Human antibody specifically binding to human SLAMF1 protein and Use thereof

Abstract

The present invention relates to a human antibody that specifically binds to the human SLAMF1 protein and its uses. The antibody or its antigen-binding fragment of the present invention enables the provision of a therapeutic technology capable of completely and permanently eliminating the inflammatory response of GvHD by targeting allograft-specifically activated T cells in GvHD patients. Furthermore, it can provide new therapeutic opportunities for treating steroid-refractory acute GvHD patients. Moreover, after verifying the potential of SLAMF1 expression as a biomarker capable of predicting GvHD induction, personalized treatment for patients can be anticipated.

Description

Technology Field

[0001] The present invention relates to a human antibody that specifically binds to the human SLAMF1 protein and the use thereof. Background Technology

[0003] During the process of hematopoietic stem cell transplantation (HSCT), there is a risk of developing graft-versus-host disease (GvHD) as the donor's immune cells recognize and attack the recipient's cells as foreign antigens. GvHD is a highly fatal disease and is the leading cause of death after hematopoietic stem cell transplantation, and the number of hematopoietic stem cell transplants and the resulting GvHD cases are on the rise every year.

[0004] Currently, for the treatment of GvHD, corticosteroids, anti-thymocyte globulin (ATG) therapy, IL-2Rα inhibitors, TNF inhibitors, calcineurin inhibitors (e.g., cyclosporin A (CsA) and tacrolimus (FK506)), and mTOR inhibitors (sirolimus and rapamycin) are used. Although high-dose corticosteroids such as prednisone are most commonly used as first-line therapy, none have yet been established as a standard treatment, and most of these are therapies developed to target other diseases.

[0005] In the treatment of GvHD, the greatest unmet medical need is to completely and permanently eliminate the inflammatory response associated with GvHD. However, to date, there are no agents available that completely eliminate inflammation in GvHD patients, and most treatments have limitations due to their short duration of action. Furthermore, there is a lack of treatment options for patients with steroid-refractory acute GvHD.

[0006] Therefore, the development of new treatments to overcome these problems is urgent. Prior art literature

[0008] Korean Published Patent No. 10-2022-0121793 (Publication Date: September 1, 2022) The problem to be solved

[0009] The inventors have made diligent research efforts to develop a fundamental GvHD treatment capable of permanently eliminating the inflammatory response of GvHD by targeting T cells specifically activated in allografts. As a result, the present invention was completed by developing an antibody treatment against a novel immunomodulatory substance called SLAMF1, whose function in T cells was previously unknown, and by identifying that it possesses excellent T cell activation inhibitory ability.

[0010] Accordingly, the object of the present invention is to provide an antibody or its antigen-binding fragment that specifically binds to SLAMF1.

[0011] Another object of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding the antibody or its antigen-binding fragment.

[0012] Another objective of the present invention is to provide a recombinant vector comprising the nucleic acid molecule.

[0013] Another objective of the present invention is to provide a host cell comprising the recombinant vector.

[0014] In addition, another objective of the present invention is to provide a pharmaceutical composition for treating GvHD (Graft-versus-host disease) comprising the antibody or antigen-binding fragment as an active ingredient.

[0015] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings. means of solving the problem

[0017] According to one aspect of the present invention, the present invention provides an antibody or its antigen-binding fragment that specifically binds to SLAMF1, comprising the following:

[0018] (a) a heavy chain variable region of CDR-H1 having the amino acid sequence of SEQ ID NO. 1, CDR-H2 having the amino acid sequence of SEQ ID NO. 2, and CDR-H3 having the amino acid sequence of SEQ ID NO. 3; and an antibody or its antigen-binding fragment having a light chain variable region of CDR-L1 having the amino acid sequence of SEQ ID NO. 4, CDR-L2 having the amino acid sequence of SEQ ID NO. 5, and CDR-L3 having the amino acid sequence of SEQ ID NO. 6;

[0019] (b) an antibody or its antigen-binding fragment comprising a heavy chain variable region of CDR-H1 having the amino acid sequence of SEQ ID NO. 7, CDR-H2 having the amino acid sequence of SEQ ID NO. 8, and CDR-H3 having the amino acid sequence of SEQ ID NO. 9; and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO. 10, CDR-L2 having the amino acid sequence of SEQ ID NO. 11, and CDR-L3 having the amino acid sequence of SEQ ID NO. 12;

[0020] (c) an antibody or its antigen-binding fragment comprising a heavy chain variable region of CDR-H1 having the amino acid sequence of SEQ ID NO. 13, CDR-H2 having the amino acid sequence of SEQ ID NO. 14, and CDR-H3 having the amino acid sequence of SEQ ID NO. 15; and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO. 16, CDR-L2 having the amino acid sequence of SEQ ID NO. 17, and CDR-L3 having the amino acid sequence of SEQ ID NO. 18;

[0021] (d) a heavy chain variable region of CDR-H1 having the amino acid sequence of SEQ ID NO. 19, CDR-H2 having the amino acid sequence of SEQ ID NO. 20, and CDR-H3 having the amino acid sequence of SEQ ID NO. 21; and an antibody or its antigen-binding fragment having a light chain variable region of CDR-L1 having the amino acid sequence of SEQ ID NO. 22, CDR-L2 having the amino acid sequence of SEQ ID NO. 23, and CDR-L3 having the amino acid sequence of SEQ ID NO. 24;

[0022] (e) an antibody or its antigen-binding fragment comprising a heavy chain variable region of CDR-H1 having the amino acid sequence of SEQ ID NO. 25, CDR-H2 having the amino acid sequence of SEQ ID NO. 26, and CDR-H3 having the amino acid sequence of SEQ ID NO. 27; and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO. 28, CDR-L2 having the amino acid sequence of SEQ ID NO. 29, and CDR-L3 having the amino acid sequence of SEQ ID NO. 30; or

[0023] (f) a heavy chain variable region of CDR-H1 having the amino acid sequence of SEQ ID NO. 31, CDR-H2 having the amino acid sequence of SEQ ID NO. 32, and CDR-H3 having the amino acid sequence of SEQ ID NO. 33; and a light chain variable region of CDR-L1 having the amino acid sequence of SEQ ID NO. 34, CDR-L2 having the amino acid sequence of SEQ ID NO. 35, and CDR-L3 having the amino acid sequence of SEQ ID NO. 36, comprising an antibody or its antigen-binding fragment.

[0024] In one embodiment of the present invention, the antibody or its antigen-binding fragment comprises: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 37; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 38; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 39; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 40; (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 41; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 42; (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 43; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 44; (e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 45; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 46; or (f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 47; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 48.

[0025] As used in this specification, the term "SLAMF1" is an abbreviation for signaling lymphocytic activation molecule family member 1, referring to a protein located primarily on the surface of lymphocytes in the immune system that plays an important role in the intercellular interactions and signaling processes of the immune system. Specifically, SLAMF1 is known to act as a co-stimulatory molecule that promotes intercellular interactions, thereby aiding in the activation and proliferation of lymphocytes. It also plays an important role in cell survival, differentiation, and memory cell formation in the immune system by regulating immune responses through the activation of intracellular signaling pathways. In particular, since the function of SLAMF1 is closely related to the immune system's response to autoimmune diseases, infectious diseases, and tumors, SLAMF1 is considered an important target not only for the regulation of immune responses but also in research regarding the treatment or prevention of autoimmune diseases and vaccine development.

[0026] Meanwhile, the aforementioned SLAMF1 protein is a protein expressed on the cell surface and is characterized by being primarily expressed only in lymphoid tissues such as the spleen, bone marrow, and thymus. As mentioned above, the SLAMF1 protein plays an important role in immune responses and is mainly expressed in immune cells such as T cells, plasma cells, and B cells.

[0027] In this regard, Graft-versus-host disease (GvHD), a unique inflammatory disease caused by allogeneic transplantation in hematopoietic stem cell transplantation, is characterized by the expression of the donor T cells used in the allogeneic transplantation as they recognize the recipient's tissue as an external antigen and become activated.

[0028] Accordingly, in one embodiment of the present invention, the antibody or antigen-binding fragment targets T cells that are specifically activated for allografts.

[0029] As used herein, the term "antibody" refers to an antibody specific to SLAMF1, comprising not only the complete antibody form but also the antigen-binding fragment of the antibody molecule.

[0030] A complete antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain being connected to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2) subclasses. Meanwhile, the invariant region of the above light chain has kappa (κ) and lambda (λ) types (Cellular and Molecular Immunology, Wonsiewicz, MJ, Ed., Chapter 45, pp. 41-50, WB Saunders Co. Philadelphia, PA (1991); Nisonoff, A., Introduction to Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, Sinauer Associates, Inc., Sunderland, MA (1984)).

[0031] As used herein, the term “antigen-binding fragment” refers to a fragment possessing an antigen-binding function and includes Fab, F(ab'), F(ab')2, chemically linked F(ab')2 and Fv, etc. Among the antibody fragments, Fab has a structure having a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab’ differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. The F(ab')2 antibody is produced when the cysteine ​​residues in the hinge region of Fab’ form a disulfide bond. Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and recombinant technology for generating Fv fragments is disclosed in PCT international published patent applications WO 88 / 10649, WO 88 / 106630, WO 88 / 07085, WO 88 / 07086, and WO 88 / 09344. In two-chain Fv, the heavy chain variable region and the light chain variable region are connected by non-covalent bonds, and in single-chain Fv, the variable region of the heavy chain and the variable region of the single chain are generally connected by covalent bonds through a peptide linker or directly at the C-terminus, so they can form a dimer-like structure like two-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restricting the whole antibody with papain and F(ab')2 fragment can be obtained by cleaving it with pepsin), or they can be produced through genetic recombination technology.

[0032] In the present invention, the antibody is specifically in the scFv form or in the complete antibody form. Additionally, the heavy chain constant region may be selected from any one isotype of gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). Preferably, the constant region is gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), and gamma 4 (IgG4), and most specifically, may be the gamma 1 (IgG1) isotype. The light chain constant region may be in the kappa (κ) or lambda (λ) form.

[0033] In one embodiment of the present invention, the antibody or its antigen-binding fragment may be a monoclonal antibody, polyclonal antibody, scFv, Fab, F(ab), F(ab)2, scFv-Fc, minibody, diabody, tribody, tetrabody, bispecific antibody, multispecific antibody, human antibody, humanized antibody, chimeric antibody, or an antigen-binding fragment thereof, comprising the heavy chain variable region and the light chain variable region.

[0034] As used herein, the term “heavy chain” refers to both the full-length heavy chain and fragments thereof, comprising a variable region domain VH and three constant region domains CH1, CH2, and CH3, which have an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen. Additionally, as used herein, the term “light chain” refers to both the full-length light chain and fragments thereof, comprising a variable region domain VL and a constant region domain CL, which have an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen.

[0035] As used herein, the term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chains (CDR-H1, CDR-H2, and CDR-H3) and light chains (CDR-L1, CDR-L2, and CDR-L3) each contain three CDRs. CDRs provide major contact residues for the antibody to bind to an antigen or epitope.

[0036] In this specification, the antibody or its antigen-binding fragment includes not only the full-length or original polyclonal or monoclonal antibody, but also its antigen-binding fragments (e.g., Fab, Fab′, F(ab′)2, Fab3, Fv and variants thereof), fusion proteins comprising one or more antibody portions, human antibodies, humanized antibodies, chimeric antibodies, minibodies, diabodies, tribodies, tetrabodies, linear antibodies, monochain antibodies (scFv), scFv-Fc, bispecific antibodies (bispecific antibodies), multispecific antibodies (multispecific antibodies), and other modified arrangement forms of immunoglobulin molecules comprising antigen recognition sites of the required specificity, such as glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Specific examples of modified antibodies and their antigen-binding fragments include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engagers), TandAbs (tandem diabodies), DAFs (dual acting Fabs), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), duobodies, and triomAbs. The list of such antibodies and their antigen-binding fragments is not limited to the above.

[0037] Specific examples of the above-mentioned modified antibodies and their antigen-binding fragments may include nanobodies, AlbudAbs, DARTs (dual affinity re-targeting), BiTEs (bispecific T-cell engager), TandAbs (tandem diabodies), DAFs (dual acting Fab), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), FynomAbs (fynomers fused to antibodies), DVD-Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), duobodies, and triomAbs, but the list of such antibodies and their antigen-binding fragments is not limited to the above.

[0038] As used herein, the terms "Framework" or "FR" refer to variable domain residues other than hypervariable region (HVR) residues. The FR of the variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, HVR and FR sequences generally appear in the following order in VH (or VL / Vk):

[0039] (a) FRH1 (Framework region 1 of Heavy chain)-CDRH1 (complementarity determining region 1 of Heavy chain)-FRH2-CDRH2-FRH3-CDRH3-FRH4; and

[0040] (b) FRL1 (Framework region 1 of Light chain)-CDRL1 (complementarity determining region 1 of Light chain)-FRL2-CDRL2-FRL3-CDRL3-FRL4.

[0041] As used herein, the terms “variable region” or “variable domain” refer to a domain of the antibody heavy chain or light chain associated with binding the antibody to an antigen. The variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) generally have similar structures, and each domain comprises four conserved framework regions (FR) and three hypervariable regions (HVR) (Kindt et al. , Kuby Immunology, 6th edition, WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Additionally, antibodies that bind to a specific antigen may be separated using the VH or VL domain from antibodies that bind to the antigen and screen a library of VL or VH domains that are complementary to the antigen.

[0042] The antibody of the present invention can be generated using various phage display methods known in the art, and [Brinkman et al. , 1995, J. Immunol. Methods, 182:41-50]; [Ames et al. , 1995, J. Immunol. Methods, 184, 177-186]; [Kettleborough et al. , 1994, Eur. J. Immunol, 24, 952-958]; [Persic et al. , 1997, Gene, 187, 9-18]; and [Burton et al., 1994, Adv. Immunol., 57, 191-280]; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; and WO 95 / 20401; and US 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; Includes those posted in 5,733,743; and 5,969,108.

[0043] As used herein, the term "specifically binds" or similar means that an antibody or its antigen-binding fragment, or other components such as scFvs, form a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1 x 10⁻⁶ -6 M or less (e.g., 9 x 10 -7 M, 8 x 10 -7 M, 7 x 10 -7 M, 6 x 10 -7 M, 5 x 10 -7 M, 4 x 10 -7 M, 3 x 10 -7 M, 2 x 10 -7 M, or 1 x 10 -7 M), preferably 1 x 10 -7 M or less (e.g., 9 x 10 -8 M, 8 x 10 -8 M, 7 x 10 -8 M, 6 x 10 -8 M, 5 x 10 -8 M, 4 x 10 -8 M, 3 x 10 -8 M, 2 x 10 -8 M, or 1 x 10 -8 M), more preferably 1 x 10 -8 M or less (e.g., 9 x 10 -9M, 8 x 10 -9 M, 7 x 10 -9 M, 6 x 10 -9 M, 5 x 10 -9 M, 4 x 10 -9 M, 3 x 10 -9 M, 2 x 10 -9 M, or 1 x 10 -9 The equilibrium dissociation constant of M) (e.g., K smaller than this). D It can be characterized as (indicating a tighter bond). Methods for determining whether two molecules specifically bind are well known in the art, including, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0044] As used herein, the term “affinity” refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., antibody) and its binding partner (e.g., antigen). Unless otherwise specified, as used herein, “binding affinity” refers to the intrinsic binding affinity reflecting the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity between molecule Y and its partner Y can generally be expressed by a dissociation constant (Kd). Affinity may be measured by conventional methods known in the art, including those described herein.

[0045] Additionally, as used herein, the term “human antibody” has an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source utilizing a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody excludes humanized antibodies containing non-human antigen-binding residues.

[0046] As used herein, the term “humanized antibody” refers to a chimeric immunoglobulin containing a minimal sequence derived from a non-human (e.g., mouse) antibody, its immunoglobulin chain, or fragment (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibody). In most cases, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the recipient’s complementarity-determining region (CDR) are replaced by residues of the CDR of a non-human species (donor antibody), e.g., mouse, rat, or rabbit, having the desired specificity, affinity, and ability. In some cases, residues of the Fv framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. Additionally, the humanized antibody may contain residues not found in the recipient antibody or in the imported CDR or framework sequence. Such modifications are made to further improve and optimize antibody performance. Generally, the humanized antibody will comprise at least one, and typically two, substantially all variable domains, wherein all or substantially all of the CDR region in the domain corresponds to the CDR region of a non-human immunoglobulin, and all or substantially all of the FR region has the sequence of the FR region of a human immunoglobulin. The humanized antibody comprises at least a portion of the immunoglobulin constant region (Fc region) or a substantial sequence of the human immunoglobulin constant region (Fc region).

[0047] The anti-SLAMF1 antibody of the present disclosure or its antigen-binding fragment may comprise variants of the amino acid sequence within a range capable of specifically recognizing SLAMF1, as recognized by a person skilled in the art. For example, changes may be made to the amino acid sequence of the antibody to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody.

[0048] The above variants are said to have "substantial similarity," meaning that when two peptide sequences are optimally aligned, such as by a program GAP or BESTFIT using default gap weights, they share at least about 90% sequence identity, more preferably at least about 95%, 98%, or 99% sequence identity. Preferably, non-identical residue positions differ by conservative amino acid substitutions. The "conservative amino acid substitution" is when an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functionality of the protein. Where two or more amino acid sequences differ from each other due to conservative substitutions, the percentage or degree of similarity may be upregulated to compensate for the conservative nature of the substitution.

[0049] These amino acid variations are based on the relative similarities of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0050] In introducing mutations, the hydropathic index of the amino acids may be considered. Each amino acid is assigned a hydropathic index based on hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0051] The hydrophobic amino acid index is very important in conferring interactive biological functions of proteins. It is a known fact that similar biological activity can be achieved by substituting with amino acids having similar hydrophobic indices. When introducing a variation based on the hydrophobic index, the substitution is preferably made between amino acids exhibiting a difference in hydrophobic index within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0052] Meanwhile, it is also well known that substitution between amino acids having similar hydrophilicity values ​​results in proteins having uniform biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspalate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); Phenylalanine (-2.5); tryptophan (-3.4).

[0053] When introducing a variation by referring to the hydrophilicity value, substitution is made between amino acids that exhibit a difference in hydrophilicity value within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0054] Amino acid exchanges in proteins that do not change the overall activity of the molecule are known in the art (H. Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0055] The antibody specific to SLAMF1 of the present invention or its antigen-binding fragment comprises an antibody or its antigen-binding fragment comprising a slight change to the amino acid sequence described above, that is, a modification that has little effect on the tertiary structure and the function of the antibody. Accordingly, in some embodiments, even if it does not match the sequence described above, it may have at least 93%, 95%, 96%, 97%, or 98% or more similarity, or 100% similarity.

[0056] In one embodiment of the present invention, the SLAMF1-specific antibody or its antigen-binding fragment comprises, but is not limited to, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies (scFv), Fab fragments, F(ab') fragments, disulfide-binding Fvs (sdFV) and anti-idiotype (anti-Id) antibodies comprising a CDR of the sequence described above, and epitope-binding fragments of said antibodies.

[0057] In a specific embodiment of the present invention, the heavy chain variable region and the light chain variable region included in the antibody or its antigen-binding fragment are connected by a linker such as (Gly-Ser)n, (Gly2-Ser)n, (Gly3-Ser)n, or (Gly4-Ser)n. Here, n is an integer from 1 to 6, specifically 3 to 4, but is not limited thereto. The light chain variable region and the heavy chain variable region of the scFv may exist, for example, in the following orientation: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.

[0058] In one embodiment of the present invention, the antibody or antigen-binding fragment that specifically binds to SLAMF1 of the present invention is represented as SLAMF1-b2-1G5(#6), SLAMF1-b2-1G12(#8), SLAMF1-b3-1E10(#11), SLAMF1-b3-1C12(#13), SLAMF1-b3-1G3(#15), and SLAMF1-b4-1B2s(#21).

[0060] According to another aspect of the present invention, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the aforementioned antibody or its antigen-binding fragment.

[0061] As used herein, the term "nucleic acid molecule" has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified (Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584(1990)).

[0062] It is obvious to those skilled in the art that the nucleotide sequence encoding the antibody or its antigen-binding fragment of the present invention is sufficient to be a nucleotide sequence encoding an amino acid sequence constituting the antibody or its antigen-binding fragment, and is not limited to any specific nucleotide sequence.

[0063] This is because even if a mutation occurs in the nucleotide sequence, there are cases where expressing the mutated nucleotide sequence as a protein does not lead to a change in the protein sequence. This is called codon degeneracy. Therefore, the nucleotide sequence includes a nucleotide sequence containing functionally equivalent codons or codons coding for the same amino acid (for example, due to codon degeneracy, there are six codons for arginine or serine), or codons coding for biologically equivalent amino acids.

[0064] Considering the variant having the aforementioned biological equivalence activity, the nucleic acid molecule of the present invention encoding the amino acid sequence constituting the antibody or its antigen-binding fragment is interpreted to also include a sequence exhibiting substantial identity therewith. The above substantial identity is, when the sequence of the present invention described above is aligned with any other sequence to correspond as much as possible and the aligned sequence is analyzed using an algorithm commonly used in the art, at least 60% homology (e.g., 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69%), more specifically 70% homology (e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%), even more specifically 80% homology (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%), and even more specifically 90% or more It refers to a sequence exhibiting homology (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%), most specifically, homology of 95% or more (e.g., 95%, 96%, 97%, 98%, or 99%). All integers between 60% and 100% and decimals existing in between are included within the scope of the present invention with respect to % homology.

[0065] Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are Smith and Waterman, Adv. Appl. Math . 2:482 (1981); Needleman and Wunsch, J. Mol. Bio. 48:443 (1970); Pearson and Lipman, Methods in Mol. Biol . 24: 307-31 (1988); Higgins and Sharp, Gene73:237-44 (1988); Higgins and Sharp, CABIOS 5:151-3 (1989); Corpet et al. , Nuc. Acids Res . 16:10881-90(1988); Huang et al. , Comp. Appl. BioSci . 8:155-65(1992) and Pearson et al. , Meth. Mol. Biol It is disclosed in . 24:307-31(1994). NCBI Basic Local Alignment Search Tool(BLAST)(Altschul et al. , J. Mol. Biol . 215:403-10(1990)) is accessible from sources such as the NBCI (National Center for Biological Information) and can be used online in conjunction with sequencing analysis programs such as blastp, blastn, blastx, tblastn, and tblastx. BLAST can be accessed through the BLAST page on the ncbi website. Methods for comparing sequence homology using this program can be found on the BLAST help page of the ncbi website.

[0067] According to another aspect of the present invention, the present invention provides a recombinant vector comprising the nucleic acid molecule described above.

[0068] As used herein, the term "vector" includes, but is not limited to, plasmid vectors, cosmid vectors, and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors as means for expressing an enemy gene in a host cell.

[0069] In one embodiment of the present invention, a nucleic acid molecule encoding a heavy chain variable region and a nucleic acid molecule encoding a light chain variable region in a vector of the present invention are operatively linked with a promoter.

[0070] As used herein, the term "operatively linked" refers to a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, thereby allowing the regulatory sequence to regulate the transcription and / or translation of the other nucleic acid sequence.

[0071] The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are as follows: Sambrook et al. , disclosed in *Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), and incorporated herein by reference.

[0072] The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. Additionally, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0073] For example, when the vector of the present invention is an expression vector and the host is a eukaryotic cell, a promoter derived from the genome of a mammalian cell (e.g., metallothionein promoter, beta-actin promoter, human hemoglobin promoter and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV TK promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Pak virus (EBV) promoter and Rhoese's sarcoma virus (RSV) promoter) may be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0074] The vector of the present invention may be fused with other sequences to facilitate the purification of antibodies expressed therefrom. Examples of fused sequences include glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (Hexahistidine; Qiagen, USA).

[0075] In addition, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified through a protein A column, etc., without the need for an additional sequence for purification.

[0076] Meanwhile, the expression vector of the present invention includes antibiotic resistance genes commonly used in the art as selection markers, for example, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0077] Optionally, the vector may additionally carry a gene encoding a reporter molecule (e.g., luciferase and γ-glucuronidase).

[0078] According to one embodiment of the present invention, the expression vector is a recombinant vector for host cell expression that includes a nucleic acid molecule encoded by the antibody of the present invention or its antigen-binding fragment described above, operatively linked to the nucleotide sequence of the nucleic acid molecule and comprises a promoter that forms an RNA molecule in a host cell and a poly A signal sequence that acts in a host cell to cause polyadenylation of the 3'-terminus of the RNA molecule.

[0080] According to another aspect of the present invention, the present invention provides an isolated host cell comprising the recombinant vector described above.

[0081] Host cells capable of stably and continuously cloning and expressing the vector of the present invention are known in the art, and any host cell may be used, for example, a suitable eukaryotic host cell for the vector is yeast ( Saccharomyce cerevisiae Includes, but is not limited to, insect cells, monkey kidney cells (COS7), NSO cells, SP2 / 0, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, multiple myeloma cell lines, HuT 78 cells and HEK-293 cells.

[0082] In this specification, the terms "transformed," "transduced," or "transfused" refer to the process in which exogenous nucleic acids are delivered or introduced into a host cell. "Transformed," "transduced," or "transfused" cells are cells that have been transformed, transduced, or transfused with exogenous nucleic acids, and said cells include said cells and progeny cells resulting from their passage.

[0083] A method for delivering the vector of the present invention into a host cell is, when the host cell is a eukaryotic cell, a microinjection method (Capecchi, MR, Cell , 22:479(1980)), calcium phosphate precipitation method (Graham, FL et al. , Virology , 52:456(1973)), electroporation (Neumann, E. et al ., EMBO J ., 1:841(1982)), liposome-mediated transfusion (Wong, TK et al ., Gene , 10:87(1980)), DEAE-dextran treatment method(Gopal, Mol. Cell Biol., 5:1188-1190(1985)), and gene bombardment (Yang et al. , Proc. Natl. Acad. Sci. The vector can be injected into a host cell by means of , 87:9568-9572(1990)), etc.

[0084] In the present invention, the recombinant vector injected into a host cell can express the above-mentioned antibody or polypeptide complex recombined within the host cell, and in such cases, a large amount of the antibody or polypeptide complex is obtained. For example, if the expression vector includes a lac promoter, gene expression can be induced by treating the host cell with IPTG.

[0085] The above culture is typically carried out under aerobic conditions, such as by shaking culture or rotation using a rotary machine. The culture temperature is preferably carried out in the range of 10 to 40°C, and the culture time is generally carried out for 5 hours to 7 days. The pH of the medium is preferably maintained in the range of 3.0 to 9.0 during culture. The pH of the medium can be adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc. During culture, if necessary, antibiotics such as ampicillin, streptomycin, chloramphenicol, kanamycin, and tetracycline may be added for the maintenance and expression of the recombinant vector. When culturing host cells transformed with a recombinant expression vector having an induction-capable promoter, an inducer suitable for the medium may be added if necessary. For example, if the expression vector contains a lac promoter, IPTG (isopropyl-beta-D-thiogalactopyranoside) can be added, and if it contains a trp promoter, indoleacrylic acid can be added to the medium.

[0087] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for the treatment of GvHD comprising the aforementioned antibody or its antigen-binding fragment as an active ingredient.

[0088] In one embodiment of the present invention, the composition is characterized by inhibiting T cell activation or removing activated T cells.

[0089] In a specific embodiment of the present invention, the T cells are characterized by expressing SLAMF1 on the cell surface specifically for allogeneic transplantation.

[0090] In addition, in a specific embodiment of the present invention, the inhibition of T cell activation is achieved by reducing T cell proliferation, reducing PD-1 expression, or reducing GzmB expression.

[0091] As used herein, the term "GvHD (Graft-versus-host disease)" or "Graft-versus-host disease" refers to a unique inflammatory disease in allogeneic transplantation that occurs when the donor's immune cells recognize the recipient's cells as foreign antigens and attack them during the process of allogeneic hematopoietic stem cell transplantation (HSCT). This occurs because the immune system can still recognize other differences between tissues even when the HLA of the donor and recipient are identical. GvHD is classically classified into acute and chronic forms. Acute GvHD typically occurs during engraftment up to 100 days after transplantation, and chronic GvHD occurs within 100 days after HSCT. While these two types of GvHD may be further subdivided based on the clinical severity of the disease, the distinction between them may be ambiguous in terms of temporal differentiation or new therapeutic approaches, as both share characteristics.

[0092] Specifically, as described above, "Acute GvHD (aGvHD)" generally occurs within the first three months after transplantation and primarily affects the skin, gastrointestinal tract, and liver. Skin lesions typically consist of macular papular rashes, and in the most severe cases, may cause blisters and ulcers, accompanied by blistering and toxic epidermal necrosis mimicking Stevens-Johnson syndrome. Gastrointestinal symptoms include abdominal cramps and pain, diarrhea, bloody stools, intestinal obstruction, loss of appetite, nausea, and vomiting. Liver disease is caused by damage to the bile canaliculi, leading to cholestasis, consequently hyperbilirubinemia, and elevated alkaline phosphatase (ALP).

[0093] The aforementioned "Chronic GvHD (cGvHD)" can also occur after allogeneic transplantation; although it rarely results in death, it can serve as a major cause of late treatment-related complications. It is typically similar to autoimmune diseases such as systemic sclerosis, and can also lead to the development of fibrosis or scar tissue similar to scleroderma. For example, typical symptoms that may occur in the skin include sclerosis, poikiloderma, and lichen-type lesions, while in the lungs, it involves obstructive bronchioles, which result from damage and obstruction of the bronchioles and can lead to a high mortality rate.

[0094] Currently, methods to prevent, treat, or suppress GvHD involve the use of immunosuppressive drugs such as calcineurin inhibitors (cyclosporine A (CsV) and tacrolimus (FK506)), antiproliferative agents (methotrexate and mycophenolate mofetil), mTOR inhibitors (sirolimus and rapamycin), and steroids such as prednisone. Recently, cyclophosphamide, anti-thymocyte globulin (ATG), extracorporeal photoapheresis, monoclonal antibodies such as rituximab, and kinase inhibitors that inhibit B-cell signaling have been used; however, corticosteroids are still used as a standard treatment because they have non-specific and broad-spectrum immunosuppressive effects, despite substantial side effects of long-term use such as immune system suppression due to high toxicity or infectious diseases caused by tumor recurrence.

[0095] The antibody and its antigen-binding fragment of the present invention are characterized by targeting T cells specifically activated in allogeneic transplant patients in GvHD patients and inhibiting the activation of said T cells.

[0096] Specifically, in an embodiment of the present invention, the therapeutic efficacy of the antibody and its antigen-binding fragment of the present invention was confirmed using a Xenogeneic GvHD mouse model. As a result, it was confirmed that the antibody not only eliminates activated human T cells but also inhibits T cell activation by reducing the expression of PD-1 in said T cells and reducing the expression of GzmB (Granzyme B), which causes cytotoxicity.

[0097] Accordingly, the present invention provides an antibody or its antigen-binding fragment targeting allogeneic transplant-specifically activated T cells in GvHD patients, specifically, an antibody or its antigen-binding fragment that specifically binds to SLAMF1 expressed on the surface of said allogeneic transplant-specifically activated T cells, and provides a pharmaceutical composition containing the same as an active ingredient, thereby presenting a more effective treatment method for GvHD patients.

[0098] Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in formulations and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0099] The pharmaceutical composition of the present invention may be administered orally or parenterally, for example, by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrasternal injection, local administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0100] In one embodiment of the present invention, the administration may be intraperitoneal administration, intravenous administration, intravitreal administration, intrathecal administration, parenteral administration, subcutaneous administration, transdermal administration, or administration by infusion.

[0101] Suitable dosages of the pharmaceutical composition of the present invention vary depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, diet, time of administration, route of administration, rate of excretion, and responsiveness, and a physician of ordinary skill can easily determine and prescribe a dosage effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg. As specified herein, the term "pharmaceutical effective dose" means an amount sufficient to prevent or treat the aforementioned disease.

[0102] In this specification, the term "prevention" means the prevention or protective treatment of a disease or diseased state. In this specification, the term "treatment" means the reduction, suppression, soothing, or eradication of a diseased state.

[0103] The pharmaceutical composition of the present invention may be prepared in a unit volume form or contained in a multi-volume container by formulation using a pharmaceutically acceptable carrier and / or excipient, according to a method that can be easily carried out by a person skilled in the art to which the invention belongs. In this case, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0104] Since the inventions according to the other embodiments of the present invention described above share common major configurations or technologies with the inventions described above, any overlapping content between the two inventions can be applied equally. Effects of the invention

[0106] The antibody or its antigen-binding fragment of the present invention enables the provision of a therapeutic technology capable of completely and permanently eliminating the inflammatory response to GvHD by targeting allograft-specifically activated T cells in GvHD patients. Furthermore, it can provide a new therapeutic opportunity to treat steroid-refractory acute GvHD patients. Moreover, after verifying the potential of SLAMF1 expression as a biomarker capable of predicting GvHD induction, personalized patient treatment can be anticipated. Brief explanation of the drawing

[0108] Figure 1 shows the screening results of the anti-human SLAMF1 effective antibody. Figure 2 shows the binding affinity (K) of the SLAMF1 effective antibody. D Displays the measurement result of value. Figure 3 shows the results of the analysis of interspecies cross-reactivity and binding specificity of the SLAMF1 effective antibody. Figure 4 shows the results of the thermal stability analysis of the SLAMF1 effective antibody. Figure 5 shows the results of the hydrophobicity analysis of the SLAMF1 effective antibody. Figures 6a and 6b show the results of the pI value analysis of the SLAMF1 effective antibody. Figures 7a and 7b show the anti-SLAMF1 antibody in vitro Displays the results of the validity evaluation. Figure 8 shows the results of the evaluation of T cell activity inhibition ability according to the concentration of anti-SLAMF1 antibody. Figure 9 shows the anti-SLAMF1 antibody in vivo Displays the results of the validity evaluation. Figures 10a to 10c show the results of the mode of action (MoA) analysis and the evaluation of the therapeutic efficacy of the anti-SLAMF1 antibody. Figure 11 is a schematic diagram showing the mechanism of action of the anti-SLAMF1 antibody. Specific details for implementing the invention

[0109] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0111] Examples

[0112] Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.

[0114] Example 1. Selection of SLAMF1-specific effective antibody group

[0115] The inventors performed bio-panning from a phage display human antibody library to select fully human antibodies that specifically bind to SLAMF1. By repeating bio-panning over four rounds, the enrichment pattern of antibodies that specifically bind to the target antigen was confirmed. Specifically, after performing bio-panning targeting SLAMF1, the output titer was calculated relative to the control group, and an enrichment pattern was confirmed in rounds 2-4. Accordingly, monoclonal antibodies (mAbs) were selected by performing ELISA on the round 2-4 pool (Fig. 1). The novelty of the effective antibody group obtained was confirmed through sequence analysis of the genes of the 16 selected antibodies.

[0117] Example 2. IgG conversion of selected antibodies, antibody production, and purification

[0118] Example 2.1. Conversion of SLAMF1 effective antibody to IgG (IgG type 1)

[0119] Insert DNA was obtained by amplifying the variable regions of the light and heavy chains of 16 selected effective SLAMF1 antibodies via PCR, and then subcloning was performed using monocistronic full-IgG (pIgG1-LC, pIgG1-HC) vectors for IgG antibody production. High-purity recombinant DNA for IgG antibody production was obtained in large quantities from Expi293 and ExpiCHO cells using the Maxi-prep kit for each of the 16 effective antibodies. DNA purity was verified using a nanodrop.

[0121] Example 2.2. Production and Purification of SLAMF1-Specific IgG (IgG 1) Antibody

[0122] Using the provisional expression method with the Expi293 system (Invitrogen), light and heavy chain IgG expression vectors were co-transfected into Expi293 cells with Expifectamine, and cultured for 6 days. To purify the antibodies, the culture medium was centrifuged, the precipitated cell pellet was removed, and the final culture medium (media) was obtained. Subsequently, the selected antibodies were purified using affinity column chromatography with HiTrap MabSelect SuRe.

[0124] Example 3. Analysis of the chemical characteristics of the SLAMF1 effective antibody

[0125] Example 3.1. Binding affinity (K D value) measurement

[0126] The inventors have identified the binding affinity (K) of a selected SLAMF1 effective antibody to a target. DTo measure the value, analysis was performed using a Biacore T200 instrument. In this example, the binding affinity to the target of six SLAMF1 effective antibodies selected from the 16 effective antibodies, specifically SLAMF1-b2-1G5 (#6), SLAMF1-b2-1G12 (#8), SLAMF1-b3-1E10 (#11), SLAMF1-b3-1C12 (#13), SLAMF1-b3-1G3 (#15), and SLAMF1-b4-1B2s (#21), was measured.

[0127] The results are shown in Figure 2 below.

[0128] The selected SLAMF1 effective antibody of the present invention is on rate (K a ) is high, and each K D The values ​​were 36 nM, 4.4 nM, 3.7 nM, 8.2 nM, 3.6 nM, and 1.0 nM, confirming that the six selected effective SLAMF1 antibodies exhibited excellent binding affinity to the target.

[0130] Example 3.2. Analysis of Intermediate Cross-Reactivity and Binding Specificity

[0131] 3.2.1. Interspecies Cross-reactivity

[0132] First, the inventors confirmed the cross-reactivity to monkey SLAMF1 in order to analyze the interspecies cross-reactivity of six selected SLAMF1 effective antibodies.

[0133] As a result, as shown in Figure 3 below, it was confirmed that interspecies cross-reactivity was exhibited for monkey SLAMF1.

[0135] 3.2.2. Binding Specificity

[0136] Next, the inventors performed an analysis of the binding specificity of the selected SLAMF1 effective antibody and confirmed whether it exhibited non-specific binding to major proteins present in the blood.

[0137] Specifically, for this example, hemoglobin (Hb), fibrinogen (bFgn), transferrin (Tf), α1-antitrypsin (AAT), and human serum albumin (HSA) were used as blood proteins. Histone, CA9 (Carbonic anhydrase 9), and skim milk were used as negative controls, and hSLAMF1 was used as a positive control.

[0138] The results are shown in Figure 3 below.

[0139] It was confirmed that the selected effective antibody against SLAMF1 did not exhibit non-specific binding to proteins in the blood, but exhibited a specific binding reaction only to hSLAMF1. However, as confirmed in the interspecies cross-reactivity of Example 3.2.1 above, it was confirmed that it exhibited a binding reaction to monkey SLAMF1 (cynoSLAMF1).

[0140] In addition, it was confirmed that proteins with high or low pI values, such as Histone and CA9, did not exhibit non-specific binding.

[0142] Example 3.3. Thermal Stability Analysis

[0143] In order to perform a thermal stability analysis of the selected SLAMF1 effective antibody, the inventors measured the Tm B value of the selected SLAMF1 effective antibody through PTS (Protein thermal shift) analysis.

[0144] The results are shown in Figure 4 below.

[0145] It was confirmed that the selected SLAMF1-specific antibody of the present invention has high thermal stability by exhibiting a Tm B value of 80.4 to 84.9°C.

[0147] Example 3.4. Hydrophobicity Analysis

[0148] The inventors performed Hydrophobic Interaction Chromatography (HIC) analysis to analyze the hydrophobicity characteristics of selected SLAMF1-specific antibodies.

[0149] According to previous studies, high RT values ​​in HIC analysis may be correlated with an increased risk of aggregation or nonspecific binding indicating surface hydrophobicity (Bailly, Marc. et al. (Jain, 2020), analysis of clinical-stage antibodies reported a lower 10% threshold RT value of 11.7 minutes or less (Jain, Tushar et al. , 2017).

[0150] The hydrophobicity analysis results of this embodiment are shown in Figure 5 below.

[0151] As shown in Figure 5 below, the selected SLAMF1-specific antibodies of the present invention showed a low risk in terms of hydrophobicity with an RT value of within 8 minutes, and all six antibodies did not show agglutination during the purification process, and it was confirmed that no non-specific binding occurred in ELISA analysis for various antigens.

[0153] Example 3.5. Analysis of pI values

[0154] The inventors performed an analysis of the pI values ​​of the selected SLAMF1 effective antibody through cIEF (Capillary isoelectric focusing) analysis.

[0155] Bailly, Marc. et al. According to (2020), if the above pI value falls outside the range of 7 to 9, losses may occur during the purification process and problems with virus inactivation may arise. In particular, it is known that when the pI value is high, such as 9 or higher, clearance via pinocytosis increases, raising concerns about a decrease in the blood half-life (Kraft, Thomas E et al. , 2020).

[0156] The pI analysis results of this embodiment are shown in Figure 6 below.

[0157] As shown in Figure 6 below, it was confirmed that all selected SLAMF1 effective antibodies of the present invention did not fall outside the range of 7 to 9.

[0159] Example 4. Anti-SLAMF1 antibody in vitro Validity evaluation

[0160] The inventors of the present invention [use] human peripheral blood mononuclear cells (PBMCs). in vitro A group of 16 effective anti-SLAMF1 antibodies based on T cell proliferation and PD-1 expression inhibition ability through activation assays in vitro Validity was evaluated.

[0161] Specifically, during the target protein selection process, 20 μg / ml of anti-SLAMF1 antibody was administered along with the activation step of stimulating human peripheral blood mononuclear cells (PBMC) with an anti-CD3 antibody, and the efficacy of the anti-SLAMF1 effective antibody group was evaluated based on T cell proliferation and PD-1 expression.

[0162] First, the T cell activation and PD-1 expression inhibition ability of the 16 anti-SLAMF1 effective antibodies prepared above was evaluated, and the results are shown in Figures 7a and 7b below.

[0163] As can be seen in Figures 7a and 7b, it was confirmed that 6 of the 16 antibodies produced above showed a T cell proliferation inhibition rate of over 90% and PD-1 expression inhibition of over 70%.

[0165] Next, six antibodies with high T cell activation inhibitory activity were selected, and their T cell activation inhibitory activity according to concentration was evaluated in conjunction with a commercially available antibody (BioLegend, Clone: ​​A12). Specifically, 10 μg / ml was set as the highest concentration, and T cell activation inhibitory activity was evaluated at different concentrations by serially diluting the antibodies by 1 / 10.

[0166] The results are shown in Figure 8 below.

[0167] As can be seen in Figure 8, the BioLegend A12 antibody exhibits T cell activation inhibitory ability only at a concentration of 10 μg / ml, whereas the novel anti-SLAMF1 antibody of the present invention exhibits superior T cell activation inhibitory ability compared to the BioLegend A12 antibody even at a concentration 100 times lower, such as 0.1 μg / ml.

[0169] Example 5. Anti-SLAMF1 antibody in vivo Validity evaluation

[0170] The inventors [discuss] the anti-SLAMF1 antibody in a xenogeneic GvHD model using human peripheral blood mononuclear cells in vivo Validity was evaluated.

[0171] Specifically, human PBMCs were intravenously injected into radiation-pretreated immunodeficient NSG (NOD / SCID / IL-2R(null)) mice to induce acute GvHD induced by xenograft. Then, starting from the time of PBMC injection, an IgG1 isotype antibody (control group) and two types of anti-SLAMF1 antibodies (experimental group) were injected intraperitoneally (IP) at 8 mpk per dose a total of four times at one-week intervals into the mice with induced acute GvHD. Subsequently, changes in the mice were observed. The antibodies used for the experimental group were derived from the six novel anti-SLAMF1 antibodies identified in Example 3, (i) in vitroTwo types of antibodies were identified as having high T cell activation inhibitory ability, (ii) possessing affinity for cynomolgus SLAMF1, and (iii) suitable developmental factors such as production yield, specifically anti-SLAMF1-b2-1G5 (#5) and anti-SLAMF1-b3-1G3 (#15)) used as the experimental group in this example.

[0172] The results are shown in Figure 9 below. First, the control group used i) In the group administered isotype antibodies, continuous body weight loss and an increase in the GvHD index were observed starting 2 weeks after intraperitoneal administration, and all mice died by day 50. On the other hand, ii) In the group administered anti-SLAMF1 b2-1G5 (#5) antibody, all patients survived until the end of the study at 56 days, body weight was maintained within a 10% loss range, and the GvHD index was also maintained at a low level. iii) In the group administered anti-SLAMF1 b3-1G3 (#15) antibody, 60% survived until the end of the study at 56 days, body weight was maintained within a 10% loss range, and the GvHD index was also maintained at a low level.

[0174] delete

[0175] delete

[0176] Example 6. Evaluation of Therapeutic Efficacy of Anti-SLAMF1 Antibody - Mode of Action (MoA) Analysis

[0177] The inventors performed a MoA analysis to evaluate the therapeutic efficacy of the novel anti-SLAMF1 antibody of the present invention in a xenogeneic GvHD mouse model.

[0178] The xenogeneic GvHD mouse model used in this embodiment was prepared in the same manner as in Example 4 above. Specifically, it was prepared by inducing acute GvHD via xenograft by intravenously injecting human PBMC into radiation-pretreated immunodeficient NSG (NOD / SCID / IL-2R(null)) mice. Then, starting from the time of PBMC injection, an IgG1 isotype antibody (control group) and two types of anti-SLAMF1 antibodies (b2-1G5, b3-1G3: experimental group) were injected intraperitoneally (IP) at 8 mpk a total of four times at one-week intervals into the mice with induced acute GvHD. Two days after the last administration, the mice were euthanized, splenocytes were obtained, and human T cell activation was measured.

[0179] The results are shown in Figures 10a to 10c below.

[0180] First, in Figure 10a, it was confirmed that the anti-SLAMF1 (b2-1G5, b3-1G3) antibody administration group maintained body weight within a 10% loss range and the GvHD index also remained low.

[0181] In addition, as a result of measuring T cell activation in each experimental group, as can be seen in Figure 10b, most CD8 and CD4 T cells disappeared in the group administered with the anti-SLAMF1 b2-1G5 antibody, and a significant decrease in CD8 and CD4 T cells was also observed in the group administered with the anti-SLAMF1 b3-1G3 antibody. In Figure 10c, the expression of PD-1 and GzmB (Granzyme B) among the remaining CD8 T cells was confirmed, and a decrease in the frequency of cells expressing PD-1 and GzmB was observed.

Claims

Claim 1 An antibody or antigen-binding fragment thereof that specifically binds to SLAMF1 comprising: (a) a heavy chain variable region of CDR-H1 comprising the amino acid sequence of SEQ ID NO. 1, CDR-H2 comprising the amino acid sequence of SEQ ID NO. 2, and CDR-H3 comprising the amino acid sequence of SEQ ID NO. 3; and a light chain variable region comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO. 4, CDR-L2 comprising the amino acid sequence of SEQ ID NO. 5, and CDR-L3 comprising the amino acid sequence of SEQ ID NO. 6; (b) a heavy chain variable region of CDR-H1 comprising the amino acid sequence of SEQ ID NO. 7, CDR-H2 comprising the amino acid sequence of SEQ ID NO. 8, and CDR-H3 comprising the amino acid sequence of SEQ ID NO. 9; (c) a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO. 10, CDR-L2 having the amino acid sequence of SEQ ID NO. 11, and CDR-L3 having the amino acid sequence of SEQ ID NO. 12; (c) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO. 13, CDR-H2 having the amino acid sequence of SEQ ID NO. 14, and CDR-H3 having the amino acid sequence of SEQ ID NO. 15; and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO. 16, CDR-L2 having the amino acid sequence of SEQ ID NO. 17, and CDR-L3 having the amino acid sequence of SEQ ID NO. 18; (d) a heavy chain variable region comprising CDR-H1 having the amino acid sequence of SEQ ID NO. 19, CDR-H2 having the amino acid sequence of SEQ ID NO. 20, and CDR-H3 having the amino acid sequence of SEQ ID NO. 21; and a light chain variable region comprising CDR-L1 having the amino acid sequence of SEQ ID NO. 22, CDR-L2 having the amino acid sequence of SEQ ID NO. 23, and CDR-L3 having the amino acid sequence of SEQ ID NO. 24;(e) a heavy chain variable region of CDR-H1 containing the amino acid sequence of SEQ ID NO. 25, CDR-H2 containing the amino acid sequence of SEQ ID NO. 26, and CDR-H3 containing the amino acid sequence of SEQ ID NO. 27; and a light chain variable region of CDR-L1 containing the amino acid sequence of SEQ ID NO. 28, CDR-L2 containing the amino acid sequence of SEQ ID NO. 29, and CDR-L3 containing the amino acid sequence of SEQ ID NO. 30; or (f) a heavy chain variable region of CDR-H1 containing the amino acid sequence of SEQ ID NO. 31, CDR-H2 containing the amino acid sequence of SEQ ID NO. 32, and CDR-H3 containing the amino acid sequence of SEQ ID NO. 33; and a light chain variable region of CDR-L1 containing the amino acid sequence of SEQ ID NO. 34, CDR-L2 containing the amino acid sequence of SEQ ID NO. 35, and CDR-L3 containing the amino acid sequence of SEQ ID NO. 36.; Claim 2 The antibody or antigen-binding fragment thereof comprising the following: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 37; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 38; (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 39; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 40; (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 41; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 42; (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 43; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 44; (e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 45; and a light chain variable region comprising the amino acid sequence of SEQ ID NO. 46; or (f) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 47; and a light chain variable region comprising the amino acid sequence of SEQ ID NO.

48. Claim 3 In claim 1, the antibody or antigen-binding fragment is an antibody or its antigen-binding fragment comprising the heavy chain variable region and the light chain variable region, a monoclonal antibody, a polyclonal antibody, scFv, Fab, F(ab), F(ab)2, scFv-Fc, a minibody, a diabody, a tribody, a tetrabody, a bispecific antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment thereof. Claim 4 In claim 1, the SLAMF1 is an antibody or its antigen-binding fragment that is expressed on the surface of allograft-specifically activated T cells. Claim 5 A nucleic acid molecule comprising a nucleotide sequence encoding an antibody or its antigen-binding fragment according to any one of claims 1 to 4. Claim 6 A recombinant vector comprising the nucleic acid molecule of claim 5. Claim 7 Isolated host cells comprising the recombinant vector of claim 6. Claim 8 A pharmaceutical composition for the treatment of graft-versus-host disease (GvHD) comprising, as an active ingredient, an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4. Claim 9 A pharmaceutical composition for the treatment of GvHD according to claim 8, wherein the composition inhibits T cell activation or removes activated T cells. Claim 10 A pharmaceutical composition for the treatment of GvHD according to claim 9, characterized in that the T cells express SLAMF1 on the cell surface specifically for allografts. Claim 11 A pharmaceutical composition for the treatment of GvHD according to claim 9, wherein the inhibition is a decrease in T cell proliferation, a decrease in PD-1 expression, or a decrease in GzmB (Granzyme B) expression compared to a control group.

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