Anti-VSIG4 antibodies or antigen-binding fragments and uses thereof

Abstract

Disclosed are novel anti-VSIG4 (V-set Ig domain-containing protein 4) antibodies or antigen-binding fragments. Also provided are uses of these antibodies, including methods of treatment.

Description

Technical Field

[0001] The present invention relates to an anti-VSIG4 (V-set Ig domain-containing 4) antibody or antigen-binding fragment and use thereof. Background Art

[0002] Cancer cells' immune evasion mechanism is achieved by binding to immune checkpoint proteins present on the surface of T cells, inactivating cytotoxic T cells with killer activity. This provides the theoretical background for immune checkpoint inhibitors, which can restore T cell function and enhance T cell activation by targeting immune checkpoints, thereby killing virus-infected cells or cancer cells.

[0003] In 2010, the Food and Drug Administration approved immune checkpoint inhibitors as third-generation anticancer immunotherapies for the first time. Beginning with clinical trials for melanoma, a series of research results have been published, demonstrating remarkable efficacy in treating lung cancer, liver cancer, and other cancers. Over the past decade, immune checkpoint inhibitors have become a major topic worldwide. Because anticancer immunotherapies are antibodies produced to induce T cells to attack cancer cells, reported research results have shown remarkable efficacy even in combination with conventional anticancer agents. To date, multiple immune checkpoint proteins are known, including CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), PD-1 (programmed cell death protein 1), TIM-3 (T cell immunoglobulin and mucin-domain containing-3), LAG-3 (lymphocyte activation gene 3), TIGIT (T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains), and VISTA (v-domain Ig-containing suppressor of T cell activation).

[0004] V-set Ig domain protein 4 (VSIG4, CRIg or Z39Ig) is an immune checkpoint protein being studied in recent years. It is a B7 related family protein. VSIG4 is known to be highly expressed in liver, dendritic cells, neutrophils and resting macrophages, but is expressed at low levels in other organs including lungs, heart, spleen and lymph nodes, and is not expressed in T cells and B cells. VSIG4 and B7 family proteins share a conserved amino acid sequence, and VSIG4 has a complete IgV type domain and a cut IgC type domain (Vogt L. et al., J Clin Invest. (2006) 116: 2817-2826; Helmy KY. et al., Cell (2006) 124: 915-927). VSIG4 is known to inhibit the alternative complement pathway of complement activity by binding to the subunit C3b of convertase. In addition, it has been reported that VSIG4 can inhibit the proliferation of CD4+ and CD8+ T cells by binding to an unknown T cell receptor. The relationship between VSIG4 and the occurrence of autoimmune and / or inflammatory diseases has been studied, and it has been shown that soluble VSIG4-Fc fusion protein appears to prevent the occurrence of experimental autoimmune arthritis, uveitis, and hepatitis (He et al., Mol. Immunol. (2008) Molecular Immunology 45 (16): 4041-4047). However, it has also been recently reported that VSIG4 expression is associated with the regulation of anti-tumor immunity, such as the occurrence of lung cancer and the poor prognosis of high-grade glioma (Liao Y. et al., Lab Invest. (2014) 94: 706-715; Xu T. et al., Am J Transl Res. (2015) 7: 1172-1180). Furthermore, according to a study by Jung et al. (Hepatology (2012) 56(5):1838-48), there are differences in the binding sites of VSIG4 between anti-inflammatory and T cell inhibition.

[0005] Antibodies against VSIG4 have been described previously (see, for example, WO 2020 / 069507). However, these antibodies bind to only one of the two forms of the protein, mediating only partial inhibition of its activity.

[0006] Therefore, there is still a need to provide novel anti-VSIG4 antibodies that can establish optimal anti-tumor immunity. Summary of the Invention

[0007] Purpose

[0008] The purpose of the present disclosure is to provide a novel antibody or antigen-binding fragment thereof against VSIG4.

[0009] Another object of the present disclosure is to provide a composition comprising the above-mentioned antibody or antigen-binding fragment for treating cancer.

[0010] Technical methods for achieving the above objectives

[0011] To achieve the above objectives, the present invention provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to VSIG4. The antibodies disclosed herein bind to both the long and short forms of VSIG4, effectively inhibiting VSIG4-mediated anti-inflammatory signaling. Thus, the anti-VSIG4 antibodies disclosed herein activate an immune response in patients in need thereof, thereby conferring protective anti-tumor immunity.

[0012] The present disclosure particularly provides an anti-VSIG4 monoclonal antibody or antigen-binding fragment thereof having three heavy chain CDRs and three light chain CDRs, wherein the sequences of the CDRs are selected from the sequences listed in SEQ ID NOs. 3 to 58. More specifically, the antibodies disclosed herein comprise three heavy chain CDRs and three light chain CDRs as listed in Table 2.

[0013] The present disclosure further provides an anti-VSIG4 monoclonal antibody or an antigen-binding fragment thereof, which comprises any heavy chain variable region selected from the following amino acid sequences: SEQ ID NO: 129, 131, 133, 135, 137, 139, 141, 143, 145, 147 and 149; and any light chain variable region selected from the following amino acid sequences: SEQ ID NO: 130, 132, 134, 136, 138, 140, 142, 144, 146, 148 and 150, and an antigen-binding fragment of the monoclonal antibody.

[0014] In addition, the present invention further provides polynucleotides encoding the heavy chain variable region and the light chain variable region of the monoclonal antibody or the antigen-binding fragment thereof.

[0015] In addition, the present invention further provides an expression vector comprising the polynucleotide.

[0016] In addition, the present invention further provides a transformant transformed with the expression vector.

[0017] In addition, the present invention further provides a method for preparing a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to VSIG4 by culturing the transformant.

[0018] In addition, the present invention further provides a composition for stimulating an immune response, which comprises a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to VSIG4 as an effective ingredient.

[0019] In addition, the present invention further provides a pharmaceutical composition for treating cancer, comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to VSIG4 as an active ingredient.

[0020] In addition, the present invention further provides a method for treating cancer, which comprises administering the pharmaceutical composition for treating cancer to an individual.

[0021] In addition, the present invention further provides an antibody-drug conjugate having a drug linked to a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to VSIG4.

[0022] In addition, the present invention further provides a CAR (chimeric antigen receptor) protein, which comprises i) the above-mentioned antibody; ii) a transmembrane domain, and; iii) a CAR (chimeric antigen receptor) having an intracellular signaling domain, characterized in that the binding of the above-mentioned i) antibody to the antigen causes T cell activation.

[0023] In addition, the present invention further provides a multispecific antibody comprising a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to VSIG4.

[0024] Beneficial effects

[0025] Since the novel antibodies and antigen-binding fragments thereof that bind to VSIG4 of the present invention can bind to VSIG4 to inhibit the activity of VSIG4, they are expected to be advantageously used in the development of various immunotherapeutic agents for VSIG4-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The structure and expression of hVSIG4(S) and hVSIG4(L) are shown. (A) Diagram illustrating the structures of the two forms of the VSIG4 protein (based on Small et al., Swiss Med Wkly. (2016) 146: w14301). (B) Western blot analysis of hVSIG4(L) and hVSIG4(S) expression in macrophages: rechVSIG4: recombinant hVSIG4 (long and short); 264, 265, and 266: donors from which PBMs were isolated. AF4646: polyclonal anti-VSIG4 antibody (R&D Systems, Minneapolis, MN, USA).

[0027] Figure 2 hVSIG4(S) and hVSIG4(L) were shown to inhibit CD4 +T cell activation. In the presence of recombinant proteins (hVSIG4(L)-Fc, hVGIG4(S)-Fc, PDL1-Fc (R&D Systems 156-B7) or isotype control hIgG1 (c9G4)), CD4 + Cells were exposed to anti-CD3 OKT3 antibody (BioxCell ref BE0001-2 clone OKT3). CD4 + T cell proliferation (A) and IFNγ release (B).

[0028] Figure 3 is a diagram illustrating the method disclosed herein for screening for a monoclonal antibody that specifically binds to VSIG4.

[0029] Figure 4 This is a diagram illustrating the expression vector for the VSIG4 antigen protein.

[0030] Figure 5 Shown are the SDS-PAGE results of the purified VSIG4 antigen protein.

[0031] Figure 6 Shown are the results of multi-phage ELISA performed to detect the specificity of the positive multi-scFv-phage antibody pools to the antigen, which were obtained through each round (ie, the first, second, and third rounds) of the panning process.

[0032] Figure 7 Shown are the results of ELISA performed to select positive phages having excellent binding properties to the antigen VSIG4.

[0033] Figure 8 Shown are the results of SDS-PAGE analysis of 11 recombinant VSIG4 single human antibodies.

[0034] Figure 9 Shown are the results of FACS analysis of transformed cells overexpressing human VSIG4 using an anti-human VSIG4 antibody linked to an APC fluorescent material.

[0035] Figure 10 The results of FACS analysis of the binding specificity of 11 human VSIG4 antibodies to cells overexpressing human VSIG4 are shown. (A) HEK293E: Non-specific binding assay. (B) hVSIG4 / HEK293E: Specific binding to cell surface VSIG4.

[0036] Figure 11Illustration of the binding of 11 human monoclonal anti-VSIG4 antibodies to hVSIG4(S) and hVSIG4(L). (A) Binding of the original scFv versions of 11 human anti-VSIG4 antibodies to hVSIG4(S) and hVSIG4(L) was determined by ELISA. (B) Binding of 11 full-length human anti-VSIG4 antibodies to hVSIG4(S) and hVSIG4(L) was determined by Western blotting. NRH: non-reduced, heated; RH: reduced, heated.

[0037] Figure 12 Murine m6H8 and its humanized version, hz6H8-A2, bind to hVSIG4(L) but not hVSIG4(S). (A) Western blot: rechVSIG4: recombinant hVSIG4 (long and short); 264, 265, and 266: donors from which PBMS were isolated. AF4646: polyclonal anti-VSIG4 antibody (R&D Systems, Minneapolis, MN, USA). (B) ELISA using hVSIG4-His (short form) and hVSIG4 Fc (long form); m9G4: isotype control, goat IgG control: negative control.

[0038] Figure 13 The results of an ELISA for epitope mapping of 11 scFv human monoclonal anti-VSIG4 antibodies using defined groups of 8 epitopes are shown. The group numbering does not relate to either the position of the sequence or the 3D structure of the antigen.

[0039] Figure 14 is a diagram illustrating the method disclosed herein for testing 11 full-length human monoclonal anti-VSIG4 antibodies in an inflammation assay.

[0040] Figure 15 is a diagram illustrating the method disclosed herein for testing 11 full-length human monoclonal anti-VSIG4 antibodies in an immunosuppression assay. DETAILED DESCRIPTION

[0041] The present invention will be more fully understood from the detailed description and accompanying drawings given herein, which are given by way of illustration only and do not limit the intended scope of the present invention.

[0042] definition

[0043] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the fields of chemistry, biochemistry, cell biology, molecular biology and medical sciences.

[0044] The term "about" or "approximately" refers to the normal error range of a given value or range known to those skilled in the art. It generally means within 20%, such as within 10%, or within 5% (or 1% or less) of a given value or range.

[0045] As used herein, "administer" or "administration" refers to the act of injecting or otherwise physically delivering a substance present outside the body (e.g., an anti-VSIG4 antibody provided herein) into a patient's body, such as by mucosal, intradermal, intravenous, intramuscular delivery and / or any other physical delivery method described herein or known in the art. When treating a disease or its symptoms, administration of the substance generally occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, administration of the substance generally occurs before the onset of the disease or its symptoms. The route of administration of the composition of the present invention can be any of a variety of routes, including oral and parenteral routes, as long as it allows the composition to be delivered to the target tissue. Specifically, administration can be performed by conventional methods of oral, colorectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intraocular or intradermal routes.

[0046] The terms "antibody" and "immunoglobulin" or "Ig" are used interchangeably herein. These terms are used herein in the broadest sense and specifically encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, chimeric antibodies, and antibody fragments of any isotype, such as IgG, IgM, IgA, IgD, and IgE, provided that the fragments retain the desired biological function. These terms are intended to include polypeptide products of B cells in the immunoglobulin class of polypeptides, which are capable of binding to a specific molecular antigen and are composed of two identical pairs of polypeptide chains interconnected by disulfide bonds, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain comprising a variable region of about 100 to about 130 or more amino acids, and each carboxyl-terminal portion of each chain comprising a constant region (see Borrebaeck (ed.) (1995) Antibody Engineering , 2nd edition, Oxford University Press.; Kuby (1997) Immunology,Third edition, WH Freeman and Company, New York). Each variable region of each heavy and light chain consists of three complementarity determining regions (CDRs, also called hypervariable regions) and four frameworks (FRs, more highly conserved parts of the variable domain), arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. In some embodiments, the antibodies provided herein can bind to specific molecular antigens, including target VSIG4 polypeptides, fragments or epitopes. Antibodies that react with specific antigens can be produced by recombinant methods, such as selecting a recombinant antibody library in a phage or similar vector, or by immunizing an animal with the antigen or a nucleic acid encoding the antigen.

[0047] Antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the above (referring to a portion of an antibody heavy chain or light chain polypeptide that retains some or all of the biological functions of the antibody from which the fragment is derived). The antibodies provided herein can be immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b).

[0048] The terms "anti-VSIG4 antibody," "antibody that binds to VSIG4," "antibody that binds to a VSIG4 epitope," and similar terms are used interchangeably herein to refer to an antibody that binds to a VSIG4 polypeptide (e.g., a VSIG4 antigen or epitope). Such antibodies include polyclonal and monoclonal antibodies, including chimeric, humanized, and human antibodies. Antibodies that bind to the VSIG4 antigen may have cross-reactivity with related antigens. In some embodiments, antibodies that bind to VSIG4 do not cross-react with other antigens (e.g., other peptides or polypeptides belonging to the B7 superfamily). Antibodies that bind to VSIG4 can be identified, for example, by immunoassays, BIAcore, or other techniques known to those skilled in the art. For example, an antibody binds to VSIG4, e.g., an antibody that specifically binds to VSIG4, when the antibody binds to VSIG4 with a higher affinity than any cross-reactive antigen, as determined using experimental techniques such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Generally, a specific or selective reaction will be at least two times the background signal or noise, and can be more than 10 times the background. For a discussion of antibody specificity, see, e.g., Paul, ed., 1989, Fundamental Immunology Second Edition , Raven Press, New York pp. 332-336. In some embodiments, the antibody " in conjunction with " the antigen of interest is such an antibody that binds to the antigen with enough affinity so that the antibody can be used as a diagnostic and / or therapeutic agent for cells or tissues that target the expression of the antigen, and does not significantly cross-react with other proteins. In such embodiments, as determined by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIPA), the degree of binding of the antibody to " non-target " protein will be less than about 10% of the binding of the antibody to its specific target protein. With regard to the combination of the antibody and the target molecule, for the epitope on a specific polypeptide or a specific polypeptide target, the term "specific binding" or "with ... specific binding" or "to ... having specificity" refers to a combination that is measurably different from non-specific interactions. For example, specific binding can be measured by comparing the combination of the molecule with the combination of a control molecule, and a control molecule is typically a molecule of a similar structure that does not have binding activity. For example, specific binding can be determined by competing with a control molecule similar to a target (such as an excess of unlabeled target). In this case, specific binding is indicated if excess unlabeled target competitively inhibits the binding of the labeled target to the probe. As used herein, the terms "specifically bind" or "binds specifically to" or "has specificity for" a particular polypeptide or epitope on a particular polypeptide target can be, for example, indicated by a molecule having a K for the target of D To show: at least about 10 -4 M, or at least about 10 -5M, or at least about 10 -6 M, or at least about 10 -7 M, or at least about 10 -8 M, or at least about 10 -9 M, or at least about 10 -10 M, or at least about 10 -11 M, or at least about 10 -12 In some embodiments, the term "specific binding" refers to binding in which a molecule binds to a specific polypeptide or an epitope on a specific polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In some embodiments, an antibody that binds to VSIG4 has a dissociation constant (K) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. D ).

[0049] As used herein, the term "antigen" refers to a predetermined antigen to which an antibody can selectively bind. The target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide, including, for example, a VSIG4 polypeptide.

[0050] The terms "antigen-binding fragment," "antigen-binding domain," "antigen-binding region," and similar terms refer to the portion of an antibody that comprises the amino acid residues that interact with the antigen and confer specificity and affinity to the binding agent for the antigen (e.g., the complementarity determining region (CDR)). The expression "antigen-binding fragment" of an antibody is intended to mean any peptide, polypeptide, or protein that retains the ability of the antibody to bind to its target (also commonly referred to as an antigen), typically to the same epitope, and comprises the following amino acid sequence: at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, or at least 200 consecutive amino acid residues of the amino acid sequence of the antibody. In a specific embodiment, the antigen-binding fragment comprises at least one CDR of the antibody from which it is derived. Still in a preferred embodiment, the antigen-binding fragment comprises 2, 3, 4 or 5 CDRs, more preferably 6 CDRs of the antibody from which it is derived.

[0051] "Antigen-binding fragments" may be selected from, but are not limited to, Fab, Fab', (Fab')2, Fv, scFv (sc stands for single chain), Bis-scFv, scFv-Fc fragments, Fab2, Fab3, minibodies, diabodies, triabodies, tetrabodies and nanobodies, and fusion proteins with disordered peptides such as XTEN (extended recombinant polypeptide) or PAS motifs, and any fragments whose half-life is increased by chemical modification, such as the addition of poly (alkylene) glycols, such as polyethylene glycol ("PEGylation") (PEGylated fragments are referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" means polyethylene glycol), or by incorporation into liposomes, wherein the fragment has at least one characteristic CDR of an antibody according to the present invention. Among antibody fragments, Fab has a structure comprising light and heavy chain variable regions, a light chain constant region and the first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' is different 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. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv is the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. Recombinant techniques for producing Fv fragments are described in International Publication WO88 / 10649, etc. In double-chain Fv (dsFv), the heavy chain variable region and the light chain variable region are linked to each other by a disulfide bond, while in single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are typically covalently linked to each other by a peptide linker. These antibody fragments can be obtained by using proteases (for example, Fab can be obtained by restrictive digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by restrictive digestion with pepsin), and can preferably be produced by genetic engineering techniques. Preferably, the "antigen-binding fragments" will be composed of or comprise a partial sequence of the heavy or light variable chain of the antibody from which they are derived, said partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, preferably at least 1 / 100, and more preferably at least 1 / 10, of the affinity of the antibody from which it is derived for the target. Antibodies:A Laboratory Manual , Cold Spring Harbor Laboratory, New York (1989); Myers (editor), Molec.Biology and Biotechnology:A Comprehensive Desk Reference, New York: VCH Publishers, Inc.; Huston et al., Cell Biophysics, 22: 189-224 (1993); Plückthun and Skerra, Meth. Enzymol., 178: 497-515 (1989) and in Day, ED, Advanced Immunochemistry A description of such antibody fragments can be found in Antibody 1990, 2nd ed., Wiley-Liss, Inc., New York, NY (1990).

[0052] As used herein, the term "binds" or "binding" refers to the interaction between molecules to form a complex that is relatively stable under physiological conditions. The interaction can be, for example, a non-covalent interaction, including hydrogen bonds, ionic bonds, hydrophobic interactions and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions or forces. The strength of the total non-covalent interaction between a single antigen binding site on an antibody and a single epitope of a target molecule (such as VSIG4) is the affinity of the antibody or functional fragment for that epitope. The association (k1) and dissociation (k2) of an antibody to a monovalent antigen are related to the affinity of the antibody or functional fragment for that epitope. -1 ) ratio (k1 / k -1 ) is the binding constant K, which is a measure of affinity. The value of K varies for different complexes of antibody and antigen and depends on k1 and k -1 . The association constant K of the antibodies provided herein can be determined using any of the methods provided herein or any other method well known to those skilled in the art. The affinity of one binding site does not always reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeated antigenic determinants (such as multivalent VSIG4) is in contact with an antibody containing multiple binding sites, the interaction between the antibody and the antigen at one site will increase the likelihood of a reaction at a second site. The strength of this multiple interaction between a multivalent antibody and an antigen is called avidity. The avidity of an antibody can be a better measure of its binding ability than the affinity of its single binding site. For example, high avidity can make up for the low affinity of pentameric IgM antibodies that are sometimes found. Pentameric IgM antibodies have an affinity lower than that of IgG, but the multivalence of IgM results in high avidity, enabling it to effectively bind to the antigen. Methods for determining whether two molecules are bound are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. In a specific embodiment, the antibody or antigen-binding fragment thereof binds to VSIG4 with an affinity that is at least two times greater than its affinity for binding to a non-specific molecule (such as BSA or casein). In a more specific embodiment, the antibody or antigen-binding fragment thereof binds only to VSIG4.

[0053] As used herein, the term "biological sample" or "sample" refers to a sample that has been obtained from a biological source, such as a patient or subject. As used herein, "biological sample" refers, inter alia, to a whole organism or a subset of its tissues, cells, or components (e.g., blood vessels (including arteries, veins, and capillaries), body fluids (including, but not limited to, blood, serum, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid, and semen). "Biological sample" further refers to a homogenate, lysate, or extract prepared from a whole organism or a subset of its tissues, cells, or components, or a fraction or portion thereof. Finally, "biological sample" refers to a culture medium containing cellular components (e.g., proteins or nucleic acid molecules), such as a nutrient broth or gel in which an organism is propagated.

[0054] As described herein, the term "biopanning" refers to the process of selecting phages from a phage library displaying peptides on the phage coat, wherein the selected phages only display peptides on the surface that have the property of binding to target molecules (e.g., antibodies, enzymes, and cell surface receptors). In one embodiment, biopanning as used herein includes four steps, wherein the first step is a step of preparing a phage library, the second step is a capture step involving contacting the phage library with the target molecule, the third step is a washing step involving removal of phages that do not bind to the target molecule, and the fourth step is an elution step of the phages of interest thereby recovered. Examples of biopanning are shown in the Examples of the present disclosure.

[0055] When used in the context of antibodies, the term "blocking" or its grammatical equivalents refers to an antibody that prevents or terminates the biological activity of the antigen to which it binds. Blocking antibodies include antibodies that bind to the antigen without causing a reaction but block subsequent binding or complexing of another protein with the antigen. The blocking effect of the antibody can result in a measurable change in the biological activity of the antigen.

[0056] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disease is a tumor or cancer. As used herein, "tumor" refers to all tumor cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. As referred to herein, the terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth. As used herein, "cancer" is any malignant tumor caused by the unwanted growth, invasion, and, under certain conditions, metastasis of damaged cells in an organism. The cells that cause cancer are genetically damaged and have typically lost the ability to control cell division, cell migration behavior, differentiation state, and / or cell death mechanisms. Most cancers form tumors, but some hematopoietic cancers (such as leukemias) do not form tumors. Therefore, "cancer" as used herein can include both benign and malignant cancers. As used herein, the term "cancer" specifically refers to any cancer that can be treated by the human antibodies of the present disclosure, without any limitation. Examples include, but are not limited to, liver cancer, breast cancer, kidney cancer, brain tumors, biliary tract cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, nasopharyngeal cancer, laryngeal cancer, lung cancer, ascending colon cancer, cervical cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, and multiple myeloma.

[0057] A "chemotherapeutic agent" is a chemical or biological agent (e.g., a pharmaceutical agent, including a small molecule drug or a biological agent (such as an antibody or cell)) that can be used to treat cancer, regardless of the mechanism of action. Chemotherapeutic agents include compounds used in targeted therapy and conventional chemotherapy. Chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenic agents, antiestrogens, antiandrogens, or immunomodulators.

[0058] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In one embodiment, a "chimeric antibody" is an antibody in which the constant region, or a portion thereof, is altered, replaced, or exchanged so that the variable region is linked to a constant region from a different species or belonging to another antibody class or subclass. In another embodiment, a "chimeric antibody" is an antibody in which the variable region, or a portion thereof, is altered, replaced, or exchanged so that the constant region is linked to a variable region from a different species or belonging to another antibody class or subclass.

[0059] As used herein, "CDR" refers to one of the three hypervariable regions (H1, H2, or H3) within the non-framework region of the immunoglobulin (Ig or antibody) VH β-sheet framework, or one of the three hypervariable regions (L1, L2, or L3) within the non-framework region of the antibody VL β-sheet framework. Thus, CDRs are variable region sequences interspersed within the framework region sequences. CDR regions are well known to those skilled in the art and have been defined by, for example, Kabat as the most hypervariable region within the variable (V) domain of an antibody (Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat, Adv. Prot. Chem. 32: 1-75 (1978)). Kabat CDRs are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers to the position of structural loops (Chothia and Lesk, J Mol. Biol. 196:901-917 (1987)). CDR region sequences are also structurally defined by Chothia as those residues that are not part of the conserved β-sheet framework and are therefore able to adopt different conformations (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). When numbered using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). Both terms are recognized in the art. CDR region sequences have also been defined by AbM, Contact, and IMGT. The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on analysis of available complex crystal structures. A universal numbering system has recently been developed and widely adopted by ImmunoGeneTics (IMGT) Information. (Lafranc et al., Dev. Comp. Immunol. 27(1):55-77(2003)). IMGT universal numbering has been defined to compare variable domains regardless of antigen receptor, chain type or species [Lefranc M.-P., Immunology Today 18, 509(1997) / Lefranc M.-P., The Immunologist, 7, 132-136(1999)]. In the IMGT universal numbering, conserved amino acids always have the same position, for example, cysteine ​​23 (1st-CYS), tryptophan 41 (CONSERVED-TRP), hydrophobic amino acid 89, cysteine ​​104 (2nd-CYS), phenylalanine or tryptophan 118 (J-PHE or J-TRP). The IMGT universal numbering provides standardized delimitations for framework regions (FR1-IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104, FR4-IMGT: 118 to 128) and complementarity determining regions (CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65, CDR3-IMGT: 105 to 117). Since gaps represent unoccupied positions, the CDR-IMGT length (shown between brackets and separated by dots, e.g., [8.8.13]) becomes key information. The IMGT universal numbering is used for 2D graphical representations, named IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)], and for 3D structures in IMGT / 3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P., T cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)]. The positions of the CDRs within the variable domains of typical antibodies have been determined by comparing a variety of structures (Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); Morea et al., Methods 20:267-279 (2000)). Since the number of residues within the hypervariable regions varies among different antibodies, additional residues associated with the typical positions are typically numbered with a, b, c, etc., next to the residue numbers in the typical variable domain numbering scheme (Al-Lazikani et al., supra (1997)).Such nomenclature is also well known to those skilled in the art.

[0060] The hypervariable region may comprise an "extended hypervariable region" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in VL, and 26-35 or 26-35A (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residue number is 25 according to Kabat et al., supra. As used herein, the terms "HVR" and "CDR" are used interchangeably.

[0061] As used herein, "check point inhibitors" refer to molecules that target immune checkpoints and block the immune checkpoint functions, such as small molecules, soluble receptors, or antibodies. More specifically, as used herein, "check point inhibitors" are molecules that can inhibit or otherwise reduce one or more biological activities of immune checkpoints, such as small molecules, soluble receptors, or antibodies. In some embodiments, inhibitors of immune checkpoint proteins (such as antagonistic antibodies provided herein) can, for example, play a role by inhibiting or otherwise reducing the activation and / or cell signaling pathways of cells (such as T cells) expressing the immune checkpoint proteins, thereby inhibiting the biological activity of the cell relative to the biological activity in the absence of an antagonist. Examples of immune checkpoint inhibitors include small molecule drugs, soluble receptors, and antibodies.

[0062] The term "constant region" or "constant domain" refers to the carboxyl-terminal portion of the light and heavy chains that is not directly involved in binding the antibody to the antigen, but exhibits various effector functions, such as interacting with Fc receptors. The term refers to a portion of the immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin (i.e., the variable domain, which contains the antigen-binding site). The constant domain contains the CH1, CH2, and CH3 domains of the heavy chain and the CL domain of the light chain.

[0063] As described herein, "cytotoxic agent" refers to an agent that, when administered to a subject, treats or prevents the development of cell proliferation, preferably cancer, in a subject by inhibiting or preventing cell function and / or causing cell death. Cytotoxic agents that can be used in the antibody drug conjugates of the present invention include any agent, portion or residue thereof that has a cytotoxic effect or inhibitory effect on cell proliferation. Examples of such agents include (i) chemotherapeutic agents that can act as tubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors or DNA chelators; (ii) protein toxins with enzymatic function; (iii) radioactive isotopes (radionuclides). Cytotoxic agents can be conjugated to antibodies (such as anti-VSIG4 antibodies) to form immunoconjugates. Preferably, the cytotoxic agent is released from the antibody under specific conditions (e.g., under acidic conditions), thereby therapeutically affecting the target cells, for example, by preventing the proliferation of target cells or by displaying a cytotoxic effect.

[0064] As used herein, the term "reduced" refers to a subject's level of a biomarker (e.g., VSIG4) that is at least 1-fold (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000-fold or more) lower than a reference value. "Reduced" in reference to a subject's level of a biomarker (e.g., VSIG4) also refers to a level that is at least 5% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%) lower than the level of a reference sample or relative to a reference value for the marker.

[0065] As used herein, the term "detecting" encompasses quantitative or qualitative detection.

[0066] As used herein, the term "detectable probe" or "detectable agent" refers to a composition that provides a detectable signal. The term refers to a substance that can be used to determine the presence or absence of a desired molecule (e.g., an antibody provided herein) in a sample or subject. A detectable agent can be a substance that can be visualized or can be determined and / or measured (e.g., by quantification) in other ways. The term includes, but is not limited to, any fluorophore, chromophore, radiolabel, enzyme, antibody, or antibody fragment that provides a detectable signal through its activity.

[0067] As used herein, "diagnosis" or "identifying a subject as having" refers to the process of identifying a disease, condition, or injury by its signs and symptoms. Diagnosis is particularly the process of determining whether an individual has a disease or condition (e.g., cancer). Cancer is diagnosed, for example, by detecting the presence of a marker associated with cancer (e.g., VSIG4).

[0068] The term "encoding" or its grammatical equivalents when used in reference to a nucleic acid molecule refers to a nucleic acid molecule that can be transcribed to produce mRNA in its native state or when manipulated by methods well known to those skilled in the art, which is then translated into a polypeptide and / or fragments thereof. The antisense strand is the complementary strand of such a nucleic acid molecule, from which the coding sequence can be deduced.

[0069] An "effective amount" or "therapeutically effective amount" of an agent (e.g., a pharmaceutical formulation) refers to an amount effective to elicit a desired biological response in a subject at the necessary dosage and time period. Such responses include: alleviating the symptoms of the disease or condition being treated, preventing, inhibiting, or delaying the recurrence of symptoms of the disease or the disease itself, increasing the lifespan of the subject compared to not receiving treatment, or preventing, inhibiting, or delaying the progression of symptoms of the disease or the disease itself. An "effective amount" is particularly an amount of an agent that is effective to achieve the desired therapeutic or preventive result. More specifically, as used herein, an "effective amount" is an amount of an agent that confers a therapeutic benefit. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects of the agent outweigh any toxic or deleterious effects.

[0070] An effective amount can be administered in one or more administrations, applications or dosages. Such delivery depends on many variables, including the time period over which a single dosage unit will be used, the bioavailability of the medicament, the route of administration, etc. In some embodiments, an effective amount also refers to the amount of the antibody provided herein (e.g., an anti-VSIG4 antibody) that achieves a specific result (e.g., inhibiting the biological activity of immune checkpoints, such as regulating T cell activation). In some embodiments, the term refers to an amount of a therapy (e.g., an immune checkpoint inhibitor, such as an anti-VSIG4 antibody) sufficient to reduce and / or improve the severity and / or duration of a given disease, illness or condition and / or symptoms associated therewith. The term also encompasses the amount required for: reducing or improving the progression or progress of a given disease, illness or condition, reducing or improving the recurrence, development or onset of a given disease, illness or condition, and / or improving or enhancing the preventive or therapeutic effect of another therapy (e.g., a therapy other than the immune checkpoint inhibitor). In the context of cancer treatment, a therapeutic benefit refers to, for example, any improvement in the cancer, including any one or a combination of the following: stopping or slowing the progression of the cancer (e.g., from one stage of the cancer to the next), stopping or delaying the aggravation or deterioration of the symptoms or signs of the cancer, reducing the severity of the cancer, inducing remission of the cancer, inhibiting tumor cell proliferation, tumor size or number of tumors, or reducing the level of a biomarker indicative of the cancer. In some embodiments, the effective amount of the antibody is from about 0.1 mg / kg (mg antibody per kg of subject body weight) to about 100 mg / kg. In some embodiments, the effective amount of the antibody provided therein is about 0.1 mg / kg, about 0.5 mg / kg, about 1 mg / kg, 3 mg / kg, 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg (or ranges therein).

[0071] As used herein, the term "epitope" refers to an antigenic region to which an antibody binds, such as a VSIG4 polypeptide or a VSIG4 polypeptide fragment. Preferably, as used herein, an epitope is a local region on the surface of an antigen, such as a VSIG4 polypeptide or a VSIG4 polypeptide fragment, which is capable of binding to one or more antigen-binding regions of an antibody and has antigenic or immunogenic activity in an animal (such as a mammal, for example, a human) that is capable of eliciting an immune response. An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. An epitope with antigenic activity is a portion of a polypeptide to which an antibody binds, the binding being determined by any method well known in the art, such as by an immunoassay. An antigenic epitope is not necessarily immunogenic. An epitope is typically composed of chemically active surface group molecules (such as amino acids) and has specific three-dimensional structural characteristics as well as specific charge characteristics. In certain embodiments, an epitope may include a determinant that is a chemically active surface group molecule (such as a sugar side chain, a phosphoryl group, or a sulfonyl group), and in certain embodiments, may have specific three-dimensional structural characteristics and / or specific charge characteristics. Epitopes can be formed by continuous residues or by non-continuous residues that are close to each other by folding of antigenic proteins. Epitopes formed by continuous amino acids are usually retained when exposed to denaturing solvents, while epitopes formed by non-continuous amino acids are usually lost under said exposure. Typically, an antigen has several or many different epitopes and reacts with many different antibodies. The epitope to which an antibody binds can be determined by any epitope mapping technique known to those skilled in the art.

[0072] The terms "full-length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to an antibody in substantially its entire form, rather than an antibody fragment. Specifically, as used herein, "full-length antibody" includes those antibodies having heavy and light chains comprising an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. In some cases, intact antibodies may have one or more effector functions.

[0073] The term "glycosylation," as used herein, refers to the process used to deliver glycosyl groups to proteins. Glycosylation is achieved through the glycosyl transferase-mediated attachment of glycosyl groups to serine, threonine, asparagine, or hydroxylysine residues of target proteins. Glycosylated proteins are not only used as building blocks for living tissues but also play an important role in cell recognition on cell surfaces. Thus, according to the present invention, by altering the glycosylation or glycosylation pattern of the monoclonal antibodies or antigen-binding fragments thereof of the present invention, an enhanced effect of the antibody can be achieved.

[0074] When used in reference to antibodies, the term "heavy chain" refers to a polypeptide chain of about 50-70 kDa, wherein the amino-terminal portion comprises a variable region of about 120 to 130 or more amino acids and the carboxyl-terminal portion comprises a constant region. Based on the amino acid sequence of the heavy chain constant region, the constant region can be one of five different types, referred to as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ). Different heavy chains have different sizes: α, δ, and γ contain approximately 450 amino acids, while μ and ε contain approximately 550 amino acids. When combined with light chains, these different types of heavy chains produce the five well-known classes of antibodies, namely IgA, IgD, IgE, IgG, and IgM, including the four subclasses of IgG, namely IgG1, IgG2, IgG3, and IgG4. The heavy chain can be a human heavy chain.

[0075] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably to refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. The nucleic acid content of the progeny may not be identical to that of the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0076] "Human antibodies" are antibodies having an amino acid sequence corresponding to an antibody produced by a human and / or produced using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using various techniques known in the art, including phage display libraries as disclosed herein. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1): 86-95 (1991) methods described can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can also be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to an antigenic challenge but has had its endogenous loci disabled, such as an immune xenomouse (referring to XENOMOUSE). TMFor human antibodies generated by human B cell hybridoma technology, see, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0077] "Humanized" antibody refers to a chimeric antibody containing the minimum sequence derived from a non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (receptor antibody), wherein the residues from the receptor CDR are replaced with residues from a non-human species (such as mouse, rat, rabbit or non-human primate) CDR (donor antibody) with desired specificity, affinity and / or ability. In some cases, according to technology known to those skilled in the art (Jones et al., Nature, 321:522-525, 1986), some skeleton fragment residues (referred to as FR, representing framework) can be modified to maintain binding affinity. In some embodiments, the FR residues of human immunoglobulin are replaced with corresponding non-human residues. In certain embodiments, a humanized antibody will substantially comprise at least one, typically all of two variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of non-human antibodies, and all or substantially all of the FRs correspond to the FRs of human antibodies. A humanized antibody optionally can comprise at least a portion of an antibody constant region (Fc), typically a portion of a human immunoglobulin. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization. The goal of humanization is to reduce the immunogenicity of a xenogeneic antibody (such as a murine antibody) for introduction into humans while maintaining the full antigen binding affinity and specificity of the antibody. For further details, see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, eg, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5: 428-433 (1994); and US Pat. Nos. 6,982,321 and 7,087,409.

[0078] As used herein, "identifying," when referring to a subject having a disorder, refers to the process of evaluating a subject and determining that the subject has the disorder (eg, has cancer).

[0079] As used herein, the term "immune checkpoint" or "immune checkpoint protein" refers to certain proteins produced by certain types of immune system cells (such as T cells) and certain cancer cells. Such proteins regulate T cell function in the immune system. Notably, they help check the immune response and can prevent T cells from killing cancer cells. The immune checkpoint proteins achieve this result by interacting with specific ligands, which send signals into T cells and essentially shut down or inhibit T cell function. Inhibiting these proteins can lead to the restoration of T cell function and an immune response to cancer cells. Examples of checkpoint proteins include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules, expressed in all NK, γδ and memory CD8+ (αβ) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, IDO1, A2aR and various B7 family ligands.

[0080] As used herein, the term "increase" refers to a subject's level of a biomarker (e.g., VSIG4) that is at least 1-fold (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000-fold or more) higher than a reference value. "Increase" in reference to a subject's level of a biomarker (e.g., VSIG4) also refers to a level that is at least 5% (e.g., 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%) higher than a reference sample's level or relative to a reference value for the marker.

[0081] As used herein, "inhibitor" or "antagonist" refers to a molecule that can inhibit or otherwise reduce one or more biological activities of a target protein (such as any of the immune checkpoint proteins mentioned above).

[0082] An "isolated" antibody is one that has been separated from components of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0083] An "isolated" nucleic acid is a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0084] The term "K D " refers to the dissociation constant of a specific antibody-antigen interaction and is used as a measure of the affinity of the antibody for the antigen. A lower K D This means that the antibody has a high affinity for the antigen.

[0085] As used herein, the "level" of a biomarker (e.g., VSIG4) consists of a quantitative value of the biomarker in a sample (e.g., in a sample collected from a patient with cancer). In some embodiments, the quantitative value is not composed of the absolute value of the actual measurement, but rather consists of a final value that takes into account the signal-to-noise ratio that occurs under the assay format used, and / or takes into account a calibration reference value used to improve the reproducibility of cancer marker level measurements between assays. In some embodiments, the "level" of a biomarker (e.g., VSIG4) is expressed as arbitrary units because it is important that the same type of arbitrary units are compared (i) between assays, or (ii) between patients with cancer and others, or (iii) between assays performed on the same patient at different time periods, or (iv) between the biomarker level measured in a patient sample and a predetermined reference value (also referred to herein as a "cutoff" value).

[0086] When used in reference to antibodies, the term "light chain" refers to a polypeptide chain of about 25 kDa, wherein the amino-terminal portion comprises a variable region of about 100 to about 110 or more amino acids and the carboxyl-terminal portion comprises a constant region. The approximate length of a light chain is 211 to 217 amino acids. Based on the amino acid sequence of the constant domain, there are two different types, referred to as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. The light chain can be a human light chain.

[0087] As used herein, the term "monoclonal antibody" refers to an antibody derived from an almost homogeneous antibody population, wherein the population comprises the same antibody, except that some possible naturally occurring mutations can be found in minimal proportions. Monoclonal antibodies are derived from the growth of individual cell clones (such as hybridomas) and are characterized by a class and subclass of heavy chains and a type of light chain. As used herein, when a monoclonal antibody is presented to a single antigenic site (i.e., a single epitope), the antibody demonstrates specific binding to the site. Monoclonal antibodies can be produced by various methods well known in the relevant technical field.

[0088] As described herein, the term "PEGylation" refers to a processing method for increasing the retention time of an antibody in the blood by introducing polyethylene glycol into the above-mentioned monoclonal antibody or its antigen-binding fragment. Specifically, by carrying out PEGylation of polymer nanoparticles with polyethylene glycol, the hydrophilicity of the nanoparticle surface is improved, and therefore rapid degradation in the living body can be prevented by the so-called stealth effect, which prevents pathogens, waste and foreign matter introduced from the outside from being recognized by immune activity (including macrophages) in the human body and causing them to be engulfed and digested. Therefore, the retention time of an antibody in the blood can be increased by PEGylation. The PEGylation adopted in the present disclosure can be carried out by a method for forming an amino group based on the bond between the carboxyl group of hyaluronic acid and the amino group of polyethylene glycol, but is not limited thereto, and can be PEGylated by various methods. At this time, for the polyethylene glycol to be used, it is preferred to use a polyethylene glycol with a molecular weight of 100 to 1000 and a straight or branched structure, but is not particularly limited thereto.

[0089] As used herein, "percent identity" or "% identity" between two nucleic acid or amino acid sequences refers to the percentage of identical nucleotides or amino acid residues between the two sequences to be compared after optimal alignment, which is purely statistical, and the differences between the two sequences are randomly distributed along their lengths. Traditionally, the comparison of two nucleic acid or amino acid sequences is performed by comparing the sequences after optimal alignment, which can be performed by segment or by using an "alignment window". In addition to manual comparison, the sequences to be compared can also be optimally aligned by methods known to those skilled in the art.

[0090] For amino acid sequences that exhibit at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a reference amino acid sequence, preferred examples include those comprising the reference sequence, certain modifications, particularly deletions, additions or substitutions of at least one amino acid, as well as truncations or extensions. In the case of substitutions of one or more consecutive or non-consecutive amino acids, preferred substitutions are those in which the substituted amino acid is replaced with an "equivalent" amino acid. As used herein, the expression "equivalent amino acid" refers to any amino acid that may replace one of the structural amino acids without modifying the biological activity of the corresponding antibody and those embodiments defined below. Equivalent amino acids can be determined based on the structural homology of the equivalent amino acid with the amino acid it replaces, or based on the results of comparative biological activity tests between various antibodies that may be generated.

[0091] As non-limiting examples, Table 1 below summarizes possible substitutions that may be made without significantly altering the biological activity of the corresponding modified antigen binding protein; reverse substitutions can naturally be made under the same conditions.

[0092] Table 1

[0093]

[0094]

[0095] As used herein, the term "pharmaceutically acceptable" refers to a pharmaceutical composition approved by a regulatory agency of a federal or state government, or listed in the U.S. Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, more particularly in humans. More specifically, when referring to a carrier, the expression "pharmaceutically acceptable" means that the carrier is compatible with the other ingredients of the composition and is harmless to its receptor. Therefore, as used herein, the expression "pharmaceutically acceptable carrier" refers to a carrier or diluent for administration without stimulating living organisms, which does not inhibit the biological activity and characteristics of the compound. The type of carrier can be selected based on the intended route of administration. The amount of each carrier used can vary within the conventional range of this area. As pharmaceutically acceptable carriers in compositions prepared as liquid solutions, physiological saline, sterile water, buffered saline, albumin injection, glucose solution, maltodextrin solution, glycerol, and mixtures of one or more thereof can be used as sterile carriers suitable for living organisms. If necessary, common additives such as antioxidants, buffers, and antibacterial agents can be added. Furthermore, by additionally adding a diluent, a dispersant, a surfactant, a binder or a lubricant, the composition can be prepared into an injection preparation such as an aqueous solution, a suspension and an emulsion, a pill, a capsule, a granule or a tablet.

[0096] As used herein, the term "polyclonal antibody" refers to an antibody produced in the presence of or among one or more other different antibodies. Polyclonal antibodies are generally produced by B lymphocytes in the presence of several other B lymphocytes that produce different antibodies. Polyclonal antibodies are usually obtained directly from immunized animals.

[0097] As used herein, the term "reference value" refers to the expression level of the biomarker (e.g., VSIG4) considered in a reference sample. As used herein, a "reference sample" refers to a sample obtained from a subject known to be free of the disease, preferably two or more subjects, optionally from the general population. Suitable reference expression levels of the biomarkers can be determined by measuring the expression levels of the biomarkers in several suitable subjects, and such reference levels can be adjusted for a specific subject population. A reference value or reference level can be an absolute value; a relative value; a value with an upper or lower limit; a range of values; an average value; a median, mean, or a value compared to a specific control or baseline value. A reference value can be based on the value of a single sample, such as a value obtained from a sample from a tested subject, but at an earlier time point. A reference value can be based on a large number of samples, such as a subject population from a chronological age-matched group, or based on a sample pool that includes or does not include the sample to be tested.

[0098] A "subject" that can be subjected to the methods described herein can be any mammal, including humans, dogs, cats, cows, goats, pigs, swine, sheep, and monkeys. Human subjects can be referred to as patients. In one embodiment, a "subject" or "subject in need thereof" refers to a mammal that has cancer or is suspected of having cancer or has been diagnosed with cancer. As used herein, a "subject with cancer" refers to a mammal that has cancer or has been diagnosed with cancer. A "control subject" refers to a mammal that does not have cancer and is not suspected of having cancer.

[0099] As used herein, "treating" a disease in a subject or "treating" a subject with a disease refers to subjecting the subject to drug therapy, such as administration of a drug, to reduce or prevent the extent of the disease. For example, treatment results in a reduction in at least one sign or symptom of the disease or condition. Treatment includes, but is not limited to, administration of a composition (such as a pharmaceutical composition), and can be performed prophylactically or after the onset of a pathological event. Treatment may require administration of a medicament and / or treatment more than once.

[0100] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of an antibody heavy or light chain. The variable domain of the heavy chain may be referred to as the "V H The variable domain of the light chain may be referred to as "V L These domains are usually the most variable parts of antibodies and contain the antigen-binding site.

[0101] The term "vector" refers to a material for introducing a nucleic acid molecule into a host cell. In particular, as used herein, a "vector" is a nucleic acid molecule that can propagate another nucleic acid molecule connected thereto. An example of a vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA fragments can be connected. Another example of a vector is a viral vector, in which additional DNA fragments can be connected to the viral genome. Some vectors can replicate autonomously in the host cell in which they are introduced (for example, bacterial vectors and episomal mammalian vectors with bacterial replication origins). After importing the host cell, other vectors (for example, non-episomal mammalian vectors) can be integrated into the genome of the host cell, thereby replicating together with the host genome. Therefore, the term "vector" includes vectors as self-replicating nucleic acid structures and vectors that are integrated into the host cell genome into which they are introduced. Applicable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers that can be stably integrated into the host cell chromosome.

[0102] Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology are in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vectors. However, the present invention is intended to include such forms of expression vectors, such as bacterial plasmids, YACs, cosmids, retroviruses, EBV-derived episomes, and all other vectors known to technicians to be conveniently used to ensure the expression of the heavy and / or light chains of the antibody of interest (e.g., anti-VSIG4 antibodies). The skilled person will recognize that the polynucleotides encoding the heavy and light chains can be cloned into different vectors or the same vector.

[0103] The vector can include one or more selective marker genes and appropriate expression control sequences. The selective marker gene that can be included, for example, provides resistance to antibiotics or toxins, supplements nutritional deficiencies, or provides key nutrients that are not in the culture medium. The expression control sequence can include constitutive and inducible promoters, transcription enhancers, transcription terminators, etc. well known in the art. When two or more nucleic acid molecules (such as antibody heavy chain and light chain) are to be co-expressed, the two nucleic acid molecules can be inserted into, for example, a single expression vector or different expression vectors. For single vector expression, the encoding nucleic acid can be operably connected with a common expression control sequence, or connected with different expression control sequences, such as an inducible promoter and a constitutive promoter. Methods well known in the art can be used to confirm that nucleic acid molecules are introduced into host cells. Such methods include, for example, nucleic acid analysis, such as Northern blot or polymerase chain reaction (PCR) amplification of mRNA, or immunoblotting for gene product expression, or other suitable analytical methods for testing the expression of the nucleic acid sequence introduced or its corresponding gene product. Those skilled in the art understand that nucleic acid molecules are expressed in amounts sufficient to produce a desired product (e.g., an anti-VSIG4 antibody provided herein), and further understand that expression levels can be optimized using methods well known in the art to achieve adequate expression.

[0104] The term "VSIG4" or "VSIG4 polypeptide" and similar terms refer to a polypeptide ("polypeptide," "peptide," and "protein" are used interchangeably herein) encoded by the human V-set and immunoglobulin domain-containing 4 (VIG4) gene, located in the pericentromeric region of human chromosome X, also known in the art as immunoglobulin superfamily protein Z39IG, Z39IG, or complement receptor CRIg of the immunoglobulin superfamily. The VSIG4 gene sequence can be represented, for example, by a sequence having the following GenBank accession numbers, such as NM_007268.2, NM_001100431.1, NM_001184831.1, NM_001184830.1, or NM_001257403.1.

[0105] VSIG4 (V-set and Ig domain 4) is a protein containing v-set and immunoglobulin domains that is structurally related to the B7 family of immunomodulatory proteins. There are two different forms of VSIG4 protein in humans. The long form contains both constant (C2 type) and variable (V type) immunoglobulin domains, while the short form contains only V type immunoglobulin domains without C2 type. These two forms are Figure 1A. In one embodiment, the human VSIG4 protein has a sequence represented by the sequence of Uniprot Accession No. Q9Y279. In one embodiment, the long form human VSIG4 protein has a sequence represented by the sequence of Uniprot Accession No. Q9Y279-1. Preferably, the long form VSIG4 has a sequence listed in SEQ ID NO.1. In one embodiment, the short form human VSIG4 protein has a sequence represented by the sequence of Uniprot Accession No. Q9Y279-3. Preferably, the short form VSIG4 has a sequence listed in SEQ ID NO.2.

[0106] VSIG4 functions as a complement receptor, functionally inhibiting complement activity by binding complement iC3b and C3b fragments, thereby mediating the clearance of C3b-opsonized pathogens. VSIG4 expression has been observed to be restricted to tissue macrophages and has been shown to be downregulated in response to lipopolysaccharide (LPS) (Vogt et al. (2006) J. of Clin. Invest. 116:2817).

[0107] VSIG4 is an immune checkpoint protein with anti-inflammatory and immunosuppressive properties. Soluble VSIG4 fusion protein inhibits inflammation (Small et al., Swiss Med Wkly. (2016) 146: w14301), while the lack of VSIG4 triggers macrophage-mediated inflammation (Liao et al. (2014) Lab. Invest. 94: 706). This VSIG4 inhibition of macrophage activation appears to be independent of C3b (Li et al. (2017) Nat Commun. 8 (1): 1322). VSIG4 has a regulatory function in T cell activation (Vogt et al. (2006) J. of Clin. Invest. 116: 2817; Xu et al. (2010) Immunol Lett. 18; 128(1): 46-50; Jung et al. (2012) Hepatology. 56(5): 1838-48; Jung et al. (2015) Immunol Lett. 165(2): 78-83; Munawara et al. (2019) Front Immunol. 10; 10: 2892). In particular, VSIG4 is a strong negative regulator of T cell proliferation and IL-2 production, which is regulated by binding to an unidentified T cell ligand receptor (Vogt et al. (2006) J. of Clin. Invest. 116: 2817).

[0108] Like many immune checkpoint proteins, VSIG4 activity promotes tumor growth by promoting immune tolerance. Vsig4-deficient mice grow smaller tumors than wild-type mice, indicating that the loss of VSIG4 activates an immune response that prevents tumor growth. In patients diagnosed with non-small cell lung cancer, a large number of macrophages expressing VSIG4 were observed to infiltrate the tumor microenvironment (Liao et al. (2014) Lab. Invest. 94: 706). The VSIG4 gene is overexpressed in a variety of cancer cells such as lung cancer, ovarian cancer, breast cancer, liver cancer and multiple melanomas, and its effect is similar to that of oncogenes that suppress immune responses and promote tumor progression. High VSIG4 expression is indeed associated with high-grade gliomas and poor patient prognosis (Xu et al. (2015) Am. J. Transl. Res. 7: 1172).

[0109] Anti-VSIG4 antibody

[0110] Immune checkpoints play a crucial role in maintaining self-tolerance and limiting immune-mediated tissue damage under physiological conditions. VSIG4 is a type I transmembrane protein belonging to the B7-related immunoglobulin superfamily and is expressed on resting macrophages. VSIG4 is a co-inhibitory ligand that inhibits CD4 + and CD8 + T cell proliferation and IL-2 production negatively regulate T cell activation. Two forms of VSIG4 are known, a long form (huVSIG4(L)) and a short form (huVSIG4(S)), which differ in that the long form contains a membrane-proximal domain that is an IgC-type immunoglobulin domain.

[0111] The inventors have now shown that both forms are expressed in macrophages. Furthermore, both forms are functional: soluble versions of huVSIG4(L) or huVSIG4(S) inhibit human CD4 T cell proliferation and IFNγ production, as demonstrated by inhibition of T cell proliferation and IFNγ production. + T cell activation. Therefore, both the long and short forms of VSIG4 contribute to the protein's regulatory activity, meaning that both must be inhibited to relieve immunosuppression.

[0112] The present disclosure provides novel monoclonal antibodies that specifically bind to human VSIG4. More specifically, the present disclosure provides novel monoclonal antibodies that can bind to both the long and short forms of the protein. In addition, the antibodies disclosed herein further induce internalization after binding to VSIG4, thereby facilitating the removal of the receptor from the cell surface. This is in contrast to antibodies in the prior art, such as the antibody described in WO2020 / 069507, which can only bind to the long form of human VSIG4 and cannot trigger internalization.

[0113] The inventors have discovered that effective blockade of VSIG4 is achieved using the anti-VSIG4 antibodies disclosed herein. Indeed, these antibodies modulate anti-inflammatory functions and inhibit the immunosuppressive properties of VSIG4, as demonstrated by their ability to trigger the release of pro-inflammatory cytokines, block the secretion of anti-inflammatory cytokines by macrophages, and promote T cell activation. Therefore, the anti-VSIG4 antibodies disclosed herein can be used to generate anti-tumor immune responses in cancer patients.

[0114] In a first aspect, the present disclosure provides a monoclonal antibody or antigen-binding fragment thereof that specifically binds to human VSIG4. In one embodiment, the antibody is capable of binding to both the long form and the short form of human VSIG4. In one embodiment, the long form of the human VSIG4 protein has the sequence set forth in SEQ ID NO. 1. In one embodiment, the short form of the human VSIG4 protein has the sequence set forth in SEQ ID NO. 2.

[0115] In one embodiment, the anti-VSIG4 antibody induces internalization upon binding to VSIG4.

[0116] In one embodiment, internalization of the antibodies according to the invention can be assessed by immunofluorescence (as exemplified herein below) or any method or procedure known to those skilled in the art that is specific for the internalization mechanism.

[0117] After the antibody binds to the extracellular domain (ECD) of VSIG4, the complex VSIG4 / antibody is internalized, resulting in a decrease in the amount of VSIG4 on the cell surface. This decrease can be quantified by any method known to those skilled in the art, such as, as non-limiting examples, Western blotting, FACS, immunofluorescence, etc.

[0118] In one embodiment, this reduction, which reflects internalization, is therefore measured by FACS and expressed as the difference or delta between the mean fluorescence intensity (MFI) measured at 4°C and the MFI measured at 37°C, in both cases after 4 hours of incubation of cells with the antibody.

[0119] This Δ can be determined, for example, based on the MFI obtained with untreated cells and cells treated with the antibody, using i) VSIG4-transfected HEK293 cells incubated for 4 hours with the antibodies described herein and ii) a secondary antibody labeled with Alexa 488. This parameter is defined as calculated using the following formula: Δ(MFI 4℃ –MFI 37℃ ).

[0120] This difference between MFIs reflects VSIG4 downregulation, as MFI is proportional to the cell surface expression of VSIG4.

[0121] Advantageously, the antibodies or any antigen-binding fragments thereof described herein are those that trigger a Δ(MFI) of at least 280, preferably at least 370, on VSIG4-transfected HEK293 cells. 4℃ –MFI 37℃ ) monoclonal antibodies.

[0122] In more detail, the above-mentioned Δ can be measured according to the following procedure, which must be considered as an illustrative but non-limiting example:

[0123] a) contacting cells of interest with an antibody of the invention in cold (4° C.) or warm (37° C.) complete culture medium;

[0124] b) contacting the cells of step a) and untreated cells with a secondary antibody in parallel;

[0125] c) measuring the MFI (indicating the amount of VSIG4 present on the surface) of the treated and untreated cells by using a second labeled antibody capable of binding to the antibody of the present invention; and

[0126] d) Calculate Δ as the MFI obtained with treated cells minus the MFI obtained with untreated cells. From this ΔMFI the percentage of internalization can be determined as:

[0127] 100×(MFI 4℃ -MFI 37℃ ) / MFI 4℃

[0128] The antibody according to the present invention or any antigen-binding fragment thereof shows an internalization percentage on VSIG4-transfected HEK293 between 60% and 99%, preferably between 61% and 96%.

[0129] In one embodiment, the antibody or antigen-binding fragment thereof is capable of quantifying the antigen binding activity of the antibody at a concentration between 10×10 -10 to 1×10 -9 EC between M 50 Combined with VSIG4.

[0130] As used herein, “EC 50 ” refers to the 50% effective concentration. More precisely, the term half maximal effective concentration (EC 50 ) corresponds to the concentration of a drug, antibody, or toxicant that induces a response halfway between baseline and maximum after some specified exposure time. It is often used as a measure of drug potency. Thus, the EC of a graded dose-response curve is 50 The EC value of a quantal dose-response curve represents the concentration of compound at which 50% of the maximal effect is observed. 50The concentration of compound at which 50% of the population exhibits a response after a specified exposure duration. Concentration measurements typically follow an S-shaped curve, increasing rapidly after a period of relatively small concentration changes. This can be determined mathematically by deriving a line of best fit.

[0131] As a preferred embodiment, the EC determined herein is 50 Characterize the potency of antibodies binding to VSIG4 ECD exposed on human HEK293 cells. EC was determined using FACS analysis. 50 Parameters. EC 50 The parameters reflect the antibody concentration that achieves 50% of maximal binding to human IGF-1R expressed on human tumor cells. Each EC 50 The value is calculated as the midpoint of the dose-response curve. This parameter was chosen to represent physiological / pathological conditions.

[0132] As used herein, anti-VSIG4 monoclonal antibodies include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the above. Anti-VSIG4 monoclonal antibodies may be of human or non-human origin. Examples of non-human anti-VSIG4 antibodies include, but are not limited to, those of mammalian origin (e.g., monkeys, rodents, goats, and rabbits). Since each structure of a human antibody is derived from humans, it is less likely to produce an immune response than conventional humanized antibodies or mouse antibodies, so it has the advantage of not causing any undesirable immune response when administered to humans. Therefore, it can be very advantageously used as a therapeutic antibody. Therefore, anti-VSIG4 monoclonal antibodies for human therapeutic use are preferably humanized or fully human. More preferably, they are fully human.

[0133] According to one embodiment of the present invention, the antibodies described herein are human antibodies that specifically bind to VSIG4, and the present inventors produced these antibodies by biopanning a naive human single-chain Fv library using a phage display method.

[0134] In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. H and V L The DNA sequence of the V domain was amplified from an animal cDNA library (e.g., a human or mouse cDNA library of the affected tissue). H and V LThe DNA of the structural domain is recombined with the scFv linker and cloned into a phagemid vector. The vector is electroporated into E. coli, and the E. coli is infected with a helper phage. The phage used in these methods is usually filamentous phage, including fd and M13, and V H and V L The domain is typically recombinantly fused to phage gene III or gene VIII. Phage expressing an antigen binding domain that binds a specific antigen can be selected or identified using antigen, for example, using labeled antigen, or antigen bound or captured to a solid surface or beads. Examples of phage display methods that can be used to prepare the antibodies provided herein include those disclosed in 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; Burton et al., 1994, Advances in Immunology 57:191-280; PCT / GB91 / 01134; WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401, and WO 97 / 13844; and U.S. Patent Nos. 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.

[0135] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to produce whole antibodies (including human antibodies or any other desired antigen-binding fragments) and expressed in any desired host (including mammalian cells, insect cells, plant cells, yeast and bacteria), for example as described below. Techniques for recombinant production of Fab, Fab' and F(ab')2 fragments can also be used using methods known in the art, such as those disclosed in PCT Publication No. WO 92 / 22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al., 1988, Science 240:1041-1043.

[0136] To generate full antibodies, the VH or VL nucleotide sequence, restriction sites, and flanking sequences protecting the restriction sites can be used to amplify the VH or VL nucleotide sequence in the scFv clone. H or V L Sequence. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a VH constant region (e.g., a human γ4 constant region), and the PCR-amplified VL domain can be cloned into a vector expressing a VL constant region (e.g., a human κ or λ constant region). The VH and VL domains can also be cloned into a single vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transiently transfected cell line expressing a full-length antibody (e.g., IgG).

[0137] The antibodies produced according to the above method are antibodies with enhanced affinity for antigens. The term "affinity" refers to the property of specifically recognizing and binding to a specific antigenic site, and high affinity and the specificity of the antibody to the antigen are both important factors in the immune response. In the present invention, humanized heavy chain library cells are produced by random mutation of the heavy chain variable region, and the library cells are subjected to colony lift assay to screen out first variant clones with high antigen binding properties. The affinity of each clone is checked by performing competitive ELISA on the selected clones. In addition to this method, various methods for determining affinity for antigens can also be used, and surface plasmon resonance technology is an example of these methods.

[0138] In one embodiment, the anti-VSIG4 monoclonal antibodies disclosed herein specifically bind to an epitope within the VSIG4 protein. Specifically, the epitope to which the antibodies of the invention bind can be identified by determining which VSIG4 residues, when mutated, abolish antibody binding. In one embodiment, VSIG4 is a long variant. In another embodiment, VSIG4 is a short variant.

[0139] Preferably, the antibodies disclosed herein are antibodies that bind to at least one amino acid in one or more epitopes selected from:

[0140] a) epitope M1 comprising residues E24, V25, E27, V29 and / or T30 of the sequence set forth in SEQ ID No. 2;

[0141] b) epitope M2 comprising residues D36, N38, L39 and / or T42 of the sequence set forth in SEQ ID No. 2;

[0142] c) epitope M3 comprising residues Q59, G61, S62, D63 and / or V65 of the sequence set forth in SEQ ID No. 2;

[0143] d) epitope M4 comprising residues I77, A80, Y82 and / or Q83 of the sequence set forth in SEQ ID No. 2;

[0144] e) epitope M5 comprising residues H87, H90, K91 and / or V92 of the sequence set forth in SEQ ID No. 2;

[0145] f) epitope M6 comprising residues S97, Q99, S101 and / or T102 of the sequence set forth in SEQ ID No. 2;

[0146] g) epitope M7 comprising residues R108, S109, H110, T112 and / or E114 of the sequence set forth in SEQ ID No. 2;

[0147] h) epitope M8 comprising residues T119, P120, D121, N123, Q124 and / or V125 of the sequence set forth in SEQ ID No. 2;

[0148] More preferably, the antibodies disclosed herein are antibodies that bind:

[0149] a) at least one amino acid in M1;

[0150] b) at least one amino acid in M4 and optionally at least one residue in M3;

[0151] c) at least one amino acid in M7;

[0152] d) at least one amino acid in M8;

[0153] e) at least one amino acid from M7 and optionally at least one amino acid from M8; or

[0154] f) at least one amino acid in M3, at least one amino acid in M7, and at least one amino acid in M8, and optionally at least one residue in M2 and / or at least one residue in M4.

[0155] Binding of an anti-VSIG4 antibody to an epitope can be determined by any method or technique known to those skilled in the art, such as but not limited to radioactivity, Biacore, ELISA, flow cytometry, etc., or according to the methods described herein.

[0156] In one embodiment, the anti-VSIG4 monoclonal antibodies disclosed herein comprise three heavy chain CDRs and three light chain CDRs. Preferably, the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises three heavy chain CDRs and the light chain comprises three light chain CDRs.

[0157] Preferably, the antibodies disclosed herein comprise three heavy chain CDRs and three heavy chain CDRs, wherein the sequence of each CDR is selected from the sequences listed in SEQ ID No. 3-58.

[0158] In one embodiment, the anti-VSIG4 antibody comprises three heavy chain CDRs comprising a sequence selected from SEQ ID NO. 3, 4, 5, 9, 10, 11, 12, 13, 17, 18, 19, 23, 24, 26, 27, 28, 32, 33, 34, 37, 38, 39, 43, 44, 45, 49, 50, 51 and 55.

[0159] In one embodiment, the anti-VSIG4 antibody comprises three light chain CDRs comprising a sequence selected from SEQ ID NO. 6, 7, 8, 14, 15, 16, 20, 21, 22, 25, 29, 30, 31, 35, 36, 40, 41, 42, 46, 47, 48, 52, 53, 54, 56, 57 and 58.

[0160] A preferred embodiment provides an anti-VSIG4 antibody having a heavy chain comprising three heavy chain CDRs, wherein the heavy chain CDRs comprise a sequence selected from SEQ ID NO. 3, 4, 5, 9, 10, 11, 12, 13, 17, 18, 19, 23, 24, 26, 27, 28, 32, 33, 34, 37, 38, 39, 43, 44, 45, 49, 50, 51 and 55.

[0161] Another preferred embodiment provides an anti-VSIG4 antibody having a light chain comprising three light chain CDRs, wherein the light chain CDRs comprise a sequence selected from SEQ ID NO. 6, 7, 8, 14, 15, 16, 20, 21, 22, 25, 29, 30, 31, 35, 36, 40, 41, 42, 46, 47, 48, 52, 53, 54, 56, 57 and 58.

[0162] In another preferred embodiment, the anti-VSIG4 antibody comprises three heavy chain CDRs and three light chain CDRs, wherein the heavy chain CDRs comprise a sequence selected from SEQ ID NO. 3, 4, 5, 9, 10, 11, 12, 13, 17, 18, 19, 23, 24, 26, 27, 28, 32, 33, 34, 37, 38, 39, 43, 44, 45, 49, 50, 51 and 55; and the light chain CDRs comprise a sequence selected from SEQ ID NO. 6, 7, 8, 14, 15, 16, 20, 21, 22, 25, 29, 30, 31, 35, 36, 40, 41, 42, 46, 47, 48, 52, 53, 54, 56, 57 and 58.

[0163] In another preferred embodiment, the anti-VSIG4 antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises three heavy chain CDRs, wherein the heavy chain CDRs comprise a sequence selected from SEQ ID NO. 3, 4, 5, 9, 10, 11, 12, 13, 17, 18, 19, 23, 24, 26, 27, 28, 32, 33, 34, 37, 38, 39, 43, 44, 45, 49, 50, 51 and 55; and the light chain comprises three light chain CDRs, wherein the light chain CDRs comprise a sequence selected from SEQ ID NO. 6, 7, 8, 14, 15, 16, 20, 21, 22, 25, 29, 30, 31, 35, 36, 40, 41, 42, 46, 47, 48, 52, 53, 54, 56, 57 and 58.

[0164] More preferably, the antibodies disclosed herein are selected from:

[0165] a) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 3, 4 and 5 and three light chain CDRs of SEQ ID Nos. 6, 7 and 8;

[0166] b) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 9, 10 and 5 and three light chain CDRs of SEQ ID Nos. 6, 7 and 8;

[0167] c) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 11, 12 and 13 and three light chain CDRs of SEQ ID Nos. 14, 15 and 16;

[0168] d) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 17, 18 and 19 and three light chain CDRs of SEQ ID Nos. 20, 21 and 22;

[0169] e) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 23, 24 and 3 and three light chain CDRs of SEQ ID Nos. 6, 7 and 25;

[0170] f) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 26, 27 and 28 and three light chain CDRs of SEQ ID Nos. 29, 30 and 31;

[0171] g) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 32, 33 and 34 and three light chain CDRs of SEQ ID Nos. 35, 36 and 16;

[0172] h) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 37, 38 and 39 and three light chain CDRs of SEQ ID Nos. 40, 41 and 42;

[0173] i) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 43, 44 and 45 and three light chain CDRs of SEQ ID Nos. 46, 47 and 48;

[0174] j) an antibody comprising three heavy chain CDRs of SEQ ID Nos. 49, 50 and 51 and three light chain CDRs of SEQ ID Nos. 52, 53 and 54;

[0175] k) an antibody comprising the three heavy chain CDRs of SEQ ID Nos. 17, 18 and 55 and the three light chain CDRs of SEQ ID Nos. 56, 57 and 58.

[0176] In a preferred but non-limiting embodiment, the antibodies of the present invention are selected from:

[0177] a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 129 or any sequence showing at least 80% identity to SEQ ID No. 129, and three light chain CDRs of sequences SEQ ID Nos. 6, 7 and 8;

[0178] b) an antibody comprising, or consisting of, a heavy chain variable domain of sequence SEQ ID No. 131 or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 131, and three light chain CDRs of sequences SEQ ID Nos. 6, 7 and 8;

[0179] c) an antibody comprising, or consisting of, a heavy chain variable domain of sequence SEQ ID No. 133, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 133, and three light chain CDRs of sequences SEQ ID Nos. 14, 15 and 16;

[0180] d) an antibody comprising, or consisting of, a heavy chain variable domain of sequence SEQ ID No. 135, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 135, and three light chain CDRs of sequences SEQ ID Nos. 20, 21 and 22;

[0181] e) an antibody comprising, or consisting of, a heavy chain variable domain of sequence SEQ ID No. 137, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 137, and three light chain CDRs of sequences SEQ ID Nos. 6, 7 and 25;

[0182] f) an antibody comprising, or consisting of, a heavy chain variable domain of sequence SEQ ID No. 139, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 139, and three light chain CDRs of sequences SEQ ID Nos. 29, 30 and 31;

[0183] g) an antibody comprising, or consisting of, a heavy chain variable domain of sequence SEQ ID No. 141, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 141, and three light chain CDRs of sequences SEQ ID Nos. 35, 36 and 16;

[0184] h) an antibody comprising, or consisting of, a heavy chain variable domain of sequence SEQ ID No. 143, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 143, and three light chain CDRs of sequences SEQ ID Nos. 40, 41 and 42;

[0185] i) an antibody comprising, or consisting of, a heavy chain variable domain of sequence SEQ ID No. 145, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 145, and three light chain CDRs of sequences SEQ ID Nos. 46, 47 and 48;

[0186] j) an antibody comprising, or consisting of, a heavy chain variable domain of sequence SEQ ID No. 147, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 147, and three light chain CDRs of sequences SEQ ID Nos. 52, 53 and 54;

[0187] k) an antibody comprising, or consisting of, a heavy chain variable domain of sequence SEQ ID No. 149, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 149, and the three light chain CDRs of sequences SEQ ID Nos. 56, 57 and 58.

[0188] “Any sequence that exhibits at least 80%, preferably 85%, 90%, 95% or 98% identity to SEQ ID No. 129” is intended to mean sequences that exhibit the three heavy chain CDRs SEQ ID Nos. 3, 4 and 5, which, in addition to the sequences corresponding to the CDRs (i.e., SEQ ID Nos. 3, 4 and 5), exhibit at least 80%, preferably 85%, 90%, 95% or 98% identity to the complete sequence SEQ ID No. 129, wherein “in addition to the sequences corresponding to the CDRs” is intended to mean “in addition to the sequences corresponding to the CDRs”.

[0189] In another preferred but non-limiting embodiment, the antibody of the present invention is selected from:

[0190] a) an antibody comprising a light chain variable domain of sequence SEQ ID No. 130, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 130, and the three heavy chain CDRs of sequences SEQ ID Nos. 3, 4 and 5;

[0191] b) an antibody comprising a light chain variable domain of sequence SEQ ID No. 132, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 132, and the three heavy chain CDRs of sequences SEQ ID Nos. 9, 10 and 5;

[0192] c) an antibody comprising a light chain variable domain of sequence SEQ ID No. 134, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 134, and the three heavy chain CDRs of sequences SEQ ID Nos. 11, 12 and 13;

[0193] d) an antibody comprising a light chain variable domain of sequence SEQ ID No. 136, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 136, and the three heavy chain CDRs of sequences SEQ ID Nos. 17, 18 and 19;

[0194] e) an antibody comprising a light chain variable domain of sequence SEQ ID No. 138, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 138, and the three heavy chain CDRs of sequences SEQ ID Nos. 23, 24 and 3;

[0195] f) an antibody comprising a light chain variable domain of sequence SEQ ID No. 140, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 140, and the three heavy chain CDRs of sequences SEQ ID Nos. 26, 27 and 28;

[0196] g) an antibody comprising a light chain variable domain of sequence SEQ ID No. 142, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 142, and the three heavy chain CDRs of sequences SEQ ID Nos. 32, 33 and 34;

[0197] h) an antibody comprising a light chain variable domain of sequence SEQ ID No. 144, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 144, and the three heavy chain CDRs of sequences SEQ ID Nos. 37, 38 and 39;

[0198] i) an antibody comprising a light chain variable domain of sequence SEQ ID No. 146, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 146, and the three heavy chain CDRs of sequences SEQ ID Nos. 43, 44 and 45;

[0199] j) an antibody comprising a light chain variable domain of sequence SEQ ID No. 148, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 148, and the three heavy chain CDRs of sequences SEQ ID Nos. 49, 50 and 51; and

[0200] k) an antibody comprising a light chain variable domain of sequence SEQ ID No. 150, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 150, and the three heavy chain CDRs of sequences SEQ ID Nos. 17, 18 and 55.

[0201] By “any sequence that exhibits at least 80%, preferably 85%, 90%, 95% or 98% identity to SEQ ID No. 130” is intended a sequence that exhibits the three light chain CDRs SEQ ID Nos. 6, 7 and 8, which furthermore exhibits, outside the sequences corresponding to the CDRs (i.e. SEQ ID Nos. 6, 7 and 8), at least 80%, preferably 85%, 90%, 95% or 98% identity to the complete sequence SEQ ID No. 130.

[0202] One embodiment of the present invention relates to an antibody that recognizes VSIG4, said antibody being selected from the group consisting of:

[0203] a) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 129, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 129, and a light chain variable domain of sequence SEQ ID No. 130, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 130;

[0204] b) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 131 or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 131, and a light chain variable domain of sequence SEQ ID No. 132 or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 132;

[0205] c) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 133, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 133, and a light chain variable domain of sequence SEQ ID No. 134, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 134;

[0206] d) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 135, or any sequence that shows at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 135, and a light chain variable domain of sequence SEQ ID No. 136, or any sequence that shows at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 136;

[0207] e) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 137, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 137, and a light chain variable domain of sequence SEQ ID No. 138, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 138;

[0208] f) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 139, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 139, and a light chain variable domain of sequence SEQ ID No. 140, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 140;

[0209] g) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 141 or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 141, and a light chain variable domain of sequence SEQ ID No. 142 or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 142;

[0210] h) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 143, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 143, and a light chain variable domain of sequence SEQ ID No. 144, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 144;

[0211] i) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 145, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 145, and a light chain variable domain of sequence SEQ ID No. 146, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 146;

[0212] j) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 147, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 147, and a light chain variable domain of sequence SEQ ID No. 148, or any sequence showing at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 148; and

[0213] k) an antibody comprising a heavy chain variable domain of sequence SEQ ID No. 149, or any sequence that shows at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 1149, and a light chain variable domain of sequence SEQ ID No. 150, or any sequence that shows at least 80%, 85%, 90%, 95% or 98% identity to SEQ ID No. 150.

[0214] The monoclonal antibody or antigen-binding fragment thereof that specifically binds to VSIG4 according to one embodiment of the present invention is preferably an antibody selected from the following:

[0215] a) an antibody comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 129 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 130;

[0216] b) an antibody comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 131 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 132;

[0217] c) an antibody comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 133 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 134;

[0218] d) an antibody comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 135 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 136;

[0219] e) an antibody comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 137 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 138;

[0220] f) an antibody comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 139 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 140;

[0221] g) an antibody comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 141 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 142;

[0222] h) an antibody comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 143 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 144;

[0223] i) an antibody comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 145 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 146;

[0224] j) an antibody comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 147 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 148; and

[0225] k) an antibody comprising a heavy chain variable region described by the amino acid sequence of SEQ ID NO: 149 and a light chain variable region described by the amino acid sequence of SEQ ID NO: 150.

[0226] For greater clarity, Table 2 below illustrates the sequences of preferred antibodies (CDRs, framework, V H and V L ) and the epitope to which each of these antibodies binds.

[0227]

[0228]

[0229]

[0230] To the extent that they are capable of specifically recognizing VSIG4, the monoclonal antibodies or antigen-binding fragments thereof of the present invention may include not only the sequences of the anti-VSIG4 antibodies of the present invention described in this specification, but also their biological equivalents. For example, in order to further improve the binding affinity and / or other biological properties of the antibody, additional changes may be made to the amino acid sequence of the antibody. For example, these modifications include deletions, insertions and / or substitutions of the antibody amino acid sequence. Those modifications of amino acids are based on the relative similarity between the amino acid side chain substituents (e.g., hydrophobicity, hydrophilicity, charge, size, etc.). Based on an analysis of the size, shape, and type of amino acid side chain substituents, it was found that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine are similar in size; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, it can be said that biologically, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are functional equivalents.

[0231] In one embodiment, the anti-VSIG4 monoclonal antibodies described herein may be in the form of full-length antibodies, multi-chain or single-chain antibodies, such antibody fragments that selectively bind to PVSIG4 (including but not limited to Fab, Fab', (Fab')2, Fv and scFv), surrobody (including surrogate light chain constructs), single domain antibodies, humanized antibodies, camelized antibodies, etc. They may also be or be derived from any isotype, including, for example, IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4), or IgM. In some embodiments, the anti-VSIG4 antibody is IgG (e.g., IgG1, IgG2, IgG3 or IgG4). In one embodiment, the antibody further comprises a human constant region. In a further embodiment, the human constant region is selected from IgG1, IgG2, IgG2, IgG3, IgG4. In a further specific embodiment, the human constant region is IgG1. In addition, the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and it has gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2) as subclasses. The light chain constant region has kappa (κ) and lambda (λ) types.

[0232] Anti-VSIG4 antibodies include labeled antibodies that can be used in diagnostic applications. Antibodies can be used for diagnosis, such as detecting the expression of a target of interest in specific cells, tissues or serum; or as part of a clinical testing procedure, such as to determine the efficacy of a given treatment regimen, monitoring the development or progression of an immune response. Detection can be facilitated by coupling the antibody to a detectable substance or "label." The label can be conjugated directly or indirectly to the anti-VSIG4 antibodies of the present disclosure. The label itself can be detectable (e.g., radioisotope labeling, isotope labeling, or fluorescent labeling), or in the case of an enzyme label, can catalyze a chemical change in a detectable substrate compound or composition. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. The detectable substance can be directly coupled or conjugated using techniques known in the art, or indirectly coupled or conjugated to the antibody (or its fragment) through an intermediate (e.g., a linker known in the art). Examples of enzyme labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, acetylcholinesterase, glucoamylase, lysozyme, carbohydrate oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, dimethylamine-1-naphthalenesulfonyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable isotopic materials include 13 C. 15 N and deuterium; examples of suitable radioactive materials include 125 I. 131 I. 111 In or 99 Tc.

[0233] Bispecific antibodies

[0234] Furthermore, the present disclosure provides multispecific antibodies comprising the monoclonal anti-VSIG4 antibodies or antigen-binding fragments thereof disclosed herein.

[0235] The above multispecific antibody of the present invention may preferably be a bispecific antibody, but is not limited thereto.

[0236] The multispecific antibody according to the present invention preferably has a form in which the anti-VSIG4 antibody described herein is bound to an antibody or fragment thereof having binding properties to an immune effector cell-specific target molecule. The immune effector cell-specific target molecule is preferably an immune checkpoint, but is not limited thereto. Examples of immune effector cell-specific target molecules include, for example, PD-1, PD-L1, CTLA-4, TIM-3, TIGIT, BTLA, KIR, A2aR, VISTA, B7-H3, TCR / CD3, CD16 (FcγRIIIa) CD44, Cd56, CD69, CD64 (FcγRI), CD89, and CD11b / CD18 (CR3).

[0237] Multispecific antibodies are antibodies that can simultaneously recognize different multiple (double or more) epitopes of the same antigen or two or more different antigens. Antibodies belonging to multispecific antibodies can be divided into scFv-based antibodies, Fab-based antibodies, IgG-based antibodies, etc. In the case of multispecific (e.g., bispecific) antibodies, two signals can be suppressed or amplified at the same time, so it can be more effective than suppressing / amplifying one signal. Compared with the case where each signal is treated with a signal inhibitor separately, low-dose administration can be achieved, and two signals can be suppressed / amplified simultaneously in the same space.

[0238] Methods for producing bispecific antibodies are well known. Typically, recombinant production of bispecific antibodies is based on the co-expression of a heavy chain / light chain pair of two immunoglobulins, where the two heavy chains have different specificities.

[0239] In the case of scFv-based bispecific antibodies, hybrid scFv-based antibodies are prepared in a heterodimeric form by combining the VL and VH of different scFvs to obtain diabodies (Holliger et al., Proc. Natl. Acad. Sci. USA, 90: 6444, 1993), and tandem ScFvs are produced by connecting different scFvs to each other. By expressing the CH1 and CL of Fab at the end of each scFv, heterodimeric miniantibodies can be produced (Muller et al., FEBSlett., 432: 45, 1998). In addition, by replacing some amino acids of the CH3 domain with the homodimer domain of Fc, the structure is changed to a "knob into hole" form to have a heterodimeric structure, and these modified CH3 domains are expressed at the end of each different scFv, thus miniantibodies in the form of heterodimeric scFvs can be produced (Merchant et al., Nat. Biotechnol., 16: 677, 1998).

[0240] In the case of Fab-based bispecific antibodies, antibodies in the form of heterodimeric Fabs can be produced by utilizing a combination of disulfide bonds or mediators based on individual Fab's directed against specific antigens, and by expressing scFvs directed against different antigens at the heavy chain or light chain end of a specific Fab, an antigen valency of 2 can be obtained. In addition, by having a hinge region between the Fab and the scFv, an antigen valency of 4 can be obtained in the form of a homodimer. In addition, a method for producing the following is known in the related art: a dual-target bibody, in which an antigen valency of 3 is obtained by fusing scFvs directed against different antigens to the light chain end and the heavy chain end of the Fab; a triple-target bibody, in which an antigen valency of 3 is obtained by fusing different scFvs to the light chain end and the heavy chain end of the Fab; and a simple form of a triple-target antibody F(ab')3, which is obtained by chemically fusing three different Fabs.

[0241] In the case of IgG-based bispecific antibodies, a method for producing bispecific antibodies by Trion Pharma is known, which is based on re-hybridizing mouse and rat hybridomas and producing by preparing hybrid hybridomas (called quadromas). In addition, a method for producing a bispecific antibody called a "Holes and Knob" format is known, in which some amino acids of the CH3 homodimer domain of Fc in different heavy chains are modified while sharing the light chain portion (Merchant et al., Nat. Biotechnol., 16: 677, 1998); and in addition to heterodimer-form bispecific antibodies, a method for producing a homodimer-form (scFv) 4-IgG is known, which is produced by fusing two different scFvs to the constant domains of the light and heavy chains of IgG instead of the variable domains and then expressing them. In addition, according to ImClone Systems, a chimeric monoclonal antibody based on IMC-1C11 for human VEGFR-2 was fused only to the amino terminus of the antibody light chain with a single variable domain of mouse platelet-derived growth factor receptor-α, thereby producing a bispecific antibody. In addition, Rossi et al. reported an antibody with high antigenicity for CD20, which was based on a method called "dock and lock (DNL)" using the dimerization and docking domain (DDD) of the protein kinase A (PKA) R subunit and the anchoring domain of PKA (Rossi et al., Proc. Natl. Acad. Sci. USA, 103: 6841, 2006).

[0242] Antibody derivatives

[0243] The anti-VSIG4 antibodies of the present invention can be further modified to contain additional non-protein moieties that are known in the art and readily available. In particular, anti-VSIG4 monoclonal antibodies that are derivatized, covalently modified, or conjugated to other molecules are included herein for diagnostic and therapeutic applications. For example, but not limited to, derivative antibodies include antibodies that have been modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a variety of chemical modifications can be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivatives may contain one or more non-classical amino acids.

[0244] In particular, the monoclonal antibodies of the present invention or antigen-binding fragments thereof can be derivatized as described above, in particular by, for example, glycosylation and / or PEGylation, to increase the residence time in the living body to which the antibody is administered.

[0245] With respect to glycosylation and / or PEGylation, various patterns of glycosylation and / or PEGylation can be modified by methods well known in the art as long as the function of the antibody of the present invention is maintained, and the antibodies of the present invention include variant monoclonal antibodies or antigen-binding fragments thereof in which various patterns of glycosylation and / or PEGylation are modified.

[0246] Preferably, the part that is suitable for antibody derivatization is a water-soluble polymer.The non-limiting example of water-soluble polymer includes but is not limited to polyethylene glycol (PEG), copolymer of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (such as glycerol), polyvinyl alcohol and its mixture.Polymer can have any molecular weight, and can be side chain or non-branched.The quantity of the polymer that is connected to the antibody may be different, and if the polymer that is connected is more than one, they can be identical or different molecules.Generally, the quantity and / or type of the polymer that is used for derivatization can be determined based on including but not limited to the following considerations: whether the specific properties or function of antibody to be improved, antibody derivatives will be used for treatment under prescribed conditions.

[0247] In specific embodiments, the anti-VSIG4 antibodies of the present disclosure can be linked to a poly(ethylene glycol) (PEG) moiety. In specific embodiments, the antibody is an antibody fragment, and the PEG moiety is linked via any available amino acid side chain or terminal amino acid functional group located in the antibody fragment, such as any free amino, imino, sulfhydryl, hydroxyl or carboxyl group. Such amino acids may be naturally present in the antibody fragment or may be engineered into the fragment using recombinant DNA methods. See, for example, U.S. Patent No. 5,219,996. Multiple sites can be used to link two or more PEG molecules. The PEG moiety can be covalently linked via the sulfhydryl group of at least one cysteine ​​residue located in the antibody fragment. When a sulfhydryl group is used as a point of attachment, an appropriately activated effector moiety, such as a sulfhydryl selective derivative (such as maleimide and cysteine ​​derivatives), can be used.

[0248] In a specific embodiment, the anti-VSIG4 antibody conjugate is a modified PEGylated Fab' fragment, i.e., having PEG (poly(ethylene glycol)) covalently attached thereto, for example, according to the methods disclosed in EP0948544. See also Poly(ethylene glycol) Chemistry, Biotechnical and Biomedical Applications, (J. Milton Harris (ed.), Plenum Press, New York, 1992); Poly(ethylene glycol) Chemistry and Biological Applications, (J. Milton Harris and S. Zalipsky, eds., American Chemical Society, Washington DC, 1997); and Bioconjugation Protein Coupling Techniques for the Biomedical Sciences, (M. Aslam and A. Dent, eds., Grove Publishers, New York, 1998); and Chapman, 2002, Advanced Drug Delivery Reviews 54:531-545. PEG can be attached to a cysteine ​​in the hinge region. In one embodiment, the PEG-modified Fab' fragment has a maleimide group covalently linked to a single sulfhydryl group in the modified hinge region. Lysine residues can be covalently linked to the maleimide group, and each amine group on the lysine residue can be linked to a methoxy poly (ethylene glycol) polymer having a molecular weight of approximately 20,000 Da. Thus, the total molecular weight of the PEG linked to the Fab' fragment can be approximately 40,000 Da.

[0249] In another embodiment, a conjugate of an antibody and a non-protein moiety is provided that can be selectively heated by exposure to radiation. In one embodiment, the non-protein moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including but not limited to, a wavelength that does not harm normal cells but heats the non-protein moiety to a temperature that kills cells in proximity to the antibody-non-protein moiety.

[0250] Immunoconjugates

[0251] In another aspect, the present disclosure provides immunoconjugates (interchangeably referred to as "antibody-drug conjugates" or "ADCs") comprising an anti-VSIG4 antibody as described herein conjugated to a cytotoxic agent.

[0252] Many cytotoxic agents have been isolated or synthesized that make it possible to inhibit cell proliferation or at least significantly, if not decisively, destroy or reduce tumor cells. However, the toxic activity of these agents is not limited to tumor cells; non-tumor cells can also be affected and destroyed. More specifically, side effects are observed on cells with rapid turnover, such as hematopoietic cells or epithelial cells, particularly mucosal cells. To limit side effects on normal cells while maintaining high cytotoxicity against tumor cells, immunoconjugates have been used to locally deliver cytotoxic agents in cancer therapy (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al. (2005) Nature Biotechnology 23(9):1137-1146; Payne, G. (2003) i 3:207-212; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Deliv. Rev. 26:151-172; U.S. Pat. No. 4,975,278). Immunoconjugates allow for the targeted delivery and intracellular accumulation of the drug moiety (i.e., cytotoxic agent) to tumors, where systemic administration of the unconjugated drug may result in unacceptable levels of toxicity to normal cells as well as the tumor cells being eliminated (Baldwin et al., Lancet (March 15, 1986) pp. 603-05; Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies '84: Biological And Clinical Applications (A. Pinchera et al., eds.) pp. 475-506). Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother. 21: 183-87).

[0253] The cytotoxic agent used in the immunoconjugates disclosed herein can be, but is not limited to, a drug (i.e., an "antibody-drug conjugate"), a toxin (i.e., an "immunotoxin" or an "antibody-toxin conjugate"), a radioisotope (i.e., a "radioimmunoconjugate" or an "antibody-radioisotope conjugate"), etc.

[0254] Preferably, the immunoconjugate is a binding protein linked to at least a drug or medicament. When the binding protein is an antibody or antigen-binding fragment thereof, such immunoconjugates are generally referred to as antibody-drug conjugates (or "ADCs").

[0255] In a first embodiment, such drugs can be described according to their mode of action. As non-limiting examples, there can be mentioned alkylating agents (such as nitrogen mustards, alkyl sulfonates, nitrosoureas, oxazophorins, aziridines or imines), antimetabolites, antitumor antibiotics, mitotic inhibitors, chromatin function inhibitors, anti-angiogenic agents, antiestrogens, antiandrogens, chelating agents, iron absorption stimulants, cyclooxygenase inhibitors, phosphodiesterase inhibitors, DNA inhibitors, DNA synthesis inhibitors, apoptosis stimulants, thymidylate inhibitors, T cell inhibitors, interferon agonists, ribonucleoside triphosphate reductase inhibitors, aromatase inhibitors, estrogen receptor antagonists, tyrosine kinase inhibitors, thiazolinone ... The present invention also includes a novel drug comprising a 5-amino acid kinase inhibitor, a cell cycle inhibitor, a taxane, a microtubule inhibitor, an angiogenesis inhibitor, a macrophage stimulator, a neurokinin receptor antagonist, a cannabinoid receptor agonist, a dopamine receptor agonist, a granulocyte-stimulating factor agonist, an erythropoietin receptor agonist, a somatostatin receptor agonist, a LHRH agonist, a calcium sensitizer, a VEGF receptor antagonist, an interleukin receptor antagonist, an osteoclast inhibitor, a free radical formation stimulator, an endothelin receptor antagonist, a vinca alkaloid, an antihormonal or immunomodulatory agent, or any other novel drug that meets the criteria for cytotoxic or toxin activity.

[0256] Such drugs are cited for example in VIDAL 2010 on the page of compounds attached to the section "Cytotoxic Agents" devoted to Oncology and Hematology, and these cytotoxic compounds cited in this document are cited here as preferred cytotoxic agents.

[0257] More specifically, the following drugs are preferred according to the present invention, but are not limited thereto: mechlorethamine, chloramine phenol, melphalan, chlorhydrate, pipobromide, prednimustine, disodium hydrogen phosphate, estramustine, cyclophosphamide, atriamine, trofosfamide, sulfaphosphamide, ifosfamide, thiotepa, triethylamine, altetramine, carmustine, streptozotocin, flumustine, lomustine, busulfan, trothiosulfate, iprosulfuron, dacarbazine, cisplatin, oxaliplatin, lobaplatin, epplatin, miplatin hydrate, carboplatin, methotrexate, pemetrexed, 5-fluorouracil, floxuridine, 5-fluorouracil, capecitabine, cytarabine, fludarabine, cytosine arabinoside, 6-mercaptopurine (6-MP), nelarabine, 6-thioguanine (6-TG), chlor Deoxyadenosine, 5-azacytidine, gemcitabine, cladribine, deoxycoformycin, tegafur, pentostatin, doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone, dactinomycin, mithramycin, plicamycin, mitomycin C, bleomycin, procarbazine, paclitaxel, docetaxel, vinblastine, vincristine, vindesine, vinorelbine, topotecan, irinotecan, etoposide, valrubicin, amrubicin hydrochloride, pirarubicin, iritinium acetate, daunorubicin, epirubicin, idarubicin and teniposide, razoxane, marimastat, batimastat, prinomastat, tanomastat, ilomastat, CGS-27023A, halofuginone, COL-3, neivastatin, thalidomide, CDC 501, DMXAA, L-651582, squalamine, endostatin, SU5416, SU6668, interferon-α, EMD121974, interleukin-12, IM862, angiostatin, tamoxifen, toremifene, raloxifene, droloxifene, iodoxifene, anastrozole, etrozole, flutamide, nilutamide, spironolactone, cyproterone acetate, finasteride, cimetidine, bortezomib, Velcade, bicalutamide, cyproterone acetate, flutamide, fulvestrant, Simestane, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, tretinoin, rexinoid, methoxsalen, aminolevulinic acid, aleucine, OCT-43, denileukin, interleukin-2, tasonamine, lentinan, cilofemidine, roquine, pidotimod, pegaptase, thymopentin, poly I:C, procodazole, TicBCG, corynebacteriumparvum), NOV-002, ukrain, levamisole, 1311-chTNT, H-101, simoleukin, interferon alpha 2a, interferon alpha 2b, interferon gamma 1a, interleukin-2, mobeleukin, Rexin-G, tesileukin, aclarubicin, actinomycin, arglabin, asparaginase, oncocytogenes, chromomycin, daunorubicin, folinic acid, masoprocol, neocarcinomatidylserine, peplomycin, sarcopeniain, australopithecin, trabectedin, streptozocin, testosterone, kunecatechins, sinecatechins, alitretinoin, belotecan hydrochloride, calutosterone, drostanolone, elixirsium acetate, ethinyl estradiol, etoposide glycosides, fluoxymesterone, formestane, fosfetrol, goserelin acetate, aminolevulinic acid hexyl ester, histrelin, hydroxyprogesterone, ixabepilone, leuprorelin, medroxyprogesterone acetate, megestrol acetate, methylprednisolone, methyltestosterone, miltefosine, mitropol, naphthalene phenylpropionate, norethindrone acetate, prednisolone, prednisone, triptorelin, vapreotide acetate, zenastatin ester, amsacrine, arsenic trioxide, bisantrene hydrochloride, chlorambucil, chlorotrianisole, cis-diaminodichloroplatinum, cyclophosphamide, diethylstilbestrol, hexamethylmelamine, hydroxyurea, lenalidomide, lonidamine, mechlorethamine, mitotane, nedaplatin, nimustine hydrochloride, pamidronate disodium , pipobroman, porfimer sodium, ranimustine, razoxane, semustine, sobuzoxane, mesylate, triethylene melamine, zoledronic acid, camostat mesylate, fadrozole hydrochloride, nafoxidine, aminoglutamine, carmofur, clofarabine, cytarabine, decitabine, docifluridine, escitabine, fludarab phosphate, fluorouracil, furouracil, uracil mustard, abarelix, bexarotene, raltiterxed, tamibarotene, temozolomide, vorinostat, megastrol, clodronate disodium, levamisole, nano iron oxide, iron isomaltoside, celecoxib, isobublast, bendamustine, atriamine, mitolactone, temsirolimus, pralatrexate, TS-1, di Citabine, bicalutamide, flutamide, letrozole, clodronate disodium, degarelix, toremifene citrate, histamine dihydrochloride, DW-166HC, nitracrine, decitabine, irinotecan hydrochloride, amsacrine, romidepsin, tretinoin, cabazitaxel, vandetanib, lenalidomide, ibandronic acid, miltefosine, vetspone, mifatide, nadroparin, granisetron, ondansetron, tosetron, alizapril, ramosetron, dolasetron mesylate, fosaprepitant dimeglumine, nabilone, aprepitant, dronabinol, TY-10721, glycyrrhizin hydrogen maleate, prisnar, defibrotide, dabigatran etexilate, filgrastim, pegfilgrastim, reditux, epoetin, molgramostimoprelvekin, sipuleucel-T, M-Vax, acetyl-L-carnitine hydrochloride, donepezil hydrochloride, 5-aminolevulinic acid, methyl aminolevulinate, cetrorelix acetate, icodextrin, leuprolide, methylphenidate, octreotide, amlexanox, plerixafor, menatetrenone, anethole dithiothione, doxorcalciferol, cinacalcet hydrochloride, alefacept, romiplostim, thymoglobulin, thymalfasin, ubenimex, imiquimod, everolimus, sirolimus, H-101, lasofoxifene, trilostane, incadronate, gangliosides, pegaptanib sodium, viltoporfin, minodronic acid, zoledronic acid, gallium nitrate, alendronate sodium, etidronate sodium, pamidronate disodium, dutasteride, sodium stibogluconate, armodafinil, dexrazoxane, amifostine, WF-10, temoporfin, alfadabepol, anacetomycin, sargramostim, palifermin, R-744, nepidermin, oprelvekin, denileukin, crisantaspase, buserelin, deslorelin, lanreotide, octreotide, pilocarpine, bosentan, calicheamicin, maytansine, and cyclonicotinamide.

[0258] For more details, those skilled in the art can refer to the Handbook entitled "Traité de chimie thérapeutique, vol. 6, Medicaments antitumouraux et perspectives dans le traitement descancers, edition TEC&DOC, 2003" edited by Enseignants de Chimie Thérapeutique".

[0259] Alternatively, the immunoconjugate may comprise at least a binding protein linked to a radioisotope. When the binding protein is an antibody or antigen-binding fragment thereof, such immunoconjugates are often referred to as antibody-radioisotope conjugates (or "ARCs").

[0260] For selective destruction of tumors, antibodies can contain highly radioactive atoms. A variety of radioisotopes can be used to produce ARCs, such as, but not limited to: At 211 、C 13 、N 15 , O 17 、Fl 19 , I 123 , I 131 , I 125 、In 111 、Y 90 、Re 186 、Re188 、Sm 153 、tc 99 m、Bi 212 、P 32 , Pb 212 , Lu, Gadolinium, manganese or iron radioactive isotopes.

[0261] Any method or process known to those skilled in the art can be used to incorporate such radioisotopes into ARCs (see, for example, "Monoclonal Antibodies in Immunoscintigraphy", Chatal, CRC Press 1989). As a non-limiting example, Tc can be linked via a cysteine ​​residue. 99 m or I 123 、Re 186 、Re 188 and In 111 Y can be linked via a lysine residue 90 IODOGEN method can be used to connect I 123 (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80:49-57).

[0262] Several examples can be mentioned to illustrate the knowledge of those skilled in the art in the field of ARC, such as It is composed of an anti-CD20 monoclonal antibody and an In 111 or Y 90 ARC composed of radioactive isotopes (Wiseman et al. (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al. (2002) Blood 99(12):4336-42; Witzig et al. (2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al. (2002) J. Clin. Oncol. 20(15):3262-69); or It is composed of an anti-CD33 antibody linked to calicheamicin (U.S. Patent Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116; 5,767,285; 5,773,001). More recently, the ADC called Adcetris (corresponding to Brentuximab vedotin) can also be mentioned, which was recently accepted by the FDA for the treatment of Hodgkin's lymphoma (Nature, vol. 476, pp. 380-381, August 25, 2011).

[0263] In yet another embodiment of the present disclosure, the immunoconjugate may comprise a binding protein linked to a toxin. When the binding protein is an antibody or an antigen-binding fragment thereof, such immunoconjugates are often referred to as antibody-toxin conjugates (or "ATCs").

[0264] Toxins are potent and specific poisons produced by living organisms. They are typically composed of chains of amino acids, and their molecular weight can vary from a few hundred daltons (peptides) to hundreds of thousands of daltons (proteins). They can also be low-molecular-weight organic compounds. Toxins are produced by many organisms, such as bacteria, fungi, algae, and plants. Many of them are highly toxic, with toxicity several orders of magnitude greater than that of nerve agents.

[0265] Toxins used in ATC may include, but are not limited to, all kinds of toxins that can exert their cytotoxic effects through mechanisms including binding to tubulin, binding to DNA, or inhibiting topoisomerase.

[0266] Enzymatically active toxins and fragments thereof that can be used include: diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-inosine, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, Curcas toxin, crotonin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0267] Also contemplated herein are small molecule toxins such as dolastatin, auristatin, trichothecenes, and CC1065, as well as derivatives of these toxins having toxin activity. Dolastatin and auristatin have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division, and have anticancer and antifungal activity.

[0268] The immunoconjugates described herein may further comprise a linker.

[0269] "Linker," "Linker unit," or "link" refers to a chemical moiety comprising a covalent bond or a chain of atoms that covalently links a binding protein to at least one cytotoxic agent.

[0270] Linkers can be made using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipate hydrochloride), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating cytotoxic agents to addressing systems. Other cross-linking reagents can be BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSB (succinimidyl-(4-vinylsulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, 111., USA).

[0271] Linkers can be "non-cleavable" or "cleavable" linkers.

[0272] Preferably, the linker is a "cleavable linker" that facilitates release of the cytotoxic agent in the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used. Preferably, the linker is cleaved under intracellular conditions, such that cleavage of the linker releases the cytotoxic agent from the binding protein in the intracellular environment.

[0273] For example, in some embodiments, the linker can be cleaved by a cleavage agent present in the intracellular environment (e.g., within a lysosome or endosome or caveolae). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease (including but not limited to a lysosomal or endosomal protease). Typically, the peptidyl linker is at least two amino acids long or at least three amino acids long. The cleavage agent can include cathepsins B, D, and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, thereby releasing the active drug within the target cell. For example, a peptidyl linker (e.g., a Phe-Leu or Gly-Phe-Leu-Gly linker) that is cleaved by the sulfhydryl-dependent protease cathepsin-B, which is highly expressed in cancer tissue, can be used. In specific embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker. One advantage of using intracellular proteolytic release of the cytotoxic agent is that the cytotoxic agent is typically attenuated upon conjugation, and the serum stability of the conjugate is typically high.

[0274] In other embodiments, cleavable joints are pH sensitive, i.e., sensitive to hydrolysis under certain pH values. Typically, pH sensitive joints are hydrolyzable under acidic conditions. For example, acid-unstable joints (e.g., hydrazones, semicarbazides, thiosemicarbazides, cis-aconitamides, orthoesters, acetals, ketals, etc.) that can be hydrolyzed in lysosomes can be used. Such joints are relatively stable under neutral pH conditions (e.g., pH conditions in blood), but unstable under conditions lower than pH 5.5 or 5.0 (lysosomal approximate pH). In certain embodiments, hydrolyzable joints are thioether joints (e.g., thioethers connected to therapeutic agents by acylhydrazone bonds).

[0275] In yet other embodiments, the linker can be cleaved under reducing conditions (e.g., a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio) propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio) butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio) cresol), SPDB, and SMPT.

[0276] In contrast, non-cleavable linkers have no obvious drug release mechanism. Immunoconjugates containing such non-cleavable linkers rely on complete lysosomal proteolytic degradation of the antibody to release the cytotoxic agent after internalization.

[0277] As an example of an immunoconjugate comprising a non-cleavable linker, one can mention the immunoconjugate trastuzumab-emtansine (TDM1), which combines trastuzumab with the linked chemotherapeutic agent maytansine (Cancer Research 2008; 68:(22). November 15, 2008).

[0278] In preferred embodiments, the immunoconjugates disclosed herein can be prepared by any method known to those skilled in the art, such as, but not limited to, i) reacting a nucleophilic group of an antigen binding protein with a divalent linker reagent followed by reaction with a cytotoxic agent, or ii) reacting a nucleophilic group of a cytotoxic agent with a divalent linker reagent followed by reaction with a nucleophilic group of an antigen binding protein.

[0279] Nucleophilic groups on antigen-binding proteins include, but are not limited to, N-terminal amine groups, side chain amine groups such as lysine, side chain sulfhydryl groups, and sugar hydroxyl or amino groups when the antigen-binding protein is glycosylated. Amine, sulfhydryl, and hydroxyl groups are nucleophilic and can react with electrophilic groups on linker moieties and linker reagents to form covalent bonds, including, but not limited to, active esters such as NHS esters, HOBt esters, haloformates, and acyl halides; alkyl halides and benzyl halides such as haloacetamides; aldehydes, ketones, carboxyls, and maleimide groups. Antigen-binding proteins may have reducible interchain disulfide bonds, i.e., cysteine ​​bridges. Antigen-binding proteins can be made reactive toward conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Thus, in theory, each cysteine ​​bridge will form two reactive sulfhydryl nucleophiles. Additional nucleophilic groups can be introduced into antigen-binding proteins by any reaction known to those skilled in the art. As a non-limiting example, reactive sulfhydryl groups can be introduced into an antigen binding protein by introducing one or more cysteine ​​residues.

[0280] Immunoconjugates can also be produced by modifying the antigen-binding protein to introduce an electrophilic moiety that can react with nucleophilic substituents on a linker reagent or cytotoxic agent. The sugar of the glycosylated antigen-binding protein can be oxidized to form an aldehyde or ketone group, which can react with the amino group of a linker reagent or cytotoxic agent. The resulting imine Schiff base group can form a stable bond or can be reduced to form a stable amine bond. In one embodiment, the reaction of the carbohydrate portion of the glycosylated antigen-binding protein with galactose oxidase or sodium metaperiodate can produce a carbonyl (aldehyde and ketone) group in the protein that can react with the appropriate group on the drug. In another embodiment, a protein containing an N-terminal serine or threonine residue can react with sodium metaperiodate to produce an aldehyde instead of the first amino acid.

[0281] Chimeric antigen receptor

[0282] The present disclosure further provides a CAR (chimeric antigen receptor) protein comprising i) an antibody of the present invention; ii) a transmembrane domain, and iii) an intracellular signaling domain, characterized in that the antibody according to the above i) binds to an antigen and causes T cell activation.

[0283] In the present invention, the CAR protein is characterized in that it is composed of the monoclonal antibody of the present invention, a known transmembrane domain, and an intracellular signaling domain.

[0284] As described herein, the term "CAR (chimeric antigen receptor)" refers to a non-natural receptor that can provide immune effector cells with specificity for a particular antigen. Typically, CAR represents a receptor for providing T cells with the specificity of a monoclonal antibody. CAR is generally composed of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains an antigen recognition region, and in the present invention, the antigen recognition site is a VSIG4-specific antibody. As described above, the antibody used in CAR is preferably in the form of an antibody fragment. It is more preferably in the form of Fab or scFv, but is not limited thereto.

[0285] In addition, the transmembrane domain of CAR has a form connected to the extracellular domain, which can be derived from a natural form or a synthetic form. When it is derived from a natural form, it may be derived from a membrane-bound protein or a transmembrane protein, which can be derived from a portion of a transmembrane domain of various proteins, such as α, β or ζ chains of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or CD8. The sequences of these transmembrane domains can be obtained from literature known in the art, in which the transmembrane domains of transmembrane proteins are well described, but are not limited thereto.

[0286] The CAR of the present invention is a part of the intracellular CAR domain, which is connected to the transmembrane domain. The intracellular domain of the present invention may include an intracellular signaling domain, characterized in that it has a characteristic of causing T cell activation (preferably T cell proliferation) when the antigen is bound to the antigen recognition site of the CAR. The intracellular signaling domain is not particularly limited in its type, as long as it can cause T cell activation when the antigen is bound to the antigen recognition site of the CAR present outside the cell, various intracellular signaling domains can be used. Examples thereof include based on immunoreceptor tyrosine activation motifs (ITAMs), which can include motifs derived from CD3 zeta (ξ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, CD66d or FcεRIγ, but are not limited thereto.

[0287] In addition, it is preferred that the intracellular domain of the CAR of the present invention further comprises a costimulatory domain together with the intracellular signaling domain, but is not limited thereto. The costimulatory domain is a portion contained in the CAR of the present invention that plays a role in transmitting signals to T cells in addition to the signal from the intracellular signaling domain, which means that the intracellular portion of the CAR includes the intracellular domain of a costimulatory molecule.

[0288] Costimulatory molecules as cell surface molecules refer to molecules required for lymphocytes to fully respond to antigens, examples of which include CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1 (lymphocyte function-associated antigen-1), CD2, CD7, LIGHT, NKG2C and B7-H3, but are not limited thereto. The costimulatory domain can be an intracellular portion of a molecule selected from those costimulatory molecules and combinations thereof.

[0289] In addition, selectively, a short oligopeptide or polypeptide linker can connect the intracellular domain and the transmembrane domain of the CAR. Although the linker can be included in the CAR of the present invention, there is no particular limitation on the length of the linker, as long as it can induce T cell activation by binding to the extracellular antibody through the intracellular domain of the antigen.

[0290] Nucleic acids and expression systems

[0291] The present disclosure encompasses polynucleotides encoding immunoglobulin light and heavy chain genes for antibodies (particularly anti-VSIG4 antibodies), vectors comprising such nucleic acids, and host cells capable of producing antibodies of the present disclosure. Also provided herein are polynucleotides that hybridize under highly stringent, moderately or less stringent hybridization conditions (e.g., as defined above) to polynucleotides encoding antibodies or modified antibodies provided herein.

[0292] In a first aspect, the present disclosure relates to one or more polynucleotides encoding antibodies (especially antibodies or fragments thereof that can specifically bind to VSIG4, as described above). The present disclosure particularly provides polynucleotides encoding heavy chain variable regions and / or light chain variable regions of monoclonal antibodies or antigen-binding fragments thereof. More specifically, in certain embodiments, the nucleic acid molecules provided herein comprise or consist of nucleic acid sequences encoding heavy chain variable regions and light chain variable regions disclosed herein, or any combination thereof (e.g., as nucleotide sequences encoding antibodies provided herein, such as full-length antibodies, heavy chains and / or light chains of antibodies, or single-chain antibodies provided herein).

[0293] In one embodiment, the polynucleotide encodes three heavy chain CDRs of an anti-VSIG4 antibody described herein. In one embodiment, the polynucleotide encodes three light chain CDRs of an anti-VSIG4 antibody described herein. In one embodiment, the polynucleotide encodes three heavy chain CDRs and three light chain CDRs of an anti-VSIG4 antibody described herein. Another embodiment provides a pair of polynucleotides, wherein the first polynucleotide encodes the three heavy chain CDRs of an anti-VSIG4 antibody described herein; and the second polynucleotide encodes the three light chain CDRs of the same anti-VSIG4 antibody described herein.

[0294] In one embodiment, the polynucleotide encodes the heavy chain variable region of an anti-VSIG4 antibody described herein. In one embodiment, the polynucleotide encodes the light chain variable region of an anti-VSIG4 antibody described herein. In one embodiment, the polynucleotide encodes the heavy chain variable region and the light chain variable region of an anti-VSIG4 antibody described herein. Another embodiment provides a pair of polynucleotides, wherein the first polynucleotide encodes the heavy chain variable region of an anti-VSIG4 antibody described herein; and the second polynucleotide encodes the light chain variable region of the same anti-VSIG4 antibody described herein.

[0295] In one embodiment, the polynucleotide encodes the heavy chain of an anti-VSIG4 antibody described herein. In one embodiment, the polynucleotide encodes the light chain of an anti-VSIG4 antibody described herein. In one embodiment, the polynucleotide encodes the heavy chain and light chain of an anti-VSIG4 antibody described herein. Another embodiment provides a pair of polynucleotides, wherein the first polynucleotide encodes the heavy chain of an anti-VSIG4 antibody described herein; and the second polynucleotide encodes the light chain of the same anti-VSIG4 antibody described herein.

[0296] In one embodiment, a polynucleotide encoding the heavy chain of the anti-VSIG4 antibody SA1956 is provided. Preferably, the heavy chain comprises three heavy chain CDRs of SEQ ID NOs. 3, 4, and 5. More preferably, the heavy chain comprises a heavy chain variable region of SEQ ID NO. 129.

[0297] In another embodiment, the polynucleotide encodes the light chain of the anti-VSIG4 antibody SA1956. Preferably, the light chain comprises three light chain CDRs of SEQ ID NOs. 6, 7, and 8. More preferably, the light chain comprises a variable region of SEQ ID NO. 130.

[0298] In another embodiment, a polynucleotide encoding the heavy chain of the anti-VSIG4 antibody SA1957 is provided. Preferably, the heavy chain comprises three heavy chain CDRs of SEQ ID NOs. 9, 10, and 5. More preferably, the heavy chain comprises a heavy chain variable region of SEQ ID NO. 131.

[0299] In another embodiment, the polynucleotide encodes the light chain of the anti-VSIG4 antibody SA1957. Preferably, the light chain comprises three light chain CDRs of SEQ ID NOs. 6, 7, and 8. More preferably, the light chain comprises a light chain variable region of SEQ ID NO. 132.

[0300] In another embodiment, a polynucleotide encoding the heavy chain of the anti-VSIG4 antibody SA 1975 is provided. Preferably, the heavy chain comprises three heavy chain CDRs of SEQ ID NOs. 11, 12, and 13. More preferably, the heavy chain comprises a variable region of SEQ ID NO. 133.

[0301] In another embodiment, the polynucleotide encodes the light chain of the anti-VSIG4 antibody SA 1975. Preferably, the light chain comprises three light chain CDRs of SEQ ID NOs. 14, 15, and 16. More preferably, the light chain comprises a variable region of SEQ ID NO. 134.

[0302] In another embodiment, a polynucleotide encoding the heavy chain of the anti-VSIG4 antibody SA2283 is provided. Preferably, the heavy chain comprises three heavy chain CDRs of SEQ ID NOs. 17, 18, and 19. More preferably, the heavy chain comprises a variable region of SEQ ID NO. 135.

[0303] In another embodiment, the polynucleotide encodes the light chain of the anti-VSIG4 antibody SA2283. Preferably, the light chain comprises three light chain CDRs of SEQ ID NOs. 20, 21, and 22. More preferably, the light chain comprises a variable region of SEQ ID NO. 136.

[0304] In another embodiment, a polynucleotide encoding the heavy chain of the anti-VSIG4 antibody SA 2285 is provided. Preferably, the heavy chain comprises three heavy chain CDRs of SEQ ID NOs. 23, 24, and 3. More preferably, the heavy chain comprises a variable region of SEQ ID NO. 137.

[0305] In another embodiment, the polynucleotide encodes the light chain of the anti-VSIG4 antibody SA 2285. Preferably, the light chain comprises three light chain CDRs of SEQ ID NOs. 6, 7, and 25. More preferably, the light chain comprises a variable region of SEQ ID NO. 138.

[0306] In another embodiment, a polynucleotide encoding the heavy chain of the anti-VSIG4 antibody SA2287 is provided. Preferably, the heavy chain comprises three heavy chain CDRs of SEQ ID NOs. 26, 27, and 28. More preferably, the heavy chain comprises a heavy chain variable region of SEQ ID NO. 139.

[0307] In another embodiment, the polynucleotide encodes the light chain of the anti-VSIG4 antibody SA2287. Preferably, the light chain comprises three light chain CDRs of SEQ ID NOs. 29, 30, and 31. More preferably, the light chain comprises a variable region of SEQ ID NO. 140.

[0308] In another embodiment, a polynucleotide encoding the heavy chain of the anti-VSIG4 antibody SA2290 is provided. Preferably, the heavy chain comprises three heavy chain CDRs of SEQ ID NOs. 32, 33, and 34. More preferably, the heavy chain comprises a heavy chain variable region of SEQ ID NO. 141.

[0309] In another embodiment, the polynucleotide encodes the light chain of the anti-VSIG4 antibody SA2290. Preferably, the light chain comprises three light chain CDRs of SEQ ID NOs. 35, 36, and 16. More preferably, the light chain comprises a variable region of SEQ ID NO. 142.

[0310] In another embodiment, a polynucleotide encoding the heavy chain of the anti-VSIG4 antibody SA2291 is provided. Preferably, the heavy chain comprises three heavy chain CDRs of SEQ ID NOs. 37, 38, and 39. More preferably, the heavy chain comprises a variable region of SEQ ID NO. 143.

[0311] In another embodiment, the polynucleotide encodes the light chain of the anti-VSIG4 antibody SA2291. Preferably, the light chain comprises three light chain CDRs of SEQ ID NOs. 40, 41, and 42. More preferably, the light chain comprises a variable region of SEQ ID NO. 144.

[0312] In another embodiment, a polynucleotide encoding the heavy chain of the anti-VSIG4 antibody SA 2386 is provided. Preferably, the heavy chain comprises three heavy chain CDRs of SEQ ID NOs. 43, 44, and 45. More preferably, the heavy chain comprises a heavy chain variable region of SEQ ID NO. 145.

[0313] In another embodiment, the polynucleotide encodes the light chain of the anti-VSIG4 antibody SA 2386. Preferably, the light chain comprises three light chain CDRs of SEQ ID NOs. 46, 47, and 48. More preferably, the light chain comprises a variable region of SEQ ID NO. 146.

[0314] In another embodiment, a polynucleotide encoding the heavy chain of the anti-VSIG4 antibody SA2390 is provided. Preferably, the heavy chain comprises three heavy chain CDRs of SEQ ID NOs. 49, 50, and 51. More preferably, the heavy chain comprises a variable region of SEQ ID NO. 147.

[0315] In another embodiment, the polynucleotide encodes the light chain of the anti-VSIG4 antibody SA2390. Preferably, the light chain comprises three light chain CDRs of SEQ ID NOs. 52, 53, and 54. More preferably, the light chain comprises a variable region of SEQ ID NO. 148.

[0316] In another embodiment, a polynucleotide encoding the heavy chain of the anti-VSIG4 antibody SA2455 is provided. Preferably, the heavy chain comprises three heavy chain CDRs of SEQ ID NOs. 17, 18, and 55. More preferably, the heavy chain comprises a variable region of SEQ ID NO. 149.

[0317] In another embodiment, the polynucleotide encodes the light chain of the anti-VSIG4 antibody SA2455. Preferably, the light chain comprises three light chain CDRs of SEQ ID NOs. 56, 57, and 58. More preferably, the light chain comprises a light chain variable region of SEQ ID NO. 150.

[0318] Due to codon degeneracy or taking into account the preferred codons in the organism in which the light chain and heavy chain of a human antibody or its fragment are to be expressed, the polynucleotides encoding the light chain and heavy chain of the monoclonal antibody of the present invention or its antigen-binding fragment can have various variations in the coding region without changing the amino acid sequence of the light chain and heavy chain of the antibody expressed from the coding region, and even in regions outside the coding region, various changes or modifications can be made without affecting gene expression. It will be readily understood by those skilled in the art that those variant genes also fall within the scope of the present invention. That is, as long as the polynucleotide encoding the present invention has a protein with equivalent activity, one or more nucleic acid bases can be changed by substitution, deletion, insertion or a combination thereof, and these also fall within the scope of the present invention. The sequence of the polynucleotide can be single-stranded or double-stranded, and can be a DNA molecule or an RNA (mRNA) molecule.

[0319] According to the present invention, can use multiple expression system to express antibody of the present invention.On the one hand, such expression system represents can produce the carrier of purpose coding sequence and purification subsequently, also represents when with suitable nucleotide coding sequence transient transfection, can the cell of in situ expression IgG antibody.

[0320] The present disclosure provides a vector comprising the above-mentioned polynucleotide. In one embodiment, the vector contains a polynucleotide encoding the heavy chain of an antibody of interest (e.g., an anti-VSIG4 antibody). In another embodiment, the polynucleotide encodes the light chain of an antibody of interest (e.g., an anti-VSIG4 antibody). In another embodiment, the polynucleotide encodes the heavy chain and light chain of an antibody of interest (e.g., an anti-VSIG4 antibody). In yet another embodiment, a pair of polynucleotides is provided, wherein the first polynucleotide encodes the heavy chain of an antibody of interest (e.g., an anti-VSIG4 antibody) and the second polynucleotide encodes the light chain of the same antibody of interest (e.g., an anti-VSIG4 antibody).

[0321] The present disclosure also provides vectors comprising polynucleotide molecules encoding fusion proteins, modified antibodies, antibody fragments and probes thereof.

[0322] In order to express the heavy chain and / or light chain of an antibody of interest (e.g., an anti-VSIG4 antibody), the polynucleotide encoding the heavy chain and / or light chain is inserted into an expression vector such that the gene is operably linked to the transcription and translation sequences. In a preferred embodiment, these polynucleotides are cloned into two vectors.

[0323] "Operably linked" sequences include expression control sequences that are contiguous with the gene of interest, and expression control sequences that control the gene of interest in trans or at a distance. As used herein, the term "expression control sequence" refers to a polynucleotide sequence necessary to achieve expression and processing of the coding sequence to which it is linked. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion when desired. The nature of such control sequences varies depending on the host organism: in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences; in eukaryotes, such control sequences include promoters and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may also include other components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0324] The polynucleotides of the present invention and vectors comprising these molecules can be used to transform suitable host cells. As used herein, the term "host cell" is intended to refer to a cell into which a recombinant expression vector has been introduced to express an antibody of interest (e.g., an anti-VSIG4 antibody). It should be understood that such terms are intended to refer not only to specific test cells, but also to the progeny of such cells. Because certain modifications may occur in progeny due to mutations or environmental influences, such progeny may actually be different from the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0325] Transformation can be performed by any known method for introducing a polynucleotide into a cellular host. Such methods are well known to those skilled in the art and include dextran-mediated transformation, calcium phosphate precipitation, polyethylene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, gene gun injection, and direct microinjection of DNA into the nucleus.

[0326] Host cells can be co-transfected with one or more expression vectors. For example, host cells can be transfected with vectors encoding both the heavy chain and the light chain of an antibody of interest (e.g., an anti-VSIG4 antibody) as described above. Alternatively, host cells can be transformed with a first vector encoding the heavy chain of an antibody of interest (e.g., an anti-VSIG4 antibody) and a second vector encoding the light chain of the antibody. Mammalian cells are commonly used to express recombinant therapeutic immunoglobulins, particularly for expressing fully recombinant antibodies. For example, mammalian cells (such as HEK293 or CHO cells) together with vectors containing expression signals (such as expression signals carrying the major intermediate early gene promoter element from human cytomegalovirus) are effective systems for expressing the humanized anti-VSIG4 antibodies of the present invention (Foecking et al., 1986, Gene 45:101; Cockett et al., 1990, Bio / Technology 8:2).

[0327] In addition, the host cell that can select to regulate the expression of insertion sequence or modify and process gene product in required ad hoc manner.This type of modification (such as glycosylation) and processing of protein product may be very important to the function of protein.Different host cells have the feature and specific mechanism of post-translational processing and modification of protein and gene product.Select appropriate cell line or host system to guarantee the interested antibody of correct modification and processing expression.Therefore, eukaryotic host cells with the cellular mechanism for suitably processing primary transcript and glycosylated gene product can be used.This type of mammalian host cell includes but is not limited to CHO, COS, HEK293, NS / 0, BHK, Y2 / 0, 3T3 or myeloma cell (all these cell lines can be obtained from public deposits, such as the National Microbiological Culture Collection in Paris, France or the American Type Culture Collection in Manassas, Virginia, USA).

[0328] In order to produce recombinant proteins for a long time and in high yield, stable expression is preferred. In one embodiment of the present invention, the cell line of the antibody can be stably expressed by engineering approaches. The host cell is transformed with DNA and selectable markers under the control of suitable expression regulatory elements (including promoters, enhancers, transcription terminators, polyadenylation sites and other sequences well known to those skilled in the art), without using expression vectors containing viral replication origins. After introducing foreign DNA, the engineered cells can be allowed to grow one to two days in enrichment medium and then moved to selective medium. The selectable marker on the recombinant plasmid confers resistance to selection and allows the cell to stably integrate the plasmid into the chromosome and amplify into a cell line. Other methods for building stable cell lines are known in the art. In particular, methods for site-specific integration have been developed. According to these methods, the transforming DNA under the control of appropriate expression regulatory elements (including promoters, enhancers, transcription terminators, polyadenylation sites and other appropriate sequences) is integrated into the host cell genome at a specific target site that has been previously cut (Moele et al., Proc. Natl. Acad. Sci. USA, 104(9):3055-3060; US 5,792,632; US 5,830,729; US 6,238,924; WO 2009 / 054985; WO 03 / 025183; WO 2004 / 067753).

[0329] A number of selection systems can be used according to the present invention, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., Proc Natl Acad Sci USA 48:202, 1992), glutamate synthase in the presence of methionine sulfoximine (Adv Drug Del Rev, 58:671, 2006, and the website or literature of Lonza Group Ltd.), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817, 1980) genes in tk, hgprt, or aprt cells, respectively. In addition, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr, which confers methotrexate resistance (Wigler et al., Proc Natl Acad Sci USA 77:357, 1980); gpt, which confers mycophenolic acid resistance (Mulligan et al., Proc Natl Acad Sci USA 78:2072, 1981); neo, which confers aminoglycoside G-418 resistance (Wu et al., Biotherapy 3:87, 1991); and hygro, which confers hygromycin resistance (Santerre et al., Gene 30:147, 1984). Methods known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clones, such as those described in Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons (1993). The expression level of the antibody can be increased by vector amplification. When the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the culture will increase the copy number of the marker gene. Since the amplified region is associated with the gene encoding the IgG antibody of the present invention, the production of the antibody will also increase (Crouse et al., Mol Cell Biol 3:257, 1983). There are alternative methods for expressing the genes of the present invention and are known to those skilled in the art. For example, modified zinc finger proteins that can bind to expression control elements upstream of the genes of the present invention can be engineered; expressing the engineered zinc finger proteins (ZFNs) in the host cells of the present invention results in increased protein production (see, for example, Reik et al., Biotechnol. Bioeng., 97(5):1180-1189, 2006). In addition, ZFNs can stimulate DNA integration into a predetermined genomic location, resulting in efficient site-specific gene addition (Moehle et al., Proc Natl Acad Sci USA, 104:3055, 2007).

[0330] The antibody of interest (e.g., anti-VSIG4 antibody) can be prepared by culturing a culture of transformed host cells under the culture conditions necessary for expression of the desired antibody. The resulting expressed antibody can then be purified from the culture medium or cell extract. The antibody of interest (e.g., anti-VSIG4 antibody) can be recovered in soluble form from the culture supernatant. The antibody can then be purified by any method known in the art for purifying immunoglobulin molecules, such as by chromatography (e.g., ion exchange, affinity, particularly by affinity of protein A for Fc, etc.), centrifugation, differential solubility, or by any other standard technique for purifying proteins. Suitable purification methods will be apparent to those of ordinary skill in the art.

[0331] Therefore, another aspect of the present invention relates to a method for producing an antibody described herein (eg, an anti-VSIG4 antibody), comprising the steps of:

[0332] a) growing the host cell in a culture medium under suitable culture conditions; and

[0333] b) recovering the antibody (eg, anti-VSIG4 antibody) from the culture medium or from the cultured cells.

[0334] The antibodies obtained by culturing the transformants can be used in an unpurified state. Impurities can be removed by various other common methods such as centrifugation or ultrafiltration, and the resulting product can be subjected to dialysis, salt precipitation, chromatography, etc., wherein the methods can be used alone or in combination. Among them, affinity chromatography is the most widely used, including ion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, etc.

[0335] Pharmaceutical composition

[0336] In another aspect, the present disclosure provides a composition comprising an anti-VSIG4 antibody or antigen-binding fragment thereof (eg, any anti-VSIG4 antibody described herein) or a conjugate thereof (ie, an immunoconjugate comprising one of the anti-VSIG4 antibodies described herein).

[0337] These compositions are particularly useful, for example, for stimulating an immune response in a subject. The antibodies of the present invention that specifically bind to VSIG4 induce T cell activation by binding to the VSIG4 protein that inhibits T cell activation, and thus the antibodies can stimulate an immune response.

[0338] The compositions described herein can also be used to treat cancer. Protective anti-tumor immunity can be established by administering such compositions comprising an anti-VSIG4 antibody, antigen-binding fragment thereof, or conjugate thereof disclosed herein.

[0339] Optionally, the composition may include one or more additional therapeutic agents, such as immune checkpoint inhibitors described below. The composition is typically provided as part of a sterile pharmaceutical composition, which typically includes a pharmaceutically acceptable carrier and / or excipient. Therefore, in another aspect, the present invention provides a pharmaceutical composition comprising an anti-VSIG4 antibody or a conjugate thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0340] Said composition can be in any suitable form (depending on the desired method for applying it to the patient). The composition used in the methods described herein can be, for example, administered as follows: intravitreal (e.g., by intravitreal injection), by eye drops, intramuscularly, intravenously, intradermally, transcutaneously, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intrathecally, intranasally, intravaginally, intrarectally, locally, intratumorally, transperitoneally, subcutaneously, subconjunctivally, intracapsularly, transmucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, orally, locally, transdermally, by inhalation, by injection, by implantation, by infusion, by continuous infusion, by local perfusion to directly bathe target cells, by catheter, by lavage, in cream or in lipid compositions. The composition utilized in the methods described herein can also be administered systemically or topically. The method of administration can vary depending on various factors (e.g., the compound or composition administered and the severity of the condition, disease or illness treated). In any given case, the most appropriate route of administration will depend on the particular antibody, the subject, the nature and severity of the disease, and the subject's physical condition.Anti-VSIG4 antibodies, antigen-binding fragments thereof, or conjugates thereof can be formulated into an aqueous solution and administered by subcutaneous injection.

[0341] The pharmaceutical composition can be conveniently presented in unit dosage form, with each dose containing a predetermined amount of anti-VSIG4, its antigen-binding fragment, or its conjugate. Such a unit may contain, for example, but not limited to, 5 mg to 5 g, such as 10 mg to 1 g, or 20 to 50 mg. The pharmaceutically acceptable carrier used in the present disclosure can take a variety of forms, depending on, for example, the condition to be treated or the route of administration.

[0342] The pharmaceutical compositions of the present disclosure can be prepared by mixing the antibodies of the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers commonly used in the art (all of which are referred to herein as "carriers," i.e., buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and other various additives) for storage as lyophilized formulations or aqueous solutions. See Remington's Pharmaceutical Sciences, 16th edition (Osol ed., 1980). Such additives must be nontoxic to the recipient at the dosages and concentrations employed.

[0343] Buffers help maintain the pH within a range close to physiological conditions. They can be present in a concentration of about 2 mM to about 50 mM. Suitable buffers for use in the present disclosure include organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, citric acid-trisodium citrate mixtures, citric acid-monosodium citrate mixtures, etc.), succinic acid buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, succinic acid-disodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, fumaric acid-fumaric acid ... In some embodiments, the present invention provides a buffer solution comprising a gluconate buffer (e.g., a gluconate-sodium gluconate mixture, a gluconate-sodium hydroxide mixture, a gluconate-potassium gluconate mixture, etc.), an oxalate buffer (e.g., an oxalic acid-sodium oxalate mixture, an oxalic acid-sodium hydroxide mixture, an oxalic acid-potassium oxalate mixture, etc.), a lactate buffer (e.g., a lactic acid-sodium lactate mixture, a lactic acid-sodium hydroxide mixture, a lactic acid-potassium lactate mixture, etc.), and an acetate buffer (e.g., an acetic acid-sodium acetate mixture, an acetic acid-sodium hydroxide mixture, etc.). In addition, a phosphate buffer, a histidine buffer, and a trimethylamine salt such as Tris can be used.

[0344] Preservatives can be added to slow down microbial growth in an amount ranging from 0.2% to 1% (w / v). Suitable preservatives for use in the present invention include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethylammonium chloride, and alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. An isotonic agent, sometimes referred to as a "stabilizer," can be added to ensure the isotonicity of the liquid composition of the present invention. The isotonic agent includes polyols, such as trivalent or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad class of excipients whose functions range from fillers to additives that make the therapeutic agent (i.e., anti-VSIG4 antibody, antigen-binding fragment thereof, or conjugate thereof) soluble or help prevent denaturation or adhesion to the container wall. Typical stabilizers can be polyols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, etc., including cyclic alcohols such as inositol; polyethylene glycol; amino acids Polymers; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides of 10 or fewer residues); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, sucrose, and trisaccharides such as raffinose; and polysaccharides such as dextran. The stabilizer may be present in an amount ranging from 0.1 to 10,000 parts by weight per part by weight of the active protein (e.g., an anti-VSIG4 antibody or a conjugate comprising such an antibody).

[0345] Nonionic surfactants or detergents (also called "wetting agents") may be added to help make the anti-VSIG4 antibody (or its conjugate) soluble and to protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxyheteroisomers (184, 188, etc.), pluronic polyols, polyoxyethylene sorbitan monoethers ( The nonionic surfactant may be present in a range of about 0.05 mg / ml to about 1.0 mg / ml, such as about 0.07 mg / ml to about 0.2 mg / ml.

[0346] Other miscellaneous excipients include fillers (eg, starch), chelating agents (eg, EDTA), antioxidants (eg, ascorbic acid, methionine, vitamin E), and solubilizers.

[0347] The present invention further relates to a pharmaceutical composition comprising at least:

[0348] i) an anti-VSIG4 antibody, an antigen-binding fragment thereof, or a conjugate thereof as disclosed herein, and

[0349] ii) a second therapeutic agent, such as an immune checkpoint inhibitor as described below,

[0350] As a combination product for simultaneous, separate or sequential use.

[0351] As used herein, "simultaneous use" means that the two compounds of the combination according to the present invention are administered in a single and identical pharmaceutical form.

[0352] As used herein, "separate use" means that the two compounds of the composition according to the present invention are administered simultaneously in different pharmaceutical forms.

[0353] As used herein, "sequential use" means that the two compounds of the composition according to the present invention are administered consecutively in respective different pharmaceutical forms.

[0354] The composition of an anti-VSIG4 antibody (or its antigen-binding fragment or its conjugate) and a second therapeutic agent (e.g., an immune checkpoint inhibitor) can be administered alone, as a mixture of one or more anti-VSIG4 antibodies (or its antigen-binding fragment or its conjugate) and / or one or more second therapeutic agents (e.g., an immune checkpoint inhibitor as described below), mixed or administered in combination with other agents useful for treating cancer, or administered as an adjuvant to other cancer therapies. Examples of suitable combinations and adjuvant therapies are provided below.

[0355] The present disclosure encompasses pharmaceutical kits containing anti-VSIG4 antibodies (or antigen-binding fragments thereof or conjugates thereof) and are described herein. The pharmaceutical kit is a package containing an anti-VSIG4 antibody (e.g., in lyophilized form or as an aqueous solution) and one or more of the following:

[0356] A second therapeutic agent, such as an immune checkpoint inhibitor as described below;

[0357] a device for administering the anti-VSIG4 antibody, such as a pen, needle, and / or syringe; and

[0358] (If the inhibitor is in the form of an antibody) Pharmaceutical-grade water or buffer for resuspending the antibody.

[0359] Each unit dose of an anti-VSIG4 antibody (or an antigen-binding fragment thereof or a conjugate thereof) can be packaged separately, and the kit can contain one or more unit doses (e.g., two unit doses, three unit doses, four unit doses, five unit doses, eight unit doses, ten unit doses or more). In a specific embodiment, the one or more unit doses are each contained in a syringe or pen.

[0360] effective dose

[0361] Anti-VSIG4 antibodies, antigen-binding fragments thereof, and conjugates thereof, optionally in combination with immune checkpoint inhibitors, are generally used in an amount effective to achieve the desired result, for example, in an amount effective to treat cancer in a subject in need thereof. Pharmaceutical compositions comprising anti-VSIG4 antibodies (or antigen-binding fragments thereof or conjugates thereof) and / or immune checkpoint inhibitors can be administered to a patient (e.g., a human subject) in a therapeutically effective dose.

[0362] Determining the effective amount is fully within the capabilities of those skilled in the art, especially according to the detailed disclosure provided herein. The toxicity and therapeutic efficacy of the compound or conjugate can be determined by standard pharmaceutical procedures in cell cultures and experimental animals. The effective amount of this combination or other therapeutic agents to be administered to the subject will depend on the stage, category and state of the disease (e.g., cancer) and the characteristics of the subject such as general health, age, sex, body weight and drug tolerance. The effective amount of this therapeutic agent to be administered or the combination will also depend on route of administration and dosage form. Dosage and interval can be adjusted separately to provide the plasma level of the active compound that is sufficient to maintain the desired therapeutic effect.

[0363] The amount of the anti-VSIG4 antibody or antigen-binding fragment thereof or conjugate thereof administered will depend on a variety of factors, including the nature and stage of the disease being treated (e.g., cancer), the form, route and site of administration, the treatment regimen (e.g., whether the therapeutic agent is used in combination with an immune checkpoint inhibitor), the age and condition of the specific subject being treated, and the sensitivity of the patient treated with the antibody or conjugate. A person skilled in the art can easily determine the appropriate dosage. Ultimately, the physician will determine the appropriate dosage to be used. The dosage can be repeated at an appropriate frequency. If side effects occur, the amount and / or frequency of the dosage can be changed or reduced according to usual clinical practice. An appropriate dosage and treatment regimen can be established by monitoring the progress of the treatment using conventional techniques known to those skilled in the art.

[0364] An effective dose can be preliminarily estimated from in vitro assays. For example, an initial dose for an animal can be formulated to achieve a circulating blood or serum concentration of an anti-VSIG4 antibody that is equal to or higher than the binding affinity of the antibody for VSIG4 measured in vitro. Taking into account the bioavailability of a particular antibody, the dose that achieves such circulating blood or serum concentration is well within the capabilities of the skilled person. For guidance, the reader is referred to Fingl & Woodbury, "General Principles" in Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, Chapter 1, latest edition, Pagamonon Press and references cited therein. The initial dose can be estimated from in vivo data (such as animal models). Animal models that can be used to test the efficacy of compounds in treating specific diseases (such as cancer) are generally well known in the art. A person of ordinary skill can routinely adjust such information to determine a dose suitable for administration to humans.

[0365] An effective dose of an anti-VSIG4 antibody as described herein can be in the range of about 0.001 to about 75 mg / kg per single (e.g., bolus) administration, multiple administrations, or continuous administration, or to a serum concentration of 0.01-5000 μg / ml per single (e.g., bolus) administration, multiple administrations, or continuous administration, or any effective range or value therein depending on the condition being treated, the route of administration, and the age, weight, and condition of the subject. In a certain embodiment, each dose can range from about 0.5 μg to about 50 μg per kilogram of body weight, for example, about 3 μg to about 30 μg per kilogram of body weight.

[0366] The amount, frequency and duration of administration will depend on a variety of factors, such as the patient's age, weight and disease condition. The treatment regimen for administration can last from 2 weeks to indefinitely, from 2 weeks to 6 months, from 3 months to 5 years, from 6 months to 1 or 2 years, from 8 months to 18 months, etc. Optionally, the treatment regimen provides repeated administration, such as once a day, twice a day, every two days, three days, five days, one week, two weeks or one month. The same dose or different doses can be repeatedly administered. It can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. A therapeutically effective amount of an anti-VSIG4 antibody or its conjugate (optionally in combination with an immune checkpoint inhibitor) can be administered in a single dose or during the course of a treatment regimen, for example, in the course of one week, two weeks, three weeks, one month, three months, six months, one year or longer.

[0367] Treatment

[0368] The ability of the anti-VSIG4 antibodies of the present invention to induce immune responses (e.g., by promoting M2 macrophage differentiation and by inhibiting VSIG4-mediated immunosuppression) makes them useful for treating a variety of VSIG4-mediated conditions, including cancer. Therefore, therapeutic intervention of the VSIG4 inhibitory pathway represents a promising approach to modulate inflammation and T cell-mediated immunity for the treatment of a variety of cancers.

[0369] Therefore, the anti-VSIG4 antibodies, antigen-binding fragments thereof, or conjugates described herein can be used in the following methods: treating cancer, inducing macrophages to release proinflammatory cytokines, inducing CD4 + T cell proliferation, induction of CD8 + T cell proliferation, induction of CD4 + T cell production of cytokines and induction of CD8 + T cells produce cytokines, wherein the method comprises administering an effective amount of an anti-VSIG4 antibody, an antigen-binding fragment thereof, or a conjugate thereof to a subject in need thereof. The treatment methods described herein may comprise administering an antibody that specifically binds to VSIG4 as described herein, or an antigen-binding fragment thereof, or a conjugate comprising these antibodies disclosed herein to a patient in need thereof. Therefore, the VSIG4 antibodies, antigen-binding fragments, and conjugates thereof disclosed herein can be used to modulate immunity (especially T cell immunity) to treat cancer.

[0370] Therefore, one aspect of the present disclosure relates to an anti-VSIG4 antibody, or an antigen-binding fragment thereof, or a conjugate thereof, for use in treating cancer in a patient. Also provided herein is a method of treating cancer in a subject in need thereof, comprising administering to the patient an anti-VSIG4 antibody, or an antigen-binding fragment thereof, or a conjugate thereof disclosed herein. The present disclosure also relates to the use of an anti-VSIG4 antibody, or an antigen-binding fragment thereof, or a conjugate thereof in the preparation of a medicament for treating cancer.

[0371] In one embodiment, the present disclosure relates to a composition comprising an anti-VSIG4 antibody, or an antigen-binding fragment or conjugate thereof, disclosed herein, for use in treating cancer in a patient. Also provided herein is a method for treating cancer in a subject in need thereof, comprising administering a composition comprising an anti-VSIG4 antibody, or an antigen-binding fragment or conjugate thereof disclosed herein to a patient. The present disclosure also relates to the use of a composition comprising an anti-VSIG4 antibody, or an antigen-binding fragment or conjugate thereof disclosed herein, in the preparation of a medicament for treating cancer.

[0372] In some embodiments, the cancer is selected from bladder cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, blood cancer (e.g., leukemia, lymphoma, or myeloma), laryngeal cancer, liver cancer, lung cancer, lymphoma, melanoma, mesothelioma, ovarian cancer, primary peritoneal cancer, salivary gland cancer, sarcoma, gastric cancer, thyroid cancer, pancreatic cancer, renal cell carcinoma, glioblastoma, and prostate cancer.

[0373] One embodiment provides an anti-VSIG4 antibody, or an antigen-binding fragment thereof, or a conjugate thereof for use in inducing an immune response in a cancer patient. Also provided herein is a method of inducing an immune response in a cancer patient in need thereof, comprising administering an anti-VSIG4 antibody, or an antigen-binding fragment thereof, or a conjugate thereof disclosed herein to the patient. The present disclosure also relates to the use of an anti-VSIG4 antibody, or an antigen-binding fragment thereof, or a conjugate thereof, for preparing a medicament for inducing an immune response in a cancer patient.

[0374] In one embodiment, the present disclosure relates to a composition comprising an anti-VSIG4 antibody or an antigen-binding fragment thereof or a conjugate thereof disclosed herein, which is used to induce an immune response in a cancer patient. Also provided herein is a method of inducing an immune response in a cancer patient in need thereof, the method comprising administering a composition comprising an anti-VSIG4 antibody or an antigen-binding fragment thereof or a conjugate thereof disclosed herein to a patient. The present disclosure also relates to the use of a composition comprising an anti-VSIG4 antibody or an antigen-binding fragment thereof or a conjugate thereof disclosed herein for the preparation of a medicament for inducing an immune response in a cancer patient.

[0375] The immune response generated by the antibodies disclosed herein includes, but is not limited to, inducing macrophages to release proinflammatory cytokines, inducing CD4 + T cell proliferation, induction of CD8 + T cell proliferation, induction of CD4 + T cells produce cytokines and induce CD8 + T cells produce cytokines.

[0376] The anti-VSIG4 antibody or its antigen-binding fragment or conjugate can be mixed with another chemotherapeutic agent, cytotoxic agent, antibody, lymphokine or hematopoietic growth factor. In particular, the treatment methods described herein may include administering an immune checkpoint inhibitor together with the anti-VSIG4 antibody or its antigen-binding fragment or conjugate. The immune checkpoint inhibitor and the anti-VSIG4 antibody or its antigen-binding fragment or conjugate can be administered simultaneously, separately or sequentially.

[0377] As used herein, "checkpoint inhibitors" refers to molecules, such as small molecules, soluble receptors, or antibodies, that target immune checkpoints and block their function. More specifically, as used herein, "checkpoint inhibitors" are molecules (such as small molecules, soluble receptors, or antibodies) that block certain proteins made by certain types of immune system cells, such as T cells and certain cancer cells.

[0378] In a first embodiment, the immune checkpoint inhibitor is any one of the following inhibitors: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules, expressed in all NK, γδ and memory CD8+ (αβ) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, IDO1, A2aR and various B-7 family ligands.

[0379] Exemplary immune checkpoint inhibitors include: anti-CTLA-4 antibodies (e.g., ipilimumab), anti-LAG-3 antibodies (e.g., BMS-986016), anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-Tim3 antibodies (e.g., TSR-022, MBG453), anti-BTLA antibodies, anti-KIR antibodies, anti-A2aR antibodies, anti-CD200 antibodies, anti-PD-1 antibodies (e.g., pembrolizumab, nivolumab, cemiplizumab, pilizumab), anti-PD-L1 antibodies (e.g., atezolizumab, avelumab, durvalumab, BMS936559), anti-VISTA antibodies (e.g., JNJ 61610588), anti-CD28 antibodies, anti-CD80 or CD86 antibodies, anti-B7RP1 antibodies, anti-B7-H3 antibodies, anti-HVEM antibodies, anti-CD137 antibodies (e.g., urelumab), anti-CD137L antibodies, anti-OX40 (e.g., 9B12, PF-04518600, MEDI6469), anti-OX40L antibodies, anti-CD40 or CD40L antibodies, anti-GAL9 antibodies, anti-IL-10 antibodies, fusion proteins of the extracellular domain of a PD-1 ligand (e.g., PDL-1 or PD-L2) and IgG1 (e.g., AMP-224), fusion proteins of the extracellular domain of an OX40 ligand (e.g., OX40L) and IgG1 (e.g., MEDI6383), IDO1 drugs (e.g., epacadostat), and A2aR drugs. Many immune checkpoint inhibitors have been approved or are undergoing clinical trials. Such inhibitors include ipilimumab, pembrolizumab, nivolumab, cemiplimab, pilizumab, atezolizumab, avelumab, durvalumab, BMS 936559, JNJ 61610588, usrelumab, 9B12, PF-04518600, BMS-986016, TSR-022, MBG453, MEDI6469, MEDI6383, and epacadostat.

[0380] Examples of immune checkpoint inhibitors are listed in, for example, Marin-Acevedo et al., Journal of Hematology & Oncology 11:8, 2018; Kavecansky and Pavlick, AJHO 13(2):9-20, 2017; Wei et al., Cancer Discov 8(9):1069–86, 2018.

[0381] Preferably, the immune checkpoint inhibitor is an inhibitor of CTLA-4, LAG-3, Tim3, PD-1, PD-L1, VISTA, CD137, OX40 or IDO1.

[0382] Diagnostic methods

[0383] VSIG4 is overexpressed in a variety of cancers, indicating that VSIG4 is a reliable biomarker for diagnosing cancer. Reagents such as labeled antibodies that bind to VSIG4 proteins provided herein can therefore be used for diagnostic purposes to detect, diagnose, or monitor cell proliferative diseases, disorders, or conditions, such as cancer.

[0384] The anti-VSIG4 antibodies provided herein can be used to detect VSIG4 in biological samples or to determine VSIG4 levels using classical immunohistological methods as described herein or known to those skilled in the art (e.g., see Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; and Jalkanen et al., 1987, J. Cell. Biol. 105:3087-3096). Other antibody-based methods that can be used to detect protein gene expression include immunoassays, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA). Suitable antibody assay labels are known in the art and include: enzyme labels, such as glucose oxidase; radioisotopes, such as iodine ( 125 I. 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 121 In) and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

[0385] Thus, in a first aspect, the present invention relates to an in vitro method for detecting a VSIG4-expressing cancer in a subject, said method comprising the following steps:

[0386] a) contacting a biological sample of the subject with an anti-VSIG4 antibody or antigen-binding fragment thereof disclosed herein; and

[0387] b) detecting binding of the reagent to the biological sample.

[0388] According to the present method, binding of VSIG4 indicates the presence of a cancer expressing VSIG4. Preferably, binding of an anti-VSIG4 antibody in an immune infiltrate of the tumor microenvironment indicates the presence of a cancer expressing VSIG4.

[0389] The present invention also relates to an in vitro method for detecting a VSIG4-expressing cancer in a subject, the method comprising the steps of:

[0390] a) contacting a biological sample of the subject with an anti-VSIG4 antibody or an antigen-binding fragment thereof; and

[0391] b) quantifying the binding of the agent to the biological sample.

[0392] According to the present method, binding of VSIG4 indicates the presence of a cancer expressing VSIG4. Preferably, binding of an anti-VSIG4 antibody in an immune infiltrate of the tumor microenvironment indicates the presence of a cancer expressing VSIG4.

[0393] As will be apparent to the skilled artisan, the level of antibodies binding to VSIG4 can be quantified by any means known to those skilled in the art, as described in detail below. Preferred methods include the use of immunoenzymatic assays such as ELISA or ELISPOT, immunofluorescence, immunohistochemistry (IHC), radioimmunoassay (RIA) or FACS.

[0394] The quantification in step b) of the present method is a direct reflection of the expression level of VSIG4 in the sample (particularly in the immune infiltrate of the tumor microenvironment). The present method thus allows for the identification of cancers expressing VSIG4 by determining the expression level of VSIG4 as described above. In a preferred embodiment, the expression level of VSIG4 in the sample (particularly in the immune infiltrate of the tumor microenvironment) is compared to a reference level.

[0395] According to a further preferred embodiment, the present invention relates to an in vitro method for detecting a VSIG4-expressing cancer in a subject, said method comprising the following steps:

[0396] a) determining the expression level of VSIG4 in a biological sample of the subject; and

[0397] b) comparing the expression level of step a) with a reference level;

[0398] wherein an increase in the determined level of VSIG4 in step a) compared to the reference level is indicative of a cancer expressing VSIG4.

[0399] The present invention also relates to an in vitro method for diagnosing a VSIG4-expressing cancer in a subject, the method comprising the steps of:

[0400] a) determining the expression level of VSIG4 in a biological sample of the subject; and

[0401] b) comparing the expression level of step a) with a reference level;

[0402] wherein an increase in the determined level of VSIG4 in step b) compared to the reference level is indicative of a cancer expressing VSIG4.

[0403] Advantageously, the expression level of VSIG4 is compared or measured relative to the level in a control cell or sample (also referred to as a "reference level" or "reference expression level"). "Reference level," "reference expression level," "control level," and "control" are used interchangeably herein. A "control level" refers to a single baseline level measured in comparable control cells, which typically do not have disease or cancer. The control cells can be from the same individual, as even in cancer patients, the tissue that serves as the tumor site still includes non-tumor healthy tissue. They can also be from another normal individual or an individual who does not have the same disease as the diseased or test sample. In the context of the present invention, the term "reference level" refers to a "control level" of VSIG4 expression, which is used to assess the test expression level of VSIG4 in a patient's sample containing cancer cells. For example, when the level of VSIG4 in a patient's biological sample is higher than a reference level of VSIG4, the cells are considered to have a high level of expression or overexpression of VSIG4. The reference level can be determined by a variety of methods. Thus, the expression level can define cells that harbor VSIG4, or alternatively, the expression level of VSIG4 independent of the number of cells expressing VSIG4. Thus, a reference level for each patient can be specified by a reference ratio of VSIG4, wherein the reference ratio can be determined by any of the methods for determining reference levels described herein.

[0404] For example, the control can be a predetermined value, which can take a variety of forms. It can be a single cutoff value, such as the median or mean. A "reference level" can be a single number, applied equally to every individual patient, or it can vary based on specific subpopulations of patients. Thus, for example, older men might have different reference levels than younger men for the same cancer, while women might have different reference levels than men for the same cancer. Alternatively, a "reference level" can be determined by measuring the expression level of VSIG4 in non-oncogenic cancer cells from the same tissue as the tumor cells being tested. Furthermore, a "reference level" can be a ratio of VSIG4 levels in a patient's tumor cells relative to the level of VSIG4 in non-tumor cells from the same patient. A "reference level" can also be the level of VSIG4 in cultured cells in vitro, manipulated to mimic tumor cells, or manipulated in any other manner that produces expression levels that accurately determine the reference level. Alternatively, a "reference level" can be established based on a comparison group, such as a group without elevated VSIG4 levels and a group with elevated VSIG4 levels. Another example of a comparison group is a group with a particular disease, condition, or symptom and a group without that disease. For example, the predetermined values ​​may be arranged into equal (or unequal) groups according to the test population, such as a low-risk group, a medium-risk group, and a high-risk group.

[0405] Reference levels can also be determined by comparing VSIG4 levels in a population of patients with the same cancer. This can be accomplished, for example, by a histogram analysis, in which the entire patient cohort is presented graphically, with the first axis representing the level of VSIG4 and the second axis representing the number of patients in the cohort whose tumor cells express a given level of VSIG4. Two or more separate patient groups can be determined by identifying subgroups of the cohort with identical or similar VSIG4 levels. Reference levels can then be determined based on the levels that best distinguish these separate groups. Reference levels can also represent the levels of two or more markers, one of which is VSIG4. For example, two or more markers can be represented by a ratio of the level values ​​of each marker.

[0406] Similarly, an apparently healthy population will have a different "normal" range than a population known to have a condition associated with VSIG4 expression. Therefore, the selection of predetermined values ​​can take into account the category to which the individual belongs. One of ordinary skill in the art can select appropriate ranges and categories using only routine experimentation. "Elevated" or "increased" means higher relative to a selected control. Typically, controls will be based on apparently healthy, normal individuals of the appropriate age range.

[0407] It will also be understood that a control according to the present invention may also be a sample of material tested in parallel with the experimental material in addition to a predetermined value. Examples include tissue or cells obtained simultaneously from the same subject, such as a portion of a single biopsy, or a portion of a single cell sample from a subject.

[0408] Preferably, the reference level of VSIG4 is the expression level of VSIG4 in a normal tissue sample (eg, from a patient not suffering from a cancer expressing VSIG4, or from the same patient before the onset of the disease).

[0409] A more definitive diagnosis of VSIG4-expressing cancers could allow health professionals to employ preventative measures or aggressive treatment earlier, thereby preventing the development or further progression of the VSIG4-expressing cancers.

[0410] The present invention will be described in detail below with reference to Examples. However, the following Examples are given only to illustrate the present invention, and it is obvious that the present invention is not limited to the following Examples.

[0411] Example

[0412] Example 1. Characteristics of long and short forms of VSIG4

[0413] 1-1. Expression of long and short forms of VSIG4 on macrophages

[0414] Macrophages are known to express VSIG4. To test whether there was a difference in expression, the presence of each of the two forms of VSIG4, VSIG4(L) and VSIG4(S), was looked for in extracts of M1 and M2 macrophages.

[0415] 50 ng / ml IFN-γ (285-IF, R&D) was added to GM-CSF-differentiated M0 macrophages to polarize them into pro-inflammatory M1 macrophages. 20 ng / ml of each of the following cytokines: IL-4 (130.093.922, Miltenyi Biotec), IL-10 (217-IL / CF, R&D), and TGF-β (130.095.066, Miltenyi Biotec) were added to M-CSF-differentiated M0 macrophages to polarize them into immunosuppressive M2 macrophages. Differentiated M0 macrophages were incubated with cytokines at 37°C, 5% CO2 for 2 days. M1 and M2 polarized macrophages were obtained on day 8. Polarized macrophages were activated with 100 ng / ml LPS (L4516, Sigma) for 4 hours at 37°C, 5% CO2. The macrophages were then harvested and washed in culture medium. Binding of target antibodies on polarized M1 and M2 macrophages was assessed by flow cytometry following LPS activation.

[0416] 15 μg of M1 and M2 protein extracts and 100 ng of hVSIG4(L)-hFc and hVSIG4(S)-hFc were electrophoresed on SDS-PAGE gels, transferred to membranes, and probed with a VSIG4-specific polyclonal antibody (AF4646, R&D Systems, Minneapolis, MN, USA) or a goat isotype control.

[0417] Two bands of the expected size were observed in extracts from M2 macrophages ( Figure 1 B). This result confirms previous data showing that VSIG4 is expressed in macrophages. Furthermore, it shows that both hVSIG4(S) and hVSIG4(L) are expressed in macrophages.

[0418] 1-2. Expression of long and short forms of VSIG4 in tumors

[0419] The expression of two VSIG4 forms in tumors was studied. The VSIG4 gene is located on chromosome X, and 7 exons are depicted in the gene model. This results in the production of two messenger RNAs through alternative splicing. A long form, Long-VSIG4 (uc004dwh.2), and a short form, Short-VSIG4 (uc004dwi.2), produce hVSIG4 (L) and hVSIG4 (S), respectively. The Cancer Genome Atlas (TCGA) contains data from the characterization of more than 20,000 primary cancers and matched normal samples across 33 cancer types. The expression patterns of the two isoforms were determined using ISOexpresso using TCGA tumor expression data (Tumor TCGA RNASeq) (Yang et al., BMC Genomics (2016) 17: 631; http: / / wiki.tgilab.org / ISOexpresso / ).

[0420] The results are shown in Table 3.

[0421] Table 3: Percentage expression of long VSIG4 isoforms and short VSIG4 isoforms according to indication

[0422]

[0423]

[0424] Both long and short VSIG4 isoforms are expressed in tumors.

[0425] 1-3. hVSIG4(S) and hVSIG4(L) inhibit CD4 + T cell activation

[0426] 96-well plates were coated with 100 μl / well of 2.5 μg / ml anti-CD3 OKT3 antibody (BioxCell ref BE0001-2 clone OKT3) for 4 hours at 37°C, washed twice with PBS and coated with 10 μg / ml recombinant proteins (VSIG4(L)-Fc (SEQ ID NO. 183), VGIG4(S)-Fc (SEQ ID NO. 184), PDL1-Fc (R&D Systems 156-B7) or isotype control hIgG1 (c9G4) and incubated overnight at 4°C. The wells were washed twice with PBS and 200,000 negatively purified and CFSE-labeled CD4 from healthy donors in 200 μl of culture medium were added. + T cells were added to each well.

[0427] After 3 days of culture, the supernatant was transferred to a new plate and analyzed for IFNγ release by MSD. In addition, the cells were analyzed by flow cytometry to assess their proliferation rate.

[0428] Figure 2 A shows that both forms of VSIG4 (VSIG4(S) and VSIG4(L)) inhibit CD4 + T cell proliferation. Similarly, both forms inhibit CD4 + T cells release IFNγ ( Figure 2 B).

[0429] Example 2. Production and purification of VSIG4 antigen

[0430] 2-1. Construction of VSIG4 antigen protein expression vector

[0431] To clone the VSIG4 protein, a Jurkat cell cDNA library (Stratagene, USA) was amplified by polymerase chain reaction (PCR) using primers for VSIG4 (Table 4), which included the restriction enzyme sites Sfi I at the 5' and 3' positions to obtain only the extracellular domain (20Arg-Ser281). The amplified PCR product was fused to human Fc (hFc) or mouse Fc (mFc) at the carboxyl terminus using the N293F vector ( Figure 4 ).

[0432] Table 4: PCR primers for cloning VSIG4

[0433]

[0434] 2-2. Expression and purification of VSIG4 antigen protein

[0435] HEK293F cells (Invitrogen, USA) were transfected with the prepared VSIG4 antigen plasmid using PEI (polyethyleneimine: #23966, Polysciences, USA). Thereafter, the cells were cultured in FreeStyle 293 expression medium (#AG100009, Thermo Fisher Scientific, USA, a serum-free medium) for 7 days. The cell culture containing the VSIG4 antigen was collected and centrifuged at 5,000 rpm for 10 minutes, and a 0.22 μm TOP-filter (Millipore, USA) was used to remove residual cells and floating materials. The first purification of the antigen was performed based on affinity chromatography using protein A agarose resin. The protein obtained after the first purification was purified for a second time using Superdex 200 (1.5 cm × 100 cm) gel filtration chromatography.

[0436] The purity of the purified protein was determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) under reducing conditions. Figure 5As shown, the purity of the purified VSIG4-hFc and VSIG4-mFc proteins was found to be 95% or higher.

[0437] Example 3. Selection of VSIG4 human antibodies

[0438] 3-1. Biopanning

[0439] VSIG4-hFc and VSIG4-mFc prepared in Example 2, VSIG4-his (12163-H08H) protein antigen purchased from Sino Biological Inc., and ITGA6-Fc (50 μg) used as a nonspecific binding indicator were coated on immunosorbent tubes and then blocked.

[0440] For human antibody library phage, 2.7 × 10 10 The human scFv (single chain variable fragment) library of 1000 kinds of bacteria was infected, and then the bacteria were cultured at 30 ° C for 16 hours. After cultivation, PEG (polyethylene glycol, Sigma) was used for centrifugation to concentrate the supernatant, and the resultant was dissolved in PBS buffer to prepare the human antibody library. The library phage was added to the immunoadsorption tube and allowed to react for 2 hours at room temperature. Then, after washing with 1×PBS-Tween20 (PBS-T) and 1×PBS, only the scFv-phage that specifically bound to the antigen was eluted.

[0441] A positive phage pool was obtained by re-infecting bacteria with the eluted phage for amplification. Using the phage amplified in the first round, the second and third rounds of panning were performed in the same manner as the first round, except for increasing the number of PBS-T wash steps. The results are shown in Table 5, confirming a slight increase in the output of phage binding to the antigen relative to the input during the third round of panning.

[0442] Table 5: Comparison of antibody titers according to panning

[0443] Panning times Input phage quantity Output phage quantity 1 <![CDATA[3×10 11 ]]> <![CDATA[1.05×10 6 ]]> 2 <![CDATA[5×10 12 ]]> <![CDATA[2.5×10 5 ]]> 3 <![CDATA[3×10 10 ]]> <![CDATA[2.4×10 7 ]]>

[0444] 3-2. Polyphage ELISA

[0445] In order to examine the antigen specificity of the positive multi-scFv-phage antibody pool obtained from each round of panning in Example 3-1, a multi-phage ELISA (enzyme-linked immunosorbent assay) was performed.

[0446] The frozen cell stock solution after each panning from the first to the third round was added to a culture medium containing 2×YTCM (10 g yeast extract, 17 g tryptone, 5 g NaCl, 34 μg / ml chloramphenicol), 2% glucose and 5 mM magnesium chloride (MgCl 2 ) to an OD of 0.1 and then cultured at 37° C. for 2 to 3 hours (OD of 0.5 to 0.7). Then, after infection with M1 helper phage, the cells were cultured at 30° C. for 16 hours in a culture medium containing 2×YTCMK (2×YTCM, 35 μg / ml kanamycin), 5 mM magnesium chloride and 1 mM IPTG. The cultured cells were centrifuged (4500 rpm, 15 minutes, 4° C.) and the supernatant was transferred to a new tube. On a 96-well immunoplate (#439454, NUNC, USA), each of the two antigens was coated with 100 ng per well of coating buffer at 4°C for 16 hours, and then each well was blocked with 4% skim milk dissolved in PBS. Each well was then washed with 0.2 ml PBS-T, and the multi-scFv-phage from the first to third panning was added to each well in an amount of 100 μl per well, followed by reaction at room temperature for 2 hours. Each well was then washed again with 0.2 ml PBS-T 4 times, and anti-M13-HRP (Amersham 27-9421-01) as a secondary antibody was diluted 1:2000 and reacted with the antibody at room temperature for 1 hour. After washing with PBS-T, OPD tablets (Sigma.8787-TAB) were prepared in PC buffer (0.1 M Na2HPO4, 0.005 M sodium citrate, pH 5.0) and added to the wells (100 μl per well) for color development for 10 minutes. The absorbance at 490 nm was then measured using a spectrophotometer (Molecular Devices, USA).

[0447] The results are as follows Figure 6 As shown, the antigen binding properties for both VSIG4 antigens were confirmed by ELISA in the third multi-scFv-phage enrichment.

[0448] 3-3. Selection of positive phage

[0449] Colonies obtained from the polyclonal phage antibody panel with high binding properties (third panning) were cultured in 1 ml of medium containing 2×YTCM, 2% glucose, and 5 mM magnesium chloride at 37°C for 16 hours in a 96-deep-well plate (#90030, Bioneer, Korea). 100-200 μl of the cultured cells were collected to an OD600 of 0.1, and then added to a medium containing 1 ml of 2×YTCM, 2% glucose, and 5 mM magnesium chloride. The cells were cultured in a 96-deep-well plate at 37°C for 2 to 3 hours to an OD600 of 0.5 to 0.7. The cells were infected with M1 helper phage at an MOI of 1:20 and then cultured at 30°C in a medium containing 2×YTCMK, 5 mM magnesium chloride, and 1 mM IPTG for 16 hours.

[0450] At 4 ° C, on a 96-well immunoplate, the antigen VSIG4 was coated in an amount of 100 ng per well for 16 hours, and then each well was blocked with 4% skim milk dissolved in PBS. Each well was then washed with 0.2 ml PBS-T, and the monoclonal scFv-phage (100 scFv-phages, respectively) cultured for 16 hours was added to each well in an amount of 100 μl and reacted at room temperature for 2 hours. Each well was then washed again with 0.2 ml PBS-T 4 times, and the anti-M13-HRP as a secondary antibody was diluted 1:2000 and reacted with the antibody at room temperature for 1 hour. After washing with PBS-T (0.2 ml), color development was allowed to occur and the absorbance at 490 nm was measured.

[0451] Depend on Figure 7 The results shown indicate that several dozen single phage clones targeting VSIG4 were obtained with high binding properties to each antigen.

[0452] 3-4. Nucleotide sequencing of positive phage antibodies

[0453] For the individual clones selected as described above, DNA was obtained by DNA-prep using a kit for DNA purification (Qiagen, Germany). DNA sequencing was performed at Macrogen, Korea. Based on the sequencing results, the CDR sites of the heavy chain variable region (VH) and light chain variable region (VL) of the selected antibody were determined. The similarity between these antibodies and the germline antibody group was then checked using the Ig BLAST program (http: / / www.ncbi.nlm.nih.gov / igblast / ) provided in the NCBI webpage. As a result, 39 VSIG4-specific phage antibodies were obtained, 11 of which were more specifically characterized. They are summarized in Table 2.

[0454] Example 4. Production of VSIG4 Human Antibody

[0455] 4-1. Converting scFv format to IgG format

[0456] In order to fully convert 39 selected monoclonal phage antibodies against VSIG4 into IgG form, DNA sequences corresponding to the heavy and light chain variable regions were subjected to PCR (iCycler iQ, BIO-RAD, USA) using primers containing SfiI / NheI and SfiI / BglII restriction enzyme sites, respectively. The heavy and light chain PCR products were digested, each of which had a corresponding restriction enzyme site in the expression vector, and the DNA was purified using a DNA gel extraction kit (Qiagen). For ligation, the vector (1 μl, 10 ng), heavy or light chain (100 to 200 ng, 15 μl), 10× buffer (2 μl), ligase (1 U / μl, 1 μl) and water were mixed with each other, kept at room temperature for 1 to 2 hours, and then added to cells (competent cells, XL1-blue) for transformation. The resultant was kept on ice for 5 minutes, and then heat-shocked at 42°C for 90 seconds. After heat shock, 1 ml of culture medium was added to the cells and incubated at 37°C for 1 hour. The cells were then plated onto LB Amp plates and incubated at 37°C for 16 hours. The resulting colonies were collected and inoculated with 5 ml of LB Amp medium. After incubation at 37°C for 16 hours, DNA prep was performed using a DNA prep kit (Nuclogen). DNA sequencing of the resulting DNA was requested (Macrogen, Korea).

[0457] The results confirmed that each heavy and light chain of 11 antibody clones against VSIG4, all of which had been converted into complete IgG, corresponded to the sequence of the phage antibody. The heavy and light chain plasmid DNA with the identified sequences was then used for antibody production.

[0458] 4-2. Production of human antibodies

[0459] The prepared expression vectors containing the heavy and light chains were co-transfected into HEK-293F cells at a ratio of 6:4. Seven days after co-transfection, the supernatant was collected, and cells and floating matter were removed by centrifugation and a 0.22 μm top filter. The supernatant was collected and subjected to protein A affinity chromatography to purify the IgG antibody. After purification, the antibody was separated using lysine buffer and the buffer was exchanged so that the final resuspension buffer was PBS. The purified antibody was quantified by BCA and Nano-drop to determine the production quantity. The antibody was then analyzed by SDS-PAGE with 5 μg of sample for each reducing and non-reducing condition. The purity and migration state of the purified protein were thus determined.

[0460] The results are as follows Figure 8As shown, 11 VSIG4 monoclonal antibodies were detected under non-reducing conditions with a size of at least 150 kDa and production amounts varying from as low as 5 mg / L to as high as 142.6 mg / L.

[0461] Example 5. VSIG4 Binding Properties of VSIG4 Human Antibodies

[0462] 5-1. Antibody binding specificity for cell surface VSIG4

[0463] To obtain a pool of transformed cells overexpressing human VSIG4, HEK293E cells were transfected with a pcDNA3.1 plasmid containing human VSIG4, and then a selection process was performed in a selection medium containing 400 μg / ml Zeocin (#R25001, Thermo Fisher Scientific). After the selection process, the cell pool overexpressing VSIG4 was isolated by determining the expression status by FACS (fluorescence-activated cell sorting) analysis using an anti-human VSIG4 antibody linked to an APC (allophycocyanin) fluorescent material (#17-5757-42, ebioscience, USA). Figure 9 B), and no basal expression of VSIG4 was confirmed by FACS analysis in HEK293E cells (used as the parent cell of the HEK293E cell pool overexpressing human VSIG4) using an anti-human VSIG4 antibody linked to an APC fluorescent material ( Figure 9 A), antibody characterization analysis of 11 anti-human VSIG4 antibodies was evaluated using these two cell lines.

[0464] To confirm that the VSIG4 antibody binds to cells, 0.5 × 10 6 cells and allowed to react with 0.08 μg / ml, 0.4 μg / ml or 2 μg / ml of antibodies at 4°C for 30 minutes. Thereafter, the cells were washed three times with a buffer containing 2% PBS and reacted with an anti-human IgG antibody (#FI-3000, Vectorlabs) linked to a FITC (fluorescein isothiocyanate) fluorescent material at 4°C for 20 minutes. The cells were washed by the same washing process as above and then suspended in 0.5 ml PBS containing 2% FPS. The cells were then analyzed by FACSCanto II flow cytometer. The results showed that all 11 human VSIG4 monoclonal antibodies bound well to cells overexpressing human VSIG4 in a concentration-dependent manner ( Figure 10 B). However, there was no binding in HEK293E cells without basal expression of VSIG4 ( Figure 10 A). The results indicate that 11 human VSIG4 monoclonal antibodies specifically bind to human VSIG4 antigen.

[0465] 5.2. FACS analysis of the binding of VSIG4 human antibodies to human native VSIG4

[0466] The binding properties of a series of anti-VSIG4 antibodies were evaluated by FACS analysis in HEK293E expressing human VSIG4 using increasing antibody concentrations. The same experiment was performed with the murine monoclonal antibody m6H8 recognizing VSIG4 described in WO 2020 / 069507. For this purpose, cells (1×10 6 Cells were incubated with each of 11 complete Ig anti-VSIG4 antibodies or m6H8 for 20 minutes. The cells were then washed three times and incubated with the appropriate Alexa 488-conjugated secondary antibody at 4°C in the dark for another 20 minutes, followed by three washes in FACS buffer. Binding of anti-VSIG4 antibodies or m6H8 was immediately performed on live cells, and binding was identified using propidium iodide (staining dead cells). The maximum signal intensity obtained with each antibody was set as B max The fluorescence intensity was expressed as mean fluorescence intensity (MFI). Nonlinear regression analysis (GraphPad Prims 4.0) was used to calculate the EC of the binding. 50 , expressed as molar concentration (M).

[0467] Titration curves for each murine or chimeric antibody showed that all generated antibodies were able to recognize the native VSIG4 form and had typical saturation curves. The binding EC value of each antibody was determined using nonlinear regression analysis. 50 .EC 50 Between 1.2×10 -9 and 9.3×10 -10 Between. EC 50 The values ​​are summarized in Table 6.

[0468] Table 6: EC of anti-VSIG4 antibodies 50

[0469]

[0470]

[0471] 5.3. Binding of VSIG4 human antibodies to long and short forms of VSIG4

[0472] In a first series of experiments, 11 scFv anti-VSIG4 antibodies were tested for binding to each of the long and short forms of VSIG4 by ELISA. Under these conditions, each of the 11 antibodies tested was detected to bind specifically to both forms ( Figure 11 A).

[0473] To confirm this result, the binding of 11 anti-VSIG4 antibodies to each of the long and short forms of VSIG4 was determined by Western blotting. In each case, 100 ng of hVSIG4 long-hFc (L) and hVSIG4 short-hFc (S) were probed with specific anti-VSIG4 antibodies. Figure 11 As shown in Figure B, two bands of the expected size were observed for each of the 11 anti-VSIG4 antibodies. In contrast, the murine monoclonal antibody m6H8, which recognizes VSIG4 and is described in WO 2020 / 069507, only binds to the long form of VSIG4 ( Figure 12 A). This result was confirmed in ELISA assay ( Figure 12 B).

[0474] 5-4. Epitope mapping of anti-VSIG4 antibodies

[0475] To characterize the epitopes recognized by each of the 11 human anti-VSIG4 antibodies, their ability to bind to a series of soluble VSIG4 proteins carrying specific mutations was determined by ELISA. The constructs used are detailed in Table 7.

[0476]

[0477] At 4 ° C, on a 96-well immunoplate, various antigens were coated in an amount of 100ng per well for 16 hours, and then each well was blocked with 4% skim milk dissolved in PBS. Each well was then washed with 0.2ml PBS-T, and each of the 11 scFv-phages cultured for 16 hours was added to each well in an amount of 100μl and reacted at room temperature for 2 hours. Each well was then washed again with 0.2ml PBS-T 4 times, and anti-M13-HRP as a secondary antibody was diluted 1:2000 and reacted with the antibody at room temperature for 1 hour. After washing with PBS-T (0.2ml), color development was allowed to occur and the absorbance at 490nm was measured.

[0478] The results are shown in Figure 13 .

[0479] The epitope recognized by antibodies SA1956, SA1957, and SA2285 comprises at least one of amino acids E24, V25, E27, V29, and T30.

[0480] The epitopes recognized by antibodies SA1975 and SA2290 include at least one of amino acids I77, A80, Y82, and Q83. At least one of residues Q59, G61, S62, D63, and V65 may also contribute to the binding of SA2290 to VSIG4.

[0481] The epitope recognized by antibody SA2283 comprises at least one of amino acids R108, S109, H110, T112, and E114.

[0482] The epitope recognized by antibody SA2287 comprises at least one of amino acids T119, P120, D121, N123, Q124, and V125.

[0483] The epitope recognized by antibody SA2291 comprises at least one of amino acids R108, S109, H110, T112, and E114, and at least one of amino acids T119, P120, D121, N123, Q124, and V125.

[0484] The epitope recognized by antibody SA2390 includes at least one of amino acids Q59, G61, S62, D63, and V65, at least one of amino acids S97, Q99, S101, and T102, at least one of amino acids R108, S109, H110, T112, and E114, and at least one of amino acids T119, P120, D121, N123, Q124, and V125. In addition, at least one of residues D36T, N38R, L39M, and T42, and at least one of residues I77, A80, Y82, and Q83 may also contribute to the binding of SA2390 to VSIG4.

[0485] The epitope recognized by antibody SA2455 includes at least one of amino acids Q59, G61, S62, D63, and V65; at least one of amino acids S97, Q99, S101, and T102; at least one of amino acids R108, S109, H110, T112, and E114; and at least one of amino acids T119, P120, D121, N123, Q124, and V125. Furthermore, at least one of residues D36T, N38R, L39M, and T42 may also contribute to the binding of SA2390 to VSIG4.

[0486] Example 6. Internalization of VSIG4 human antibody

[0487] Eleven full Ig anti-VSIG4 antibodies were evaluated in the internalization assay.

[0488] For this assay, HEK-VSIG4 cells (i.e., HEK293 cells transfected and expressing VSIG4 on the surface) at 80% confluence were detached in trypsin and counted in a ViCells counter. 100,000 HEK-VSIG4 cells were incubated at 4°C for 20 minutes in the presence of 10 μg / ml of each antibody: anti-VSIG4, m6H8 antibody, and its humanized version A2 (WO 2020 / 069507), or anti-IGF1R (Hz208F2-4) in a total of 100 μl of cold culture medium. The cells were then centrifuged at 2000 rpm and washed twice with 200 μl of cold culture medium.

[0489] Time T0: cells were directly incubated with 200 μl of 1 / 500 diluted goat anti-human Alexa 488 secondary antibody for 20 minutes at 4° C. Cells were then washed twice in cold culture medium and analyzed by FACS.

[0490] Time 4h: Incubate cells for 4 hours in 100 μl of cold culture medium (4°C) or warm culture medium (37°C). Each batch of cells was spun at 2000 rpm, washed twice with 200 μl of cold culture medium, and incubated with goat anti-human Alexa 488 secondary antibody in cold culture medium for 20 minutes at 4°C. The cells were then washed twice in cold culture medium and analyzed by FACS.

[0491] The level of internalization was determined by calculating the ΔMFI corresponding to (MFI (at 4°C) - MFI (at 37°C)), and the percentage of internalization was determined by calculating the percentage decrease in MFI between 4°C and 37°C. (All MFIs were calculated after subtracting the isotype MFI value).

[0492] Since HEK cells express IGF-1R on the cell surface, the internalizing anti-IGF-1R antibody Hz208F2-4 (WO 2015 / 162292) was used as a positive control in this assay.

[0493] The anti-VSIG4 antibodies of the present invention showed different levels of internalization (see Table 8). On the other hand, the m6H8 antibody and its humanized version A2 did not induce any type of internalization.

[0494] Table 8: Internalization of 11 anti-VSIG4 human antibodies

[0495] T4h 4℃-isotype T4h 37℃-isotype T0-isotype Internalization% ΔMFI SA1956 2336 1735 2635 26 602 SA1957 1645 582 2213 65 1064 SA1975 1493 952 1932 36 542 SA2283 2254 2259 2595 0 -5 SA2285 955 578 1280 39 376 SA2287 1033 742 1461 28 291 SA2290 1844 984 2319 47 860 SA2291 546 161 813 70 384 SA2386 502 116 1060 77 385 SA2390 1240 843 1433 32 397 SA2455 1888 1546 2342 18 342 Hz208F2-4 113 56 139 50 57 A2 1580 2019 1727 0 -439 m6H8 970 1107 972 0 -138

[0496] Example 7. Inhibitory effects of VSIG4 human antibodies on the anti-inflammatory and immunosuppressive functions of VSIG4

[0497] 7-1. Inflammation assay

[0498] To evaluate the ability of anti-VSIG4 antibodies to modulate the inflammatory phenotype of macrophages, cytokine release assays were performed on macrophages treated with each fully Ig human anti-VSIG4 antibody. Figure 14 .

[0499] EFS (Etablissement Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using a cell separation loop provided by Sigma-Aldrich (Du Sang). Monocytes were then purified from PBMCs by positive immunomagnetic cell selection according to the manufacturer's instructions (130-050-201, Miltenyi Biotec).

[0500] Fresh monocytes were seeded into 96-well flat-bottom treated culture plates (353072, Falcon) in RPMI 1640 medium (1% penicillin-streptomycin, 1% sodium pyruvate, 1% L-glutamine, and 10% fetal bovine serum) supplemented with 50 ng / ml M-CSF (130-096-492, Miltenyi Biotec). The cells were cultured at 37°C, 5% CO₂, for 6 days to differentiate into macrophages. Differentiated M0 macrophages were obtained on day 6.

[0501] The binding of the target antibody on differentiated M0 macrophages was assessed by flow cytometry on the 6th day. LPS (L4516, Sigma) was added to differentiated M0 macrophages at a final concentration of 100 ng / ml. Test antibodies or corresponding isotypes were added to differentiated M0 macrophages at three concentrations (2.5 μg / ml, 5 μg / ml and 10 μg / ml). Murine antibody m6H8 and its humanized form A2 (both described in WO 2020 / 069507) were also tested in the assay. As a control, a control antibody (R&D, RefMAB2078, clone 287219, mIgG2a) known to stimulate the release of cytokines from M0 macrophages and the conversion to a pro-inflammatory phenotype was used at a final concentration of 5 μg / ml. For SA1956, SA2386, SA2390 and SA2455, 50 μg / ml C3b (A114, Complement Technology) was added to the culture medium.

[0502] Differentiated M0 macrophages were incubated with LPS and test antibodies for 24 hours at 37°C, 5% CO2. Cell culture supernatants were harvested on day 7 and transferred to new V-bottom 96-well plates for cytokine analysis. Concentrations of IL-10, IL-6, IL-1β, IL-12 / 23p40, and TNF-α were measured. Quantification was performed using Meso Scale Discovery technology according to the manufacturer's instructions (K15UQK-4 and K151AOH-4, Meso Scale Discovery).

[0503] At least five donors were evaluated to account for heterogeneity among healthy donors. Each experimental condition was performed in triplicate within a single experiment.

[0504] The measurement results are shown in Table 9.

[0505] Table 9: Human monocyte-derived macrophages modulate cytokine release in response to VSIG4 antibody treatment

[0506]

[0507]

[0508] Except for SA2285, all anti-VSIG4 antibodies led to increased pro-inflammatory cytokine release and / or decreased anti-inflammatory cytokine secretion by macrophages.

[0509] Therefore, these antibodies are able to modulate the phenotype of human macrophages.

[0510] 7-2. Immunosuppression assay

[0511] EFS (Etablissement Peripheral blood mononuclear cells (PBMCs) were isolated from human blood using a cell separation loop provided by du Sang. Monocytes and CD4 + T cells: Monocytes were purified by positive immunomagnetic cell selection according to the manufacturer's instructions (130-050-201, Miltenyi Biotec), while CD4 T cells were isolated from the non-positive fraction of the monocyte purification by negative immunomagnetic cell selection according to the manufacturer's instructions (19052, STEMCELL Technologies). + T cells were cultured in 1 ml of freezing medium (07930, STEMCELL Technologies) at a rate of 15 × 10 cells per cryovial. 6 Freeze CD4 cells + T cells for further co-culture.

[0512] Fresh monocytes were seeded into 96-well flat-bottom treated culture plates (353072, Falcon) in culture medium (RPMI 1640 medium + 1% penicillin-streptomycin + 1% sodium pyruvate + 1% L-glutamine + 10% fetal bovine serum) containing either 50 ng / ml M-CSF (130-096-492, Miltenyi Biotec) for further M2 macrophage polarization or 50 ng / ml GM-CSF (130-093-866, Miltenyi Biotec) for further M1 macrophage polarization. The cells were cultured at 37°C, 5% CO2 for 6 days to differentiate into macrophages. Differentiated M0 macrophages were obtained on day 6.

[0513] 50 ng / ml IFN-γ (285-IF, R&D) was added to GM-CSF-differentiated M0 macrophages to polarize them into pro-inflammatory M1 macrophages. 20 ng / ml of each of the following cytokines: IL-4 (130.093.922, Miltenyi Biotec), IL-10 (217-IL / CF, R&D), and TGF-β (130.095.066, Miltenyi Biotec) were added to M-CSF-differentiated M0 macrophages to polarize them into immunosuppressive M2 macrophages. Differentiated M0 macrophages were incubated with cytokines at 37°C, 5% CO2 for 2 days. M1 and M2 polarized macrophages were obtained on day 8. Polarized macrophages were activated with 100 ng / ml LPS (L4516, Sigma) for 4 hours at 37°C, 5% CO2. The macrophages were then harvested and washed in culture medium. Binding of target antibodies on polarized M1 and M2 macrophages was assessed by flow cytometry following LPS activation.

[0514] M1 and M2 macrophages were seeded at 20,000 cells / well in a classic flat-bottom 96-well plate in culture medium. Cells were cultured for 24 hours at 37°C and 5% CO2. + T cells were added to macrophages at a ratio of 1 macrophage to 5 CD4 T cells. CD3 / CD28 beads (111-32D, Gibco) were added to the co-culture to activate CD4 + T cells.

[0515] The test antibody or the corresponding isotype was added to the co-culture at a final concentration of 10 μg / ml. The anti-PD-L1 monoclonal antibody avelumab was used as a positive control. For SA2386, 50 μg / ml C3b (A114, Complement Technology) was added to the culture medium. The co-cultured macrophages and CD4 + T cells were cultured for 5 days. On day 14, cell culture supernatants were harvested and transferred to fresh V-bottom 96-well plates for cytokine analysis. IFN-γ concentrations were measured and quantified using Meso Scale Discovery technology according to the manufacturer's instructions (K151AEB-4, Meso Scale Discovery).

[0516] At least five donors were evaluated to account for heterogeneity among healthy donors. Each experimental condition was performed in triplicate within one experiment.

[0517] The measurement results are shown in Table 10.

[0518] Table 10: M2 macrophage-mediated immunosuppression is reversed in response to VSIG4 antibody treatment Quantification of IFN-γ secretion was used as a surrogate for T cell activation.

[0519]

[0520] Except SA2285, all anti-VSIG4 antibodies induced CD4 + T cells release IFN-γ, indicating that they trigger T cell activation. Therefore, these antibodies can inhibit the immunosuppressive function of VSIG4. Sequence Listing <110> Y Biotech Co., Ltd. Pierre Fabre Pharmaceuticals <120> Anti-VSIG4 antibodies or antigen-binding fragments and uses thereof <130> B378459PCTD40837 <150> KR10-2019-0109365 <151> 2019-09-04 <160> 184 <170> PatentIn version 3.5 <210> 1 <211> 399 <212> PRT <213> people <400> 1 Met Gly Ile Leu Leu Gly Leu Leu Leu Leu Gly His Leu Thr Val Asp 1 5 10 15 Thr Tyr Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro 20 25 30 Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly 35 40 45 Tyr Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro 50 55 60 Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala 65 70 75 80 Lys Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val 85 90 95 Ser Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr 100 105 110 Cys Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp 115 120 125 Lys Ile Thr Glu Leu Arg Val Gln Lys Leu Ser Val Ser Lys Pro Thr 130 135 140 Val Thr Thr Gly Ser Gly Tyr Gly Phe Thr Val Pro Gln Gly Met Arg 145 150 155 160 Ile Ser Leu Gln Cys Gln Ala Arg Gly Ser Pro Pro Ile Ser Tyr Ile 165 170 175 Trp Tyr Lys Gln Gln Thr Asn Asn Gln Glu Pro Ile Lys Val Ala Thr 180 185 190 Leu Ser Thr Leu Leu Phe Lys Pro Ala Val Ile Ala Asp Ser Gly Ser 195 200 205 Tyr Phe Cys Thr Ala Lys Gly Gln Val Gly Ser Glu Gln His Ser Asp 210 215 220 Ile Val Lys Phe Val Val Lys Asp Ser Ser Lys Leu Leu Lys Thr Lys 225 230 235 240 Thr Glu Ala Pro Thr Thr Met Thr Tyr Pro Leu Lys Ala Thr Ser Thr 245 250 255 Val Lys Gln Ser Trp Asp Trp Thr Thr Asp Met Asp Gly Tyr Leu Gly 260 265 270 Glu Thr Ser Ala Gly Pro Gly Lys Ser Leu Pro Val Phe Ala Ile Ile 275 280 285 Leu Ile Ile Ser Leu Cys Cys Met Val Val Phe Thr Met Ala Tyr Ile 290 295 300 Met Leu Cys Arg Lys Thr Ser Gln Gln Glu His Val Tyr Glu Ala Ala 305 310 315 320 Arg Ala His Ala Arg Glu Ala Asn Asp Ser Gly Glu Thr Met Arg Val 325 330 335 Ala Ile Phe Ala Ser Gly Cys Ser Ser Asp Glu Pro Thr Ser Gln Asn 340 345 350 Leu Gly Asn Asn Tyr Ser Asp Glu Pro Cys Ile Gly Gln Glu Tyr Gln 355 360 365 Ile Ile Ala Gln Ile Asn Gly Asn Tyr Ala Arg Leu Leu Asp Thr Val 370 375 380 Pro Leu Asp Tyr Glu Phe Leu Ala Thr Glu Gly Lys Ser Val Cys 385 390 395 <210> 2 <211> 305 <212> PRT <213> Human <400> 2 Met Gly Ile Leu Leu Gly Leu Leu Leu Leu Gly His Leu Thr Val Asp 1 5 10 15 Thr Tyr Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro 20 25 30 Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly 35 40 45 Tyr Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro 50 55 60 Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala 65 70 75 80 Lys Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val 85 90 95 Ser Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr 100 105 110 Cys Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp 115 120 125 Lys Ile Thr Glu Leu Arg Val Gln Lys His Ser Ser Lys Leu Leu Lys 130 135 140 Thr Lys Thr Glu Ala Pro Thr Thr Met Thr Tyr Pro Leu Lys Ala Thr 145 150 155 160 Ser Thr Val Lys Gln Ser Trp Asp Trp Thr Thr Asp Met Asp Gly Tyr 165 170 175 Leu Gly Glu Thr Ser Ala Gly Pro Gly Lys Ser Leu Pro Val Phe Ala 180 185 190 Ile Ile Leu Ile Ile Ser Leu Cys Cys Met Val Val Phe Thr Met Ala 195 200 205 Tyr Ile Met Leu Cys Arg Lys Thr Ser Gln Gln Glu His Val Tyr Glu 210 215 220 Ala Ala Arg Ala His Ala Arg Glu Ala Asn Asp Ser Gly Glu Thr Met 225 230 235 240 Arg Val Ala Ile Phe Ala Ser Gly Cys Ser Ser Asp Glu Pro Thr Ser 245 250 255 Gln Asn Leu Gly Asn Asn Tyr Ser Asp Glu Pro Cys Ile Gly Gln Glu 260 265 270 Tyr Gln Ile Ile Ala Gln Ile Asn Gly Asn Tyr Ala Arg Leu Leu Asp 275 280 285 Thr Val Pro Leu Asp Tyr Glu Phe Leu Ala Thr Glu Gly Lys Ser Val 290 295 300 Cys 305 <210> 3 <211> 10 <212> PRT <213> Human <400> 3 Gly Phe Ser Leu Thr Thr Ser Gly Val Ala 1 5 10 <210> 4 <211> 7 <212> PRT <213> Human <400> 4 Ile Tyr Trp Asp Gly Asp Glu 1 5 <210> 5 <211> 19 <212> PRT <213> Human <400> 5 Ala Arg Ile Arg Gly Tyr Tyr Asp Trp Gly Ser Tyr Tyr Ser Tyr Gly 1 5 10 15 Met Asp Val <210> 6 <211> 6 <212> PRT <213> Human <400> 6 Asn Ile Gly Ser Lys Asn 1 5[[ID=L19]] <210> 7 <211> 3 <212> PRT <213> Human <400> 7 Arg Asp Ser 1 <210> 8 <211> 10 <212> PRT <213> Human <400> 8 Gln Val Trp Asp Ser Ser Thr His Val Val 1 5 10 <210> 9 <211> 10 <212> PRT <213> Human <400> 9 Gly Phe Ser Phe Asn Thr Pro Gly Glu Gly 1 5 10 <210> 10 <211> 7 <212> PRT <213> Human <400> 10 Ile Tyr Trp Asp Asp Glu Lys 1 5 <210> 11 <211> 8 <212> PRT <213> Human <400> 11 Gly Tyr Ile Phe Thr Asn Tyr Tyr 1 5 <210> 12 <211> 8 <212> PRT <213> Human <400> 12 Ile Asp Pro Ser Gly Gly Ser Thr 1 5 <210> 13 <211> 10 <212> PRT <213> Human <400> 13 Ala Arg Asp Tyr Trp Gly Ser Leu Asp Tyr 1 5 10 <210> 14 <211> 8 <212> PRT <213> Human <400> 14 Ser Gly Ser Ile Ala Ser Asn Tyr 1 5 <210> 15 <211> 3 <212> PRT <213> Human <400> 15 Glu Asn Tyr 1 <210> 16 <211> 11 <212> PRT <213> Human <400> 16 Gln Ser Tyr Asp Ser Arg Asn Arg Asn Tyr Val 1 5 10 <210> 17 <211> 8 <212> PRT <213> Human <400> 17 Gly Phe Thr Phe Ser Asp Tyr Tyr 1 5 <210> 18 <211> 8 <212> PRT <213> human <400> 18 Ile Ser Ser Ser Gly Ser Thr Ile 1 5 <210> 19 <211> 14 <212> PRT <213> human <400> 19 Ala Arg Arg Tyr Ser Ala Tyr Glu Thr Gly Tyr Phe Asp Phe 1 5 10 <210> 20 <211> 6 <212> PRT <213> human <400> 20 Gln Gly Ile Ser Thr Tyr 1 5 <210> 21 <211> 3 <212> PRT <213> human <400> 21 Ser Thr Ser 1 <210> 22 <211> 9 <212> PRT <213> human <400> 22 Gln Gln Ser Tyr Ser Ala Pro Pro Thr 1 5 <210> 23 <211> 10 <212> PRT <213> human <400> 23 Gly Phe Ser Leu Asn Thr Pro Gly Met Gly 1 5 10 <210> 24 <211> 7 <212> PRT <213> Human <400> 24 Val Phe Trp Asp Asp Asp Lys 1 5 <210> 25 <211> 11 <212> PRT <213> Human <400> 25 Gln Val Trp Asp Ser Asn Ser Asp Gln Tyr Val 1 5 10 <210> 26 <211> 8 <212> PRT <213> Human <400> 26 Gly Phe Thr Phe Ser Ser Arg Gly 1 5 <210> 27 <211> 8 <212> PRT <213> Human <400> 27 Ile Trp Tyr His Gly Ser Asp Asp 1 5 <210> 28 <211> 11 <212> PRT <213> Human <400> 28 Ala Asn Leu Gly Ala Thr Asp Gly Phe Asp Ile 1 5 10 <210> 29 <211> 9 <212> PRT <213> Human <400> 29 Ser Ser Asp Val Ser Ala Tyr Asn Tyr 1 5 <210> 30 <211> 3 <212> PRT <213> Human <400> 30 Gly Val Ser 1 <210> 31 <211> 10 <212> PRT <213> Human <400> 31 Asn Ser Tyr Thr Thr Ser Asn Thr Trp Val 1 5 10 <210> 32 <211> 8 <212> PRT <213> Human <400> 32 Gly Tyr Thr Phe Thr Ser Tyr Gly 1 5 <210> 33 <211> 8 <212> PRT <213> Human <400> 33 Ile Ser Ala Tyr Asn Gly Asn Thr 1 5 <210> 34 <211> 10 <212> PRT <213> Human <400> 34 Ala Arg Asp Tyr Trp Gly Ser Leu Asp His 1 5 10 <210> 35 <211> 8 <212> PRT <213> Human <400> 35 Ser Gly Ser Ile Asp Ile Asn Tyr 1 5 <210> 36 <211> 3 <212> PRT <213> Human <400> 36 Glu Asp Ser 1 <210> 37 <211> 8 <212> PRT <213> Human <400> 37 Gly Phe Thr Phe Ser Asp His Tyr 1 5 <210> 38 <211> 8 <212> PRT <213> Human <400> 38 Ile Ser Ser Gly Gly Gly Thr Ile 1 5 <210> 39 <211> 15 <212> PRT <213> Human <400> 39 Ala Arg Arg Glu Tyr Asp Ser Asp Gly His Tyr Tyr Phe Asp Tyr 1 5 10 15 <210> 40 <211> 6 <212> PRT <213> Human <400> 40 Ala Leu Pro Lys Gln Tyr 1 5 <210> 41 <211> 3 <212> PRT <213> Human <400> 41 Lys Asp Asn 1 <210> 42 <211> 11 <212> PRT <213> Human <400> 42 Gln Ser Val Asp Ser Ser Asp Thr Ser Val Val 1 5 10 <210> 43 <211> 8 <212> PRT <213> Human <400> 43 Gly Ser Thr Phe Arg Val Ala Trp 1 5 <210> 44 <211> 10 <212> PRT<00017​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<213> people <400> 47 Asp Val Ser 1 <210> 48 <211> 11 <212> PRT <213> people <400> 48 Ser Ser Tyr Ala Ser Ser Ser Thr Leu Tyr Val 1 5 10 <210> 49 <211> 8 <212> PRT <213> people <400> 49 Gly Phe Thr Phe Asp Asp Tyr Ala 1 5 <210> 50 <211> 8 <212> PRT <213> people <400> 50 Ile Ser Trp Asn Ser Gly Asn Ile 1 5 <210> 51 <211> 10 <212> PRT <213> people <400> 51 Ala Arg Glu Thr Ala Leu Ala Phe Asp Met 1 5 10 <210> 52 <211> 6 <212> PRT <213> people <400> 52 Gln Gly Ile Ser Tyr Trp 1 5 <210> 53 <211> 3 <212> PRT <213> people <400> 53 Ala Ser Ser 1 <210> 54 <211> 9 <212> PRT <213> Human <400> 54 Leu Gln Ala Thr Ser Phe Pro Tyr Thr 1 5 <210> 55 <211> 11 <212> PRT <213> Human <400> 55 Ala Arg Pro Gly Tyr Tyr Tyr Gly Leu Asp Val 1 5 10 <210> 56 <211> 6 <212> PRT <213> Human <400> 56 Gln Ser Ile Ser Ser Trp 1 5 <210> 57 <211> 3 <212> PRT <213> Human <400> 57 Gln Ala Ser 1 <210> 58 <211> 8 <212> PRT <213> Human <400> 58 Gln Gln Tyr Ser Ser Leu Trp Thr 1 5 <210> 59 <211> 25 <212> PRT <213> Human <400> 59 Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser 20 25 <210> 60 <211> 17 <212> PRT <213> Human <400> 60 Val Ala Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala 1 5 10 15 Leu <210> 61 <211> 38 <212> PRT <213> Human <400> 61 Arg Tyr Ser Ser Ser Leu Lys Asn Arg Leu Thr Ile Thr Lys Asp Thr 1 5 10 15 Ser Lys Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp 20 25 30 Thr Ala Thr Tyr Tyr Cys 35 <210> 62 <211> 11 <212> PRT <213> Human <400> 62 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 63 <211> 25 <212> PRT <213> Human <400> 63 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn 20 25 <210> 64 <211> 17 <212> PRT <213> Human <400> 64 Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile 1 5 10 15 Tyr <210> 65 <211> 36 <212> PRT <213> Human <400> 65 Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly 1 5 10 15 Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly Asp Glu Ala 20 25 30 Asp Tyr Tyr Cys 35 <210> 66 <211> 10 <212> PRT <213> Human <400> 66 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 1 5 10 <210> 67 <211> 25 <212> PRT <213> Human <400> 67 Gln Val Thr Leu Arg Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser 20 25 <210> 68 <211> 17 <212> PRT <213> human <400> 68 Val Thr Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala 1 5 10 15 Leu <210> 69 <211> 38 <212> PRT <213> human <400> 69 His Tyr Asn Pro Ser Leu Asn Gly Arg Leu Thr Ile Thr Lys Asp Thr 1 5 10 15 Ser Lys Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp 20 25 30 Thr Ala Thr Tyr Tyr Cys 35 <210> 70 <211> 11 <212> PRT <213> human <400> 70 Trp Gly Gln Gly Thr Thr Ile Thr Val Ser Ser 1 5 10 <210> 71 <211> 36 <212> PRT <213> Person <400> 71 Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly 1 5 10 15 Asp Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Gly Asp Glu Ala 20 25 30 Asp Tyr Tyr Cys 35 <210> 72 <211> 25 <212> PRT <213> Person <400> 72 Gln Val Gln Leu Val Glu Ser Gly Ala Glu Ile Lys Lys Pro Gly Ala 1 5 10 15 Ser Met Asn Val Ser Cys Lys Ala Ser 20 25 <210> 73 <211> 17 <212> PRT <213> Person <400> 73 Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 15 Ile <210> 74 <211> 38 <212> PRT <213> Person <400> 74 Thr Tyr Ala Gln Lys Phe Gln His Arg Val Thr Met Thr Ser Asp Thr 1 5 10 15 Ser Thr Ser Thr Val Tyr Met Glu Leu Ile Gly Leu Gly Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 75 <211> 11 <212> PRT <213> Human <400> 75 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 76 <211> 25 <212> PRT <213> Human <400> 76 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser 20 25 <210> 77 <211> 17 <212> PRT <213> Human <400> 77 Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val Ile 1 5 10 15 Tyr <210> 78 <211> 38 <212> PRT <213> Human <400> 78 Leu Arg Pro Ser Glu Val Pro Asp Arg Phe Ser Gly Ser Ile Asp Ser 1 5 10 15 Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly Leu Lys Thr Glu Asp 20 25 30 Glu Gly Asp Tyr Tyr Cys 35 <210> 79 <211> 10 <212> PRT <213> Human <400> 79 Phe Gly Thr Gly Thr Lys Val Thr Val Leu 1 5 10 <210> 80 <211> 25 <212> PRT <213> Human <400> 80 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 81 <211> 17 <212> PRT <213> Human <400> 81 Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Tyr <210> 82 <211> 38 <212> PRT <213> Human <400> 82 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 83 <211> 26 <212> PRT <213> Human <400> 83 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 84 <211> 17 <212> PRT <213> Human <400> 84 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 85 <211> 36 <212> PRT <213> Human <400> 85 Thr Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly Gly Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 86 <211> 10 <212> PRT <213> Human <400> 86 Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 87 <211> 25 <212> PRT <213> Human <400> 87 Gln Val Thr Leu Arg Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Arg 20 25 <210> 88 <211> 17 <212> PRT <213> Human <400> 88 Val Ala Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Gly 1 5 10 15 Leu <210> 89 <211> 38 <212> PRT <213> Human <400> 89 Thr Tyr Arg Pro Ser Leu Arg Asn Arg Leu Thr Ile Thr Lys Glu Thr 1 5 10 15 Ser Lys Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val Asp 20 25 30 Thr Ala Thr Tyr Tyr Cys 35 <210> 90 <211> 36 <212> PRT <213> human <400> 90 Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly 1 5 10 15 Asn Thr Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly Asp Glu Ala 20 25 30 Asp Tyr Tyr Cys 35 <210> 91 <211> 10 <212> PRT <213> human <400> 91 Phe Gly Thr Gly Ser Lys Val Thr Val Leu 1 5 10 <210> 92 <211> 25 <212> PRT <213> human <400> 92 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 93 <211> 17 <212> PRT <213> Person <400> 93 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala 1 5 10 15 Leu <210> 94 <211> 38 <212> PRT <213> Person <400> 94 Ser Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ser Lys Asn Met Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 95 <211> 11 <212> PRT <213> Person <400> 95 Trp Gly Gln Gly Thr Val Val Thr Val Ser Ser 1 5 10 <210> 96 <211> 25 <212> PRT <213> Person <400> 96 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr 20 25 <210> 97 <211> 17 <212> PRT <213> Human <400> 97 Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile 1 5 10 15 Tyr <210> 98 <211> 36 <212> PRT <213> Human <400> 98 Asp Arg Pro Ser Gly Val Ser Phe Arg Phe Ser Gly Ser Lys Ser Gly 1 5 10 15 Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala 20 25 30 Tyr Tyr Tyr Cys 35 <210> 99 <211> 25 <212> PRT <213> Human <400> 99 Gln Met Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser 20 25 <210> 100 <211> 17 <212> PRT <213> Human <400> 100 Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 15 Trp <210> 101 <211> 38 <212> PRT <213> Human <400> 101 Asn Tyr Ala Gln Lys Leu Gln Gly Arg Val Thr Met Thr Thr Asp Thr 1 5 10 15 Ser Thr Ser Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 102 <211> 11 <212> PRT <213> Human <400> 102 Trp Gly Gln Gly Ala Leu Val Thr Val Ser Ser 1 5 10 <210> 103 <211> 25 <212> PRT <213> Human <400> 103 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Gly Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser 20 25 <210> 104 <211> 17 <212> PRT <213> Human <400> 104 Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Ile Ile 1 5 10 15 Tyr <210> 105 <211> 38 <212> PRT <213> Human <400> 105 Gln Arg Thr Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Ile Asp Arg 1 5 10 15 Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly Leu Lys Thr Glu Asp 20 25 30 Glu Gly Asp Tyr Tyr Cys 35 <210> 106 <211> 25 <212> PRT <213> Human <400> 106 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 107 <211> 17 <212> PRT <213> Human <400> 107 Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 1 5 10 15 Ser <210> 108 <211> 11 <212> PRT <213> Human <400> 108 Trp Gly Gln Gly Ile Leu Val Thr Val Ser Ser 1 5 10 <210> 109 <211> 25 <212> PRT <213> Human <400> 109 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Leu Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp 20 25 <210> 110 <211> 17 <212> PRT <213> Human <400> 110 Ala Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Met 1 5 10 15 Tyr <210> 111 <211> 36 <212> PRT <213> Human <400> 111 Val Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Ser Ser Gly 1 5 10 15 Thr Thr Val Thr Leu Thr Ile Ser Arg Val Gln Ala Glu Asp Glu Ala 20 25 30 Asp Tyr Tyr Cys 35 <210> 112 <211> 17 <212> PRT <213> Human <400> 112 Val Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 1 5 10 15 Arg <210> 113 <211> 38 <212> PRT <213> Human <400> 113 Asp Tyr Ala Thr Pro Met Lys Asp Arg Phe Ile Ile Ser Arg Asp Asp 1 5 10 15[[ID=2​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<212> PRT <213> Human <400> 116 Asp Arg Pro Ser Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Ala 1 5 10 15 Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala 20 25 30 Asp Tyr Tyr Cys 35 <210> 117 <211> 25 <212> PRT <213> Human <400> 117 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 <210> 118 <211> 17 <212> PRT <213> Human <400> 118 Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ser 1 5 10 15 Gly <210> 119 <211> 38 <212> PRT <213> Human<D002399><400> 119 Val Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 1 5 10 15 Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 120 <211> 11 <212> PRT <213> Human <400> 120 Trp Gly Pro Gly Thr Met Val Thr Val Ser Ser 1 5 10 <210> 121 <211> 26 <212> PRT <213> Human <400> 121 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 122 <211> 17 <212> PRT <213> Human <400> 122 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Ile Ala Pro Lys Leu Leu Ile 1 5 10 15 Phe <210> 123 <211> 36 <212> PRT <213> Human <400> 123 Arg Leu His Ser Gly Val Pro Ser Arg Phe Asn Gly Ser Gly Ser Gly 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 124 <211> 10 <212> PRT <213> Human <400> 124 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 125 <211> 26 <212> PRT <213> Human <400> 125 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 126 <211> 17 <212> PRT <213> Human <400> 126 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Ile Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 127 <211> 36 <212> PRT <213> Human <400> 127 Ile Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 128 <211> 10 <212> PRT <213> Human <400> 128 Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 1 5 10 <210> 129 <211> 127 <212> PRT <213> Human <400> 129 Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Thr Thr Ser 20 25 30 Gly Val Ala Val Ala Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Leu Ile Tyr Trp Asp Gly Asp Glu Arg Tyr Ser Ser Ser 50 55 60 Leu Lys Asn Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Ser Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ile Arg Gly Tyr Tyr Asp Trp Gly Ser Tyr Tyr Ser Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 130 <211> 107 <212> PRT <213> Human <400> 130 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Arg Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Thr His Val 85 90 95 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 131 <211> 127 <212> PRT <213> human <400> 131 Gln Val Thr Leu Arg Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Phe Asn Thr Pro 20 25 30 Gly Glu Gly Val Thr Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala Leu Ile Tyr Trp Asp Asp Glu Lys His Tyr Asn Pro Ser 50 55 60 Leu Asn Gly Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Ser Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ile Arg Gly Tyr Tyr Asp Trp Gly Ser Tyr Tyr Ser Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Ile Thr Val Ser Ser 115 120 125 <210> 132 <211> 107 <212> PRT <213> Human <400> 132 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Arg Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asp Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Thr His Val 85 90 95 Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 133 <211> 117 <212> PRT <213> Human <400> 133 Gln Val Gln Leu Val Glu Ser Gly Ala Glu Ile Lys Lys Pro Gly Ala 1 5 10 15 Ser Met Asn Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Asp Pro Ser Gly Gly Ser Thr Thr Tyr Ala Gln Lys Phe 50 55 60 Gln His Arg Val Thr Met Thr Ser Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ile Gly Leu Gly Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Trp Gly Ser Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 134 <211> 112 <212> PRT <213> Human <400> 134 Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu Ser Pro Gly Lys 1 5 10 15 Thr Val Thr Ile Ser Cys Thr Arg Ser Ser Gly Ser Ile Ala Ser Asn 20 25 30 Tyr Val Gln Trp Tyr Gln Gln Arg Pro Gly Ser Ala Pro Thr Ile Val 35 40 45 Ile Tyr Glu Asn Tyr Leu Arg Pro Ser Glu Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ile Asp Ser Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Arg Asn Arg Asn Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 135 <211> 121 <212> PRT <213> Human <400> 135 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Tyr Ser Ala Tyr Glu Thr Gly Tyr Phe Asp Phe Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 136 <211> 107 <212> PRT <213> Human <400> 136 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Thr Ser Thr Leu Gln Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro Pro 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 100 105 <210> 137 <211> 127 <212> PRT <213> Human <400> 137 Gln Val Thr Leu Arg Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Arg Gly Phe Ser Leu Asn Thr Pro 20 25 30 Gly Met Gly Val Ala Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Gly Leu Val Phe Trp Asp Asp Asp Lys Thr Tyr Arg Pro Ser 50 55 60 Leu Arg Asn Arg Leu Thr Ile Thr Lys Glu Thr Ser Lys Ser Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ile Arg Gly Tyr Tyr Asp Trp Gly Ser Tyr Tyr Ser Tyr 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 138 <211> 108 <212> PRT <213> human <400> 138 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Arg Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Val Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Asp Gln 85 90 95 Tyr Val Phe Gly Thr Gly Ser Lys Val Thr Val Leu 100 105 <210> 139 <211> 118 <212> PRT <213> human <400> 139 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Arg 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Tyr His Gly Ser Asp Asp Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Leu Gly Ala Thr Asp Gly Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Val Val Thr Val Ser Ser 115 <210> 140 <211> 110 <212> PRT <213> Human <400> 140 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Ser Ala Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Gly Val Ser Asp Arg Pro Ser Gly Val Ser Phe Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Tyr Tyr Tyr Cys Asn Ser Tyr Thr Thr Ser 85 90 95 Asn Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 141 <211> 117 <212> PRT <213> Human <400> 141 Gln Met Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Gly Ser Ile Asp Arg Ser Ser Asn Ser Ala Ser Leu Thr Ile Ser Gly 65 70 75 80 Leu Lys Thr Glu Asp Glu Gly Asp Tyr Tyr Cys Gln Ser Tyr Asp Ser 85 90 95 Arg Asn Arg Asn Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 143 <211> 122 <212> PRT <213> Human <400> 143 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Gly Gly Gly Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Glu Tyr Asp Ser Asp Gly His Tyr Tyr Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Ile Leu Val Thr Val Ser Ser 115 120 <210> 144 <211> 108 <212> PRT <213> human <400> 144 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Leu Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Gln Tyr Ala 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Met Tyr 35 40 45 Lys Asp Asn Val Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Ser Arg Val Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Val Asp Ser Ser Asp Thr Ser 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 145 <211> 124 <212> PRT <213> Person <400> 145 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Arg Val Ala 20 25 30 Trp Val Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Ser Asn Ser Asp Gly Gly Thr Thr Asp Tyr Ala Thr 50 55 60 Pro Met Lys Asp Arg Phe Ile Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Val Phe Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asp Ala Asn Ala Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Lys Val Thr Val Ser Ser 115 120 <210> 146 <211> 111 <212> PRT <213> Person <400> 146 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Ile Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Asp Val Ser Asp Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Ala Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Ala Ser Ser 85 90 95 Ser Thr Leu Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 147 <211> 117 <212> PRT <213> Human <400> 147 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Asn Ile Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Thr Ala Leu Ala Phe Asp Met Trp Gly Pro Gly Thr Met 100 105 110 Val Thr Val Ser Ser 115 <210> 148 <211> 107 <212> PRT <213> Human <400> 148 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Tyr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Ile Ala Pro Lys Leu Leu Ile 35 40 45 Phe Ala Ser Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Asn Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Ala Thr Ser Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 149 <211> 118 <212> PRT <213> Human <400> 149 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Gly Tyr Tyr Tyr Gly Leu Asp Val Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 150 <211> 106 <212> PRT <213> Human <400> 150 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Ile Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gln Ala Ser Ile Leu Asp Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Ser Leu Trp Thr 85 90 95 Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 151 <211> 22 <212> DNA <21 <220> <223> Primer <400> 151 cgtcccatcc tggaagtgcc ag 22 <210> 152 <211> 23 <212> DNA <213> Artificial sequence <220> <223> Primer <400> 152 y gctctttcct ggcccagcac tgg 23 <210> 153 <211> 792 <212> DNA <213> Artificial sequence <220> <223> Fusion protein (hVSIG4(L)-Fc) <400> 153 cgtcccatcc tggaagtgcc agagagtgta acaggacctt ggaaagggga tgtgaatctt 60 ccctgcacct atgaccccct gcaaggctac acccaagtct tggtgaagtg gctggtacaa 120 cgtggctcag accctgtcac catctttcta cgtgactctt ctggagacca tatccagcag 180 gcaaagtacc agggccgcct gcatgtgagc cacaaggttc caggagatgt atccctccaa 240 ttgagcaccc tggagatgga tgaccggagc cactacacgt gtgaagtcac ctggcagact 300 cctgatggca accaagtcgt gagagataag attactgagc tccgtgtcca gaaactctct 360 gtctccaagc ccacagtgac aactggcagc ggttatggct tcacggtgcc ccagggaatg 420 aggattagcc ttcaatgcca ggctcggggt tctcctccca tcagttatat ttggtataag 480 caacagacta ataaccagga acccatcaaa gtagcaaccc taagtacctt actcttcaag 540 cctgcggtga tagccgactc aggctcctat ttctgcactg ccaagggcca ggttggctct 600 gagcagcaca gcgacattgt gaagtttgtg gtcaaagact cctcaaagct actcaagacc 660 aagactgagg cacctacaac catgacatac cccttgaaag caacatctac agtgaagcag 720 tcctgggact ggaccactga catggatggc taccttggag agaccagtgc tgggccagga 780 aagagcctgc ct 792 <210> 154 <211> 264 <212> PRT <213> Artificial sequence <220> <223> Fusion protein (hVSIG4(L)-Fc) <400> 154 Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp Lys Gly 1 5 10 15 Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr Thr Gln 20 25 30 Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val Thr Ile 35 40 45 Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys Tyr Gln 50 55 60 Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser Leu Gln 65 70 75 80 Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys Glu Val 85 90 95 Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys Ile Thr 100 105 110 Glu Leu Arg Val Gln Lys Leu Ser Val Ser Lys Pro Thr Val Thr Thr 115 120 125 Gly Ser Gly Tyr Gly Phe Thr Val Pro Gln Gly Met Arg Ile Ser Leu 130 135 140 Gln Cys Gln Ala Arg Gly Ser Pro Pro Ile Ser Tyr Ile Trp Tyr Lys 145 150 155 160 Gln Gln Thr Asn Asn Gln Glu Pro Ile Lys Val Ala Thr Leu Ser Thr 165 170 175 Leu Leu Phe Lys Pro Ala Val Ile Ala Asp Ser Gly Ser Tyr Phe Cys 180 185 190 Thr Ala Lys Gly Gln Val Gly Ser Glu Gln His Ser Asp Ile Val Lys 195 200 205 Phe Val Val Lys Asp Ser Ser Lys Leu Leu Lys Thr Lys Thr Glu Ala 210 215 220 Pro Thr Thr Met Thr Tyr Pro Leu Lys Ala Thr Ser Thr Val Lys Gln 225 230 235 240 Ser Trp Asp Trp Thr Thr Asp Met Asp Gly Tyr Leu Gly Glu Thr Ser 245 250 255 Ala Gly Pro Gly Lys Ser Leu Pro 260 <210> 155 <211> 517 <212> PRT <213> Synthetic sequence <220> <223> Fusion protein + Signal sequence (NLS-hVSIG4(L)-Fc) <400> 155 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Ala Asp 1 5 10 15 Val His Ser Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro 20 25 30 Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly 35 40 45 Tyr Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro 50 55 60 Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala 65 70 75 80 Lys Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val 85 90 95 Ser Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr 100 105 110 Cys Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp 115 120 125 Lys Ile Thr Glu Leu Arg Val Gln Lys Leu Ser Val Ser Lys Pro Thr 130 135 140 Val Thr Thr Gly Ser Gly Tyr Gly Phe Thr Val Pro Gln Gly Met Arg 145 150 155 160 Ile Ser Leu Gln Cys Gln Ala Arg Gly Ser Pro Pro Ile Ser Tyr Ile 165 170 175 Trp Tyr Lys Gln Gln Thr Asn Asn Gln Glu Pro Ile Lys Val Ala Thr 180 185 190 Leu Ser Thr Leu Leu Phe Lys Pro Ala Val Ile Ala Asp Ser Gly Ser 195 200 205 Tyr Phe Cys Thr Ala Lys Gly Gln Val Gly Ser Glu Gln His Ser Asp 210 215 220 Ile Val Lys Phe Val Val Lys Asp Ser Ser Lys Leu Leu Lys Thr Lys 225 230 235 240 Thr Glu Ala Pro Thr Thr Met Thr Tyr Pro Leu Lys Ala Thr Ser Thr 245 250 255 Val Lys Gln Ser Trp Asp Trp Thr Thr Asp Met Asp Gly Tyr Leu Gly 260 265 270 Glu Thr Ser Ala Gly Pro Gly Lys Ser Leu Pro Ala Ser Glu Pro Lys 275 280 285 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 290 295 300 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 305 310 315 320 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 325 330 335 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 340 345 350 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 355 360 365 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 370 375 380 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 385 390 395 400 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 405 410 415 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 420 425 430 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 435 440 445 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 450 455 460 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 465 470 475 480 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 485 490 495 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 500 505 510 Leu Ser Pro Gly Lys 515 <210> 156 <211> 318 <212> DNA <213> Artificial sequence <220> <223> Fusion protein (hVSIG4(S)-V-Fc) <400> 156 cgtcccatcc tggaagtgcc agagagtgta acaggacctt ggaaagggga tgtgaatctt 60 ccctgcacct atgaccccct gcaaggctac acccaagtct tggtgaagtg gctggtacaa 120 cgtggctcag accctgtcac catctttcta cgtgactctt ctggagacca tatccagcag 180 gcaaagtacc agggccgcct gcatgtgagc cacaaggttc caggagatgt atccctccaa 240 ttgagcaccc tggagatgga tgaccggagc cactacacgt gtgaagtcac ctggcagact 300 cctgatggca accaagtc 318 <210> 157 <211> 106 <212> PRT <213> Artificial sequence <220> <223> Fusion protein (hVSIG4(S)-V-Fc) <400> 157 Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp Lys Gly 1 5 10 15 Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr Thr Gln 20 25 30 Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val Thr Ile 35 40 45 Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys Tyr Gln 50 55 60 Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser Leu Gln 65 70 75 80 Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys Glu Val 85 90 95 Thr Trp Gln Thr Pro Asp Gly Asn Gln Val 100 105 <210> 158 <211> 359 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein + Signal Sequence (NLS-hVSIG4(S)-V-Fc) <400> 158 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Ala Asp 1 5 10 15 Val His Ser Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro 20 25 30 Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly 35 40 45 Tyr Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro 50 55 60 Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala 65 70 75 80 Lys Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val 85 90 95 Ser Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr 100 105 110 Cys Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Ala Ser Glu 115 120 125 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 130 135 140 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 145 150 155 160 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 165 170 175 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 180 185 190 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 195 200 205 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 210 215 220 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 225 230 235 240 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 245 250 255 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 260 265 270 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 275 280 285 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 290 295 300 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 305 310 315 320 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 325 330 335 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 340 345 350 Leu Ser Leu Ser Pro Gly Lys 355 <210> 159 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> Fusion Protein (hVSIG4(S)-M1-Fc) <400> 159 cgtcccatcc tgaccgcccc ccacagcctg gccggacctt ggaaagggga tgtgaatctt 60 ccctgcacct atgaccccct gcaaggctac acccaagtct tggtgaagtg gctggtacaa 120 cgtggctcag accctgtcac catctttcta cgtgactctt ctggagacca tatccagcag 180 gcaaagtacc agggccgcct gcatgtgagc cacaaggttc caggagatgt atccctccaa 240 ttgagcaccc tggagatgga tgaccggagc cactacacgt gtgaagtcac ctggcagact 300 cctgatggca accaagtcgt gagagataag attactgagc tccgtgtt 348 <210> 160 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein (hVSIG4(S)-M1-Fc) <400> 160 Arg Pro Ile Leu Thr Ala Pro His Ser Leu Ala Gly Pro Trp Lys Gly 1 5 10 15 Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr Thr Gln 20 25 30 Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val Thr Ile 35 40 45 Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys Tyr Gln 50 55 60 Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser Leu Gln 65 70 75 80 Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys Glu Val 85 90 95 Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys Ile Thr 100 105 110 Glu Leu Arg Val 115 <210> 161 <211> 380 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein + Signal Sequence (NLS-hVSIG4(S)-M1-Fc) <400> 161 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Asp 1 5 10 15 Val His Ser Gln Gly Ala Val Gly Ala Arg Pro Ile Leu Thr Ala Pro 20 25 30 His Ser Leu Ala Gly Pro Trp Lys Gly Asp Val Asn Leu Pro Cys Thr 35 40 45 Tyr Asp Pro Leu Gln Gly Tyr Thr Gln Val Leu Val Lys Trp Leu Val 50 55 60 Gln Arg Gly Ser Asp Pro Val Thr Ile Phe Leu Arg Asp Ser Ser Gly 65 70 75 80 Asp His Ile Gln Gln Ala Lys Tyr Gln Gly Arg Leu His Val Ser His 85 90 95 Lys Val Pro Gly Asp Val Ser Leu Gln Leu Ser Thr Leu Glu Met Asp 100 105 110 Asp Arg Ser His Tyr Thr Cys Glu Val Thr Trp Gln Thr Pro Asp Gly 115 120 125 Asn Gln Val Val Arg Asp Lys Ile Thr Glu Leu Arg Val Leu Ala Ala 130 135 140 Ser Ala Ala Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 145 150 155 160 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 165 170 175 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 180 185 190 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 195 200 205 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 210 215 220 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 225 230 235 240 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 245 250 255 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 260 265 270 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 275 280 285 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 290 295 300 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 305 310 315 320 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 325 330 335 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 340 345 350 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 355 360 365 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <210> 162 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Fusion protein (hVSIG4(S)-M2-Fc) <400> 162 cccatcctgg aagtgccaga gagtgtaaca ggaccttgga aagggaccgt gcggatgccc 60 tgcagctatg accccctgca aggctacacc caagtcttgg tgaagtggct ggtacaacgt 120 ggctcagacc ctgtcaccat ctttctacgt gactcttctg gagaccatat ccagcaggca 180 aagtaccagg gccgcctgca tgtgagccac aaggttccag gagatgtatc cctccaattg 240 agcaccctgg agatggatga ccggagccac tacacgtgtg aagtcacctg gcagactcct 300 gatggcaacc aagtcgtgag agataagatt actgagctcc gtgtt 345 <210> 163 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Fusion protein (hVSIG4(S)-M2-Fc) <400> 163 Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp Lys Gly Thr 1 5 10 15 Val Arg Met Pro Cys Ser Tyr Asp Pro Leu Gln Gly Tyr Thr Gln Val 20 25 30 Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val Thr Ile Phe 35 40 45 Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys Tyr Gln Gly 50 55 60 Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser Leu Gln Leu 65 70 75 80 Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys Glu Val Thr 85 90 95 Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys Ile Thr Glu 100 105 110 Leu Arg Val 115 <210> 164 <211> 379 <212> PRT <213> Synthetic Sequence <220> <223> Fusion Protein + Signal Sequence (NLS-hVSIG4(S)-M2-Fc) <400> 164 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Asp 1 5 10 15 Val His Ser Gln Gly Ala Val Gly Ala Pro Ile Leu Glu Val Pro Glu 20 25 30 Ser Val Thr Gly Pro Trp Lys Gly Thr Val Arg Met Pro Cys Ser Tyr 35 40 45 Asp Pro Leu Gln Gly Tyr Thr Gln Val Leu Val Lys Trp Leu Val Gln 50 55 60 Arg Gly Ser Asp Pro Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp 65 70 75 80 His Ile Gln Gln Ala Lys Tyr Gln Gly Arg Leu His Val Ser His Lys 85 90 95 Val Pro Gly Asp Val Ser Leu Gln Leu Ser Thr Leu Glu Met Asp Asp 100 105 110 Arg Ser His Tyr Thr Cys Glu Val Thr Trp Gln Thr Pro Asp Gly Asn 115 120 125 Gln Val Val Arg Asp Lys Ile Thr Glu Leu Arg Val Leu Ala Ala Ser 130 135 140 Ala Ala Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 145 150 155 160 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 165 170 175 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 180 185 190 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 195 200 205 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 210 215 220 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 225 230 235 240 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 245 250 255 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 260 265 270 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 275 280 285 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 290 295 300 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 305 310 315 320 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 325 330 335 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 340 345 350 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 355 360 365 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 165 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Fusion protein (hVSIG4(S)-M3-Fc) <400> 165 cccatcctgg aagtgccaga gagtgtaaca ggaccttgga aaggggatgt gaatcttccc 60 tgcacctatg accccctgca aggctacacc caagtcttgg tgaagtggct ggtactgcgg 120 aactaccacc ccgccaccat ctttctacgt gactcttctg gagaccatat ccagcaggca 180 aagtaccagg gccgcctgca tgtgagccac aaggttccag gagatgtatc cctccaattg 240 agcaccctgg agatggatga ccggagccac tacacgtgtg aagtcacctg gcagactcct 300 gatggcaacc aagtcgtgag agataagatt actgagctcc gtgtc 345 <210> 166 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Fusion protein (hVSIG4(S)-M3-Fc) <400> 166 Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp Lys Gly Asp 1 5 10 15 Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr Thr Gln Val 20 25 30 Leu Val Lys Trp Leu Val Leu Arg Asn Tyr His Pro Ala Thr Ile Phe 35 40 45 Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys Tyr Gln Gly 50 55 60 Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser Leu Gln Leu 65 70 75 80 Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys Glu Val Thr 85 90 95 Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys Ile Thr Glu 100 105 110 Leu Arg Val 115 <210> 167 <211> 379 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein + Signal Sequence (NLS-hVSIG4(S)-M3-Fc) <400> 167 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Asp 1 5 10 15 Val His Ser Gln Gly Ala Val Gly Ala Pro Ile Leu Glu Val Pro Glu 20 25 30 Ser Val Thr Gly Pro Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr 35 40 45 Asp Pro Leu Gln Gly Tyr Thr Gln Val Leu Val Lys Trp Leu Val Leu 50 55 60 Arg Asn Tyr His Pro Ala Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp 65 70 75 80 His Ile Gln Gln Ala Lys Tyr Gln Gly Arg Leu His Val Ser His Lys 85 90 95 Val Pro Gly Asp Val Ser Leu Gln Leu Ser Thr Leu Glu Met Asp Asp 100 105 110 Arg Ser His Tyr Thr Cys Glu Val Thr Trp Gln Thr Pro Asp Gly Asn 115 120 125 Gln Val Val Arg Asp Lys Ile Thr Glu Leu Arg Val Leu Ala Ala Ser 130 135 140 Ala Ala Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro 145 150 155 160 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 165 170 175 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 180 185 190 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 195 200 205 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 210 215 220 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 225 230 235 240 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 245 250 255 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 260 265 270 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 275 280 285 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 290 295 300 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 305 310 315 320 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 325 330 335 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 340 345 350 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 355 360 365 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 168 <211> 345 <212> DNA <213> Artificial sequence <220> <223> Fusion protein (hVSIG4(S)-M4-Fc) <4OO> 168 cccatcctgg aagtgccaga gagtgtaaca ggaccttgga aaggggatgt gaatcttccc 60 tgcacctatg accccctgca aggctacacc caagtcttgg tgaagtggct ggtacaacgt 120 ggctcagacc ctgtcaccat ctttctacgt gactcttctg gagaccatgt gcagcagacc 180 aagttccggg gccgcctgca tgtgagccac aaggttccag gagatgtatc cctccaattg 240 agcaccctgg agatggatga ccggagccac tacacgtgtg aagtcacctg gcagactcct 300 gatggcaacc aagtcgtgag agataagatt actgagctcc gtgtt 345 <210> 169 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Fusion protein (hVSIG4(S)-M4-Fc) <400> 169 Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp Lys Gly Asp 1 5 10 15 Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr Thr Gln Val 20 25 30 Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val Thr Ile Phe 35 40 45 Leu Arg Asp Ser Ser Gly Asp His Val Gln Gln Thr Lys Phe Arg Gly 50 55 60 Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser Leu Gln Leu 65 70 75 80 Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys Glu Val Thr 85 90 95 Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys Ile Thr Glu 100 105 110 Leu Arg Val 115 <210> 170 <211> 379 <212> PRT <213> Artificial Sequence <220> <223> Fusion Protein + Signal Sequence (NLS-hVSIG4(S)-M4-Fc) <400> 170 Met Gly Trp Ser Tyr Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Asp 1 5 10 15 Val His Ser Gln Gly Ala Val Gly Ala Pro Ile Leu Glu Val Pro Glu 20 25 30 Ser Val Thr Gly Pro Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr 35 40 45 Asp Pro Leu Gln Gly Tyr Thr Gln Val Leu Val Lys Trp Leu Val Gln 50 55 60 Arg Gly Ser Asp Pro Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp 65 70 75 80 His Val Gln Gln Thr Lys Phe Arg Gly Arg Leu His Val Ser His Lys 85 90 95 Val Pro Gly Asp Val Ser Leu ...

Claims

1. A monoclonal anti-VSIG4 antibody or an antigen-binding fragment thereof, wherein the antibody is selected from: a) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 of SEQ ID No.3, a heavy chain CDR2 of SEQ ID No.4, and a heavy chain CDR3 of SEQ ID No.5; and the light chain variable region comprises a light chain CDR1 of SEQ ID No.6, a light chain CDR2 of SEQ ID No.7, and a light chain CDR3 of SEQ ID No.8; b) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 of SEQ ID No.9, a heavy chain CDR2 of SEQ ID No.10, and a heavy chain CDR3 of SEQ ID No.5; and the light chain variable region comprises a light chain CDR1 of SEQ ID No.6, a light chain CDR2 of SEQ ID No.7, and a light chain CDR3 of SEQ ID No.8; c) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 of SEQ ID No.11, a heavy chain CDR2 of SEQ ID No.12, and a heavy chain CDR3 of SEQ ID No.13; and the light chain variable region comprises a light chain CDR1 of SEQ ID No.14, a light chain CDR2 of SEQ ID No.15, and a light chain CDR3 of SEQ ID No.16; d) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 of SEQ ID No.17, a heavy chain CDR2 of SEQ ID No.18, and a heavy chain CDR3 of SEQ ID No.19; and the light chain variable region comprises a light chain CDR1 of SEQ ID No.20, a light chain CDR2 of SEQ ID No.21, and a light chain CDR3 of SEQ ID No.22; e) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 of SEQ ID No.23, a heavy chain CDR2 of SEQ ID No.24, and a heavy chain CDR3 of SEQ ID No.3; and the light chain variable region comprises a light chain CDR1 of SEQ ID No.6, a light chain CDR2 of SEQ ID No.7, and a light chain CDR3 of SEQ ID No.25; f) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 of SEQ ID No.26, a heavy chain CDR2 of SEQ ID No.27, and a heavy chain CDR3 of SEQ ID No.28; and the light chain variable region comprises a light chain CDR1 of SEQ ID No.29, a light chain CDR2 of SEQ ID No.30, and a light chain CDR3 of SEQ ID No.31; g) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 of SEQ ID No.32, a heavy chain CDR2 of SEQ ID No.33, and a heavy chain CDR3 of SEQ ID No.34; and the light chain variable region comprises a light chain CDR1 of SEQ ID No.35, a light chain CDR2 of SEQ ID No.36, and a light chain CDR3 of SEQ ID No.16; h) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 of SEQ ID No.37, a heavy chain CDR2 of SEQ ID No.38, and a heavy chain CDR3 of SEQ ID No.39; and the light chain variable region comprises a light chain CDR1 of SEQ ID No.40, a light chain CDR2 of SEQ ID No.41, and a light chain CDR3 of SEQ ID No.42; i) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 of SEQ ID No.43, a heavy chain CDR2 of SEQ ID No.44, and a heavy chain CDR3 of SEQ ID No.45; and the light chain variable region comprises a light chain CDR1 of SEQ ID No.46, a light chain CDR2 of SEQ ID No.47, and a light chain CDR3 of SEQ ID No.48; j) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 of SEQ ID No.49, a heavy chain CDR2 of SEQ ID No.50, and a heavy chain CDR3 of SEQ ID No.51; and the light chain variable region comprises a light chain CDR1 of SEQ ID No.52, a light chain CDR2 of SEQ ID No.53, and a light chain CDR3 of SEQ ID No.54; or k) an antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 of SEQ ID No.17, a heavy chain CDR2 of SEQ ID No.18, and a heavy chain CDR3 of SEQ ID No.55; and the light chain variable region comprises a light chain CDR1 of SEQ ID No.56, a light chain CDR2 of SEQ ID No.57, and a light chain CDR3 of SEQ ID No.58; The antibody is selected from the group consisting of a chimeric antibody, a humanized antibody and a human antibody. 2 . The monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody is a human antibody.

3. The monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is selected from the group consisting of IgA1 antibody, IgA2 antibody, IgD antibody, IgE antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, IgG4 antibody and IgM antibody.

4. The monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab')2, Fv, scFv (sc means single chain), scFv-Fc fragment and miniantibody. 5 . The monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1 , wherein the antigen-binding fragment is a scFv.

6. The monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is selected from: a) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 129 or any sequence showing at least 80% identity with SEQ ID No. 129, CDR1-3 of the heavy chain variable region as shown in a) of claim 1, and a light chain variable region comprising a light chain CDR1 of SEQ ID No. 6, a light chain CDR2 of SEQ ID No. 7 and a light chain CDR3 of SEQ ID No. 8; b) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 131 or any sequence showing at least 80% identity with SEQ ID No. 131, CDR1-3 of the heavy chain variable region as shown in b) of claim 1, and a light chain variable region comprising a light chain CDR1 of SEQ ID No. 6, a light chain CDR2 of SEQ ID No. 7 and a light chain CDR3 of SEQ ID No. 8; c) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 133 or any sequence showing at least 80% identity with SEQ ID No. 133, CDR1-3 of the heavy chain variable region as shown in c) of claim 1, and a light chain variable region comprising a light chain CDR1 of SEQ ID No. 14, a light chain CDR2 of SEQ ID No. 15 and a light chain CDR3 of SEQ ID No. 16; d) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 135 or any sequence showing at least 80% identity with SEQ ID No. 135, CDR1-3 of the heavy chain variable region as shown in d) of claim 1, and a light chain variable region comprising a light chain CDR1 of SEQ ID No. 20, a light chain CDR2 of SEQ ID No. 21 and a light chain CDR3 of SEQ ID No. 22; e) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 137 or any sequence showing at least 80% identity with SEQ ID No. 137, CDR1-3 of the heavy chain variable region as shown in e) of claim 1, and a light chain variable region comprising a light chain CDR1 of SEQ ID No. 6, a light chain CDR2 of SEQ ID No. 7 and a light chain CDR3 of SEQ ID No. 25; f) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 139 or any sequence showing at least 80% identity with SEQ ID No. 139, CDR1-3 of the heavy chain variable region as shown in f) of claim 1, and a light chain variable region comprising a light chain CDR1 of SEQ ID No. 29, a light chain CDR2 of SEQ ID No. 30 and a light chain CDR3 of SEQ ID No. 31; g) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 141 or any sequence showing at least 80% identity to SEQ ID No. 141, CDR1-3 of the heavy chain variable region as shown in g) of claim 1, and a light chain variable region comprising a light chain CDR1 of SEQ ID No. 35, a light chain CDR2 of SEQ ID No. 36 and a light chain CDR3 of SEQ ID No. 16; h) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 143 or any sequence showing at least 80% identity with SEQ ID No. 143, CDR1-3 of the heavy chain variable region as shown in h) of claim 1, and a light chain variable region comprising a light chain CDR1 of SEQ ID No. 40, a light chain CDR2 of SEQ ID No. 41 and a light chain CDR3 of SEQ ID No. 42; i) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 145 or any sequence showing at least 80% identity with SEQ ID No. 145, CDR1-3 of the heavy chain variable region as shown in i) of claim 1, and a light chain variable region comprising a light chain CDR1 of SEQ ID No. 46, a light chain CDR2 of SEQ ID No. 47 and a light chain CDR3 of SEQ ID No. 48; j) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 147 or any sequence showing at least 80% identity to SEQ ID No. 147, CDR1-3 of the heavy chain variable region as shown in j) of claim 1, and a light chain variable region comprising a light chain CDR1 of SEQ ID No. 52, a light chain CDR2 of SEQ ID No. 53 and a light chain CDR3 of SEQ ID No. 54; or k) An antibody comprising a heavy chain variable region having a sequence of SEQ ID No.149 or any sequence showing at least 80% identity with SEQ ID No.149, CDR1-3 of the heavy chain variable region being as shown in k) of claim 1, and a light chain variable region comprising a light chain CDR1 of SEQ ID No.56, a light chain CDR2 of SEQ ID No.57 and a light chain CDR3 of SEQ ID No.

58.

7. The monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is selected from: a) an antibody comprising a light chain variable region of sequence SEQ ID No. 130 or any sequence showing at least 80% identity with SEQ ID No. 130, CDR1-3 of the light chain variable region as shown in a) of claim 1, and a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID No. 3, a heavy chain CDR2 of SEQ ID No. 4 and a heavy chain CDR3 of SEQ ID No. 5; b) an antibody comprising a light chain variable region of sequence SEQ ID No. 132 or any sequence showing at least 80% identity with SEQ ID No. 132, CDR1-3 of the light chain variable region as shown in b) of claim 1, and a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID No. 9, a heavy chain CDR2 of SEQ ID No. 10 and a heavy chain CDR3 of SEQ ID No. 5; c) an antibody comprising a light chain variable region of sequence SEQ ID No. 134 or any sequence showing at least 80% identity with SEQ ID No. 134, CDR1-3 of the light chain variable region as shown in c) of claim 1, and a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID No. 11, a heavy chain CDR2 of SEQ ID No. 12 and a heavy chain CDR3 of SEQ ID No. 13; d) an antibody comprising a light chain variable region of sequence SEQ ID No. 136 or any sequence showing at least 80% identity with SEQ ID No. 136, CDR1-3 of the light chain variable region as shown in d) of claim 1, and a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID No. 17, a heavy chain CDR2 of SEQ ID No. 18 and a heavy chain CDR3 of SEQ ID No. 19; e) an antibody comprising a light chain variable region of sequence SEQ ID No. 138 or any sequence showing at least 80% identity with SEQ ID No. 138, CDR1-3 of the light chain variable region as shown in e) of claim 1, and a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID No. 23, a heavy chain CDR2 of SEQ ID No. 24 and a heavy chain CDR3 of SEQ ID No. 3; f) an antibody comprising a light chain variable region of sequence SEQ ID No. 140 or any sequence showing at least 80% identity with SEQ ID No. 140, CDR1-3 of the light chain variable region as shown in f) of claim 1, and a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID No. 26, a heavy chain CDR2 of SEQ ID No. 27 and a heavy chain CDR3 of SEQ ID No. 28; g) an antibody comprising a light chain variable region of sequence SEQ ID No. 142 or any sequence showing at least 80% identity with SEQ ID No. 142, CDR1-3 of the light chain variable region as shown in g) of claim 1, and a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID No. 32, a heavy chain CDR2 of SEQ ID No. 33 and a heavy chain CDR3 of SEQ ID No. 34; h) an antibody comprising a light chain variable region of sequence SEQ ID No. 144 or any sequence showing at least 80% identity with SEQ ID No. 144, CDR1-3 of the light chain variable region as shown in h) of claim 1, and a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID No. 37, a heavy chain CDR2 of SEQ ID No. 38 and a heavy chain CDR3 of SEQ ID No. 39; i) an antibody comprising a light chain variable region of sequence SEQ ID No. 146 or any sequence showing at least 80% identity with SEQ ID No. 146, CDR1-3 of the light chain variable region as shown in i) of claim 1, and a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID No. 43, a heavy chain CDR2 of SEQ ID No. 44 and a heavy chain CDR3 of SEQ ID No. 45; j) an antibody comprising a light chain variable region of sequence SEQ ID No. 148 or any sequence showing at least 80% identity to SEQ ID No. 148, CDR1-3 of said light chain variable region as shown in j) of claim 1, and a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID No. 49, a heavy chain CDR2 of SEQ ID No. 50 and a heavy chain CDR3 of SEQ ID No. 51; or k) An antibody comprising a light chain variable region having a sequence of SEQ ID No. 150 or any sequence showing at least 80% identity with SEQ ID No. 150, CDR1-3 of the light chain variable region being as shown in k) of claim 1, and a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID No. 17, a heavy chain CDR2 of SEQ ID No. 18 and a heavy chain CDR3 of SEQ ID No.

55.

8. The monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is selected from: a) an antibody comprising a heavy chain variable region having a sequence of SEQ ID No. 129 or any sequence showing at least 80% identity with SEQ ID No. 129, and a light chain variable region having a sequence of SEQ ID No. 130 or any sequence showing at least 80% identity with SEQ ID No. 130, wherein CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region are as shown in a) of claim 1; b) an antibody comprising a heavy chain variable region having a sequence of SEQ ID No. 131 or any sequence showing at least 80% identity with SEQ ID No. 131, and a light chain variable region having a sequence of SEQ ID No. 132 or any sequence showing at least 80% identity with SEQ ID No. 132, wherein CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region are as shown in b) of claim 1; c) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 133 or any sequence showing at least 80% identity with SEQ ID No. 133, and a light chain variable region of sequence SEQ ID No. 134 or any sequence showing at least 80% identity with SEQ ID No. 134, wherein CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region are as shown in c) of claim 1; d) an antibody comprising a heavy chain variable region having a sequence of SEQ ID No. 135 or any sequence showing at least 80% identity with SEQ ID No. 135, and a light chain variable region having a sequence of SEQ ID No. 136 or any sequence showing at least 80% identity with SEQ ID No. 136, wherein CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region are as shown in d) of claim 1; e) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 137 or any sequence showing at least 80% identity with SEQ ID No. 137, and a light chain variable region of sequence SEQ ID No. 138 or any sequence showing at least 80% identity with SEQ ID No. 138, wherein CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region are as shown in claim 1 e); f) an antibody comprising a heavy chain variable region of sequence SEQ ID No.139 or any sequence showing at least 80% identity with SEQ ID No.139, and a light chain variable region of sequence SEQ ID No.140 or any sequence showing at least 80% identity with SEQ ID No.140, wherein CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region are as shown in f) of claim 1; g) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 141 or any sequence showing at least 80% identity with SEQ ID No. 141, and a light chain variable region of sequence SEQ ID No. 142 or any sequence showing at least 80% identity with SEQ ID No. 142, wherein CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region are as shown in g) of claim 1; h) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 143 or any sequence showing at least 80% identity with SEQ ID No. 143, and a light chain variable region of sequence SEQ ID No. 144 or any sequence showing at least 80% identity with SEQ ID No. 144, wherein CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region are as shown in h) of claim 1; i) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 145 or any sequence showing at least 80% identity with SEQ ID No. 145, and a light chain variable region of sequence SEQ ID No. 146 or any sequence showing at least 80% identity with SEQ ID No. 146, wherein CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region are as shown in i) of claim 1; j) an antibody comprising a heavy chain variable region of sequence SEQ ID No. 147 or any sequence showing at least 80% identity with SEQ ID No. 147, and a light chain variable region of sequence SEQ ID No. 148 or any sequence showing at least 80% identity with SEQ ID No. 148, wherein CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region are as shown in j) of claim 1; or k) An antibody comprising a heavy chain variable region having a sequence of SEQ ID No. 149 or any sequence showing at least 80% identity with SEQ ID No. 149, and a light chain variable region having a sequence of SEQ ID No. 150 or any sequence showing at least 80% identity with SEQ ID No. 150, wherein CDR1-3 of the heavy chain variable region and CDR1-3 of the light chain variable region are as shown in k) of claim 1. 9 . The monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1 , wherein the antibody is an internalizing antibody. 10 . A polynucleotide encoding the VL of the monoclonal anti-VSIG4 antibody or an antigen-binding fragment thereof according to claim 1 and the VH of the monoclonal anti-VSIG4 antibody or an antigen-binding fragment thereof according to claim 1 . An expression vector comprising the polynucleotide according to claim 10 .

12. A host cell transformed with the expression vector according to claim 11.

13. A method for producing the monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1, the method comprising: a) cultivating the host cell according to claim 12 under suitable conditions, and b) recovering the monoclonal anti-VSIG4 antibody or the antigen-binding fragment thereof from the culture medium or the cultured cells. 14 . A pharmaceutical composition comprising the monoclonal anti-VSIG4 antibody or an antigen-binding fragment thereof according to claim 1 , and a pharmaceutically acceptable carrier and / or excipient.

15. Use of the monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1 in preparing a kit for use in an in vitro method for detecting a cancer expressing VSIG4 in a subject, the method comprising the following steps: a) contacting a biological sample of the subject with the monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof according to claim 1; and b) detecting the binding of the antibody to the biological sample, The binding of VSIG4 indicates the presence of a VSIG4-expressing cancer.

16. The use according to claim 15, wherein the monoclonal anti-VSIG4 antibody or antigen-binding fragment thereof is labeled with a detectable label.

Images