Anti-tigit antibody specifically binding to tigit, fusion protein comprising same and interleukin-10, and uses thereof

Abstract

Provided are: an anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to TIGIT; a fusion protein comprising the antibody or antigen-binding fragment and interleukin-10 (IL-10); and uses of the antibody or antigen-binding fragment, and / or the fusion protein.

Description

Anti-TIGIT antibodies that specifically bind to TIGIT, fusion proteins comprising the antibody and interleukin-10, and uses thereof

[0001] Cross-citation with related applications

[0002] This application claims the benefit of priority from Republic of Korea Patent Application No. 10-2024-0011242, dated January 24, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to an anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to TIGIT; a fusion protein comprising the antibody or antigen-binding fragment and interleukin-10 (IL-10); and a use of the antibody or antigen-binding fragment and / or fusion protein.

[0004]

[0005] Autoimmune and anti-inflammatory immunotherapy are methods for treating autoimmunity and inflammation by altering the body's inappropriate immune response and alleviating associated pathologies. Autoimmune immunotherapy involves mechanisms that suppress the immune system, thereby reducing inappropriate recognition and response to healthy tissue. Immune system activation is a complex mechanism involving the functions of various cells, such as antigen-presenting cells, which are crucial for initiating antigen-specific responses, and effector cells, which are responsible for autoimmune and inflammatory pathologies.

[0006] Representative examples of the above effector cells include T cells and NK cells.

[0007] Meanwhile, TIGIT (T-cell immunoglobulin and ITIM (immunoreceptor tyrosine-based inhibitory motif) domain), also known as WUCAM, VSIG9, or Vstm3, is a corepressor receptor preferentially expressed on NK, CD8+, and CD4+ T cells, and regulatory T cells ("Treg"). TIGIT is a transmembrane protein containing an intracellular ITIM domain, a transmembrane domain, and an immunoglobulin variable domain.

[0008] TIGIT expression is increased in tissue-infiltrating lymphocytes in disease settings such as autoimmune, inflammatory, and infectious conditions. TIGIT expression may be a marker of activated T cells with enhanced effector function compared to TIGIT-negative cells. Furthermore, TIGIT-expressing Treg cells exhibit enhanced immunosuppressive activity compared to TIGIT-negative Treg populations.

[0009] Based on this, drugs targeting TIGIT may selectively act on effector T and NK cells.

[0010]

[0011] One example provides an anti-TIGIT antibody or antigen-binding fragment thereof that specifically recognizes and / or specifically binds to TIGIT.

[0012] Another example provides a nucleic acid molecule encoding the anti-TIGIT antibody or antigen-binding fragment thereof.

[0013] Another example provides a recombinant vector comprising a nucleic acid molecule encoding the anti-TIGIT antibody or antigen-binding fragment thereof.

[0014] Another example provides a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0015] Another example provides a composition for delivering a biologically active substance (a biologically active substance delivery vehicle or a biologically active substance delivery system) comprising at least one selected from the group consisting of the anti-TIGIT antibody or an antigen-binding fragment thereof, a nucleic acid molecule, a recombinant vector, and a recombinant cell. The composition for delivering a biologically active substance may further comprise the biologically active substance to be delivered, and in this case, the biologically active substance may be linked to the antibody or antigen-binding fragment, but is not limited thereto.

[0016] Another example provides a method for preparing a composition for delivering a biologically active substance, comprising the step of conjugating a biologically active substance to the anti-TIGIT antibody or an antigen-binding fragment thereof. The conjugation may be performed recombinantly, chemically, or physically, but is not limited thereto.

[0017] Another example provides a method for delivering a biologically active substance, comprising administering the composition for delivering the biologically active substance to a subject in need of delivery of the biologically active substance.

[0018] Another example provides a use for delivering one or more biologically active substances selected from the group consisting of the anti-TIGIT antibody or antigen-binding fragment thereof, nucleic acid molecules, recombinant vectors, and recombinant cells and / or a use for preparing a composition for delivering a biologically active substance.

[0019] Another example provides a composition for detecting TIGIT comprising the anti-TIGIT antibody or an antigen-binding fragment thereof.

[0020] Another example provides a method for detecting TIGIT, comprising the steps of treating a biological sample with the anti-TIGIT antibody or an antigen-binding fragment thereof; and determining whether an antigen-antibody reaction (binding) occurs.

[0021] Another example provides a use of the anti-TIGIT antibody or antigen-binding fragment thereof for detecting TIGIT.

[0022] Another example provides a composition for diagnosing a disease associated with TIGIT expression or overexpression, comprising the anti-TIGIT antibody or antigen-binding fragment thereof.

[0023] Another example provides a method for diagnosing a disease associated with TIGIT expression or overexpression or a method for providing information for diagnosing the disease, comprising the step of contacting the anti-TIGIT antibody or an antigen-binding fragment thereof with a biological sample and the step of determining whether there is an antigen-antibody reaction (binding) in the sample.

[0024] Another example provides a use of the anti-TIGIT antibody or antigen-binding fragment thereof for diagnosing a disease associated with TIGIT expression or overexpression.

[0025] Another example provides a fusion protein comprising (1) the anti-TIGIT antibody or an antigen-binding fragment thereof and (2) IL-10.

[0026] In one example, in the fusion protein, the IL-10 may be linked to the C-terminus and / or N-terminus of the heavy chain (heavy chain variable region or heavy chain constant region) or light chain (light chain variable region or light chain constant region) of the anti-TIGIT antibody or antigen-binding fragment thereof via a linker or directly without a linker.

[0027] Another example provides a nucleic acid molecule encoding the above fusion protein.

[0028] Another example provides a recombinant vector comprising a nucleic acid molecule encoding the fusion protein.

[0029] Another example provides a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0030] Another example provides an immunosuppressant or a pharmaceutical composition for immunosuppression, comprising as an active ingredient at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0031] Another example provides a pharmaceutical composition for preventing and / or treating an immune-related disease, comprising as an active ingredient at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0032] Another example provides a pharmaceutical composition for preventing and / or treating an autoimmune disease and / or an inflammatory disease, comprising as an active ingredient at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0033] Another example provides a method of immunosuppression, comprising administering to a subject in need of immunosuppression a pharmaceutically effective amount of at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0034] Another example provides a method for preventing and / or treating an immune-related disease, comprising administering to a subject in need of prevention and / or treatment of an immune-related disease a pharmaceutically effective amount of at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0035] Another example provides a method for preventing and / or treating an autoimmune disease and / or inflammation, comprising administering to a subject in need of prevention and / or treatment of an autoimmune disease and / or inflammatory disease a pharmaceutically effective amount of at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0036] Another example provides an immunosuppressive use or use for manufacturing an immunosuppressant selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0037] Another example provides a use for preventing and / or treating one or more immune-related diseases selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector, or a use for preparing a drug for preventing and / or treating an immune-related disease.

[0038] Another example provides a use for preventing and / or treating one or more autoimmune diseases and / or inflammatory diseases selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector, or a use for preparing a drug for preventing and / or treating autoimmune diseases and / or inflammation.

[0039]

[0040] The present application provides an anti-TIGIT antibody or antigen-binding fragment thereof that binds to TIGIT; a fusion protein comprising (1) the anti-TIGIT antibody or antigen-binding fragment thereof and (2) IL-10; and pharmaceutical uses thereof. The anti-TIGIT antibody or antigen-binding fragment thereof has excellent binding affinity to TIGIT and can have the activity of delivering another useful substance (e.g., IL-10) to a site where TIGIT exists, such as a cell expressing (overexpressing) TIGIT on its surface. The fusion protein (1) targets T cells and NK cells activated by the anti-TIGIT antibody or antigen-binding fragment thereof due to increased TIGIT expression, thereby delivering IL-10, thereby suppressing immunity, and (2) has the advantage of having excellent binding affinity to a receptor by IL-10 while having low immunogenicity, and thus can be applied as a variety of immunosuppressant and / or immunotherapeutic agent.

[0041]

[0042] Below, it is explained in more detail.

[0043]

[0044] Definition of terms

[0045] In this specification, the expression that a nucleic acid molecule (which may be used interchangeably with “polynucleotide” or “gene”) or a polypeptide (which may be used interchangeably with “protein” or “antibody”) “contains or consists of or is expressed by a specific nucleic acid sequence or amino acid sequence” may mean that the nucleic acid molecule or polypeptide essentially includes the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including (or not excluding) a “substantially equivalent sequence” in which a mutation (deletion, substitution, modification, and / or addition) is added to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or the desired function of the nucleic acid molecule or polypeptide is maintained.

[0046] In one example, the nucleic acid sequence or amino acid sequence provided herein may include a modification by conventional mutagenesis, such as directed evolution and / or site-directed mutagenesis, to the extent that it maintains its original function or desired function. In one example, a nucleic acid molecule or polypeptide “comprises, consists of, or is represented by a particular nucleic acid sequence or amino acid sequence” can mean that the nucleic acid molecule or polypeptide (i) essentially comprises the particular nucleic acid sequence or amino acid sequence, or (ii) essentially comprises or consists of an amino acid sequence that is at least 60%, at least 70%, 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%, at least 99%, at least 99.5%, or at least 99.9% homologous to the particular nucleic acid sequence or amino acid sequence and retains its original function and / or desired function.

[0047] The nucleic acid sequence described herein may be modified in various ways in the coding region within a range that does not change the amino acid sequence and / or function of the protein expressed from the coding region, taking into account the codons preferred in the microorganism that is to express the protein due to the degeneracy of the codons.

[0048] As used herein, the term "homology" refers to the degree of identity with a given nucleic acid sequence or amino acid sequence, and may be expressed as a percentage (%). Homology for nucleic acid sequences can be determined, for example, using well-known algorithms such as BLAST or FASTA, and programs called BLASTN or BLASTX have been developed based on the BLAST algorithm (see: http: / www.ncbi.nlm.nih.gov).

[0049]

[0050] anti-TIGIT antibody or antigen-binding fragment thereof

[0051] An example of the present application provides an anti-TIGIT antibody or antigen-binding fragment thereof having the activity of specifically recognizing and / or specifically binding to TIGIT.

[0052] The above anti-TIGIT antibody or antigen-binding fragment thereof,

[0053] A polypeptide comprising the amino acid sequence of sequence number 1 (CDR-H1),

[0054] A polypeptide comprising the amino acid sequence of sequence number 2 (CDR-H2),

[0055] A polypeptide comprising the amino acid sequence of sequence number 3 (CDR-H3),

[0056] A polypeptide comprising the amino acid sequence of sequence number 4 (CDR-L1),

[0057] A polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and

[0058] A polypeptide comprising the amino acid sequence of sequence number 6 (CDR-L3)

[0059] It may include:

[0060] In this specification, the complementarity determining region (CDR) is determined based on the CDR definition according to the Kabat system.

[0061] In one specific example, the six CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) that can be included in the anti-TIGIT antibody or antigen-binding fragment thereof provided herein are summarized in Table 1 below:

[0062] Amino acid sequence SEQ ID NO: CDR-H1SDYAWN1CDR-H2YITYSGSTSYNPSLKS2CDR-H3RQVGLGFAY3CDR-L1KASQDVSNTVA4CDR-L2SASYRYT5CDR-L3QQHYSTPWT6

[0063] (In Table 1 above, CDR-H1, CDR-H2, and CDR-H3 represent the heavy chain complementarity determining region, and CDR-L1, CDR-L2, and CDR-L3 represent the light chain complementarity determining region)

[0064]

[0065] In a specific example, the anti-TIGIT antibody or antigen-binding fragment thereof is

[0066] A heavy chain variable region comprising CDR-H1 of SEQ ID NO: 1, CDR-H2 of SEQ ID NO: 2, and CDR-H3 of SEQ ID NO: 3, and

[0067] A light chain variable region comprising CDR-L1 of SEQ ID NO: 4, CDR-L2 of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6

[0068] It may include:

[0069] More specifically, the anti-TIGIT antibody or antigen-binding fragment thereof,

[0070] A heavy chain variable region comprising an amino acid sequence selected from among SEQ ID NO: 7 and SEQ ID NO: 29 to 34, and

[0071] A light chain variable region comprising an amino acid sequence selected from among SEQ ID NO: 8 and SEQ ID NOs: 35 to 41

[0072] It may include:

[0073] In some cases (e.g., when produced recombinantly), the heavy chain variable region and / or light chain variable region may additionally comprise an appropriate signal sequence at the N terminus.

[0074] The amino acid sequences of the heavy chain variable region and light chain variable region that can be included in the anti-TIGIT antibody or antigen-binding fragment thereof provided herein are exemplified in Table 2 below:

[0075] 중쇄가변영역아미노산 서열(N→C)서열번호VH0DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCASRQVGLGFAYWGQGTLVTVSA29VH1DVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMAYITYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTIEDTATYYCASRQVGLGFAYWGQGTLVTVSS30VH6DVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMAYITYSGSTSYNPSLKSRITISRDTSKNQFFLKLSSVTIEDTATYYCASRQVGLGFAYWGQGTLVTVSS31VH7DVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTIEDTATYYCASRQVGLGFAYWGQGTLVTVSS32VH5QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSS33VH8QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFFLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSS34VH9QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSS7경쇄가변영역아미노산서열(N→C)서열번호Vk0DIVMTQSHKFMSTSVGDRVSITCKASQDVSNTVAWYQQRPGQSPKPLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIK35Vk1DIVMTQSHSFLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK36Vk2DIVMTQSPSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK37Vk7DIVMTQSPSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQAPRPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK38Vk11DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK8Vk5DIQMTQSPSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQHYSTPWTFGGGTKVEIK39Vk10DIQMTQSPSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQAPRPLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQHYSTPWTFGGGTKVEIK40Vk14DIQMTQSPSFLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQHYSTPWTFGGGTKVEIK41

[0076] (표 2에서, 밑줄로 표시된 영역은 순서대로 중쇄 및 경쇄의 CDR1, CDR2, 및 CDR3을 나타냄)

[0077]

[0078] In one example, the anti-TIGIT antibody or antigen-binding fragment thereof provided herein may bind to (bind / recognize) a TIGIT protein, such as human TIGIT protein (NCBI Reference Sequence NP_776160.2; UniProtKB / SwissProt Q495A1-1), and may bind to one or more or two or more (e.g., consecutively positioned) amino acids in the protein, but is not limited thereto.

[0079] [Human TIGIT protein (UniProtKB / SwissProt Q495A1-1)]]

[0080] 1 MRWCLLLIWA QGLRQAPLAS GMMTGTIETT GNISAEKGGS IILQCHLSST TAQVTQVNWE

[0081] 61 QQDQLLAICN ADLGWHISPS FKDRVAPGPG LGLTLQSLTV NDTGEYFCIY HTYPDGTYTG

[0082] 121 RIFLEVLESS VAEHGARFQI PLLGAMAATL VVICTAVIVV VALTRKKKAL RIHSVEGDLR

[0083] 181 RKSAGQEEWS PSAPSPPGSC VQAEAAPAGL CGEQRGEDCA ELHDYFNVLS YRSLGNCSFF

[0084] 241 TETG (SEQ ID NO: 42)

[0085]

[0086] As used herein, the term "antibody" refers to a general term for a protein that specifically binds to a specific antigen. It may be a protein produced by antigen stimulation within the immune system, or a protein produced chemically synthesized or recombinantly, and the type thereof is not particularly limited. The antibody may be non-naturally produced, for example, recombinantly or synthetically produced. The antibody may be an animal antibody (e.g., mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody. The antibody may be a monoclonal antibody or a polyclonal antibody.

[0087] The anti-TIGIT antibody or antigen-binding fragment thereof provided herein, except for the heavy chain CDR and light chain CDR regions, or the heavy chain variable region and the light chain variable region as defined above, may be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, etc.), and may be derived from, for example, the framework region, and / or the light chain constant region and / or the heavy chain constant region of any of the above subtypes of immunoglobulin. In one example, the anti-TIGIT antibody provided herein may be a human IgG type antibody, such as, but not limited to, an IgG1, IgG2, IgG3, or IgG4 type antibody.

[0088] A complete antibody (e.g., IgG type) has a structure with two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2) subclasses. The constant region of the light chain has kappa (κ) and lambda (λ) types.

[0089] In one example, the anti-TIGIT antibody provided herein may include, but is not limited to, an IgG constant region as the heavy chain constant region and a kappa constant region as the light chain constant region.

[0090] In one specific embodiment, the constant region of the IgG (e.g., human IgG1) may be wild-type. In another specific embodiment, the constant region of the IgG may be a mutant comprising one or more mutations selected from the group consisting of S240D (wherein S at position 240 is substituted with D, and the amino acid mutations are expressed in the same manner hereinafter), A331L, I333E, N298A, S299A, E334A, K335A, L235A, L236A, P330G, and K478 deletion (ΔK478), based on human IgG1, and for example, may be a mutant comprising the following mutations:

[0091] (1) S240D, A331L, and I333E;

[0092] (2) N298A;

[0093] (3) S299A, E334A, and K335A;

[0094] (4) L235A, L236A, and P330G; or

[0095] (5) K478 fruit.

[0096] The term "heavy chain" refers to a variable region domain V containing an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. H and three invariant region domains C H1 , C H2 and C H3 The term "light chain" is interpreted to mean a full-length heavy chain and fragments thereof, including a hinge. In addition, the term "light chain" refers to a variable region domain V comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. L and invariant region domain C L It is interpreted to mean including both full-length light chains and fragments thereof.

[0097] The term "CDR (complementarity determining region)" refers to a region among the variable regions of an antibody that confers binding specificity to an antigen, and refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulins. The heavy and light chains may each include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDRs may provide key contact residues for binding of the antibody to an antigen or epitope. Meanwhile, in the present specification, the terms "specifically bind" or "specifically recognize" have the same meaning as that commonly known to those skilled in the art, and mean that an antigen and an antibody specifically interact to cause an immunological reaction.

[0098] The complementarity determining region (CDR) described in this specification is determined based on the CDR definition according to the Kabat system.

[0099] In this specification, an antibody may be understood to include an antigen-binding fragment of an antibody having antigen-binding ability, unless otherwise specified.

[0100] The term "antigen-binding fragment" refers to any form of polypeptide that comprises a portion capable of binding an antigen (e.g., six CDRs as defined herein). Examples include, but are not limited to, scFv, scFv-Fc, (scFv)2, Fab, Fab', or F(ab')2 of an antibody.

[0101] Among the above antigen-binding fragments, Fab is composed of variable regions of light and heavy chains, constant region of light chain, and first constant region of heavy chain (C H1 ) has one antigen binding site.

[0102] Fab' is a medium chain C H1 It differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the domain.

[0103] F(ab')2 antibodies are produced by disulfide bonds between cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment containing only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art.

[0104] In a two-chain Fv, the heavy chain variable region and the light chain variable region are connected non-covalently, and in a single-chain Fv, the heavy chain variable region and the single chain variable region are generally connected covalently through a peptide linker or directly at the C-terminus, so that they can form a dimer-like structure like a two-chain Fv.

[0105] The above antigen-binding fragment can be obtained using a protein hydrolytic enzyme (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), and can be produced through genetic recombination technology.

[0106] The term "hinge region" refers to a region contained in the heavy chain of an antibody, which exists between the CH1 and CH2 regions and functions to provide flexibility to the antigen-binding site within the antibody.

[0107] The antibodies provided herein may be monoclonal antibodies. Monoclonal antibodies may be produced using methods widely known in the art. For example, they may be produced using phage display techniques. Alternatively, the antibodies may be produced as monoclonal antibodies derived from animals (e.g., mice) using conventional methods.

[0108] Meanwhile, individual monoclonal antibodies can be screened based on their binding affinity to the receptor-binding domain of TIGIT using a typical Enzyme-Linked ImmunoSorbent Assay (ELISA) format. Inhibitory activity can be tested using functional assays, such as competitive ELISA to examine molecular interactions between the binders, or cell-based assays. Then, the affinity (Kd values) for the receptor-binding domain of TIGIT can be tested for each monoclonal antibody selected based on its strong inhibitory activity.

[0109]

[0110] The final selected antibodies can be manufactured and used as humanized antibodies, as well as antibodies in which the remainder, excluding the antigen-binding region, is converted to human immunoglobulin. Methods for manufacturing humanized antibodies are well known in the art.

[0111] The antigen-binding fragment of the anti-TIGIT antibody provided herein refers to a fragment derived from the anti-TIGIT antibody and having binding ability to the antigen (TIGIT), and may be any polypeptide including six CDRs of the anti-TIGIT antibody, for example, scFv, scFv-Fc, scFv-Ck (kappa constant region), scFv-Cλ (lambda constant region), (scFv)2, Fab, Fab' or F(ab')2, but is not limited thereto. In one example, the antigen-binding fragment may be, but is not limited to, a scFv, or a fusion polypeptide (scFv-Fc) in which the scFv is fused with the Fc region of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, etc.) or a fusion polypeptide (scFv-Ck or scFv-Cλ) in which the scFv is fused with the constant region of a light chain (e.g., kappa or lambda).

[0112] In the present specification, the phrase “an antibody (e.g., a CDR, a variable region, or a heavy / light chain, an antigen-binding fragment, etc.) comprises, consists of, or is represented by a specific amino acid sequence” may mean both cases where the antibody essentially comprises the amino acid sequence, and cases where a meaningless mutation (e.g., substitution, deletion, and / or addition of an amino acid residue) that does not significantly affect antibody activity (e.g., antigen affinity, pharmacological activity, etc.) is introduced into the amino acid sequence.

[0113] The anti-TIGIT antibody or antigen-binding fragment thereof provided herein has a binding affinity (K) for TIGIT (e.g., human TIGIT) D) may be, for example, 10 mM or less, 5 mM or less, 1 mM or less, 0.5 mM or less, 0.2 mM or less, 0.1 mM or less, 0.05 mM or less, 0.01 mM or less, 0.005 mM or less, or 0.001 mM or less, as measured by surface plasmon resonance (SPR), for example, 0.0001 nM to 10 mM, 0.0005 nM to 10 mM, 0.001 nM to 10 mM, 0.005 nM to 10 mM, 0.01 nM to 10 mM, 0.05 nM to 10 mM, 0.1 nM to 10 mM, 0.5 nM to 10 mM, 1 nM to 10 mM, 0.0001 nM to 5 mM, 0.0005 nM to 5 mM, 0.001 nM to 5 mM, 0.005 nM to 5 mM, 0.01 nM to 5 mM, 0.05 nM to 5 mM, 0.1 nM to 5 mM, 0.5 nM to 5 mM, 1 nM to 5 mM, 0.0001 nM to 1 mM, 0.0005 nM to 1 mM, 0.001 nM to 1 mM, 0.005 nM to 1 mM, 0.01 nM to 1 mM, 0.05 nM to 1 mM, 0.1 nM to 1 mM, 0.5 nM to 1 mM, 1 nM to 1 mM, 0.0001 nM to 0.5 mM, 0.0005 nM to 0.5 mM, 0.001 nM to 0.5 mM, 0.005 nM to 0.5 mM, 0.01 nM to 0.5 mM, 0.05 nM to 0.5 mM, 0.1 nM to 0.5 mM, 0.5 nM to 0.5 mM, 1 nM to 0.5 mM, 0.0001 nM to 0.2 mM, 0.0005 nM to 0.2 mM, 0.001 nM to 0.2 mM, 0.005 nM to 0.2 mM, 0.01 nM to 0.2 mM, 0.05 nM to 0.2 mM, 0.1 nM to 0.2 mM, 0.5 nM to 0.2 mM, 1 nM to 0.2 mM, 0.0001 nM to 0.1 mM, 0.0005 nM to 0.1 mM, 0.001 nM to 0.1 mM, 0.005 nM to 0.1 mM, 0.01 nM to 0.1 mM, 0.05 nM to 0.1 mM, 0.1 nM to 0.1 mM, 0.5 nM to 0.1 mM, 1 nM to 0.1 mM, 0.0001 nM to 0.05 mM, 0.0005 nM to 0.05 mM, 0.001 nM to 0.05 mM, 0.005 nM to 0.05 mM, 0.01 nM to 0.05 mM, 0.05 nM to 0.05 mM, 0.1 nM to 0.05 mM, 0.5 nM to 0.05 mM, 1 nM to 0.05 mM, 0.0001 nM to 0.01 mM, 0.0005 nM to 0.01 mM, It may be, but is not limited to, 0.001 nM to 0.01 mM, 0.005 nM to 0.01 mM, 0.01 nM to 0.01 mM, 0.05 nM to 0.01 mM, 0.1 nM to 0.01 mM, 0.5 nM to 0.01 mM, or 1 nM to 0.01 mM.

[0114]

[0115] Another example of the present application provides a nucleic acid molecule encoding the antibody or antigen-binding fragment.

[0116] Another example provides a recombinant vector comprising a nucleic acid molecule encoding the antibody or antigen-binding fragment.

[0117] Another example provides a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0118]

[0119] The above anti-TIGIT antibody or antigen-binding fragment thereof has a characteristic of having excellent binding affinity to TIGIT while not inhibiting interaction (binding) with a protein (e.g., CD155) that interacts with TIGIT. Due to this characteristic, the anti-TIGIT antibody or antigen-binding fragment thereof can have the activity of binding (fusion / conjugating) with a biologically active substance (e.g., IL-10) having a desired effect, and effectively delivering the biologically active substance to a site where TIGIT exists, e.g., a cell expressing (overexpressing) TIGIT on the surface (e.g., an immune cell such as a T cell or NK (natural killer) cell) without affecting the function / activity / action of TIGIT.

[0120] Accordingly, another example of the present application provides a composition for delivering a biologically active substance (a biologically active substance delivery vehicle or a biologically active substance delivery system) comprising at least one selected from the group consisting of the antibody or antigen-binding fragment, a nucleic acid molecule, a recombinant vector, and a recombinant cell. The composition for delivering a biologically active substance may further comprise the biologically active substance to be delivered. In this case, the biologically active substance may be linked to the antibody or antigen-binding fragment (in the form of an antibody-biologically active substance conjugate; for example, an antibody-drug conjugate (ADC), etc.), but is not limited thereto.

[0121] Another example provides a method for preparing a composition for delivering a biologically active substance, comprising the step of conjugating a biologically active substance to the antibody or antigen-binding fragment. The conjugation may be performed recombinantly, chemically, or physically, but is not limited thereto.

[0122] Another example provides a method for delivering a biologically active substance, comprising administering the composition for delivering the biologically active substance to a subject in need of delivery of the biologically active substance.

[0123] Another example provides a use for delivering one or more biologically active substances selected from the group consisting of antibodies or antigen-binding fragments, nucleic acid molecules, recombinant vectors, and recombinant cells and / or a use for preparing a composition for delivering a biologically active substance.

[0124] The above biologically active substance may be at least one selected from the group consisting of all biocompatible substances that can exhibit beneficial effects or desired effects, such as immunosuppression, immune activation, visualization, etc., in cells (e.g., immune cells such as T cells and NK (natural killer) cells) expressing (overexpressing) TIGIT at a site delivered by the above-described anti-TIGIT antibody or antigen-binding fragment thereof, for example, small molecule compounds, proteins, peptides, nucleic acid molecules (e.g., antisense oligonucleotides, siRNA, shRNA, microRNA, aptamers, etc.), labeling substances such as radioactive isotopes, etc.

[0125] For example, the biologically active substance may be at least one selected from the group consisting of, but is not limited to, chemical drugs having an immunosuppressive or immunoactivating effect on immune cells; proteins having an immunosuppressive or immunoactivating effect on immune cells (e.g., cytokines, chemokines, etc.); ligands binding to receptors on the surface of immune cells; antibodies binding to immune-related proteins (e.g., receptors on the surface of immune cells, ligands binding thereto, etc.).

[0126] Another example provides a composition for detecting TIGIT comprising the antibody or antigen-binding fragment thereof. Another example provides a method for detecting TIGIT, comprising the steps of treating a biological sample with the anti-TIGIT antibody or antigen-binding fragment thereof; and determining whether an antigen-antibody reaction (binding) occurs. In the detection method, if an antigen-antibody reaction is detected, it can be determined that TIGIT is present in the biological sample. Another example provides a use of the anti-TIGIT antibody or antigen-binding fragment thereof for detecting TIGIT. The detection of TIGIT refers to determining whether TIGIT is present, expressed, or the degree of presence or expression of TIGIT in a biological sample.

[0127] The anti-TIGIT antibody or antigen-binding fragment thereof may be conjugated to a detectable label. If the detectable label is visualizable, the anti-TIGIT antibody or antigen-binding fragment thereof may be used to visualize TIGIT in a biological sample. In one example, the detectable label may be at least one selected from the group consisting of, but not limited to, a radioisotope (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 1251, 1311, 177Lu, 166Ho, 153Sm, etc.), an enzyme, a fluorescent label, a luminescent label (e.g., a bioluminescent label), a magnetic label, a chemical substance such as biotin, etc.

[0128] The above biological sample may be selected from the group consisting of cells (e.g., immune cells such as T cells and NK cells), tissues, body fluids (e.g., blood, serum, etc.) obtained from mammals such as primates including humans, monkeys, etc., rodents including mice, rats, etc., and may be isolated from a living body.

[0129] Another example provides a composition for diagnosing a disease associated with TIGIT expression or overexpression, comprising the anti-TIGIT antibody or antigen-binding fragment thereof.

[0130] Another example provides a method for diagnosing a disease associated with TIGIT expression or overexpression or a method for providing information for diagnosing the disease, comprising the step of contacting the anti-TIGIT antibody or an antigen-binding fragment thereof with a biological sample and the step of determining whether there is an antigen-antibody reaction (binding) in the sample.

[0131] Another example provides a use of the anti-TIGIT antibody or antigen-binding fragment thereof for diagnosing a disease associated with TIGIT expression or overexpression.

[0132] The above TIGIT expression or overexpression-related disease may be any disease caused by TIGIT expression or overexpression, and may be, for example, an autoimmune disease. The autoimmune disease may be, but is not limited to, rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, Behcet's syndrome, lupus (systemic lupus erythematosus; SLE), Sjogren's syndrome, and may be, but is not limited to, one or more autoimmune diseases selected from the group consisting of myasthenia gravis, scleroderma, hypothyroidism, hyperthyroidism, psoriasis, vitiligo, multiple sclerosis, autoimmune hepatitis, autoimmune nephritis, autoimmune pancreatitis, autoimmune encephalitis, cytokine storm, etc.

[0133] In the above diagnostic method, the biological sample may be selected from the group consisting of cells (e.g., immune cells such as T cells and NK cells), tissues, body fluids (e.g., blood, serum, etc.) obtained from mammals such as humans, primates including monkeys, rodents including mice, rats, etc., which require diagnosis of a disease related to TIGIT expression or overexpression, and may be isolated from a living body.

[0134] The step of confirming whether the antigen-antibody reaction occurs can be performed through various methods known in the art. For example, it can be measured through conventional enzyme reaction, fluorescence, luminescence and / or radiation detection, and specifically, it can be measured by a method selected from the group consisting of immunochromatography, immunohistochemistry, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescence immunoassay (LIA), Western blotting, microarray, etc., but is not limited thereto.

[0135]

[0136] fusion protein

[0137] In the present specification, an example provides a fusion protein comprising (1) an anti-TIGIT antibody or an antigen-binding fragment thereof that specifically recognizes TIGIT and (2) interleukin-10 (IL-10).

[0138] The anti-TIGIT antibody or antigen-binding fragment thereof is as described above.

[0139] The above fusion protein may have both the function of the anti-TIGIT antibody or its antigen-binding fragment (TIGIT recognition / binding, targeting, activation, etc.) and the function of IL-10 (immunosuppression, IL-10 receptor (IL-10R) recognition / binding, etc.).

[0140]

[0141] The above interleukin-10 (IL-10) is

[0142] (a) a polypeptide comprising IL-10,

[0143] (b) a polypeptide having a homology of 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the polypeptide (a) above;

[0144] It may be selected from the group consisting of .

[0145] Interleukin-10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), is encoded by the IL-10 gene and participates in signal transduction by binding to two IL10Rα (also known as IL10R1) and two IL10Rβ (also known as IL10R2). For example, the interleukin-10 may be of human origin, such as, but not limited to, NCBI Accession No. NP_000563.1 (amino acid residues 1-18: signal sequence; amino acid residues 19-178: mature segment).

[0146] In one specific example, the IL-10 may be represented by the following amino acid sequence (from N-terminus to C-terminus):

[0147] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (wild type; SEQ ID NO: 9)

[0148]

[0149] The polypeptide (a) comprising the IL-10 may be in the form of a monomer comprising one IL-10 or in the form of a polymer comprising two or more IL-10s (e.g., a dimer comprising two IL-10s). When the IL-10 is in the form of a polymer (e.g., a dimer), two or more (e.g., two) IL-10 monomers may be linked via a linker or directly linked without a linker. When the IL-10 is linked via a linker, the linker (second linker) may be selected from among conventional peptide linkers, and may be, for example, GGGSGGG (SEQ ID NO: 16), but is not limited thereto.

[0150]

[0151] The above polypeptide (a) may include additional oligopeptides for additional purposes, such as, but not limited to, a Tag for labeling at the N-terminus (e.g., MHSSALLCCLVLLTGVRA (SEQ ID NO: 45)) for convenience of protein purification.

[0152] The above interleukin-10 is of human origin and is characterized by low immunogenicity when administered into the human body.

[0153] The IL-10 may have binding activity to a receptor, for example, IL-10 receptor alpha chain (IL10RA, CD210, CD210a, CDW210A, HIL-10R, IL-10R1, IL10R, interleukin 10 receptor, alpha subunit, interleukin 10 receptor subunit alpha) and / or IL-10 receptor beta chain (IL10RB, CDW210B, CRF2-4, CRFB4, D21S58, D21S66, IL-10R2, interleukin 10 receptor, beta subunit, interleukin 10 receptor subunit beta), and / or may inhibit cytokine production and / or secretion activity in immune cells (e.g., T cells (CD4+, CD8+), NK cells, etc.).

[0154]

[0155] In one specific example, the IL-10 is

[0156] (a) (a-1) A polypeptide (monomer) comprising one amino acid sequence of sequence number 9,

[0157] (a-2) A polypeptide (dimer) containing two amino acid sequences of sequence number 9,

[0158] (b) a polypeptide having 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with the polypeptide (a) above, and inhibiting / inducing binding activity to the IL-10 receptor and / or cytokine production and / or secretion activity in immune cells;

[0159] It may be selected from the group consisting of. The polypeptide of (a-2) may be a polypeptide in which two amino acid sequences of SEQ ID NO: 9 are linked with or without a peptide linker (e.g., SEQ ID NO: 16, etc.), but is not limited thereto.

[0160] The above IL-10 may have an activity such as binding activity to an excellent IL-10 receptor, and / or inhibiting cytokine production and / or secretion in immune cells, and / or inhibiting cytokine production and / or secretion in non-immune cells (e.g., tissue cells).

[0161]

[0162] In the above fusion protein, the anti-TIGIT antibody or antigen-binding fragment thereof may have the function of delivering IL-10 to effector cells (e.g., effector T cell function and / or Treg cells) and / or NK cells. In addition, by maintaining the native sequence of the antibody, it enables Fc receptor binding. Therefore, the anti-TIGIT antibody can also target TIGIT-negative effector cells important in autoimmunity and / or inflammation, including monocytes and macrophages. The IL-10 is a safe polypeptide with strong receptor binding affinity and low immunogenicity, and when administered in vivo, it has an excellent immunosuppressive effect while having a low risk of inducing an immune response recognizing it as an immunogen. Therefore, the fusion protein can be utilized as a variety of immunosuppressive and immunotherapeutic agents.

[0163] In one example, in the fusion protein, the IL-10 may be linked to the C-terminus and / or N-terminus of the heavy chain (heavy chain variable region or heavy chain constant region) or light chain (light chain variable region or light chain constant region) of the anti-TIGIT antibody or antigen-binding fragment thereof via a linker or may be linked directly as a monomer or dimer without a linker.

[0164] More specifically, the fusion protein comprises (1) an anti-TIGIT antibody (e.g., an IgG type antibody comprising two heavy chains and two light chains) and (2) one IL-10, wherein the IL-10 may be linked as a monomer or dimer to the C-terminus (C-terminus of the constant region) or N-terminus of either one of the two heavy chains or one of the two light chains of the anti-TIGIT antibody via a peptide linker (first linker) or without a linker.

[0165] In another specific embodiment, the fusion protein comprises (1) an anti-TIGIT antibody (e.g., an IgG type antibody comprising two heavy chains and two light chains) and (2) two IL-10s, wherein the two IL-10s may be linked to the C-terminus or N-terminus of the two heavy chains or the two light chains of the anti-TIGIT antibody as a monomer or a dimer, respectively, via a linker (first linker) or without a linker.

[0166] For example, in the above fusion protein,

[0167] (i) one of the two IL-10s is linked to the C-terminus (i.e., the C-terminus of the heavy chain constant region (e.g., Fc)) or N-terminus (i.e., the N-terminus of the heavy chain variable region) of one of the two heavy chains of the anti-TIGIT antibody, and the other is linked to the C-terminus (i.e., the C-terminus of the heavy chain constant region (e.g., Fc)) or N-terminus (i.e., the N-terminus of the heavy chain variable region) of the other heavy chain of the anti-TIGIT antibody, as a monomer or dimer, with or without a linker, or

[0168] (ii) Either one of the two IL-10s can be linked to the C-terminus (i.e., the C-terminus of the light chain constant region) or the N-terminus (i.e., the N-terminus of the light chain variable region) of one of the two light chains of the anti-TIGIT antibody, and the other one can be linked to the C-terminus (i.e., the C-terminus of the light chain constant region) or the N-terminus (i.e., the N-terminus of the light chain variable region) of the other light chain of the anti-TIGIT antibody, as a monomer or dimer, with or without a linker.

[0169] More specifically, in the fusion protein,

[0170] (i) one of the two IL-10s is linked to the C-terminus of the Fc of one of the two heavy chains of the anti-TIGIT antibody, and the other is linked to the C-terminus of the Fc of the other of the anti-TIGIT antibody, as a monomer or dimer, with or without a linker, or

[0171] (ii) Either one of the two IL-10s may be linked to the C-terminus of the constant region of one of the two light chains of the anti-TIGIT antibody, and the other may be linked to the C-terminus of the constant region of the other light chain of the anti-TIGIT antibody, as a monomer or dimer, with or without a linker.

[0172]

[0173] The linker (first linker) connecting the antibody and IL-10 may be, but is not limited to, a rigid peptide linker (e.g., [EAAAK]n (n is the number of [EAAAK] and is an integer from 1 to 5)), a flexible peptide linker (e.g., [(G)mS]l (m is the number of G and is an integer from 1 to 5, l is the number of [(G)mS] and is an integer from 1 to 5)), etc.

[0174]

[0175] Linker

[0176] The linker (first linker) connecting the antibody and IL-10 may be a peptide linker, for example, [EAAAK]n (n is the number of [EAAAK] (SEQ ID NO: 77) and is an integer from 1 to 5), [(G)mS]l (m is the number of G and is an integer from 1 to 5, l is the number of [(G)mS] and is an integer from 1 to 5), A EAAAK A, A EAAAK EAAAK A, A EAAAK EAAAK EAAAK EAAAK A, A EAAAK EAAAK EAAAK EAAAK A, A EAAAK EAAAK EAAAK EAAAK A, AEAAAKEAAAAKAG, AEAAAKEAAAAKAG, GGGG, GGGGG, GGGGGGG, GAGAGAGAGA, RPLSYRPPPPFFPFGFPSVRP, YPRSYIRRHHPSSLTT, TPSHLSHILPSFGLPTFWN, RPVSPFTFPPRLSNSWLPA, SPAAHPRSIPRPPGPIRT, APGPSAPSHRSLPSRAAFG, PRNSHFLHPLLVAPLGA, MPSLSGVLQVRYLSPPDL, SPQYPSPLTLTLPPHPSL, NPSLNPPSYLHRAPSRIS, LPWRTSLPSLPLPRRPP, PPLFAKGPVGLLSSRSFPP, VPPAPVVSSLRSAHARPPY, LRPTPPRVRSYTCCPP, PNVAHVLPLL TVPWDNLR, CNPLLPLCARSPAVRTFP, LGTPTPTPTTGEF, EDFTRGKL, L EAAAR EAAAR EAAAR EAAAR EAAAR EAAAR EAAAR, L EAAAR EAAAR EAAAR EAAAR EAAAR, L EAAR, L EAAR, EAAR EAAR EAAR EAAR, EAAAR EAAAR EAAAR, EAAAR, LTEEQQEGGG, TEEQQEGGG, LAKLKQKTEQLQDRIAGGG, LELKTPLGDT THTCPRCPEP KSCDTPPPCP RCPEPKSCDT PPPCPRCPEP KSCDTPPPCP RCPGG,And LEPKSSDKTHTSPPSPGG, etc., but may be at least one selected from the group consisting of, but is not limited thereto. In one specific example, the peptide linker may be a rigid linker such as [EAAAK]n (where n is the number of [EAAAK] and is an integer from 1 to 5; for example, SEQ ID NOs: 14, 46 to 49, etc.) or a flexible linker such as [(G)mS]l (where m is the number of G and is an integer from 1 to 5, and l is the number of [(G)mS] and is an integer from 1 to 5; for example, SEQ ID NOs: 15, 98 to 101, etc.), but is not limited thereto.

[0177]

[0178] Nucleic acid molecules, recombinant vectors, and recombinant cells

[0179] Another example provides a nucleic acid molecule encoding the antibody, antigen-binding fragment, or fusion protein.

[0180] Another example provides a recombinant vector comprising the nucleic acid molecule. The vector can be used as an expression vector for the nucleic acid molecule.

[0181] Another example provides a recombinant cell comprising the nucleic acid molecule or a recombinant vector comprising the same. The cell can be used for producing the fusion protein.

[0182] Another example provides a method for producing the fusion protein, comprising expressing the nucleic acid molecule in a suitable host cell. The step of expressing the nucleic acid molecule in a suitable host cell may comprise culturing the recombinant cell described above under conventional conditions.

[0183] The term "vector" refers to a means for expressing a target gene in a host cell. For example, it includes a plasmid vector, a cosmid vector, a bacteriophage vector, an adenovirus vector, a retrovirus vector, and a viral vector such as an adeno-associated virus vector. Vectors that can be used as the recombinant vector can be produced by manipulating plasmids (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13, etc.) or viruses (e.g., SV40, etc.) that are frequently used in the art.

[0184] In the recombinant vector, the nucleic acid molecule encoding the fusion protein may be operably linked to a promoter. The term "operably linked" refers to a functional linkage between a nucleotide expression regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. By being "operably linked," the regulatory sequence can regulate the transcription and / or translation of the other nucleotide sequence.

[0185] The recombinant vector may be constructed typically as a cloning vector or an expression vector. The expression vector may be any vector commonly used in the art to express foreign proteins in plants, animals, or microorganisms. The recombinant vector may be constructed using various methods known in the art.

[0186] The above recombinant vector can be constructed using prokaryotic or eukaryotic cells as hosts. For example, if the vector used is an expression vector and uses prokaryotic cells as hosts, a strong promoter capable of driving transcription (e.g., pL) λ It usually includes a ribosome binding site for translation initiation and a transcription / translation terminator sequence (e.g., promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.). In the case of using a eukaryotic cell as a host, the replication origin that operates in the eukaryotic cell included in the vector includes, but is not limited to, the f1 replication origin, the SV40 replication origin, the pMB1 replication origin, the adeno replication origin, the AAV replication origin, and the BBV replication origin. In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0187] The recombinant cell may be obtained by introducing the recombinant vector into an appropriate host cell. Any host cell known in the art that can stably and continuously clone or express the recombinant vector may be used as the host cell, and examples of prokaryotic cells include E. coliJM109, E. coliBL21, E. coliRR1, E. coliLE392, E. coliB, E. coliX1776, E. Examples of the host cells that can be used include, but are not limited to, strains of the genus Bacillus, such as Bacillus coliW3110, Bacillus subtilis, and Bacillus thuringiensis, and strains of enterobacteria, such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. In the case of transforming eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, plant cells, and animal cells, such as Sp2 / 0, CHO (Chinese hamster ovary) K1, CHO, DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines.

[0188] The delivery (introduction) of the nucleic acid molecule or the recombinant vector containing the same into a host cell can be accomplished using a delivery method widely known in the art. For example, if the host cell is a prokaryotic cell, the CaCl2 method or electroporation can be used, and if the host cell is a eukaryotic cell, the microinjection method, calcium phosphate precipitation method, electroporation method, liposome-mediated transfection method, and gene bombardment can be used, but are not limited thereto.

[0189] The method for selecting the host cell transformed (introduced with a recombinant vector) can be easily performed using a method widely known in the art, utilizing a phenotype expressed by a selectable marker. For example, if the selectable marker is a specific antibiotic resistance gene, recombinant cells introduced with the recombinant vector can be easily selected by culturing them in a medium containing the antibiotic.

[0190]

[0191] Medicinal uses

[0192] The anti-TIGIT antibody or antigen-binding fragment thereof included in the fusion protein according to the present invention has the ability to deliver IL-10 to effector immune cells to suppress immune cell activity (e.g., suppress effector T cell function, promote Treg activity, reduce secretion of inflammatory cytokines, etc.). In addition, IL-10 has excellent binding affinity to its receptor (IL-10 receptor) and low immunogenicity, so that when administered in vivo, it has an excellent immunosuppressive effect such as reducing the secretion of cytokines (inflammatory cytokines), but does not act as an immunogen, so that the risk of inducing an immune response recognizing it as an immunogen is low, thereby ensuring safety, and it has excellent anti-autoimmune and anti-inflammatory effects (e.g., suppression of pro-inflammatory cytokine production and / or secretion, suppression of effector immune cell proliferation, etc.). Therefore, the above fusion peptide has the advantages of both anti-TIGIT antibody or antigen-binding fragment thereof and IL-10, and can be usefully applied to immunosuppression, prevention and / or treatment of immune-related diseases, and particularly prevention and / or treatment of autoimmune and / or inflammatory diseases.

[0193] One example provides an immunosuppressant or a pharmaceutical composition for immunosuppression, which comprises as an active ingredient at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0194] Another example provides a pharmaceutical composition for preventing and / or treating an immune-related disease, comprising as an active ingredient at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector. The immune-related disease may be selected from the group consisting of autoimmune autoimmune diseases, inflammation, and inflammatory diseases.

[0195] Another example provides a pharmaceutical composition (or anti-autoimmune agent and / or anti-inflammatory agent) for preventing and / or treating autoimmunity (or autoimmune disease) and / or inflammation (or inflammatory disease), comprising as an active ingredient at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0196] Another example provides a method of immunosuppression, comprising administering to a subject in need of immunosuppression a pharmaceutically effective amount of at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector. The method of immunosuppression may further comprise, prior to the administering step, a step of identifying the subject in need of immunosuppression.

[0197] Another example provides a method for preventing and / or treating an immune-related disease, comprising administering to a subject in need of prevention and / or treatment of an immune-related disease a pharmaceutically effective amount of at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector. The method for preventing and / or treating an immune-related disease may further comprise, prior to the administering step, a step of identifying a subject in need of prevention and / or treatment of an immune-related disease.

[0198] Another example provides a method for preventing and / or treating autoimmunity (or autoimmune disease) and / or inflammation (or inflammatory disease), comprising administering to a subject in need of prevention and / or treatment of autoimmunity (or autoimmune disease) and / or inflammation (or inflammatory disease) a pharmaceutically effective amount of at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector. The method for preventing and / or treating cancer may further comprise, prior to the administering step, a step of identifying a subject in need of prevention and / or treatment of autoimmunity (or autoimmune disease) and / or inflammation (or inflammatory disease).

[0199] Another example provides a use for immunosuppression, prevention and / or treatment of an immune-related disease, and / or prevention and / or treatment of autoimmunity (or autoimmune disease) and / or inflammation (or inflammatory disease), or a use for manufacturing a pharmaceutical composition for immunosuppression, prevention and / or treatment of an immune-related disease, and / or prevention and / or treatment of autoimmunity (or autoimmune disease) and / or inflammation (or inflammatory disease), selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0200]

[0201] In this specification, the term 'immunosuppression' may mean reducing the initial immune response to an antigen or reducing an existing immune response, and may be used interchangeably with terms such as immunosuppression, immune deactivation, etc.

[0202] As used herein, the term "immune-related disease" includes all diseases caused by a disorder and / or inappropriate activity of the immune system, and may be, for example, one or more selected from the group consisting of autoimmune diseases, inflammatory diseases, etc., but is not limited thereto. The inflammation may be inflammation (e.g., chronic inflammation or acute inflammation), and may include, but is not limited to, inflammation due to infection (e.g., local inflammation, systemic inflammation, etc.).

[0203] The above autoimmune disease means all symptoms and / or diseases caused by autoimmunity or autoimmunity, and includes, but is not limited to, rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, Behcet's syndrome, lupus, and Sjogren's syndrome, and may be at least one selected from the group consisting of myasthenia gravis (e.g., myasthenia gravis), scleroderma, hypothyroidism, hyperthyroidism, psoriasis, vitiligo, multiple sclerosis, autoimmune hepatitis, autoimmune nephritis, autoimmune pancreatitis, autoimmune encephalitis, cytokine storm, and the like, but is not limited thereto.

[0204] The above inflammatory disease means inflammation (e.g., chronic inflammation or acute inflammation) or a disease caused by inflammation, such as heart inflammation (e.g., coronary artery disease, angina pectoris, myocardial infarction, pericarditis, myocarditis, etc.), vascular inflammation (e.g., atherosclerosis, arteriosclerosis, myocarditis, etc. collectively referred to as atherosclerosis, vasculitis, disseminated intravascular coagulation (DIC), immune thrombocytopenia (ITP), thrombotic thrombocytopenia (TTP), anemia), upper respiratory tract inflammation (e.g., acute nasopharyngitis, allergic rhinitis, sinusitis, pharyngitis, tonsillitis, laryngitis), lower respiratory tract and / or lung inflammation (e.g., bronchitis, bronchiectasis, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, interstitial lung disease, tuberculosis), upper gastrointestinal inflammation (e.g., gastritis, esophagitis), lower gastrointestinal inflammation (e.g., small intestine disease, ulcerative colitis, Crohn's disease, celiac disease, diverticulitis, irritable bowel syndrome, appendicitis, fistula, etc.), inflammation of the liver, biliary tract and / or pancreas (e.g., hepatitis, fatty liver, cholangitis, cholecystitis, pancreatitis, type 1 diabetes, etc.), inflammation of the kidney (upper urinary tract) (e.g., pyelonephritis, glomerulonephritis, urinary tract infection), inflammation of the lower urinary tract (e.g., ureteritis, urethritis, cystitis, prostatitis / chronic pelvic pain syndrome, etc.), inflammation of the thyroid and / or parathyroid glands (e.g., thyroiditis, parathyroiditis, etc.), inflammation of the adrenal glands (e.g., adrenalitis, etc.), inflammation of the reproductive system (e.g., pelvic inflammatory disease, oophoritis, orchitis, epididymitis, etc.), inflammation of the bones and / or joints (e.g., osteoarthritis, rheumatoid arthritis, osteomyelitis, synovitis, etc.), inflammation of the skin (e.g., skin: soft tissue infection, tinea versicolor, athlete's foot, acne, etc.), inflammation of the muscles (e.g., myositis, etc.), inflammation of the brain (e.g., encephalitis, major depressive disorder) disorders, etc.), neuroinflammation (e.g., neuritis of various parts including the eyes and ears, complex regional pain syndrome, Guillain-Barré syndrome, etc.), eye inflammation (e.g., blepharitis, uveitis, conjunctivitis, etc.), ear inflammation (otitis media, mastoiditis, etc.), oral inflammation (stomatitis, periodontitis, gingivitis, etc.), systemic inflammation (systemic inflammatory response syndrome (e.g., sepsis, etc.), metabolic syndrome-related diseases, etc.), peritonitis, reperfusion injury, transplant rejection,It may include, but is not limited to, one or more selected from the group consisting of hypersensitivity reactions, etc. The preventive and / or therapeutic effect of the above autoimmune disease and / or inflammatory disease includes, but is not limited to, suppressing the occurrence and / or increase of inflammation, as well as suppressing the aggravation of autoimmunity and / or inflammation due to migration, invasion, infection, etc.

[0205] The systemic inflammation caused by the above infection may be selected from any infectious disease that can be treated, alleviated and / or prevented through immunosuppression as described above, such as immunosuppression (e.g., suppression of function (e.g., inactivation) and / or reduction in proliferation of immune cells (e.g., NK cells, effector T cells, cytotoxic T cells, CD3+ T cells, CD4+ T cells, CD8+ T cells, etc.), enhancement and / or stabilization of regulatory T cell (Treg) activity, reduction in production and / or secretion of inflammatory proteins (e.g., cytokines (TNF-alpha, IFN-gamma, etc.)), etc.). For example, the infectious disease may be an infection by one or more pathogens selected from the group consisting of viruses (e.g., influenza viruses, coronaviruses (e.g., severe acute respiratory syndrome (SARS) virus (SARS-CoV, SARS-CoV-2, etc.), Middle East respiratory syndrome (MERS) virus (MERS-CoV), etc.), bacteria, fungi, parasites, etc.), or a disease (infectious disease) caused by the infection. there is.

[0206]

[0207] The subject of administration of the fusion protein and / or pharmaceutical composition provided herein may be any animal or cell, and may be, for example, an animal selected from mammals including humans, primates such as monkeys, rodents such as rats and mice, or cells, tissues, body fluids (e.g., serum) derived (isolated) from the animal, or a culture thereof, and may be, for example, a human or cells, tissues, body fluids (e.g., serum) isolated from a human.

[0208] The pharmaceutical composition may further comprise, in addition to the fusion protein, nucleic acid molecule, vector and / or cell as an active ingredient, a pharmaceutically acceptable carrier, and the carrier may be one or more selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc., which are commonly used in the formulation of protein drugs, but are not limited thereto. The pharmaceutical composition may further comprise one or more selected from the group consisting of diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc., which are commonly used in the preparation of pharmaceutical compositions.

[0209] The content of the fusion protein, nucleic acid molecule, vector and / or cell as an active ingredient in the pharmaceutical composition may be, but is not limited to, 0.00001 to 99 wt%, 0.0001 to 99 wt%, 0.001 to 99 wt%, 0.01 to 99 wt%, 0.1 to 99 wt%, 1 to 99 wt%, 10 to 99 wt%, 15 to 99 wt%, 20 to 99 wt%, 25 to 99 wt%, 30 to 99 wt%, 35 to 99 wt%, 40 to 99 wt%, 45 to 99 wt%, or 50 to 99 wt%, based on the total composition weight.

[0210] The above fusion proteins, nucleic acid molecules, vectors, cells, and / or pharmaceutical compositions may be administered via oral or parenteral routes. Parenteral administration may include intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. Since proteins or peptides are digested when administered orally, oral compositions must be formulated to coat the active agent or protect it from degradation in the stomach.

[0211] Additionally, the fusion protein, nucleic acid molecule, vector, cell, and / or pharmaceutical composition may be formulated in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, injection, etc., and may additionally include a dispersing agent or stabilizer for the formulation.

[0212] The content of the fusion protein, nucleic acid molecule, vector, and / or cell, which are active ingredients in the pharmaceutical composition, can be varied depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration interval, administration route, excretion rate, and response sensitivity. The daily dosage of the pharmaceutical composition is 0.00001 to 1000 mg / kg, 0.00001 to 500 mg / kg, 0.00001 to 100 mg / kg, 0.00001 to 50 mg / kg, 0.0001 to 1000 mg / kg, 0.0001 to 500 mg / kg, 0.0001 to 500 mg / kg, 0.0001 to 100 mg / kg, 0.0001 to 50 mg / kg, 0.001 to 1000 mg / kg, 0.001 to 500 mg / kg, 0.001 to 100 mg / kg, 0.001 to 50 mg / kg, 0.01 to 1000 mg / kg, 0.01 to The dosage may be, but is not limited to, 500 mg / kg, 0.01 to 100 mg / kg, 0.01 to 50 mg / kg, 0.1 to 1000 mg / kg, 0.1 to 500 mg / kg, 0.1 to 100 mg / kg, or 0.1 to 50 mg / kg. The above daily dosage may be formulated as a single preparation in the form of a unit dose, formulated in an appropriate amount, or manufactured by being placed in a multi-dose container. In addition, the pharmaceutically effective amount in the present specification means the amount of the active ingredient that can exhibit the desired pharmaceutical activity, and may be in the dosage range described above.

[0213]

[0214] Another example provides an immunosuppressive health functional food comprising as an active ingredient at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0215] Another example provides a health functional food for preventing and / or improving an immune-related disease, comprising as an active ingredient at least one selected from the group consisting of the antibody or antigen-binding fragment, the fusion protein, a nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, a recombinant vector comprising the nucleic acid molecule, and a recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0216] "Health functional foods" refer to any food manufactured (processed) using nutrients that are easily deficient in daily meals or raw materials or ingredients (hereinafter referred to as "functional raw materials") that have beneficial functions for the human body. These foods help maintain health or prevent and / or improve certain diseases or symptoms. There are no specific restrictions on the final product form. For example, the health functional foods may be selected from, but are not limited to, various foods, beverages, food additives, etc.

[0217] The above food composition may include acceptable food additives and may further include suitable carriers, excipients and diluents commonly used in the manufacture of health functional foods.

[0218] When the above food composition is used as a food additive, the composition may be added as is or used together with other foods or food ingredients, and may be used appropriately according to a conventional method.

[0219] The content of the effective ingredient contained in the food composition can be appropriately determined without any special limitation depending on the form of the food, the desired effect, the purpose, etc., and may be, for example, 0.00001 to 99 wt%, 0.0001 to 99 wt%, 0.001 to 99 wt%, 0.01 to 99 wt%, 0.1 to 99 wt%, 1 to 99 wt%, or 10 to 99 wt% of the total food weight, but is not limited thereto.

[0220]

[0221] The fusion protein provided herein has the advantages of both the anti-TIGIT antibody or antigen-binding fragment thereof described above and IL-10, and may be usefully applied to the prevention and / or treatment of immunosuppression and immune-related diseases.

[0222]

[0223] FIG. 1 is a graph showing the results of ELISA measurement of the degree of binding of anti-TIGIT antibody MMB0 and fusion protein (MMB5) to human TIGIT according to one embodiment.

[0224] Figure 2 is a graph showing the results of ELISA measurement of the binding degree of anti-TIGIT antibody (MMB0) and fusion protein (MMB5) to IL-10Ra.

[0225] Figures 3a to 3c are graphs showing the results of measuring the binding affinity of fusion proteins to TIGIT-expressing effector T cells and NK cells by FACS according to one embodiment (3a: CD4 + T cell; 3b: CD8 + T cell; 3c: NK cell).

[0226] Figure 4 is a graph showing the results of measuring whether fusion proteins block TIGIT / CD155 interaction according to one embodiment using FACS.

[0227] Figures 5a and 5b are graphs showing the degree of STAT3 signal induction of fusion proteins according to one embodiment, as confirmed by STAT3 phosphorylation (5a: CD4+ T cell; 5b: CD8+ T cell).

[0228] Figure 6 is a graph showing the transport effect of IL-10 of a fusion protein to immune cells (effector T cells, NK cells) according to one embodiment.

[0229] Figure 7 is a graph showing the inhibitory effect of fusion proteins on the production of inflammatory cytokines (TNF-α, GranzymeB) according to one embodiment.

[0230] Figures 8a to 8c are graphs showing the T cell proliferation inhibitory effect of fusion proteins according to one embodiment (8a: CD4 + T cell, 8b: CD8 + T cell, 8c: NK cell).

[0231] Figures 9a to 9c are graphs showing the inhibitory effect of fusion proteins according to one embodiment on the expression of inflammatory cytokines (TNF-α) in PBMCs derived from patients with autoimmune diseases.

[0232] Figure 10 is a graph showing the anti-inflammatory effect of a fusion protein according to one embodiment as a result of measuring the DAI score (Disease activity index score) in a DSS (Dextran sulfate sodium)-induced colitis model.

[0233]

[0234] The present invention will be described in more detail below through examples. However, these are merely illustrative and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that modifications to the examples described below may be made without departing from the essential spirit of the invention.

[0235]

[0236] Example 1: Production of monoclonal anti-TIGIT antibodies

[0237] 1.1. Production of mouse monoclonal anti-TIGIT antibody

[0238] Four BALB / c mice were immunized with His-tagged human TIGIT protein (R&D systems, UniProtKB / SwissProt Q495A1-1) by four consecutive cross-injections over 14 days. Lymphocytes were collected from the four mice, pooled, purified, and fused with SP2 / 0 myeloma cells. The fused cells were expanded in HAT selective single-step cloning media, and 2 × 920 hybridoma clones obtained were selected with ClonePix2 (Molecular Devices), transferred to 96-well plates, and cultured.

[0239] Using an indirect ELISA, supernatants of hybridoma tissue cultures were screened for antibodies to recombinant human TIGIT (His) (R&D systems). Positive clones were further subjected to indirect ELISA against the screening antigen (recombinant human TIGIT Fc chimeric protein (R&D systems)) and cells to confirm Ig secretion and specificity. CHO-K1 (ATCC, CCL-61) cell line was used as a negative control.

[0240] ELISA was performed under the following conditions:

[0241] - ELISA plates were coated at 4°C with a coating solution containing 0.1 μg / well of recombinant human TIGIT (rhTIGIT) protein in Carbonate Coating Buffer (pH 9.6) O / N at a concentration of 100 μL / well.

[0242] - Block with 3% skim milk powder in PBS for 1 hour at room temperature

[0243] - Treatment with secondary antibody 1:10000 Goat anti-mouse IgG / M(H+L)-HRP (100uL / well) in PBS-Tween for 1 hour (37℃, w / shaking).

[0244] - All washing steps were performed with PBS-Tween for 3 minutes.

[0245] - Add TMB substrate at a concentration of 50uL / well, keep under dark conditions (5-10 mins), and terminate with an equal volume of 1M HCl.

[0246] By isotyping, IgM expressing clones were isolated and eliminated, and IgG expressing clones were collected.

[0247] The collected positive clones were subcloned to identify stable expression clones. The culture supernatant containing the selected clones was eluted through a Protein A column, and the buffer was replaced with PBS. The purified antibodies were tested by indirect ELISA against the screening antigen (Recombinant Human TIGIT (T103) Fc Chimera Protein; R&D systems) and a negative control antigen (His peptide).

[0248] Among the collected positive clones, the MMB0 clone (mouse antibody based on mouse backbone (mIgG1)) was selected as the leading antibody.

[0249] The CDRs and variable regions of the selected antibodies are listed in Table 3 below.

[0250] Amino acid sequence SEQ ID NO: CDR-H1SDYAWN1 CDR-H2YITYSGSTSYNPSLKS2 CDR-H3RQVGLGFAY3 CDR-L1KASQDVSNTVA4 CDR-L2SASYRYT5 CDR-L3QQHYSTPWT6 Heavy chain variable region (VH0) DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCASRQVGLGFAYWGQGTLVTVSA29 Light chain variable region (VK0) DIVMTQSHKFMSTSVGDRVSITCKASQDVSNTVAWYQQRPGQSPKPLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIK35

[0251]

[0252] 1.2. Humanization of mouse anti-TIGIT antibodies

[0253] To identify amino acid residues critical for antibody structure and binding, we analyzed protein structural models of the mAb V regions. This information, along with in silico design of the human antibody structure, was used to select a large pool of preliminary sequence fragments for use in the construction of humanized variants of MMB0, and peptide binding to human MHC class II alleles was analyzed. Where possible, sequence fragments identified as significant non-human germline binders to human MHC class II were discarded. This reduced the set of fragments, and these combinations were reanalyzed using the same method to ensure that the junctions between fragments did not contain potential T-cell epitopes. The selected sequence fragments were assembled to generate a complete variable region (V region) that did not contain significant T-cell epitopes or had a reduced number of significant T-cell epitopes, thereby designing a human variable region that evades potential T-cell epitope recognition (deimmunization).

[0254] For use in gene synthesis and expression in mammalian cells, six heavy chain variable region (VH1, VH6, VH7, VH5, VH8, VH9) and seven light chain variable region (Vk1, Vk2, Vk7, Vk11, Vk5, Vk10, Vk14) sequences were selected for production of humanized antibodies.

[0255] The sequences of the six heavy chain variable regions and seven light chain variable regions for the production of the selected humanized antibodies and the heavy chain variable region (VH0) and light chain variable region (Vk0) of the mouse antibody MMB0 are summarized in Table 4 below:

[0256] 중쇄가변영역아미노산 서열(N→C)서열번호VH0DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTIEDTATYYCASRQVGLGFAYWGQGTLVTVSA29VH1DVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMAYITYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTIEDTATYYCASRQVGLGFAYWGQGTLVTVSS30VH6DVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMAYITYSGSTSYNPSLKSRITISRDTSKNQFFLKLSSVTIEDTATYYCASRQVGLGFAYWGQGTLVTVSS31VH7DVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRITISRDTSKNQFSLKLSSVTIEDTATYYCASRQVGLGFAYWGQGTLVTVSS32VH5QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSS33VH8QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFFLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSS34VH9QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSS7경쇄가변영역아미노산서열(N→C)서열번호Vk0DIVMTQSHKFMSTSVGDRVSITCKASQDVSNTVAWYQQRPGQSPKPLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYSTPWTFGGGTKLEIK35Vk1DIVMTQSHSFLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK36Vk2DIVMTQSPSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK37Vk7DIVMTQSPSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQAPRPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK38Vk11DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK8Vk5DIQMTQSPSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQHYSTPWTFGGGTKVEIK39Vk10DIQMTQSPSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQAPRPLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQHYSTPWTFGGGTKVEIK40Vk14DIQMTQSPSFLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDVATYYCQQHYSTPWTFGGGTKVEIK41

[0257] 또한, 인간화 항체 제조에 사용된 인간 IgG 유래의 불변영역을 아래의 표 5에 정리하였다:

[0258] amino acid Sequence (N → C) Sequence number Heavy chain constant region ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK10Light Chain Immutable AreaRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC12

[0259] Based on the variable region sequences described in Tables 4 and 5 above, various combinations of humanized antibodies were produced using the sequences of six heavy chain variable regions (VH1, VH6, VH7, VH5, VH8, VH9), seven light chain variable regions (Vk1, Vk2, Vk7, Vk11, Vk5, Vk10, Vk14), and the human constant region sequences of Table 5, and chimeric antibodies were produced using the combination of the variable regions of mouse antibodies, VH0 / VK0, and the human constant regions of Table 5. Among these, a humanized antibody produced using a combination of VH9 (SEQ ID NO: 7) and Vk11 (SEQ ID NO: 8) (hereinafter referred to as “MMB0” or “MMB0_VH9 / Vk11”) was selected and used in the following examples.

[0260]

[0261] Example 2. Preparation of [anti-TIGIT monoclonal antibody]-[wild-type IL-10] fusion protein

[0262] A fusion protein in which the humanized anti-TIGIT antibody prepared in Example 1.2 and IL-10 are fused with or without a linker was designed to have the structure shown in Table 6 below.

[0263] Light and heavy chain TIGIT mAb-wild type IL-10 fusion proteinsFusion protein nameStructureLinkerMMB0-IL-10 monomer(Fusion protein of TIGIT mAbMMB0 with wild type IL-10 at LC linking)TIGIT mAb-(LC-(EAAAK)3-(IL-10)2Wild type IL-10 fused to LC C-terminus (LC variant) with rigid linkerTIGIT mAb-(LC-(GGGGS)3-(IL-10)2Wild type IL-10 fused to LC C-terminus (LC variant) with flexible linkerMMB0-IL-10(monomer)-Fc(Fusion protein of TIGIT mAbMMB0 with wild type IL-10 at FC linking)TIGIT mAb-Fc-((EAAAK)3-(IL-10)2Wild type IL-10 fused to Fc C-terminus (Fc variant) with rigid linkerTIGIT mAb-Fc-((GGGGS)3-(IL-10)2Wild type IL-10 fused to Fc C-terminus (Fc variant) with flexible linkerTIGIT mAb-Fc-(IL-10)2Wild type IL-10 fused to Fc C-terminus (Fc variant) without linkerMMB0-IL-10 dimer(Fusion protein of TIGIT mAbMMB0 with wild type IL-10 dimer at LC linking)TIGIT mAb-(LC-(EAAAK)3-(IL-10 dimer)2Wild type IL-10 dimer fused to LC C-terminus (LC variant) with rigid linkerTIGIT mAb-(LC-(GGGGS)3-(IL-10dimer)2Wild type IL-10 dimer fused to LC C-terminus (LC variant) with flexible linkerMMB0-IL-10(dimer)-Fc(Fusion protein of TIGIT mAbMMB0 with wild type IL-10 dimer at FC linking)TIGIT mAb-Fc-((EAAAK)3-(IL-10 dimer)2Wild type IL-10 dimer fused to Fc C-terminus (Fc variant) with rigid linkerTIGIT mAb-Fc-((GGGGS)3-(IL-10 dimer)2Wild type IL-10 dimer fused to Fc C-terminus (Fc variant) with flexible linkerTIGIT mAb-Fc-(IL-10 dimer)2Wild type IL-10 dimer fused to Fc C-terminus (Fc variant) without linker

[0264] (상기 표 6에서,

[0265] 항-TIGIT 단클론 항체(TIGIT mAb): MMB0_VH9 / Vk11(이하, ‘MMB0’);

[0266] IL-10: wild type IL-10;

[0267] Two linkers or no linker: LC variants (fused to LC(light chain) C-terminus with rigid and flexible linker) & Fc variants (ΔK478) (fused to Fc C-terminus without linker and rigid and flexible linker))

[0268]

[0269] The gene encoding TIGIT mAb (see Table 7) was synthesized and cloned into a modified pcDNA3.4 vector (Biointron) containing a 'linker-wild type IL-10 element'. CHO-K1 cells (ATCC) were seeded on plates and cultured to 70-90% confluency, and the gene DNA was transfected into CHO-K1 cells by electrophoresis. The cells were then cultured at 37°C for transient expression. The culture supernatant was purified by Protein A affinity chromatography and preparative SEC. The produced protein was buffer-exchanged with PBS, quantified by measuring OD280 nm, and analyzed by SDS-PAGE.

[0270]

[0271] Sequences of anti-TIGIT mAb and fusion protein MMB0-IL-10ComponentsAmino acid sequence (N→C) or nucleic acid sequence (5'→3')Sequence numberanti-TIGIT heavy chain variable regionQVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSS7anti-TIGIT heavy chain constant regionASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK10Anti-TIGIT heavychainQVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK11Anti-TIGIT Light Chain Variable RegionDIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIK8Anti-TIGIT Light Chain Constant regionRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC12Anti-TIGIT light chainsDIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC13IL-10PolypeptidesSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN9Linker (V-1)EAAAKEAAAKEAAAK14Linker (V-2)GGGGSGGGGSGGGGS15Linker (D-1)GGGSGGG16IL-10 monomer (2개) is linked without a linker to the respective C terminus of the light chains (2개) of the anti-TIGIT antibody MMB0Heavy chain strand (anti-TIGIT heavy chain (IgG1))QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK11Light chain strand ([Anti-TIGIT light chain(kappa)]-[IL-10])DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN17IL-10 monomer (2) is linked via linker V-1 to the respective C terminus of the light chains (2) of the anti-TIGIT antibody MMB0Heavy chain strand (anti-TIGIT heavy chain(IgG1))QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK11Light chain strand ([Anti-TIGIT light chain (kappa)]-[Linker (V-1)]-[IL-10])DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAKEAAAKEAAAKSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN18IL-10 monomer (2) is linked via linker V-2 to each C-terminus of the lightchain (2) of the anti-TIGIT antibody MMB0 (MMB2)Heavy chain strand (anti-TIGIT heavy chain (IgG1))QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK11Light chain strand ([Anti-TIGIT light chain (kappa)]-[Linker(V-2)]-[IL-10])DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN19IL-10 monomer (2) is linked without a linker to each C-terminus of the heavy chain (2) of the anti-TIGIT antibody MMB0Heavy chain strand ([anti-TIGIT heavy chain(IgG1)]-[IL-10])QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN20Light chain strand (Anti-TIGIT light chain (kappa))DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC13IL-10 monomer (2) is linked via linker V-1 to each C-terminus of the heavy chain (2) of the anti-TIGITantibody MMB0Heavy chain strand ([anti-TIGIT heavy chain (IgG1)]-[linker (V-1)]-[IL-10])QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKEAAAKEAAAKEAAAKSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN21Light chain strand (Anti-TIGIT light chain (kappa))DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC13IL-10 monomer (2) islinked via linker V-2 to each C-terminus of the light chain (2) of the anti-TIGIT antibody MMB0 (MMB1)Heavy chain strand ([anti-TIGIT heavy chain (IgG1)]-[linker (V-2)]-[IL-10])QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN22Light chain strand (Anti-TIGIT light chain(kappa))DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC13IL-10 dimer (2) is linked without a linker to the respective C terminus of the light chains (2) of the anti-TIGIT antibody MMB0Heavy chain strand (anti-TIGIT heavy chain (IgG1))QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK11Light chain strand ([Anti-TIGIT light chain(kappa)]-[IL-10]-[D-1]-[IL-10])DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGSGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN23IL-10 dimer (2) is linked via linker V-1 to the respective C terminus of the light chains (2) of the anti-TIGIT antibody MMB0 (MMB5)Heavy chain strand (anti-TIGIT heavy chain(IgG1))QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK11Light chain strand ([Anti-TIGIT light chain (kappa)]-[Linker(V-1)]-[IL-10]-[D-1]-[IL-10])DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEAAAKEAAAKEAAAKSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGSGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN24IL-10 dimer (2) is linked via linker V-2 to each C-terminus of the light chain (2) of the anti-TIGIT antibody MMB0 (MMB6)Heavy chain strand (anti-TIGIT heavy chain(IgG1))QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK11Light chain strand ([Anti-TIGIT light chain (kappa)]-[Linker(V-2)]-[IL-10]-[D-1]-[IL-10])DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGSGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN25IL-10 dimer (2) is linked without a linker to each C-terminus of the heavy chain (2) of the anti-TIGIT antibody MMB0Heavy chain strand ([anti-TIGIT heavy chain (IgG1)]-[linker(V-1)]-[IL-10]-[D-1]-[IL-10])QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGSGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN26Light chain strand (Anti-TIGIT light chain(kappa))DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC13IL-10 dimer (2) is linked via linker V-1 to each C-terminus of the heavy chain (2) of the anti-TIGIT antibody MMB0 (MMB3)Heavy chain strand ([anti-TIGIT heavy chain (IgG1)]-[linker(V-1)]-[IL-10]-[D-1]-[IL-10])QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKEAAAKEAAAKEAAAKSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGSGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN27Light chain strand (Anti-TIGIT light chain(kappa))DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC13IL-10 dimer (2) is linked via linker V-2 to each C-terminus of the light chain (2) of the anti-TIGIT antibody MMB0 (MMB4)Heavy chain strand ([anti-TIGIT heavy chain (IgG1)]-[linker (V-2)]-[IL-10]-[D-1]-[IL-10])QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMAYITYSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCASRQVGLGFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSGGGGSGGGGSSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRNGGGSGGGSPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN28Light chain strand (Anti-TIGIT light chain(kappa))DIVMTQSHSSLSASVGDRVSITCKASQDVSNTVAWYQQKPGQPPKPLIYSASYRYTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPWTFGGGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC13

[0272] (In Table 7, the IL-10 dimer is IL-10 of sequence number 9 linked via a D-1 linker (GGGSGGG; sequence number 16))

[0273]

[0274] Hereinafter, the fusion proteins with the above-mentioned anti-TIGIT antibody MMB0 and IL-10 (see Table 6) were named as shown in Table 8 below:

[0275] Description of fusion proteinIL-10 monomer (2) is linked via flexible linker V-2 to each C-terminus of the heavy chain (2) of the anti-TIGIT antibody MMB0MMB1IL-10 monomer (2) is linked via flexible linker V-2 to each C terminus of the light chains (2) of the anti-TIGIT antibody MMB0MMB2IL-10 dimer (2) is linked via rigid linker V-1 to each C-terminus of the heavy chain (2) of the anti-TIGIT antibody MMB0MMB3IL-10 dimer (2) is linked via flexible linker V-2 to each C-terminus of the light chain (2) of the anti-TIGIT antibody MMB0MMB4IL-10 dimer (2) is linked via rigid linker V-1 to each C terminus of the light chains (2) of the anti-TIGIT antibody MMB0MMB5IL-10 dimer (2) is linked via flexible linker V-2 to each C-terminus of the light chain (2) of the anti-TIGIT antibody MMB0MMB6

[0276] Hereinafter, the fusion proteins of anti-TIGIT antibody MMB0 and IL-10 prepared in this example are indicated according to the names in Table 8 above.

[0277]

[0278] Example 3. Protein binding activity of MMB0-IL-10 fusion protein

[0279] 3.1. TIGIT binding affinity

[0280] Recombinant human TIGIT protein (Acro Biosystems - Cat# TIT-H52H5) was diluted to a concentration of 1 mg / ml in coating buffer and coated on an ELISA plate (using an ELISA buffer kit (Invitrogen™, CNB0011)). Coating was performed by incubation at 37°C for 2 hours. The plate was blocked with blocking buffer (1X assay buffer) for 1 hour, and anti-TIGIT monoclonal antibody (MMB0), MMB0-IL-10 fusion protein (MMB5), or human IgG4 (Biolegend, Cat# 403701) was serially diluted at a 1:3 ratio starting from 62.5 nM using the assay buffer. The serially diluted drugs were added to the TIGIT-coated plate and incubated. Afterwards, anti-Human IgG-HRP antibody (Promega, Cat#W4031) diluted 1:10,000 was added to each well. The plate was fluoresced with tetramethylbenzidine (TMB) solution, and the reaction was stopped using ELISA stop solution (Invitrogen, Cat#SS04). OD values ​​were measured at a wavelength of 450 nM using a GloMax® Explorer.

[0281] The binding affinities of the obtained anti-TIGIT antibody (MMB0) and MMB0-IL-10 fusion protein (MMB5) to TIGIT are shown in Fig. 1. Fig. 1 shows that the MMB0-IL-10 fusion protein (MMB5) has a similar affinity for human TIGIT as the parental antibody (MMB0). These results demonstrate that the addition (fusion) of IL-10 to the MMB0-IL-10 fusion protein MMB5 does not interfere with the TIGIT binding site of the parental antibody (MMB0) and / or inhibit the binding ability, and that the fusion protein maintains the functional quality of the parental antibody.

[0282]

[0283] 3.2.IL-10 receptor binding affinity

[0284] Recombinant human IL-10RA His-tag protein (Sino Biological, 10419-H08H) was diluted to a concentration of 0.1 μg / ml using coating buffer B (Invitrogen, CB01100) and coated on an ELISA plate. 200 μl of 1X Assay Buffer (Invitrogen, CNB0011) was added to each well and incubated at room temperature (RT) for 1 hour to block. 100 μl of each sample (anti-TIGIT monoclonal antibody (MMB0), MMB0-IL-10 fusion protein (MMB5), human IL-10-Fc (Human IgG1 AdipoGen, Cat#CHI-HF-22010), and human IgG4 (Biolegend, Cat#403701)) was added to the blocked plate and incubated at 37°C for 1 h with shaking (200 rpm). The samples were diluted with assay buffer to a concentration of 200 nM and processed. Detection Antibody (HRP-conjugated anti-Human IgG (H+L) antibody (Promega, Cat#W4031)) was diluted 1:10000 in assay buffer and added 100 μl to each well, and incubated at 37°C for 1 h with shaking (200 rpm). Afterwards, TMB solution was added to each well. After adding 100 μl of each solution and inducing luminescence, the reaction was stopped by adding 100 μl of ELISA Stop solution (Invitrogen, Cat#SS04). The OD value was measured at a wavelength of 450 nM using GloMax® Explorer.

[0285] The binding affinity of the obtained anti-TIGIT antibody (MMB0) and fusion protein (MMB5) to IL-10Ra is shown in Fig. 2. As shown in Fig. 2, the parent antibody MMB0 did not bind to IL-10RA, whereas the fusion protein (MMB5) in which IL-10 was linked to the anti-TIGIT antibody showed binding affinity to IL-10Ra, and showed a stronger binding affinity to IL-10 than IL-10-Fc.

[0286]

[0287] Example 4. Cell binding activity of fusion proteins

[0288] 4.1. CD4 + T cells, CD8 + T cells and CD56 + Binding affinity for TIGIT on NK cells

[0289] In this example, immune cells expressing TIGIT (CD4 + T cells, CD8 + T cells and CD56 + The binding of the MMB0-IL-10 fusion protein (MMB5) to each cell population (NK cells) was tested using an anti-TIGIT antibody labeled with PE / Cy7. Since the fusion protein (MMB5) competitively binds to TIGIT expressed on the surface of the immune cells, including the anti-TIGIT antibody labeled with PE / Cy7, the binding percentage of the anti-TIGIT antibody labeled with PE / Cy7 to TIGIT on the surface of the immune cells will decrease as the fusion protein (MMB5) binds to the immune cells via TIGIT expressed on the surface of the immune cells. Therefore, the degree of decrease in the binding percentage of the anti-TIGIT antibody labeled with PE / Cy7 was measured to test the binding ability of the fusion protein (MMB5) to the immune cells.

[0290] Peripheral blood mononuclear cells (PBMC; STEMCELL Technologies) from healthy human donors were counted and resuspended at 5 million cells per ml of phosphate-buffered saline (PBS) (SPL, Cat#34296, performed in a 96-well plate (round bottom)). The prepared cells (PBMC) were bound to MMB0-IL-10 fusion proteins MMB1, MMB3, MMB5 (68.7 nM each) or their parental antibody MMB0 (68.7 nM) for 15 minutes in a 37°C incubator. The plate was centrifuged at 1500 rpm for 3 minutes at room temperature to remove unbound antibodies, and then stained with an antibody cocktail. The antibody cocktail was composed of anti-CD3 APC (Biolegend, 1:50), anti-CD4 AF700 (Biolegend, 1:50), anti-CD8 FITC (Biolegend, 1:50), anti-CD56 BV421 (Biolegend, 1:50), and anti-hTIGIT PE / Cy7 (Biolegend, 1:50). After the staining was performed for 10 min at room temperature in the dark, 100 μl of PBS was added and centrifuged at 1500 rpm for 3 min at room temperature to wash. The supernatant was then removed, and the cells were resuspended in 100 μl of PBS. Flow cytometry was then performed using CytoFLEX (Beckman Coulter) at a speed of 60 μl / min (abort rate 5% or less). This allows the measurement of the TIGIT level bound to the anti-hTIGIT PE / Cy7 antibody (anti-hTIGIT antibody labeled with PE / Cy7). For comparison, the same test was performed on an unstimulated group that was not treated with the fusion protein or parent antibody.

[0291] The percentage of TIGIT on the surface of immune cells labeled by the binding of the anti-hTIGIT PE / Cy7 antibody obtained above (expressed as “% TIGIT expression”) is shown in Figures 3a to 3c (3a: CD4 + T cell; 3b: CD8 + T cell; 3c: NK cell).

[0292] As described above, the lower the level of TIGIT labeled by the binding of anti-hTIGIT PE / Cy7 antibody, the higher the cell binding affinity through TIGIT of the treated sample, and therefore, the results of Figures 3a to 3c show that the anti-TIGIT antibody-IL-10 fusion protein or parent antibody (anti-TIGIT antibody) provided herein binds to effector T cells and NK cells with excellent binding affinity.

[0293]

[0294] Example 5. Maintaining TIGIT / CD155 interaction / signaling function

[0295] In order to determine whether the MMB0-IL-10 fusion protein provided herein maintains the TIGIT / CD155 interaction / signaling function, the TIGIT / CD155 Blockade was tested according to the TIGIT / CD155 Blockade Bioassay instructions from Promega. More specifically, TIGIT Effector cells (Human TIGIT-expressing Jurkat cell line; Promega) were thawed, transferred to assay buffer (Promega), seeded in a white 96-well plate, and rested in an incubator for one day. The following day, the MMB0-IL-10 fusion proteins MMB1, MMB3, MMB5, and their parent antibody MMB0 were treated at the concentrations described in Figure 4. Thereafter, CD155-CHO-K1 (Promega) was added to all experimental groups, including the negative control group (drug (fusion protein or antibody) untreated group). After culturing for 6 hours in a 37℃ incubator, it was left at room temperature for 10-15 minutes. Finally, Bio-Glo TM After adding the reagent to all experimental groups, including the negative control group, and culturing at room temperature, luminescence was measured three times using a luminescence plate reader (luminescence plate reader (BHS LumiStar)).

[0296] For comparison, the same experiment was performed using an antagonistic anti-TIGIT antibody (designated “other anti-TIGIT mAb” in Figure 4) as a reference antibody. The sequence information of the reference antibody used is as follows:

[0297] 중쇄: QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (서열번호 43)

[0298] 경쇄: DIQMTQSPSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (서열번호 44)

[0299]

[0300] The results obtained above are shown in Fig. 4. As shown in Fig. 4, the MMB0-IL-10 fusion proteins MMB1, MMB3, MMB5, and their parent antibody MMB0 did not block the interaction (binding) of TIGIT and CD155. This demonstrates that the fusion protein provided in the present application can utilize the benefits of CD155 signaling that occurs naturally in the immune microenvironment of immune cells, and also utilize the benefits of the TIGIT stimulatory signal of the included anti-TIGIT monoclonal antibody.

[0301]

[0302] Example 6. Induce IL-10R (IL-10 receptor) signaling (measured by STAT3 phosphorylation)

[0303] The immunostimulatory effect of the MMB0-IL-10 fusion protein on human CD3 + T cells (CD4 + and CD8 + STAT3 phosphorylation was tested in T cells.

[0304] 5x10 PBMCs (STEMCELL Technologies) from healthy human donors 5Cells were dispensed into plates at a concentration of 10 cells / well. Drugs (MMB0-IL-10 fusion proteins MMB1, MMB2, MMB3, MMB4, MMB5, MMB6) and control drug human IL-10 (Miltenyi Biotec, 130-093-948) were treated at a concentration of 68.7 nM each and stimulated in a 37°C incubator for 15 minutes. After centrifugation (1500 rpm, 3 min, 4°C), the supernatant was removed, and the cells were stained with 50 μl of surface marker antibody at 4°C for 10 minutes. The above surface marker antibodies refer to Live / Dead-AV750 (eBioscience, 65-0865-12, 1:500), anti-CD3-BV150 (Biolegend, 344828, 1:50), anti-CD4-A700 (Biolegend, 344622, 1:50), anti-CD8-FITC (Biolegend, 980908, 1:50). The markers were diluted in PBS at the indicated ratios and used. After centrifugation (1500 rpm, 3 min, 4℃), the supernatant was removed, and the cells were incubated with 100 ul of Fix buffer (Biolegend, 420801) for 15 minutes at room temperature. After the reaction was completed, centrifugation (1500 rpm, 3 min, room temperature) was performed, the supernatant was removed, and the cells were incubated at -20℃ for 1 hour with 100 ul of True-Phos perm buffer (Biolegend, 425401). After centrifugation (1500 rpm, 3 min), the supernatant was removed, and the cells were stained with pSTAT3 antibody. The staining was performed using pSTAT3-APC (Biolegend, 651008, 1:50) antibody diluted in PBS and was carried out in a light-shielded state. After staining, the supernatant was removed by centrifugation (1500 rpm, 3 min), and the cells were resuspended in 100 ul of PBS.Fluorescence of antibodies detecting surface marker labeling and phosphorylated STAT3 (pSTAT3) of each cell was detected using a flow cytometer (CytoFLEX, Beckman Coulter). Data were analyzed using Flowjo software (BD Biosciences) and plotted using GraphPad Prism 9 software (Dotmatics).

[0305] The above-obtained results (signaling transduction) are shown in Figures 5a and 5b (5a: CD4 + cell; 5b: CD8 + cell; The signal for each drug concentration was normalized by taking the signal at 68.7 nM as 100%.

[0306] As shown in Figures 5a and 5b, phosphorylation of STAT3 in both CD4+ T cells and CD8+ T cells was highly observed at 68.7 nM for both tested fusion proteins, while IL-10 (free human IL-10, not a fusion protein; a control drug) began to lose binding efficiency to IL-10R at a concentration of 0.687 nM. This experiment suggests that the MMB fusion antibody can induce pSTAT3 signaling at a lower concentration than free hIL-10, and that IL-10 bound to the antibody sustains signaling.

[0307]

[0308] Example 7. IL-10 delivery

[0309] In this example, the effect of the MMB0-IL-10 fusion protein provided herein on transporting IL-10 to immune cells (effector T cells, NK cells) was tested.

[0310] 2x10 PBMCs (STEMCELL Technologies) obtained from healthy human donors 5After dispensing into a 96-well plate at a concentration of 10 cells / well, the MMB0-IL-10 fusion protein MMB1 was injected and reacted in a 37℃ incubator for 15 minutes. After centrifugation (1500 rpm, 3 minutes), the supernatant was removed, and the cells were resuspended in 50 μl of antibody master mix and reacted in a light-blocked state. The above antibody master mix is ​​anti-CD3-BV510 (Biolegend, 344828, 1:50), anti-CD4-AF700 (Biolegend, 344622, 1:50), anti-CD8-FTIC (Biolegend, 980908, 1:50), anti-CD56-BV421 (Biolegend, 318328, 1:50), anti-IL-10-PE (Miltenyi Biotec, 130-112-728, 1:50), Live / Dead-A750 (ebioscience, 1:50) diluted in PBS buffer. The obtained reactant was centrifuged (1500 rpm, 3 min), the supernatant was removed, and the resuspended in 100 μl of PBS was used to measure the fluorescence value using a flow cytometer (CytoFLEX, Beckman Coulter), and the ratio of cells with PE (Phycoerythrin) color was determined. The ratio of cells with the PE color represents the ratio of cells to which IL-10 was bound (transported). The data were analyzed using Flowjo software (BD Biosciences) and graphed using GraphPad Prism 9 software (Dotmatics).

[0311] The results obtained above (the proportion of cells bound (transported) with IL-10) are shown in Fig. 6. As shown in Fig. 6, IL-10 binding increases up to a concentration of 6.87 nM of the MMB0-IL-10 fusion protein (MMB1), but slightly decreases at 68.7 nM. This suggests that the MMB0-IL-10 fusion protein provided herein can deliver IL-10 to effector T cells and NK cells in proportion to the concentration.

[0312]

[0313] Example 8. Suppressive function (in vitro)

[0314] 8.1. Inhibition of pro-inflammatory cytokine production

[0315] In this example, the inhibitory effect of the MMB0-IL-10 fusion proteins provided herein on the production of pro-inflammatory cytokines was tested.

[0316] PBMC from healthy human donors (STEMCELL Technologies; 2x10 5 Cells / well) were stimulated for 72 hours with anti-CD3 antibody (BD, 1 ug / ml), anti-CD28 antibody (BD, 0.00032 ug / ml), and drugs. Here, the drugs refer to the MMB0-IL-10 fusion proteins MMB3, MMB5, and their parent antibody MMB0. Each drug was stimulated at a concentration of 68.7 nM at 37°C. Afterwards, the supernatant was used to measure pro-inflammatory cytokines TNF-α and Granzyme B using LegendPlex. For comparison, the same test was performed using anti-CD3 antibody / anti-CD28 antibody without the above drug treatment (denoted as 'anti-CD3 / CD28').

[0317] LegendPlex is LEGENDplex TM Buffer set A(Biolegend, 740368), LEGENDplex TM Human CD8 / NK Panel Standard (Biolegend, 740281), LEGENDplex TM Human CD8 / NK Panel Detection Antibodies(Biolegend, 740282), LEGENDplex TM Human TNF-α Capture Bead A10 (Biolegend, 740273), LEGENDplex TM Human Granzyme B Capture Bead B6 (Biolegend, 740278) was used according to the manufacturer's manual.

[0318] More specifically, the supernatant was diluted 1:1 with assay buffer (Invitrogen), mixed with beads, and diluted in a shaker at 8000 rpm for 2 hours at room temperature in a light-protected state. After centrifugation (1050 rpm, 5 min), the supernatant was completely removed, wash buffer was added, and incubated at RT (room temperature) for 1 minute. After centrifugation (1050 rpm, 5 min), the supernatant was removed. Afterwards, the detection antibody was added and shaken at room temperature for 1 hour in a light-protected state. Afterwards, SA-PE (Streptavidin-Phycoerythrin Conjugate) was added and shaken at room temperature for 30 minutes in a light-protected state. Afterwards, centrifugation (1050 rpm, 5 min) was performed, and the supernatant was removed. After resuspending with 150 μl of wash buffer, TNF-α and Granzyme B were quantified by measuring fluorescence using LegendPlex.

[0319] The results obtained above are shown in Fig. 7. As shown in Fig. 7, all tested MMB0-IL-10 fusion proteins reduced the pro-inflammatory cytokines TNF-α and Granzyme (GZMB) increased by anti-CD3 / CD28 stimulation. These results suggest that the MMB0-IL-10 fusion protein provided herein maintains the anti-inflammatory effect of IL-10.

[0320]

[0321] 8.2. Inhibition of T cell proliferation

[0322] In this example, the T cell proliferation inhibitory effect of the MMB0-IL-10 fusion proteins provided herein was tested.

[0323] 2x10 healthy human donor PBMCs ((STEMCELL Technologies) labeled with CellTrace Violet (Invitrogen, C34557) 6Cells were stimulated for 72 hours with anti-CD3 antibody (BD, 0.1 ug / ml), anti-CD28 antibody (BD, 0.00032 ug / ml) and drugs at a concentration of 10 cells / ml. Here, the drugs refer to the MMB0-IL-10 fusion proteins MMB3, MMB5 and their parent antibody MMB0, and each drug was treated at a concentration of 68.7 nM. Afterwards, the plate was centrifuged (400 g, 3 min, room temperature) and the supernatant was removed. The cells were incubated with Viability Dye eFluor 780 (1 / 1000) diluted in PBS for 10 minutes at room temperature in a light-protected state. The supernatant was removed after centrifugation (400 g, 3 min, room temperature). The obtained cells were permeabilized with FOXP3 Perm buffer (eBioscience) for 15 minutes at room temperature in a light-protected state. After centrifugation (400g, 3min, room temperature), the supernatant was removed and stained with internal staining antibodies. Internal staining antibodies refer to AF700 anti-CD4 (Biolegend, 344622, 1 / 50), APC anti-Ki67 (Biolegend, 350504, 1 / 50), BV510 anti-CD3 (Biolegend, 344828, 1 / 50), PE anti-CD56 (Biolegend, 318306, 1 / 50), PerCP anti-CD8 (Biolegend, 980916, 1 / 50). After centrifugation to remove the supernatant, 100ul of PBS was added, resuspended, and the fluorescence value was measured using a flow cytometer (CytoFLEX, Beckman Coulter). Data were analyzed using Flowjo software (BD Biosciences) and plotted using GraphPad Prism 9 software (Dotmatics). For comparison, the same experiment was performed using anti-CD3 / anti-CD28 antibodies without the above drug treatment (indicated as 'anti-CD3 / CD28' in Figures 8a-8c).

[0324] The results obtained above are shown in Fig. 8a (CD4 + T cell), 8b (CD8 + As shown in Figures 8a-8c, all tested MMB0-IL-10 fusion proteins induced CD4 cell death by stimulation with anti-CD3 / CD28 antibodies. + T cell, CD8 + It was found that the MMB0-IL-10 fusion protein inhibited the proliferation of T cells and NK cells. On the other hand, the parental antibody (MMB0) was confirmed to have a weak or low statistical significance. This indicates that the inhibition of proliferation of T cells and NK cells is due to IL-10 signaling rather than TIGIT signaling of the MMB0-IL-10 fusion protein. In conclusion, the MMB0-IL-10 fusion proteins provided herein inhibited CD4 cell proliferation due to anti-CD3 / CD28. + T cell, CD8 + It suggests that it can suppress the proliferation effect of T cells and NK cells.

[0325]

[0326] 8.3. Inhibition of pro-inflammatory cytokine production (expression) in PBMCs derived from patients with autoimmune diseases

[0327] 5x10 each of PBMC (STEMCELL Technologies) from patients with autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and ulcerative colitis (UC). 5Cells were seeded at a concentration of 10 cells / well. Cells were stimulated with anti-CD3 antibody (BD, 555336, 1 ug / ml) and anti-CD28 (BD, 555725, 1 ug / ml), and treated with 68.7 nM drugs. Here, drugs refer to MMB0-IL-10 fusion proteins MMB1, MMB3, MMB5 and their parental antibody MMB0, and control anti-TIGIT antibodies A15153G (Biolegend, 372702) and 22G2 (BPS, 71340). After drug treatment and incubation for 4 hours in a 5% CO2 incubator at 37°C, Brefeldin A (Sigma-aldrich, B7651) was treated at a concentration of 10 ug / ml to block cytokine release, and then reacted in a 37°C incubator for 15 hours. In conclusion, cells (PBMCs derived from patients with autoimmune diseases) reacted with drugs and stimulants (anti-CD3 / CD28) for 19 hours. After the reaction was completed, the supernatant was removed by centrifugation (1500 rpm, 5 min, 4℃). Zombie Aqua Fixable dead cell dye solution (Biolegend, 77143) was added for Live / Dead staining, and the reaction was performed for 20 minutes at room temperature in a light-shielded state. After the reaction was completed, the supernatant was removed by centrifugation (1500 rpm, 5 min, 4℃), and 150 µl of surface marker antibody master mix was added and stained at 4℃ for 30 minutes in a light-shielded state. Surface marker antibody master mix is ​​anti-CD3-BV421 (Biolegend, 300434, 1:100), anti-CD4-PerCP-Cy5.5 (Biolegend, 3446081:100), anti-CD8-AF700 (Biolegend, 301028, 1:200), anti-CD56-APC (Biolegend, 362504, 1:100) into FACS buffer (0.This refers to dilution in PBS containing 1% FBS. After staining, centrifuge (1500 rpm, 5 min, 4℃) to remove the supernatant, add 100 ul of Fix / perm buffer (eBioscience, 00-5523-00), and fix the cells at room temperature in a light-shielded state for 40 minutes. After centrifugation (1500 rpm, 5 min, 4℃), remove the supernatant, and stain with intracellular staining antibody at room temperature in a light-shielded state for 30 minutes. Intracellular staining antibody refers to anti-TNF-α-PE-Cy7 (Biolegend, 502930, 1:50) diluted in Perm buffer (eBioscience, 00-5523-00). After staining, the supernatant was removed by centrifugation (1500 rpm, 5 min, 4°C), 200 μl of PBS was added, and the fluorescence value was measured using a flow cytometer (CytoFLEX, Beckman Coulter). The data were analyzed using Flowjo software (BD Biosciences) and graphed using GraphPad Prism 9 software (Dotmatics). For comparison, the same test was performed using anti-CD3 antibody / anti-CD28 antibody without the above drug treatment (indicated as 'Stim' in Figures 9a-9c).

[0328] The results obtained above (TNF-α expressing CD4+ T cell ratio) are shown in Fig. 9a (PBMC derived from SLE patients), 9b (PBMC derived from RA patients), and 9c (PBMC derived from UC patients). As shown in Fig. 9a-9c, the ratio of CD4+ T cells expressing TNF-α, a pro-inflammatory cytokine involved in the pathological symptoms of autoimmune diseases, increased due to anti-CD3 / CD28 antibody stimulation, whereas this increased TNF-α expressing CD4+ T cell ratio was decreased by treatment with all tested MMB0-IL-10 fusion proteins. This effect of reducing TNF-α expressing CD4+ T cells by the MMB0-IL-10 fusion protein was observed in all PBMCs obtained from patients with the tested autoimmune diseases, i.e., systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and ulcerative colitis (UC). In addition, the effect of reducing TNF-α-expressing CD4+ T cells by this MMB0-IL-10 fusion protein is superior to that of A15153G, an antibody known as an anti-TIGIT agonistic function used as a control drug. 22G2, which has an anti-TIGIT antagonistic function used as a control drug, can be seen to increase CD4+ T cells expressing TNF-α. Therefore, it can be seen that the MMB0-IL-10 fusion protein provided herein has an excellent anti-inflammatory effect by reducing CD4+ T cells that produce TNF-α, a pro-inflammatory cytokine, in patients with various autoimmune diseases.

[0329]

[0330] Example 9. Anti-inflammatory effect (in vivo)

[0331] In this example, the anti-inflammatory effect of the MMB0-IL-10 fusion protein was confirmed by measuring the DAI score in the DSS-induced colitis model.

[0332] More specifically, human TIGIT KI balb / c mice were administered drugs intraperitoneally or orally to intestinal inflammation induced with 3% DSS (Dextran sulfate sodium) and observed for 11 days. Here, the drugs refer to 5 mg / kg of MMB0-IL-10 fusion protein MMB5, 40 mg / kg of Cyclosporine A (MCE, 34526), ​​a drug mainly used for inflammatory bowel disease (IBD), and 0.2 mg / kg of human IL-10 (Peprotech, 200-10) as a control. Cyclosporine A was administered orally, and the other drugs were administered intraperitoneally. The DAI score (Disease activity index score) was measured daily during the experimental period and calculated according to the following formula (DAI score = (Body mass drop fraction + feces property score + fecal occult blood score) / 3).

[0333] The results obtained above are shown in Fig. 10. As shown in Fig. 10, in the DSS-induced colitis animal model experiment, the Soluble human IL-10 administration group did not show an anti-inflammatory effect (reducing DAI score), whereas the MMB0-IL-10 fusion protein (MMB5) reduced the DAI score to a level similar to that of the positive control, Cyclosporine A, and showed an anti-inflammatory effect higher than Cyclosporin A for up to 8 days after administration. These results show that the MMB0-IL-10 fusion protein provided herein has a strong anti-inflammatory effect on inflammatory diseases.

Claims

1. A polypeptide comprising the amino acid sequence of sequence number 1 (CDR-H1), A polypeptide comprising the amino acid sequence of sequence number 2 (CDR-H2), A polypeptide comprising the amino acid sequence of sequence number 3 (CDR-H3), A polypeptide comprising the amino acid sequence of sequence number 4 (CDR-L1), A polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and A polypeptide comprising the amino acid sequence of sequence number 6 (CDR-L3) An anti-TIGIT antibody or an antigen-binding fragment thereof, comprising:

2. In paragraph 1, A heavy chain variable region comprising an amino acid sequence selected from among SEQ ID NO: 7 and SEQ ID NO: 29 to 34, and A light chain variable region comprising an amino acid sequence selected from among SEQ ID NO: 8 and SEQ ID NOs: 35 to 41 An anti-TIGIT antibody or an antigen-binding fragment thereof, comprising:

3. In the first paragraph, the anti-TIGIT antibody is an animal antibody, a chimeric antibody, or a humanized antibody, an anti-TIGIT antibody or an antigen-binding fragment thereof.

4. In the first paragraph, the antigen-binding fragment is an anti-TIGIT antibody or an antigen-binding fragment thereof, which is scFv, scFv-Fc, (scFv)2, Fab, Fab', or F(ab')2 of the anti-TIGIT antibody. 5.(1) Anti-TIGIT antibody or antigen-binding fragment thereof and (2) Interleukin-10 (IL-10) A fusion protein containing .

6. In paragraph 5, the anti-TIGIT antibody or antigen-binding fragment thereof, A polypeptide comprising the amino acid sequence of sequence number 1 (CDR-H1), A polypeptide comprising the amino acid sequence of sequence number 2 (CDR-H2), A polypeptide comprising the amino acid sequence of sequence number 3 (CDR-H3), A polypeptide comprising the amino acid sequence of sequence number 4 (CDR-L1), A polypeptide comprising the amino acid sequence of SEQ ID NO: 5 (CDR-L2), and A polypeptide comprising the amino acid sequence of sequence number 6 (CDR-L3) A fusion protein comprising:

7. In paragraph 5, the anti-TIGIT antibody or antigen-binding fragment thereof, A heavy chain variable region comprising an amino acid sequence selected from among SEQ ID NO: 7 and SEQ ID NO: 29 to 34, and A light chain variable region comprising an amino acid sequence selected from among SEQ ID NO: 8 and SEQ ID NOs: 35 to 41 A fusion protein comprising:

8. In the fifth paragraph, the IL-10 is a fusion protein that is a monomer or a dimer.

9. A fusion protein according to claim 5, which does not contain a peptide linker between the anti-TIGIT antibody or antigen-binding fragment thereof and interleukin-10.

10. A fusion protein according to claim 5, comprising a peptide linker between the anti-TIGIT antibody or antigen-binding fragment thereof and interleukin-10.

11. In the 10th paragraph, the peptide linker is expressed as [EAAAK]n or [(G)mS]l, The above n is an integer from 1 to 5 as the number of [EAAAK], The above m is an integer from 1 to 5 as the number of G, and l is an integer from 1 to 5 as the number of [(G)mS]. fusion protein.

12. A nucleic acid molecule encoding an anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4 or a fusion protein according to any one of claims 5 to 11.

13. A recombinant vector comprising the nucleic acid molecule of paragraph 12.

14. A recombinant cell comprising the nucleic acid molecule of paragraph 12 or a recombinant vector comprising the same.

15. A pharmaceutical composition for preventing or treating an immune-related disease, comprising an anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4 or a fusion protein according to any one of claims 5 to 11.

16. A pharmaceutical composition for preventing or treating an immune-related disease, wherein the immune-related disease in paragraph 15 is an autoimmune disease or an inflammatory disease.

17. A pharmaceutical composition for preventing or treating an immune-related disease, wherein the autoimmune disease in claim 16 is rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, Behcet's syndrome, lupus, Sjogren's syndrome, myasthenia gravis, scleroderma, hypothyroidism, hyperthyroidism, psoriasis, vitiligo, multiple sclerosis, autoimmune hepatitis, autoimmune nephritis, autoimmune pancreatitis, autoimmune encephalitis, or cytokine storm.

18. A pharmaceutical composition for preventing or treating an immune-related disease, wherein the inflammatory disease is inflammation or a disease caused by inflammation, in accordance with paragraph 16.

19. An immunosuppressive pharmaceutical composition comprising an anti-TIGIT antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4 or a fusion protein according to any one of claims 5 to 11.

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