbit loop structure between the D and E β-sheet chains: graft gH6 also includes C36S and C51S mutations to remove the disulfide bond between CDRH1 and CDRH2. In light chain grafts gL6 and gL7, additional donor residues from the rabbit light chain structure were introduced at positions 10 (serine, S10), 42 (glutamine, Q42), 83 (alanine, A83), 106 (glutamate, E106), and 108 (valine, V108): graft gL7 also includes the C90S mutation to remove the free cysteine ​​residue in CDRL3. Using the human V-region IGKV4-1 plus IGKJ4 J-region (IMGT, http: / / www.imgt.org / ) as the acceptor framework to design additional humanized light chains ( Figure 2Donor residues from the rabbit antibody light chain framework were retained at five or more positions selected from the group consisting of the following: residues 2 (valine, V2), 4 (leucine, L4), 12 (serine, S12), 19 (valine, V19), 60 (serine, S60), 63 (lysine, K63), 70 (glutamate, E70), 83 (alanine, A83), 85 (threonine, T85), 106 (glutamate, E106), and 108 (valine, V108). Grafts gL15 and gL16 also included a mutant C90S to remove free cysteine ​​residues in CDRL3. The humanized heavy and light chains were expressed as hIgG1LALA antibodies in different graft combinations, and the resulting antibodies were tested for functional activity and SPR binding affinity to CD45 in a T lymphocyte depletion assay (Table 5 below).

[0526] Table 5

[0527]

[0528]

[0529] The deletion of residue 78 in frame 3 of the humanized heavy chain graft gH5 increases the binding affinity for CD45 (Table 5, gL1gH1 (418.5 nM) compared to gL1gH5 (279.9 nM)), but leads to a decrease in potency and efficacy in T cell exhaustion assays. Consistent with the unexpected increase in binding affinity observed after the removal of disulfide bonds in the humanized heavy chain graft gH2, the binding affinity for CD45 is further increased in graft gH6 (gL1gH6 179.2 nM).

[0530] By increasing the donor residue content in the light chain grafts gL6 and gL7, the T cell exhaustion activity of the humanized 17415 antibody containing grafts gH5 and gH6 was restored (gL1gH5 EC50 19.17 nM, Emax 58.75%, compared to gL6gH5 EC50 1.99 nM, Emax 88.45% or gL7gH5 EC50 2.4 nM, Emax 88.15%, and gL6gH6 EC50 1.76 nM, Emax 76.15% or gL7gH6 EC50 0.63 nM, Emax 106%). Similarly, when paired with heavy chain grafts gH5 and gH6, grafting light chain CDRs onto the alternative human recipient framework IGKV4-1 plus JK4 J-region (grafts gL13 to gL16) provides increased T cell exhaustion potency and efficacy (relative to gL1gH5 and gL1gH6) while maintaining affinity for CD45 (Table C).

[0531] Further description of some of the experiments that constitute part of the humanization of the original 17415 antibody is provided in Example 6 below.

[0532] Example 5: Humanization of antibody 17552

[0533] Rabbit antibody 17552, obtained and evaluated using the method described in Example 2, was humanized by grafting a CDR from the rabbit V-region onto the human antibody V-region framework. To restore the activity of the antibody, many framework residues from the rabbit V-region were also retained in the humanized sequence. These residues were selected using the protocol outlined by Adair et al. (1991) (Humanisedantibodies. WO91 / 09967 - ibid.). Alignment of the rabbit antibody (donor) V-region sequence with the human (recipient) V-region sequence, along with the designed humanized sequence, is shown in the figure. Figure 4 The CDR grafted from the donor sequence to the recipient sequence is as defined by Kabat (Kabat et al., 1987, ibid.), except for CDR-H1, which uses the combined Chothia / Kabat definition (see Adair et al.). , 1991 Humanisedantibodies, WO91 / 09967).

[0534] Human V-region IGKV1-8 and IGKJ4-1 J-region (IMGT, http: / / www.imgt.org / ) were selected as recipients for the light chain CDR of antibody 17552. The light chain framework residues in the humanized graft variant are all derived from human germline genes, except for one or more residues selected from the group including residues 2, 3, and 63 (see SEQ ID NO: 3 gL1), where donor residues leucine (L2), valine (V3), and glutamate (E63) are retained, respectively. Figure 4 The free cysteine ​​residue at position 77 (C77) in framework 3 is mutated to serine (C77S).

[0535] The human V-region IGHV4-4 and IGHJ4-1 J-region (IMGT, http: / / www.imgt.org / ) were selected as acceptors for the heavy chain CDR of antibody 17552. Like many rabbit antibodies, the VH gene of antibody 17552 is shorter than the selected human acceptor. When compared with the human acceptor sequence, the VH region of antibody 17552 lacks N-terminal residues in construct 1, which are retained in the humanized antibody. Figure 4 The structure 3 of the VH region of rabbit 17552 is missing two residues (75 and 76, see SEQ ID NO: 17, gH1) in the loop between D and E of the β-sheet chain: in the humanized grafted variant, this gap is filled by the corresponding residues from the selected human acceptor sequence (lysine 75, K75; asparagine 76, N76). Figure 4 In the humanized grafted variants, the heavy chain framework residues are all derived from human germline genes, except for residues 23, 47, 67, 71, 73, 78, and 96 (see SEQ ID NO: 17, gH1), which retain donor residues threonine (T23), tyrosine (Y47), phenylalanine (F67), lysine (K71), serine (S73), valine (V78), and threonine (T96), respectively. In some humanized grafted variants, the potential aspartic acid isomerization site in CDRH3 is modified by replacing the aspartic acid residue at position 101 with glutamic acid (D101E) or by replacing the glycine residue at position 102 with serine (G102S) or alanine (G102A).

[0536] Genes encoding the humanized V-region sequences of the modified heavy and light chains were designed and constructed by ATUM (Newark, CA) using an automated synthesis method. For transient expression in mammalian cells, the humanized light chain V-region gene was cloned into the UCB light chain expression vector pMhCK, which contains DNA encoding the human κ chain constant region (Km3 allotype). The humanized heavy chain V-region gene was cloned into the UCB human γ-1 heavy chain expression vector pMhg1 L234A L235A, which contains DNA encoding the human γ-1 heavy chain constant region (G1m17,1 allotype) with L234A and L235A mutations to reduce binding to the Fc γ receptor. The rabbit V-region gene of antibody 17552 was also cloned into a human antibody expression vector: the light chain V-region was cloned into a modified version of the human κ vector containing the S171C mutation to recreate the additional disulfide bonds found in the rabbit VK light chain. The resulting heavy and light chain vectors were co-transfected into CHOS-XE suspension cells to produce expression of humanized and chimeric recombinant antibodies in the form of human IgG1 LALA. The antibodies were evaluated by SPR for their binding affinity to CD45 (Table 6).

[0537] The humanized 17552 gL1gH1 antibody retained its binding affinity for CD45 relative to the chimeric parent rabbit antibody (gL1gH1, 7.9 nM, compared to cLcH, 5.6 nM). Modifying the potential aspartic acid isomerization site in the humanized heavy chain graft gH4 by mutating the glycine residue 102 to alanine (G102A) retained affinity compared to gL1gH1 (gL1gH4, 6.5 nM), while mutations in D101E (gH2) and G102S (gH3) both resulted in decreased binding affinity (gL1gH2, 72.3 nM; and gL1gH3, 20.7 nM).

[0538] Table 6

[0539]

[0540] Example 6: Human PBMC cell exhaustion assay using supernatant of monospecific antibodies with different arrangements of 17415 light and heavy chain grafted variants.

[0541] introduction:

[0542] This embodiment provides a further description of the experiments involved in the humanization of the original rabbit 17415 antibody, which were also described in Example 3 above.

[0543] Materials and Methods - PBMC Depletion Determination Method:

[0544] Human PBMCs derived from blood apheresis cones (NHSBT Oxford) were used as frozen aliquots for library construction. Before the assay, one vial of frozen cells (each containing 5 × 10⁶ cells in 1 ml) was prepared. 7 Cells were thawed in a 37°C water bath and then added to 50 ml of complete medium (RPMI 1640 + 2 mM GlutaMAX + 1% Pen / Strep, all supplied by Invitrogen, + 10% fetal bovine serum (FBS), Sigma-Aldrich). Cells were centrifuged (300 g, 5 min, at RT) and resuspended in 50 ml of complete medium for washing, and centrifuged again. Cells were resuspended in 10 ml of complete medium and then counted using a ChemoMetec NucleoCounter NC-3000. Cells were then counted in 50 μl at a concentration of 1×10⁻⁶ cells. 5 One cell / well was added to each well of a CorningCostar 96-well microplate with a U-shaped bottom treated with cell culture (catalog number 07-200-95). PBMCs from seven donors were used in this assay: UCB Cone 032, 034, 036, 037, 045, 858, and 1031.

[0545] Stock solutions were prepared in Greiner 96-well unbound microplates with 400 nM supernatant of each humanized 17415 IgG1 LALA graft in complete medium. The reagents were serially diluted seven times 1:5 in complete medium to generate an 8-point dose profile. 50 μl of each dilution (final well concentration 200–0.0026 nM) was added to the cells and incubated at 37°C, 5% CO2 for 4 h. After incubation, the plates were centrifuged at 300 g for 5 min at RT, buffer was aspirated using a BioTek ELx405 microplate washer, and the cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma Aldrich) for washing, followed by centrifugation and buffer aspiration, leaving the cells in 20 μl of residual medium. For lymphocyte analysis, 20 μl of LIVE / DEAD TMFixable Near-IR Dead Cell Stain (Invitrogen, 1:5000 dilution) was added to the wells and incubated at 4°C for 15 minutes. For CD3 staining, 20 μl of a solution containing anti-human CD3 FITC (BDBiosciences, catalog number 561806, 1:50 dilution) and LIVE / DEAD was used. TM Cells were stained with an antibody solution of Fixable Near-IR Dead CellStain (Invitrogen, 1:5000 dilution). After incubation, plates were centrifuged at 300 g for 5 minutes at RT, buffer was aspirated using a BioTek ELx405 microplate washer, and cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma-Aldrich) for washing, followed by centrifugation and buffer aspiration, leaving cells in 20 μl of residual culture medium. Cell viability was analyzed using Intellicyt iQue Screener PLUS and iQUE 3. Viable cell counts were extracted as a metric and graphical representations were generated using Graphpad Prism version 8.1 (Graphpad). Asymmetric (4-parameter) curve fitting was applied to derive EC50 and Emax values.

[0546] result:

[0547] The percentage reduction in lymphocytes in a PBMC population treated with 17415 IgG1 LALA heavy chain H1 and five light chain grafts (L1-5) is shown in Figure 5(A). The percentage reduction in lymphocytes in a PBMC population treated with 17415 light chain L1 and four heavy chain grafts (H1-4) is shown in Figure 5(B). Data from a single donor, Cone 1031, are shown. Humanization alterations in both the heavy and light chains, compared to the chimeric conjugate (cLcH), reduced the EC50 and Emax of the 17415 IgG1 LALA antibody.

[0548] To identify humanized mutations that reduce the functional killing effect of 17415 IgG1 antibodies, each light chain graft (L1-5) was paired with a chimeric heavy chain (cH), and the percentage reduction in lymphocytes in the PBMC population is shown in Figure 6(A). All humanized light chain grafts showed reduced EC50 and Emax for 17415 IgG1 LALA antibodies compared to the chimeric heavy chain (cLcH). Each heavy chain graft (H1-4) was paired with a chimeric light chain (cL), and the percentage reduction in lymphocytes in the PBMC population is shown in Figure 6(B). Data from a single donor, Cone 858, are shown. Humanized heavy chain grafts showed a smaller effect on EC50 and Emax for 17415 IgG1 LALA antibodies compared to the chimeric heavy chain (cLcH), suggesting that the humanized light chain contributes more to the loss of functional killing.

[0549] To enhance the functional killing effect of humanized 17415 IgG1 LALA, further heavy chains (H5 and H6) were tested with further light chains (L6, 7, 13, 14, 15, 16). The percentage reduction in T cells in PBMC populations treated with these humanized grafts is shown in Figure 7. Humanized 17415 grafts with heavy chain H5 are shown in Figure 7(A), and humanized 17415 grafts with heavy chain H6 are shown in Figure 7(B). Data from Cone 036 are shown as representative of data from three donors. Compared to H5, humanized 17415 grafts containing heavy chain H6 showed similar Emax and EC50 values ​​to chimeric (cLcH) 17415. Among the grafts containing H6, L7, L15, and L16, the light chain grafts showed the best functional and humanized properties.

[0550] Example 7: Human Jurkat cell exhaustion assay using supernatants of monospecific antibodies with different arrangements of 17415 light and heavy chain grafted variants.

[0551] Jurkat Depletion Test

[0552] Before performing the assay, one vial of Jurkat (acute T-cell leukemia cell line) was thawed in a 37°C water bath and then added to 20 ml of complete medium (RPMI 1640 + 2 mM 25 GlutaMAX + 1% Pen / Strep, all supplied by Invitrogen, + 10% fetal bovine serum (FBS), Sigma Aldrich). The cells were centrifuged (300 g, 5 min, at RT) and resuspended in 20 ml of complete medium for washing, and centrifuged again. The cells were resuspended in 10 ml of complete medium and counted using a ChemoMetec NucleoCounter NC-3000. Then, 1×10⁻⁶ cells were added to 50 μl of complete medium. 5 One cell was added to each well of a Corning Costar 96-well microculture plate with a U-shaped bottom and pre-treated cell culture (catalog number 07-200-95).

[0553] Stock solutions were prepared in Greiner 96-well unbound microplates with 400 nM supernatant of each humanized 17415 IgG1 LALA graft in complete medium. The reagents were serially diluted seven times 1:5 in complete medium to generate an 8-point dose profile. 50 μl of each dilution (final well concentration 200–0.0026 nM) was added to the cells and incubated at 37°C, 5% CO2 for 4 h. After incubation, the plates were centrifuged at 300 g for 5 min at RT, buffer was aspirated using a BioTek ELx405 microplate washer, and the cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma Aldrich) for washing, followed by centrifugation and buffer aspiration, leaving the cells in 20 μl of residual medium. 10 μL of Sytox Blue (Invitrogen, 1:500 dilution) was added to each well, and the cells were incubated at RT for 10 min. Cell viability analysis was performed using Intellicyt iQueScreener PLUS and iQUE 3. Viable cell counts were extracted as a metric and graphical representations were generated using Graphpad Prism version 8.1 (Graphpad). Asymmetric (4-parameter) curve fitting was applied to derive EC50 and Emax values.

[0554] result:

[0555] The results obtained for different arrangements of the 17415 light chain and heavy chain grafted variants are shown in parts (A) to (D) of Figure 8.

[0556] Example 8: Human PBMC cell exhaustion assay using purified monospecific antibodies with 17415 light chain grafted variants 7, 15, and 16

[0557] Materials and Methods - Determination of PBMC Depletion

[0558] Human PBMCs derived from blood apheresis cones (NHSBT Oxford) were used as frozen aliquots for library construction. Before the assay, one vial of frozen cells (each containing 5 × 10⁶ cells in 1 ml) was prepared. 7 Cells were thawed in a 37°C water bath and then added to 50 ml of complete medium (RPMI 1640 + 2 mM GlutaMAX + 1% Pen / Strep, all supplied by Invitrogen, + 10% fetal bovine serum (FBS), Sigma-Aldrich). Cells were centrifuged (300 g, 5 min, at RT) and resuspended in 50 ml of complete medium for washing, and centrifuged again. Cells were resuspended in 10 ml of complete medium and then counted using a ChemoMetec NucleoCounter NC-3000. Cells were then counted in 50 μl at a concentration of 1×10⁻⁶ cells. 5 One cell / well was added to each well of a CorningCostar 96-well microplate with a U-shaped bottom and pre-treated cell culture (catalog number 07-200-95). PBMCs from three donors were used in this assay: UCB-Cone 1033, 1001, and 1000.

[0559] Stock solutions were prepared in Greiner 96-well unbound microplates at 500 nM each of the purified humanized 17415 IgG1 LALA graft, 17552 IgG1 LALA graft, 17552 IgG1, and isotype control IgG1 (5604) in complete medium. The reagents were serially diluted seven times at a 1:5 ratio in complete medium to generate an 8-point dose profile, using one concentration (final well concentration 250 nM) except for 17552 IgG1 and 5604 IgG1. 50 μl of each dilution (final well concentration 250–0.0032 nM) was added to cells and incubated at 37°C, 5% CO2 for 4 h. After incubation, the plates were centrifuged at 300 g for 5 minutes at RT. Buffer was aspirated using a BioTek ELx405 microplate washer, and the cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2mM EDTA, Sigma Aldrich) for washing. The plates were then centrifuged again, and buffer was aspirated, leaving 20 μl of residual culture medium for the cells. For CD3 staining, 20 μl of a solution containing anti-human CD3 FITC (BDBiosciences, catalog number 561806, 1:50 dilution) and LIVE / DEAD was used at 4°C. TM Cells were stained with an antibody solution of Fixable Near-IR Dead CellStain (Invitrogen, 1:5000 dilution) for 30 minutes. After incubation, plates were centrifuged at 300 g for 5 minutes at RT, buffer was aspirated using a BioTek ELx405 microplate washer, and cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma-Aldrich) for washing, followed by centrifugation. Cells were washed again, and buffer was aspirated to leave cells in 20 μl of residual culture medium. Cell viability was analyzed using Intellicyt iQue 3. Viable cell counts were extracted as a metric and graphical representations were generated using Graphpad Prism version 8.1 (Graphpad). Asymmetric (4-parameter) curve fitting was applied to derive EC50 and Emax values.

[0560] result:

[0561] The percentage reduction in T cells in PBMC populations treated with humanized 17415 IgG1 LALA grafts containing 17415 light chain modifications 7, 15, and 16, and with humanized 17552 IgG1 LALA grafts containing 17552 heavy chain modifications 1 and 4, was shown in [data missing]. Figure 9 In the middle. Data from Cone 1000 are shown as representative of data from three donors. All grafts showed T-cell reduction, with an Emax range of 63.4-83.3% and an EC50 range of 0.21-0.85 nM. The 17552 IgG1 LALA graft did not deplete T-cells.

[0562] Example 9: Jurkat cell exhaustion assay using purified monospecific antibodies with 17415 light chain grafted variants 7, 15, and 16

[0563] Jurkat depletion assay:

[0564] Before performing the assay, one vial of Jurkat (acute T-cell leukemia cell line) was thawed in a 37°C water bath and then added to 20 ml of complete medium (RPMI 1640 + 2 mM 25 GlutaMAX + 1% Pen / Strep, all supplied by Invitrogen, + 10% fetal bovine serum (FBS), Sigma Aldrich). The cells were centrifuged (300 g, 5 min, at RT) and resuspended in 20 ml of complete medium for washing, and centrifuged again. The cells were resuspended in 10 ml of complete medium and counted using a ChemoMetec NucleoCounter NC-3000. Then, 1×10⁻⁶ cells were added to 50 μl of complete medium. 5 One cell was added to each well of a Corning Costar 96-well microculture plate with a U-shaped bottom and pre-treated cell culture (catalog number 07-200-95).

[0565] Stock solutions were prepared in Greiner 96-well unbound microplates for each humanized 17415 IgG1 LALA graft, 17552 IgG1, and allotype control IgG1 (5604) at 500 nM in complete medium. The reagents were serially diluted seven times at a 1:5 ratio in complete medium to generate an 8-point dose profile, using one concentration (final well concentration 250 nM) except for 17552 IgG1 and 5604 IgG1. 50 μl of each dilution (final well concentration 250–0.0032 nM) was added to cells and incubated at 37°C, 5% CO2 for 4 hours. After incubation, plates were centrifuged at 300g for 5 minutes at RT. Buffer was aspirated using a BioTek ELx405 microplate washer, and cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma Aldrich) for washing, followed by centrifugation and buffer aspiration, leaving cells in 20 μl of residual culture medium. 10 μL of Sytox Blue (Invitrogen, 1:500 dilution) was added to the wells, and the plates were incubated at RT for 10 minutes. Cell viability was analyzed using Intellicyt iQue 3. Live cell counts were extracted as a metric, and a graphical representation was generated using GraphpadPrism version 8.1 (Graphpad). Asymmetric (4-parameter) curve fitting was applied to derive EC50 and Emax values.

[0566] result:

[0567] The percentage reduction of Jurkat cells in a PBMC population treated with humanized 17415 IgG1 LALA grafts containing 17415 light chain modifications 7, 15, and 16 was shown in [data missing]. Figure 10 In the 17415 grafts, all showed a reduction in Jurkat cells, with an Emax range of 87.9-89.4% and an EC50 range of 0.08-0.59 nM. The 17552 IgG1 did not deplete Jurkat cells.

[0568] Example 10: Evaluation of the binding of a monospecific CD45 antibody to ExpiHEK cells expressing human CD45

[0569] Materials and methods:

[0570] Use Lipofectamine TMRNAiMAX transfection reagent (RNAiMAX, Invitrogen), using human CD45 ROECD mRNA to transfect Expi293F TM (Gibco) cells. Prior to transfection, in Expi293... TM Expression Medium (Gibco) uses 5×10 5 Expi293F cells were prepared at 100 μL / mL. A total of 2 μg of mRNA was added to 100 μL of Opti-MEM. TM Dilute in Reduced Serum Medium (Opti-MEM, Invitrogen) and mix with 3 μL of RNAiMAX pre-diluted in 100 μL of Opti-MEM. After incubating at room temperature for 10–20 minutes, add 50 μL of the mixture to each well of a 24-well plate (Corning) to a final volume of 0.5 mL of medium. Incubate cells at 37°C and 5% CO2 for 18–24 hours to allow protein expression. Collect cells from the 24-well plates and dilute in 10 mL of FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma Aldrich) and centrifuge (200 x g, 6 min). Resuspend cells in 10 mL of FACS buffer and count using a ChemoMetec NucleoCounter NC-3000. Then, 20,000 cells / well in 25 μl were added to each well of a Corning Costar 96-well microculture plate with a U-shaped bottom treated with cell culture (catalog number 07-200-95).

[0571] Stockpile was prepared in Greiner 96-well unbound microplates for each purified humanized 17415 IgG1 LALA graft and control molecule at 500 nM in FACS buffer. The reagents were serially diluted seven times at a 1:5 ratio in FACS buffer to generate an 8-point dose profile. 25 μl of each dilution (final well concentration 250–0.0032 nM) was added to cells and incubated at 4°C on a shaker for 1 h. Cells were then incubated at 4°C with 25 μl of goat anti-human IgG Fcγ containing the anti-human IgG AF488Fab fragment (Jackson ImmunoResearch, catalog number 109-547-008, 1:200 dilution) and LIVE / DEAD. TMCells were stained with secondary antibody solution of Fixable Near-IR Dead Cell Stain (Invitrogen, 1:5000 dilution) for 1 hour. After incubation, plates were centrifuged at 300 g for 5 minutes at RT. Buffer was aspirated using a BioTek ELx405 microplate washer, and cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma Aldrich) for washing, followed by centrifugation. Cells were washed again, and buffer was aspirated to leave cells in 20 μl of residual culture medium. Cell viability was analyzed using Intellicyt iQue 3. The fluorescence geometric mean was extracted as a metric and a graphical representation was generated using Graphpad Prism version 8.1 (Graphpad).

[0572] Protein sequence:

[0573] >Hu_CD45_RO ECD (SEQ ID NO: 136)

[0574] MTMYLWLKLLAFGFAFLDTEVFVTGQSPPTPSPTDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVH NLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNAS KIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTV SMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSKALIAFLAFLIIVTSIALLVVLYKIYDL

[0575] result:

[0576] The mean fluorescence intensity (MFI, geometric mean) of the binding of humanized 17415 IgG1 LALA graft containing 17415 light chain modifications 7, 15, and 16 to HEK cells expressing human CD45 was shown in the figure. Figure 11 In the middle, together with the isotype control IgG1 (5604).

[0577] Example 11: Generation of dual complementary site pattern antibodies

[0578] introduction :

[0579] Various double-complementary-site antibodies were generated and then evaluated in subsequent examples. Therefore, this example describes possible methods for generating double-complementary-site antibodies and their initial evaluation.

[0580] Parental antibody expression:

[0581] For the KiH heterodimerization technique, the KiH mutation (T366W) and KiH mutation (T366S L368AY407V) were introduced into their respective heavy chain constant domains to promote the formation of dual complementary antibody sites. As a first step, the parental antibody was expressed. Genes encoding the respective light and heavy chain V-regions of the antibody were constructed using an automated synthesis method (ATUM). Following the supplier's instructions, the DNA was amplified using the QIAGEN Plasmid Plus Giga Kit (catalog number 12991), and finally, ExpiCHO was used. TM The Expression System Kit (A29133) was used to transfect the cells into CHO-SXE cells. The supernatant was collected after 7 days.

[0582] Protein exchange methods:

[0583] The supernatant of CD45-specific IgG1 was purified using 2 × 5 mL MabSelect Sure (Cytiva) columns attached to an AKTA Pure purification system (GE Healthcare Life Sciences). Once the column was equilibrated with PBS, the clarified culture supernatant was loaded onto the column at a flow rate of 10 ml / min. After loading, the column was washed with 4 CV of PBS, and the bound product was eluted from the column with 0.1 M sodium citrate (pH 3.4). The eluent was neutralized with 1 / 5 of the elution volume of 2 M Tris-HCl (pH 8.5).

[0584] Once eluted and neutralized, the concentration of the affinity-treated material was determined, and heavy chains 1 through 2 were combined. Cysteine ​​(Sigma-M9768) was added to the mixture to give a final concentration of 5 mM (from a 500 mM stock in PBS), and the mixture was incubated at room temperature for 4 hours. The material was then concentrated to approximately 4 mL using Amicon® Ultra-15 Centrifugal Filter Units (Merck-UFC903024) and loaded onto a HiLoad 26 / 600 Superdex 200 pg column (Cytiva) and eluted with PBS. The desired fractions were collected and subjected to endotoxin removal using a Proteus NoEndoHC column (Protein Ark), and finally treated with Steriflip. TM The vacuum filtration unit (Merck) performs sterile filtration.

[0585] Characterization and quality control:

[0586] Protein concentration was calculated after measuring A280 absorbance readings using the Lunatic system (Unchained Labs). Monomer percentage was determined by loading 2 μg of protein onto an ACQUITY BEH200 column attached to a Waters ACQUITY UPLC system. HIC analysis was performed using a DionexProPac HIC-10 column attached to an Agilent 1200 binary HPLC system with a fluorescence detector. SDS-PAGE was performed using a 4-20% Tris-glycine gel electrophoresis run at 180 V for 45 min. Endotoxin was determined using an Endosafe nexgen-MCS system (Charles River). Determination was performed using NuPAGE according to the supplier's instructions. TM Sample Reducing Agent (10X) (Invitrogen-NP0004) and / or Rapid TM PNGase F (Neb-P0710s) was used to treat the samples to determine the quality of the constructs. The samples were then loaded onto an XEVO G2 QTof (Waters) column equipped with a BioResolveT RP mAb Polyphenyl, 450 Å, 2.7 μm column.

[0587] result:

[0588] The results of specific experiments on the generated double complementary site antibodies are described in the following examples.

[0589] Example 12: Evaluation of the binding of a dual complementary CD45 antibody to ExpiHEK cells expressing human and cynomolgus monkey CD45

[0590] introduction:

[0591] As discussed further below, regarding its relationship with Expi293F... TM The ability of (Gibco) cells to bind to human or cynomolgus monkey CD45 RO ECD was evaluated in various bispecific humanized 17415-17552 IgG1 LALA grafts containing 17415 light chain modification. The ability to bind to both cynomolgus monkeys and humans is useful because the former means the antibody can be studied in cynomolgus monkeys prior to human trials.

[0592] Materials and Methods - Cell-based Approach

[0593] The same cell binding assay described above for monospecific CD45 antibodies was used to evaluate the results compared to those achieved using Lipofectamine. TM RNAiMAX transfection reagent (RNAiMAX, Invitrogen) uses Expi293F transfection kit containing human or cynomolgus monkey CD45 ROECD mRNA. TM (Gibco) binding to CD45 on the surface of cells.

[0594] As described, stockpiles were prepared in 500 nM of each purified humanized 17415-17552 IgG1 LALA and control molecule grafts in FACS buffer.

[0595] Protein sequence:

[0596] >Hu_CD45_RO ECD (SEQ ID NO: 136)

[0597] MTMYLWLKLLAFGFAFLDTEVFVTGQSPTPSPTDAYLNASETTTLSPSGSAVISTTTIATTPSKPTCDEKYANITVDYLYNKETKLFTAKLNVNENVECGNNTCTNNEVHNLTECKNASVSISHNSCTAPDKTLILDVPPGVEKFQLHDCTQVEKADTTICLKWKNIETFTCDTQNITYRFQCGNMIFDNKEIKLENLEPEHEYKCDSEILYNNHKFTNASKIIKTDFGSPGEPQIIFCRSEAAHQGVITWNPPQRSFHNFTLCYIKETEKDCLNLDKNLIKYDLQNLKPYTKYVLSLHAYIIAKVQRNGSAAMCHFTTKSAPPSQVWNMTVSMTSDNSMHVKCRPPRDRNGPHERYHLEVEAGNTLVRNESHKNCDFRVKDLQYSTDYTFKAYFHNGDYPGEPFILHHSTSYNSKALIAFLAFLIIVTSIALLVVLYKIYDL

[0598] >Cyno_CD45_RO ECD(SEQ ID NO: 137)

[0599] MTMCLWLKLLAFVFAFLDTEVFVTGQGSTLSPTVSYLNASETTTPSPSGSTVISTPTIATTTSKPTCAEKYATIPVDYLYNNKTKLFTAKLNVNENVECTNNNHTHNICTNNEVLNLPECKEMNVFVSHNSCTDRHKELKLDVPPEVEKFQLDDCTPDVEANTTICLKWKIIETFACDKSKITYRFQCGNKTYNKEGIYLENLEPEYEYKCDSEILYNNHKYINITKLIKTDFGIPGQPQNVVCRHEDAHQGVITWNPPQRSFHNFTLCYVNKPAKKCLILDKHLTTYHLQNLKPYTNYSLSLHAYIIAKVQRNGTAATCNFTTESAPPSQVQNMIVSTSDNSMHVKCEVPRDVNGPTGLYHLEVEAGNTLVRNLSQSKCDFSVNNLQYSTYYNLKAYYHNGKYSGEPVILRESTSYNSKALIAFLAFLIIVTSIALLVVLYKIYDL

[0600] result

[0601] The mean fluorescence intensity (MFI, geometric mean) of the binding of humanized 17415-17552 IgG1 LALA graft containing 17415 light chain modifications 7 (A), 15 (B), and 16 (C) to HEK cells expressing human CD45 is shown in Figure 12, along with relevant control molecules. The MFI of the binding of humanized 17415-17552 graft containing 17415 light chain modifications 7 (A), 15 (B), and 16 (C) to HEK cells expressing cynomolgus monkey CD45 is shown in Figure 13, along with relevant control molecules. Each light chain is paired with 17552 heavy chain grafts H1 and H4. The 17415-17552 grafts show a variation range of 4 × 10⁻⁶. 6 -6.8×10 6 (Regarding human CD45 binding) and 10×10 6 -11.3×10 6 Emax MFI (regarding cynomolgus CD45 binding) indicates that all bicomponent antibody grafts bind well to human and cynomolgus CD45 expressed on cells.

[0602] Example 13: Human T-cell exhaustion assay using antibodies with dual complementary sites of 17415 light chain grafted variants 7, 15, and 16

[0603] Materials and Methods - Determination of PBMC Depletion

[0604] The human PBMC depletion assay used was the same as that used in earlier examples, but the assay was performed using a stock of purified humanized 17415-17552 IgG1 LALA grafts at 500 nM each and a control antibody. PBMCs from three donors were used in this assay: UCB-Cone 1001, 1000, and 947.

[0605] result:

[0606] The percentage reduction in T cells in a PBMC population treated with humanized 17415-17552 IgG1 LALA grafts containing 17415 light chain modifications 7 (A), 15 (B), and 16 (C) is shown in Figure 14, along with relevant control molecules. Data from Cone 1000 are shown as representative of data from three donors. Each light chain was paired with 17552 heavy chain grafts H1 and H4. All grafts showed T cell reduction with an Emax range of 60.2–88.2% and an EC50 range of 0.02–0.21 nM, demonstrating successful cell exhaustion. The most effective T cell exhaustion double-complementary graft was 17415gL7gH6-17552gL1gH4 IgG1 LALA.

[0607] Example 14: Jurkat cell exhaustion assay using antibodies with dual complementary sites of 17415 light chain grafted variants 7, 15, and 16

[0608] introduction

[0609] Jurkat cells were used again as a model for cell exhaustion in cancer cells targeting CD45.

[0610] Materials and methods:

[0611] The method employed is as described in earlier embodiments. As described in other embodiments, stockpiles are prepared with each humanized 17415-17552 IgG1 LALA graft and control antibody at 500 nM in complete culture medium.

[0612] result:

[0613] The percentage reduction in Jurkat cells treated with humanized 17415-17552 IgG1 LALA grafts containing 17415 light chain modifications 7 (A), 15 (B), and 16 (C) is shown in Figure 15, along with relevant control molecules. Each light chain was paired with 17552 heavy chain grafts H1 and H4. All grafts showed Jurkat cell reduction, with an Emax range of 83–95.7% and an EC50 range of 0.14–0.33 nM. The most efficient bicomponent molecules for depleting Jurkat cells were 17415gL15gH6-17552gL1gH1 IgG1 LALA.

[0614] Example 15: Cell exhaustion assay in cynomolgus monkey PBMCs

[0615] introduction

[0616] This embodiment describes a method for evaluating the ability of both monospecific and dual complementary CD45 antibodies to deplete cynomolgus monkey cells.

[0617] Materials and Methods - Assay for T-cell exhaustion in cynomolgus monkeys

[0618] Cynomolgus monkey PBMCs were obtained from Primacyt as frozen aliquots. Before performing the assay, one vial of frozen cells (each containing 2 × 10⁶ cells in 1 ml) was prepared. 7 Cells were thawed in a 37°C water bath and then added to 50 ml of complete medium (RPMI 1640 + 2 mM GlutaMAX + 1% Pen / Strep, all supplied by Invitrogen, + 10% fetal bovine serum (FBS), Sigma Aldrich). Cells were centrifuged (300 g, 5 min, at RT) and resuspended in 50 ml of complete medium for washing, and centrifuged again. Cells were resuspended in 10 ml of complete medium and then counted using a ChemoMetec NucleoCounter NC-3000. Cells were then counted in 50 μl at a concentration of 1×10⁻⁶ cells. 5 One cell / well was added to each well of a Corning Costar 96-well microculture plate with a U-shaped bottom and pre-treated cell culture (catalog number 07-200-95).

[0619] Stock solutions were prepared in Greiner 96-well unbound microplates for each purified 17415 IgG1, 17552 IgG1, 17415-17552 IgG1, 4133-6294 IgG1, and isotype IgG1 antibody at 500 nM in complete medium. The reagents were serially diluted seven times at a 1:5 ratio in complete medium to generate an 8-point dose profile. 50 μl of each dilution (final well concentration 250–0.0032 nM) was added to cells and incubated at 37°C and 5% CO2 for 24 h. After incubation, the plates were centrifuged at 300 g for 5 minutes at RT. Buffer was aspirated using a BioTek ELx405 microplate washer, and the cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma Aldrich) for washing. The plates were then centrifuged again, and buffer was aspirated, leaving 20 μl of residual culture medium for the cells. For CD3 staining, 20 μl of a solution containing anti-human / NHP CD3BV605 (BD Biosciences, catalog number 562994, 1:50 dilution) and LIVE / DEAD was used at 4°C. TM Cells were stained with an antibody solution of Fixable Near-IR Dead Cell Stain (Invitrogen, 1:5000 dilution) for 30 minutes. After incubation, plates were centrifuged at 300 g for 5 minutes at RT, buffer was aspirated using a BioTek ELx405 microplate washer, and cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma Aldrich) for washing, followed by centrifugation. Cells were washed again, buffer was aspirated, leaving cells in 20 μl of residual culture medium. Cell viability was analyzed using Intellicyt iQue 3. Viable cell counts were extracted as a metric and graphical representations were generated using Graphpad Prism version 8.1 (Graphpad). Asymmetric (4-parameter) curve fitting was applied to derive EC50 and Emax values.

[0620] result:

[0621] The percentage reduction in T cells in a cynomolgus monkey PBMC population treated with 17415-17552 IgG1, 17552 IgG1, 17415 IgG1, 4133-6294 IgG1, and isotype IgG1 was shown in Figure 16Among the antibodies for which the results are shown in the figure, only the bicomplementary site 17415-17552 IgG1 depleted cynomolgus monkey T cells at a high level (Emax 75%, EC50 1.5 nM).

[0622] Example 16: Comparison of the ability of 17415-17552 with other known dual complementary antibodies to kill cells expressing human CD45

[0623] introduction:

[0624] This embodiment investigated the ability of antibodies 17415-17552 to kill CD45-expressing target cells, compared to the IgG1 patterns of antibodies 4133-6294 and YTH24.5-YTH54.12 described in international application PCT / EP2021 / 078516 (published as WO 2022 / 079199 A1). The variable region (V-region) sequence of the anti-CD45 antibody YTH24.5-YTH54.12 was taken from international application PCT / GB2021 / 052458 (published as WO 2022 / 064191 A1). None of these CD45 dual-complementary site antibodies were conjugated to cytotoxic agents.

[0625] PBMC depletion determination method:

[0626] The human PBMC depletion assay used was the same as that used in the earlier examples, but the assay was performed using reserves of 4133-6294 IgG1, 17415-17552 IgG1, YTH24.5-YTH54.12 IgG1, and 5604 IgG1 at 500 nM each. In this assay, PBMCs from four donors, UCB-Cone 1001, 1000, 1033, and 947, were used to compare 4133-6294 IgG1 and 17415-17552 IgG1, and PBMCs from one donor, UCB-Cone 924, were used to compare 17415-17552 IgG1 and YTH24.5-YTH54.12 IgG1.

[0627] Jurkat depletion assay:

[0628] The method performed is as described in earlier embodiments. Stocks of 4133-6294 IgG1, 17415-17552 IgG1, YTH24.5-YTH54.12 IgG1, and 5604 IgG1, each at 500 nM, were used.

[0629] result:

[0630] The percentage reduction in T cells in PBMC populations treated with 4133-6294 IgG1 or 17415-17552 IgG1 was shown in Figure 17 In comparison, both 4133-6294 IgG1 and 17415-17552 IgG1 showed T cell reduction, compared to the isotype (5604 IgG1) control. 4133-6294 IgG1 had 80.7-89.9% of the Emax range and an EC50 range of 0.17-0.53 nM, while 17415-17552 IgG1 had 74.4-83.5% of the Emax range and an EC50 range of 0.08-0.4 nM.

[0631] The percentage reduction in T cells in PBMC populations treated with 17415-17552 IgG1 or YTH24.5-YTH54.12 IgG1 was shown in Figure 18 In comparison, the isotype (5604 IgG1) was compared with the control. Only 17415-17552 IgG1 showed T cell reduction. 17415-17552 IgG1 had an Emax of 88.8% and an EC50 of 0.05 nM. The Emax and EC50 of YTH24.5-YTH54.12 IgG1 were indeterminate.

[0632] The percentage reduction in Jurkat cells treated with 4133-6294 IgG1 or 17415-17552 IgG1 is shown in the figure. Figure 19 In comparison, the 4133-6294 IgG1 and 17415-17552 IgG1 strains showed a reduction in Jurkat cells, compared to the isotype (5604 IgG1) control. The 4133-6294 IgG1 strain had 91.9% Emax and an EC50 of 0.47 nM, while the 17415-17552 IgG1 strain had 92.7% Emax and an EC50 of 0.06 nM.

[0633] The percentage reduction in Jurkat cells treated with 17415-17552 IgG1 or YTH24.5-YTH54.12 IgG1 is shown in the figure. Figure 21 In comparison, the IgG1 strain (5604 IgG1) was compared with the isotype (5604 IgG1) control. Only IgG1 strains 17415–17552 showed a reduction in Jurkat cells. IgG1 strains 17415–17552 had an Emax of 90.9% and an EC50 of 0.05 nM. The Emax and EC50 of YTH24.5–YTH54.12 IgG1 were indeterminate.

[0634] In both PBMCs and Jurkat cells, the EC50 of the IgG1 antibody with bicomplementary sites 17415-17552 was lower than that of the antibody with bicomplementary sites 4133-6294. The IgG1 antibody with bicomplementary sites YTH24.5-YTH54.12 could not induce killing of T cells or Jurkat cells.

[0635] in conclusion:

[0636] In summary, this data shows that the claimed dual complementary CD45 antibody has increased potency compared to previously known dual complementary CD45 antibodies.

[0637] Example 17: Cytokine release in whole blood at 24 hours, measured by Meso Scale Discovery (MSD) assay.

[0638] introduction:

[0639] This study investigated the ability of CD45 antibodies to induce cytokine release in whole blood. Cytokine release can drive unwanted inflammation in treated subjects.

[0640] Materials and methods:

[0641] Human whole blood (heparinized lithium tubes) was collected from two donors at UCB Pharma Slough, UK, in accordance with the approved ethical sample collection procedure.

[0642] Stocks of purified 17415gL15gH6-17552gL1gH4 IgG1 LALA, 17415gL15gH6 IgG1 LALA, YTH24.5-YTH54.12 IgG1, and 5604 IgG1 LALA antibodies were prepared in PBS at 8000 nM in Greiner 96-well non-binding microplates. The reagents were then serially diluted seven times at a 1:5 ratio in PBS to generate an 8-point dose profile. Camphor was diluted in PBS to produce a 200 μg / ml stock.

[0643] Transfer 12.5 μl of reagent diluent to a Corning Costar 96-well microplate with a U-shaped bottom and pre-treated with cell culture (catalog number 3799), and add 237.5 μl of whole blood to each well. The final antibody concentration was 400–0.00512 nM. Campath (diluted from 10 mg / ml stock to 0.2 mg / ml in PBS, lot number CHV0387) was used as a positive control at a final concentration of 10 μg / ml. Seal the plates with a breathable adhesive sealing film and recap them. Then, incubate the plates in an undisturbed position at 37°C and 5% humidified CO2 for 24 hours.

[0644] After 24 hours of incubation, the plates were centrifuged at 1000 g for 10 minutes, and 50 μl of plasma was transferred to separate plates and stored at -80°C until cytokine quantification analysis.

[0645] Following the manufacturer's instructions, cytokine measurements were performed using the V-PLEX Human Proinflammatory Panel I (which includes interferon (IFN)-γ, interleukin (IL)-6, and tumor necrosis factor (TNF)-α, catalog number K15052D, Meso Scale Discovery). In short, plasma samples were thawed at RT and diluted 1:2 with Diluent 2 (catalog number R51BB-3, Meso Scale Discovery). A standard curve calibrator was prepared using 500 μl of Diluent 2 (catalog number R51BB-3, Meso Scale Discovery). The Proinflammatory Panel I plates were washed with PBS (supplemented with 0.05% Tween-20) using a BioTek ELx405 microplate washer, and 50 μl of sample or standard curve calibrator was added to each well. The plates were sealed with an adhesive sealing film and incubated at RT on a plate shaker (750 rpm) for 2 hours. Wash the plates as before and add 25 μL of detection antibody to each well. Incubate the plates on a plate shaker at RT for 2 hours. Wash the plates as before and add 150 μL of read buffer (diluted 1:2 in dH2O) to each well. Then analyze the plates on a SECTOR Imager 6000 (MesoScale Discovery).

[0646] result:

[0647] The levels of the inflammatory cytokines detected are shown in Figure 20: (A) IFN-γ, (B) IL-6, and (C) TNF-α. Notably, little or no induction of inflammatory cytokines was observed by 17415gL15gH6-17552gL1gH4 and 17415gL15gH6 IgG1 LALA, with levels matching those observed in wells treated with PBS and 5604 IgG1 LALA.

[0648] in conclusion:

[0649] In summary, these data show that the claimed antibody induces cell death without inducing a significant increase in cytokine release. The absence of cytokines reduces the likelihood of unwanted cytokine-driven inflammation in treated subjects.

[0650] Example 18: Exhaustion assay in leukemia T-cell and leukemia B-cell lines

[0651] introduction:

[0652] This embodiment investigates the ability of the claimed CD45 antibody to deplete immune cells from different leukemia cell lines.

[0653] Materials and Methods - Cell Line Exhaustion Assay:

[0654] Prior to the assays, one vial each of (a) leukemia T-cell lines (SUDHL1, SUPT11, and Peers) and (b) leukemia B-cell lines (DOHH2 and Ramos) were thawed in a 37°C water bath and then added to 5 ml of complete culture medium (RPMI 1640, Corning, +20% fetal bovine serum (FBS), Gibco, for Ramos, DOHH2, and SUDHL1; RPMI 1640 +10% FBS, for SUPT11 and Peers) for culture. For the exhaustion assay, cells were centrifuged (200 g, 5 min, at RT) and resuspended in 5 ml of complete culture medium for washing, followed by centrifugation again. Cells were resuspended in 5 ml of complete culture medium and counted using a Biorad TC20™ automated cell counter. Then, 1×10⁶ cells were added to 90 μl of complete culture medium. 5 One cell was added to each well of a Corning Costar 96-well microculture plate with a U-shaped bottom and pre-treated cell culture (catalog number 3879).

[0655] Stockpile was prepared in Sarstedt 96-well microplates (catalog number 83.3924.005) to contain 2 μM of purified 17415gL15gH6-17552gL1gH4 IgG1 LALA, 17415gL15gH6 IgG1 LALA, and isotype control IgG1 LALA (5604) in complete medium. The antibodies were serially diluted nine times at a ratio of 1:3.16 in complete medium to generate a 10-point dose profile. 10 μL of each dilution (final well concentration 200–0.002 nM) was added to the cells and incubated at 37°C, 5% CO2 for 2 h. After incubation, 150 μL of PBS was added to each well, and the plates were centrifuged at 400 g for 5 min at RT. The supernatant was discarded by inverting the plates and blotting them dry on thin paper. Resuspend the cells in 150 μl of PBS, centrifuge the plate again, and discard the supernatant. For viability staining, use 50 μl of a solution containing LIVE / DEAD at 4°C. TM Cells were stained with Fixable Near-IR Dead Cell Stain (Invitrogen, 1:1000 dilution) for 30 minutes. After incubation, 150 μl of FACS buffer (PBS + 1% bovine serum albumin (BSA), Fisher BioReagents) was added to each well, and the plates were centrifuged at 400 g for 5 minutes at RT, discarding the supernatant. Cells were washed again with FACS buffer and then resuspended in 150 μl of FACS lysis buffer (BDBiosciences, catalog number 349202). For flow cytometry analysis, 100 μl / well volume was obtained on an Attune NxT flow cytometer (Invitrogen). Viable single-cell counts were extracted as a metric and graphical representations were generated using GraphpadPrism version 9.0 (Graphpad). Asymmetric (4-parameter) curve fitting was applied to derive EC50 and Emax values.

[0656] result:

[0657] The percentage reductions in Peers, SUPT11, and SUDHL1 T-cells treated with 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA are shown in the figures below. Figure 22In A, 22B, and 22C, along with the isotype control (5604 IgG1 LALA), the percentage reduction in Ramos and DOHH2 B- cells treated with 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA is shown in the figure. Figure 23 In A and 23B, together with the isotype control (5604 IgG1 LALA).

[0658] Among the cell lines tested, the monospecific antibody 17415gL15gH6 IgG1LALA showed significant reductions in certain T-cell lines (Peers and SUPT11) and certain B-cell lines (Ramos) compared to the control. The Emax ranged from 28.4% to 66.8%, and the EC50 ranged from 0.45% to 18.58 nM.

[0659] Compared to the control, the dual complementary antibody 17415gL15gH6-17552gL1gH4 IgG1 LALA showed a significant reduction in all tested T-cell and B-cell lines (Peers, SUPT11, SUDHL1, Ramos, and DOHH2). The Emax ranged from 29.3% to 85.7%, and the EC50 ranged from 0.03% to 0.30 nM.

[0660] in conclusion:

[0661] These data demonstrate the ability of both the monospecific killer arm and the dual complementary antibody to successfully deplete T-cell and B-cell lines, with the dual complementary antibody showing increased potency and efficacy compared to the monospecific killer arm.

[0662] Example 19: Cell depletion of PBMCs derived from healthy and leukemia patients

[0663] introduction:

[0664] This embodiment investigated the ability of CD45 antibodies to deplete immune cells in healthy and diseased subjects.

[0665] Materials and Methods - PBMC Depletion Determination Method:

[0666] Blood samples were collected from healthy volunteers (University of Leicester) and from patients with T- / B-cell leukemia (Leicester Royal Hospital) to isolate human PBMCs using a Lymphoprep (StemCell) density gradient. PBMCs were resuspended in 10 ml of complete medium (RPMI 1640, Corning, + 10% fetal bovine serum (FBS) + 2 mM GlutaMAX, both provided by Gibco, + 1% Pen / Strep, Fisher, 0.004 μl / ml β-mercaptoethanol (BME) (Sigma)) and counted using a Biorad TC20™ automated cell counter. Then, 1×10⁻⁶ cells were transferred to 90 μl of medium. 5 Cells / well were added to each well of a Corning Costar 96-well microplate with a U-shaped bottom treated with cell culture (catalog number 3879). PBMCs from 8 donors were used in this assay (6 healthy volunteers (330CD, 334ES, 335AC, 336BB, 365DS, 370EE), 1 T-cell leukemia patient (4386POS, Cezari syndrome), and 1 B-cell leukemia patient (4650ADG, mantle cell lymphoma)).

[0667] Stock solutions were prepared in Sarstedt 96-well microplates (catalog number 83.3924.005) to contain 2 μM of purified 17415gL15gH6-17552gL1gH4 IgG1 LALA, 17415gL15gH6 IgG1 LALA, and isotype control IgG1 LALA (5604) in complete medium. The reagents were serially diluted 10 times at a ratio of 1:3.16 in complete medium to generate an 11-point dose profile. 10 μL of each dilution (final well concentration 200–0.002 nM) was added to the cells and incubated at 37°C, 5% CO2 for 22 h. After incubation, 150 μL of PBS was added to each well, and the plates were centrifuged at 400 g for 5 min at RT. The supernatant was discarded by inverting the plates and blotting them dry on thin paper. Resuspend the cells in 150 μl of PBS, centrifuge the plate again, and discard the supernatant. For viability staining, use 50 μl of a solution containing LIVE / DEAD at 4°C. TMCells were stained with Fixable Violet Dead Cell Stain (Invitrogen, 1:1000 dilution) for 30 minutes. After incubation, 150 μl of FACS buffer (PBS + 1% bovine serum albumin (BSA), Fisher BioReagents) was added to each well, and the plates were centrifuged at 400 g for 5 minutes at RT, discarding the supernatant. Cells were washed again in FACS buffer, and the supernatant was discarded. For extracellular marker staining, cells were stained for 30 minutes at 4°C with 50 μl of antibody solution containing anti-human CD16 BV480 (BD Biosciences, catalog number 566171, 1:100 dilution), CD19 Alexafluor 700 (BD Biosciences, catalog number 557921, 1:200 dilution), CD3 Alexafluor 594 (Biolegend, catalog number 300446, 1:250 dilution), CD4 PerCP Cy5.5 (Biolegend, catalog number 300530, 1:100 dilution), CD8 APC Cy7 (Biolegend, catalog number 301016, 1:400 dilution), HLA-DR PECy7 (Thermofisher, 25-9956-42, 1:200 dilution), and CD56 APC (Biolegend, 318310, 1:50 dilution). After incubation, 150 μl of FACS buffer was added to each well, and the plates were centrifuged at 400 g for 5 minutes at RT, discarding the supernatant. Cells were washed again with FACS buffer and then resuspended in 150 μl of FACS lysis buffer (BD Biosciences, catalog number 349202). For flow cytometry analysis, 100 μl / well volume was obtained on an Attune NxT flow cytometer (Invitrogen). Viable single-cell counts for each leukocyte population were extracted as a metric and graphical representations were generated using Graphpad Prism version 9.0 (Graphpad). Asymmetric (4-parameter) curve fitting was applied to derive EC50 and Emax values.

[0668] result:

[0669] PBMC Depletion Determination Method

[0670] The percentage reduction in T cells in PBMC populations derived from healthy volunteers (A) and patients with T-cell leukemia (B) treated with 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA is shown in the figure. Figure 24 In, together with the isotype control (5604 IgG1 LALA). The percentage reduction of B cells in PBMC populations derived from healthy volunteers (A) and patients with B-cell leukemia (B) treated with 17415gL15gH6-17552gL1gH4IgG1 LALA and 17415gL15gH6 IgG1 LALA is shown in Figure 25 In the middle, together with the isotype control (5604 IgG1LALA). As representative data from 6 healthy donors, data from healthy donors 336BB and 330CD are shown.

[0671] In healthy volunteers, both 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA showed T-cell reduction, with an Emax range of 74.2-96.7% and an EC50 range of 0.05-8.94 nM. In patients with T-cell leukemia (4368 POS), both 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA showed similar T-cell reduction capacity, with an Emax range of 74.2-97% and an EC50 range of 0.03-8.94 nM. Although the dual complementary antibody (17415gL15gH6-17552gL1gH4 IgG1 LALA) was more effective at depleting T cells in both healthy and diseased subjects than the monospecific antibody (17415gL15gH6 IgG1 LALA), the T cell reduction achieved using the monospecific antibody (17415gL15gH6 IgG1 LALA) was still significant compared to the control.

[0672] In healthy volunteers, both 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA showed B-cell reduction, with an Emax range of 36.5–80.2% and an EC50 range of 0.57–3.1 nM. In patients with B-cell leukemia (4650 ADG), both 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA showed similar B-cell reduction capabilities, with an Emax range of 71.2–79% and an EC50 range of 0.11–0.84 nM. A similar trend was observed in B-cell depletion, because although the dual complementary antibody (17415gL15gH6-17552gL1gH4 IgG1 LALA) was more effective at depleting B-cells in both healthy and diseased subjects than the monospecific antibody (17415gL15gH6 IgG1 LALA), the B-cell reduction achieved using the monospecific antibody (17415gL15gH6 IgG1 LALA) was still significant compared to the control.

[0673] in conclusion:

[0674] These data demonstrate successful depletion of CD45-expressing target cells in both healthy and diseased subjects. It further demonstrates the therapeutic potential of both monospecific and dual complementary antibody groups in different populations of CD45-expressing target cells.

[0675] Numbered implementation plan

[0676] The following represent other numbered embodiments of the invention, but do not necessarily constitute claims:

[0677] 1. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one variable domain specific to CD45, said variable domain comprising variable regions of the light and heavy chains:

[0678] (a) A light chain variable region comprising: CDR1 comprising the sequence of SEQ ID NO: 33, CDR2 comprising the sequence of SEQ ID NO: 34, and CDR3 comprising a sequence selected from any one of SEQ ID NO: 35 and 39 to 42; and

[0679] (b) A heavy chain variable region comprising: CDR1 comprising a sequence selected from any one of SEQ ID NO: 46, 52, 53 and 54, CDR2 comprising a sequence selected from any one of SEQ ID NO: 47, 55, 56 and 57, and CDR3 comprising a sequence of SEQ ID NO: 48.

[0680] 2. The antibody or antigen-binding fragment according to 1, wherein the light chain variable region of (a) comprises a combination of any one of the following CDR1, CDR2, and CDR3:

[0681] SEQ ID NO: 33, 34 and 35;

[0682] SEQ ID NO: 33, 34 and 39;

[0683] SEQ ID NO: 33, 34 and 40;

[0684] SEQ ID NO: 33, 34 and 41; and

[0685] SEQ ID NO: 33, 34 and 42.

[0686] 3. The antibody or antigen-binding fragment according to 1 or 2, wherein the heavy chain variable region of (b) comprises a combination of any one of the following CDR1, CDR2 and CDR3:

[0687] SEQ ID NO: 46, 47 and 48;

[0688] SEQ ID NO: 52, 55 and 48;

[0689] SEQ ID NO: 53, 56 and 48; and

[0690] SEQ ID NO: 54, 57 and 48.

[0691] 4. An antibody or antigen-binding fragment according to any one of 1 to 3, wherein:

[0692] (i) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ ID NO: 33, 34, and 35, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any of the following: (aa) SEQ ID NO: 46, 47, and 48; (bb) SEQ ID NO: 52, 55, and 48; (cc) SEQ ID NO: 53, 56, and 48; and (dd) SEQ ID NO: 54, 57, and 48; or

[0693] (ii) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ ID NO: 33, 34, and 39, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any of the following: (aa) SEQ ID NO: 46, 47, and 48; (bb) SEQ ID NO: 52, 55, and 48; (cc) SEQ ID NO: 53, 56, and 48; and (dd) SEQ ID NO: 54, 57, and 48; or

[0694] (iii) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ ID NO: 33, 34, and 40, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any of the following: (aa) SEQ ID NO: 46, 47, and 48; (bb) SEQ ID NO: 52, 55, and 48; (cc) SEQ ID NO: 53, 56, and 48; and (dd) SEQ ID NO: 54, 57, and 48; or

[0695] (iv) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ ID NO: 33, 34, and 41, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any of the following: (aa) SEQ ID NO: 46, 47, and 48; (bb) SEQ ID NO: 52, 55, and 48; (cc) SEQ ID NO: 53, 56, and 48; and (dd) SEQ ID NO: 54, 57, and 48; or

[0696] (v) The light chain variable region of (a) comprises a combination of CDR1, CDR2 and CDR3 of SEQ ID NO: 33, 34 and 42, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2 and CDR3 selected from any of the following: (aa) SEQ ID NO: 46, 47 and 48; (bb) SEQ ID NO: 52, 55 and 48; (cc) SEQ ID NO: 53, 56 and 48; and (dd) SEQ ID NO: 54, 57 and 48.

[0697] 5. An antibody or antigen-binding fragment according to any one of 1 to 4, wherein the antibody or antigen-binding fragment comprises: a light chain having a CDR set of LCDR1, LCDR2 and LCDR3 comprising sequences of SEQ ID NO: 33, 34 and 39 respectively; and a CDR set of HCDR1, HCDR2 and HCDR3 comprising sequences of SEQ ID NO: 52, 55 and 48 respectively for the heavy chain variable region.

[0698] 6. The antibody or antigen-binding fragment according to any one of 1 to 5, wherein the light chain and heavy chain variable regions are humanized.

[0699] 7. The antibody or antigen-binding fragment according to 6, wherein the light chain variable region of (a) is humanized using an IGKV4-1 or IGKV1-9 framework as the acceptor sequence for the framework region, but optionally wherein one or more amino acid residues of the acceptor framework sequence are replaced with one or more corresponding residues of the donor framework sequence, preferably wherein the acceptor framework sequence is derived from IGKV4-1.

[0700] 8. The antibody or antigen-binding fragment according to any one of 1 to 7, wherein the light chain variable region of (a) comprises a sequence selected from one of SEQ ID NO: 11 to 14 and SEQ ID NO: 3 to 9, preferably selected from one of SEQ ID NO: 11 to 14.

[0701] 9. An antibody or antigen-binding fragment according to any one of 6 to 8, wherein the heavy chain variable region of (b) is humanized by using the IGHV3-72 framework as the acceptor sequence for the framework region, but optionally wherein one or more amino acid residues of the acceptor framework sequence are replaced with one or more corresponding residues of the donor framework sequence.

[0702] 10. An antibody or antigen-binding fragment according to any one of 1 to 9, wherein the heavy chain variable region of (b) comprises a sequence selected from any one of SEQ ID NO: 17 to 22.

[0703] 11. An antibody or antigen-binding fragment according to any one of 1 to 10, wherein the at least one antigen-binding site comprises a light chain variable region sequence selected from any one of SEQ ID NO: 11 to 14 and SEQ ID NO: 3 to 9 and a heavy chain variable region selected from any one of SEQ ID NO: 17 to 22, preferably wherein the light chain variable region is selected from any one of SEQ ID NO: 11 to 14.

[0704] 12. The antibody or antigen-binding fragment according to any one of 1 to 11, wherein the at least one antigen-binding site comprises:

[0705] (i) The light chain variable region having the sequence of SEQ ID NO: 9 and the heavy chain variable region having the sequence of SEQ ID NO: 22; or

[0706] (ii) The light chain variable region having the sequence of SEQ ID NO: 13 and the heavy chain variable region having the sequence of SEQ ID NO: 22; or

[0707] (iii) The light chain variable region having the sequence of SEQ ID NO: 14 and the heavy chain variable region having the sequence of SEQ ID NO: 22; or

[0708] (iv) The light chain variable region sequence of SEQ ID NO: 11 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or

[0709] (v) The light chain variable region sequence of SEQ ID NO: 12 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or

[0710] (vi) The light chain variable region sequence of SEQ ID NO: 13 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or

[0711] (vii) The light chain variable region sequence of SEQ ID NO: 14 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or

[0712] (viii) The light chain variable region sequence of SEQ ID NO: 3 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or

[0713] (ix) The light chain variable region sequence of SEQ ID NO: 4 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or

[0714] (x) The light chain variable region sequence of SEQ ID NO: 5 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or

[0715] (xi) The light chain variable region sequence of SEQ ID NO: 6 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or

[0716] (xii) The light chain variable region sequence of SEQ ID NO: 7 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or

[0717] (xiii) The light chain variable region sequence of SEQ ID NO: 8 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or

[0718] (xiv) The light chain variable region sequence of SEQ ID NO: 9 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22.

[0719] 13. The antibody or antigen-binding fragment according to claim 12, wherein the at least one antigen-binding site comprises:

[0720] (i) The light chain variable region having the sequence of SEQ ID NO: 9 and the heavy chain variable region having the sequence of SEQ ID NO: 22; or

[0721] (ii) The light chain variable region having the sequence of SEQ ID NO: 13 and the heavy chain variable region having the sequence of SEQ ID NO: 22; or

[0722] (iii) The light chain variable region having the sequence of SEQ ID NO: 14 and the heavy chain variable region having the sequence of SEQ ID NO: 2.

[0723] 14. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one variable domain specific to CD45, said variable domain comprising variable regions of the light and heavy chains:

[0724] (a) A light chain variable region comprising: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and

[0725] (b) A heavy chain variable region comprising: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence selected from any one of SEQ ID NO: 102 to 105.

[0726] 15. The antibody or antigen-binding fragment according to 14, wherein:

[0727] (i) The light chain variable region of (a) comprises: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and the heavy chain variable region of (b) comprises: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence of SEQ ID NO: 102; or

[0728] (ii) The light chain variable region of (a) comprises: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and the heavy chain variable region of (b) comprises: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence of SEQ ID NO: 103; or

[0729] (iii) The light chain variable region of (a) comprises: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and the heavy chain variable region of (b) comprises: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence of SEQ ID NO: 104; or

[0730] (iv) The light chain variable region of (a) comprises: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and the heavy chain variable region of (b) comprises: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequences of SEQ ID NO: 102 and 105.

[0731] 16. The antibody or antigen-binding fragment according to 15, wherein:

[0732] (i) The light chain variable region of (a) comprises: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequen...

Claims

1. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one variable domain specific to CD45, said variable domain comprising the following light chain and heavy chain variable regions: (a) A light chain variable region comprising: CDR1 comprising the sequence of SEQ ID NO: 33, CDR2 comprising the sequence of SEQ ID NO: 34, and CDR3 comprising a sequence selected from any one of SEQ ID NO: 35 and 39 to 42; and (b) A heavy chain variable region comprising: CDR1 comprising a sequence selected from any one of SEQ ID NO: 46, 52, 53 and 54, CDR2 comprising a sequence selected from any one of SEQ ID NO: 47, 55, 56 and 57, and CDR3 comprising a sequence of SEQ ID NO:

48.

2. The antibody or antigen-binding fragment according to claim 1, wherein: (i) The light chain variable region of (a) contains a combination of any of the following CDR1, CDR2 and CDR3: SEQ ID NO: 33, 34 and 35; SEQ ID NO: 33, 34 and 39; SEQ ID NO: 33, 34 and 40; SEQ ID NO: 33, 34 and 41; and SEQ ID NO: 33, 34 and 42, and / or (ii) The heavy chain variable region of (b) contains a combination of any of the following CDR1, CDR2 and CDR3: SEQ ID NO: 46, 47 and 48; SEQ ID NO: 52, 55 and 48; SEQ ID NO: 53, 56 and 48; and SEQ ID NO: 54, 57 and 48.

3. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein: (i) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ ID NO: 33, 34, and 35, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any of the following: (aa) SEQ ID NO: 46, 47, and 48; (bb) SEQ ID NO: 52, 55, and 48; (cc) SEQ ID NO: 53, 56, and 48; and (dd) SEQ ID NO: 54, 57, and 48; or (ii) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ ID NO: 33, 34, and 39, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any of the following: (aa) SEQ ID NO: 46, 47, and 48; (bb) SEQ ID NO: 52, 55, and 48; (cc) SEQ ID NO: 53, 56, and 48; and (dd) SEQ ID NO: 54, 57, and 48; or (iii) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ ID NO: 33, 34, and 40, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any of the following: (aa) SEQ ID NO: 46, 47, and 48; (bb) SEQ ID NO: 52, 55, and 48; (cc) SEQ ID NO: 53, 56, and 48; and (dd) SEQ ID NO: 54, 57, and 48; or (iv) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ ID NO: 33, 34, and 41, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any of the following: (aa) SEQ ID NO: 46, 47, and 48; (bb) SEQ ID NO: 52, 55, and 48; (cc) SEQ ID NO: 53, 56, and 48; and (dd) SEQ ID NO: 54, 57, and 48; or (v) The light chain variable region of (a) comprises a combination of CDR1, CDR2 and CDR3 of SEQ ID NO: 33, 34 and 42, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2 and CDR3 selected from any of the following: (aa) SEQ ID NO: 46, 47 and 48; (bb) SEQ ID NO: 52, 55 and 48; (cc) SEQ ID NO: 53, 56 and 48; and (dd) SEQ ID NO: 54, 57 and 48.

4. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment comprises: a light chain having a CDR set of LCDR1, LCDR2 and LCDR3 comprising sequences of SEQ ID NO: 33, 34 and 39 respectively; and a CDR set of HCDR1, HCDR2 and HCDR3 comprising sequences of SEQ ID NO: 52, 55 and 48 respectively for the heavy chain variable region.

5. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein: (i) The light chain and heavy chain variable regions are humanized, optionally wherein the light chain variable region of (a) is humanized by using an IGKV4-1 or IGKV1-9 framework as the acceptor sequence for the framework region, but optionally wherein one or more amino acid residues of the acceptor framework sequence are replaced with one or more corresponding residues of the donor framework sequence, preferably wherein the acceptor framework sequence is derived from IGKV4-1; (ii) The light chain variable region of (a) comprises a sequence selected from one of SEQ ID NO: 11 to 14 and SEQ ID NO: 3 to 9, preferably selected from one of SEQ ID NO: 11 to 14; (iii) The heavy chain variable region of (b) is humanized by using the IGHV3-72 framework as the acceptor sequence for the framework region, but optionally one or more amino acid residues of the acceptor framework sequence are replaced with one or more corresponding residues of the donor framework sequence. (iv) The heavy chain variable region of (b) contains a sequence selected from any one of SEQ ID NO: 17 to 22; and / or (v) The at least one antigen binding site comprises a light chain variable region sequence selected from any one of SEQ ID NO: 11 to 14 and SEQ ID NO: 3 to 9 and a heavy chain variable region selected from any one of SEQ ID NO: 17 to 22, preferably wherein the light chain variable region is selected from any one of SEQ ID NO: 11 to 14.

6. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the at least one antigen-binding site comprises: (i) The light chain variable region having the sequence of SEQ ID NO: 9 and the heavy chain variable region having the sequence of SEQ ID NO: 22; or (ii) The light chain variable region having the sequence of SEQ ID NO: 13 and the heavy chain variable region having the sequence of SEQ ID NO: 22; or (iii) The light chain variable region having the sequence of SEQ ID NO: 14 and the heavy chain variable region having the sequence of SEQ ID NO: 22; or (iv) The light chain variable region sequence of SEQ ID NO: 11 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or (v) The light chain variable region sequence of SEQ ID NO: 12 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or (vi) The light chain variable region sequence of SEQ ID NO: 13 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or (vii) The light chain variable region sequence of SEQ ID NO: 14 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or (viii) The light chain variable region sequence of SEQ ID NO: 3 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or (ix) The light chain variable region sequence of SEQ ID NO: 4 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or (x) The light chain variable region sequence of SEQ ID NO: 5 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or (xi) The light chain variable region sequence of SEQ ID NO: 6 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or (xii) The light chain variable region sequence of SEQ ID NO: 7 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or (xiii) The light chain variable region sequence of SEQ ID NO: 8 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22; or (xiv) The light chain variable region sequence of SEQ ID NO: 9 and the heavy chain variable region sequence selected from any one of SEQ ID NO: 17 to 22. Preferably, the at least one antigen binding site comprises: (i) The light chain variable region having the sequence of SEQ ID NO: 9 and the heavy chain variable region having the sequence of SEQ ID NO: 22; or (ii) The light chain variable region having the sequence of SEQ ID NO: 13 and the heavy chain variable region having the sequence of SEQ ID NO: 22; or (iii) The light chain variable region having the sequence of SEQ ID NO: 14 and the heavy chain variable region having the sequence of SEQ ID NO:

2.

7. The antibody or antigen-binding fragment according to any one of claims 1 to 5, wherein the antibody comprises a heavy chain having the sequence of SEQ ID NO: 140 or 147 and a light chain having the sequence of SEQ ID NO: 141, preferably wherein the antibody comprises a heavy chain having the sequence of SEQ ID NO:

147.

8. An antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising at least one variable domain specific to CD45, said variable domain comprising the following light chain and heavy chain variable regions: (a) A light chain variable region comprising: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and (b) A heavy chain variable region comprising: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence selected from any one of SEQ ID NO: 102 to 105. Choose one of them: (i) The light chain variable region of (a) comprises: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and the heavy chain variable region of (b) comprises: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence of SEQ ID NO: 102; or (ii) The light chain variable region of (a) comprises: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and the heavy chain variable region of (b) comprises: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence of SEQ ID NO: 103; or (iii) The light chain variable region of (a) comprises: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and the heavy chain variable region of (b) comprises: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence of SEQ ID NO: 104; or (iv) The light chain variable region of (a) comprises: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and the heavy chain variable region of (b) comprises: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence of SEQ ID NO: 102105.

9. The antibody or antigen-binding fragment according to claim 8, wherein: (i) The light chain variable region of (a) comprises: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and the heavy chain variable region of (b) comprises: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence of SEQ ID NO: 102; or (ii) The light chain variable region of (a) comprises: CDR1 containing the sequence of SEQ ID NO: 94, CDR2 containing the sequence of SEQ ID NO: 95, and CDR3 containing the sequence of SEQ ID NO: 96; and the heavy chain variable region of (b) comprises: CDR1 containing the sequence of SEQ ID NO: 100, CDR2 containing the sequence of SEQ ID NO: 101, and CDR3 containing the sequence of SEQ ID NO:

105.

10. The antibody or antigen-binding fragment according to claim 8 or 9, wherein: (i) The light chain and heavy chain variable regions are humanized, optionally wherein the light chain variable region of (a) is humanized by using the IGKV1-8 framework as the acceptor sequence for the framework region, optionally wherein one or more amino acid residues of the acceptor framework sequence are replaced with one or more corresponding residues of the donor framework sequence. (ii) The light chain variable region of (a) contains sequences selected from SEQ ID NO: 25 and 26; (iii) The heavy chain variable region of (b) is humanized by using the IGHV4-4 framework as the acceptor sequence for the framework region, optionally wherein one or more amino acid residues of the acceptor framework sequence are replaced with one or more corresponding residues of the donor framework sequence; and / or (iv) The heavy chain variable region of (b) contains a sequence selected from any one of SEQ ID NO: 29 to 32.

11. The antibody or antigen-binding fragment according to any one of claims 8 to 10, wherein the antibody or antigen-binding fragment comprises a light chain variable region sequence selected from SEQ ID NO: 25 and 26 and a heavy chain variable region sequence selected from any one of SEQ ID NO: 29 to 32.

12. The antibody or antigen-binding fragment of claim 11, wherein the antibody comprises: (i) the light chain variable region sequence of SEQ ID NO: 25 and the heavy chain variable region sequence of SEQ ID NO: 29; or (ii) The light chain variable region sequence of SEQ ID NO: 26 and the heavy chain variable region sequence of SEQ ID NO: 32; or (iii) The light chain variable region sequence of SEQ ID NO: 25 and the heavy chain variable region sequence of SEQ ID NO: 30; or (iv) The light chain variable region sequence of SEQ ID NO: 25 and the heavy chain variable region sequence of SEQ ID NO: 31; or (v) The light chain variable region sequence of SEQ ID NO: 25 and the heavy chain variable region sequence of SEQ ID NO: 32; or (vi) The light chain variable region sequence of SEQ ID NO: 26 and the heavy chain variable region sequence of SEQ ID NO: 29; or (vii) The light chain variable region sequence of SEQ ID NO: 26 and the heavy chain variable region sequence of SEQ ID NO: 30; or (viii) The light chain variable region sequence of SEQ ID NO: 26 and the heavy chain variable region sequence of SEQ ID NO: 31, Preferably, the antibody comprises: (i) the light chain variable region sequence of SEQ ID NO: 25 and the heavy chain variable region sequence of SEQ ID NO: 29; or (ii) The light chain variable region sequence of SEQ ID NO: 25 and the heavy chain variable region sequence of SEQ ID NO:

32.

13. The antibody or antigen-binding fragment according to any one of claims 8 to 11, wherein the antibody comprises a heavy chain having the sequence of SEQ ID NO: 142 and a light chain having the sequence of SEQ ID NO:

143.

14. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is: (i) Single-specific for CD45; or (ii) For CD45, it is a double complementary bit.

15. The antibody or antigen-binding fragment of claim 14, wherein the antibody or antigen-binding fragment is dually complementary to CD45, wherein the antibody or antigen-binding fragment includes a first variable domain specific to CD45 as defined in any one of claims 1 to 7 and a second variable domain specific to CD45 as defined in any one of claims 8 to 14.

16. The antibody or antigen-binding fragment of claim 15, wherein the antibody or antigen-binding fragment is double complementary to CD45 and has a CDR set from one of the following specific pairs: (a) 17415gL7gH6 x 17552gL1gH1 double complementary site molecules (the CDRs specific to 17415gL7gH6 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 52, 55, and 48, respectively; the CDRs specific to 17552gL1gH1 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 100, 101, and 102, respectively); or (b) 17415gL7gH6 x 17552gL1gH4 double complementary site molecules (the CDRs specific to 17415gL7gH6 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 52, 55, and 48, respectively; the CDRs specific to 17552gL1gH4 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 100, 101, and 105, respectively); or (c) 17415gL15gH6 x 17552gL1gH1 double complementary site molecules (the CDRs specific to 17415gL15gH6 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 52, 55, and 48, respectively; the CDRs specific to 17552gL1gH1 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 100, 101, and 102, respectively); or (d) 17415gL15gH6 x 17552gL1gH4 double complementary site molecules (the CDRs specific to 17415gL15gH6 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 52, 55, and 48, respectively; the CDRs specific to 17552gL1gH4 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 100, 101, and 105, respectively); or (e) 17415gL16gH6 x 17552gL1gH1 double complementary site molecules (the CDRs specific to 17415gL16gH6 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 52, 55, and 48, respectively; the CDRs specific to 17552gL1gH1 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 100, 101, and 102, respectively); or (f) 17415gL16gH6 x 17552gL1gH4 double complementary site molecules (the CDRs specific to 17415gL16gH6 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 52, 55, and 48, respectively; the CDRs specific to 17552gL1gH4 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 100, 101, and 105, respectively); or (g) 17415gL7gH6 x 17552gL1gH1 double complementary site molecules (the CDRs specific to 17415gL7gH6 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 52, 55, and 48, respectively; the CDRs specific to 17552gL1gH1 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 100, 101, and 102, respectively); or (h) 17415gL7gH6 x 17552gL1gH4 double complementary site molecules (the CDRs specific to 17415gL7gH6 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 52, 55, and 48, respectively; the CDRs specific to 17552gL1gH4 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 100, 101, and 105, respectively); or (i) 17415gL15gH6 x 17552gL1gH1 double complementary site molecules (the CDRs specific to 17415gL15gH6 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 52, 55, and 48, respectively; the CDRs specific to 17552gL1gH1 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 100, 101, and 102, respectively); or (j) 17415gL15gH6 x 17552gL1gH4 double complementary site molecules (the CDRs specific to 17415gL15gH6 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 52, 55, and 48, respectively; the CDRs specific to 17552gL1gH1 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 100, 101, and 105, respectively); or (k) 17415gL16gH6 x 17552gL1gH1 double complementary site molecules (the CDRs specific to 17416gL16gH6 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 52, 55, and 48, respectively; the CDRs specific to 17552gL1gH1 are LCDR1, LCDR2, and LCDR3 of SEQ ID NO: 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 100, 101, and 102, respectively); or (l) 17415gL16gH6 x 17552gL1gH4 double complementary site molecules (the CDRs specific to 17416gL16gH6 are LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 33, 34 and 39, respectively, and HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 52, 55 and 48, respectively; the CDRs specific to 17552gL1gH4 are LCDR1, LCDR2 and LCDR3 of SEQ ID NO: 94, 95 and 96, respectively, and HCDR1, HCDR2 and HCDR3 of SEQ ID NO: 100, 101 and 105, respectively).

17. The antibody or antigen-binding fragment of claim 16, wherein the antibody or antigen-binding fragment is bicomplementary to CD45, and wherein the antibody or antigen-binding fragment has a pair of light and heavy chain variable regions specific to each of the following: (a) 17415gL7gH6 x 17552gL1gH1 double complementary site molecule (with sequences of SEQ ID NO: 9 and 22 for the light and heavy chain variable regions specific to 17415gL7gH6, respectively, and sequences of SEQ ID NO: 25 and 29 for the light and heavy chain variable regions specific to 17552gL1gH1, respectively); (b) 17415gL7gH6 x 17552gL1gH4 double complementary site molecule (with sequences of SEQ ID NO: 9 and 22 for the light and heavy chain variable regions specific to 17415gL7gH6, respectively, and sequences of SEQ ID NO: 25 and 32 for the light and heavy chain variable regions specific to 17552gL1gH4, respectively); (c) 17415gL15gH6 x 17552gL1gH1 double complementary site molecule (with sequences of SEQ ID NO: 13 and 22 for the light chain and heavy chain variable regions specific to 17415gL15gH6, respectively, and sequences of SEQ ID NO: 25 and 29 for the light chain and heavy chain variable regions specific to 17552gL1gH1, respectively); (d) 17415gL15gH6 x 17552gL1gH4 double complementary site molecule (with sequences of SEQ ID NO: 13 and 22 for the light and heavy chain variable regions specific to 17415gL15gH6, respectively, and sequences of SEQ ID NO: 25 and 32 for the light and heavy chain variable regions specific to 17552gL1gH4, respectively). (e) 17415gL16gH6 x 17552gL1gH1 double complementary site molecule (with sequences of SEQ ID NO: 14 and 22 for the light and heavy chain variable regions specific to 17415gL16gH6, respectively, and sequences of SEQ ID NO: 25 and 29 for the light and heavy chain variable regions specific to 17552gL1gH1, respectively). (f) 17415gL16gH6 x 17552gL1gH4 double complementary site molecule (with sequences of SEQ ID NO: 14 and 22 for the light and heavy chain variable regions specific to 17415gL16gH6, respectively, and sequences of SEQ ID NO: 25 and 32 for the light and heavy chain variable regions specific to 17552gL1gH1, respectively); (g) 17415gL7gH6 x 17552gL1gH1 double complementary site molecule (with sequences of SEQ ID NO: 9 and 22 for the light chain and heavy chain variable regions specific to 17415gL7gH6, respectively, and sequences of SEQ ID NO: 25 and 29 for the light chain and heavy chain variable regions specific to 17552gL1gH1, respectively); (h) 17415gL7gH6 x 17552gL1gH4 double complementary site molecule (with sequences of SEQ ID NO: 9 and 22 for the light and heavy chain variable regions specific to 17415gL7gH6, respectively, and sequences of SEQ ID NO: 25 and 32 for the light and heavy chain variable regions specific to 17552gL1gH4, respectively); (i) 17415gL15gH6 x 17552gL1gH1 double complementary site molecule (with sequences of SEQ ID NO: 13 and 22 for the light chain and heavy chain variable regions specific to 17415gL15gH6, respectively, and sequences of SEQ ID NO: 25 and 29 for the light chain and heavy chain variable regions specific to 17552gL1gH1, respectively); (j) 17415gL15gH6 x 17552gL1gH4 double complementary site molecule (with sequences of SEQ ID NO: 13 and 22 for the light and heavy chain variable regions specific to 17415gL15gH6, respectively, and sequences of SEQ ID NO: 25 and 32 for the light and heavy chain variable regions specific to 17552gL1gH1, respectively); (k) 17415gL16gH6 x 17552gL1gH1 double complementary site molecule (with sequences of SEQ ID NO: 14 and 22 for the light and heavy chain variable regions specific to 17415gL16gH6, respectively, and sequences of SEQ ID NO: 25 and 29 for the light and heavy chain variable regions specific to 17552gL1gH1, respectively); (l) 17415gL16gH6 x 17552gL1gH4 double complementary site molecule (with SEQ ID NO: 14 and 22 for the light chain and heavy chain variable regions specific to 17415gL16gH6, respectively, and with SEQ ID NO: 25 and 32 for the light chain and heavy chain variable regions specific to 17552gL1gH1, respectively).

18. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the constant region of the antibody or the antigen-binding fragment thereof comprises one or more modifications to reduce or eliminate binding to the Fc receptor.

19. The antibody or antigen-binding fragment according to any one of the preceding claims, wherein the constant region of said antibody comprises heavy chain constant region modifications 234A and 235A in EU numbering manner. Optional, of which (i) The constant region is the IgG1 constant region, and the modification is an LALA double mutation in the constant region; or (ii) The constant region is the IgG4 constant region, and the modification is a FALA double mutation in the constant region.

20. The antibody or antigen-binding fragment according to any one of claims 1 to 19, wherein the antibody is a dual complementary site antibody, comprising: (i) a heavy chain constant region comprising a first heavy chain constant region sequence having the sequence of SEQ ID NO: 146 and a second heavy chain constant region sequence having the sequence of SEQ ID NO: 148; and (ii) A light chain constant region sequence having the sequence of SEQ ID NO:

145.

21. The antibody according to any one of the preceding claims, wherein: (i) The antibody is VR17415gL15gH6 x VR17552gL1gH4 IgG1 LALA, wherein the heavy and light chain sequences of the VR17415gL15gH6 portion of the dual complementary site molecule comprise those of SEQ ID NO: 147 and 141, and the heavy and light chain sequences of the VR17552gL1gH4 portion of the dual complementary site molecule comprise those of SEQ ID NO: 142 and 143; and / or (ii) The antibody or its antigen-binding fragment is capable of specifically binding to both human CD45 and cynomolgus monkey CD45.

22. A monospecific antibody specific for CD45, comprising: a heavy chain comprising the sequence of SEQ ID NO: 140, and a light chain comprising the sequence of SEQ ID NO:

141.

23. A dual complementary site antibody, comprising: (a) A heavy chain containing the sequence of SEQ ID NO: 147 and a light chain containing the sequence of SEQ ID NO: 141, which impart first specificity for CD45; and (b) A heavy chain containing the sequence of SEQ ID NO: 142 and a light chain containing the sequence of SEQ ID NO: 143, which give first specificity for CD45.

24. One or more nucleic acid molecules encoding an antibody or an antigen-binding fragment thereof as defined in any of the preceding claims.

25. One or more vectors encoding an antibody as defined in any one of claims 1 to 23, or comprising one or more nucleic acid molecules as described in claim 24.

26. A pharmaceutical composition comprising: (a) The antibody according to any one of claims 1 to 23, one or more nucleic acid molecules according to claim 24, or one or more vectors according to claim 25; and (b) Pharmaceutically acceptable carriers or diluents.

27. The pharmaceutical composition according to claim 26, for use in a treatment method.

28. The pharmaceutical composition according to claim 27, for use in the following aspects: (i) A method for killing or depleting CD45-expressing cells in subjects; (ii) Methods for treating blood cancers such as leukemia, lymphoma, or multiple myeloma; methods for treating autoimmune diseases such as multiple sclerosis or scleroderma; and / or (iii) The method further includes transferring the cells to the subject after the cells have been depleted.

29. A method of killing or depleting CD45-expressing cells in a subject, the method comprising administering the subject the pharmaceutical composition according to claim 26.

30. The method of claim 29, wherein the method: (i) Used to treat blood cancers such as leukemia, lymphoma, multiple myeloma, and autoimmune diseases such as multiple sclerosis or scleroderma; (ii) Further includes transferring the cells to the subject after cell killing or depletion.

31. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 23, one or more nucleic acid molecules according to claim 24, or one or more carriers according to claim 25 in the preparation of a medicament for killing or depleting CD45-expressing cells in a subject.

32. The use according to claim 31, wherein the drug: (i) For the treatment of blood cancers such as leukemia, lymphoma, multiple myeloma, autoimmune diseases such as multiple sclerosis or scleroderma; and / or (ii) For use in a method that further includes transferring cells to the subject after cell killing or depletion.

33. An in vitro method for depleting or killing target cells expressing CD45 in a cell population, tissue, or organ, the method comprising contacting the cells, tissue, or organ with an antibody or antigen-binding fragment according to any one of claims 1 to 23.

34. The antibody according to any one of claims 1 to 23, used in a method for treating or preventing graft-versus-host disease (GVHD) in a subject, the method comprising: (a) Ex vivo contact of a cell population, tissue, or organ with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 to kill target cells expressing CD45; and (b) Transplanting the treated cell population, tissue or organ into the subject.

35. A method for treating or preventing graft-versus-host disease (GVHD), the method comprising: (a) Ex vivo contact of a cell population, tissue, or organ with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 23 to kill target cells expressing CD45; and (b) Transplanting treated cell populations, tissues or organs to subjects who require such transplants.

36. Use of the antibody according to any one of claims 1 to 23 in the preparation of a medicament, wherein the medicament is used to treat or prevent graft-versus-host disease (GVHD) in a method comprising the steps of: (a) Ex vivo contact of a cell population, tissue, or organ with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 38 to kill target cells expressing CD45; and (b) Transplanting treated cell populations, tissues or organs to subjects who require such transplants.

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