antibodies

Abstract

The present invention provides an antibody or an antigen-binding fragment thereof that is particularly specific to CD45. The antibody includes a monospecific antibody to CD45 as well as a biparatopic antibody to CD45. The antibody can generally bind to both human and cynomolgus monkey CD45. The antibody of the present invention has various uses, including the targeting, depletion, and killing of CD45-expressing cells, and is particularly used for the treatment of cancer or autoimmune diseases mediated by CD45-positive cells or prior to cell transplantation.

Description

Technology Field

[0001] The present invention relates to an antibody specific to CD45. The antibody comprises a monospecific or bipartopic antibody against CD45. For example, the antibody may be used to kill or deplete CD45-positive target cells, and may be used, in particular, for the treatment of cancer or autoimmune diseases mediated by CD45-positive cells or prior to cell transplantation. Background Technology

[0002] CD45 is the original and prototypical receptor-like protein tyrosine phosphatase, expressed in nucleated hematopoietic cells, and plays a key role in regulating cellular responses. CD45 has also been known as PTPRC, T200, Ly5, Common Leukocyte Antigen (LCA), and B220. CD45 is the most abundantly expressed cell surface protein on the surfaces of T cells and B cells and is essential for their development and activation. Studies on CD45 mutant cell lines, CD45-deficient mice, and CD45-deficient humans first demonstrated that CD45 plays an essential role in antigen receptor signaling and lymphocyte development in T cells and B cells. It is now known that CD45 also regulates signals originating from integrin and cytokine receptors. In contrast to its positive role in antigen receptor signaling, CD45 acts as a negative regulator of integrin-mediated signaling, for example, in macrophages. CD45 may also play a role in regulating hematopoiesis and interferon-dependent antiviral responses. In addition, CD45 can also be involved in cell survival.

[0003] CD45 comprises a highly diverse glycosylated extracellular domain consisting of approximately 400 to 550 amino acids, one transmembrane domain, and a long intracellular domain consisting of 705 amino acids, including two consecutive repeating phosphatase domains. The regulation of CD45 expression and the expression of various alternative splicing isoforms (selectively spliced ​​exons 4, 5, and 6 of the CD45 gene, designated as A, B, and C) significantly regulate phosphatase activity and differential signaling. CD45 influences cellular responses by regulating the relative threshold of sensitivity to external stimuli. Disruption of these functions can be a cause of autoimmune diseases, immunodeficiency, and malignancies.

[0004] All CD45 isoforms exhibit tyrosine phosphatase activity, which is mediated by the cytoplasmic domain of the molecule consisting of two consecutive repeat sequences of phosphatase domains D1 and D2, and each domain contains a highly conserved H C It contains the (X)5R motif. All tyrosine phosphatase activity of CD45 is thought to originate from the D1 domain, while the D2 domain is likely involved in regulation. One of the major targets of CD45 tyrosine phosphatase is Src-family kinases, which reflects the role of CD45 in cell signaling. Depending on the site of action of CD45's phosphatase activity, it can either activate or downregulate the activity of these Src-family kinases.

[0005] CD45 is an attractive target in cancer and other therapeutic fields. Given the importance of CD45, continuous research is needed on drugs that can be used to treat the aforementioned diseases by targeting CD45.

[0006] Summary of the Invention

[0007] The present invention provides antibodies specific to CD45. The antibodies provided include monospecific and biparatopic antibodies specific to CD45. Both of these can be used for the killing and / or depletion of cells expressing CD45.

[0008] In particular, the present invention provides an antibody or an antigen-binding fragment thereof comprising at least one variable domain specific to CD45, comprising the following light chain and heavy chain variable regions:

[0009] (a) a light chain variable region comprising a CDR1 having the sequence of SEQ ID NO. 33, a CDR2 having the sequence of SEQ ID NO. 34, and a CDR3 having a sequence selected from any one of SEQ ID NOs. 35 and 39 to 42; and

[0010] (b) a heavy chain variable region comprising a CDR1 having a sequence selected from any one of sequence numbers 46, 52, 53 and 54, a CDR2 having a sequence selected from any one of sequence numbers 47, 55, 56 and 57, and a CDR3 having a sequence of sequence number 48.

[0011] In addition, the present invention provides an antibody or an antigen-binding fragment thereof comprising at least one variable domain specific to CD45, comprising the following light chain and heavy chain variable regions:

[0012] (a') a light chain variable region comprising a CDR1 having the sequence of SEQ ID NO. 94, a CDR2 having the sequence of SEQ ID NO. 95, and a CDR3 having the sequence of SEQ ID NO. 96; and

[0013] (b') A heavy chain variable region comprising a CDR1 having the sequence of sequence number 100, a CDR2 having the sequence of sequence number 101, and a CDR3 having a sequence selected from any one of sequence numbers 102 to 105.

[0014] The light and heavy chain variable regions may be humanized. The antibody or antigen-binding fragment may be biparatopic by comprising a first variable domain specific to CD45 of the first antibody group presented above (i.e., antibodies with chains (a) and (b)) and a second variable domain specific to CD45 of the second antibody group presented above (i.e., antibodies with chains (a') and (b')). The constant region of the antibody or its antigen-binding fragment may include modifications or alterations 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.

[0015] The present invention also provides a pharmaceutical composition comprising the antibody presented herein and a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition may be used to kill or deplete CD45-expressing cells in a subject and may be used to treat blood cancers, for example, leukemia, lymphoma, or multiple myeloma. Brief explanation of the drawing

[0016] Fig. 1 This study compares the ability of monospecific (monoclonal) antibodies 17415, 17552, and 4133 to kill human PBMCs (peripheral blood mononuclear cells) expressing CD45. A VR5604 isotype control antibody that does not bind to CD45 was also included. Fig. 2This provides alignments of various sequences related to the humanization of the light chain variable region of the original rabbit 17415 antibody. The CDR of the rabbit antibody was transplanted into a human IGKV1-9 receptor framework (top alignment set) or an IGKV4-1 receptor framework (bottom alignment set). Several donor framework residues were also transposed, including some CDR variants. The CDR is indicated in bold underline. Amino acid residues different from the original CDR or human donor framework are indicated in bold italics with shading. The resulting transplant variants are shown below the receptor sequences. Fig. 3 This provides the alignment of various sequences related to the humanization of the variable region of the heavy chain of the original rabbit 17415 antibody. The CDR of the rabbit antibody was transplanted into the human IGHV3-72 receptor framework. Several donor framework residues were also translocated, and some CDR variants were included. The CDR is indicated in bold underline. Amino acid residues that differ from the original CDR or the human donor framework are indicated in bold italics with shading. The resulting transplant variants are shown below the receptor sequence. Fig. 4 This provides alignments of various sequences related to the humanization of the original rabbit 17552 antibody. The top alignment set represents the light chain humanized to the human IGKV1-8 receptor framework, and the bottom alignment set represents the heavy chain humanized to the human IGHV4-4 receptor framework. Several donor framework residues were also transposed, and some CDR variants were included. The CDRs are indicated in bold underline. Amino acid residues that differ from the original CDR or human donor framework are indicated in bold italics with shading. The resulting transgenic variants are shown below the receptor sequences. Fig. 5Figure 2 shows the results of a human lymphocyte depletion analysis comparing the following: (a) 17415 light chain transplant variants L1 to L5 paired with 17415 IgG LALA heavy chain transplant variant H1 and (b) 17415 light chain transplant variant L1 paired with 17415 IgG LALA heavy chain transplant variants H1 to H4. Fig. 6 It shows the results of the human lymphocyte analysis for the following: (a) Chimera 17415 heavy chain comprising an original rabbit 17415 heavy chain variable region paired with 17415 light chain transplant variants L1 to L5 versus original rabbit 17415 light chain and chimera 17415 light chain and heavy chain comprising a heavy chain variable region, and (b) Original rabbit 17415 light chain variable region including chimera 17415 light chain and paired 17415 heavy chain transplant variants H1 to H4 versus original rabbit 17415 light chain and chimera 17415 light chain and heavy chain including heavy chain variable region. Fig. 7 It shows the results of the human T cell depletion analysis for the following: (a) 17415 light chain transplant variants L6, L7, L13, L14, L15, and L16 paired with 17415 heavy chain transplant variant H5, and (b) 17415 The same light chain transplant variant paired with the heavy chain transplant variant H6. Fig. 8 The following shows the results of human Jurkat cell killing against IgG1 LALA type antibodies: (a) 17415 light chain variable region transplant variants L1 to L5 paired with 17415 heavy chain transplant variant H1, (b) 17415 light chain variable region transplant variant L1 paired with 17415 heavy chain transplant variants H1 to H4, (c)When 17415 chimeric light and heavy chain antibodies containing the original variable region of the original rabbit 17415 antibody were compared with antibodies paired with the chimeric light chain and 17415 heavy chain transplant variants H1 to H4, respectively, and (d) When 17415 chimeric light and heavy chain antibodies containing the original variable region of the original rabbit 17415 antibody were compared with 17415 light chain variants L1 to L5 and chimeric heavy chain paired antibodies. Fig. 9 It shows the results of a human T cell depletion assay for 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. Fig. 10 This shows the results of a human Jurkat cell depletion assay for IgG1 LALA antibodies containing 17415 light chain graft variant 7, 15, or 16 and 17415 heavy chain graft variant H6. Fig. 11 This shows the binding results of humanized 17415 IgG1 LALA grafts to human CD45, including 17415 heavy chain graft variant 6 paired with 17415 light chain graft variants 7, 15, and 16. Fig. 12 is 17415 light chain transplant variant 7 (a) , 15 (b) , and 16 (c) For the various antibodies indicated, including those containing, the binding results for human CD45 of various biparatopic 17415 / 17552, 17415 / 5604, and 5604 / 17552 antibody variants and the monospecific 5604 antibody are shown. Fig. 13 Silver 17415 light chain transplant variant 7 (a) , 15 (b) , and 16 (c)For the various antibodies indicated, including those containing, the binding results for various biparatopic 17415 / 17552, 17415 / 5604, and 5604 / 17552 antibody variants and the monospecific 5604 antibody to Sino CD45 are shown. Fig. 14 is light chain transplant variant 7 (a) , 15 (b) , and 16 (c) Shows the results of human T cell depletion analysis for various biparatopic antibody variants including 17415 / 17552, 17415 / 5604, and 5604 / 17552. Fig. 15 is light chain transplant variant 7 (a) , 15 (b) , and 16 (c) Shows the results of human Jurkat cell depletion assays for various biparatopic 17415 / 17552, 17415 / 5604, and 5604 / 17552 antibody variants including Fig. 16 This shows the results of a synomolgus T cell depletion assay comparing 17415 and 17552 monospecific (monoclonal) IgG1 antibodies with the biparatopic 17415 / 17552 IgG1 antibody and the biparatopic 4133 / 6294 IgG1 antibody. Fig. 17 This shows a comparison of the ability of dual-paratopic 17415-17552 IgG1 and 4133-6294 IgG1 antibodies to kill human PBMCs (peripheral blood mononuclear cells) expressing CD45. A VR5604 isotype control antibody that does not bind to CD45 was also included. Fig. 18 This shows the ability of dual-paratopic 17415-17552 IgG1 and YTH24.5-YTH54.12 IgG1 antibodies to reduce T cell numbers in a PBMC population. Results for an isotype (5604 IgG1) control group are also included. Fig. 19This shows the results of human Jurkat cell killing with dual-paratopic 4133-6294 IgG1 and 17415-17552 IgG1 antibodies. A VR5604 isotype control antibody that does not bind to CD45 was also included. Fig. 20 This shows the levels of cytokines (a) IFNγ, (b) IL-6, and (c) TNFα in whole blood induced by the biparatopic 17415gL15gH6-17552gL1gH4 IgG1 LALA antibody, or the monospecific (monoclonal) 17415gL15gH6 IgG1 LALA antibody, compared to a control with unrelated specificity (5604 IgG1 LALA), Campath, or PBS. Fig. 21 This shows a comparison of the ability of dual-paratopic 17415-17552 IgG1 and YTH24.5-YTH54.12 IgG1 antibodies to kill Jurkat cells. A VR5604 isotype control antibody that does not bind to CD45 was also included. Fig. 22 This shows the T cell reduction rates in T cell lines (A) Peers, (B) SUPT11, and (C) SUDHL1 against the biparatopic 17415gL15gH6-17552gL1gH4 IgG1 LALA antibody and the monospecific (monoclonal) 17415gL15gH6 IgG1 LALA antibody. Fig. 23 This shows the T cell reduction rates in B cell lines (A) Ramos and (B) DOHH2 against the biparatopic 17415gL15gH6-17552gL1gH4 and monospecific (monoclonal) 17415gL15gH6 IgG1 LALA antibodies. Fig. 24 This shows the T cell reduction rate induced by the dual-paratopic 17415gL15gH6-17552gL1gH4 IgG1 LALA antibody or the monospecific (monoclonal) 17415gL15gH6 IgG1 LALA antibody in PBMCs derived from (A) healthy volunteers 336BB + 330CD and (B) T-cell leukemia patient 4368POS, compared to the control group. Fig. 25This shows the B cell reduction rate induced by the dual-paratopic 17415gL15gH6-17552gL1gH4 IgG1 LALA antibody or the monospecific (monoclonal) 17415gL15gH6 IgG1 LALA antibody in PBMCs derived from (A) healthy volunteers 336BB + 330CD and (B) B-cell leukemia patient 4650ADG, compared to the control group. Fig. 26 and 27 Each provides a summary of experimental results for some of the preferred monospecific antibodies and biparatopic antibodies of the present invention. Figs. 28, 29, and 30 The amino acid sequences of the full length of human CD45 (Sequence No. 127), the extracellular domains D1 to D4 of human CD45 (Sequence No. 128), and the full length of synomolgus monkey CD45 (Sequence No. 129) are provided, respectively. Fig. 31 The full heavy chain (top) and light chain (bottom) sequences of the VR17415gL15gH6 IgG1 LALA antibody are provided, with the sequences of each constant region 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). Fig. 32 This shows the full heavy and light chain sequences of the VR17415gL15gH6 x VR17552gL1gH4 IgG1 LALA dual-paratopic antibodies. The heavy and light chain sequences for VR17415gL15gH6 are the sequences shown at the top and second from the top. The heavy and light chain sequences for VR17552gL1gH4 specificity are shown as the second from the bottom and the bottom sequence. In all sequences, the constant region sequences 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 "knob-into-hole" variants, and the corresponding mutations are indicated in shading. Fig. 33 The table provides examples of preferred heavy and light chain constant region sequences to be used in the antibodies of the present invention, wherein the constant region is an IgG1 LALA modified constant region. The table provides heavy chain amino acid sequences with and without the "knob-into-hole" modification. The "knob-into-hole" modification can be used to promote the formation of biparatopic antibodies compared to the formation of monospecific antibodies by promoting heteromer formation in the case of biparatopic antibodies. Specific details for implementing the invention

[0017] The present invention provides antibodies that are particularly specific to CD45, specifically monospecific antibodies to CD45 and biparatopic antibodies to CD45. These antibodies are useful for targeting cells expressing CD45, and are particularly useful for depleting and / or killing cells expressing CD45. In a preferred embodiment, the provided antibodies can specifically bind to both human and monkey CD45. In a particularly preferred embodiment, the provided antibodies can specifically bind to both human CD45 and cynomolgus monkey CD45. These 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, that is, has a single specificity for CD45. In a particularly preferred embodiment, the provided antibody is a biparatopic antibody to CD45, combining the two monospecific binding specificities to CD45 presented herein within a single molecule. In another preferred embodiment, a biparatopic antibody that binds to human and cynomolgus monkey cells is provided. In a particularly preferred embodiment, a biparatopic antibody against CD45 that binds to and kills both human cells and synomolgus monkey cells is provided.

[0018] Detailed information regarding the above antibody and its uses is provided below.

[0019] CD45 molecule

[0020] The antibody of the present invention is specific to CD45. As previously described, CD45 belongs to the protein tyrosine phosphatase (PTP) family. PTPs are known as signaling molecules that regulate various cellular processes, including cell growth, differentiation, the mitotic cycle, and neoplastic transformation. CD45 belongs to the receptor type PTP because it contains an extracellular domain, a single transmembrane segment, and two consecutive intracytoplasmic catalytic domains. Various isoforms of CD45 exist, including CD45RA, CD45RB, CD45RC, CD45RAB, CD45RAC, CD45RBC, CD45RO, and CD45R(ABC). CD45 splice variant isoforms A, B, and C are differentially expressed in various leukocyte subtypes. Although CD45 exists in various isoforms, they share a common sequence, which means that all isoforms can be targeted with a single antibody.

[0021] The intracellular (COOH-terminal) region of CD45 contains two PTP catalytic domains, while the extracellular region is highly variable due to the alternative splicing of exons 4, 5, and 6 (designated A, B, and C, respectively) and varying levels of glycosylation. The detected CD45 isoform proteins are specific to cell type, maturity, and activation state. Generally, the long forms of the protein (A, B, or C) are naive ( CD45(RO) is expressed in ) or resting B cells, and mature or truncation type CD45(RO) is expressed in activated or mature / memory B cells.

[0022] The sequence of human CD45 is available in UniProt registration number P08575 and is provided herein as sequence number 127 or amino acids 24-1304 of sequence number 127 (excluding the signal peptide). The amino acid sequence of the extracellular domain human CD45 domain 1-4 is provided in sequence number 128.

[0023] The mouse version of CD45 is provided under UniProt registration number P06800.

[0024] A synomolgus monkey version of CD45 is provided herein as sequence number 129 (Fig. 21).

[0025] In one embodiment, the CD45 to which the antibody of the present invention binds 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 may bind to all CD45 isoforms expressed in a particular species. For example, the antibody may bind to all human CD45 isoforms. Preferably, the antibody of the present invention may bind to both human and cynomolgus monkey CD45, and preferably may bind to all isoforms of human and cynomolgus monkey CD45.

[0026] Antibodies - Overview

[0027] The antibody of the present invention has at least one specificity for CD45. The "specificity" of an antibody refers to the target to which the antibody binds. The part of the antibody that binds to the target may be the antigen-binding site or, in some cases, the paratope of the antibody. The part of the antigen to which the antibody binds may be the epitope. The specificity of an antibody can be described in terms of the antigen to which it binds or at the level of which epitope of the antigen it binds to. Biparatopic antibodies are a subset of bispecific antibodies, which can recognize two different epitopes of different antigens or two different epitopes of the same antigen. In the latter case, they are biparatopic antibodies. The number of binding sites an antibody possesses is called the antibody's valency, and each valency represents one antigen-binding site of the antibody.

[0028] The antibody provided in the present invention is specific to CD45. Accordingly, the antibody of the present invention comprises one or more antigen-binding sites, namely paratopes, that are specific to CD45. An antibody that recognizes a single epitope of CD45 can be referred to as a monospecific antibody against CD45. An antibody that recognizes two different epitopes of CD45 can be referred to as a biparatopic antibody against CD45. The present invention provides both a monospecific antibody against CD45 and a biparatopic antibody against CD45. The CD45-specific biparatopic antibody of the present invention may be monovalent or multivalent for each epitope.

[0029] In a preferred embodiment, the antibody of the present invention specifically binds to CD45 but does not significantly bind to proteins other than CD45. In one embodiment, this specificity relates only to the antigen-binding site of the antibody that recognizes CD45, but the antibody may have other antigen-binding sites having different specificities. For example, in one embodiment, the antibody of the present invention has at least one antigen-binding site specific to CD45 as well as an antigen-binding site specific to molecules other than CD45. In one embodiment, additional specificity is for serum albumin. In another embodiment, all specificity of the antibody of the present invention is for CD45.

[0030] In one embodiment, the antibody specifically binds to CD45 of at least one species, but does not necessarily bind to CD45 of 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 specifically binds to CD45 of at least one other species. Preferably, the antibody specifically binds to CD45 of a species used in animal studies that will aid in the development of antibodies as therapeutic agents. 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 another particularly preferred embodiment, the antibody of the present invention kills and / or depletes both human cells expressing CD45 and cynomolgus monkey cells expressing CD45.

[0031] In one embodiment, the antibody of the present invention exhibits a trans bond, which means simultaneously binding to two or more CD45 molecules. Such a trans bond generally induces cross-linking of CD45 and thus represents one of the preferred embodiments of the present invention. In one embodiment, the antibody of the present invention exhibits a cis bond to CD45, specifically binding to only one CD45 molecule through the binding site.

[0032] In a particularly preferred embodiment, the antibody of the present invention specifically binds to CD45 and induces the multimerization of CD45. In one embodiment, the antibody of the present invention can induce the multimerization of CD45 on the surface of a target cell. A CD45 multimer is a higher-order structure, particularly one composed of two or more CD45 molecules. In one embodiment, the antibody of the present invention specifically binds to the extracellular portion of CD45 to induce the formation of CD45 multimers on the surface of a target cell. In a particularly preferred embodiment, the CD45 multimer comprises at least three CD45 molecules. In one embodiment, the CD45 multimer may comprise at least three, four, five, six, or seven or more CD45 molecules bound together by the antibody of the present invention. Using a technique such as mass spectrometry, the CD45 multimers that have formed a complex with the antibody of the present invention can be identified, and thus the ability of the antibody of the present invention to produce CD45 multimers can be measured.

[0033] The specificity (or specificity) for the target molecule used herein, particularly CD45, means that the interaction partners or their related parts recognize each other only or have a significantly higher affinity for each other compared to non-partners, for example, having an affinity that is 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 than background-level binding or binding to other unrelated proteins (e.g., chicken egg white lysozyme). In one embodiment, this specificity is for CD45. In another embodiment, this specificity is for a specific epitope of CD45, particularly a paratope, that is bound by the antigen-binding site of the antibody, compared not only to CD45 but also to other epitopes of CD45.

[0034] In one embodiment, the dissociation constant (K DThe affinity of the antibody measured by ) is about 100 nM or less, for example, about 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, 100 pM or less. In one embodiment, K D is 50 pM or less. In one embodiment, at least one paratop of the antibody has this affinity for CD45. In another embodiment, the antibody has two paratops, each having different specificities for CD45, and all paratops individually have this affinity for CD45. In one embodiment, this is the total binding affinity of the antibody for CD45. In one embodiment, K of the paratop for CD45 D may 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, K D is about 0.1 pM to about 1 μM. In one embodiment, the antibody of the present invention has this level of affinity for CD45. Affinity is generally measured using surface plasmon resonance assays such as Biacore assay.

[0035] 17415-derived antibody

[0036] (a) Overview of 17415-derived antibody and variable region

[0037] A preferred antibody of the present invention is an antibody comprising one or more antigen-binding sites derived from the original rabbit 17415 antibody described herein, in particular an antibody comprising a pair of humanized light chain 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 CDRs of the original rabbit 17415 antibody are LCDR1, LCDR2, and LCDR3, each having the sequences of SEQ ID NOs. 33, 34, and 35. The heavy chain variable region CDRs of the original rabbit 17415 antibody are HCDR1, HCDR2, and HCDR3, each having the sequences of SEQ ID NOs. 46, 47, and 48.

[0038] References to “antibodies derived from 17415” include specific 17415-derived sequences presented herein as well as variants of such antibodies. References to such “17415-derived” sequences include humanized light and heavy chain variable regions shown in FIGS. 2 and 3, as well as variable regions including the set of CDRs CDR1, CDR2, and CDR3 shown in the figures. Specific transplant variants shown in FIGS. 2 and 3 are particularly preferred. Pairs of light and heavy chain variable region transplant variants produced in the embodiments of the present application are also preferred.

[0039] The original rabbit 17415 antibody produced herein and the antibody comprising the transplant variant of the 17415 antibody described herein are particularly effective in killing or depleting cells expressing CD45. Additionally, they have the additional advantage of being able to specifically bind to both human CD45 and cynomolgus monkey CD45, making them particularly suitable for development as therapeutic agents. Preferably, they can kill or deplete both human cells and cynomolgus monkey cells expressing CD45.

[0040] In one embodiment, the antibody comprises one or more 17415-derived antigen-binding sites specific to CD45. In a particularly preferred embodiment, the antibody comprises one or more antigen-binding sites formed from a pair of humanized 17415-derived light chain and heavy chain variable regions.

[0041] In one embodiment, the antibody has monospecificity for CD45, so the 17415-derived antigen-binding sites or sites present in the antibody are the only sites specific to CD45 contained in the antibody. In another embodiment, the antibody of the present invention has a plurality of paratopes that bind to CD45, and one of these paratopes specific to CD45 is provided by the 17415-derived antigen-binding site. In a particularly preferred embodiment, the antibody of the present invention is biparatopic for CD45, and one of these paratopes is the 17415-derived antigen-binding site. A particularly preferred antibody of the present invention that is biparatopic for CD45 is an antibody comprising one paratope of 17415-derived and another paratope of 17552-derived that is specific to CD45.

[0042] Accordingly, in a particularly preferred embodiment, the present invention provides an antibody or an antigen-binding fragment thereof comprising at least one variable domain specific to CD45, comprising the following light chain and heavy chain variable regions:

[0043] (a) a light chain variable region comprising a CDR1 having the sequence of SEQ ID NO. 33, a CDR2 having the sequence of SEQ ID NO. 34, and a CDR3 having a sequence selected from any one of SEQ ID NOs. 35 and 39 to 42; and

[0044] (b) a heavy chain variable region comprising a CDR1 having a sequence selected from any one of sequence numbers 46, 52, 53 and 54, a CDR2 having a sequence selected from any one of sequence numbers 47, 55, 56 and 57, and a CDR3 having a sequence of sequence number 48.

[0045] In a preferred embodiment, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of SEQ NOs 33, 34, and 35, respectively. In another embodiment, they comprise the sequences of SEQ NOs 33, 34, and 39. In another embodiment, they comprise the sequences of SEQ NOs 33, 34, and 40. In another embodiment, they comprise the sequences of SEQ NOs 33, 34, and 41. In another embodiment, they comprise the sequences of SEQ NOs 33, 34, and 42.

[0046] In a preferred embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 46, 47, and 48, respectively. In another embodiment, they comprise the sequences of SEQ ID NOs 52, 55, and 48. In another embodiment, they comprise the sequences of SEQ ID NOs 53, 56, and 48. In another embodiment, they comprise the sequences of SEQ ID NOs 54, 57, and 48.

[0047] The present invention describes the humanization of the original rabbit 17415 antibody. In one preferred embodiment, the 17415-derived antigen-binding site is the humanized antigen-binding site. In a particularly preferred embodiment, the 17415-derived paratope is generated by transferring the CDR of the original rabbit 17415 variable region (donor sequence) to the framework region of a second antibody (receptor sequence). Humanization may include the step of transferring not only the CDR of the donor sequence but also some framework residues.

[0048] Preferred human light chain variable regions acting as receptor sequences of the framework include IGKV1-9 and IGKV4-1. A particularly preferred receptor sequence is a sequence derived from IGKV4-1. For the heavy chain, a particularly preferred receptor sequence for the CDR is the IGHV3-72 receptor framework. In a preferred embodiment, the 17415-derived paratop includes a light chain variable region based on the IGKV4-1 receptor framework and a heavy chain variable region based on the IGHV3-72 receptor framework.

[0049] Table 1 below summarizes the light and heavy chain variable regions of the original 17415, the framework receptor sequences used, and specific transplant variants generated from the original 17415 antibody. Figures 2 and 3 also show the alignment of various original donor and receptor sequences with the generated specific transplant variants. Individual amino acids shown in shaded and italics represent residues that show changes beyond a simple transfer of the CDR sequence of the original 17415 antibody to the human receptor framework. For example, these residues are either donor framework residues transferred along with the CDR sequence, or CDR residues different from those of the original 17415 CDR.

[0050] [Table 1]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] Various examples of desirable 17415-derived sequences are presented below.

[0059] (b) A preferred 17415 light chain transplant variant containing an IGKV4-1 / IGKJ4-derived framework

[0060] In one preferred embodiment, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the rabbit 17415 antibody. In another preferred embodiment, LCDR1, LCDR2, and LCDR3 are identical to the rabbit 17415 antibody except that LCDR3 contains a C90S mutation.

[0061] In a preferred embodiment, the humanized 17415 light chain variable region transplant variant comprises a set of LCDR1, LCDR2, and LCDR3 derived from a rabbit 17415 antibody having an IGKV4-1 / IGKJ4 derived framework. In one preferred embodiment, the light chain variable region comprises a CDR1 having the sequence of SEQ ID NO. 33, a CDR2 having the sequence of SEQ ID NO. 34, and a CDR3 having a sequence selected from any one of SEQ ID NOs. 35 and 39 to 42, and the framework region is an IGKV4-1 / IGKJ4 derived framework.

[0062] In a preferred embodiment, 17415 light chain LCDR1, LCDR2, and LCDR3, as well as one or more donor residues derived from the 17415 rabbit antibody light chain framework, are maintained at one or more positions from the group comprising 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).

[0063] 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 one embodiment, donor residues (valine, V2), 4 (leucine, L4), 12 (serine, S12), and 19 (valine, V19) are retained in FWR1 from 17415. In another embodiment, FWR1 corresponds to the FWR1 receptor sequence of IGKV4-1 / IGKJ4.

[0064] In a preferred embodiment, FWR2 corresponds to the FWR2 receptor sequence of IGKV4-1 / IGKJ4.

[0065] In one embodiment, donor residues 60 (serine, S60), 63 (lysine, K63), 83 (alanine, A83) and 85 (threonine, T85) (and optionally (glutamic acid, E70)) are retained in the light chain variable region FWR3 from the rabbit 17415 antibody. In one embodiment, donor residues 60 (serine, S60), 63 (lysine, K63) and 85 (threonine, T85) are retained in FWR3 from 17415.

[0066] In one embodiment, donor residues 106 (glutamic acid, E106) and 108 (valine, V108) are retained in the light chain variable region FWR4 from rabbit 17415 antibody. In another embodiment, FWR4 corresponds to the FWR4 receptor sequence of IGKV4-1 / IGKJ4.

[0067] Examples of preferred light chain frameworks based on IGKV4-1 receptor sequences include having FWR1, FWR2, FWR3, and FWR4 of sequence numbers 77, 74, 79, and 81, respectively. In another preferred embodiment, they have the sequences of sequence numbers 78, 74, 80, and 76.

[0068] Examples of particularly desirable 17415-derived light chain variable region transplant variants that can be used include 17415gL13 (Sequence No. 11), 17415gL14 (Sequence No. 12), 17415gL15 (Sequence No. 13), and 17415gL16 (Sequence No. 14).

[0069] (c) A preferred 17415 light chain transplant variant containing an IGKV1-9 / IGKJ4-derived framework

[0070] In a preferred embodiment, the light chain variable region derived from the original rabbit 17415 comprises a CDR1 comprising the sequence of SEQ ID NO. 33, a CDR2 comprising the sequence of SEQ ID NO. 34, and a CDR3 comprising a sequence selected from any one of SEQ ID NOs. 35 and 39 to 42, and the framework region is derived from the IGKV1-9 receptor framework.

[0071] In one preferred embodiment, the light chain variable region comprises LCDR1, LCDR2, and LCDR3 of the rabbit 17415 antibody. In another preferred embodiment, LCDR1, LCDR2, and LCDR3 are identical to the rabbit 17415 antibody except that LCDR3 contains a C90S mutation. Alternatively, C90 may be mutated to A (alanine), V (valine), or Q (glutamine).

[0072] In one embodiment, donor residues of 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) of the light chain variable region. In one embodiment, all of these residues are retained.

[0073] In another embodiment, donor residues of 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 (glutamic acid, E106), and 108 (valine, V108). In a preferred embodiment, all of these donor framework residues are retained.

[0074] In one embodiment, in FWR1, the donor residues of the framework are maintained at positions 2 (valine, V2) and 3 (valine, V3). In a particularly preferred embodiment, in FWR1, the donor residues of the framework are maintained at positions 2 (valine, V2), 3 (valine, V3), and 10 (serine, S10).

[0075] In one embodiment, in FWR2, the donor residue is maintained at position 42 (glutamine, Q42). In another embodiment, FWR2 is identical to the receptor sequence and no donor residue is maintained in FWR2.

[0076] In one embodiment, in FWR3, the donor residue is maintained at positions 63 (lysine, K63) and 83 (alanine, A83).

[0077] In one embodiment, in FWR4, the donor residues are maintained at positions 106 (glutamic acid, E106) and 108 (valine, V108). In another embodiment, the sequence of FWR4 is identical to that of the receptor sequence FWR4.

[0078] Examples of desirable donor framework regions to be maintained in particular are all locations at positions 2 (valine, V2), 3 (valine, V3), 10 (serine, S10), 42 (glutamine, Q42), 63 (lysine, K63), 83 (alanine, A83), 106 (glutamic acid, E106), and 108 (valine, V108). In another preferred embodiment, LCDR3 contains the C90S mutation. In another embodiment, LCDR3 does not contain it.

[0079] In a preferred embodiment, the receptor framework comprises FWR1, FWR2, FWR3, and FWR4 corresponding to sequence numbers 58, 59, 60, and 61, respectively. In another embodiment, these FWRs each comprise the sequences of sequence numbers 66, 63, 70, and 65. In another embodiment, the FWRs each comprise the sequences of sequence numbers 67, 69, 71, and 72. In another embodiment, the FWRs each comprise the sequences of sequence numbers 68, 69, 71, and 72.

[0080] Examples of preferred light chain variable region transplant variants include variants 17415gL1 to 17415gL7 (each SEQ NOs 3 to 9). A particularly preferred example of a transplant variant light chain is the transplant variant of SEQ NO 8 (17415gL6). Another particularly preferred example of a transplant variant light chain is the transplant variant of SEQ NO 9 (17415gL6). Additionally, transplant variants of SEQ NOs 3 to 7 (17415gL1 to 17415gL5) are provided.

[0081] (d) A preferred 17415 heavy chain transplant variant containing an IGV3-72-derived framework

[0082] In another preferred embodiment, a 17415-derived heavy chain transplant variant is generated using an IGHV3-72 / IGHJ4 J region receptor framework. Thus, in a preferred embodiment, the heavy chain variable region comprises a CDR1 containing a sequence selected from any one of SEQ NOs 46, 52, 53 and 54, a CDR2 containing a sequence selected from any one of SEQ NOs 47, 55, 56 and 57, and a CDR3 containing the sequence of SEQ NO. 48, and the receptor framework is derived from IGHV3-72 / IGHJ4.

[0083] In one embodiment, the 17415-derived heavy chain variable region comprises HCDR1 of the rabbit 17415 antibody. In another embodiment, the last residue of HCDR1 is changed from C (cysteine) to S (serine), A (alanine), or V (valine).

[0084] In one embodiment, the 17415-derived heavy chain variable region comprises HCDR2 of the rabbit 17415 antibody. In another embodiment, the first residue of HCDR2 is changed from C (cysteine) to S (serine), A (alanine), or V (valine).

[0085] In one embodiment, the derived heavy chain variable region comprises the original HCDR3 of the rabbit 17415 antibody.

[0086] Particularly desirable CDR combinations are shown in FIG. 3. Any of the three heavy chain CDR combinations indicated for individual variants can be used for the 17415-derived heavy chain variable region variant. In particular, in one embodiment, the 17415-derived heavy chain variant has a CDR of 17415gH1 (SEQ No. 17). In another embodiment, it has a CDR of 17415gH2 (SEQ No. 18). In yet another embodiment, it has a CDR of 17415gH3 (SEQ No. 19). In yet another embodiment, it has a CDR of 17415gH4 (SEQ No. 20). In yet another embodiment, it has a CDR of 17415gH5 (SEQ No. 21). In yet another embodiment, it has a CDR of 17415gH6 (SEQ No. 22).

[0087] In a preferred embodiment, the 17415-derived heavy chain variant has FWR1 of the IGHV3-72 receptor, but retains the donor residue of the rabbit 17415 antibody heavy chain framework at position 23 (threonine, T23).

[0088] In a preferred embodiment, the 17415-derived heavy chain variant has FWR2 of the IGHV3-72 receptor, but retains a donor residue at position 49 (isoleucine, I49).

[0089] In a preferred embodiment, the 17415-derived heavy chain variant has FWR3 of the IGHV3-72 receptor, 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).

[0090] In a preferred embodiment, the 17415-derived heavy chain variant has FWR4 of the IGHV3-72 / IGHJ4 receptor, but no additional sequence changes.

[0091] A particularly desirable framework is illustrated in FIG. 3. Any combination of FWR1, FWR2, FWR3, and FWR4 for the individual variants shown in FIG. 3 can be used in the 17415-derived heavy chain variable region.

[0092] Accordingly, in a preferred embodiment, the receptor framework of the 17415-derived heavy chain variable region has FWR1, FWR2, FWR3, and FWR4, each comprising the sequences of SEQ NOs 90, 91, 92, and 89. In another embodiment, they each comprise the sequences of SEQ NOs 90, 91, 93, and 89.

[0093] Particularly preferred 17415-derived heavy chain transplant variants are illustrated in FIG. 3. In one preferred embodiment, a heavy chain variable region of 17415gH1 (SEQ No. 17) may be used. In another embodiment, a heavy chain variable region of 17415gH2 (SEQ No. 18) may be used. In yet another embodiment, a heavy chain variable region of 17415gH3 (SEQ No. 19) may be used. In yet another embodiment, a heavy chain variable region of 17415gH4 (SEQ No. 20) may be used. In another embodiment, a heavy chain variable region of 17415gH5 (SEQ No. 21) may be used. In yet another embodiment, a heavy chain variable region of 17415gH6 (SEQ No. 22) may be used.

[0094] (e) Another desirable 17415-derived light and heavy chain pair

[0095] 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 any one of those illustrated in FIG. 2, and the heavy chain variable region is any one of those illustrated in FIG. 3. Particularly preferred pairs of light chain and heavy chain variable regions are those of the embodiments of the present application. Additionally, a set of six CDRs (LCDR1, LCDR2, LCDR3 and HCDR1, HCDR2, HCDR3) of specific light chain and heavy chain variable region pairs used in the embodiments of the present application is preferred.

[0096] In a particularly preferred embodiment, the light chain variable region is a transplant variant of 17415gL6 (SEQ No. 8), 17415gL7 (SEQ No. 9), or 17415gL13 to 17415gL16 (each SEQ No. 11 to 14). In a particularly preferred embodiment, the heavy chain variable region is a transplant variant of 17415gH1 to H6 (each SEQ No. 17 to 22).

[0097] In one embodiment, 17415gL6 (Sequence No. 8) is paired with any one of 17415gH1 to H6 (each Sequence No. 17 to 22). In another embodiment, 17415gL7 (Sequence No. 9) is paired with any one of 17415gH1 to H6 (each Sequence No. 17 to 22). In another embodiment, 17415gL13 (Sequence No. 11) is paired with any one of 17415gH1 to H6 (each Sequence No. 17 to 22). In another embodiment, 17415gL14 (Sequence No. 12) is paired with any one of 17415gH1 to H6. In another embodiment, 17415gL15 (Sequence No. 13) is paired with any one of 17415gH1 to H6. In another embodiment, 17415gL16 (Sequence No. 14) is paired with any one of 17415gH1 to H6 (each Sequence No. 17 to 22).

[0098] In one embodiment, 17415gH1 (Sequence No. 17) is paired with any one of 17415gL6 (Sequence No. 8), 17415gL7 (Sequence No. 9), or 17415gL13 to 17415gL16 (each Sequence No. 11 to 14). In one embodiment, 17415gH2 (Sequence No. 18) is paired with any one of 17415gL6 (Sequence No. 8), 17415gL7 (Sequence No. 9), or 17415gL13 to 17415gL16 (each Sequence No. 11 to 14). In one embodiment, 17415gH3 (Sequence No. 19) is paired with any one of 17415gL6, 17415gL7, or 17415gL13 to 17415gL16. In one embodiment, 17415gH4 (Sequence No. 20) is paired with any one of 17415gL6 (Sequence No. 8), 17415gL7 (Sequence No. 9), or 17415gL13 to 17415gL16 (each Sequence No. 11 to 14). In one embodiment, 17415gH5 (Sequence No. 21) is paired with any one of 17415gL6 (Sequence No. 8), 17415gL7 (Sequence No. 9), or 17415gL13 to 17415gL16 (each Sequence No. 11 to 14). In one embodiment, 17415gH6 (Sequence No. 22) is paired with any one of 17415gL6 (Sequence No. 8), 17415gL7 (Sequence No. 9), or 17415gL13 to 17415gL16 (each Sequence No. 11 to 14).

[0099] In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL7 (SEQ No. 9) and 17415gH6 (SEQ No. 22). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL15 (SEQ No. 13) and 17415gH6 (SEQ No. 22).

[0100] In one embodiment, the 17415-derived antigen binding site comprises a pair of light and heavy chain variable regions of 17415gL16 (SEQ No. 14) and 17415gH6 (SEQ No. 22).

[0101] In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL1 (SEQ No. 3) and 17415gH1 (SEQ No. 17). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL1 (SEQ No. 3) and 17415gH4 (SEQ No. 20).

[0102] In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL2 (SEQ No. 4) and 17415gH1 (SEQ No. 17). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL3 (SEQ No. 5) and 17415gH1 (SEQ No. 17). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL4 (SEQ No. 6) and 17415gH1 (SEQ No. 17). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL5 (SEQ No. 7) and 17415gH1 (SEQ No. 17).

[0103] In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL1 (SEQ No. 3) and 17415gH2 (SEQ No. 18). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL1 (SEQ No. 3) and 17415gH3 (SEQ No. 19). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL1 (SEQ No. 3) and 17415gH4 (SEQ No. 20).

[0104] In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL1 (SEQ No. 3) and 17415gH5 (SEQ No. 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL6 (SEQ No. 8) and 17415gH5 (SEQ No. 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL7 (SEQ No. 9) and 17415gH5 (SEQ No. 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL13 (SEQ No. 11) and 17415gH5 (SEQ No. 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL14 (SEQ No. 12) and 17415gH5 (SEQ No. 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL15 (SEQ No. 13) and 17415gH5 (SEQ No. 21). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL16 (SEQ No. 14) and 17415gH5 (SEQ No. 21).

[0105] In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL7 (SEQ No. 9) and 17415gH6 (SEQ No. 22). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL15 (SEQ No. 13) and 17415gH6 (SEQ No. 22). In one embodiment, the 17415-derived antigen binding site comprises a pair of light chain and heavy chain variable regions of 17415gL16 (SEQ No. 14) and 17415gH6.

[0106] (f) Variants and derivatives

[0107] In addition to the specific 17415-derived sequence presented herein, variants and derivatives of specific sequences described in other parts of this invention are also provided. These variants will retain the ability to specifically bind to CD45. Preferably, they will retain the ability to kill or deplete CD45-expressing cells.

[0108] 17552-derived antibody

[0109] (a) Overview of 17552-derived antibodies and variable regions

[0110] Another preferred antibody of the present invention is an antibody comprising one or more antigen-binding sites derived from the rabbit 17552 antibody described herein, in particular an antibody comprising a pair of humanized light chain and heavy chain variable regions derived from the rabbit 17552 antibody described herein. The original light chain and heavy chain variable region sequences of the rabbit 17552 antibody are provided as sequence no. 23 and 27, respectively. The light chain variable regions LCDR1, LCDR2, and LCDR3 of the original rabbit antibody are provided as sequence no. 94, 56, and 96, respectively. The heavy chain variable regions HCDR1, HCDR2, and HCDR3 of the original rabbit antibody are provided as sequence no. 100, 101, and 102, respectively.

[0111] In a preferred embodiment, the 17552-derived antibody has a light chain and heavy chain CDR set of one of the antibodies of the embodiments of the present application and includes the 17552-derived specificity in the dual-paratopic antibody shown in FIG. 17. In another preferred embodiment, it includes the light chain and heavy chain variable regions of the 17552-derived specificity shown in FIG. 17.

[0112] The generated original rabbit 17552 antibody and the antibody comprising the transplant variant described herein have the advantage of being able to specifically bind to both human CD45 and cynomolgus monkey CD45, which makes them particularly suitable for therapeutic development. The 17552-derived antigen-binding site also has the additional advantage that, when used in conjunction with a second specificity for CD45, the 17552 specificity can act as a "auxiliary" specificity to potentially enhance the efficacy of the other specificity by killing or depleting CD45. Thus, the 17552-derived specificity is particularly effective in antibodies having more than one paratopic or specificity for CD45. In a more preferred embodiment, the 17552-derived specificity is used as part of a dual-paratopic antibody for CD45. In a particularly preferred embodiment, the present invention provides a dual-paratopic antibody comprising 17415 and 17552 derived specificities, wherein the 17415 specificity acts as a “killing” specificity and the 17552 specificity acts as an “assistant” specificity.

[0113] The present invention describes the humanization of the original rabbit 17552 antibody. In a preferred embodiment, the 17552-derived paratope present in the antibody of the present invention is a humanized antigen-binding site. In a particularly preferred embodiment, the 17552-derived paratope was generated by transferring the CDR of the 17552 variable region (donor sequence) to the framework region of a second antibody (receptor sequence). Humanization may include the transfer of some framework residues as well as the CDR of the donor sequence.

[0114] Accordingly, in a particularly preferred embodiment, the present invention provides an antibody or an antigen-binding fragment thereof comprising at least one variable domain specific to CD45, comprising the following light chain and heavy chain variable regions:

[0115] (a) a light chain variable region comprising a CDR1 having the sequence of SEQ ID NO. 94, a CDR2 having the sequence of SEQ ID NO. 95, and a CDR3 having the sequence of SEQ ID NO. 96; and

[0116] (b) a heavy chain variable region comprising a CDR1 having the sequence of sequence number 100, a CDR2 having the sequence of sequence number 101, and a CDR3 having a sequence selected from any one of sequence numbers 102 to 105.

[0117] Preferred human light chain variable regions acting as receptor sequences of the framework include IGKV1-8 / IGKJ4. For the heavy chain, a particularly preferred receptor sequence for CDR is the IGHV4-4 / IGHJ4 receptor framework. In a preferred embodiment, the 17552-derived paratop includes a light chain variable region based on the IGKV1-8 receptor framework and a heavy chain variable region based on the IGHV4-4 receptor framework.

[0118] Table 2 below summarizes the light and heavy chain variable regions of the original 17552, the framework receptor sequences used, and specific transplant variants generated from the original 17552 antibody. Figure 4 also shows the alignment of various original donor and receptor sequences with the generated specific transplant variants. Individual amino acids shown in shaded and italics represent residues that show changes beyond a simple transfer of the CDR sequence of the original 17552 antibody to the human receptor framework. For example, these residues are either donor framework residues transferred along with the CDR sequence, or CDR residues different from those of the original 17552 CDR.

[0119] [Table 2]

[0120]

[0121]

[0122]

[0123]

[0124] (b) A preferred 17552 light chain transplant variant containing an IGKV11-8 derived framework

[0125] In a particularly preferred embodiment, the 17552-derived light chain variable region comprises the original CDRs of the 17552 light chain variable region. Specifically, it comprises 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. This set of CDRs may be combined with any one of the framework regions presented below.

[0126] In a preferred embodiment, the 17552-derived light chain variable region has an IGKV1-8 / IGKJ4 framework region, provided that one or more residues 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 one embodiment, leucine (L2), valine (V3), and glutamic acid (E63) are retained.

[0127] In one embodiment, the free cysteine ​​residue (C77) at position 77 of FWR3 is mutated to serine (C77S). Thus, 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.

[0128] In a preferred embodiment, the FWR1, FWR2, FWR3, and FWR4 framework sequences of the light chain variable region each include sequence numbers 114, 111, 115, and 113.

[0129] In another preferred embodiment, the FWR1, FWR2, FWR3, and FWR4 framework sequences of the light chain variable region each include sequence numbers 114, 111, 112, and 113.

[0130] A particularly desirable 17552-derived light chain transplant variant is 17552gL1 (sequence number 25). Another desirable 17552-derived light chain transplant variant is 17552gL2 (sequence number 26).

[0131] (d) A preferred 17552 heavy chain transplant variant containing an IGHV4-4 derived framework

[0132] 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. Or, in another preferred embodiment, HCDR3 has a sequence change from aspartic acid (D) to glutamic acid (E) at the fourth amino acid of HCDR3. In another preferred embodiment, HCDR3 has an amino acid sequence change from glycine (G) to serine (S) or alanine (A) at the fifth amino acid of HCDR.

[0133] In a preferred embodiment, the 17552-derived heavy chain variable region comprises a CDR1 comprising the sequence of sequence number 100, a CDR2 comprising the sequence of sequence number 101, and a CDR3 comprising a sequence selected from any one of sequence numbers 102 to 105.

[0134] In one embodiment, the framework region of the 17552-derived heavy chain variable region is FWR1 identical to the FWR1 of the IGHV4-4 receptor, but the first amino acid, glutamine (Q1), is substituted with glutamic acid (E1), and threonine (T23) is retained from the donor sequence.

[0135] In one embodiment, FWR2 is identical to the receptor sequence, but tyrosine (Y47) is retained from the donor sequence.

[0136] In one embodiment, FWR3 is identical to the receptor sequence except that phenylalanine (F67), lysine (K71), serine (S73), valine (V78) and threonine (T96) are retained.

[0137] In one embodiment, FWR4 is identical to the IGHV4-4 / IGHJ4 receptor sequence.

[0138] In one embodiment, one or more of the following residues are retained from the donor 17552 sequence: threonine (T23), tyrosine (Y47), phenylalanine (F67), lysine (K71), serine (S73), valine (V78), and threonine (T96). In a preferred embodiment, all of these residues are retained. In another preferred embodiment, all of these residues are retained and glutamic acid (E1) is substituted with glutamine (Q1).

[0139] In a preferred embodiment, the FWR1, FWR2, FWR3, and FWR4 regions each have the amino acid sequences of sequence numbers 123, 124, 125, and 126.

[0140] A particularly desirable heavy chain variable region derived from 17552 is 17552gH1 (Sequence No. 29). Another desirable variant is 17552gH2 (Sequence No. 30). Another desirable variant is 17552gH3 (Sequence No. 31). Another desirable variant is 17552gH4 (Sequence No. 32).

[0141] (c) Desirable 17552-derived light and heavy chain pairs

[0142] In a preferred embodiment, the antibody of the present invention comprises a 17552-derived light chain and heavy chain variable region, for example, a pair of 17552-derived light chain and heavy chain variable regions presented above.

[0143] Accordingly, in one embodiment, the antibody of the present invention or its antigen-binding fragment comprises at least one variable domain specific to CD45, comprising the following light chain and heavy chain variable regions:

[0144] (a) a light chain variable region comprising a CDR1 having the sequence of SEQ ID NO. 94, a CDR2 having the sequence of SEQ ID NO. 95, and a CDR3 having the sequence of SEQ ID NO. 96; and

[0145] (b) a heavy chain variable region comprising a CDR1 having the sequence of sequence number 100, a CDR2 having the sequence of sequence number 101, and a CDR3 having a sequence selected from any one of sequence numbers 102 to 105.

[0146] Preferred pairs include the 17552gL1 light chain variable region (Sequence No. 25) and the 17552gH1 heavy chain variable region (Sequence No. 29). Another preferred pair is 17552gL1 (Sequence No. 25) and 17552gH2 (Sequence No. 30). Another preferred pair is 17552gL1 (Sequence No. 25) and 17552gH3 (Sequence No. 31). Another example of a preferred pair is 17552gL1 (Sequence No. 25) and 17552gH4 (Sequence No. 32).

[0147] Another preferred pair includes a light chain variable region of 17552gL2 (sequence number 26) and a heavy chain variable region of 17552gH1 (sequence number 29). Another preferred pair is 17552gL2 (sequence number 26) and 17552gH2 (sequence number 30). Another preferred pair is 17552gL2 (sequence number 26) and 17552gH3 (sequence number 31). Another example of a preferred pair is 17552gL2 (sequence number 26) and 17552gH4 (sequence number 32).

[0148] (d) Variants and derivatives

[0149] In addition to the specific 17552-derived sequence described herein, variants and derivatives of the specific sequence described in other parts of this invention are also provided. These 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.

[0150] Exemplary antibody format

[0151] The term “antibody” is not limited to conventional 4-chain IgG antibodies having two identical light chains and two identical heavy chains. It includes all forms having at least one antigen-binding site formed by a set of six CDRs, in particular. The antibodies of the present invention may be complete antibodies having full-length heavy and light chains or fragments thereof. Unless otherwise specified, all references to antibodies herein also include antigen-binding fragments used in place of antibodies. Examples of types of antibodies and antibody fragments include Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, single-domain antibodies (e.g., VH or VL or VHH), scFv, divalent, trivalent or sacral antibodies, Bis-scFv, diabodies, tribodies, tetrabodies, or epitope-binding fragments of said antibodies (e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3) , (See 209-217). In one embodiment, the antibody of the present invention is not a single-domain antibody. Methods for generating and manufacturing antibody fragments are well known in the art (e.g., Verma et al(See *., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in international patent applications WO 2005 / 003169, WO 2005 / 003170, and WO 2005 / 003171. Multivalent antibodies may have multiple specificities, such as bispecific antibodies, or may be monospecific (see, e.g., WO 92 / 22853, WO 05 / 113605, WO 2009 / 040562, and WO 2010 / 035012). In one particularly preferred embodiment, the antibody of the present invention is a monospecific antibody for CD45. In another particularly preferred embodiment, the antibody of the present invention is biparatopic for CD45. The term "antibody" includes monospecific antibodies, bispecific antibodies, and multispecific antibodies. Accordingly, for example, the term “antibody” specifically includes forms such as Fab-X / Fab-Y, BYbe, and TrYbe. The term “antibody” includes antibody fragments, preferably the antibody fragments mentioned herein. Wherever antibody is referred to herein, antigen-binding antibody fragments may also be used, unless otherwise required in a particular context.

[0152] Examples of possible antibody formats are known in the technical field, for example, the review paper incorporated herein by reference “The coming of Age of Engineered Multivalent Antibodies, Nunez-Prado et al Drug Discovery Today Vol 20 Number 5 Mar 2015, pages 588-594, D. Holmes, Nature Rev Drug Disc Nov 2011:10; 798, Chan and Carter, Nature Reviews Immunology As disclosed in [vol. 10, May 2010, 301]. In one embodiment, the antibody of the present invention may comprise, be essentially composed of, or be composed of any of the forms presented below. An antibody based on a sequence derived from the rabbit 17415 and 17552 antibodies described herein is particularly preferred. Accordingly, for any one of the forms presented below, in a preferred embodiment, the antibody will comprise at least one antigen-binding site comprising a humanized light chain and heavy chain variable region derived from the rabbit 17415 antibody. In another preferred embodiment, for any one of the forms presented below, the antibody will comprise at least one antigen-binding site comprising a humanized light chain and heavy chain variable region derived from the rabbit 17552 antibody. In a particularly preferred embodiment, such a form having at least two antigen-binding sites will comprise at least one antigen-binding site comprising a humanized light chain and heavy chain variable region derived from the rabbit 17415 antibody and at least one antigen-binding site comprising a humanized light chain and heavy chain variable region derived from the rabbit 17552 antibody.

[0153] One of the particularly desirable antibody types is the IgG type antibody.

[0154] As used herein, "binding fragment" means a fragment capable of binding to a target peptide or antigen with sufficient affinity to be characterized as specific to the target peptide or antigen. As used herein, the term "Fab fragment" refers to the V of the light chain L (Light chain mutable) domains and immutable domains (C L A light chain fragment containing ), and V of the heavy chain H It refers to an antibody fragment comprising a (heavy chain variable) domain and a first constant domain (CH1). The term "Fv" refers to two variable domains, for example, a cognate pair or an affinity maturation variable domain (i.e., VH and V L It refers to cooperative variable domains such as a pair). In one embodiment, such fragments are used as antibody molecules of the present invention. As used herein, cooperative variable domains complement each other and / or both contribute to antigen binding, so that Fv(V H / V L It is a variable domain that makes the pair specific to the corresponding antigen.

[0155] As used herein, “antigen binding site” refers to a binding region capable of binding to a target antigen, generally a polypeptide, having, for example, sufficient affinity to characterize that the region is specific to the antigen. In one embodiment, the binding site comprises at least one variable domain or a derivative thereof, for example, a pair of homologous variable domains or derivatives thereof. Generally, this is a VH / VL pair. The variable region (also referred to herein as a variable domain) generally comprises three CDRs and an appropriate framework. In one embodiment, the antigen binding site comprises two variable regions, namely 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 to CD45, particularly specificity in terms of the location on CD45 where the binding site binds. In a particularly preferred embodiment, the VH / VL pair is humanized. The six CDRs provided by the light chain and heavy chain variable region pair may be referred to as a “CDR set.”

[0156] Amino acid residues of the antibody variable domain are conventionally numbered according to a system devised by Kabat et al. This system [Kabat et al. , 1987, in Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services National Institutes of Health (NIH) (hereinafter "Kabat et alThis is described in detail in the aforementioned literature. Kabat residue designations do not always directly correspond to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or more amino acids than in the strict Kabat numbering scheme, corresponding to the shortening or insertion of structural components, whether in the framework of the basic variable domain structure or the complementarity determining region (CDR). The precise Kabat numbering scheme of residues for a given antibody can be determined by aligning the antibody's sequence with homologous residues to the "standard" Kabat-numbered sequence. 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) according to the Kabat numbering scheme. However, according to Chothia (Chothia, C. and Lesk, AMJ Mol. Biol., 196, 901-917 (1987)), the loop corresponding to CDR-H1 extends from residue 26 to residue 32. Therefore, unless otherwise specified, 'CDR-H1' as used herein refers to residues 26 through 35, described by combining the Kabat numbering system and Chothia's topological loop definition. The CDRs of the light chain variable domain are located at residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) according to the Kabat numbering system.

[0157] Since this application discusses humanization, linear amino acid numbers are used instead of Kabat numbers. Accordingly, discussions regarding the amino acid positions of transplant variants follow the linear amino acid numbering system.

[0158] Derivatives, modifications, and humanization

[0159] In addition to the specific antibody described herein, variants and derivatives of this antibody are also provided. These variants and derivatives will retain the ability to specifically bind to at least CD45. In a preferred embodiment, they will retain the biological function(s) described herein, e.g., the ability to kill or deplete target cells expressing CD45. All specific assays and target cells used herein may be used, for example, to verify these activities. In a particularly preferred embodiment, the variant or derivative will retain the ability to specifically bind to both human and cynomolgus monkey CD45. This ability may be verified using assays such as Biacore or cell-based assays. Likewise, all specific methods used in the embodiments of this application may be used to verify the ability to specifically bind to human and cynomolgus CD45.

[0160] As used herein, a “variant” or “derivative” may have one, two, four, four, or five or more amino acid sequence changes compared to a specific sequence described herein, for example. In one embodiment, the paratop or antigen-binding site may include one of the six specific CDR sets presented herein, and there may be a total of up to seven amino acid sequence changes across all six CDRs compared to a specific sequence. In one embodiment, there are up to six amino acid sequence changes. In another embodiment, there are up to five amino acid sequence changes. In another embodiment, there are up to four amino acid sequence changes. In a preferred embodiment, there are up to three amino acid sequence changes. In a more preferred embodiment, there are up to two amino acid sequence changes. In a particularly preferred embodiment, there is only one amino acid sequence change compared to the six specific CDRs presented. All such variants will retain the ability to specifically bind to human CD45. Preferably, the variants will retain the ability to specifically bind to both human and cynomolgus monkey CD45. In another embodiment, the number of such sequence changes may occur in the entire variable region of the paratop compared to the variable region of the specific antibody presented herein.

[0161] For example, modifications to the CDR may involve replacing one or more cysteine ​​residues, for instance, with serine residues. Asn can be a substrate for deamination, and this tendency can be reduced by replacing Asn and / or adjacent amino acids with other amino acids (e.g., conservative substitution). The Asp in the CDR may be isomerized. Isomeration can be minimized by replacing Asp and / or adjacent amino acids with other amino acids (e.g., conservative substitution). Undesirable characteristics can be removed or reduced using the amino acid sequence, while characteristic characteristic(s) are retained. Examples of modifications include removing glycosylation sites, GPI anchors, or solvent-exposed lysines. These modifications can be achieved by replacing the relevant amino acid residues with conservative amino acid substitutions.

[0162] Antibody constant region and Fc region functions

[0163] In one preferred embodiment, the antibody of the present invention does not include an Fc domain. In another preferred embodiment, the antibody of the present invention includes a modified Fc domain as described below. In a preferred embodiment, the antibody of the present invention includes an Fc domain, but the sequence of the Fc domain is modified to remove one or more Fc effector functions.

[0164] In another embodiment, the Fc region of the antibody of the present invention is modified to optimize a specific characteristic of the antibody, such as any one of those discussed herein.

[0165] 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 effector functions or functions associated with a normal Fc region.

[0166] As used herein, the Fc domain generally refers to -(CH2CH3)2 unless otherwise specified in the context.

[0167] In one embodiment, the antibody of the present invention does not contain a -CH2CH3 fragment.

[0168] In one embodiment, the antibody of the present invention does not include a CH2 domain.

[0169] In one embodiment, the antibody of the present invention does not include a CH3 domain.

[0170] In one embodiment, the antibody of the present invention does not bind to the Fc receptor.

[0171] In one embodiment, the antibody of the present invention does not bind to complement. In a preferred embodiment, the antibody of the present invention does not bind to the first complement factor, C1q or C1. In one embodiment, the antibody of the present invention does not bind to these factors, for example, because it lacks an Fc region. In another embodiment, the antibody of the present invention does not bind to these factors because there is a modification within the invariant region that interferes with the ability to bind to these factors. In another embodiment, the antibody of the present invention does not bind to FcγR but binds to complement.

[0172] For example, in one embodiment, the antibody of the present invention does not bind to FcγR but binds to C1q and / or C1.

[0173] In one embodiment, the antibody of the present invention does not include an active Fc region in the sense that the normal Fc region does not induce the release of one or more cytokines. For example, the Fc region of the antibody according to the present invention may not induce cytokine release when binding to an Fc receptor, or even if it does, the effect may be negligible.

[0174] In one embodiment, the antibody according to the present invention may generally include a modification that alters the serum half-life of the antibody. Accordingly, in another embodiment, the antibody according to the present invention may have Fc region modification(s) that alter the half-life of the antibody. Such modifications may coexist with modifications that alter the function of the Fc region. In one embodiment, the antibody according to the present invention has modification(s) that increase or decrease the serum half-life of the antibody compared to an antibody without such modifications. In another embodiment, the antibody according to the present invention includes all modification(s) that silence the Fc region and increase or decrease the serum half-life of the antibody compared to an antibody without such modifications.

[0175] The antibody constant region domain of the antibody of the present invention, if present, may be selected in consideration of the proposed function of the antibody molecule, particularly the effector function that may be required. In a preferred embodiment, the antibody is an antibody that lacks Fc, or lacks one or more effector functions of the Fc region, preferably all of which are lacking. In another embodiment of the present invention, the effector function(s) of the Fc region of the antibody may still be present. In one embodiment, the antibody of the present invention may include a human constant region, e.g., an IgA, IgD, IgE, IgG, or IgM constant region. In particular, if the antibody molecule is intended for a therapeutic use where antibody effector function is required, a human IgG constant region domain, particularly the constant region domains of IgG1 and IgG3 isotypes, may be used. Alternatively, if the antibody molecule is intended for a therapeutic use 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 be modified so that the antibody does not have an effector function. Therefore, it will be understood that sequence variants of these invariant region domains can also be used. For example, the literature [Angal et alAs described in [., 1993, Molecular Immunology, 1993, 30:105-108], an IgG4 molecule in which the serine at position 241 is changed to proline can be used. Accordingly, in the present embodiment, if the antibody is an IgG4 antibody, the antibody may contain the S241P mutation. In another embodiment, the antibody of the present invention may lack the Fc region.

[0176] In one embodiment, the antibody of the present invention may have the Fc region silenced. As used herein, the terms “silenced,” “silenced,” or “silencing” mean an antibody having the modified Fc region described herein, in which binding to the Fc gamma receptor (FcγR) is reduced compared to binding to the FcγR when the same antibody contains the unmodified Fc region (e.g., when the binding affinity to FcγR is reduced by 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% or more, or 100% compared to binding to the FcγR when the same antibody contains the unmodified Fc region as measured by BLI). In some embodiments, the Fc-silenced antibody does not show any binding to FcγR. The binding of antibodies having a modified Fc region to FcγR can be measured using various techniques known in the art, for example, equilibration methods (e.g., enzyme-linked immunosorbent assay (ELISA); KinExA, Rathanaswami et alAnalytical Biochemistry, Vol. 373:52-60, 2008; or radioimmunoassay (RIA)), or surface plasmon resonance assay or other kinetic-based assay mechanisms (e.g., BIACORE™ assay or Octet™ assay (forteBIO)), and other methods, e.g., indirect binding assay, competitive binding assay, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration), are included but not limited thereto. In other embodiments, the antibody of the present invention may be modified to allow complement activation while reducing or removing binding to FcγR. In other embodiments, the Fc region of the antibody of the present invention may be modified to allow complement activation without activating cytokine release.

[0177] In one embodiment, the antibody heavy chain comprises a CH1 domain, and the antibody light chain comprises a kappa or lambda CL domain. In one embodiment, the antibody heavy chain comprises a CH1 domain, a CH2 domain, and a CH3 domain, and the antibody light chain comprises a kappa or lambda CL domain. A kappa light chain is preferred.

[0178] The four human IgG isotypes bind to activating Fcγ receptors (FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa), repressor FcγRIIb receptors, and the first complement component (C1q) with different affinities, exhibiting highly diverse 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 ResearchRefer to [2007 Sep 15; 67(18):8882-90]. In one embodiment, the antibody of the present invention does not bind to an Fc receptor. In another embodiment, the antibody of the present invention binds to one or more types of Fc receptors.

[0179] Binding of IgG to FcγR or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are important for FcγR and C1q binding and have unique sequences in IgG2 and IgG4. Substituting human IgG1 with IgG2 residues at positions 233–236 and IgG4 residues at positions 327, 330, and 331 was 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 R.L. 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 R. J Biol Chem. 276:9-6591, 604). In addition, Idusogie et al. demonstrated that alanine substitution at various sites, including K322, significantly reduces complement activation (Idusogie EE. et al., 2000. Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc. J Immunol. 164(8):4178-84). Similarly, mutations within the CH2 domain of mouse IgG2A were shown to reduce binding to FcγRI and C1q (Steurer W. et al ., 1995. Ex vivo coating of islet cell allografts with murine CTLA4 / Fc promotes graft tolerance. J Immunol. 155(3):1165-74).

[0180] In one embodiment, the Fc region used is mutated, specifically by the mutations described herein. In one embodiment, said mutation is to remove binding and / or effector function. In a preferred embodiment, the antibody of the present invention is mutated so as not to bind to the Fc receptor. In another preferred embodiment, the antibody of the present invention does not contain the Fc region and thus does not exhibit Fc effector activity. In one embodiment, the Fc mutation is selected from the group comprising mutations that remove or enhance binding between the Fc region and the Fc receptor, mutations that increase or remove effector function, mutations that increase or decrease the half-life of the antibody, and combinations thereof. In a preferred embodiment, said modification removes or reduces binding with the Fc receptor. In another preferred embodiment, said modification removes or reduces Fc effector function. In another preferred embodiment, said modification increases or decreases serum half-life. In another preferred embodiment, the constant region of the antibody includes one or more modifications that reduce or remove Fc receptor binding and Fc effector function, and increase or decrease serum half-life. In one embodiment, when referring to the effect of the modification, this can be demonstrated through a comparison with an equivalent antibody without modification.

[0181] In another embodiment of the present invention, the antibody may have a heavy chain modification that modifies the protein A binding ability, particularly by removing the protein A binding. As discussed herein, this approach may be preferably used to facilitate the purification of bispecific antibodies. However, in another embodiment, all antibodies of the present invention may be modified to alter protein A binding if they possess an Fc region. For example, modifications may be included in both heavy chains. Or, there may be no modifications in either heavy chain. However, in a preferred embodiment, one has a modification and the other does not.

[0182] Some antibodies that selectively bind to FcRn at pH 6.0 but not at pH 7.4 exhibit longer half-lives in various animal models. 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), M252Y / S254T / T256E + H433K / N434F (Vaccaro C. et al ., 2005. Engineering the Fc region of immunoglobulin G to modulate in vivo antibody levels. Nat Biotechnol. 23(10):1283-8) and M428L / N434S (Zalevsky et al ., 2010, Enhanced antibody half-life improves in vivo As in *activity. Nature Biotech 28:157-159*), several mutations located at the interface between the CH2 and CH3 domains have been shown to increase binding affinity to FcRn and the half-life of IgG1 in vivo. Therefore, modifications in M252 / S254 / T256 + H44 / N434 may exist to alter serum half-life, and specifically in M252Y / S254T / T256E + H433K / N434F. However, there is not always a direct relationship 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, it is desirable to increase the half-life. In another embodiment, it may be practically desirable to decrease the serum half-life of the antibody, and thus variations that decrease the serum half-life may exist.

[0183] The IgG4 subclass exhibits reduced binding to the Fc receptor (FcγRIIIa). Antibodies of other IgG subclasses generally exhibit strong binding. Reduced receptor binding in these other IgG subtypes can be affected by altering, for example, substituting, one or more amino acids selected from the group comprising Pro238, Aps265, Asp270, Asn270 (Fc carbohydrate loss), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435. In one embodiment, a molecule according to the present invention has an Fc of an IgG subclass, for example, IgG1, IgG2, or IgG3, wherein one, two, or all of the positions S228, L234, and / or D265 of the Fc are mutated. In one embodiment, mutations in the Fc region are independently selected from S228P, L234A, L235A, L235A, L235E and combinations thereof.

[0184] In one embodiment, the antibody of the present invention may include modifications that affect whether the antibody induces cytokine release. In particular, L234F and K274Q modifications have been shown to reduce the antibody's ability to induce cytokine release. Accordingly, in one embodiment, the antibody of the present invention may include modifications of L234 and / or K274 that alter cytokine release, particularly L234F and K274Q modifications. Additionally, L234 residues may affect platelet activation, and these residues may be additionally or alternatively modified. In one embodiment of the present invention, for example, L234 modifications that alter platelet binding, particularly L234F modifications, may be introduced. Since P331 has been shown to be involved in C1q binding as well, in one embodiment, P331 may not be modified to maintain complement activation. In another embodiment, it may be modified to reduce or eliminate complement activation. For example, the heavy chain may include the P331S modification. In another embodiment, a P329 variant, particularly a P329A variant, is present to reduce or eliminate complement binding. In another embodiment, the antibody may include one or more variants at positions P329, P331, K332, and / or D265. In a preferred embodiment, the antibody may include variants at P329A, P331S, K332A, and D265A to affect complement binding and, in particular, reduce C1q binding.

[0185] It may be desirable to reduce or increase the effector function of the Fc region. In a preferred embodiment, it is desirable to reduce this effector function. In another embodiment, it is desirable to optimize it. For antibodies targeting cell surface molecules, particularly surface molecules of immune cells, it is generally necessary to completely eliminate the effector function. In other cases, particularly when the goal is to deplete cells, it may be desirable to eliminate the Fc effector function or reduce it to the lowest possible level. For example, in a particularly preferred embodiment, the antibody of the present invention can induce apoptosis 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 not physically possess an Fc region or may include 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 may induce less or no release of one or more cytokines that an antibody with an unmodified Fc region would typically induce release of. In a preferred embodiment, the antibody of the present invention may stimulate apoptosis but does not exhibit Fc function. Other examples of Fc function include stimulation of mast cell degranulation, and this function may also be reduced or absent in the antibody of the present invention. The reduction rate of Fc function may be, for example, 65% or more, for example, 75% or more. In one embodiment, the reduction rate is 80% or more. In another embodiment, the reduction rate is 90% or more. The reduction rate may be, for example, 95% or more. In a preferred embodiment, the reduction rate is 99% or more. In another embodiment, the reduction rate may be 100%, which means that in this case, the Fc function is completely eliminated.

[0186] Numerous mutations were introduced into the CH2 domain of human IgG1, and their effects on ADCC and CDC were tested in vitro (Idusogie EE. et al ., 2001. Engineered antibodies with increased activity to recruit complement. J Immunol. 166(4):2571-5). In particular, alanine substitution at position 333 has been reported to increase both ADCC and CDC. Therefore, in one embodiment, a modification may exist at position 333, and in particular, a modification that alters complement mobilization ability may exist. Lazar et al. described a triple mutant (S239D / I332E / A330L) with enhanced ADCC, having a higher affinity for FcγRIIIa and a lower affinity for FcγRIIb (Lazar GA. et al ., 2006). Therefore, variants of S239 / I332 / A330, particularly variants that alter the affinity for the Fc receptor, especially S239D / I332E / A330L variants, may exist (Engineered antibody Fc variants with enhanced effector function. PNAS 103(11): 4005-4010). Antibodies with increased ADCC were generated using the same mutations (Ryan MC. et al ., 2007. Antibody targeting of B-cell maturation antigen on malignant plasma cells. Mol. Cancer Ther., 6: 3009 - 3018). Richards et al. studied a slightly different triple mutant (S239D / I332E / G236A) in which FcγRIIIa affinity and the FcγRIIa / FcγRIIb ratio were improved, mediating enhanced phagocytosis of target cells by macrophages (Richards JO et al(2008) Optimization of antibody binding to Fcgamma RIIa enhances macrophage phagocytosis of tumor cells. Mol Cancer Ther. 7(8):2517-27). Accordingly, in one embodiment, the S239D / I332E / G236A variant may be present.

[0187] IgG4 antibodies are an IgG subclass suitable for receptor blockade due to a lack of effector function. IgG4 molecules can exchange half-molecules through a dynamic process called Fab-arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous IgG4. In a preferred embodiment, the antibody of the present invention has a modification in S228, particularly S228P. The S228P mutation has been shown to prevent this recombination process, thereby enabling the design of therapeutic IgG4 antibodies with low unpredictability (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 produce bispecific antibody molecules. The modifications presented herein may be utilized in relation to IgG4 in preferred embodiments.

[0188] WO 2008 / 145142 discloses examples of modifications that can be utilized in the present invention, in particular modifications to IgG4 isotype antibodies.

[0189] In one embodiment, the heavy chain of the antibody of the present invention may comprise a human IgG4 constant region substituted with 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 comprises a modification at position 409, specifically one selected from the introduction of a Lys, Ala, Thr, Met, or Leu residue at that position. In one embodiment, the modification is the introduction of a Lys, Ala, 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 comprise 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 excluding proline. In one embodiment, the antibody of the present invention may utilize the ability to exhibit specific characteristics of a particular antibody class, antibody isotype, or antibody allotype. Such natural variability may be used to confer specific characteristics. For example, IgG1 has R409 at position 409 of the heavy chain, whereas IgG4 has K409, which may naturally affect the ability of the antibody. A review of various naturally occurring sequence variations is in the literature [Jefferis et al

[2009] mAbs, 1(4): 332-338] is provided, the full content of which is included by reference, particularly in relation to the sequence variations discussed therein.

[0190] Furthermore, those skilled in the art will understand that antibodies can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the antibody. These modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A common modification is the loss of carboxy-terminal basic residues (e.g., lysine or arginine) due to the action of carboxypeptidase (described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Consequently, the C-terminal lysine of the antibody heavy chain may be absent.

[0191] In one embodiment, the antibody of the present invention may be a non-glycolytic IgG to induce, for example, a reduction in Fc function, particularly a nearly Fc-null phenotype. In one embodiment, the antibody of the present invention has a modification in N297, particularly N297A. In one embodiment, the antibody of the present invention has a modification in F243 and / or F244, in particular, such modification implies that the antibody is a non-glycolytic IgG. In one embodiment, the antibody of the present invention may include F243A and / or F244A heavy chain modifications. In another embodiment, one or more of F241, F243, V262, and V264 may be modified, in particular, to amino acids that affect glycosylation. In one embodiment, the antibody of the present invention may have modifications in F241A, F243A, V262E, and V264E. These modifications [Yu et al

[2013] 135(26): 9723-9732] is discussed, the entire content of which is incorporated by reference, particularly with respect to the modifications discussed therein. These modifications provide, for example, a method for regulating Fc receptor binding. Modifications affecting the glycosylation of the antibody may exist. Additionally, the antibody of the present invention may be produced in a cell type that affects glycosylation as another approach for glycosylation engineering. In one embodiment, the fucosylation, sialylation, galactosylation, and / or mannosylation of the antibody of the present invention may be altered through sequence modification and / or the cell type used for antibody production.

[0192] In one embodiment, the antibody of the present invention may have a modification at position 297 and / or 299. For example, in one embodiment, the antibody of the present invention comprises an N297A modification in the heavy chain, preferably N297Q, or a mutation to another residue of Ser or Thr at position 299. In one embodiment, the antibody of the present invention has both of these modifications.

[0193] Examples of particularly desirable constant region modifications include the Leu234Ala and Leu235Ala modifications (based on the EU numbering system), which are also known as the LALA modifications. In particular, an IgG1 antibody form having the LALA modification is preferred as a form. In another preferred embodiment, the antibody of the present invention is an IgG4 antibody having the FALA modification.

[0194] In another embodiment, the antibody of the present invention may have a modified hinge region and / or a CH1 region. Alternatively, the isotype used may be selected to have a specific hinge region. Literature [White et al (2015) Cancer CellAs described in [27(1): 138-148], IgG2 CH1 and the hinge region impart specific characteristics, particularly in relation to disulfide crosslinking between the heavy and light chains. The use of modifications to increase or decrease the flexibility of the hinge region can also be utilized, for example, in the antibodies of the present invention. Approaches to changing the flexibility of the hinge region [Liu et al (2019) Nature Communications It is disclosed in [10: 4206]. Literature [White et al (2015) and Liu et al

[2019] is incorporated by reference in its entirety, particularly in relation to the variations discussed. In one embodiment, the heavy chain of the antibody of the present invention has an IgG2 CH1 and / or hinge region, and in another embodiment, both heavy chains do. 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 having a hinge or CH1 modification, particularly one having a modified hinge, for example, designed to alter disulfide bond formation. In another embodiment, an IgG2 or IgG4 isotype antibody is used because the hinge region of these isotype antibodies is less flexible than that of an IgG3 isotype antibody. In one embodiment, an IgG4 isotype antibody is used in a form capable of inducing CD32 crosslinking.

[0195] In another embodiment, the antibody exhibits the most stereochemically superior ability to induce cross-linking of the CD45 molecule.

[0196] Examples of particularly desirable heavy chain invariant region sequences containing LALA modifications are provided as sequence numbers 144, 146, and 148, respectively.

[0197] The heavy chain constant region of SEQ ID NO. 148 has the so-called "knob" variant T336W. The heavy chain constant region of SEQ ID NO. 146 has the so-called "hole" variants T366S, L366S, and Y407. These "knob-into-hole" variants promote heterodimer formation, thereby promoting the formation of bispecific antibodies rather than monospecific antibodies. Accordingly, in a particularly preferred embodiment, these heavy chain constant regions are used in the biparatopic antibodies of the present invention.

[0198] Examples of particularly desirable light chain invariant region sequences are provided as sequence number 145, respectively.

[0199] In a particularly preferred embodiment, the constant regions of the heavy chain and light chain in the monospecific antibody of the present invention are the constant regions of SEQ ID NOs. 144 and 145. In one embodiment, these constant regions are used together with the VR17415gL15gH6 variable regions.

[0200] In another particularly preferred embodiment, the heavy chain and light chain constant regions of the biparatopic antibody of the present invention are the constant regions of SEQ ID NOs. 146 and 148 for the heavy chain and the constant region of SEQ ID NO. 145 for the light chain. In one embodiment, the constant region used in the VR17552gL1gH4 portion of the biparatopic antibody is the constant region of SEQ ID NOs. 148 and 145. In one embodiment, the constant region used in the VR17552gL1gH4 portion of the biparatopic antibody is the constant region of SEQ ID NOs. 146 and 145.

[0201] In one embodiment, the constant region used in the VR17415gL15gH6 portion of the dual-paratopic antibody is the constant region of SEQ ID NOs 148 and 145. In one embodiment, the constant region used in the VR17415gL15gH6 portion of the dual-paratopic antibody is the constant region of SEQ ID NOs 146 and 145.

[0202] In one embodiment, the constant region used in the VR17552gL1gH4 portion of the dual-paratopic antibody is the constant region of SEQ ID NOs 148 and 145, and the constant region used in the VR17415gL15gH6 portion of the dual-paratopic antibody is the constant region of SEQ ID NOs 148 and 145. In one embodiment, the constant region used in the VR17552gL1gH4 portion of the dual-paratopic antibody is the constant region of SEQ ID NOs 146 and 145, and the constant region used in the VR17415gL15gH6 portion of the dual-paratopic antibody is the constant region of SEQ ID NOs 146 and 145.

[0203] Bispecific and biparatopic antibodies

[0204] In a preferred embodiment, the antibody of the present invention is bispecific. In a preferred embodiment, it is a biparatopic antibody for CD45, i.e., has two specificities for CD45. Various bispecific antibody formulations are available that promote the formation or purification of bispecific antibodies more than monospecific antibodies when different heavy and light chains for specificity are expressed together, and these can be utilized in the present invention.

[0205] In one embodiment, the antibody of the present invention may have a modification that promotes the formation of the antibody of the present invention over an unwanted species. Such modification is particularly preferred when the antibody of the present invention comprises at least two different antigen-binding sites that recognize different epitopes. In particular, such modification is particularly preferred for bispecific antibodies and biparatopic antibodies. For example, in one embodiment, the production of the antibody of the present invention may comprise two different antigen-binding sites located on different polypeptide chains and associated with each other, particularly two different paratopes. Thus, it may be preferable to form a heterodimer containing both specificities rather than a homodimer containing only one of the two specificities. An example of one approach to promote heterodimer formation is to utilize a heavy chain modification that promotes the binding of two different heavy chains rather than the binding of two identical heavy chains. In one embodiment, one (or more than one) of the binding partners cannot form a homodimer. For example, the amino acid sequence of the binding partner is modified to eliminate or minimize homodimer formation. Examples of such variations include the so-called "knob-into-hole" variation. Possible knob-into-hole variations are, for example, the literature [Merchant et al (1998) Nature Biotechnology 16(7): 677-681 and Carter et al (2001) J Immunol Methods This is presented in [ , 248(1-2): 7-15], and these two documents are included by reference in particular with regard to the knob-into-hole modification discussed therein. Charge modification may be used alternatively or additionally to promote heterodimer formation rather than homodimer formation, for example, such modification may be present in the heavy chain. In another embodiment, charge modification is used to induce pairing of a specific light chain and a specific heavy chain.

[0206] In one embodiment, this approach that promotes heterodimer formation is used in conjunction with a common light chain approach. In other embodiments, rather than promoting heterodimer formation over homodimers, there may be a modification that allows heterodimers to be separated more easily from homodimers, for example, by chromatography. Additionally, this approach may be used in conjunction with a common light chain approach in some embodiments. In other embodiments, a portion of the antibody having a specific paratope for CD45 may bind only to a portion of the antibody constituting a different paratope of the antibody.

[0207] As used herein, "unable to form homodimers" means that the tendency to form homodimers is low or zero. As used herein, "low" means aggregates of 20%, 10%, 5% or less, e.g., 4%, 3%, 2%, 1%, 0.5% or less.

[0208] Heavy chain modifications may be used to cause one heavy chain to have a different affinity for a specific binder compared to another heavy chain. For example, two different heavy chains may have different affinities for Protein A. In one embodiment, one heavy chain has a modification that removes Protein A binding or is an isotype that does not bind to Protein A, while the other heavy chain still binds to Protein A. This approach does not change the rate of heterodimer formation but enables the purification of heterodimer antibodies from homodimer antibodies based on Protein A affinity. The antibody of the present invention may have modifications affecting Protein A binding at positions 95 and 96 of one heavy chain. Examples of such modifications that may be used include applying an H95R modification to one heavy chain or using both H95R and Y96F modifications in the IMGT exon numbering system. These modifications are the H435R modification and the H435R and Y436F modifications in the EU numbering system. In one embodiment, the antibody of the present invention may also have modifications to D16, L18, N44, K52, V57, and V82. In one embodiment, these modifications are present in the heavy chain, and one or more of the modifications D16E, L18M, N44S, K52N, V57M, and V82I in the IMGT numbering scheme are present. In one embodiment, these modifications are used when the IgG is IgG1, IgG2, or IgG4. In a particularly preferred embodiment, when both heavy chains are IgG4 isotypes, these modifications are used in one of the two heavy chains. An approach to these modifications affecting protein A binding is described, for example, in US 2010 / 0331527 A1, the entire contents of which are incorporated by reference, particularly with respect to modifications related to protein A binding.

[0209] In another embodiment, the isotype of the heavy chain used may be selected based on protein A binding ability. For example, in humans, IgG1, IgG2, and IgG4 all bind to protein A in the wild type, whereas wild-type human IgG3 does not bind to protein A. In a particularly preferred embodiment, both heavy chains are IgG4, but one has modification(s) to reduce or eliminate protein A binding. This means that the heteromeric form of the antibody can be more easily separated from the unwanted homomeric form based on protein A affinity.

[0210] In one embodiment, a modification that promotes heterodimer formation can be combined with a modification that enables the purification of the heterodimer. In one embodiment, the modification may be at positions F405 and K409. For example, one example of a pair of modifications that can be introduced into two heavy chains to promote heterodimer formation is F405L and K409R. These modifications may be used alone or in combination with heavy chain modifications that enable the 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, and the other heavy chain 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. An example of this approach is the literature [Steinhardt et al (2020) PharmaceuticsIt is described in [ , 12, 3], the entire content of which is incorporated by reference in relation to the bispecific antibody formats and heavy chain modifications described in particular. In other embodiments, an approach involving light chains may be used, particularly in addition to the approach for heavy chains discussed above. In one embodiment, the Roche Cross-Mab approach is applied. In other embodiments, a common light chain may be used to use the same light chain for both specificities. Various bispecific antibody formats are described in the literature [Spiess et al (2015) Molecular Immunology It is reviewed in [67: 95-106] and can be used in the present invention.

[0211] In a particularly preferred embodiment, the dual-paratopic antibody of the present invention is an IgG-type antibody. In a preferred embodiment, it is an IgG1 or IgG4 antibody. In a particularly preferred embodiment, it is an IgG1-type antibody in which the constant region contains a LALA modification. In a particularly preferred embodiment, it is an IgG4-type antibody in which the constant region contains a FALA modification.

[0212] 17415 x 17552 derived biparatopic antibody

[0213] In a particularly preferred embodiment, the antibody of the present invention is biparatopic for CD45. In one embodiment, the antibody may also include at least one specificity for a molecule other than CD45 in addition to the two specificities for CD45. For example, the antibody may include additional specificity specific to a blood protein or a cell-specific surface protein. A particularly preferred specificity is for serum albumin. In another embodiment, the antibody of the present invention is biparatopic for CD45 and does not include additional specificity.

[0214] In a particularly preferred embodiment, the antibody of the present invention is biparatopic for CD45, because it includes 17415-derived specificity for CD45 and also includes 17552-derived specificity for CD45. The 17552-derived specificity may be considered as an "auxiliary" specificity, and the 17415-derived specificity may be considered as a "killing" specificity.

[0215] Any pair of transplant variants derived from 17415 and 17552 may be used. For example, any pair of FIGS. 2 through 4 may be used. In one embodiment, the 17415 specificity follows Table 1, and the 17552 specificity follows Table 2. Particularly preferred combinations are those used in the embodiments and drawings of this application.

[0216] In one embodiment, the preferred biparatopic is a 17415gL7gH6 x 17552gL1gH1 biparatopic. In another embodiment, the preferred biparatopic is a 17415gL7gH6 x 17552gL1gH4 biparatopic. In one embodiment, the biparatopic comprises a CDR of this combination. In another embodiment, it will comprise the entire light chain and heavy chain variable region of this combination.

[0217] In one embodiment, the preferred biparatopic is a 17415gL15gH6 x 17552gL1gH1 biparatopic. In another embodiment, the preferred biparatopic is a 17415gL15gH6 x 17552gL1gH4 biparatopic. In one embodiment, the biparatopic comprises a CDR of this combination. In another embodiment, it will comprise the entire light chain and heavy chain variable region of this combination.

[0218] In one embodiment, the preferred biparatopic is a 17415gL16gH6 x 17552gL1gH1 biparatopic. In another embodiment, the preferred biparatopic is a 17415gL16gH6 x 17552gL1gH4 biparatopic. In one embodiment, the biparatopic comprises a CDR of this combination. In another embodiment, it will comprise the entire light chain and heavy chain variable region of this combination.

[0219] In one embodiment, the preferred biparatopic is a 17415gL7gH6 x 17552gL1gH1 biparatopic. In one embodiment, the preferred biparatopic is a 17415gL7gH6 x 17552gL1gH4 biparatopic. In one embodiment, the biparatopic comprises a CDR of this combination. In another embodiment, it will comprise the entire light chain and heavy chain variable region of this combination.

[0220] In one embodiment, the preferred biparatopic is a 17415gL15gH6 x 17552gL1gH1 biparatopic. In one embodiment, the preferred biparatopic is a 17415gL15gH6 x 17552gL1gH4 biparatopic. In one embodiment, the biparatopic comprises a CDR of this combination. In another embodiment, it will comprise the entire light chain and heavy chain variable region of this combination.

[0221] In one embodiment, the preferred biparatope is a 17415gL16gH6 x 17552gL1gH1 biparatope. In one embodiment, the preferred biparatope is a 17415gL16gH6 x 17552gL1gH4 biparatope. In one embodiment, the biparatope comprises a CDR of this combination. In another embodiment, it will comprise the entire light chain and heavy chain variable region of this combination.

[0222] In a particularly preferred embodiment, the biparatopic antibody of the present invention is an IgG-type antibody. In a preferred embodiment, it is an IgG1 or IgG4 antibody. In a particularly preferred embodiment, it is an IgG1-type antibody in which the constant region comprises a LALA modification. In a particularly preferred embodiment, it is an IgG4-type antibody in which the constant region comprises a FALA modification. Accordingly, these types may be used for any 17415 and 17552 derived antibody presented herein, comprising any combination of humanized variable regions, particularly light chain and heavy chain variable regions presented above.

[0223] The sequence numbers for the light chain and heavy chain variable region sequences of the above-mentioned dual-paratopic antibody can be identified by referring to Tables 1 and 2, in particular the sequence numbers of the transplant variants presented in those tables. Additionally, a dual-paratopic antibody comprising a CDR set or a variable region pair of a specific dual-paratopic antibody described in the embodiments of the present application is preferred.

[0224] A particularly preferred biparatopic antibody of the present invention is an antibody having a set of CDRs of the biparatopic antibody shown in FIG. 17. Additionally, a particularly preferred biparatopic antibody of the present invention is an antibody having light chain and heavy chain variable regions of the biparatopic antibody shown in FIG. 17.

[0225] In a particularly preferred embodiment, when providing VR17415-derived specificity, a VR17415gL15gH6 IgG1 LALA antibody having the light and heavy chain sequences of FIG. 31 is provided. Accordingly, 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 the two heavy chains and the two light chains. In one embodiment, when the antibody is biparatopic, one specificity of the antibody will comprise the heavy and light chain pair of SEQ ID NO. 140 and 141. Variants of such specific antibodies as discussed herein are also provided.

[0226] In a particularly preferred embodiment, when using VR17552-derived specificity, a VR17552gL1gH4 IgG1 LALA antibody is used. Accordingly, 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, if the antibody is a monospecific VR17552gL1gH4 IgG1 LALA antibody, the antibody will comprise the two heavy chains and the two light chains. In one embodiment, if the antibody is biparatopic, one specificity of the antibody will comprise the heavy chain and light chain pair of SEQ ID NO. 142 and 143. Variants of such specific biparatopic antibodies as discussed herein are also provided.

[0227] In a particularly preferred embodiment, a biparatopic antibody having the light and heavy chain sequences shown in FIG. 32 is provided such that VR17415gL15gH6 specificity is provided by the heavy and light chain sequences of SEQ ID NOs. 147 and 141, respectively, and VR17552gL1gH4 specificity is provided by the heavy and light chain sequences of SEQ ID NOs. 142 and 143, respectively. Variants of such specific biparatopic antibodies as discussed herein are also provided.

[0228] Antibody generation and screening

[0229] This section describes various methods for generating variants that can be used. For a biparatopic antibody for CD45, a biparatopic antibody containing both 17415-derived and 17552-derived CD45 specificities is preferred, but the method described in this section can also be used to identify additional CD45 specificities paired with 17415-derived specificity or 17552-derived specificity.

[0230] In one embodiment, the antibody of the present invention or its antibody / fragment component has enhanced efficacy compared to other existing antibodies against CD45, and optionally, the antibody of the present invention or its antibody / fragment component also has enhanced efficacy. "Enhanced efficacy" includes the meaning that the antibody of the present invention can achieve the same level of cell killing at a lower concentration / titer compared to the CD45 antibody of the prior art. In one embodiment of the present invention, "enhanced efficacy" means that the antibody of the present invention can achieve a higher maximum effect compared to the CD45 antibody of the prior art.

[0231] In one embodiment, the antibody of the present invention or its antibody / fragment components are treated to provide enhanced affinity for a target antigen or antigens, particularly CD45. Such variants are CDR mutations (Yang et al ., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al ., Bio / Technology, 10, 779-783, 1992), E. E. coli( E. coli ) Use of mutant 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 It can be obtained by a number of affinity maturation protocols, including Nature, 391, 288-291, 1998). Vaughan et al (See above) discusses such affinity maturation methods. Binding domains for use in the present invention may be generated by suitable methods known in the art, for example, CDRs may be obtained from non-human antibodies, including commercially available antibodies, and transplanted into a human framework, or chimeric antibodies having a non-human variable region and a human constant region, etc.

[0232] Examples of CD45 antibodies are known in the art, and the paratops of such antibodies may be used in the antibodies of the present invention having one or more specificities for CD45, or may be subsequently modified, for example, by humanizing using the methods described herein, after screening for suitability using the methods described herein. Therapeutic anti-CD45 antibodies are described in the art, for example, the anti-CD45 antibody disclosed in US2011 / 0076270.

[0233] Those skilled in the art may generate antibodies for use in the antibodies of the present invention using appropriate methods known in the art. For example, antigen polypeptides for use in generating antibodies for host immunization or for use in panning, such as in phage display, may be produced from genetically engineered host cells containing expression systems through processes well known in the art or recovered from natural biological resources. In this application, the term “polypeptide” includes peptides, polypeptides, and proteins. Unless otherwise specified, these are used interchangeably. An antigen polypeptide may, in some cases, be part of a larger protein, such as a fusion protein fused to an affinity tag or similar. In one embodiment, a host may be immunized with cells transfected with CD45, for example, expressing CD45 on its surface.

[0234] If animal immunization is required, antibodies generated against the antigen polypeptide can be obtained by administering the polypeptide to animals (preferably non-human animals) using well-known conventional protocols; refer, for example, to the literature [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 produced using hybridoma technology (Kohler & Milstein, 1975, Nature, 256:495-497), trioma technology, and human B-cell hybridoma technology (Kozbor). et al 1983, Immunology Today, 4:72) and EBV hybridoma technology (Cole et al It can be prepared using any method known in the art, such as Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985). In addition, antibodies can be prepared, for example, from the literature [Babcook, J. et alAntibodies for use in the present invention may be produced using a single lymphocyte antibody method in which immunoglobulin variable region cDNA generated from a single lymphocyte selected for specific antibody production is cloned and expressed by the method described in [1996, Proc. Natl. Acad. Sci. USA 93(15):7843-7848l; WO 92 / 02551; WO 2004 / 051268 and WO 2004 / 106377]. Antibodies for use in the present invention may also be produced using various phage display methods known in the art. In a preferred embodiment, the antibody of the present invention has at least two different paratopes specific to CD45, and an antibody that recognizes one paratope of CD45 is first produced, and then, for example, two of these antibodies are used to produce the antibody of the present invention capable of specifically binding to at least two different paratopes of CD45. For example, several antibodies against CD45 can be generated using the method discussed herein and then screened for desirable characteristics such as binding affinity. Then, the antibody of the present invention can be generated using the most suitable candidate.

[0235] For example, the antigen-binding site, in particular the variable region, of the antibody according to the present invention is humanized. As used herein, humanization (including CDR-transplanted 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 understood that it may be sufficient to transfer only the specificity determining residues of the CDR, rather than the entire CDR (e.g., Kashmiri et al(See ., 2005, Methods, 36, 25-34). However, in a preferred embodiment, the entire CDR or CDRs are transplanted. The humanizing antibody may optionally further comprise one or more framework residues derived from the non-human species from which the CDR originated. As used herein, the term “humanizing antibody molecule” means an antibody molecule comprising one or more CDRs (including, optionally, one or more modified CDRs) derived from a donor antibody (e.g., mouse monoclonal antibody) in which the heavy chain and / or light chain is transplanted into the heavy chain and / or light chain variable region framework of a receptor antibody (e.g., human antibody). For review, the literature [Vaughan et al Refer to [Nature Biotechnology, 16, 535-539, 1998]. In one embodiment, instead of the entire CDR being transferred, only one or more specificity determining residues from any one of the CDRs described herein are transferred to a human antibody framework (e.g., Kashmiri et al (See ., 2005, Methods, 36, 25-34). In one embodiment, only specificity determining residues from one or more of the CDRs described herein are transferred to a human antibody framework. In another embodiment, only specificity determining residues from each of the CDRs described herein are transferred to a human antibody framework.

[0236] When a CDR or specificity determining residue is transplanted, an appropriate receptor variable domain framework sequence including mouse, primate, and human framework regions may be used, taking into account the class / type of the donor antibody from which the CDR originated. Suitably, the humanized antibody according to the present invention has a human receptor framework region and a variable domain including one or more CDRs provided herein. Examples of human frameworks that may be used in the present invention include KOL, NEWM, REI, EU, TUR, TEI, LAY, and POM (Kabat et al.(See above). For example, KOL and NEWM can be used for heavy chains, REI for light chains, and EU, LAY, and POM for both heavy and light chains. Alternatively, human germline sequences may be used. This information is available on the following website: http: / / www2.mrc-lmb.cam.ac.uk / vbase / list2.php.

[0237] In the humanized antibody molecule according to the present invention, the receptor heavy chain and light chain do not necessarily have to be derived from the same antibody and, if desired, may comprise a complex chain having a framework region derived from different chains. The framework region does not have to have exactly the same sequence as the framework region of the receptor antibody. For example, a specific residue may be changed to a residue that appears more frequently in the corresponding receptor chain class or type. Alternatively, a selected residue of the receptor framework region may be changed to correspond to a residue found at the same position in the donor antibody (Reichmann et al (See Nature, 1998, 332, 323-324). These modifications should be made within the minimum range necessary to restore the affinity of the donor antibody. A protocol for selecting residues in the receptor framework region that may require modification is presented in WO 91 / 09967. Framework derivatives may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids replaced with other amino acids, for example, donor residues. The donor residue is a residue of the antibody from which the donor antibody, i.e., the CDR, originated, and specifically, a residue at the corresponding position in the donor sequence is adopted. The donor residue may be replaced with an appropriate residue (receptor residue) derived from the human receptor framework.

[0238] In one embodiment, the present invention is extended to the antibody sequence disclosed herein, in particular to the humanization sequence disclosed herein.

[0239] For example, the combined domain is humanized.

[0240] For example, one or more CDRs provided herein may be modified to remove undesirable residues or sites, such as cysteine ​​residues, aspartic acid (D) isomerization sites, or asparagine (N) deamidation sites. For example, asparagine deamidation sites may be removed from one or more CDRs by mutating the asparagine residue (N) and / or adjacent residues to other suitable amino acids.

[0241] A person skilled in the art can test CDR variants or humanized sequences using appropriate analytical methods such as those described herein to determine whether activity is maintained.

[0242] Specific binding to an antigen can be tested using any suitable assay, including ELISA or surface plasmon resonance methods such as BIAcore, capable of measuring binding to the antigen (CD45). These assays may use isolated natural or recombinant CD45 or suitable fusion proteins / polypeptides. For example, binding is measured by surface plasmon resonance methods, such as BIAcore, using recombinant CD45 (SEQ No. 127 or amino acids 23-1304 of SEQ No. 127). Alternatively, the protein may be expressed in cells such as HEK cells, and affinity may be measured using a flow cytometry-based affinity assay. In one embodiment, if the characteristics of a single antigen binding site, particularly a paratop, are to be determined alone, an antibody containing only that paratop is produced. For example, an antibody is produced that is of the same form as the antibody of the present invention having two different specificities but having only one specificity for CD45. In one embodiment, antibodies for each paratop of CD45 can be generated from the antibody of the present invention having at least two paratops, thereby determining, for example, the affinity of each paratop or whether the paratops exhibit cross-blocking of 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 can be generated to perform a comparison.

[0243] (Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, DW, ed., 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., M Stockton Press, New York, 1991, BLAST™ Software from NCBI (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).

[0244] The present invention extends to the novel polypeptide sequences disclosed herein and to sequences having at least 80% similarity or identity therewith, e.g., at least 85%, at least 90%, particularly at least 95%, 96%, 97%, 98%, or 99% similarity or identity. In one embodiment, a sequence may have at least 99% sequence identity with at least one of the specific sequences provided herein. As used herein, “identity” indicates that amino acid residues at specific positions of aligned sequences are identical between sequences. As used herein, “similarity” indicates that amino acid residues at specific positions of aligned sequences are of a similar type between sequences. For example, leucine may be substituted for isoleucine or valine. Other amino acids that may often be substituted for one another include, but are not limited to:

[0245] - Phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains);

[0246] - Lysine, arginine, and histidine (amino acids with basic side chains);

[0247] - Aspartic acid and glutamic acid (amino acids with acidic side chains);

[0248] - Asparagine and glutamine (amino acids having amide side chains); and

[0249] - Cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0250] It will be understood that this aspect of the invention also extends to variants of such anti-CD45 antibodies, including variants in which amino acids in the CDR are modified to remove one or more isomerized, deamidated, glycosylated sites, or cysteine ​​residues as described herein.

[0251] Preferred antibodies having FALA, LALA, and Knob-into-hole modifications

[0252] Note that the amino acid residue positions presented in this section are indicated using EU numbers rather than Kabat numbers.

[0253] In a particularly preferred embodiment of the present invention, the antibody used comprises a heavy chain having a FALA or LALA modification. In particular, the FALA and LALA modifications alter Fc receptor binding.

[0254] In another preferred embodiment, the antibody includes a modification in the hinge region of the antibody, particularly a modification at position 228, preferably 228P. In one embodiment, the antibody has a heavy chain including modifications at positions 228, 234, and 235. In a particularly preferred embodiment, the heavy chain of the antibody of the present invention will include S228P, F234A, and L235A FALA modifications. In a particularly preferred embodiment of the present invention, the provided antibody is an IgG4(P) isotype antibody and will include these modifications.

[0255] In another particularly preferred embodiment, the antibody of the present invention will contain so-called "knob-in-hole" modifications. In one embodiment, one heavy chain of the antibody contains a modification to T366, and the other heavy chain contains modifications to T366, L368, and Y407, particularly so that the two different heavy chains have a complementary form, which means that instead of two identical heavy chains forming a pair, two different heavy chains preferentially form a pair. In particular, the heavy chain for one specificity may have the T366W "knob" modification, and the other heavy chain may have the T366S, L368A, and Y407V "hole" modifications. In a particularly preferred embodiment, the antibody of the present invention is an IgG4 isotype antibody and has these modifications.

[0256] In another particularly preferred embodiment of the invention, FALA, hinge, and "knob-in-hole" variants are combined. In a preferred embodiment, they are combined in the form of an IgG4 isotype antibody. In one embodiment, one heavy chain of the antibody has variants at positions 228, 234, 235, and 355. In another embodiment, one heavy chain includes variants at positions 228, 234, 235, 366, 368, and 407. For example, in one embodiment, one heavy chain has S228P, F234A, L235A, T366W variants (i.e., both FALA and "knob" variants), and preferably, another heavy chain has S228P, F234A, L235A, T366S, L368A, Y407V variants (i.e., both FALA and "hole" variants).

[0257] In a particularly preferred embodiment, the antibody of the present invention is a FALA IgG4(P) antibody. In another particularly preferred embodiment, it is a FALA Knobb-in-Hole IgG4(P) antibody. In another embodiment, it is a LALA Knobb-in-Hole IgG4(P) antibody.

[0258] In other embodiments, the forms may be combined with other forms / modifications discussed herein. For example, it may include the modifications discussed herein to remove protein A binding at positions 95 and 96. In yet another embodiment, it may include a common light chain and may also include protein A binding modifications.

[0259] Additional preferred antibody types including BYbe and TrYbe

[0260] In one aspect, an antibody molecule comprising or composed of the following is provided:

[0261] a) Polypeptide chain of formula (VII):

[0262] ;

[0263] b) polypeptide chain of formula (VIII):

[0264] ;

[0265] Here,

[0266] V H represents a heavy chain variable domain;

[0267] CH1 represents a domain of the heavy chain invariant region, e.g., domain 1;

[0268] W represents a linker or linker, e.g., an amino acid linker, provided that if p or q is 0, these will also be 0;

[0269] Z represents a linker or linker, e.g., an amino acid linker;

[0270] V1 represents dab, scFv, dsscFv, or dsFv;

[0271] V L represents a variable domain, for example, a light chain variable domain;

[0272] C L represents a domain of invariants, for example, a light chain invariant domain (e.g., C-kappa);

[0273] V2 represents dab, scFv, dsscFv, or dsFv;

[0274] p is 0 or 1 and;

[0275] q is 0 or 1 and;

[0276] When p is 1, q is 0 or 1, and when q is 1, p is 0 or 1. That is, p and q do not both represent 0, and

[0277] Here, at least two of the antigen-binding sites of the antibody are different paratopes for CD45, each recognizing a different epitope for CD45, and at least one of VH and VL is a variable region pair derived from 17415 or a variable region pair derived from 17552.

[0278] In one example, the binding domain specific to CD45 is selected from at least two of V1, V2, or VH / VL.

[0279] In one embodiment, q is 0 and p is 1.

[0280] In one embodiment, q is 1 and p is 1.

[0281] In one embodiment, V1 is dab and V2 is dab, and together they form a single bond domain of a cooperating pair of variable regions such as a homologous VH / VL pair, which are optionally connected by a disulfide bond.

[0282] In one embodiment, VH and VL are specific to CD45.

[0283] In one embodiment, V1 is specific to CD45.

[0284] In one embodiment, V2 is specific to CD45.

[0285] In one embodiment, V1 and V2 together (e.g., as binding domains) are specific to CD45, and VH and VL are specific to CD45.

[0286] In one embodiment, V1 is specific to CD45.

[0287] In one embodiment, V2 is specific to CD45.

[0288] In one embodiment, V1 and V2 together (e.g., as a single binding domain) are specific to CD45, and VH and VL are specific to CD45.

[0289] In one embodiment, V1 is specific to CD45, V2 is specific to CD45, and VH and VL are specific to CD45.

[0290] In the above structure, V1, V2, VH, and VL may each represent a binding domain and may include any sequence provided herein.

[0291] W and Z may represent appropriate linkers, for example, W and Z may independently be SGGGGSGGGGS (Sequence No. 67) or SGGGGTGGGGS (Sequence No. 114).

[0292] In one embodiment, where V1 and / or V2 is dab, dsFv, or dsscFv, the disulfide bond between the variable domains VH and VL of V1 and / or V2 is at position V H 44 and V L It is formed between 100.

[0293] In a preferred embodiment of the present invention, the antibody of the present invention is in the BYbe antibody format. The BYbe format antibody is WO 2013 / 068571 and literature [Dave et al , (2016) Mabs As described in [ , 8(7): 1319-1335], it contains a Fab linked to a single scFv or dsscFv. Thus, for example, in a preferred embodiment of the given formula, one of (V1)p and (V2)q will be a scFv or dsscFv and the other will be nothing, so the BYbe-type antibody contains only a Fab and a single scFv or dsscFv. If either (V1)p or (V2)q is 0, the corresponding W or Z will also be nothing and the other will be a binding or linker. Preferably, the BYbe-type antibody contains a Fab and a dsscFv. In such a BYbe-type antibody, the two antigen-binding sites may preferably both be specific to CD45, and these two sites correspond to two different paratopes for different epitopes of CD45.

[0294] In another particularly preferred embodiment of the present invention, the antibody is of the TrYbe format. The TrYbe format is configured such that Fab is connected 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 the other scFv or dsscFv binds to albumin, etc., to increase the half-life). Such antibody fragments are described in WO 2015 / 197772. With respect to the given formula, for the TrYbe antibody, p and q will both be 1, and V1 and V2 are each independently selected from ScFv and dsscFv. In a preferred embodiment, V1 and V2 will both be ScFvs. In another embodiment, V1 and V2 will both be dsscFvs. In another embodiment, one of V1 and V2 will be an ScFv and the other a dsscFv. At least two of the antigen-binding sites of TrYbe will be specific to CD45, and the antibody includes two different paratopes, each specific to a different epitope of CD45. In a particularly preferred embodiment, a third antigen-binding site will be specific to albumin, and in particular, one of V1 and V2 will be specific to albumin. For example, VH / VL may be specific to CD45 (e.g., the first epitope of CD45), one of V1 and V2 may be specific to CD45 (e.g., the second epitope of CD45), and the other of V1 and V2 may be specific to albumin.

[0295] In a preferred embodiment, the antibody of the present invention will comprise at least one paratope specific to albumin. In one embodiment, the antibody will be a TrYbe type antibody comprising two paratopes specific to different epitopes of CD45 and a third paratope specific to albumin. Examples of albumin-binding antibody sequences that can be used to specifically bind to albumin include the sequences disclosed in WO 2017 / 191062, the entire contents of said patent, in particular the portion relating to albumin-binding sequences, are incorporated by reference. Accordingly, the antibody of the present invention may comprise one paratope of the albumin-specific antibodies of WO 2017 / 191062.

[0296] In another embodiment, the antibody discussed above does not have at least two different specificities, but has only one specificity for CD45. For example, one of the antigen-binding sites of the antibody may be specific to CD45. In another embodiment, two of the antigen-binding sites may be specific to CD45 but have the same specificity. In yet another embodiment, all three antigen-binding sites of the antibody presented 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 antigen-binding site is specific to serum albumin.

[0297] In a particularly preferred embodiment, at least one of the variable regions of the molecules will be a 17415 or 17552 variable region. In one embodiment, at least one antigen-binding domain comprising light chain and heavy chain variable regions derived from 17415 will be present. In one embodiment, at least one antigen-binding domain comprising light chain and heavy chain variable regions derived from 17552 will be present. In one embodiment, both an antigen-binding site derived from 17415 and an antigen-binding site derived from 17552 will be present.

[0298] Disulfide bridge

[0299] In the antibody of the present invention, if one or more variable region pairs contain a disulfide bond between VH and VL, this bond may be located at any suitable position, such as between two of the residues listed below (unless otherwise specified in the context, Kabat numbers are used in the list below). Whenever a Kabat number is mentioned, the relevant reference is [Kabat et al ., 1987, in Sequences of Proteins of Immunological Interest].

[0300] In one embodiment, where V1 and / or V2 is dsFv or dsscFv in the formula 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 (unless otherwise specified in the context, Kabat numbers are used in the list below). Whenever a Kabat number is mentioned, the relevant reference is [Kabat et al ., 1987, in Sequences of Proteins of Immunological Interest].

[0301] In one embodiment, the disulfide bond is located at a position selected from the group comprising the following:

[0302] ● V H 37 + V L 95C (e.g., Protein Science 6, 781-788 Zhu et al (1997) See reference);

[0303] ● V H 44 + V L 100 (e.g., Biochemistry 33 5451-5459 Reiter et al(1994); or Journal of Biological Chemistry Vol. 269No. 28 pp.18327-18331 Reiter et al (1994); or Protein Engineering, vol.10 no.12 pp.1453-1459 Rajagopal et al (1997) See reference);

[0304] ● V H 44 + V L 105 (e.g., J Biochem. 118, 825-831 Luo et al (1995) See reference);

[0305] ● V H 45 + V L 87 (e.g., Protein Science 6, 781-788 Zhu et al (1997) See reference);

[0306] ● V H 55 + V L 101 (e.g., FEBS Letters 377 135-139 Young et al (1995) See reference);

[0307] ● V H 100 + V L 50 (e.g., Biochemistry 29 1362-1367 Glockshuber et al (1990) See reference);

[0308] ● V H 100b + V L 49;

[0309] ● V H 98 + V L 46 (e.g., Protein Science 6, 781-788 Zhu et al (1997) See reference);

[0310] ● V H 101 + V L 46;

[0311] ● V H 105 + VL 43 (e.g., Proc. Natl. Acad. Sci. USA Vol. 90 pp.7538-7542 Brinkmann et al (1993); or see Proteins 19, 35-47 Jung et al (1994)), and

[0312] ● V H 106 + V L 57 (e.g., FEBS Letters 377 135-139 Young et al (1995) See also

[0313] and the corresponding position in the variable region pair located in the molecule.

[0314] In one embodiment, the disulfide bond is at position V H 44 and V L It is formed between 100.

[0315] The amino acid pairs listed above are located at positions suitable for substitution with cysteine, so that disulfide bonds can be formed. Cysteine ​​can be manipulated at these desired positions using known techniques. In one embodiment, the manipulated cysteine ​​according to the present disclosure refers to a case where a naturally occurring residue at a specific amino acid position is substituted with a cysteine ​​residue.

[0316] The introduction of engineered cysteine ​​may be performed by any method known in the art. Such methods include, but are not limited to, PCR extension nested mutagenesis, site-specific mutagenesis, or cassette mutagenesis. Cassette mutagenesis [Wells et al This can be performed according to [1985, Gene, 34:315-323]. Alternatively, mutants can be produced by whole gene synthesis through annealing, ligation, PCR amplification, and cloning of nested oligonucleotides.

[0317] WO 2015 / 197772 details preferred locations for disulfide crosslinks in relation to BYbe and TrYbe type antibodies.

[0318] As discussed herein, altering the ability of residues in the hinge region of an antibody is one of the potential methods to affect binding with CD45 and can be utilized in the present invention.

[0319] Tether-type antibody

[0320] In one embodiment, the antibody of the present invention may consist of two parts joined by a heteromeric tether. For example, the antibody of the present invention may consist of two parts each comprising different antibody fragments having different paratopes for CD45, and a tether region that enables the formation of a whole antibody molecule with the other half of the antibody. In one embodiment, the antibody of the present invention follows, for example, the Fab-X / Fab-Y antibody format (also called the Fab-Kd-Fab format) described in WO 2017 / 093402 (see, e.g., FIG. 3). The Fab-X / Fab-Y antibody format is particularly useful for screening because it can rapidly screen combinations of different paratopes for CD45.

[0321] Accordingly, in one embodiment, the antibody molecule according to the present invention is an antibody comprising at least two different paratopes specific to different epitopes of CD45, and its formula is AX:YB. Here,

[0322] AX is the first fusion protein and;

[0323] YB is the second fusion protein;

[0324] X:Y is a heterodimer tether;

[0325] : is the coupling interaction between X and Y;

[0326] A is the first protein component of an antibody selected from the Fab or Fab' fragment;

[0327] B is the second protein component of an antibody selected from Fab or Fab';

[0328] X is a first binding partner of a binding pair independently selected from an antigen, an antibody, or a binding fragment thereof;

[0329] Y is a second binding partner of a binding pair independently selected from an antigen, an antibody, or a binding fragment thereof;

[0330] Provided, where X is an antigen, Y is an antibody specific to the antigen labeled X or a binding fragment thereof, and where Y is an antigen, X is an antibody specific to the antigen labeled Y or a binding fragment thereof.

[0331] Examples of albumin antibodies and sequences for explanation

[0332] The antibody having an antigen-binding site specific to albumin used in the present invention may have the following CDR sequence:

[0333] Sequence No. 130 - CDRH1 GIDLSNYAIN

[0334] Sequence No. 131 - CDRH2 IIWASGTTFYATWAKG

[0335] Sequence No. 132 - CDRH3 TVPGYSTAPYFDL

[0336] Sequence No. 133 - CDRL1 QSSPSVWSNFLS

[0337] Sequence No. 134 - CDRL2 EASKLTS

[0338] Sequence No. 135 - CDRL3 GGGYSSISDTT

[0339] Examples of albumin binding specificities that may be suitable for use include those disclosed in WO 05 / 117984 and WO 2017 / 191062, which are incorporated by reference in their entirety and with respect to albumin binding antibody specificities.

[0340] Effector molecule

[0341] The antibody of the present invention may be conjugated to an effector molecule. Accordingly, the antibody intended for use in the present invention may be conjugated to one or more effector molecule(s). It will be understood that the effector molecule may comprise a single effector molecule or two or more such effector molecules linked to form a single moiety that can be attached to the antibody. If one wishes to obtain an antibody according to the present invention with an effector molecule attached, this may be prepared using standard chemical or recombinant DNA methods in which the antibody is connected to the effector molecule directly or through a coupling agent. Techniques for conjugating such effector molecules to antibodies are well known in the art (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(See ., 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemical procedures include, for example, the procedures described in WO 93 / 06231, WO 92 / 22583, WO 89 / 00195, WO 89 / 01476, and WO 03 / 031581. Alternatively, if the effector molecule is a protein or polypeptide, linkage may be achieved using recombinant DNA methods, for example, as described in WO 86 / 01533 and EP0392745. In one embodiment, the antibody of the present invention may comprise an effector molecule. As used herein, the term effector molecule includes reporter groups such as, for example, antineoplastic agents, drugs, toxins, biologically active proteins (e.g., enzymes), antibodies or antibody fragments, synthetic or natural polymers, nucleic acids and fragments thereof (e.g., DNA, RNA and fragments thereof), radionuclides (particularly radioactive iodides), radioisotopes, chelate metals, nanoparticles, and fluorescent compounds or compounds detectable by NMR or ESR spectroscopy.

[0342] Examples of effector molecules may include cytotoxins or cytotoxic agents, as well as any substance harmful to cells (e.g., killing cells). Examples include combrestatin, dolastatin, epotillone, staurosporine, mytansinoids, spongestatin, lyzoxin, halicondrin, loridine, hemiasterline, taxol, cytocalcin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and their analogs or homologues. In addition, effector molecules include anmetatars (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepah chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclotosfamide, busulfan, dibromomannitol, streptozosin, mitomycin C, and cis-dichlorodiamine platinum(II) (DDP), cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mitramycin, antramycin (AMC), caliceamycin, or duocarmycin) and Antimitotic agents (e.g., vincristine and vinblastine) are included.

[0343] In other effector molecules 111 In and 90 Y, Lu 177 , bismuth 213 , Californium 252 , iridium 192 and tungsten 188 / rhenium 188Chelating radionuclides such as or drugs such as alkylphosphocholine, topoisomerase I inhibitors, taxoids, and suramin may be included. Other effector molecules include proteins, peptides, and enzymes. Enzymes of interest include, but are not limited to, proteases, hydrolases, degrading enzymes, isomerases, and transferases. Proteins, polypeptides, and peptides of interest include, but are not limited to, toxins such as immunoglobulins, abrine, lysine 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, thrombogenic agents or anti-angiogenic agents (e.g., angiostatin or endostatin), or biological response modulators 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.

[0344] Other effector molecules may include, for example, detectable substances useful for diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radionuclides, positron-emitting metals (used in positron emission tomography), and non-radioactive paramagnetic metal ions. For metal ions that may be conjugated to antibodies for use as diagnostic agents, refer to U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, danyl chloride, and phycoerythrin; Suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aquorin; suitable radionuclides include 125 I, 131 I, 111 In and 99 Tc is included.

[0345] In other embodiments, the effector molecule may increase or decrease the half-life of the antibody in vivo, decrease immunogenicity, or enhance delivery to the immune system by crossing the epithelial barrier. Examples of suitable effector molecules of this type include polymers, albumin, albumin-binding proteins, or albumin-binding compounds such as those described in WO 05 / 117984. If the effector molecule is a polymer, it may generally be a synthetic or natural polymer, for example, an optionally substituted straight-chain or branched-chain polyalkylene, polyalkenylene, or polyoxyalkylene polymer, or a branched or unbranched polysaccharide, for example, a homologous or heterologous polysaccharide. Specific optional substituents that may be present in the aforementioned synthetic polymer include one or more hydroxyl, methyl, or methoxy groups. Specific examples of synthetic polymers include selectively substituted straight-chain or branched-chain poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), or derivatives thereof, particularly selectively substituted poly(ethylene glycol) or derivatives thereof such as methoxypoly(ethylene glycol).

[0346] The antibody of the present invention may be conjugated to a molecule that modifies or alters serum half-life. The antibody of the present invention may, for example, specifically bind to albumin to modulate serum half-life. In one embodiment, the antibody of the present invention will also include a paratope specific to albumin. In another embodiment, the antibody of the present invention may include a peptide linker that is an albumin-binding peptide. Examples of albumin-binding peptides are contained in WO2015 / 197772 and WO2007 / 106120, the entirety of which is incorporated by reference.

[0347] 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 in a manner such as being conjugated to a toxin through 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 an imaging agent.

[0348] In a preferred embodiment, it is not the ability of the conjugated effector molecule to induce apoptosis, but the ability of the antibody of the present invention to specifically bind to CD45.

[0349] Cell death and killing

[0350] In a particularly preferred embodiment, the antibody of the present invention can induce apoptosis in target cells expressing CD45. Types of apoptosis that can be induced to kill target cells include intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeable transition (MPT)-induced necrosis, necrotic apoptosis, iron-dependent apoptosis, pyroptosis, partanatos, entotic apoptosis, NETotic apoptosis, lysosomal-dependent apoptosis, autophagy-dependent apoptosis, immunogenic apoptosis, cellular senescence, and mitotic catastrophe. In one embodiment, the antibody of the present invention is used to kill target cells.

[0351] In one embodiment, the target cells will be cells that specifically express CD45 on their cell surface. In a preferred embodiment, the antibody of the present invention can induce apoptosis in at least T cells. In another preferred embodiment, the antibody of the present invention can induce apoptosis in at least B cells. In another preferred embodiment of the present invention, the antibody of the present invention can induce apoptosis in B cells and T cells. In one preferred embodiment of the present invention, the antibody of the present invention can induce apoptosis in hematopoietic stem cells. In one embodiment, the antibody of the present invention does not induce apoptosis in all immune cells. For example, it does not induce apoptosis in granulocytes, macrophages, and monocytes. In one embodiment of the present invention, the antibody of the present invention induces apoptosis in all immune cells except granulocytes, macrophages, and monocytes. In one embodiment, the effect of inducing apoptosis in hematopoietic stem cells is to enable effective replacement of all hematopoietic cells. In one embodiment, the antibody of the present invention is used to kill the aforementioned target cells by inducing apoptosis.

[0352] In one embodiment, the antibody of the present invention may have different selectivity for different CD45-expressing cells. In a preferred embodiment, the antibody of the present invention may induce T cell apoptosis more efficiently than B cell apoptosis.

[0353] In another particularly preferred embodiment, the antibody of the present invention induces apoptosis but does not induce significant cytokine release. In another preferred embodiment, the antibody of the present invention induces apoptosis but does not exhibit Fc effector function, for example, because the antibody lacks an Fc region or has a silencing modification in the Fc region.

[0354] cytokines

[0355] 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 can induce apoptosis in target cells but does not induce significant cytokine release. The reduction or absence of cytokine release may mean that the subject does not undergo unwanted cytokine-induced inflammation. For example, the treatment of the present invention can kill the subject's target cells without inducing inflammation and, in particular, without the so-called "cytokine storm" associated with some treatments.

[0356] In one embodiment, the antibody of the present invention does not significantly induce the release of one or more of interferon gamma, IL-6, TNF-alpha, IL-1 beta, MCP1, and IL-8. In a preferred embodiment, the antibody of the present invention does not significantly induce the release of any of these 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 yet another preferred embodiment, it does not significantly induce the release of any of the cytokines. In one embodiment, such levels will be for one or more of interferon gamma, IL-6, TNF-alpha, IL-1 beta, MCP1, and IL-8. In another embodiment, such levels will be for one or more of CCL2, IL-1RA, IL-6, and IL-8. In another embodiment, such levels will appear for at least one of CCL2, IL-1RA, IL-6, IL-8, IL-10, and IL-11. In another embodiment, this level will be for at least one of CCL2, IL-1RA, IL-6, and IL-8.

[0357] Cytokine release can be measured using appropriate analytical methods. For example, the ability of the antibody of the present invention to induce cytokine release can be determined by incubating cells with the antibody in vitro and measuring cytokine release. In one embodiment, whole blood is incubated with the antibody, and then the level of a cytokine, such as any of the cytokines mentioned above, is measured. In another embodiment, leukocytes isolated from a whole blood sample are incubated with the antibody of the present invention, and then the level of the cytokine(s) can be measured. Alternatively, the level of the cytokine(s) can be measured in a sample of a subject administered the antibody of the present invention, and in particular, the level of the cytokine(s) can be measured in a serum sample of the subject.

[0358] In one embodiment, not "significantly inducing" cytokine release means that the antibody of the present invention does not induce cytokine release exceeding 5, 4, 3, or 2 times that observed in a negative control, for example, when compared to a negative control treated in vitro with PBS alone. In some embodiments, cytokine release levels will be compared to a positive control (e.g., treated in vitro with Campath). In one embodiment, the antibody of the present invention will induce cytokine release of 50%, 40%, 30%, 20%, 10% or less compared to that observed upon treatment with Campath. In one embodiment, the cytokine release level observed when using the antibody of the present invention will be less than 1 / 10 of the level observed with Campath. In one embodiment, the cytokine release level after incubation with Campath will be at least 2, 3, 4, 5, or 10 times higher than the level observed after incubation with the antibody of the present invention. In one embodiment, the level observed with Campath after incubating whole blood for 24 hours will be such a level compared with the antibody of the present invention.

[0359] In another embodiment, the comparative control for defining that it is not significantly induced will be another antibody. For example, if the antibody of the present invention includes a modification designed to reduce cytokine release, the comparative control will be an equivalent antibody without such modification. In another embodiment, if the antibody has an Fc region modification to reduce cytokine release or lacks an Fc region, the comparison performed is made with an equivalent antibody without such modification or having an Fc region.

[0360] In other embodiments, a comparison to confirm that no significant release of cytokines is made will be performed in vivo. For example, when the antibody of the present invention is administered to a subject, it will exhibit any cytokine release level described above compared to the comparative control group discussed above. In other embodiments, not significantly inducing cytokine release may be defined based on the level of cytokine or cytokine(s) compared to before the administration of the antibody of the present invention. For example, cases where the level of cytokine increases tenfold, fivefold or less, or less than that after the administration of the antibody of the present invention may be included. Measurements may be performed, for example, immediately before or simultaneously with the administration of the antibody, and, for example, after one day, one week, or two weeks or more have elapsed after administration. In one embodiment, measurements are performed between one day and one week after administration. In other embodiments, the antibody of the present invention does not significantly induce cytokine release in the sense that the treated subject does not experience adverse effects associated with unwanted cytokine release, for example, fever, hypotension, or irregular or rapid heartbeat.

[0361] Functional analysis

[0362] In one embodiment, a functional analysis may be used to determine whether the antibody of the present invention has a specific characteristic, e.g., any characteristic mentioned herein. Accordingly, a functional analysis may be used to evaluate the antibody of the present invention. As used herein, "functional analysis" means an analysis that may be used to determine one or more desirable characteristics or activities of the antibody or molecules of the present invention.

[0363] In a particularly preferred embodiment, the functional analysis measures the ability to bind to CD45. In a preferred embodiment, the ability to bind to human CD45 may be measured. In another embodiment, the ability to bind to synomolgus monkey CD45 may be measured. In a preferred embodiment, the ability to bind to both human CD45 and synomolgus monkey CD45 may be measured. Such binding may be measured, for example, on the CD45 protein. Or, it may be measured relative to CD45 expressed on the surface of target cells expressing CD45. Preferred measurement techniques include techniques described in the embodiments of this application, such as SPR or flow cytometry. Another preferred approach that may be used includes the related approach presented herein, which involves cross-blocking but is performed without cross-blocking antibodies. Such analysis may relate to the binding of monospecific, monovalent antibodies. Or, it may relate to any whole antibody format and specific antibody presented herein.

[0364] Additionally, suitable functional analyses may include binding analysis, apoptosis (e.g., cellular death) analysis, antibody-dependent cytotoxicity (ADCC) analysis, complement-dependent cytotoxicity (CDC) analysis, inhibition of cell growth or proliferation (cell proliferation inhibitory effect) analysis, cytotoxicity (cytotoxic effect) analysis, cell signaling analysis, cytokine production analysis, antibody production and isotype conversion, and cell differentiation analysis. In one embodiment, the degree of cell depletion may be measured for a specific cell type, for example, using the antibody of the present invention. In a preferred embodiment, the analysis may measure the ability of the antibody of the present invention to induce apoptosis in target cells expressing CD45. In another preferred embodiment, the functional analysis may measure the ability of the antibody of the present invention to induce cytokine release. In a preferred embodiment, the functional analysis method may be used to determine whether the antibody of the present invention kills cells but does not significantly induce cytokines.

[0365] Preferred functional analysis methods include the methods presented in the Examples. For example, the PBMC cell killing assay described in the Examples may be used. The Jurkat cell killing assay presented in the Examples of this Application may be used. In one preferred embodiment, the target cell is a human cell expressing CD45. In another embodiment, the target cell is a synomolgus monkey cell expressing CD45.

[0366] Functional analysis methods can be repeated as many times as necessary to increase the reliability of the results. Various statistical tests known to those skilled in the art can be used to confirm statistically significant results and thereby identify antibodies with biological functions. In one embodiment, multiple antibodies are tested in parallel or essentially simultaneously. As used herein, the term “simultaneously” means that samples / molecules / complexes are analyzed in the same analysis, for example, in the same “run.” In one embodiment, “simultaneously” means accompanying analyses in which the instrument analyzes signal outputs essentially at the same time. Deconvolution of these signals may be required to interpret the obtained results. Advantageously, testing multiple biparatopic protein complexes allows for more efficient screening of multiple antibodies and the identification of novel and interesting relationships. Clearly, the various variable regions of the interesting CD45 target antigens allow access to subtle differences in biological function.

[0367] In one embodiment, if the antibody of the present invention possesses two or more specificities for CD45, functional analysis may be used to compare the characteristics of the antibody with, for example, an antibody having the same binding affinity but possessing only one of the specificities of the antibody of the present invention. In one embodiment, through such analysis, it may be demonstrated that the antibody of the present invention having at least two different specificities for CD45 is superior to the comparison antibody. Accordingly, in a preferred embodiment, the efficacy of the antibody of the present invention having two or more different specificities for CD45, particularly the antibody according to the present invention, may be compared with an individual "comparison" antibody, particularly a "comparison" antibody having only one of the specificities for CD45 of the antibody of the present invention. For example, when performing analysis to study the cross-linking or cross-linking effects of CD45, an antibody having the same binding affinity but possessing only one specificity may be used as the comparison antibody. In one embodiment, the antibody of the present invention may be compared with an antibody containing the same single paratope of the antibody of the present invention at all antigen-binding sites of the antibody. In one embodiment, the antibody of the present invention may be compared with an antibody having the same binding valence and structure as the antibody of the present invention, but having the same single paratope of the antibody of the present invention present at all antigen-binding sites. In one embodiment, a bivalent antibody containing two different paratopes specific to different epitopes of CD45 may be compared with each of two possible bivalent antibodies containing only one of these paratopes. In one embodiment, this comparison is performed with a single comparison antibody for each different specificity of the antibody of the present invention specific to CD45, particularly for each different paratope. In one embodiment, the antibody of the present invention will show a superior result for this single comparison antibody. In another embodiment, the antibody of the present invention will show a better result than all comparison antibodies for each specificity of the antibody specific to CD45, particularly for each paratope.

[0368] In another embodiment in which the antibody of the present invention has at least two different specificities for CD45, a monospecific antibody is first evaluated, and the selected candidates are used to generate the antibody of the present invention having at least two different specificities for CD45. In one embodiment, several antibodies are tested using the multiplex defined above, and one or more functional analyses are performed.

[0369] As used herein, the term “biological function” refers to the inherent activity or purpose of a biological entity under test, e.g., the natural activity of a cell, protein, or similar entity. Ideally, the presence of function may be tested using in vitro function assays, including assays utilizing living mammalian cells. As used herein, natural function includes abnormal function, such as function associated with cancer.

[0370] In one embodiment, the antibody of the present invention may be able to crosslink more CD45 than the comparative antibody, particularly the one discussed above. For example, the ability of the antibody of the present invention to form a CD45 multimer of antibody:CD45 ECD can be studied by mixing the two, for example, by mixing them in equal amounts. The multimer may be a structure having at least two antibody:CD45 ECD units. One suitable technique is mass spectrometry, in which the antibody is mixed with equal concentrations of CD45 ECD (e.g., SEQ ID NO. 128) and then mass spectrometry is performed on the test sample. Control experiments using the antibody alone and CD45 ECD alone can be performed. The antibody of the present invention may produce more multimers than the comparative antibody. The antibody of the present invention may produce a greater amount of multimers having two, three, four, or more antibody:CD45 ECD units than the comparative antibody. This can be applied to all possible comparative controls for each specificity specific to CD45 (particularly paratop). Another technique suitable for such comparisons is analytical ultracentrifugation (AUC). Likewise, the comparison performed may be between an antibody mixture and the individual antibodies of each type within that mixture.

[0371] In another embodiment, a comparison may be made in terms of the ability of the antibody of the present invention to induce apoptosis. For example, the antibody of the present invention may induce more target cells expressing CD45 to undergo cell killing than a comparative control, e.g., a comparative antibody. It may induce a higher amount of cell killing when measured using T cells. 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 also induce a higher level of cell killing in CD8+ T cells. It may also induce a higher level of cell killing in CD4+ memory T cells. It may also induce a higher level of cell killing in CD4+ naive T cells. In another embodiment, the total number of cells in whole blood may be measured after incubation with the antibody of the present invention and compared with the results observed for a comparative control. In one embodiment, the total number of cells may be measured and compared for the antibody of the present invention and a control antibody.

[0372] In one embodiment, to test the antibody of the present invention, in vivo assays may be used, such as animal models including mouse tumor models, autoimmune disease models, rodent or primate models infected with viruses or bacteria, etc. In another embodiment, the degree of depletion of a specific cell type may be measured in vivo, for example. In one embodiment, the antibody of the present invention will induce a higher level of cell depletion than a comparative control in an animal model of disease, and in a preferred embodiment in an animal cancer model.

[0373] In one embodiment, the antibody molecule according to the present invention has a novel function or a synergistic function. As used herein, the term "synergistic function" refers to biological activity that is not observed or is higher than observed when using the comparative control(s) instead. Thus, "synergy" includes a novel biological function. In one embodiment, the antibody of the present invention comprising at least two specificities for CD45 exhibits synergy in that it is more effective than an antibody comprising only each specificity for CD45, as with the comparative control discussed above. In a preferred embodiment, this synergy appears in relation to the cross-linking of CD45. In one embodiment, the antibody mixture exhibits synergy compared to each individual single antibody constituting the mixture.

[0374] In one embodiment, "novel biological function" as used herein means a function that does not appear or exist, or a previously unidentified function, until two or more synergistic substances [Protein A and Protein B] are present together. "Higher" as used herein includes an increase from zero, also referred to herein as novel activity or novel biological function, i.e., the appearance of some activity in an antibody or molecule when a comparative control does not show activity in a relevant function analysis. The term "higher" as used herein also includes the function of the antibody in a relevant function analysis being greater than additive compared to individual paratops, for example, an increase in relevant activity of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300% or more.

[0375] In one embodiment, the new synergistic function is higher inhibitory activity.

[0376] In a particularly preferred embodiment of the present invention, the synergy is associated with cell depletion of a target cell type expressing CD45. In one embodiment, the synergy is associated with cytotoxicity.

[0377] Binding domains suitable for use in the present invention may also be identified by testing one or more pairs of binding domains in a functional analysis. For example, antibodies comprising at least one binding site specific to antigen CD45, for example, one antibody, may be tested in one or more functional analyses.

[0378] In one embodiment, the ability of an antibody to kill CD45-expressing cancer cell lines can be analyzed. The method for evaluating the ability of an antibody to kill such cell lines used in the embodiments of this application can be used to study the ability of a given antibody to kill cells. In one embodiment, a variant antibody of the present invention will have an ability to kill cancer cells in this analysis equal to or greater than the ability of one of the specific antibodies presented herein. In one embodiment, it will have at least 50%, 75%, 80%, 90%, or 100% of the activity of one of the specific antibodies presented herein to kill one of the aforementioned cancer cell lines in this analysis. In one embodiment, the antibody of the present invention will kill at least 25%, 40%, 50%, 60%, or 75% of cancer cells in this analysis. In another embodiment, the antibody of the present invention will kill 100% of cancer cells in this analysis.

[0379] In one embodiment, the antibody of the present invention will have activity similar to that of the antibody shown in FIG. 17 and 18.

[0380] For example, in one embodiment, the dual-paratopic 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 dual-paratopic may have a KD value for human CD45 measured by SPR Biocore of 5 to 20 nM, e.g., 5 to 15 nM. In one embodiment, the dual-paratopic may have a KD value for cynomolgus monkey CD45 measured by SPR Biocore of 0.10 to 0.50 nM. In one embodiment, the dual paratopic of the present invention may have an EC50 value for binding to human CD45 on cells of 0.5 nM to 10 nM, for example, 0.5 to 5 nM. In one embodiment, the dual paratopic of the present invention may have an EC50 value for binding to cynomolgus monkey CD45 on cells of 0.5 nM to 10 nM, for example, 1 to 10 nM. In a preferred embodiment, the technique used in the example is used to perform these measurements. In a much more preferred embodiment, the technique used to obtain the results illustrated in FIG. 18 is used.

[0381] Pathological conditions, medical uses, and cell depletion

[0382] The present invention provides an antibody of the present invention for use in a treatment method for humans or animals. The antibody of the present invention may be used in any situation where targeting CD45 can provide a therapeutic benefit, particularly in situations where killing these cells can provide a therapeutic benefit. The antibody of the present invention may also be used for the diagnosis or detection of CD45. The present invention also provides a pharmaceutical composition of the present invention for such uses. The present invention also provides nucleic acid molecule(s) and vector(s) of the present invention for such uses.

[0383] Accordingly, the antibody of the present invention can be used therapeutically. In one embodiment, instead of administering the antibody of the present invention, the nucleic acid molecule(s) or vector(s) of the present invention may be administered to induce antibody expression within target cells. In another embodiment, the pharmaceutical composition of the present invention is a preferred therapeutic agent to be administered. While the antibody is presented below as a preferred therapeutic agent, the pharmaceutical composition, nucleic acid molecule(s), and vector(s) of the present invention may also be used in any of the presented embodiments. In a preferred embodiment, the antibody or the pharmaceutical composition containing them is a preferred therapeutic agent.

[0384] In a particularly preferred embodiment, the present invention may be used to deplete target cells expressing CD45. In a particularly preferred embodiment, the present invention may be used to deplete disease-causing cell types expressing CD45. In particular, the present invention may be used to deplete target cells expressing CD45 on the cell surface. In a particularly preferred embodiment, the antibody used is an antibody having at least two different specificities for CD45, namely, a biparatopic antibody for CD45.

[0385] In a preferred embodiment in which the antibody of the present invention is used, the induction of apoptosis in target cells by the antibody of the present invention may mean that the antibody of the present invention does not need to exhibit one or more Fc region effector functions that antibodies typically exhibit. Accordingly, in a particularly preferred embodiment, the antibody of the present invention can induce apoptosis in target cells but does not have an active Fc region. In a particularly preferred embodiment, the antibody induces apoptosis but does not induce significant cytokine release.

[0386] In a particularly preferred embodiment, cells or tissues are delivered to a subject after cell depletion according to the present invention. In another particularly preferred embodiment, the delivered cells or tissues replace the cells or tissues depleted using the present invention. Thus, the therapeutics discussed herein do not target the actual mechanism of the disorder, but rather involve wholly or partially replacing the cell types involved in the disorder or cell types whose killing (particularly replacement) can simply provide a therapeutic benefit. Accordingly, in one embodiment, the present invention provides a method of cell depletion comprising using the present invention. In another embodiment, the method of the present invention may include both cell depletion and subsequent delivery of cells or tissues. Cell depletion can be used in various therapeutic contexts to effectively kill target cells.

[0387] In a preferred case, the antibody of the present invention is used to kill immune cells. As used herein, the term “immune cell” includes, but is not limited to, cells of hematopoietic origin that are involved 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 T cells and B cells. 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 B cells and T cells but not 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, the present invention is used to deplete HSCs in a subject before delivering HSCs to reconstitute the subject’s immune system. In another embodiment, the present invention depletes specific cell types but does not deplete hematopoietic stem cells. In another embodiment, the aforementioned cell types are killed using the present invention. Therefore, in all embodiments described herein for cell depletion, the aforementioned cells can be killed using the present invention.

[0388] In one embodiment, the target subject to treatment according to the present invention is a subject with an autoimmune disease, a blood disease, a metabolic disorder, cancer, or immunodeficiency. The fact that the disease can be treated by first depleting cells and then replacing them implies that the antibody of the present invention is particularly useful for cancer treatment. Accordingly, in a particularly preferred embodiment, the target disease to be treated is cancer. Accordingly, in one embodiment, the present invention is used to deplete cancer cells, e.g., cancer cells derived from immune system cells. In a preferred embodiment, the present invention provides a method for treating cancer comprising the step of administering the present invention to deplete cancer cells expressing CD45. The method may further comprise the step of transplanting cells to a patient. In one embodiment, the delivered cells replace the depleted cells. In one embodiment, the delivered cells are hematopoietic stem cells.

[0389] In a particularly preferred embodiment, the disease to be treated is blood cancer.

[0390] In a preferred embodiment, the cancer is a cancer associated with the bone marrow, particularly a cancer associated with hematopoietic cells.

[0391] 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.

[0392] In one embodiment of the present invention, the blood cancer to be treated may be lymphoma.

[0393] In one embodiment, the blood cancer to be treated is multiple myeloma.

[0394] In another embodiment, the treatment subject has an autoimmune disease.

[0395] In one embodiment, the disease to be treated is a disease known to be associated with abnormal CD45 expression. In a particularly preferred embodiment, the treatment depletes the CD45-expressing cell types involved in the subject's disease.

[0396] In one embodiment, since the present invention is used to deplete cells prior to cell transplantation, the method of the present invention may, in some embodiments, include a step of delivering cells to a subject to help replace the depleted cells after the depletion step using the therapeutic agent of the present invention, particularly an antibody. In one embodiment, such delivery may be the delivery of allogeneic cells. In another embodiment, such delivery may be the delivery of autologous cells. In one embodiment, the cells delivered may be cells expressing a chimeric antigen receptor (CAR). In some embodiments, the subject requires chimeric antigen receptor T cell (CART) therapy. For example, such therapy may constitute part of the method of the present invention.

[0397] In another preferred embodiment, the present invention provides a method for promoting the engraftment of a cell population in a subject, the method further comprising the step of depleting cells using the antibody of the present invention prior to the engraftment of the cell population. Accordingly, the present invention provides a method for promoting the engraftment of delivered cells comprising the step of depleting cells expressing CD45 by administering the antibody of the present invention to a subject, and then delivering cells of interest. In one embodiment, the present invention provides a method for promoting the engraftment of stem cells, particularly hematopoietic stem cells. In one embodiment, hematopoietic stem cells are administered to a subject having a defect or deficiency in one or more cell types of the hematopoietic lineage to reconstitute or partially reconstitute the defective or deficient cell population in vivo. In one embodiment, the present invention is used to treat a stem cell deficiency, for example, by using the present invention to deplete target cells and replace them with transplanted cells, so that the transplanted cells resolve the stem cell deficiency. In one embodiment, the re-injected cells are genetically modified. In one embodiment, cells are taken from a subject and genetically modified, and then the present invention is used to kill target cells, e.g., unmodified cells of the corresponding type still present in the subject, and then the cells are reinjected into the subject. In a preferred embodiment, the genetically modified cells delivered are hematopoietic stem cells.

[0398] In a preferred embodiment, the depleted cells and the delivered cells are of the same cell type or include the same cell type. In a preferred embodiment, the depleted cells are hematopoietic cells, particularly hematopoietic stem cells. In one embodiment, the present invention is used to deplete cells prior to bone marrow transplantation. In another embodiment, the present invention is used to deplete cells instead of irradiation. In another embodiment, the present invention is used to deplete cells in addition to irradiation.

[0399] In another embodiment of the present invention, the present invention provides a method that helps reduce the possibility of rejection of transplanted cells, comprising the step of administering the therapeutic agent of the present invention to deplete the cells before cell delivery. In another embodiment, the present invention may be used to promote the acceptance of transplanted immune cells in a subject by depleting target cells expressing CD45 before immune cell delivery. The target cells may be any of the cells discussed herein. In one embodiment, the cells transplanted or delivered to the subject are stem cells.

[0400] Any method discussed herein to eliminate cells expressing CD45 may be used for cell depletion or killing. However, in a particularly preferred embodiment of the present invention, the invention may be used to deplete these cells by inducing apoptosis of cells expressing CD45.

[0401] In one embodiment, bone marrow may be administered to the subject as part of cell delivery. In another embodiment, umbilical cord blood or cells isolated from umbilical cord blood may be provided to the subject as a method of cell delivery. In another embodiment, the transplanted cells may be derived from differentiated stem cells, for example, in which stem cells are differentiated in vitro and then transplanted.

[0402] In one embodiment of using the antibody of the present invention to deplete or kill cells, additional cell depleting agents or killing agents may be used in combination. 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 depletion of target cells is sufficient to be effective, for example, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% or more of the target cells. For example, in one embodiment, more than 50% of the target cells are depleted. In another embodiment, more than 75% of the target cells are depleted. In another embodiment, more than about 90% of the target cells are depleted. In another embodiment, more than about 95% of the target cells are depleted.

[0403] As discussed above, in a particularly preferred embodiment, the antibody of the present invention can be used to induce killing of CD45-expressing cells. Any suitable method may be used to evaluate cell viability and cell killing.

[0404] In another embodiment, the present invention may be used in connection with graft-versus-host disease (GVHD), particularly in connection with treating a cell population, tissue, or organ with a cell-depleting antibody.

[0405] In another embodiment, the present invention provides a treatment method comprising the step of first performing such ex vivo treatment and then performing transplantation. In another embodiment, the present invention is used to deplete or kill the cells of a subject prior to transplantation, thereby reducing the number of host cells capable of attacking the transplanted material as a method of reducing the likelihood of GVHD development. Accordingly, the present invention provides a method for treating or preventing GVHD comprising the step of administering the antibody of the present invention to deplete cells within a cell population, tissue, or organ prior to transplanting the cell population, tissue, or organ. The method may further include the transplantation itself. The depleted cells and the transplanted cells, tissue, or organ may be any of those mentioned herein. In one preferred embodiment, the transplanted cells are hematopoietic stem cells. In another preferred embodiment, the depleted cells are T cells. In another preferred embodiment, the ability of the present invention to treat or prevent GVHD is utilized for heart, lung, kidney, or liver transplantation.

[0406] In another embodiment, the present invention provides a method of depleting and / or killing cells within a cell population, tissue, or organ in vitro by applying the antibody of the present invention before transplantation, instead of treating the recipient. Accordingly, the present invention also provides a method for removing target cells from a cell population, tissue, or organ in vitro before transplantation, comprising the step of treating the cell population, tissue, or organ before transplantation and then performing the transplantation.

[0407] In one embodiment, it may be used to deplete immune cells, particularly in organs or tissues where existing treatments are difficult to access or induce an excessive inflammatory response due to unique mechanisms. In one embodiment, the present invention may be used to deplete cells in enclosed organs, such as the brain, spinal cord, eyes, or testes, for example. In one embodiment, CD45 in immune-privileged organs +It can be used to deplete cells. Since the antibody of the present invention can deplete CD45+ cells without using Fc-mediated function, it can help avoid unwanted side effects and damage. In one embodiment, it can be used to deplete cells without inducing an immune response and without requiring an antibody effector mechanism. This can provide the advantage of minimizing or at least reducing unwanted damage, for example, because occluded organs may contain delicate and often non-dividing 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 eliminate CD45-positive cells without causing or reducing additional damage to the tissue or inflammation. In one embodiment, the target cells within the occluded organ are selected from lymphocytes, B cells, and T cells. In one embodiment, the target cells within the occluded organ are CD4+ T cells or include CD4+ T cells. In another embodiment, the target cells are CD8+ T cells or include CD8+ T cells.

[0408] In another preferred embodiment, the pathological condition to which the present invention applies is characterized by the infiltration of CD8+ T cells.

[0409] Pharmaceutical composition

[0410] In one aspect of the present invention, a pharmaceutical composition is provided comprising: (a) an antibody, nucleic acid molecule or molecules of the present invention, or a vector or vectors; and (b) a pharmaceutically acceptable carrier or diluent. In a particularly preferred embodiment, it comprises the antibody or antibodies of the present invention. The composition may include various components, including a pharmaceutically acceptable carrier, excipients, and / or diluent. Optionally, the composition may include additional molecules capable of altering the properties of the molecule(s) of the present invention, for example, reducing, stabilizing, delaying, regulating, and / or activating the function of the molecule. The composition may be in solid or liquid form, and in particular may be in powder, tablet, solution, or aerosol form.

[0411] The present invention also provides a pharmaceutical composition or a diagnostic composition comprising a combination of the molecule of the present invention and one or more pharmaceutically acceptable excipients, diluents, or carriers. Accordingly, the present invention provides a use of the antibody of the present invention for use in the treatment of pathological conditions or disorders and in the manufacture of therapeutic medicines. In one embodiment in which a therapeutic agent of the present invention is administered to a subject to whom a second therapeutic agent is also provided, the two therapeutic agents may be administered, for example, simultaneously, sequentially, or separately. In one embodiment, the two therapeutic agents are administered as the same pharmaceutical composition. In another embodiment, the two therapeutic agents are administered as separate pharmaceutical compositions. In one embodiment, the present invention provides an antibody of the present invention for use in a method in which the subject is also treated with a second therapeutic agent. In another embodiment, the present invention provides a second therapeutic agent for use in a method in which the subject is treated with the antibody of the present invention. The nucleic acid molecule(s) and vector(s) of the present invention may also be administered in such combinations.

[0412] The composition of the present invention will generally be provided as a sterile pharmaceutical composition. The pharmaceutical composition of the present invention may additionally include a pharmaceutically acceptable adjuvant. In other embodiments, such an adjuvant is not present in the composition of the present invention. The present invention also provides a method for preparing a pharmaceutical composition or a diagnostic composition comprising the step of adding and mixing the antibody of the present invention with one or more of a pharmaceutically acceptable excipient, a diluent, or a carrier.

[0413] As used herein, the term “pharmaceuticalally acceptable excipient” means a pharmaceutically acceptable formulation carrier, solution, or additive for enhancing the desirable properties of the composition of the present invention. Excipients are well known in the art and include buffers (e.g., citric acid buffer, phosphate buffer, acetic acid 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. Generally, the formulation will be provided in a substantially sterile form using a sterile manufacturing process.

[0414] This may include the preparation of a buffer solvent solution used in the formulation and sterilization by filtration according to methods familiar to those skilled in the art, the process of aseptically suspending antibodies in a sterile buffer solvent solution, and the process of dispensing the formulation into a sterile container.

[0415] Pharmaceutically acceptable carriers must not induce the production of antibodies harmful to the individual receiving the composition and must not be toxic. Suitable carriers may include large, slow-metabolizing macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive viral particles.

[0416] Pharmaceutically acceptable salts may be used, such as inorganic salts like hydrochloride, hydrobromide, phosphate, and sulfate, or organic salts like acetate, propionate, malonate, and benzoate. Pharmaceutically acceptable carriers within the therapeutic composition may additionally contain liquids such as water, physiological saline, glycerol, and ethanol. Through these carriers, the pharmaceutical composition can be formulated into tablets, pills, drase, capsules, liquids, gels, syrups, slurries, and suspensions so that the patient can take them.

[0417] Detailed information on pharmaceutically acceptable carriers can be found in the literature [Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ 1991)].

[0418] As used herein, the term "therapeutic effective dose" refers to the amount of therapeutic agent required to treat, alleviate, or prevent a target disease or condition, or to produce a detectable therapeutic or preventive effect. For any antibody, the therapeutic effective dose can first be estimated through cell culture analysis or animal models (typically rodents, rabbits, dogs, pigs, or primates). Animal models can be used to determine the appropriate concentration range and route of administration. Subsequently, this information can be used to determine the useful dose and route of administration for the human body.

[0419] The composition may be administered to the patient individually or in combination with other agents, drugs, or hormones (e.g., simultaneously, sequentially, or separately). The dosage of the present invention depends on the characteristics of the disease to be treated, the degree of inflammation present, and whether the antibody is used for prophylactic purposes or to treat an existing disease.

[0420] The present invention also provides a method for preparing a pharmaceutical composition or a diagnostic composition comprising the step of adding and mixing an antibody with one or more of pharmaceutically acceptable excipients, diluents, or carriers.

[0421] An antibody, nucleic acid molecule, or vector may be the sole active ingredient of a pharmaceutical composition or diagnostic composition, or may be accompanied by other active ingredients including an antibody component or a non-antibody component such as a steroid or other drug molecule.

[0422] The pharmaceutical composition comprises, suitably, 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, alleviate, or prevent a specific disease or condition, or to produce a detectable therapeutic or preventive effect. The "therapeutically effective amount" may be the amount required to induce a desired level of cellular depletion. For any antibody, the therapeutically effective amount may first be estimated through cell culture analysis or animal models (typically rodents, rabbits, dogs, pigs, or primates). Animal models may also be used to determine appropriate concentration ranges and routes of administration. This information may then be used to determine a useful dose and route of administration for the human body.

[0423] The precise therapeutic effective dose for human subjects will depend on the severity of the disease state, the subject's overall health status, the subject's age, weight and sex, diet, time and frequency of administration, concomitant drug(s), response sensitivity, and tolerance / response to treatment. Generally, the therapeutic effective dose will be 0.01 mg / kg to 50 mg / kg per day, e.g., 0.1 mg / kg to 20 mg / kg. In one embodiment, the amount within a given dose is sufficient to induce at least a specific function.

[0424] The pharmaceutical composition may be conveniently provided in a unit dosage form containing a predetermined amount of the active agent of the present invention per dose. The pharmaceutical composition of the present invention may be provided contained in a container that provides a means for administration to a subject. The pharmaceutical composition of the present invention may be provided contained in a pre-filled syringe. Accordingly, the present invention provides a syringe loaded with such a pharmaceutical composition. Furthermore, the present invention provides an automatic injector loaded with the pharmaceutical composition of the present invention.

[0425] The composition may be administered to the patient individually or in combination with other agents, drugs, or hormones (e.g., simultaneously, sequentially, or separately).

[0426] The agents used here refer to substances that produce physiological effects upon administration. The drugs used here refer to chemical substances that produce appropriate physiological effects at therapeutic doses.

[0427] After formulation, the composition of the present invention may be administered directly to a subject. The subject of treatment may be an animal. However, in one or more embodiments, the composition is suitable for administration to a human subject.

[0428] In one embodiment, the antibody of the present invention may be used to functionally alter the activity of an antigen of interest or antigens, and in particular may be used to modulate CD45. For example, the present invention may directly or indirectly neutralize, antagonize, or promote the activity of said antigen or antigens.

[0429] The present invention is also extended to a kit comprising an antibody of the present invention. In one embodiment, a kit comprising any antibody of the present invention is optionally provided with administration instructions.

[0430] In another embodiment, the kit further includes one or more reagents for performing one or more functional analyses.

[0431] In one embodiment, molecules of the present invention comprising the antibody of the present invention are provided for use as laboratory reagents.

[0432] Additional side

[0433] In another aspect, nucleotide sequences or sequences such as DNA sequences or sequences encoding the antibody molecule of the present invention are provided, for example. In one embodiment, said nucleotide sequences are collectively present in one or more polynucleotides, but all of them together may code for the antibody of the present invention.

[0434] The present invention also extends to vectors comprising the nucleotide sequence defined above. As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying other connected nucleic acids. An example of a vector is a "plasmid," which is a circular double-stranded DNA loop to which additional DNA segments can be connected. Another type of vector is a viral vector, to which additional DNA segments can be connected within the viral genome. Certain vectors can replicate autonomously within the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episosomal mammalian vectors) can be incorporated into the host cell genome and subsequently replicated along with the host genome. In this specification, the terms "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector. General methods for constructing vectors, methods of transfection, and methods of culture are well known to those skilled in the art. In this regard, refer to the literature [“Current Protocols in Molecular Biology”, 1999, FM Ausubel (ed), Maniatis Manual published by Wiley Interscience, New York and Cold Spring Harbor Publishing].

[0435] As used herein, the term vector also includes, for example, particles constituting the vector, such as LNP (lipid nanoparticle) particles, in particular LNP-mRNA particles. This also includes viral particles used to deliver the vector of the present invention.

[0436] As used herein, the term “selection marker” refers to a protein expressed to identify cells transformed or transfected with a vector containing a marker gene. Various selection markers are known in the art. For example, selection marker genes generally confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which a vector has been introduced. Selection markers may also be visually identifiable markers, such as fluorescent markers. Examples of fluorescent markers include Rhodamine, FITC, TRITC, Alexa Fluor, and various conjugates thereof.

[0437] In one embodiment, the present invention provides a vector encoding an antibody of the present invention. In another embodiment, the present invention provides vectors encoding an antibody of the present invention collectively.

[0438] In addition, a host cell comprising one or more cloning or expression vectors comprising one or more DNA sequences encoding the antibody of the present invention is provided. Any suitable host cell / vector system may be used for the expression of the DNA sequence encoding the antibody molecule of the present invention. For example, E. coli ( E. coli Bacterial and other microbial systems such as ) can be used, or eukaryotic host cell expression systems such as mammalian cells can be used. Suitable mammalian host cells include CHO cells, myeloma cells, or hybridoma cells. Host cells comprising the nucleic acid molecule or vector of the present invention are also provided.

[0439] The present invention also provides a method for producing a molecule or a component thereof according to the present invention, comprising the steps of culturing a host cell containing a vector of the present invention under conditions suitable for inducing protein expression from DNA encoding the molecule of the present invention, and isolating the molecule.

[0440] A method for producing an antibody containing a heterodimeric tether may further include the step of mixing two parts of the antibody and allowing the binding partners of the heterodimeric tether to bind. Additionally, the method may further include a purification step to remove any species other than the desired heterodimeric, for example.

[0441] The antibody of the present invention can be used in a diagnostic / detection kit. In one embodiment, the antibody of the present invention is immobilized on a solid surface. The solid surface may be, for example, a chip or an ELISA plate.

[0442] The antibody of the present invention may be conjugated, for example, with a fluorescent marker that facilitates the detection of a conjugated antibody-antigen complex. This may be used in immunofluorescence microscopy. Alternatively, the antibody may also be used in Western blotting or ELISA.

[0443] 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 step of performing anion exchange chromatography in a non-binding mode so that impurities remain in the column and the antibody is retained in the non-binding fraction. This step may be performed, for example, at a pH of about 6-8. The method may further comprise an initial capture step using cation exchange chromatography, for example, performed at a pH of about 4 to 5. The method may further comprise additional chromatography step(s) to ensure that the product and process-related impurities are adequately separated from the product stream. The purification method may also include one or more ultrafiltration steps, such as a concentration and dialysis filtration step.

[0444] The "refined form" used above is intended to refer to a purity of at least 90%, e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99% w / w or higher.

[0445] In the context of this specification, "comprising" should be interpreted as "including." An aspect of the invention comprising a specific element is also intended to extend to other embodiments that are "consisted of" or "essentially constituted" by the relevant element. For example, a CDR described as "comprising" a sequence of a specific sequence number may be "consisted of" that sequence; that is, it contains only that sequence and has no additional sequences.

[0446] Embodiments explicitly listed herein may be used as requirements for exclusion from claims.

[0447] Where used herein, the singular is included unless otherwise specified or evident. In particular, the singular forms "a," "an," and "the," etc., include plural objects unless the context clearly indicates otherwise.

[0448] All references cited in this document are specifically included by reference.

[0449] The subheadings of this specification are used to aid in the organization of the specification and are not intended to be used to interpret the meaning of technical terms herein.

[0450] The sequence of the present invention is provided below.

[0451] In the context of this specification, "comprising" should be interpreted as "including".

[0452] Aspects of the present invention comprising specific elements are also intended to extend to other embodiments that are "consisted of" or "essentially constituted" by the related elements.

[0453] The embodiments positively described herein may also be used as grounds for exclusion from the scope of rights.

[0454] All references mentioned herein are specifically included by reference in their entirety. Furthermore, such literature is included in relation to the specific context in which it was cited.

[0455] Examples

[0456] Example 1: Evaluation of antibody binding to human CD45 using Biacore

[0457] This embodiment describes the methodology used in other embodiments to measure binding affinity using Biacore.

[0458] The human CD45 binding kinetics of humanized IgG grafts or biparatopic IgG molecules were evaluated using surface plasmon resonance with a Biacore T200 or 8K+ instrument (Cytiva).

[0459] Chlorine anti-human IgG Fc specific F(ab')2 (Jackson ImmunoResearch) was immobilized on a CM5 sensor chip (Cytiva) via an amine coupling chemical reaction at a level typically ranging from 5,000 to 7,000 RU. Each analysis cycle consisted of capturing anti-CD45 IgG molecules on the anti-Fc surface, followed by injecting human CD45 (in-house produced) at a flow rate of 30 or 50 µl / min at 25°C. At the end of each cycle, the surface was regenerated at a flow rate of 10 µl / min using an injection of 50 mM HCl for 60 seconds, followed by an injection of 5 mM NaOH for 30 seconds, and finally an injection of 50 mM HCl for 60 seconds. Human CD45 was injected into captured samples and reference flow cells at concentrations of 800 nM to 3.3 nM (5 3-fold serial dilutions) or 800 nM to 3.13 nM (4 4-fold serial dilutions) in HBS-EP+ running buffer (Cytiva). The binding reaction of the reference flow cells was subtracted from the binding reaction of the active flow cells, and instrument noise and drift were subtracted by including buffer blank injection.

[0460] The dynamic parameters were determined using a 1:1 dynamic coupling model with Biacore T200 Evaluation software (version 3.0) or Biacore Insight Evaluation (version 4.0.8.20368).

[0461] Example 2: Generation and Characterization of CD45 Antibody

[0462] Introduction:

[0463] This embodiment describes the initial generation and characterization of an antibody specific to CD45.

[0464] Immunization:

[0465] Rabbits were inoculated with a mixture of rabbit fibroblasts expressing human CD45RO (terminal form of CD45, SEQ No. 136) or the corresponding cynomolgus monkey CD45RO (SEQ No. 137). After 3 to 5 inoculations, the animals were sacrificed and PBMCs, spleens, bone marrow, and lymph nodes were collected. Binding to human and cynomolgus CD45RO proteins in serum was monitored by ELISA.

[0466] Antibody Discovery:

[0467] B cell culture medium is from the literature [Zubler RH, Erard F., Lees RK, et al It was prepared using a method similar to that described in [Mutant EL-4 Thymoma Cells Polyclonally Activate Murine and Human B Cells via Direct Cell Interaction. J. Immunol. 1985, 134, (6), 3662-3668]. Briefly, immunized rabbit spleen or PBMC-derived B cells were cultured for 7 days in 200 µl / well of RPMI 1640 medium (Gibco BRL) 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 splenocyte culture supernatant, and gamma-irradiated mutant EL4 mouse thymoma cells (5×10⁴ per well) at a cell density of approximately 2000–5000 cells per well in a barcode-labeled 96-well tissue culture plate at 37°C and 5% CO₂.

[0468] 1st Screening:

[0469] B cell cultures were established, and 34 supernatants were first screened for binding ability to human and Sino CD45RO using the bead-based mirror ball FMAT assay. This is a homogeneous assay using biotinylated human and Sino CD45RO coated on streptavidin beads and a goat anti-rabbit Fcγ fragment-specific FITC conjugate as a detection reagent. After the first screening, positive supernatants were collected in barcode-labeled 96-well master plates, and the B cells in the cell culture plates were frozen at -80°C. To confirm cell binding and interspecies cross-reactivity, the master plate supernatants were screened for binding to HEK293 cells transfected with human and Sino CD45RO.

[0470] Fluorescence Focusing Method:

[0471] A deconvolution step was performed to recover antibody variable region genes from antigen-specific B cells in wells containing a heterogeneous B cell population. This was done using the fluorescence focusing method (Clargo et al This was achieved using ., 2014. Mabs 2014 Jan. 1: 6(1) 143-159; EP1570267B1). Briefly, immunoglobulin-secreting B cells obtained from positive wells were mixed with biotinylated human CD45RO-coated streptavidin beads (New England Biolabs) and goat anti-rabbit Fcγ fragment-specific FITC conjugates (Jackson) final diluted to 1:1200. After incubation at 37°C for 1 hour, antigen-specific B cells could be identified by the appearance of a fluorescent halo around the corresponding B cells. Then, some of these individual B cell clones were selected and placed into PCR tubes. Fluorescence focusing was also used to identify antigen-specific B cells from a heterogeneous B cell population obtained directly from the bone marrow of immunized rabbits.

[0472] Antibody V region detected:

[0473] The antibody variable region gene was recovered from single cells via reverse transcription (RT)-PCR. cDNA was synthesized from individual B cells using SuperScript IV VILO Master Mix (Life Technologies) in the presence of 0.5% Nonidet P-40 (Roche). Two PCRs were performed using heavy and light chain variable region-specific primers, and restriction enzyme sites were introduced at the 3' and 5' ends via nested secondary PCR to clone the variable region into Fab X and Fab Y mammalian expression vectors allowing for Fab-X / Fab-Y expression (e.g., as described in WO 2015 / 181282 and WO 2017 / 093402).

[0474] Fab X and Fab Y constructs for the expression vector were co-transfected into Expi293 cells using Expifectamine (Life Technologies), and the recombinant antibody was expressed in 30 ml tissue culture flasks. After 5-7 days of expression, the supernatant was collected. To confirm the specificity of the recombinant antibody, the supernatant was tested using a uniform fluorescence-based binding assay in human and HEK293 cells transfected with Sino CD45RO.

[0475] Evaluation of antibody binding and Jurkat cell killing

[0476] Antibody binding to CD45 and the ability to kill Jurkat cells were evaluated using the method described in other examples.

[0477] result:

[0478] Several clones were screened for KD values ​​for human and cino CD45RO binding measured by surface plasmon resonance (SPR) and for their ability to kill Jurkat cells as monospecific antibodies, and two clones suitable for further study and development were identified. Antibodies 17415 and 17552 bound to both human and cino CD45. The rabbit antibody 17415 potently killed Jurkat cells expressing CD45. Subsequently, a biparatopic antibody was developed using the sequence derived from 17415 as the "killing arm" of the biparatopic antibody, while the sequence derived from 17552 was developed as the "non-killing arm" or "assistant arm" of the biparatopic antibody. Both the 17415 and 17552 sequences were humanized as further described below.

[0479] Example 3: Comparison of the killing ability of 17415 and 4133 antibodies on human CD45-expressing cells

[0480] Introduction:

[0481] The killing ability of the 17415 antibody on CD45-expressing target cells was compared with the killing ability of the 4133 antibody described in international patent application PCT / EP2021 / 078516 (published as WO 2022 / 079199 A1).

[0482] PBMC depletion analysis

[0483] Human PBMCs derived from blood leukocyte-platelet apheresis cones (NHSBT Oxford) were stored as frozen aliquots. Before performing the analysis, 5 x 10⁶ were placed in each 1 ml. 7One frozen cell vial containing cells was thawed in a 37°C water bath and added to 50 ml of complete medium (RPMI 1640 + 2 mM GlutaMAX + 1% penicillin / streptomycin, all supplied by Invitrogen) + 10% fetal bovine serum (FBS, Sigma Aldrich). The cells were centrifuged (300 g, 5 min, room temperature), resuspended in 50 ml of complete medium, washed, and centrifuged again. The cells were resuspended in 10 ml of complete medium and counted using a ChemoMetec NucleoCounter NC-3000. Subsequently, 1 x 10⁶ cells per well in 50 µl were added. 5 Canine cells were added to each well of a cell culture-treated Corning Costar 96-well U-bottom microplate (catalog number 07-200-95). PBMC obtained from UCB-Cone 1014 was used for this analysis.

[0484] 400 nM stocks of purified VR17415, VR4133, and 17552 IgG1 antibodies and isotype control IgG1 (5604) in complete medium were prepared in Greiner 96-well unconjugated microplates. An 8-point dose curve was constructed by serially diluting the reagents 1:5 seven times (2 times for the isotype control) in complete medium. 50 µl of each dilution (final well concentration 200-0.0026 nM) was added to the cells and incubated for 4 hours at 37°C and 5% CO₂. After incubation, the plates were centrifuged at 300 g for 5 minutes at room temperature, and the buffer was aspirated using a BioTek ELx405 microplate washer. The cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma Aldrich), washed, centrifuged again, and the buffer was aspirated to leave 20 µl of residual medium. The cells were analyzed in a live state using Intellicyt iQue 3. Lymphocytes were gated using an FSC versus SSC plot, and the number of live cells was extracted as a measure; graphs were generated using Graphpad Prism version 8.1 (Graphpad). EC50 and Emax values ​​were derived by applying asymmetric (4-parameter) curve fitting.

[0485] result:

[0486] The lymphocyte reduction rate in a PBMC population treated with CD45 IgG1 antibodies is shown in Figure 1. The EC50 of VR17415 is 0.71 and the EC50 of VR4133 is 44.13, showing a difference of >60 times between the EC50s of the two antibodies. VR17552 IgG1 was unable to deplete lymphocytes.

[0487] Example 4: Humanization of Antibody 17415

[0488] Rabbit antibody 17415, obtained and evaluated using the method presented in Example 2, was humanized by transplanting the CDR of the rabbit V region into the human germ cell antibody V region framework. Additionally, to restore antibody activity, some of the framework residues of the rabbit V region were retained in the humanized sequence. These residues were selected using the protocol outlined by Adair et al. (1991) (Humanized antibodies - WO91 / 09967, incorporated by reference in its entirety and in particular with regard to the protocol for selecting retention residues during the humanization process). The alignment of the rabbit antibody (donor) V region sequence and the human germ cell antibody (receptor) V region sequence is shown in Figures 2 and 3 along with the designed humanized sequence. The CDR transplanted from the donor to the receptor sequence is Kabat (Kabat et al ..., as defined above), and in the case of CDR-H1, the combined definition of Chothia / Kabat is used (Adair et al ., see 1991 Humanised antibodies, WO91 / 09967).

[0489] Initially, the human V region IGKV1-9 and IGKJ4-1 J region (IMGT, http: / / www.imgt.org / ) were selected as the receptors for the light chain CDR of antibody 17415. Donor residues of the rabbit antibody light chain framework were maintained at positions 2 (valine, V2), 3 (valine, V3), and 63 (lysine, K63) (Fig. 2). An unpaired / free cysteine ​​residue is present at position 90 (C90) of CDRL3 of antibody 17415: free cysteine ​​residues can undergo post-translational modifications such as cysteineation and may contribute to covalent aggregation and instability. Since the cysteine ​​amino acid side chain has both polar and hydrophobic characteristics, in humanized transplant variants gL2 to gL5, the C90 residue was removed by modifying it into an amino acid with a polar (serine (C90S), glutamine (Q90V)) or hydrophobic (alanine (C90A), valine (C90V)) side chain (Fig. 2).

[0490] The human V region IGHV3-72 and IGHJ4-1 J region (IMGT, http: / / www.imgt.org / ) were selected as receptors for the heavy chain CDR of antibody 17415. Common to many rabbit antibodies, framework 3 of the 17415 rabbit VH region lacks one residue (78, based on sequence number 17gH1) in the loop between beta sheet strands D and E. In the initial humanized transplant variant, this gap was filled with the corresponding residue (lysine 78, K78) of the selected human receptor sequence (Fig. 3). The donor residues of the rabbit antibody heavy chain framework were retained at positions 23 (threonine, T23), 49 (isoleucine, I49), 74 (lysine, K74), 76 (serine, S76), 79 (threonine, T79), 81 (valine, V81), 99 (glutamic acid, E99), and 100 (leucine, L100). In some humanized transplant variants (gH2 to gH4), the disulfide bond formed between the cysteine ​​residues at positions 36 (C36) and 51 (C51) was removed by mutating both residues to serine (S36 and S51), alanine (A36 and A51), or valine (V36 and V51) (Fig. 3).

[0491] Genes encoding humanized heavy and light chain V region sequences were designed and constructed using an automated synthesis method at ATUM (Newark, California, USA). For transient expression in mammalian cells, humanized light chain V region genes were cloned into the UCB light chain expression vector pMhCK, which contains DNA encoding the human kappa chain constant region (Km3 allotype). Humanized heavy chain V region genes were cloned into the UCB human gamma-1 heavy chain expression vector pMhg1 L234A L235A, which contains DNA encoding the human gamma-1 heavy chain constant region (G1m17,1 allotype), including L234A and L235A mutations, to reduce binding to the Fc gamma receptor. The rabbit V region genes of antibody 17415 were also cloned into the human antibody expression vector. The light chain V region was cloned into a modified version of the human kappa vector containing the S171C mutation to regenerate additional disulfide bonds found in the rabbit VK light chain. The heavy and light chain vectors obtained in this way were co-transfected into CHOS-XE suspension cells to express humanized and chimeric recombinant antibodies of the human IgG1 LALA format. The antibodies were evaluated for their activity as well as binding characteristics in an in vitro T lymphocyte depletion assay. All humanized antibodies showed reduced efficacy (EC50) and potency (Emax) compared to the chimeric parent antibody 17415 (see Table 3 below).

[0492] [Table 3]

[0493]

[0494] To determine whether the humanized heavy chain or light chain was the cause of the loss of functional activity, humanized light chain and heavy chain grafts gL1 and gH1 were expressed in combination with their respective chimeric antibody chains (cL and cH). Subsequently, the antibodies were screened for activity in the T lymphocyte depletion assay and binding affinity to human CD45 by SPR using the method described in the examples, and the results are presented in Table 4 below. The humanized heavy chain graft (cLgH1) paired with the chimeric light chain showed reduced efficacy and potency in T cell depletion and reduced binding affinity to CD45. On the other hand, the combination of the humanized light chain and the chimeric antibody heavy chain (gL1cH) maintained binding affinity to CD45 and potency in the T cell depletion assay, but showed reduced potency compared to the parent antibody 17415.

[0495] When humanized heavy chain variants gH2 to gH4 were combined with chimeric light chains, mutating the cysteine ​​residue to serine to remove the disulfide bond formed between C36 and C51 (cLgH2) resulted in an unexpected increase in binding affinity for CD45 compared to cLgH1 and improved efficacy and function in the T lymphocyte depletion assay. Conversely, substituting the cysteine ​​residue with valine (cLgH4) further diminished binding affinity and function in the T cell depletion assay (compared to cLgH1), while substituting it with alanine (cLgH3) had little effect on binding or T cell depletion activity (Table B).

[0496] When humanized light chain transplant variants gL2 to gL5 were combined with chimeric heavy chains, it was found that removing free cysteine ​​residues by mutating them to serine (C90S, gL2cH) maintained binding affinity, but functional activity was partially lost in the T lymphocyte depletion analysis, and efficacy and potency were reduced compared to gL1cH. When C90 was mutated to alanine (gL3cH), valine (gL4cH), or glutamine (gL5cH), binding affinity for CD45 was lost and T cell depletion activity was reduced (Table 4).

[0497] [Table 4]

[0498]

[0499] To restore functional activity, the humanized heavy and light chains were further modified as described herein. In heavy chain implants gH5 and gH6, residue 78 (lysine, K78) was deleted to restore the gap in Framework 3 and recreate the rabbit loop structure between beta sheet strands D and E. Implant gH6 also included C36S and C51S mutations to remove the disulfide bond between CDRH1 and CDRH2. In light chain implants gL6 and gL7, additional donor residues of the rabbit light chain framework were introduced at positions 10 (serine, S10), 42 (glutamine, Q42), 83 (alanine, A83), 106 (glutamic acid, E106), and 108 (valine, V108). Implant gL7 also included a C90S mutation to remove the free cysteine ​​residue of CDRL3. Additional humanized light chains were designed using the human V region IGKV4-1 and IGKJ4 J region (IMGT, http: / / www.imgt.org / ) as the receptor framework (Fig. 2). The donor residues of the rabbit antibody light chain framework were maintained at five or more positions from a 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). Transplants gL15 and gL16 also included a C90S mutation to remove a free cysteine ​​residue from CDRL3. Humanized heavy and light chains were expressed as hIgG1 LALA antibodies in various graft combinations, and the generated antibodies were tested for functional activity in a T lymphocyte depletion assay and for CD45 binding affinity in SPR (see Table 5 below).

[0500] [Table 5]

[0501]

[0502]

[0503] Deleting residue 78 in framework 3 of the humanized heavy chain graft gH5 increased binding affinity for CD45 (Table 5, gL1gH1 (418.5 nM) compared to gL1gH5 (279.9 nM)), but reduced efficacy and potency in the T cell depletion assay. Consistent with the unexpected increase in binding affinity observed when the disulfide bond was removed from the humanized heavy chain graft gH2, the binding affinity for CD45 also increased further in the graft gH6 (gL1gH6 179.2 nM).

[0504] The T cell depletion activity of humanized 17415 antibodies containing grafts gH5 and gH6 was restored by increasing the donor residue content in light chain grafts gL6 and gL7 (gL1 gH5 EC50 19.17 nM, Emax 58.75% versus gL6 gH5 EC50 1.99 nM, Emax 88.45% or gL7 gH5 EC50 2.4 nM, Emax 88.15%, gL6 gH6 EC50 1.76 nM, Emax 76.15% or gL7 gH6 EC50 0.63 nM, Emax 106%). Similarly, when light chain CDRs were transplanted into the alternative human receptor framework IGKV4-1 and JK4 J regions (transplants gL13 to 16), T cell depletion efficacy and potency were enhanced when paired with heavy chain transplants gH5 and gH6 (compared to gL1gH5 and gL1gH6), while affinity for CD45 was maintained (Table C).

[0505] Further description of some experiments involved in the humanization process of the original 17415 antibody is provided in Example 6 below.

[0506] Example 5: Humanization of Antibody 17552

[0507] Rabbit antibody 17552, obtained and evaluated using the method presented in Example 2, was humanized by transplanting the CDR of the rabbit V region into the human germ cell antibody V region framework. Additionally, to restore antibody activity, some of the framework residues of the rabbit V region were retained in the humanized sequence. These residues were selected using Adair et al. (1991) (Humanized antibodies. WO91 / 09967 - see above). The alignment of the rabbit antibody (donor) V region sequence and the human germ cell antibody (receptor) V region sequence is shown in Fig. 4 along with the designed humanized sequence. The CDR transplanted from the donor to the receptor sequence is Kabat (Kabat et al As defined by ., 1987 (see above), and for CDR-H1, the combined definition of Chothia / Kabat is used (Adair et al ..., see 1991 Humanized antibodies, WO91 / 09967). Human V region IGKV1-8 and IGKJ4-1 J region (IMGT, http: / / www.imgt.org / ) were selected as the receptors for antibody 17552 light chain CDR. All light chain framework residues of the humanized transplant variant were derived from human germline genes, provided that at least one residue from the group consisting of residues 2, 3, and 63 (based on sequence number 3 gL1) retained the donor residues leucine (L2), valine (V3), and glutamate (E63), respectively (Fig. 4). The free cysteine ​​residue (C77) at position 77 of framework 3 was mutated to serine (C77S).

[0508] The human V region IGHV4-4 and IGHJ4-1 J region (IMGT, http: / / www.imgt.org / ) were selected as receptors for the heavy chain CDR of antibody 17552. As with many rabbit antibodies, the VH gene of antibody 17552 is shorter than the selected human receptor. When aligned with the human receptor sequence, Framework 1 of the VH region of antibody 17552 lacks an N-terminal residue, which is retained in the humanized antibody (Fig. 4). Framework 3 of the 17552 rabbit VH region lacks two residues (75 and 76, based on sequence number 17gH1) in the loop between beta sheet strands D and E. In the humanized transplant variant, this gap is filled with the corresponding residues of the selected human receptor sequence (lysine 75, K75; arginine 76, N76) (Fig. 4). All heavy chain framework residues of the humanized transplant variants were derived from human germline genes, except that residues 23, 47, 67, 71, 73, 78, and 96 (based on sequence number 17, gH1) retained the donor residues threonine (T23), tyrosine (Y47), phenylalanine (F67), lysine (K71), serine (S73), valine (V78), and threonine (T96), respectively. In some humanized transplant variants, the potential aspartate isomerization site of CDRH3 was modified by substituting the aspartate residue at position 101 with glutamate (D101E) or the glycine residue at position 102 with serine (G102S) or alanine (G102A).

[0509] Genes encoding humanized heavy and light chain V region sequences were designed and constructed using an automated synthesis method at ATUM (Newark, California, USA). For transient expression in mammalian cells, humanized light chain V region genes were cloned into the UCB light chain expression vector pMhCK, which contains DNA encoding the human kappa chain constant region (Km3 allotype). Humanized heavy chain V region genes were cloned into the UCB human gamma-1 heavy chain expression vector pMhg1 L234A L235A, which contains DNA encoding the human gamma-1 heavy chain constant region (G1m17,1 allotype), including L234A and L235A mutations, to reduce binding to the Fc gamma receptor. The rabbit V region genes of antibody 17552 were also cloned into the human antibody expression vector. The light chain V region was cloned into a modified version of the human kappa vector containing the S171C mutation to regenerate additional disulfide bonds found in the rabbit VK light chain. The heavy and light chain vectors obtained in this way were co-transfected into CHOS-XE suspension cells to express humanized and chimeric recombinant antibodies of the human IgG1 LALA format. The CD45 binding affinity of the antibodies was evaluated by SPR (Table 6).

[0510] The humanized 17552 gL1gH1 antibody maintained binding affinity for CD45 compared to the chimeric parent rabbit antibody (gL1gH1, 7.9nM versus cLcH, 5.6nM). As a result of modifying the aspartate isomerization site by mutating residue glycine 102 to alanine in the humanized heavy chain graft gH4 (G102A), affinity was maintained compared to gL1gH1 (gL1gH4, 6.5nM), whereas the D101E (gH2) and G102S (gH3) mutations both reduced binding affinity (gL1gH2, 72.3nM and gL1gH3, 20.7nM).

[0511] [Table 6]

[0512]

[0513] Example 6: Analysis of human PBMC cell depletion using the supernatant of monospecific antibodies having various combinations of 17415 light and heavy chain transplant variants

[0514] Introduction:

[0515] This embodiment provides additional explanation regarding experiments related to the humanization of the original rabbit 17415 antibody also described in Example 3 above.

[0516] Materials and Methods - PBMC Depletion Analysis:

[0517] Human PBMCs derived from blood leukocyte-platelet apheresis cones (NHSBT Oxford) were stored as frozen aliquots. Before performing the analysis, 5 x 10⁶ were placed in each 1 ml. 7 One frozen cell vial containing cells was thawed in a 37°C water bath and added to 50 ml of complete medium (RPMI 1640 + 2 mM GlutaMAX + 1% penicillin / streptomycin, all supplied by Invitrogen) + 10% fetal bovine serum (FBS, Sigma Aldrich). The cells were centrifuged (300 g, 5 min, room temperature), resuspended in 50 ml of complete medium, washed, and centrifuged again. The cells were resuspended in 10 ml of complete medium and counted using a ChemoMetec NucleoCounter NC-3000. Subsequently, 1 x 10⁶ cells per well in 50 µl were added. 5 Canine cells were added to each well of a cell culture-treated Corning Costar 96-well U-bottom microplate (catalog number 07-200-95). PBMCs obtained from 7 donors were used for analysis (UCB Cone 032, 034, 036, 037, 045, 858, and 1031).

[0518] 400 nM stocks of the supernatant of each humanized 17415 IgG1 LALA graft in complete medium were prepared in Greiner 96-well unconjugated microplates. An 8-point dose curve was constructed by serially diluting the reagents 1:5 seven times in complete medium. 50 µl of each dilution (final well concentration 200-0.0026 nM) was added to the cells, and the plates were incubated for 4 hours at 37°C and 5% CO₂. After incubation, the plates were centrifuged at 300 g for 5 minutes at room temperature, the 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) and washed. The plates were then centrifuged again and the buffer was aspirated to leave 20 µl of residual medium. For lymphocyte analysis, 20 µl of LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (Invitrogen, 1:5000 dilution) was added to each well and incubated at 4°C for 15 minutes. For CD3 staining, cells were stained with 20 µl of an antibody solution containing anti-human CD3 FITC antibody (BD Biosciences, Catalog No. 561806, 1:50 dilution) and LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (Invitrogen, 1:5000 dilution). After incubation, the plates were centrifuged at 300 g for 5 minutes at room temperature, 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) and washed. The plates were then centrifuged again and buffer was aspirated to leave 20 µl of residual medium. The cells were analyzed in a live state using Intellicyt iQue Screener PLUS and iQUE 3.The number of live cells was extracted as a measurement indicator, and a graph was generated using Graphpad Prism version 8.1 (Graphpad). EC50 and Emax values ​​were derived by applying asymmetric (4-parameter) curve fitting.

[0519] result:

[0520] The lymphocyte reduction rate in a PBMC population treated with 17415 IgG1 LALA heavy chain H1 and five light chain grafts (L1-5) is shown in Fig. 5(a). The lymphocyte reduction rate in a PBMC population treated with 17415 light chain L1 and four heavy chain grafts (H1-4) is shown in Fig. 5(b). Data from one donor (Cone 1031) are shown. As humanization modification occurred in both the heavy and light chains, the EC50 and Emax of the 17415 IgG1 LALA antibody decreased compared to the chimeric antibody (cLcH).

[0521] To identify humanized mutations that reduce the functional killing of 17415 IgG1 antibodies, each light chain graft (L1-5) was paired with a chimeric heavy chain (cH), and the lymphocyte reduction rates in the PBMC population are shown in Figure 6(a). All humanized light chain grafts showed reduced EC50 and Emax of 17415 IgG1 LALA antibodies compared to the chimeric (cLcH). Each heavy chain graft (H1-4) was paired with a chimeric light chain (cL), and the lymphocyte reduction rates in the PBMC population are shown in Figure 6(b). Data from one donor (Cone 858) are shown. Humanized heavy chain grafts showed less effect on the EC50 and Emax of 17415 IgG1 LALA antibodies compared to the chimeric (cLcH), suggesting that the humanized light chain has a greater impact on the loss of functional killing ability.

[0522] To enhance the functional cytotoxicity of humanized 17415 IgG1 LALA, additional heavy chains (H5 and H6) and additional light chains (L6, 7, 13, 14, 15, 16) were tested. The T cell reduction rates in PBMC populations treated with these humanized grafts are shown in Fig. 7. Humanized 17415 grafts containing heavy chain H5 are shown in Fig. 7(a), and humanized 17415 grafts containing heavy chain H6 are shown in Fig. 7(b). Data from Cone 036 are presented as representative of data from three donors. Humanized 17415 grafts containing heavy chain H6 exhibit Emax and EC50 values ​​similar to chimeric (cLcH) 17415 compared to H5. Among the grafts containing H6, the L7, L15, and L16 light chain grafts demonstrate the most superior functional and humanized characteristics.

[0523] Example 7: Analysis of human Jurkat cell depletion using the supernatant of monospecific antibodies having various combinations of 17415 light and heavy chain transplant variants

[0524] Jurkat depletion analysis

[0525] Before performing the analysis, one vial of Jurkat (acute T-cell leukemia cell line) was thawed in a 37°C water bath and added to 20 ml of complete medium (RPMI 1640 + 2 mM GlutaMAX + 1% penicillin / streptomycin, all supplied by Invitrogen, + 10% fetal bovine serum (FBS), Sigma Aldrich). The cells were centrifuged (300 g, 5 min, room temperature), resuspended in 20 ml of complete medium, washed, and centrifuged again. The cells were resuspended in 10 ml of complete medium and counted using a ChemoMetec NucleoCounter NC-3000. Subsequently, 1 x 10⁶ cells in 50 µl of complete medium 5 Dog cells were added to each well of a cell culture-treated Corning Costar 96-well U-bottom microplate (catalog number 07-200-95).

[0526] 400 nM stocks of the supernatant of each humanized 17415 IgG1 LALA graft in complete medium were prepared in Greiner 96-well unconjugated microplates. An 8-point dose curve was constructed by serially diluting the reagents 1:5 seven times in complete medium. 50 µl of each dilution (final well concentration 200-0.0026 nM) was added to the cells, and the plates were incubated for 4 hours at 37°C and 5% CO₂. After incubation, the plates were centrifuged at 300 g for 5 minutes at room temperature, the 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) and washed. The plates were then centrifuged again and the buffer was aspirated to leave 20 µl of residual medium. 10 µl of Sytox Blue (Invitrogen, 1:500 dilution) was added to each well and incubated at room temperature for 10 minutes. Live cells were analyzed using Intellicyt iQue Screener PLUS and iQUE 3. The number of live cells was extracted as a measurement indicator, and a graph was generated using Graphpad Prism version 8.1 (Graphpad). EC50 and Emax values ​​were derived by applying asymmetric (4-parameter) curve fitting.

[0527] result:

[0528] The results obtained for various combinations of light and heavy chain transplant variants are shown in parts (a) to (d) of Figure 8.

[0529] Example 8: Analysis of human PBMC cell depletion using purified monospecific antibodies containing 17415 light chain transplant variants 7, 15, and 16

[0530] Materials and Methods - PBMC Depletion Analysis

[0531] Human PBMCs derived from blood leukocyte-platelet apheresis cones (NHSBT Oxford) were stored as frozen aliquots. Before performing the analysis, 5 x 10⁶ were placed in each 1 ml. 7One frozen cell vial containing cells was thawed in a 37°C water bath and added to 50 ml of complete medium (RPMI 1640 + 2 mM GlutaMAX + 1% penicillin / streptomycin, all supplied by Invitrogen) + 10% fetal bovine serum (FBS, Sigma Aldrich). The cells were centrifuged (300 g, 5 min, room temperature), resuspended in 50 ml of complete medium, washed, and centrifuged again. The cells were resuspended in 10 ml of complete medium and counted using a ChemoMetec NucleoCounter NC-3000. Subsequently, 1 x 10⁶ cells per well in 50 µl were added. 5 Canine cells were added to each well of a cell culture-treated Corning Costar 96-well U-bottom microplate (catalog number 07-200-95). For this analysis, PBMCs obtained from three donors UCB-Cone 1033, 1001, and 1000 were used.

[0532] 500 nM stocks of each purified humanized 17415 IgG1 LALA graft, 17552 IgG1 LALA graft, 17552 IgG1, and isotype control IgG1 (5604) in complete medium were prepared in Greiner 96-well unconjugated microplates. An 8-point dose curve was constructed by serially diluting the reagents 1:5 seven times in complete medium. However, for 17552 IgG1 and 5604 IgG1, a single concentration (final well concentration 250 nM) was used. 50 µl of each dilution (final well concentration 250-0.0032 nM) was added to the cells and incubated for 4 hours at 37°C and 5% CO₂. After incubation, the plates were centrifuged at 300 g for 5 minutes at room temperature, 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) and washed. The plates were then centrifuged again and buffer was aspirated to leave 20 µl of residual medium. For CD3 staining, cells were stained for 30 minutes at 4°C with 20 µl of antibody solution containing anti-human CD3 FITC antibody (BD Biosciences, catalog number 561806, 1:50 dilution) and LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (Invitrogen, 1:500 dilution). After incubation, the plates were centrifuged at 300 g for 5 minutes at room temperature, 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), washed, and centrifuged again. The cells were washed again and buffer was aspirated to leave 20 µl of residual medium. The cells were analyzed in a live state using Intellicyt iQue 3.The number of live cells was extracted as a measurement indicator, and a graph was generated using Graphpad Prism version 8.1 (Graphpad). EC50 and Emax values ​​were derived by applying asymmetric (4-parameter) curve fitting.

[0533] result:

[0534] Figure 9 shows the T cell reduction rates in PBMC populations treated with humanized 17415 IgG1 LALA grafts containing 17415 light chain modifications 7, 15, and 16, and humanized 17552 IgG1 LALA grafts containing 17552 heavy chain modifications 1 and 4. Cone 1000 data are presented as representative of data from three donors. All grafts exhibited 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 grafts do not deplete T cells.

[0535] Example 9: Human Jurkat cell depletion analysis using purified monospecific antibodies containing 17415 light chain transplant variants 7, 15, and 16

[0536] Jurkat depletion analysis

[0537] Before performing the analysis, one vial of Jurkat (acute T-cell leukemia cell line) was thawed in a 37°C water bath and added to 20 ml of complete medium (RPMI 1640 + 2 mM GlutaMAX + 1% penicillin / streptomycin, all supplied by Invitrogen, + 10% fetal bovine serum (FBS), Sigma Aldrich). The cells were centrifuged (300 g, 5 min, room temperature), resuspended in 20 ml of complete medium, washed, and centrifuged again. The cells were resuspended in 10 ml of complete medium and counted using a ChemoMetec NucleoCounter NC-3000. Subsequently, 1 x 10⁶ cells in 50 µl of complete medium 5 Dog cells were added to each well of a cell culture-treated Corning Costar 96-well U-bottom microplate (catalog number 07-200-95).

[0538] 500 nM stocks of each humanized 17415 IgG1 LALA graft, 17552 IgG1, and isotype control IgG1 (5604) in complete medium were prepared in Greiner 96-well unconjugated microplates. An 8-point dose curve was constructed by serially diluting the reagents 1:5 seven times in complete medium. However, for 17552 IgG1 and 5604 IgG1, a single concentration (final well concentration 250 nM) was used. 50 µl of each dilution (final well concentration 250-0.0032 nM) was added to the cells and incubated for 4 hours at 37°C and 5% CO₂. After incubation, the plates were centrifuged at 300g for 5 minutes at room temperature, and the buffer was aspirated using a BioTek ELx405 microplate washer. The cells were resuspended in FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma Aldrich), washed, centrifuged again, and the buffer was aspirated to leave 20 µl of residual medium. 10 µl of Sytox Blue (Invitrogen, diluted 1:500) was added to each well, and the plates were incubated at room temperature for 10 minutes. Cells were analyzed in a live state using Intellicyt iQue 3. The number of live cells was extracted as a measurement indicator, and graphs were generated using Graphpad Prism version 8.1 (Graphpad). EC50 and Emax values ​​were derived by applying asymmetric (4-parameter) curve fitting.

[0539] result:

[0540] Figure 10 shows the Jurkat cell reduction rates in a PBMC population treated with humanized 17415 IgG1 LALA grafts containing 17415 light chain modifications 7, 15, and 16. All 17415 grafts showed Jurkat cell reduction in the Emax range of 87.9–89.4% and the EC50 range of 0.08–0.59 nM. 17552 IgG1 did not deplete Jurkat cells.

[0541] Example 10: Evaluation of binding of a monospecific CD45 antibody to ExpiHEK cells expressing human CD45

[0542] Materials and Methods:

[0543] Expi293F™ (Gibco) cells were transfected with human CD45 RO ECD mRNA using Lipofectamine™ RNAiMAX transfection reagent (RNAiMAX, Invitrogen). Prior to transfection, Expi293F cells were deposited in Expi293™ Expression Medium (Gibco) at a rate of 5 x 10⁶ 5The mixture was prepared at 1 cell / mL. A total of 2 μg of mRNA was diluted in 100 µl of Opti-MEM™ I Reduced Serum Medium (Opti-MEM, Invitrogen) and mixed with 3 µl of RNAiMAX pre-diluted in 100 µl of Opti-MEM. After incubation at room temperature for 10-20 minutes, 50 µl of the mixture was added to the cells in each well of a 24-well plate (Corning) to achieve a final volume of 0.5 mL of medium. Cells were incubated at 37°C and 5% CO₂ for 18-24 hours for protein expression. Cells were collected from the 24-well plate, diluted in 10 ml of FACS buffer (PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3 + 2 mM EDTA, Sigma Aldrich), and centrifuged (200xg, 6 min). Cells were resuspended in 10 ml of FACS buffer and counted using a ChemoMetec NucleoCounter NC-3000. Then, 20,000 cells per well (25 µl) were added to each well of a cell culture-treated Corning Costar 96-well U-bottom microplate (catalog number 07-200-95).

[0544] 500 nM stocks of each purified humanized 17415 IgG1 LALA graft and control molecule in FACS buffer were prepared in Greiner 96-well unconjugated microplates. An 8-point dose curve was constructed by serially diluting the reagents 1:5 in FACS buffer seven times. 25 µl of each dilution (final well concentration 250–0.0032 nM) was added to cells and incubated in a shaker at 4°C for 1 hour. Cells were stained with 25 µl of a secondary antibody solution containing anti-human IgG AF488 Fab fragment goat anti-human IgG Fcγ (Jackson ImmunoResearch, catalog number 109-547-008, 1:200 dilution) and LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (Invitrogen, 1:5000 dilution) for 1 hour at 4°C. After incubation, the plates were centrifuged at 300g for 5 minutes at room temperature, 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), washed, and centrifuged again. The cells were washed again and the buffer was removed, leaving the cells in 20 µl of residual medium. The cells were analyzed in a live state using Intellicyt iQue 3. The fluorescence geometric mean was extracted as a measurement indicator, and graphs were generated using Graphpad Prism version 8.1 (Graphpad).

[0545] Protein sequence:

[0546]

[0547] result:

[0548] The binding mean fluorescence intensity (MFI, geometric mean) of humanized 17415 IgG1 LALA grafts containing 17415 light chain modifications 7, 15, and 16 for HEK cells expressing human CD45 is shown in Fig. 11 along with isotype control IgG1 (5604).

[0549] Example 11: Production of biparatopic type antibody

[0550] Introduction:

[0551] After generating various biparatopic antibodies, they were evaluated in subsequent examples. Accordingly, this example describes a possible method for generating biparatopic antibodies and an initial evaluation method.

[0552] Maternal antibody expression:

[0553] For the Knob-in-Hole (KiH) heteromerization technology, Knob mutations (T366W) and Hall mutations (T366S, L368A, Y407V) were introduced into the respective heavy chain constant regions to promote the formation of biparatopic antibodies. As a first step, the parent antibodies were expressed. The genes encoding the light chain and heavy chain V regions of the antibodies were constructed using automated synthesis (ATUM). DNA was amplified using the QIAGEN Plasmid Plus Giga Kit (Product No. 12991) according to the manufacturer's instructions, and finally, CHO-SXE cells were transfected using the ExpiCHO™ Expression system Kit (A29133). The supernatant was collected after 7 days.

[0554] Protein exchange method:

[0555] The supernatant of CD45-specific IgG1 was purified using a 2x5 mL MabSelect Sure column (Cytiva) attached to an AKTA Pure purification system (GE Healthcare Life Sciences). The column was equilibrated with PBS, and the cleared culture supernatant was loaded onto the column at a flow rate of 10 ml / min. After loading, the column was washed with PBS 4 CV, and the bound product was eluted with a 0.1 M sodium citrate pH 3.4 solution. The eluent was neutralized with a 2 M Tris-HCl pH 8.5 solution equivalent to 1 / 5 of the elution volume.

[0556] After elution and neutralization, the concentration of the substance was measured following affinity binding, and heavy chain 1 and heavy chain 2 were combined. Cysteamine (Sigma-M9768) was added to the mixture to achieve a final concentration of 5 mM (using 500 mM stock in PBS), and the mixture was incubated at room temperature for 4 hours. Then, the substance was concentrated to approximately 4 mL using an Amicon® Ultra-15 centrifugation filter unit (Merck-UFC903024), loaded onto a HiLoad 26 / 600 Superdex 200 pg column (Cytiva), and eluted with PBS. The desired fractions were collected, endotoxins were removed using a Proteus NoEndoHC column (Protein Ark), and finally, sterile filtration was performed using a Steriflip™ vacuum filter unit (Merck).

[0557] Characterization and Quality Management:

[0558] Protein concentration was calculated after measuring A280 absorbance using the Lunatic system (Unchained labs). Monomer ratios were measured by loading 2 μg of protein onto an ACQUITY BEH200 column attached to a Waters ACQUITY UPLC system. HIC analysis was performed using a Dionex ProPac HIC-10 column attached to an Agilent 1200 binary HPLC equipped with a fluorescence detector. SDS-PAGE was performed using a 4 to 20% Tris-glycine gel by transferring at 180 V for 45 minutes. Endotoxins were measured using an Endosafe nexgen-MCS system (Charles River). Mass verification of constructs was performed by treating samples with NuPAGE™ Sample Reducing Agent (10X) (Invitrogen-NP0004) and / or Rapid™ PNGase F (Neb-P0710s) according to the manufacturer's instructions. Samples were loaded into an XEVO G2 QTof (Waters) equipped with a BioResolveT RP mAb polyphenyl, 450 Å, 2.7 μm column.

[0559] result:

[0560] Specific experimental results regarding the generated dual-paratopic antibody are described in subsequent examples.

[0561] Example 12: Evaluation of binding of a dual-paratopic CD45 antibody to ExpiHEK cells expressing human and synomolgus CD45

[0562] Introduction:

[0563] Various bispecific humanized 17415-17552 IgG1 LALA grafts containing 17415 light chain modifications were evaluated for their binding ability to human or cino CD45 RO ECDs expressed in human Expi293F™ (Gibco) cells, which is discussed in more detail below. The ability to bind to both cino and human is useful as it means that cino antibodies can be studied in cino monkeys prior to human clinical trials.

[0564] Materials and Methods - Cell Binding Method

[0565] With respect to monospecific CD45 antibodies, binding to CD45 on the surface of Expi293F™ (Gibco) cells transfected with human or sino CD45 RO ECD mRNA using Lipofectamine™ RNAiMAX transfection reagent (RNAiMAX, Invitrogen) was evaluated using the same cell binding assay described above.

[0566] 500 nM stocks of each purified humanized 17415-17552 IgG1 LALA and control molecule in FACS buffer were prepared as described.

[0567] Protein sequence:

[0568]

[0569]

[0570] result

[0571] The binding mean fluorescence intensity (MFI, geometric mean) of humanized 17415-17552 IgG1 LALA grafts, including 17415 light chain modifications 7(a), 15(b), and 16(c), to HEK cells expressing human CD45 is shown in Fig. 12 along with the corresponding control molecules. The binding MFI of humanized 17415-17552 grafts, including 17415 light chain modifications 7(a), 15(b), and 16(c), to HEK cells expressing cino CD45 is shown in Fig. 13 along with the corresponding control molecules. Each light chain was paired with 17552 heavy chain grafts H1 and H4. The 17415-17552 grafts showed 4x10 for human CD45 binding. 6 -6.8x10 6 Emax MFI of the range, 10x10 for Sino CD45 combination 6 -11.3x10 6It exhibits an Emax MFI within the range, meaning that all biparatopic antibody grafts bind well to human and cino CD45 expressed in cells.

[0572] Example 13: Human T cell depletion analysis using biparatopic antibodies containing 17415 light chain transplant variants 7, 15, and 16

[0573] Materials and Methods - PBMC Depletion Analysis

[0574] The human PBMC depletion assay used was the same as that used in the previous example, but the assay was performed using 500 nM stocks of each purified humanized 17415-17552 IgG1 LALA graft and control antibody. For this assay, PBMCs obtained from three donors UCB-Cone 1001, 1000, and 947 were used.

[0575] result:

[0576] Figure 14 shows the T cell reduction rates in a PBMC population treated with humanized 117415-17552 IgG1 LALA grafts, including 17415 light chain modifications 7(a), 15(b), and 16(c), along with the corresponding control molecules. Cone 1000 data are presented as representative of data from three donors. Each light chain was paired with 17552 heavy chain grafts H1 and H4. All grafts demonstrated successful cell depletion by showing T cell reduction in the Emax range of 60.2–88.2% and the EC50 range of 0.02–0.21 nM. The dual-paratopic grafts that most effectively depleted T cells were 17415gL7gH6–17552gL1gH4 IgG1 LALA.

[0577] Example 14: Jurkat cell depletion analysis using dual-paratopic antibodies containing 17415 light chain transplant variants 7, 15, and 16

[0578] Introduction

[0579] Jurkat cells were reused as a model for cancer cell depletion through CD45 targeting.

[0580] Materials and Methods:

[0581] The method performed was as described in the previous example. 500 nM stocks of each humanized 17415-17552 IgG1 LALA implant and control antibody in complete medium were prepared in Greiner 96-well unconjugated microplates.

[0582] result:

[0583] Figure 15 shows the Jurkat cell reduction rates treated with humanized 117415-17552 IgG1 LALA grafts, including 17415 light chain modifications 7(a), 15(b), and 16(c), along with the corresponding control molecules. Each light chain was paired with 17552 heavy chain grafts H1 and H4. All grafts exhibited Jurkat cell reduction in the Emax range of 83–95.7% and the EC50 range of 0.14–0.33 nM. The biparatopic antibodies that most effectively depleted Jurkat cells were 17415gL15gH6–17552gL1gH1 IgG1 LALA.

[0584] Example 15: Cell depletion analysis for Sino PBMC

[0585] Introduction

[0586] This embodiment describes a method for evaluating the ability of both single-specific and dual-paratopic CD45 antibodies to deplete Sino monkey cells.

[0587] Materials and Methods - Syno T-cell Depletion Analysis

[0588] Synomolgus PBMC was purchased from Primacyt as a frozen dispensing solution. Before performing the analysis, 2 x 10⁶ were added to each 1 ml. 7One frozen cell vial containing cells was thawed in a 37°C water bath and added to 50 ml of complete medium (RPMI 1640 + 2 mM GlutaMAX + 1% penicillin / streptomycin, all supplied by Invitrogen) + 10% fetal bovine serum (FBS, Sigma Aldrich). The cells were centrifuged (300 g, 5 min, room temperature), resuspended in 50 ml of complete medium, washed, and centrifuged again. The cells were resuspended in 10 ml of complete medium and counted using a ChemoMetec NucleoCounter NC-3000. Subsequently, 1 x 10⁶ cells per well in 50 µl were added. 5 Dog cells were added to each well of a cell culture-treated Corning Costar 96-well U-bottom microplate (catalog number 07-200-95).

[0589] 500 nM stocks of each purified 7415 IgG1, 17552 IgG1, 17415-17552 IgG1, 4133-6294 IgG1, and isotype IgG1 antibody in complete medium were prepared in Greiner 96-well unconjugated microplates. An 8-point dose curve was constructed by serially diluting the reagents 1:5 seven times in complete medium. 50 µl of each dilution (final well concentration 250-0.0032 nM) was added to the cells and incubated for 24 hours at 37°C and 5% CO₂. After incubation, the plates were centrifuged at 300g for 5 minutes at room temperature, 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) and washed. The plates were then centrifuged again and buffer was aspirated to leave 20 µl of residual medium. For CD3 staining, the cells were stained with 20 µl of antibody solution containing anti-human NHP CD3 BV605 (BD Biosciences, catalog number 562994, 1:50 dilution) and LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (Invitrogen, 1:500 dilution) at 4°C for 30 minutes. After incubation, the plates were centrifuged at 300 g for 5 minutes at room temperature, 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), washed, and centrifuged again. The cells were washed again and buffer was aspirated to leave 20 µl of residual medium. The cells were analyzed in a live state using Intellicyt iQue 3. The number of live cells was extracted as a measurement indicator, and a graph was generated using Graphpad Prism version 8.1 (Graphpad).EC50 and Emax values ​​were derived by applying asymmetric (4-parameter) curve fitting.

[0590] result:

[0591] Figure 16 shows the T cell reduction rates in a population of Sino PBMCs treated with 17415-17552 IgG1, 17552 IgG1, 17415 IgG1, 4133-6294 IgG1, and isotype IgG1. Among the antibodies whose results are presented in the figure, only the ambiparatopic 17415-17552 IgG1 depleted Sino T cells to a high level (Emax 75%, EC50 1.5 nM).

[0592] Example 16: Comparison of the ability of antibodies 17415-17552 and other known biparatopic antibodies to kill human CD45-expressing cells

[0593] Introduction:

[0594] This example investigates the ability of the 17415-17552 antibodies to kill CD45-expressing target cells compared to the 4133-6294 antibodies and the IgG1-type YTH24.5-YTH54.12 antibodies described in International Patent Application No. PCT / EP2021 / 078516 (published in WO 2022 / 079199 A1). The variable region (V region) sequence of the anti-CD45 antibodies YTH24.5-YTH54.12 was taken from International Patent Application No. PCT / GB2021 / 052458 (published in WO 2022 / 064191 A1). None of these CD45 biparatopic antibodies were conjugated with a cytotoxic agent.

[0595] PBMC Depletion Analysis:

[0596] The human PBMC depletion assay used was the same as that used in the previous example, but the assay was performed using 500 nM stocks of 4133-6294 IgG1, 17415-17552 IgG1, YTH24.5-YTH54.12 IgG1, and 5604 IgG1, respectively. In this assay, 4133-6294 IgG1 and 17415-17552 IgG1 were compared using PBMCs obtained from four donors UCB-Cone 1001, 1000, 1033, and 947, and 17415-17552 IgG1 and YTH24.5-YTH54.12 IgG1 were compared using PBMCs obtained from one donor (UCB-Cone 924).

[0597] Jurkat depletion analysis

[0598] The method performed was as described in the previous example. 500 nM stocks of 4133-6294 IgG1, 17415-17552 IgG1, YTH24.5-YTH54.12 IgG1 and 5604 IgG1 were prepared, respectively.

[0599] result:

[0600] Figure 17 shows the T cell reduction rates in PBMC populations treated with 4133-6294 IgG1 or 17415-17552 IgG1, along with the isotype (5604 IgG1) control. Both 4133-6294 IgG1 and 17415-17552 IgG1 exhibited T cell reduction. 4133-6294 IgG showed an Emax range of 80.7-89.9% and an EC50 range of 0.17-0.53 nM, while 17415-17552 IgG1 showed an Emax range of 74.4-83.5% and an EC50 range of 0.08-0.4 nM.

[0601] Figure 18 shows the T cell reduction rates in PBMC populations treated with 17415-17552 IgG1 or YTH24.5-YTH54.12 IgG1, along with the isotype (5604 IgG1) control. Only 17415-17552 IgG1 showed T cell reduction. 17415-17552 IgG1 showed an Emax of 88.8% and an EC50 of 0.05 nM. The Emax and EC50 of YTH24.5-YTH54.12 IgG1 could not be measured.

[0602] The reduction rates of Jurkat cells treated with 4133-6294 IgG1 or 17415-17552 IgG1 are shown in Fig. 19 along with the isotype (5604 IgG1) control. Both 4133-6294 IgG1 and 17415-17552 IgG1 showed a reduction in Jurkat cells. 4133-6294 IgG1 showed an Emax of 91.9% and an EC50 of 0.47 nM, while 17415-17552 IgG1 showed an Emax of 92.7% and an EC50 of 0.06 nM.

[0603] The reduction rates of Jurkat cells treated with 17415-17552 IgG1 or YTH24.5-YTH54.12 IgG1 are shown in Figure 21 along with the isotype (5604 IgG1) control. Only 17415-17552 IgG1 showed T cell reduction. 17415-17552 IgG1 showed an Emax of 90.9% and an EC50 of 0.05 nM. The Emax and EC50 of YTH24.5-YTH54.12 IgG1 could not be measured.

[0604] In both PBMC and Jurkat cells, the EC50 of the dual-paratopic 17415-17552 IgG1 antibodies was lower than that of the dual-paratopic 4133-6294 IgG1 antibodies. The dual-paratopic YTH24.5-YTH54.12 IgG1 antibodies could not induce T cell or Jurkat cell killing.

[0605] conclusion

[0606] Overall, this data shows that the claimed dual-paratopic CD45 antibody has increased efficacy compared to previously known dual-paratopic CD45 antibodies.

[0607] Example 17: Cytokine release in whole blood after 24 hours as measured by Meso Scale Discovery analysis

[0608] Introduction:

[0609] This embodiment investigates the ability of a CD45 antibody to induce cytokine release in whole blood. Cytokine release can cause unwanted inflammation in the treated subject.

[0610] Materials and Methods:

[0611] Human whole blood (lithium heparin tubes) was collected from two donors in accordance with the approved ethical sample collection protocol at UCB Pharma (Slau, UK).

[0612] Stocks of purified 17415gL15gH6-17552gL1gH4 IgG1 LALA, 17415gL15gH6 IgG1 LALA, YTH24.5-YTH54.12 IgG1, and 5604 IgG1 LALA antibodies were prepared in Greiner 96-well unconjugated microplates at 8000 nM in PBS. Subsequently, an 8-point dose curve was constructed by serially diluting the reagents 1:5 seven times in PBS buffer. Campath was diluted in PBS to prepare a 200 μg / ml stock.

[0613] 12.5 µl of the reagent diluent was transferred to a cell culture-treated Corning Costar 96-well U-bottom microplate (Catalog No. 3799), and 237.5 µl of whole blood was added to each well. The final well concentration of the antibody was 400–0.00512 nM. Campath (10 mg / ml stock diluted to 0.2 mg / ml in PBS, preparation No. CHV0387) was used as a positive control at a final concentration of 10 µg / ml. The plates were sealed with gas-permeable adhesive seals and the lids were closed. The plates were then placed in an undisturbed location and incubated for 24 hours in CO2 at 37°C and 5% humidity.

[0614] After 24 hours of incubation, the plate was centrifuged at 1000 g for 10 minutes, and 50 µl of plasma was transferred to a separate plate and stored at -80℃ until cytokine quantification analysis.

[0615] Cytokine measurements were performed using the V-PLEX Human Proinflammatory Panel I (containing interferon (IFN)-γ, interleukin (IL)-6, and tumor necrosis factor (TNF)-α, catalog number K15052D, Meso Scale Discovery) according to the manufacturer's instructions. Briefly, plasma samples were thawed at room temperature and diluted with Diluent 2 (catalog number R51BB-3, Meso Scale Discovery) at a 1:2 ratio. Standard curve calibration solution was prepared using 500 µl of Diluent 2 (catalog number R51BB-3, Meso Scale Discovery). Proinflammatory Panel I plates were washed with PBS (added with 0.05% Tween-20) using a BioTek ELx405 microplate washer, after which 50 µl of sample or standard curve calibration solution was added to each well. The plate was sealed with an adhesive seal and incubated at room temperature for 2 hours using a plate shaker (750 rpm). After washing the plate as before, 25 µl of detection antibody was added to each well. The plate was incubated at room temperature for an additional 2 hours using a plate shaker. After washing the plate as before, 150 µl of readout buffer (diluted 1:2 with distilled water) was added to each well. Then, analysis was performed using a SECTOR Imager 6000 (Meso Scale Discovery).

[0616] result:

[0617] The levels of detected inflammatory cytokines are shown in Fig. 20 (a) IFN-γ, (b) IL-6, and (c) TNF-α. Notably, little to no induction of inflammatory cytokines by 17415gL15gH6-17552gL1gH4 and 17415gL15gH6 IgG1 LALA was observed, and the levels were consistent with wells treated with PBS and 5604 IgG1 LALA.

[0618] conclusion

[0619] Collectively, these data demonstrate that the claimed antibodies induce apoptosis without significantly increasing cytokine release. The absence of cytokine release reduces the likelihood of unwanted cytokine-induced inflammation in treated subjects.

[0620] Example 18: Depletion analysis of leukemia T cell lines and leukemia B cell lines

[0621] Introduction:

[0622] This embodiment investigates the ability of the claimed CD45 antibodies to deplete immune cells in various leukemia cell lines.

[0623] Materials and Methods - Cell Line Depletion Analysis:

[0624] Before performing the analysis, one vial each of (a) leukemia T cell lines (SUDHL1, SUPT11, and Peers) and (b) leukemia B cell lines (DOHH2 and Ramos) was thawed in a 37°C water bath and then added to 5 ml of complete medium for culture (RPMI 1640, Corning + 20% fetal bovine serum (FBS) and Gibco for Ramos, DOHH2, and SUDHL1; RPMI 1640 + 10% FBS for SUPT11 and Peers). For the depletion assay, cells were centrifuged (200 g, 5 min, room temperature), resuspended in 5 ml of complete medium, washed, and centrifuged again. Cells were resuspended in 5 ml of complete medium and counted using a Biorad TC20™ automated cell counter. Subsequently, 1 x 10⁶ cells were added to 90 µl of complete medium. 5 Dog cells were added to each well of a cell culture-treated Corning Costar 96-well U-bottom microplate (catalog number 3879).

[0625] 2 μM stocks of purified 17415gL15gH6-17552gL1gH4 IgG1 LALA, 17415gL15gH6 IgG1 LALA, and isotype control IgG1 LALA (5604) in complete medium were prepared in Sarstedt 96-well microplates (catalog number 83.3924.005). The antibodies were serially diluted 1:3.16 nine times in complete medium to create a 10-point dose curve. 10 μL of each dilution (final well concentration 200-0.002 nM) was added to the cells and incubated for 2 hours at 37°C and 5% CO2. After incubation, 150 μL of PBS was added to each well, the plates were centrifuged at 400 g for 5 minutes at room temperature, the plates were inverted and blotted onto tissue paper, and the supernatant was discarded. Cells were resuspended in 150 µl of PBS, the plate was centrifuged again, and the supernatant was discarded. For viability staining, cells were stained with 50 µl of a solution containing LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (Invitrogen, 1:1000 dilution) at 4°C for 30 minutes. After incubation, 150 µl of FACS buffer (PBS + 1% bovine serum albumin (BSA), Fisher BioReagents) was added to each well, the plate was centrifuged at 400 g for 5 minutes at room temperature, and the supernatant was discarded. Cells were washed once more with FACS buffer and resuspended in 150 µl of FACS Lysing Solution (BD Biosciences, Catalog No. 349202). For flow cytometry analysis, a volume of 100 µl per well was measured using an Attune NxT flow cytometer (Invitrogen). The number of single live cells was extracted as a measurement indicator, and a graph was generated using Graphpad Prism version 9.0 (Graphpad). EC50 and Emax values ​​were derived by applying asymmetric (4-parameter) curve fitting.

[0626] result:

[0627] The reduction rates of Peers, SUPT11, and SUDHL1 T cells treated with 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA are shown in Figures 22a, 22b, and 22c, respectively, along with the isotype control (5604 IgG1 LALA). The reduction rates of Ramos and DOHH2 B cells treated with 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA are shown in Figures 23a and 23b, respectively, along with the isotype control (5604 IgG1 LALA).

[0628] Among the cell lines tested, the monospecific antibody 17415gL15gH6 IgG1 LALA showed a significant decrease compared to the control group in certain T cell lines (Peers and SUPT11) and certain B cell lines (Ramos). The Emax range was 28.4-66.8%, and the EC50 range was 0.45-18.58 nM.

[0629] The dual-paratopic antibody 17415gL15gH6-17552gL1gH4 IgG1 LALA showed a significant reduction compared to the control group in all tested T cell lines and B cell lines (Peers, SUPT11, SUDHL1, Ramos, and DOHH2). The Emax range was 29.3-85.7%, and the EC50 range was 0.03-0.30 nM.

[0630] conclusion

[0631] This data demonstrates that both monospecific killer arms and biparatopic antibodies can successfully deplete T and B cell lines, and that biparatopic antibodies show increased efficacy and potency compared to monospecific killer antibodies.

[0632] Example 19: Cell depletion of PBMCs derived from healthy subjects and leukemia patients

[0633] Introduction:

[0634] This embodiment investigates the ability of CD45 antibodies to deplete immune cells in healthy subjects and subjects with disease.

[0635] Materials and Methods - PBMC Depletion Analysis:

[0636] Blood samples were collected from healthy volunteers (University of Leicester) and patients with T / B cell leukemia (Royal Hospital of Leicester), and human PBMCs were isolated using Lymphoprep (StemCell) density gradient centrifugation. PBMCs were resuspended in 10 ml of complete medium (RPMI 1640, Corning, + 10% fetal bovine serum (FBS) + 2 mM GlutaMAX, all supplied by Gibco, + 1% penicillin / streptomycin, Fisher, 0.004 µL / ml beta-mercaptoethanol (BME) (Sigma)) and counted using a Biorad TC20™ automated cell counter. Subsequently, 1 x 10⁶ in 90 µL of medium 5 Cells were added to each well of a cell culture-treated Corning Costar 96-well U-bottom microplate (catalog number 3879). PBMCs obtained from 8 donors—6 healthy volunteers (330CD, 334ES, 335AC, 336BB, 365DS, 370EE), 1 patient with T-cell leukemia (4386POS, three-digit syndrome), and 1 patient with B-cell leukemia (4650ADG, mantle cell lymphoma)—were used in this analysis.

[0637] 2 μM stocks of purified 17415gL15gH6-17552gL1gH4 IgG1 LALA, 17415gL15gH6 IgG1 LALA, and isotype control IgG1 LALA (5604) in complete medium were prepared in Sarstedt 96-well microplates (catalog number 83.3924.005). The reagents were serially diluted 10 times to 1 / 3.16 in complete medium to generate an 11-point dose curve. 10 μL of each dilution (final well concentration 200-0.002 nM) was added to the cells and incubated for 22 hours at 37°C and 5% CO2. After incubation, 150 μL of PBS was added to each well, the plate was rotated at 400g and RT for 5 minutes, and the supernatant was removed by flipping the plate and blotting it onto tissue paper. Cells were resuspended in 150 µl of PBS, the plate was spun again, and the supernatant was discarded. For viability staining, cells were stained with 50 µl of a solution containing LIVE / DEAD™ Fixable Violet Dead Cell Stain (Invitrogen, 1:1000 dilution) at 4°C for 30 minutes. After incubation, 150 µl of FACS buffer (PBS + 1% bovine serum albumin (BSA), Fisher BioReagents) was added to each well, the plate was centrifuged at 400 g for 5 minutes at room temperature, and the supernatant was discarded. Cells were washed once more with FACS buffer, and the supernatant was discarded. For extracellular marker staining, cells were stained with anti-human CD16 BV480 (BD Biosciences, Catalog No. 566171, 1:100 dilution), CD19 Alexafluor 700 (BD Biosciences, Catalog No. 557921, 1:200 dilution), CD3 Alexafluor 594 (Biolegend, Catalog No. 300446, 1:250 dilution), and CD4 PerCP Cy5.The cells were stained with 50 µl of antibody solution containing 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) at 4°C for 30 minutes. After incubation, 150 µl of FACS buffer was added to each well, the plate was centrifuged at 400 g for 5 minutes at room temperature, and the supernatant was discarded. The cells were washed once more with FACS buffer and resuspended in 150 µl of FACS Lysing Solution (BD Biosciences, catalog number 349202). For flow cytometry analysis, a volume of 100 µl per well was measured using an Attune NxT flow cytometer (Invitrogen). The number of single live cells for each leukocyte population was extracted as a measurement indicator, and graphs were generated using Graphpad Prism version 9.0 (Graphpad). EC50 and Emax values ​​were derived by applying asymmetric (4-parameter) curve fitting.

[0638] result:

[0639] PBMC depletion analysis

[0640] Figure 24 shows the reduction rate of T cells in a population of PBMCs derived from healthy volunteers (a) and T-cell leukemia patients (b) treated with 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA, along with the isotype control (5604 IgG1 LALA). Figure 25 shows the reduction rate of B cells in a population of PBMCs derived from healthy volunteers (a) and B-cell leukemia patients (b) treated with 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA, along with the isotype control (5604 IgG1 LALA). Data from healthy donors 336BB and 330CD are presented as representative data from six healthy donors.

[0641] 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA both showed T cell reduction in healthy volunteers with an Emax range of 74.2-96.7% and an EC50 range of 0.05-8.94nM. 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA both showed a similar ability to reduce T cells in patients with T-cell leukemia (4368POS) with an Emax range of 74.2-97% and an EC50 range of 0.03-8.94nM. The dual-paratopic antibody (17415gL15gH6-17552gL1gH4 IgG1 LALA) depleted T cells more effectively in healthy and diseased subjects than the monospecific antibody (17415gL15gH6 IgG1 LALA), but the T cell reduction achieved using the monospecific antibody (17415gL15gH6 IgG1 LALA) was still significant compared to the control group.

[0642] 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA both showed a reduction in healthy volunteers with an Emax range of 36.5–80.2% and an EC50 range of 0.57–3.1 nM. 17415gL15gH6-17552gL1gH4 IgG1 LALA and 17415gL15gH6 IgG1 LALA both demonstrated a similar ability to reduce B cells in patients with B-cell leukemia (4650ADG), 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. In other words, the dual-paratopic antibody (17415gL15gH6-17552gL1gH4 IgG1 LALA) depleted B cells more effectively in healthy and diseased subjects than the monospecific antibody (17415gL15gH6 IgG1 LALA), but the reduction in B cells achieved using the monospecific antibody (17415gL15gH6 IgG1 LALA) was still significant compared to the control group.

[0643] conclusion

[0644] This data demonstrates the successful depletion of CD45-expressing target cells in both healthy and diseased subjects. This further demonstrates the therapeutic potential of both monospecific and biparatopic antibodies in various populations of CD45-expressing target cells.

[0645] Numbered embodiments

[0646] The following describes additional numbered embodiments of the present invention, but does not represent claims at this time:

[0647] 1. An antibody or its antigen-binding fragment comprising at least one variable domain specific to CD45, comprising the following light chain and heavy chain variable regions:

[0648] (a) a light chain variable region comprising a CDR1 having the sequence of SEQ ID NO. 33, a CDR2 having the sequence of SEQ ID NO. 34, and a CDR3 having a sequence selected from any one of SEQ ID NOs. 35 and 39 to 42; and

[0649] (b) a heavy chain variable region comprising a CDR1 having a sequence selected from any one of sequence numbers 46, 52, 53 and 54, a CDR2 having a sequence selected from any one of sequence numbers 47, 55, 56 and 57, and a CDR3 having a sequence of sequence number 48.

[0650] 2. In 1, the light chain variable region of (a) is an antibody or antigen-binding fragment comprising any one of the following combinations of CDR1, CDR2, and CDR3:

[0651] Sequence numbers 33, 34, and 35;

[0652] Sequence numbers 33, 34, and 39;

[0653] Sequence numbers 33, 34, and 40;

[0654] Sequence numbers 33, 34, and 41; and

[0655] Sequence numbers 33, 34, and 42.

[0656] 3. In 1 or 2, the heavy chain variable region of (b) comprises any combination of CDR1, CDR2, and CDR3, an antibody or antigen-binding fragment:

[0657] Sequence numbers 46, 47, and 48;

[0658] Sequence numbers 52, 55, and 48;

[0659] Sequence numbers 53, 56 and 48; and

[0660] Sequence numbers 54, 57, and 48.

[0661] 4. In any one of 1 to 3,

[0662] (i) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ NOs 33, 34, and 35, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any one of (aa) SEQ NOs 46, 47, and 48; (bb) SEQ NOs 52, 55, and 48; (cc) SEQ NOs 53, 56, and 48; and (dd) SEQ NOs 54, 57, and 48; or;

[0663] (ii) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ NOs 33, 34, and 39, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any one of (aa) SEQ NOs 46, 47, and 48; (bb) SEQ NOs 52, 55, and 48; (cc) SEQ NOs 53, 56, and 48; and (dd) SEQ NOs 54, 57, and 48; or;

[0664] (iii) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ NOs 33, 34, and 40, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any one of (aa) SEQ NOs 46, 47, and 48; (bb) SEQ NOs 52, 55, and 48; (cc) SEQ NOs 53, 56, and 48; and (dd) SEQ NOs 54, 57, and 48; or;

[0665] (iv) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ NOs 33, 34, and 41, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any one of (aa) SEQ NOs 46, 47, and 48; (bb) SEQ NOs 52, 55, and 48; (cc) SEQ NOs 53, 56, and 48; and (dd) SEQ NOs 54, 57, and 48; or;

[0666] (v) The light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ NOs 33, 34, and 42, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any one of (aa) SEQ NOs 46, 47, and 48; (bb) SEQ NOs 52, 55, and 48; (cc) SEQ NOs 53, 56, and 48; and (dd) SEQ NOs 54, 57, and 48.

[0667] Antibody or antigen-binding fragment.

[0668] 5. An antibody or antigen-binding fragment comprising, in any one of 1 to 4, a light chain having a set of CDRs of LCDR1, LCDR2, and LCDR3 each comprising the sequences of SEQ ID NOs 33, 34, and 39, and a heavy chain variable region having a set of CDRs of HCDR1, HCDR2, and HCDR3 each comprising the sequences of SEQ ID NOs 52, 55, and 48.

[0669] 6. In any one of 1 to 5, the light chain and heavy chain variable regions are humanized antibody or antigen-binding fragments.

[0670] 7. In 6, the light chain variable region of (a) is humanized using an IGKV4-1 or IGKV1-9 framework as the receptor sequence of the framework region, but optionally one or more amino acid residues of the receptor framework sequence are substituted with one or more corresponding residues of the donor framework sequence, and preferably the receptor framework sequence is derived from IGKV4-1, an antibody or antigen binding fragment.

[0671] 8. An antibody or antigen-binding fragment, wherein in any one of 1 to 7, the light chain variable region of (a) comprises a sequence selected from one of SEQ ID NOs 11 to 14 and SEQ ID NOs 3 to 9, preferably one of SEQ ID NOs 11 to 14.

[0672] 9. An antibody or antigen-binding fragment in any one of 6 to 8, wherein the heavy chain variable region of (b) is humanized using the IGHV3-72 framework as the receptor sequence of the framework region, but optionally one or more amino acid residues of the receptor framework sequence are substituted with one or more corresponding residues of the donor framework sequence.

[0673] 10. An antibody or antigen-binding fragment in which, in any one of 1 to 9, the heavy chain variable region of (b) comprises a sequence selected from any one of SEQ ID NOs 17 to 22.

[0674] 11. An antibody or antigen-binding fragment, wherein in any one of 1 to 10, at least one antigen-binding site comprises a light chain variable region sequence selected from any one of SEQ ID NOs 11 to 14 and SEQ ID NOs 3 to 9 and a heavy chain variable region sequence selected from any one of SEQ ID NOs 17 to 22, and preferably the light chain variable region is selected from any one of SEQ ID NOs 11 to 14.

[0675] 12. An antibody or antigen-binding fragment in which, in any one of 1 to 11, at least one antigen-binding site comprises the following:

[0676] (i) a light chain variable region having the sequence of SEQ ID NO. 9 and a heavy chain variable region having the sequence of SEQ ID NO. 22; or

[0677] (ii) a light chain variable region having the sequence of SEQ ID NO. 13 and a heavy chain variable region having the sequence of SEQ ID NO. 22; or

[0678] (iii) a light chain variable region having the sequence of SEQ ID NO. 14 and a heavy chain variable region having the sequence of SEQ ID NO. 22; or

[0679] (iv) a light chain variable region sequence of SEQ ID NO. 11 and a heavy chain variable region sequence selected from any one of SEQ ID NOs. 17 to 22; or

[0680] (v) a light chain variable region sequence of SEQ ID NO. 12 and a heavy chain variable region sequence selected from any one of SEQ ID NOs. 17 to 22; or

[0681] (vi) a light chain variable region sequence of SEQ ID NO. 13 and a heavy chain variable region sequence selected from any one of SEQ ID NOs. 17 to 22; or

[0682] (vii) a light chain variable region sequence of SEQ ID NO. 14 and a heavy chain variable region sequence selected from any one of SEQ ID NOs. 17 to 22; or

[0683] (viii) a light chain variable region sequence of SEQ ID NO. 3 and a heavy chain variable region sequence selected from any one of SEQ ID NOs. 17 to 22; or

[0684] (ix) a light chain variable region sequence of SEQ ID NO. 4 and a heavy chain variable region sequence selected from any one of SEQ ID NOs 17 to 22; or

[0685] (x) a light chain variable region sequence of SEQ ID NO. 5 and a heavy chain variable region sequence selected from any one of SEQ ID NOs. 17 to 22; or

[0686] (xi) a light chain variable region sequence of SEQ ID NO. 6 and a heavy chain variable region sequence selected from any one of SEQ ID NOs. 17 to 22; or

[0687] (xii) a light chain variable region sequence of SEQ ID NO. 7 and a heavy chain variable region sequence selected from any one of SEQ ID NOs. 17 to 22; or

[0688] (xiii) a light chain variable region sequence of SEQ ID NO. 8 and a heavy chain variable region sequence selected from any one of SEQ ID NOs. 17 to 22; or

[0689] (xiv) Light chain variable region sequence of sequence number 9 and heavy chain variable region sequence selected from any one of sequence numbers 17 to 22.

[0690] 13. The antibody or antigen-binding fragment of 12, wherein at least one antigen-binding site comprises the following:

[0691] (i) a light chain variable region having the sequence of SEQ ID NO. 9 and a heavy chain variable region having the sequence of SEQ ID NO. 22; or

[0692] (ii) a light chain variable region having the sequence of SEQ ID NO. 13 and a heavy chain variable region having the sequence of SEQ ID NO. 22; or

[0693] (iii) a light chain variable region having the sequence of sequence number 14 and a heavy chain variable region having the sequence of sequence number 2.

[0694] 14. An antibody or antigen-binding fragment thereof comprising at least one variable domain specific to CD45, comprising the following light chain and heavy chain variable regions:

[0695] (a) a light chain variable region comprising a CDR1 having the sequence of SEQ ID NO. 94, a CDR2 having the sequence of SEQ ID NO. 95, and a CDR3 having the sequence of SEQ ID NO. 96; and

[0696] (b) a heavy chain variable region comprising a CDR1 having the sequence of sequence number 100, a CDR2 having the sequence of sequence number 101, and a CDR3 having a sequence selected from any one of sequence numbers 102 to 105.

[0697] 15. In 14,

[0698] (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;

[0699] (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;

[0700] (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;

[0701] (iv) the light chain variable region of (a) comprises CDR1 containing the sequence of SEQ NO 94, CDR2 containing the sequence of SEQ NO 95, and CDR3 containing the sequence of SEQ NO 96, and the heavy chain variable region of (b) comprises CDR1 containing the sequence of SEQ NO 100, CDR2 containing the sequence of SEQ NO 101, and CDR3 containing the sequence of SEQ NO 105.

[0702] Antibody or antigen-binding fragment.

[0703] 16. Regarding 15,

[0704] (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;

[0705] (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.

[0706] Antibody or antigen-binding fragment.

[0707] 17. In 15 or 16, the light chain and heavy chain variable regions are humanized antibody or antigen-binding fragments.

[0708] 18. An antibody or antigen-binding fragment in which, in 17, the light chain variable region of (a) is humanized using the IGKV1-8 framework as the receptor sequence of the framework region, and optionally one or more amino acid residues of the receptor framework sequence are substituted with one or more corresponding residues of the donor framework sequence.

[0709] 19. An antibody or antigen-binding fragment in which, in any one of 15 to 18, the light chain variable region of (a) comprises a sequence selected from sequence no. 25 and 26.

[0710] 20. An antibody or antigen-binding fragment in any one of 15 to 19, wherein the heavy chain variable region of (b) is humanized using the IGHV4-4 framework as the receptor sequence of the framework region, and optionally one or more amino acid residues of the receptor framework sequence are substituted with one or more corresponding residues of the donor framework sequence.

[0711] 21. An antibody or antigen-binding fragment in which, in any one of 15 to 20, the heavy chain variable region of (b) comprises a sequence selected from any one of SEQ ID NOs 29 to 32.

[0712] 22. An antibody or antigen-binding fragment comprising, in any one of 15 to 21, a light chain variable region sequence selected from SEQ ID NOs 25 and 26 and a heavy chain variable region sequence selected from any one of SEQ ID NOs 29 to 32.

[0713] 23. In 22, the antibody comprises an antibody or antigen-binding fragment including the following:

[0714] (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

[0715] (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

[0716] (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

[0717] (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

[0718] (v) the light chain variable region sequence of SEQ No. 25 and the heavy chain variable region sequence of SEQ No. 32; or

[0719] (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

[0720] (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 ...

Claims

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

48. Claim 2 An antibody or antigen-binding fragment according to claim 1, wherein (i) the light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of any one of SEQ ID NOs 33, 34, and 35; SEQ ID NOs 33, 34, and 39; SEQ ID NOs 33, 34, and 40; SEQ ID NOs 33, 34, and 41; and SEQ ID NOs 33, 34, and 42; and / or: (ii) the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 of any one of SEQ ID NOs 46, 47, and 48; SEQ ID NOs 52, 55, and 48; SEQ ID NOs 53, 56, and 48; and SEQ ID NOs 54, 57, and 48. Claim 3 In claim 1 or 2, (i) the light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ NOs 33, 34, and 35, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from any one of (aa) SEQ NOs 46, 47, and 48; (bb) SEQ NOs 52, 55, and 48; (cc) SEQ NOs 53, 56, and 48; and (dd) SEQ NOs 54, 57, and 48; or (ii) the light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ NOs 33, 34, and 39, and the heavy chain variable region of (b) comprises (aa) SEQ NOs 46, 47, and 48; (bb) SEQ NOs 52, 55, and 48; (cc) Sequence Nos. 53, 56, and 48; and (dd) a combination of CDR1, CDR2, and CDR3 selected from any one of Sequence Nos. 54, 57, and 48; or (iii) the light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of Sequence Nos. 33, 34, and 40, and the heavy chain variable region of (b) comprises (aa) Sequence Nos. 46, 47, and 48; (bb) Sequence Nos. 52, 55, and 48; (cc) Sequence Nos. 53, 56, and 48; and (dd) a combination of CDR1, CDR2, and CDR3 selected from any one of SEQ Nos. 54, 57, and 48; or (iv) the light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ Nos. 33, 34, and 41, and the heavy chain variable region of (b) comprises (aa) SEQ Nos. 46, 47, and 48; (bb) SEQ Nos. 52, 55, and 48; (cc) SEQ Nos. 53, 56, and 48; and (dd) a combination of CDR1, CDR2, and CDR3 selected from any one of SEQ Nos. 54, 57, and 48;(v) an antibody or antigen-binding fragment in which the light chain variable region of (a) comprises a combination of CDR1, CDR2, and CDR3 of SEQ ID NOs 33, 34, and 42, and the heavy chain variable region of (b) comprises a combination of CDR1, CDR2, and CDR3 selected from (aa) SEQ ID NOs 46, 47, and 48; (bb) SEQ ID NOs 52, 55, and 48; (cc) SEQ ID NOs 53, 56, and 48; and (dd) SEQ ID NOs 54, 57, and 48.; Claim 4 An antibody or antigen-binding fragment according to any one of claims 1 to 3, comprising a light chain having a set of CDRs of LCDR1, LCDR2, and LCDR3 each comprising the sequences of SEQ ID NOs 33, 34, and 39, and a heavy chain variable region having a set of CDRs of HCDR1, HCDR2, and HCDR3 each comprising the sequences of SEQ ID NOs 52, 55, and 48. Claim 5 In any one of claims 1 to 4, (i) the light chain variable region and the heavy chain variable region are humanized, optionally the light chain variable region of (a) is humanized using an IGKV4-1 or IGKV1-9 framework as the receptor sequence of the framework region, but optionally one or more amino acid residues of the receptor framework sequence are substituted with one or more corresponding residues of the donor framework sequence, preferably the receptor framework sequence is derived from IGKV4-1 and / or; (ii) the light chain variable region of (a) comprises a sequence selected from one of SEQ NOs 11 to 14 and SEQ NOs 3 to 9, preferably one of SEQ NOs 11 to 14 and / or; (iii) the heavy chain variable region of (b) is humanized using an IGHV3-72 framework as the receptor sequence of the framework region, but optionally one or more amino acid residues of the receptor framework sequence are substituted with one or more corresponding residues of the donor framework sequence and / or; (iv) the heavy chain variable region of (b) is derived from any one of SEQ NOs 17 to 22 An antibody or antigen-binding fragment comprising a selected sequence and / or; (v) at least one antigen-binding site comprising a light chain variable region sequence selected from any one of SEQ NOs 11 to 14 and SEQ NOs 3 to 9 and a heavy chain variable region sequence selected from any one of SEQ NOs 17 to 22, preferably the light chain variable region is selected from any one of SEQ NOs 11 to 14. Claim 6 In any one of claims 1 to 5, at least one antigen binding site comprises: (i) a light chain variable region having the sequence of SEQ ID NO. 9 and a heavy chain variable region having the sequence of SEQ ID NO. 22; or (ii) a light chain variable region having the sequence of SEQ ID NO. 13 and a heavy chain variable region having the sequence of SEQ ID NO. 22; or (iii) a light chain variable region having the sequence of SEQ ID NO. 14 and a heavy chain variable region having the sequence of SEQ ID NO. 22; or (iv) a light chain variable region sequence of SEQ ID NO. 11 and a heavy chain variable region sequence selected from any one of SEQ ID NO. 17 to 22; or (v) a light chain variable region sequence of SEQ ID NO. 12 and a heavy chain variable region sequence selected from any one of SEQ ID NO. 17 to 22; or (vi) a light chain variable region sequence of SEQ ID NO. 13 and a heavy chain variable region sequence selected from any one of SEQ ID NO. 17 to 22; or (vii) a light chain variable region sequence of SEQ ID NO. 14 and a heavy chain variable region sequence selected from any one of SEQ ID NOs 17 to 22; or (viii) a light chain variable region sequence of SEQ ID NO. 3 and a heavy chain variable region sequence selected from any one of SEQ ID NOs 17 to 22; or (ix) a light chain variable region sequence of SEQ ID NO. 4 and a heavy chain variable region sequence selected from any one of SEQ ID NOs 17 to 22; or (x) a light chain variable region sequence of SEQ ID NO. 5 and a heavy chain variable region sequence selected from any one of SEQ ID NOs 17 to 22; or (xi) a light chain variable region sequence of SEQ ID NO. 6 and a heavy chain variable region sequence selected from any one of SEQ ID NOs 17 to 22; or (xii) a light chain variable region sequence of SEQ ID NO. 7 and a heavy chain variable region sequence selected from any one of SEQ ID NOs 17 to 22; or (xiii) a light chain variable region sequence of SEQ ID NO. 8 and a heavy chain variable region sequence selected from any one of SEQ ID NOs. 17 to 22;or (xiv) an antibody or antigen-binding fragment comprising a light chain variable region sequence of SEQ ID NO. 9 and a heavy chain variable region sequence selected from any one of SEQ ID NOs. 17 to 22, wherein preferably, at least one antigen-binding site comprises (i) a light chain variable region having the sequence of SEQ ID NO. 9 and a heavy chain variable region having the sequence of SEQ ID NO. 22; or (ii) a light chain variable region having the sequence of SEQ ID NO. 13 and a heavy chain variable region having the sequence of SEQ ID NO. 22; or (iii) a light chain variable region having the sequence of SEQ ID NO. 14 and a heavy chain variable region having the sequence of SEQ ID NO. 2.; Claim 7 An 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, and preferably the antibody comprises a heavy chain having the sequence of SEQ ID NO.

147. Claim 8 (a) a light chain variable region comprising CDR1 having the sequence of SEQ ID NO. 94, CDR2 having the sequence of SEQ ID NO. 95, and CDR3 having the sequence of SEQ ID NO. 96; and (b) an antibody or antigen-binding fragment thereof comprising at least one variable domain specific to CD45, comprising a heavy chain variable region including a CDR1 comprising the sequence of SEQ NO. 100, a CDR2 comprising the sequence of SEQ NO. 101, and a CDR3 comprising a sequence selected from any one of SEQ NOs 102 to 105, wherein optionally (i) the light chain variable region of (a) comprises a CDR1 comprising the sequence of SEQ NO. 94, a CDR2 comprising the sequence of SEQ NO. 95, and a CDR3 comprising the sequence of SEQ NO. 96, and the heavy chain variable region of (b) comprises a CDR1 comprising the sequence of SEQ NO. 100, a CDR2 comprising the sequence of SEQ NO. 101, and a CDR3 comprising the sequence of SEQ NO. 102; or (ii) the light chain variable region of (a) comprises a CDR1 comprising the sequence of SEQ NO. 94, a CDR2 comprising the sequence of SEQ NO. 95, and a CDR3 comprising the sequence of SEQ NO.

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

105. Claim 10 An antibody or antigen-binding fragment, wherein (i) the light chain variable region and the heavy chain variable region are humanized, optionally the light chain variable region of (a) is humanized using the IGKV1-8 framework as the receptor sequence of the framework region, optionally one or more amino acid residues of the receptor framework sequence are substituted with one or more corresponding residues of the donor framework sequence; (ii) the light chain variable region of (a) comprises a sequence selected from SEQ ID NOs 25 and 26 and / or; (iii) the heavy chain variable region of (b) is humanized using the IGHV4-4 framework as the receptor sequence of the framework region, optionally one or more amino acid residues of the receptor framework sequence are substituted with one or more corresponding residues of the donor framework sequence; and (iv) the heavy chain variable region of (b) comprises a sequence selected from any one of SEQ ID NOs 29 to 32. Claim 11 An antibody or antigen-binding fragment according to any one of claims 8 to 10, comprising a light chain variable region sequence selected from SEQ ID NOs 25 and 26 and a heavy chain variable region sequence selected from any one of SEQ ID NOs 29 to 32. Claim 12 In claim 11, 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; (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) comprising the light chain variable region sequence of SEQ No. 26 and the heavy chain variable region sequence of SEQ No. 31, preferably the antibody comprises (i) the light chain variable region sequence of SEQ No. 25 and the heavy chain variable region sequence of SEQ No. 29; or (ii) the light chain variable region sequence of SEQ No. 25 and the heavy chain variable region sequence of SEQ No. 32, an antibody or antigen binding fragment. Claim 13 An 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. Claim 14 In any one of claims 1 to 13, the antibody or its antigen-binding fragment is (i) monospecific to CD45; or (ii) the antibody or its antigen-binding fragment is bipatopic to CD45. Claim 15 An antibody or antigen-binding fragment according to claim 14, which is biparatopic to CD45 and comprises 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. Claim 16 In claim 15, an antibody or antigen-binding fragment that is biparatopic to CD45 and has a set of CDRs of one of the following specificity pairs: (a) 17415gL7gH6 x 17552gL1gH1 biparatopic (the CDRs for 17415gL7gH6 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 52, 55, and 48, respectively, and the CDRs for 17552gL1gH1 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 100, 101, and 102, respectively); or (b) 17415gL7gH6 x 17552gL1gH4 biparatopic (the CDRs for 17415gL7gH6 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 52, 55, and 48, respectively, and the CDRs for 17552gL1gH4 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 100, 101, and 105, respectively); or (c) 17415gL15gH6 x 17552gL1gH1 double paratopic (the CDRs for 17415gL15gH6 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 52, 55, and 48, respectively, and the CDRs for 17552gL1gH1 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 100, 101, and 102, respectively);or (d) 17415gL15gH6 x 17552gL1gH4 biparatopic (the CDRs for 17415gL15gH6 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 52, 55, and 48, respectively, and the CDRs for 17552gL1gH4 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 100, 101, and 105, respectively); or (e) 17415gL16gH6 x 17552gL1gH1 double paratopic (the CDRs for 17415gL16gH6 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 52, 55, and 48, respectively, and the CDRs for 17552gL1gH1 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 100, 101, and 102, respectively); or (f) 17415gL16gH6 x 17552gL1gH4 biparatopic (the CDRs for 17415gL16gH6 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 52, 55, and 48, respectively, and the CDRs for 17552gL1gH4 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 100, 101, and 105, respectively);or (g) 17415gL7gH6 x 17552gL1gH1 biparatopic (the CDRs for 17415gL7gH6 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 52, 55, and 48, respectively, and the CDRs for 17552gL1gH1 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 100, 101, and 102, respectively); or (h) 17415gL7gH6 x 17552gL1gH4 biparatopic (the CDRs for 17415gL7gH6 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 52, 55, and 48, respectively, and the CDRs for 17552gL1gH4 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 100, 101, and 105, respectively); or (i) 17415gL15gH6 x 17552gL1gH1 double paratopic (the CDRs for 17415gL15gH6 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 52, 55, and 48, respectively, and the CDRs for 17552gL1gH1 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 100, 101, and 102, respectively);or (j) 17415gL15gH6 x 17552gL1gH4 biparatopic (the CDRs for 17415gL15gH6 specificity are LCDR1, LCDR2, and LCDR3 of sequence numbers 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of sequence numbers 52, 55, and 48, respectively, and the CDRs for 17552gL1gH1 specificity are LCDR1, LCDR2, and LCDR3 of sequence numbers 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of sequence numbers 100, 101, and 105, respectively); or (k) 17415gL16gH6 x 17552gL1gH1 double paratopic (the CDRs for 17416gL16gH6 specificity are LCDR1, LCDR2, and LCDR3 of sequence numbers 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of sequence numbers 52, 55, and 48, respectively, and the CDRs for 17552gL1gH1 specificity are LCDR1, LCDR2, and LCDR3 of sequence numbers 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of sequence numbers 100, 101, and 102, respectively); or (l) 17415gL16gH6 x 17552gL1gH4 biparatopic (CDRs for 17416gL16gH6 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 33, 34, and 39, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 52, 55, and 48, respectively, and CDRs for 17552gL1gH4 specificity are LCDR1, LCDR2, and LCDR3 of SEQ ID NOs 94, 95, and 96, respectively, and HCDR1, HCDR2, and HCDR3 of SEQ ID NOs 100, 101, and 105, respectively).; Claim 17 In claim 16, an antibody or antigen-binding fragment that is biparatopic to CD45 and has a pair of light chain variable region and heavy chain variable region for each of the following specificities: (a) 17415gL7gH6 x 17552gL1gH1 biparatopic (the light chain variable region and heavy chain variable region for the 17415gL7gH6 specificity have the sequences of SEQ ID NOs. 9 and 22, respectively, and the light chain variable region and heavy chain variable region for the 17552gL1gH1 specificity have the sequences of SEQ ID NOs. 25 and 29, respectively); (b) 17415gL7gH6 x 17552gL1gH4 biparatopic (the light chain variable region and heavy chain variable region for 17415gL7gH6 specificity have the sequences of SEQ No. 9 and 22, respectively, and the light chain variable region and heavy chain variable region for 17552gL1gH4 specificity have the sequences of SEQ No. 25 and 32, respectively); (c) 17415gL15gH6 x 17552gL1gH1 biparatopic (the light chain variable region and heavy chain variable region for 17415gL15gH6 specificity have the sequences of SEQ No. 13 and 22, respectively, and the light chain variable region and heavy chain variable region for 17552gL1gH1 specificity have the sequences of SEQ No. 25 and 29, respectively); (d) 17415gL15gH6 x 17552gL1gH4 biparatopic (the light chain variable region and heavy chain variable region for 17415gL15gH6 specificity have the sequences of SEQ No. 13 and 22, respectively, and the light chain variable region and heavy chain variable region for 17552gL1gH4 specificity have the sequences of SEQ No. 25 and 32, respectively); (e) 17415gL16gH6 x 17552gL1gH1 biparatopic (the light chain variable region and heavy chain variable region for 17415gL16gH6 specificity have the sequences of SEQ No. 14 and 22, respectively, and the light chain variable region and heavy chain variable region for 17552gL1gH1 specificity have the sequences of SEQ No. 25 and 29, respectively);(f) 17415gL16gH6 x 17552gL1gH4 biparatopic (the light chain variable region and heavy chain variable region for 17415gL16gH6 specificity have the sequences of SEQ No. 14 and 22, respectively, and the light chain variable region and heavy chain variable region for 17552gL1gH1 specificity have the sequences of SEQ No. 25 and 32, respectively); (g) 17415gL7gH6 x 17552gL1gH1 biparatopic (the light chain variable region and heavy chain variable region for 17415gL7gH6 specificity have the sequences of SEQ No. 9 and 22, respectively, and the light chain variable region and heavy chain variable region for 17552gL1gH1 specificity have the sequences of SEQ No. 25 and 29, respectively); (h) 17415gL7gH6 x 17552gL1gH4 biparatopic (the light chain variable region and heavy chain variable region for 17415gL7gH6 specificity have the sequences of SEQ No. 9 and 22, respectively, and the light chain variable region and heavy chain variable region for 17552gL1gH4 specificity have the sequences of SEQ No. 25 and 32, respectively); (i) 17415gL15gH6 x 17552gL1gH1 biparatopic (the light chain variable region and heavy chain variable region for 17415gL15gH6 specificity have the sequences of SEQ No. 13 and 22, respectively, and the light chain variable region and heavy chain variable region for 17552gL1gH1 specificity have the sequences of SEQ No. 25 and 29, respectively); (j) 17415gL15gH6 x 17552gL1gH4 biparatopic (the light chain variable region and heavy chain variable region for 17415gL15gH6 specificity have the sequences of SEQ Nos. 13 and 22, respectively, and the light chain variable region and heavy chain variable region for 17552gL1gH1 specificity have the sequences of SEQ Nos. 25 and 32, respectively);(k) 17415gL16gH6 x 17552gL1gH1 biparatopic (the light chain variable region and heavy chain variable region for 17415gL16gH6 specificity have the sequences of SEQ No. 14 and 22, respectively, and the light chain variable region and heavy chain variable region for 17552gL1gH1 specificity have the sequences of SEQ No. 25 and 29, respectively); (l) 17415gL16gH6 x 17552gL1gH4 biparatopic (the light chain variable region and heavy chain variable region for 17415gL16gH6 specificity have the sequences of SEQ No. 14 and 22, respectively, and the light chain variable region and heavy chain variable region for 17552gL1gH1 specificity have the sequences of SEQ No. 25 and 32, respectively).; Claim 18 An antibody or antigen-binding fragment according to any one of claims 1 to 17, wherein the invariant region of the antibody or its antigen-binding fragment comprises a modification or modifications for reducing or eliminating binding to an Fc receptor. Claim 19 An antibody or antigen-binding fragment wherein the constant region of the antibody comprises a 234A and 235A heavy chain constant region modification according to the EU numbering system, optionally (i) the constant region is an IgG1 constant region and the modification is a LALA double mutation of the constant region; or (ii) the constant region is an IgG4 constant region and the modification is a FALA double mutation of the constant region. Claim 20 An antibody or antigen-binding fragment that is a biparatopic antibody comprising (i) a heavy chain constant region having the sequence of SEQ ID NO. 146 and a second heavy chain constant region having the sequence of SEQ ID NO. 148; and (ii) a light chain constant region having the sequence of SEQ ID NO.

145. Claim 21 In any one of claims 1 to 20, (i) the antibody is VR17415gL15gH6 x VR17552gL1gH4 IgG1 LALA, wherein the heavy chain and light chain sequences of the VR17415gL15gH6 portion of the double paratopic comprise the sequences of SEQ ID NOs 147 and 141, and the heavy chain and light chain sequences of the VR17552gL1gH4 portion of the double paratopic comprise the sequences of SEQ ID NOs 142 and 143; and / or (ii) the antibody or the antigen-binding fragment thereof is an antibody or antigen-binding fragment capable of specifically binding to both human CD45 and cynomolgus monkey CD45. Claim 22 A monospecific antibody specific to CD45 comprising a heavy chain containing the sequence of SEQ ID NO. 140 and a light chain containing the sequence of SEQ ID NO.

141. Claim 23 A biparatopic antibody comprising: (a) a heavy chain comprising the sequence of SEQ ID NO. 147 and a light chain comprising the sequence of SEQ ID NO. 141, providing a first specificity for CD45; and (b) a heavy chain comprising the sequence of SEQ ID NO. 142 and a light chain comprising the sequence of SEQ ID NO. 143, providing a first specificity for CD45. Claim 24 Nucleic acid molecules or molecules encoding an antibody or an antigen-binding fragment thereof as defined in any one of claims 1 to 23. Claim 25 A vector or vectors comprising an antibody coding for any one of claims 1 to 23, or a nucleic acid molecule or molecules according to claim 24. Claim 26 A pharmaceutical composition comprising: (a) an antibody according to any one of claims 1 to 23, a nucleic acid molecule or molecules according to claim 24, or a vector or vectors according to claim 25; and (b) a pharmaceutically acceptable carrier or diluent. Claim 27 In paragraph 26, a pharmaceutical composition for use in a treatment method. Claim 28 A pharmaceutical composition for use in the method of claim 27, further comprising: (i) a method of killing or depleting CD45-expressing cells in a subject; (ii) a method of treating a blood cancer, e.g., leukemia, lymphoma, or multiple myeloma, or an autoimmune disease, e.g., multiple sclerosis or scleroderma; and / or (iii) a step of delivering cells to a subject after cell depletion. Claim 29 A method for killing or depleting CD45-expressing cells in a subject, comprising the step of administering a pharmaceutical composition according to claim 26 to the subject. Claim 30 In paragraph 29, the method is (i) intended to treat a blood cancer, e.g., leukemia, lymphoma, or multiple myeloma, or an autoimmune disease, e.g., multiple sclerosis or scleroderma; and (ii) further comprises the step of delivering cells to a subject after cell killing or depletion. Claim 31 The use of an antibody or its antigen-binding fragment according to any one of claims 1 to 23, a nucleic acid molecule or molecules according to claim 24, or a vector or vectors according to claim 25 in the manufacture of a drug for killing or depleting CD45-expressing cells in a subject. Claim 32 In paragraph 31, the medicine is (i) intended to treat a blood cancer, e.g., leukemia, lymphoma, multiple myeloma, or an autoimmune disease, e.g., multiple sclerosis or scleroderma; and / or (ii) intended to be used in a method further comprising the step of delivering cells to a subject after cell killing or depletion. Claim 33 An in vitro method for depleting or killing target cells expressing CD45 in a cell population, tissue, or organ, comprising the step of contacting said cell, tissue, or organ with an antibody or antigen-binding fragment according to any one of claims 1 to 23. Claim 34 An antibody for use in a method for treating or preventing graft-versus-host disease (GVHD) in a subject according to any one of claims 1 to 23, wherein the method comprises: (a) contacting a cell population, tissue, or organ in vitro with the antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof to kill target cells expressing CD45; and (b) transplanting the treated cell population, tissue, or organ into said subject. Claim 35 A method for treating or preventing graft-versus-host disease (GVHD), comprising: (a) contacting a cell population, tissue, or organ with an antibody according to any one of claims 1 to 23 or an antigen-binding fragment thereof to kill target cells expressing CD45 in vitro; and (b) transplanting the treated cell population, tissue, or organ to a subject requiring such transplantation. Claim 36 (a) contacting a cell population, tissue, or organ with an antibody according to any one of claims 1 to 38 or an antigen-binding fragment thereof to kill target cells expressing CD45 in vitro; and (b) transplanting the treated cell population, tissue, or organ to a subject requiring such transplantation, wherein the use of an antibody according to any one of claims 1 to 23 in the manufacture of a drug for the treatment or prevention of graft-versus-host disease (GVHD).

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