Human antibody specifically binding to human slamf1 protein, and use thereof

Abstract

The present invention relates to an anti-SLAMF7 antibody specifically binding to SLAMF7 protein, or an antigen-binding fragment thereof. The antibody or antigen-binding fragment thereof inhibits the proliferation or activation of immune cells or reduces the number of activated immune cells in patients with graft-versus-host disease (GvHD), particularly, reduces the number of T cells specifically activated by transplantation, and thus can be effectively used for preventing or treating GvHD. In addition, the anti-SLAMF7 antibody or an antigen-binding fragment thereof is used to predict or diagnose the possibility of developing GvHD, and thus can be effectively used for the diagnosis or personalized treatment of GvHD.

Description

Human antibody specifically binding to human SLAMF7 protein and its uses

[0001] The present invention relates to an anti-SLAMF7 antibody or an antigen-binding fragment thereof that specifically binds to the SLAMF7 protein; a composition for the prevention or treatment of graft-versus-host disease comprising the same; and a composition for the diagnosis of graft-versus-host disease using the same.

[0002]

[0003] Graft-versus-host disease (GvHD) is a condition that occurs after an allogeneic hematopoietic stem cell transplant or blood transfusion, in which the donor's immune cells recognize the recipient's cells as foreign antigens and attack them. GvHD is a highly fatal disease and is the leading cause of death following hematopoietic stem cell transplantation; furthermore, the number of hematopoietic stem cell transplants and the resulting GvHD cases are on the rise every year.

[0004] Currently, treatments for graft-versus-host disease include corticosteroids, anti-thymocyte globulin (ATG) agents, IL-2Rα inhibitors, TNF inhibitors, calcineurin inhibitors (e.g., cyclosporin A (CsA) and tacrolimus (FK506), and mTOR inhibitors (sirolimus and rapamycin). However, while high-dose corticosteroids such as prednisone are the most widely used, none have yet been established as a standard treatment, and most of these agents were developed to target other diseases. Therefore, the development of treatments for graft-versus-host disease is urgent.

[0005] Accordingly, the inventors completed the present invention by developing a novel antibody that specifically binds to the SLAMF7 protein and confirming its therapeutic efficacy against graft-versus-host disease.

[0006]

[0007] The object of the present invention is to provide an anti-SLAMF7 antibody that specifically binds to the SLAMF7 protein or an antigen-binding fragment thereof.

[0008] Another object of the present invention is to provide a nucleic acid molecule encoding the anti-SLAMF7 antibody or its antigen-binding fragment.

[0009] Another objective of the present invention is to provide a recombinant expression vector comprising the nucleic acid molecule.

[0010] Another objective of the present invention is to provide a host cell transformed with the recombinant expression vector.

[0011] Another object of the present invention is to provide a pharmaceutical composition comprising the anti-SLAMF7 antibody or its antigen-binding fragment; and a pharmaceutically acceptable carrier.

[0012] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of graft-versus-host disease comprising the anti-SLAMF7 antibody or an antigen-binding fragment thereof as an active ingredient.

[0013] Another object of the present invention is to provide a method for preventing or treating graft-versus-host disease comprising the step of administering a pharmaceutically effective amount of the anti-SLAMF7 antibody or its antigen-binding fragment to an individual.

[0014] Another objective of the present invention is to provide a composition for diagnosing graft-versus-host disease comprising the anti-SLAMF7 antibody or an antigen-binding fragment thereof.

[0015] Another objective of the present invention is to provide a diagnostic kit for graft-versus-host disease comprising the above-mentioned composition for diagnosing graft-versus-host disease.

[0016] Another objective of the present invention is to provide a method for providing information for the diagnosis of graft-versus-host disease, comprising the step of measuring the expression or activity level of SLAMF7 protein in a biological sample isolated from a subject using the anti-SLAMF7 antibody or its antigen-binding fragment.

[0017]

[0018] To achieve the above objective, the present invention provides an anti-SLAMF7 antibody that specifically binds to the SLAMF7 protein or an antigen-binding fragment thereof.

[0019] In addition, the present invention provides a nucleic acid molecule encoding the anti-SLAMF7 antibody or an antigen-binding fragment thereof.

[0020] In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0021] In addition, the present invention provides a host cell transformed with the recombinant expression vector.

[0022] In addition, the present invention provides a pharmaceutical composition comprising the anti-SLAMF7 antibody or an antigen-binding fragment thereof; and a pharmaceutically acceptable carrier.

[0023] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of graft-versus-host disease comprising the anti-SLAMF7 antibody or an antigen-binding fragment thereof as an active ingredient.

[0024] In addition, the present invention provides a method for preventing or treating graft-versus-host disease comprising the step of administering a pharmaceutically effective amount of the anti-SLAMF7 antibody or its antigen-binding fragment to an individual.

[0025] In addition, the present invention provides a composition for diagnosing graft-versus-host disease comprising the anti-SLAMF7 antibody or an antigen-binding fragment thereof.

[0026] In addition, the present invention provides a diagnostic kit for graft-versus-host disease comprising the above-mentioned composition for diagnosing graft-versus-host disease.

[0027] In addition, the present invention provides a method for providing information for the diagnosis of graft-versus-host disease, comprising the step of measuring the expression or activity level of an SLAMF7 protein in a biological sample isolated from a subject using the anti-SLAMF7 antibody or an antigen-binding fragment thereof.

[0028]

[0029] The anti-SLAMF7 antibody or its antigen-binding fragment according to the present invention can be usefully employed for the prevention or treatment of graft-versus-host disease (GvHD) by inhibiting the proliferation or activation of immune cells in patients with GvHD or by reducing the number of activated immune cells, particularly by reducing the number of graft-specifically activated T cells. Furthermore, the anti-SLAMF7 antibody or its antigen-binding fragment can be used to predict or diagnose the likelihood of developing GvHD, and personalized treatment may be possible for patients with GvHD induced by SLAMF7 expression.

[0030]

[0031] Figure 1 is a schematic diagram showing the mechanism of action of the anti-SLAMF7 antibody.

[0032] Figure 2 shows the results of screening effective antibody groups from a phage display human antibody library and confirming the enrichment pattern of antibodies binding to target antigens.

[0033] Figure 3 shows the results of measuring the binding affinity for the anti-SLAMF7 antibody.

[0034] Figure 4 shows the results of analyzing the binding specificity for the anti-SLAMF7 antibody.

[0035] Figure 5 shows the results of analyzing the thermal stability of the anti-SLAMF7 antibody.

[0036] Figure 6 shows the results of analyzing the hydrophobicity characteristics of the anti-SLAMF7 antibody.

[0037] Figures 7a to 7d show the results of analyzing the isoelectric point (pI) values ​​for the anti-SLAMF7 antibody.

[0038] Figures 8a and 8b show the results of confirming changes in PD-1 expression and T cell proliferation in response to the anti-SLAMF7 antibody.

[0039] Figure 9 shows the results of evaluating the T cell activity inhibitory ability of the anti-SLAMF7 antibody.

[0040] Figure 10 shows the results of analyzing the incidence of graft-versus-host disease after administering anti-SLAMF7 antibodies to a xenogeneic graft-versus-host disease mouse model.

[0041] Figure 11 shows the results of evaluating the incidence of graft-versus-host disease after administering various concentrations of anti-SLAMF7 antibodies to a xenograft graft-versus-host disease mouse model.

[0042] Figure 12 shows the results of analyzing CD8 T cells and CD4 T cells in the blood after administering anti-SLAMF7 antibodies to a xenograft graft-versus-host disease mouse model.

[0043]

[0044] The present invention will be described in detail below.

[0045] The terms used in this invention have been selected based on currently widely used general terms whenever possible, taking into account the functions of the invention; however, these terms may vary depending on the intent of those skilled in the art or the emergence of new technologies. Additionally, in specific cases, terms may be selected arbitrarily, and in such cases, their meanings will be described in detail in the description section of the relevant embodiments. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall content of the invention.

[0046] In the present invention, when a component or step is described as "comprising," this means that, unless specifically stated otherwise, it does not exclude other components or steps but may include additional components or steps.

[0047]

[0048] Antibody

[0049] The present invention provides an anti-SLAMF7 antibody that specifically binds to the SLAMF7 (SLAM family member 7) protein or an antigen-binding fragment thereof.

[0050] The term "SLAMF7" in this invention, also known as CS1, CRACC, or CD319, is a protein encoded by the SLAMF7 gene and is a type of SLAM (Signaling Lymphocytic Activation Molecule) family. SLAMF7 is known to be a protein that is not expressed in antigen-uncontacted T cells but is expressed in T cells activated by an antigen.

[0051] In the present invention, the SLAMF7 protein may be expressed on the surface of T cells that are specifically activated for allografts.

[0052] In the present invention, the anti-SLAMF7 antibody or the antigen-binding fragment thereof may comprise any one selected from the group consisting of (a) to (g) below: (a) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 1, a CDR2 having the amino acid sequence of SEQ ID NO. 2, and a CDR3 having the amino acid sequence of SEQ ID NO. 3; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 4, a CDR2 having the amino acid sequence of SEQ ID NO. 5, and a CDR3 having the amino acid sequence of SEQ ID NO. 6; (b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 7, a CDR2 having the amino acid sequence of SEQ ID NO. 8, and a CDR3 having the amino acid sequence of SEQ ID NO. 9; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 10, a CDR2 having the amino acid sequence of SEQ ID NO. 11, and a CDR3 having the amino acid sequence of SEQ ID NO. 12; (c) a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO. 7, CDR2 having the amino acid sequence of SEQ ID NO. 13, and CDR3 having the amino acid sequence of SEQ ID NO. 3; and a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO. 14, CDR2 having the amino acid sequence of SEQ ID NO. 15, and CDR3 having the amino acid sequence of SEQ ID NO. 16; (d) a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO. 17, CDR2 having the amino acid sequence of SEQ ID NO. 13, and CDR3 having the amino acid sequence of SEQ ID NO. 3; and a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO. 18, CDR2 having the amino acid sequence of SEQ ID NO. 19, and CDR3 having the amino acid sequence of SEQ ID NO. 20;(e) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 21, a CDR2 having the amino acid sequence of SEQ ID NO. 22, and a CDR3 having the amino acid sequence of SEQ ID NO. 23; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 24, a CDR2 having the amino acid sequence of SEQ ID NO. 25, and a CDR3 having the amino acid sequence of SEQ ID NO. 26; (f) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 7, a CDR2 having the amino acid sequence of SEQ ID NO. 27, and a CDR3 having the amino acid sequence of SEQ ID NO. 28; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 29, a CDR2 having the amino acid sequence of SEQ ID NO. 30, and a CDR3 having the amino acid sequence of SEQ ID NO. 31; and (g) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 32, a CDR2 having the amino acid sequence of SEQ ID NO. 33, and a CDR3 having the amino acid sequence of SEQ ID NO. 34; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 35, a CDR2 having the amino acid sequence of SEQ ID NO. 36, and a CDR3 having the amino acid sequence of SEQ ID NO. 37.;

[0053] In the present invention, the anti-SLAMF7 antibody or the antigen-binding fragment thereof may comprise any one selected from the group consisting of (i) to (vii) below: (i) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 38; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 39; (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 40; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 41; (iii) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 42; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 43; (iv) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 44; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 45; (v) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 46; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 47; (vi) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 48; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 49; and (vii) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 50; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 51.

[0054] The term "antibody" in the present invention refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen, and includes antibody fragments (e.g., Fab, F(ab')2, and Fv) as well as single monoclonal antibodies (including agonist and antagonist antibodies) and antibody compositions having polyepitope specificity, insofar as they exhibit the desired biological activity. In some embodiments, the antibody may be monoclonal, polyclonal, chimeric, single-chain, quadruple-specific or dual-effective, primate, human, and humanized antibodies, as well as their active fragments. Examples of active fragments of molecules that bind to known antigens include the Fab, F(ab')2, scFv, and Fv fragments, which comprise the product of the Fab immunoglobulin expression library and the epitope-binding fragments of any of the aforementioned antibodies and fragments. In some embodiments, the antibody may comprise an immunoglobulin molecule and a molecule containing an immunologically active portion of the immunoglobulin molecule, namely a binding site that binds immunospecifically to an antigen. The immunoglobulin may be any type (IgG, IgM, IgD, IgE, IgA, and IgY) or a class or subclass of immunoglobulin molecules (IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In one embodiment, the antibody may be a whole antibody and any antigen-binding fragment derived from the whole antibody. A typical antibody refers to a heterotemeric protein comprising two heavy chains (H or HC, Heavy chain) and two light chains (L or LC, Light chain). Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (VL) and a light chain constant region.The VH and VL regions can be further subdivided into a hypervariable complementarity determining region (CDR) domain and a more conserved region called the framework region (FR). Each variable domain (VH or VL) typically consists of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Within the variable regions of the light and heavy chains, there are binding regions that interact with the antigen.

[0055] The above terms, “antigen binding fragment” or “antibody fragment,” comprise a portion of an antibody that is deficient in some amino acids compared to the full chain but is capable of specifically binding to an antigen. Such fragments may be considered to have biological activity in that they can specifically bind to a target antigen or compete with other antibodies or antigen-binding fragments to bind to a specific epitope. In one aspect, such fragments comprise at least one CDR present in the full-length light chain or heavy chain, and in some embodiments, this comprises the light chain and / or heavy chain of the short chain, or a portion thereof. Such biologically active fragments may be produced by recombinant DNA technology or, for example, by enzymatically or chemically cleaving an intact antibody.

[0056] In the present invention, the antigen-binding fragment may be selected from the group consisting of Fab, Fab', F(ab')2, scFab, dsFv, Fv, scFv and combinations thereof, but is not limited thereto.

[0057] In the present invention, the anti-SLAMF7 antibody may comprise an Fc domain, and the Fc domain may be selected from the group consisting of human IgG1, IgG2, IgG3, and IgG4, but is not limited thereto.

[0058] In the present invention, the anti-SLAMF7 antibody or the antigen-binding fragment thereof 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.

[0059] The above term, "human antibody," possesses an amino acid sequence corresponding to the amino acid sequence 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. Human antibodies exclude humanized antibodies containing non-human antigen-binding residues.

[0060] The "humanized antibody" above is 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).

[0061] The antibodies of the present invention may be produced using various phage display methods known in the art, [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; It may be manufactured by referring to 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; 5,733,743; and 5,969,108.

[0062] The term "specifically binds" in this invention 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 -9 M, 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 It can be characterized by the equilibrium dissociation constant of M) (e.g., a KD smaller than this indicates a tighter bond). Methods for determining whether two molecules specifically bind are well known in the art and may include, for example, equilibrium dialysis, surface plasmon resonance, etc.

[0063] The term "affinity" in the present invention 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, "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 as a dissociation constant (Kd). Affinity may be measured by conventional methods known in the art, including those described herein.

[0064] The anti-SLAMF7 antibody of the present invention or its antigen-binding fragment may comprise variants of amino acid sequences within a range capable of specifically recognizing SLAMF7. 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, deletion, insertion, and / or substitution of amino acid sequence residues of the antibody.

[0065] 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.

[0066] 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.

[0067] 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).

[0068] 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.

[0069] 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).

[0070] 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.

[0071] 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.

[0072] The anti-SLAMF7 antibody specific to SLAMF7 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.

[0073] In the present invention, the heavy chain variable region and the light chain variable region included in the anti-SLAMF7 antibody or the antigen-binding fragment thereof may be connected by a linker. The linker may be a linker such as (Gly-Ser)n, (Gly2-Ser)n, (Gly3-Ser)n, or (Gly4-Ser)n, where n is an integer from 1 to 6, specifically from 3 to 4, but is not limited thereto. The light chain variable region and the heavy chain variable region of the scFv may be connected in, for example, the following order: heavy chain variable region - linker - light chain variable region; or light chain variable region - linker - heavy chain variable region.

[0074] In the present invention, the anti-SLAMF7 antibody or its antigen-binding fragment may inhibit the expression of an immune checkpoint protein expressed on the surface of immune cells. The immune checkpoint protein may be selected from the group consisting of PD-1 (Programmed cell death protein 1), PD-L1 (Programmed Death-Ligand 1), and CTLA-4 (Cytotoxic T-lymphocyte antigen 4), and preferably inhibits the expression of PD-1, but is not limited thereto.

[0075] In the present invention, the anti-SLAMF7 antibody or its antigen-binding fragment may (i) inhibit the proliferation or activation of immune cells, or (ii) reduce the number of activated immune cells. The immune cells are CD8 + T cells, CD4 + The immune cells may be selected from the group consisting of T cells, dendritic cells, B cells, natural killer (NK) cells, and macrophages, and preferably, the immune cells are CD8 + T cells, CD4 + It may be T cells, but is not limited to them.

[0076]

[0077] In addition, the present invention provides a nucleic acid molecule encoding the anti-SLAMF7 antibody or an antigen-binding fragment thereof.

[0078] The term "nucleic acid molecule" in this invention 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)).

[0079] In addition, the present invention provides a recombinant expression vector comprising the nucleic acid molecule.

[0080] The term "vector" in the present invention is understood as a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell and recombined and inserted into the host cell genome, or spontaneously replicated as an episome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses.

[0081] In addition, the present invention provides a host cell transformed with the recombinant expression vector.

[0082] In the present invention, suitable host cells may be transformed or transfected with the DNA sequence of the present invention and may be used for the expression and / or secretion of a target protein. Currently preferred host cells that can be used in the present invention include immortal hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, CapT cells (human amniotic fluid-derived cells), and COS cells.

[0083] The terms "transformation" and "transfection" of the present invention refer to the introduction of nucleic acids (e.g., vectors) into a cell by many techniques known in the art.

[0084]

[0085] <Pharmaceutical Composition>

[0086] In addition, the present invention provides a pharmaceutical composition comprising the anti-SLAMF7 antibody or an antigen-binding fragment thereof; and a pharmaceutically acceptable carrier.

[0087] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of graft-versus-host disease comprising the anti-SLAMF7 antibody or an antigen-binding fragment thereof as an active ingredient.

[0088] In the present invention, the composition may inhibit the proliferation or activation of immune cells. The immune cells are CD8 + T cells, CD4 + The immune cells may be selected from the group consisting of T cells, dendritic cells, B cells, natural killer (NK) cells, and macrophages, and preferably, the immune cells are CD8 + T cells, CD4 + It may be T cells, but is not limited to them.

[0089] In the present invention, it was confirmed that the anti-SLAMF7 antibody or its antigen-binding fragment has the effect of inhibiting the expression of PD-1 expressed on the surface of T cells and inhibiting the proliferation and activation of T cells, and can be usefully used for the prevention, improvement, or treatment of graft-versus-host disease (see Fig. 1).

[0090] In the present invention, the anti-SLAMF7 antibody or its antigen-binding fragment may be included in the composition at 1 to 50 mg / kg, preferably at 2 to 50 mg / kg, 3 to 50 mg / kg, 4 mg / kg to 50 mg / kg, 4 mg / kg to 45 mg / kg, 4 mg / kg to 40 mg / kg, 4 mg / kg to 35 mg / kg, 4 mg / kg to 30 mg / kg, 4 mg / kg to 25 mg / kg, or 4 mg / kg to 20 mg / kg, but is not limited thereto.

[0091] The pharmaceutical composition according to the present invention may be formulated into a suitable form with a pharmaceutically acceptable carrier and may additionally contain excipients or diluents. In the above, "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and, when administered to humans, does not typically cause allergic reactions or similar reactions such as gastrointestinal disorders or dizziness.

[0092] Pharmaceutically acceptable carriers may additionally include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Additionally, they may include various drug delivery materials used for oral administration of peptide preparations. Furthermore, carriers for parenteral administration may include water, suitable oils, saline solution, aqueous glucose and glycol, etc., and may additionally include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol. In addition to the above components, the pharmaceutical composition of the present invention may additionally include lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. Other pharmaceutically acceptable carriers and formulations may be referenced in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0093] The composition of the present invention may be administered to mammals, including humans, by any method. For example, it may be administered orally or parenterally. Parenteral administration methods may include, but are not limited to, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, rectal, mucosal delivery, or administration in the form of ophthalmic drops, and preferably may be mucosal delivery, transdermal, local, or ophthalmic delivery.

[0094] The pharmaceutical composition of the present invention can be formulated into an oral or parenteral administration formulation according to the administration route described above, and preferably into a parenteral administration formulation. For example, in the case of a parenteral administration formulation, it can be formulated in the form of an injection, a cream, a lotion, a topical ointment, an oil, a moisturizer, a gel, an aerosol, a nasal inhaler, and an eye drop by methods known in the art. These formulations are described in the literature (Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995), which is a prescription generally known in all pharmaceutical chemistry.

[0095] The total effective amount of the composition of the present invention may be administered to a patient as a single dose, or administered via a fractionated treatment protocol involving multiple doses administered over a long period. The content of the active ingredient in the pharmaceutical composition of the present invention may vary depending on the severity of the disease. Preferably, the preferred total dose of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg per kg of patient body weight per day, most preferably 0.1 μg to 500 mg. However, since the effective dose for a patient is determined by considering various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate, a person of ordinary knowledge in the art would be able to determine an appropriate effective dose of the composition of the present invention in light of these factors. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

[0096]

[0097] Treatment Methods

[0098] In addition, the present invention provides a method for preventing or treating graft-versus-host disease comprising the step of administering a pharmaceutically effective amount of the anti-SLAMF7 antibody or its antigen-binding fragment to an individual.

[0099] The term "pharmaceuticalally effective dose" above refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause adverse effects. The effective dose level can be readily determined by a person skilled in the art based on factors including the patient's gender, age, weight, health status, type and severity of the disease, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration, and elimination rate, duration of treatment, drugs used in combination or simultaneously, and other factors well known in the medical field. The above recommended dose may be administered once a day or divided into several doses.

[0100] The term "individual" above refers to all animals, including humans, rats, mice, and livestock. Preferably, the animal may be not only humans but also mammals such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require treatment for similar symptoms, but is not limited thereto.

[0101]

[0102] <Diagnostic Composition>

[0103] In addition, the present invention provides a composition for diagnosing graft-versus-host disease comprising the anti-SLAMF7 antibody or an antigen-binding fragment thereof.

[0104] The term "diagnosis" in this invention means confirming whether graft-versus-host disease has progressed or developed.

[0105] In addition, the present invention provides a diagnostic kit for graft-versus-host disease comprising the above-mentioned composition for diagnosing graft-versus-host disease.

[0106] In the present invention, the kit may be selected from the group consisting of ELISA kits, protein chip kits and rapid kits, but is not limited thereto.

[0107] The above ELISA kit may include a monoclonal antibody, a polyclonal antibody, or a recombinant antibody specific to a protein, and may also include reagents capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e.g., conjugated with an antibody) and its substrate, or other substances capable of binding to the antibody.

[0108] The above protein chip kit may include a substrate, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, a chromogenic substrate, etc. for the immunological detection of antibodies. In the above, the substrate may be a nitrocellulose membrane, a 96-well plate synthesized from polyvinyl resin, a 96-well plate synthesized from polystyrene resin, and a glass slide glass, etc., the chromogenic enzyme may be peroxidase or alkaline phosphatase, the fluorescent substance may be FITC, RITC, etc., and the chromogenic substrate solution may be ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (o-phenylenediamine), or TMB (tetramethylbenzidine).

[0109] The above rapid kit is a kit capable of rapidly performing a test using a small amount of sample, and may use a substance capable of binding to an analyte contained in the sample, namely an antibody capable of specifically binding to a protein.

[0110] In addition, the present invention provides a method for providing information for the diagnosis of graft-versus-host disease, comprising the step of measuring the expression or activity level of an SLAMF7 protein in a biological sample isolated from a subject using the anti-SLAMF7 antibody or an antigen-binding fragment thereof.

[0111] In the present invention, the biological sample may be selected from the group consisting of tissue, cell, blood, serum, plasma, lymph fluid, saliva, and urine, but is not limited thereto.

[0112] In the present invention, the method may further include a step of determining that there is a high probability of developing graft-versus-host disease when the SLAMF7 protein is higher than the reference value obtained from a control sample.

[0113]

[0114] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0115]

[0116] Example 1. Selection of an effective antibody group targeting SLAMF7

[0117] In the present invention, a group of antibodies that specifically bind to SLAMF7 was screened from a phage display human antibody library through biopanning. Biopanning was repeated up to 4 rounds, and after performing biopanning targeting SLAMF7 and calculating the Output Titer relative to the control group, it was confirmed that an enrichment pattern appeared in rounds 3-4 (Fig. 2). Subsequently, ELISA was performed on the 3-4 round pool to select a total of 13 monoclonal antibodies, and among the selected antibodies, a total of 7 antibodies were selected through the following in vitro efficacy evaluation. The sequence information for the 7 selected antibodies is as shown in Tables 1 and 2 below.

[0118]

[0119] Clone Name CDR-L1 CDR-L2 CDR-L3 CDR-H1 CDR-H2 CDR-H3 SLAMF7-b3-C5 RASQSISSYLN(Sequence No. 1) AASSLQS(Sequence No. 2) QQSYSTPWT(Sequence No. 3) GYAIH(Sequence No. 4) GIIPILGETNYAQKFQG(Sequence No. 5) ELSWYYGFDF(Sequence No. 6) SLAMF7-b3-E12 RASQSISNYLN(Sequence No. 7) GASRLQS(Sequence No. 8) QQSYSFPFT(Sequence No. 9) SYYIS(Sequence No. 10) GIIPLTGTTNYAQKFQG(Sequence No. 11) EAEWGWYEVFDY(Sequence No. 12) SLAMF7-b3-B10 RASQSISNYLN(Sequence No 7)DASTLQS(SEQ No. 13)QQSYSTPWT(SEQ No. 3)SYGMH(SEQ No. 14)IIIPIFGTTNYAQKFQG(SEQ No. 15)DESYGVEAAFDL(SEQ No. 16)SLAMF7-b4-2E7RASQTISNWLN(SEQ No. 17)DASTLQS(SEQ No. 13)QQSYSTPWT(SEQ No. 3)EYAMN(SEQ No. 18)WIIPIFGSTNYAQKFQG(SEQ No. 19)DFVVSYTEAMDI(SEQ No. 20)SLAMF7-b4-2G9sRASQDISNYLN(SEQ No. 21)ATSSLQS(SEQ No. 22)QQAYSYPYT(SEQ No. 23)NYEMH(SEQ No 24)SISSSGSTYYADSVKG(Sequence No. 25)EFTEYGGYWFDY(Sequence No. 26)SLAMF7-b4-A7sRASQSISNYLN(Sequence No. 7)DASSLQS(Sequence No. 27)QQSYSYPLT(Sequence No. 28)TYEMH(Sequence No. 29)SISSSGSNTYYADSVKG(Sequence No. 30)ERSEYGSFYFDY(Sequence No. 31)SLAMF7-b4-E11sRASQSISSWLN(Sequence No. 32)ATSNLQS(Sequence No. 33)QQSYSYPFT(Sequence No. 34)TYAMS(Sequence No. 35)AIYSSGSTYYADSVKG(Sequence No. 36)EEEAWYYSAFDV(Sequence No. 37)

[0120] Clone NameVL (light chain variable region)VH (heavy chain variable region)SLAMF7-b3-C5DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIK(서열번호 38)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSGYAIHWVRQAPGQGLEWMGGIIPILGETNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARELSWYYGFDFWGQGTLVTVSS(서열번호 39)SLAMF7-b3-E12DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYGASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPFTFGQGTKVEIK(서열번호 40)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYYISWVRQAPGQGLEWMGGIIPLTGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAREAEWGWYEVFDYWGQGTLVTVSS(서열번호 41)SLAMF7-b3-B10DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIK(서열번호 42)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGMHWVRQAPGQGLEWMGIIIPIFGTTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDESYGVEAAFDLWGQGTLVTVSS(서열번호 43)SLAMF7-b4-2E7DIQMTQSPSSLSASVGDRVTITCRASQTISNWLNWYQQKPGKAPKLLIYDASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIK(서열번호44)QVQLVQSGAEVKKPGSSVKVSCKASGGTFSEYAMNWVRQAPGQGLEWMGWIIPIFGSTNYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDFVVSYTEAMDIWGQGTLVTVSS (SEQ ID NO: 45) SLAMF7-b4-2G9sDIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYSYPYTFGQGTKVEIK (SEQ ID NO: 46)EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYEMHWVRQAPGKGLEWVSSISSSSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEFTEYGGYWFDYWGQGTLVTVSS (SEQ ID NO: 47) SLAMF7-b4-A7sDIQMTQSPSSLSASVGDRVSITCRASQSISNYLNWYQQKPGKAPKLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSYPLTFGQGTKVEIK (SEQ ID NO: 48)EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYEMHWVRQAPGKGLEWVSSISSSSGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKERSEYGSFYFDYWGQGTLVTVSS (SEQ ID NO. 49) SLAMF7-b4-E11sDIQMTQSPSSLSASVGDRVTITCRASQSISSWLNWYQQKPGKAPKLLIYATSNLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSYPFTFGQGTKVEIK (SEQ ID NO: 50) EVQLVESGGGLVQPGGSLRLSCAASGFTFSTYAMSWVRQAPGKGLEWVSAIYSSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKEEEAWYYSAFDVWGQGTLVTVSS (SEQ ID NO: 51)

[0121]

[0122] Example 2. Analysis results of the novel anti-SLAMF7 antibody

[0123] 2.1. Binding Affinity Analysis

[0124] Experiments were performed to analyze the binding affinity of selected anti-SLAMF7 antibodies. Briefly, the binding affinity of the antibodies was analyzed for the SLAMF7-b3-B10, SLAMF7-b4-2E7, and SLAMF7-b4-E11s clones among the selected antibodies using a Biacore T200 instrument.

[0125] As a result, the anti-SLAMF7 antibodies of the SLAMF7-b3-B10, SLAMF7-b4-2E7, and SLAMF7-b4-E11s clones showed a binding rate (on rate (K). a )) is 10 6 It was very high at / Ms, and K D The value was confirmed to be in the range of 0.08–2.15 nM (Fig. 3). Thus, it was confirmed that the anti-SLAMF7 antibody of the present invention has a high binding affinity for the target antigen SLAMF7.

[0126]

[0127] 2.2. Analysis of Binding Specificity

[0128] Experiments were conducted to analyze the binding specificity of selected anti-SLAMF7 antibodies. Briefly, non-specific binding was determined for major blood proteins including hemoglobin (Hb), fibrinogen (bFgn), human serum albumin (HSA), transferrin (Tf), alpha-antitrypsin (AAT1), and insulin. Additionally, non-specific binding was checked for proteins or molecules with high or low pI values, such as histones, DNA, LPS, and CA9.

[0129] As a result, it was confirmed that the anti-SLAMF7 antibodies of the SLAMF7-b3-B10, SLAMF7-b4-2E7, and SLAMF7-b4-E11s clones specifically bind to the SLAMF7 protein, and no non-specific binding was observed to other major proteins in the blood or other proteins (Fig. 4). Thus, it was confirmed that the anti-SLAMF7 antibodies of the present invention have high binding specificity to SLAMF7.

[0130]

[0131] 2.3. Thermal Stability Analysis

[0132] To analyze the thermal stability of the selected anti-SLAMF7 antibodies, the denaturation transition temperature (Tm) was measured using protein thermal shift (PTS) analysis. As a result, the anti-SLAMF7 antibodies of the SLAMF7-b3-B10, SLAMF7-b4-2E7, and SLAMF7-b4-E11s clones exhibited Tm B values ​​in the range of 76.8 to 84.5°C (Fig. 5). Thus, it was confirmed that the anti-SLAMF7 antibodies of the present invention possess high thermal stability.

[0133]

[0134] 2.4. Hydrophobicity Analysis

[0135] To analyze the hydrophobicity characteristics of the selected anti-SLAMF7 antibodies, hydrophobic interaction chromatography (HIC) analysis was performed. As a result, the anti-SLAMF7 antibodies of the SLAMF7-b3-B10, SLAMF7-b4-2E7, and SLAMF7-b4-E11s clones were confirmed to have RT values ​​of 7.256 to 8.263, within 8.3 minutes (Fig. 6).

[0136] According to previous studies, high RT values ​​in hydrophobic interaction chromatography (HIC) analysis may be correlated with an increased risk of aggregation or nonspecific binding indicating surface hydrophobicity (Bailly, Marc. et al., 2020), and in an analysis of clinical-stage antibodies, a lower 10% threshold RT value was reported to be 11.7 minutes or less (Jain, Tushare et al., 2017).

[0137] Thus, it was confirmed that the anti-SLAMF7 antibody of the present invention has a low risk in terms of hydrophobicity and that no agglutination occurs during the antibody purification process.

[0138]

[0139] 2.5. Analysis of Isoelectric Point (pI) Values

[0140] To analyze the isoelectric point (pI) values ​​of the selected anti-SLAMF7 antibodies, capillary isoelectric focusing (cIEF) analysis was performed. As a result, it was confirmed that the anti-SLAMF7 antibodies of the SLAMF7-b3-B10, SLAMF7-b4-2E7, and SLAMF7-b4-E11s clones all had pI values ​​within the range of 7 to 9 (Figs. 7a to 7d).

[0141] According to previous studies, if the pI value falls outside the range of 7 to 9, losses may occur during the purification process and problems with viral inactivation may arise (Bailly, Marc et al. (2020). In particular, when the pI value is high, such as 9 or higher, concerns have been reported regarding a decrease in blood half-life due to increased clearance through pinocytosis (Kraft, Thomas et al., 2020).

[0142] Thus, it was confirmed that the anti-SLAMF7 antibody of the present invention has low loss during the purification process and possesses advantageous characteristics in terms of in vivo half-life.

[0143]

[0144] Example 3. Evaluation of the in vitro immunosuppressive ability of a novel anti-SLAMF7 antibody

[0145] Experiments were conducted to identify antibodies with immunosuppressive ability from a group of 13 selected effective antibodies using the human T cell in vitro activation assay. Briefly, human peripheral blood mononuclear cells (PBMCs) were stimulated and activated with an anti-CD3 antibody, and then treated with a total of 13 anti-SLAMF7 antibodies (20 μg / mL). The efficacy of the anti-SLAMF7 antibodies was evaluated based on the inhibition of T cell proliferation and PD-1 expression.

[0146] As a result, it was confirmed that 7 out of a total of 13 antibodies (SLAMF7-b3-C5, SLAMF7-b3-E12, SLAMF7-b3-B10, SLAMF7-b4-2E7, SLAMF7-b4-2G9s, SLAMF7-b4-A7s, and SLAMF7-b4-E11s clones) had significant T cell proliferation inhibition and PD-1 expression inhibition effects (Figs. 8a and 8b).

[0147] Next, to evaluate the in vitro efficacy of the seven selected antibodies (SLAMF7-b3-C5, SLAMF7-b3-E12, SLAMF7-b3-B10, SLAMF7-b4-2E7, SLAMF7-b4-2G9s, SLAMF7-b4-A7s, and SLAMF7-b4-E11s), the T cell activation inhibitory ability was compared after treating the seven selected antibodies with a commercial antibody (BioLegend, Clone: ​​162.1) at different concentrations. Briefly, each antibody was prepared by setting the maximum concentration to 10 μg / mL and serially diluting it by 1 / 10.

[0148] As a result, the commercial antibody BioLegend 162.1 showed an inhibitory effect on T cell activation only at the highest concentration of 10 μg / mL, whereas the seven types of antibodies showed a significantly greater inhibitory effect on T cell activation compared to the commercial antibody (BioLegend) even at a concentration 100 times lower, 0.1 μg / mL (Fig. 9).

[0149] From the above results, it was confirmed that the anti-SLAMF7 antibody of the present invention inhibits the expression of PD-1 expressed on the surface of T cells and has the effect of inhibiting the proliferation and activation of T cells.

[0150]

[0151] Example 4. Evaluation of the therapeutic efficacy of a novel anti-SLAMF7 antibody for graft-versus-host disease

[0152] To determine whether the anti-SLAMF7 antibody of the present invention can be used as a treatment for graft-versus-host disease, an experiment was conducted to evaluate the therapeutic efficacy after administering the antibody to a xenograft-based acute graft-versus-host disease (GvHD) mouse model. Briefly, acute graft-versus-host disease was induced in the GvHD mouse model by intravenously injecting human peripheral mononuclear cells (PBMCs) into radiation-pretreated immunodeficient NSG (NOD / SCID / IL-2R(null)) mice. Subsequently, at one-week intervals from the time of PBMC injection, an IgG1 isotype antibody (control) or three types of antibodies (SLAMF7-b3-B10, SLAMF7-b4-2E7, SLAMF7-b4-E11s) were injected intraperitoneally (IP) at a dose of 8 mg / kg a total of four times, and the incidence of graft-versus-host disease was analyzed.

[0153] As a result, in the control group administered with IgG1 isoform antibodies, body weight continuously decreased starting 2 weeks after PBMC injection, the disease index score increased, and all mice died on day 51. In contrast, in the groups administered with SLAMF7-b4-2E7 and SLAMF7-b4-E11s antibodies, body weight improved by more than 20% and 15%, respectively, compared to the control group as of day 40, and the disease index also improved by more than 1.5 points and 1 point, respectively (Fig. 10). Additionally, in the groups administered with SLAMF7-b3-B10, SLAMF7-b4-2E7, and SLAMF7-b4-E11s antibodies, survival rates were significantly increased to 30%, 35%, and 70%, respectively, compared to the control group as of day 58 (Fig. 10).

[0154] From the above results, it was confirmed that the anti-SLAMF7 antibody of the present invention has effects of improving body weight, improving disease index, and increasing survival rate in individuals with graft-versus-host disease.

[0155]

[0156] Example 5. Confirmation of effective dose by novel anti-SLAMF7 antibody

[0157] To determine the effective dose of the anti-SLAMF7 antibody as a treatment for graft-versus-host disease, an experiment was conducted to evaluate therapeutic efficacy after administering the antibody at various doses to a xenograft-based acute graft-versus-host disease mouse model. Briefly, the IgG1 isotype antibody (control) or the SLAMF7-b4-E11s anti-SLAMF7 antibody at doses of 2 mg / kg, 4 mg / kg, 8 mg / kg, or 16 mg / kg was injected intraperitoneally into the GvHD mouse model at weekly intervals starting from the time of PBMC infusion, for a total of four injections. Cyclosporine A (CsA), known as a treatment for graft-versus-host disease, was used as a positive control; cyclosporine was injected daily from day 5 to day 18 of PBMC infusion, and then intraperitoneally at a dose of 10 mg / kg three times a week thereafter.

[0158] As a result, in the control group administered with IgG1 isoform antibodies, body weight continued to decrease and the disease index increased starting 2 weeks after PBMC injection, and all mice died on day 48. In addition, in the group administered with cyclosporine (CsA), body weight decreased and the disease index increased during the initial 2 weeks after PBMC injection, but after 2 weeks, body weight increased again and the disease index decreased compared to the control group, showing an improved effect, and a survival rate of 40% was observed on day 51, demonstrating efficacy in improving graft-versus-host disease (Fig. 11).

[0159] However, in the group administered anti-SLAMF7 antibody (SLAMF7-b4-E11s), there was a dose-dependent improvement in body weight and disease index, and in particular, on day 48, 100% survival was observed at 4 mg / kg, 8 mg / kg, and 16 mg / kg, and on day 51, 80% survival was observed at 4 mg / kg, and 100% survival was observed at 8 mg / kg and 16 mg / kg, thereby demonstrating significant therapeutic efficacy against graft-versus-host disease (Fig. 11).

[0160] From the above results, it was confirmed that the anti-SLAMF7 antibody of the present invention has a significant therapeutic effect on graft-versus-host disease at a dose of 4 mg / kg or higher.

[0161]

[0162] Example 6. Confirmation of the Mechanism of Action (MoA) of the Novel Anti-SLAMF7 Antibody

[0163] To confirm the mechanism of action of the anti-SLAMF7 antibody of the present invention, mouse blood was collected on day 23, which is day 2 after the end of antibody administration in the effective dose experiment of the antibody using the xenograft-based acute graft-versus-host disease mouse model of Example 5, and T cell analysis was performed.

[0164] As a result, it was confirmed that while the CD8 T cell ratios were similar in the control group administered with IgG1 isoforms and the cyclosporine group, the CD8 T cell ratios were significantly reduced at 4 mg / kg, 8 mg / kg, and 16 mg / kg in the anti-SLAMF7 antibody (SLAMF7-b4-E11s) group (Fig. 12). In addition, it was confirmed that the total number of T cells was significantly reduced at 4 mg / kg, 8 mg / kg, and 16 mg / kg in the anti-SLAMF7 antibody (SLAMF7-b4-E11s) group compared to the control group and the cyclosporine group (Fig. 12).

[0165] From the above results, it was confirmed that the anti-SLAMF7 antibody of the present invention is effective in preventing, improving, or treating graft-versus-host disease by reducing the number of activated T cells that cause graft-versus-host disease.

[0166]

[0167] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0168] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

1. An anti-SLAMF7 antibody or an antigen-binding fragment thereof that specifically binds to the SLAMF7 (SLAM family member 7) protein.

2. In Paragraph 1, The above SLAMF7 protein is an anti-SLAMF7 antibody or an antigen-binding fragment thereof that is expressed on the surface of transplant-specifically activated T cells.

3. In Paragraph 1, The anti-SLAMF7 antibody or its antigen-binding fragment comprises any one selected from the group consisting of (a) to (g) below: (a) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 1, a CDR2 having the amino acid sequence of SEQ ID NO. 2, and a CDR3 having the amino acid sequence of SEQ ID NO. 3; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 4, a CDR2 having the amino acid sequence of SEQ ID NO. 5, and a CDR3 having the amino acid sequence of SEQ ID NO. 6; (b) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 7, a CDR2 having the amino acid sequence of SEQ ID NO. 8, and a CDR3 having the amino acid sequence of SEQ ID NO. 9; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 10, a CDR2 having the amino acid sequence of SEQ ID NO. 11, and a CDR3 having the amino acid sequence of SEQ ID NO. 12; (c) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 7, a CDR2 having the amino acid sequence of SEQ ID NO. 13, and a CDR3 having the amino acid sequence of SEQ ID NO. 3; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 14, a CDR2 having the amino acid sequence of SEQ ID NO. 15, and a CDR3 having the amino acid sequence of SEQ ID NO. 16; (d) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 17, a CDR2 having the amino acid sequence of SEQ ID NO. 13, and a CDR3 having the amino acid sequence of SEQ ID NO. 3; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 18, a CDR2 having the amino acid sequence of SEQ ID NO. 19, and a CDR3 having the amino acid sequence of SEQ ID NO. 20; (e) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 21, a CDR2 having the amino acid sequence of SEQ ID NO. 22, and a CDR3 having the amino acid sequence of SEQ ID NO. 23; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 24, a CDR2 having the amino acid sequence of SEQ ID NO. 25, and a CDR3 having the amino acid sequence of SEQ ID NO. 26; (f) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 7, a CDR2 having the amino acid sequence of SEQ ID NO. 27, and a CDR3 having the amino acid sequence of SEQ ID NO. 28; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 29, a CDR2 having the amino acid sequence of SEQ ID NO. 30, and a CDR3 having the amino acid sequence of SEQ ID NO. 31; and (g) a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 32, a CDR2 having the amino acid sequence of SEQ ID NO. 33, and a CDR3 having the amino acid sequence of SEQ ID NO. 34; and a heavy chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO. 35, a CDR2 having the amino acid sequence of SEQ ID NO. 36, and a CDR3 having the amino acid sequence of SEQ ID NO.

37.

4. In Paragraph 1, The anti-SLAMF7 antibody or its antigen-binding fragment comprises any one selected from the group consisting of (i) to (vii) below: (i) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 38; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 39; (ii) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 40; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 41; (iii) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 42; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 43; (iv) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 44; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 45; (v) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 46; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 47; (vi) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 48; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 49; and (vii) a light chain variable region comprising the amino acid sequence of SEQ ID NO. 50; and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO.

51.

5. In Paragraph 1, The above anti-SLAMF7 antibody or its antigen-binding fragment inhibits the expression of immune checkpoint proteins expressed on the surface of immune cells.

6. In Paragraph 5, The above immune checkpoint protein is selected from the group consisting of PD-1 (Programmed cell death protein 1), PD-L1 (Programmed Death-Ligand 1), and CTLA-4 (Cytotoxic T-lymphocyte antigen 4), an anti-SLAMF7 antibody or an antigen-binding fragment thereof.

7. In Paragraph 1, The above anti-SLAMF7 antibody or its antigen-binding fragment is an anti-SLAMF7 antibody or its antigen-binding fragment that (i) inhibits the proliferation or activation of immune cells, or (ii) reduces the number of activated immune cells.

8. In Paragraph 5 or Paragraph 7, The above immune cells are CD8 + T cells, CD4 + An anti-SLAMF7 antibody or an antigen-binding fragment thereof selected from the group consisting of T cells, dendritic cells, B cells, natural killer (NK) cells, and macrophages.

9. In Paragraph 1, The anti-SLAMF7 antibody or its antigen-binding fragment is an anti-SLAMF7 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.

10. A nucleic acid molecule encoding the anti-SLAMF7 antibody of any one of claims 1 to 9 or the antigen-binding fragment thereof.

11. A recombinant expression vector comprising a nucleic acid molecule according to claim 10.

12. Host cells transformed with a recombinant expression vector according to claim 11.

13. An anti-SLAMF7 antibody of any one of claims 1 to 9 or an antigen-binding fragment thereof; and a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

14. A pharmaceutical composition for the prevention or treatment of graft-versus-host disease comprising, as an active ingredient, an anti-SLAMF7 antibody of any one of claims 1 to 9 or an antigen-binding fragment thereof.

15. In Paragraph 14, The above composition is a pharmaceutical composition that inhibits the proliferation or activation of immune cells.

16. A composition for diagnosing graft-versus-host disease comprising an anti-SLAMF7 antibody of any one of claims 1 to 9 or an antigen-binding fragment thereof.

17. A kit for diagnosing graft-versus-host disease comprising a composition for diagnosing graft-versus-host disease according to claim 16.

18. In Paragraph 17, The above kit is a kit selected from the group consisting of ELISA kits, protein chip kits, and rapid kits.

19. A method for providing information for the diagnosis of graft-versus-host disease, comprising the step of measuring the expression or activity level of SLAMF7 protein in a biological sample isolated from a subject using an anti-SLAMF7 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9.

20. In Paragraph 19, A method for providing information, wherein the biological sample is selected from the group consisting of tissue, cell, blood, serum, plasma, lymph fluid, saliva, and urine.

21. In Paragraph 19, A method for providing information that further includes a step of determining that there is a high probability of developing graft-versus-host disease when the above-mentioned SLAMF7 protein is higher than the reference value obtained from a control sample.

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