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

Abstract

Provided are an anti-TIGIT antibody or 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

Technical Field

[0001] Cross-references to related applications

[0002] This application claims the benefit and priority of Korean Patent Application No. 10-2024-0011242, filed on January 24, 2024, the entire contents of which are incorporated herein by reference.

[0003] This invention relates to anti-TIGIT antibodies that specifically bind to TIGIT or antigen-binding fragments thereof, fusion proteins comprising antibodies or antigen-binding fragments thereof and interleukin-10 (IL-10), and the use of said antibodies or antigen-binding fragments thereof and / or said fusion proteins. Background Technology

[0004] Autoimmune and anti-inflammatory immunotherapy are methods of treating autoimmune and inflammatory conditions by altering the body's inappropriate immune response to alleviate related pathological conditions. Autoimmune immunotherapy involves suppressing mechanisms of the immune system to reduce inappropriate recognition or response to healthy tissues. 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 responsible for autoimmune and inflammation-related pathologies.

[0005] Representative examples of the aforementioned effector cells include T cells and NK cells.

[0006] Meanwhile, TIGIT (T cell immunoglobulin and ITIM (immunoreceptor tyrosine repressor motif) domains) are also known as WUCAM, VSIG9, or Vstm3, and are co-inhibitory receptors preferentially expressed in 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.

[0007] TIGIT expression is increased in lymphocyte-infiltrating tissues within disease settings such as autoimmunity, inflammation, and infection. Compared to TIGIT-negative cells, TIGIT expression can serve as a marker for activated T cells with greater effector function. Furthermore, Treg cells expressing TIMIT exhibit enhanced immunosuppressive activity compared to the TIMIT-negative Treg population.

[0008] Based on this, drugs targeting TIGIT can selectively act on effector T cells and NK cells. Summary of the Invention

[0009] [Technical Issues]

[0010] In one aspect, anti-TIGIT antibodies or antigen-binding fragments thereof that specifically recognize TIGIT and / or specifically bind to TIGIT are provided.

[0011] On the other hand, it provides nucleic acid molecules encoding anti-TIGIT antibodies or their antigen-binding fragments.

[0012] On the other hand, a recombinant vector carrying a nucleic acid molecule encoding the anti-TIGIT antibody or its antigen-binding fragment is provided.

[0013] On the other hand, recombinant cells containing the nucleic acid molecule or the recombinant vector were provided.

[0014] On the other hand, a composition for delivering a bioactive substance (also referred to as a delivery carrier for a bioactive substance or a delivery system for a bioactive substance) is provided, comprising at least one selected from the group consisting of an anti-TIGIT antibody or its antigen-binding fragment as described above, a nucleic acid molecule, a recombinant vector, and recombinant cells. The composition for delivering a bioactive substance may further comprise the bioactive substance to be delivered, in which case the bioactive substance may be conjugated to the antibody or its antigen-binding fragment, but is not limited thereto.

[0015] On the other hand, a method for preparing a composition for delivering a bioactive substance is provided, the method comprising the step of conjugating the bioactive substance to an anti-TIGIT antibody or an antigen-binding fragment thereof. This conjugation can be recombinant, chemical, or physical, but is not limited thereto.

[0016] On the other hand, a method for delivering a bioactive substance is provided, comprising the step of applying a composition for delivering the bioactive substance to a subject who requires delivery of the bioactive substance.

[0017] On the other hand, it provides the use of at least one selected from the group consisting of anti-TIGIT antibody or its antigen-binding fragment, nucleic acid molecule, recombinant vector and recombinant cell for delivering a bioactive substance and / or preparing a composition for delivering a bioactive substance.

[0018] On the other hand, a composition for detecting TIGIT is provided, comprising an anti-TIGIT antibody or an antigen-binding fragment thereof.

[0019] On the other hand, a method for detecting TIGIT is provided, comprising: contacting an anti-TIGIT antibody or an antigen-binding fragment thereof with a biological sample; and determining whether an antigen-antibody reaction (binding) has occurred.

[0020] On the other hand, an anti-TIGIT antibody or its antigen-binding fragment is provided for use in detecting TIGIT.

[0021] On the other hand, a composition for diagnosing diseases associated with TIGIT expression or overexpression is provided, comprising an anti-TIGIT antibody or an antigen-binding fragment thereof.

[0022] On the other hand, a method for diagnosing diseases associated with TIGIT expression or overexpression, or a method for providing information for diagnosing diseases, is provided, comprising: contacting an anti-TIGIT antibody or an antigen-binding fragment thereof with a biological sample; and determining whether an antigen-antibody reaction (binding) has occurred in the sample.

[0023] On the other hand, it provides the use of anti-TIGIT antibodies or their antigen-binding fragments for the diagnosis of diseases associated with TIGIT expression or overexpression.

[0024] On the other hand, a fusion protein is provided comprising: (1) an anti-TIGIT antibody or an antigen-binding fragment thereof; and (2) IL-10.

[0025] In the fusion protein according to the embodiments, IL-10 can be linked, either via a linker or directly without a linker, 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 its antigen-binding fragment.

[0026] On the other hand, a nucleic acid molecule encoding the fusion protein is provided.

[0027] On the other hand, a recombinant vector comprising a nucleic acid molecule encoding the fusion protein is provided.

[0028] On the other hand, a recombinant cell comprising the nucleic acid molecule or the recombinant vector is provided.

[0029] On the other hand, an immunosuppressant or pharmaceutical composition for immunosuppression is provided, comprising at least one selected from the group consisting of: an antibody or an antigen-binding fragment thereof, a fusion protein, a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof or a fusion protein, a recombinant vector comprising a nucleic acid molecule, and a recombinant cell comprising a nucleic acid molecule or a recombinant vector.

[0030] On the other hand, a pharmaceutical composition for the prevention and / or treatment of immune-related diseases is provided, comprising at least one active ingredient selected from the group consisting of: an antibody or an antigen-binding fragment thereof, a fusion protein, a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof or a fusion protein, a recombinant vector comprising a nucleic acid molecule, and a recombinant cell comprising a nucleic acid molecule or a recombinant vector.

[0031] On the other hand, a pharmaceutical composition for the prevention and / or treatment of autoimmune diseases and / or inflammatory diseases is provided, comprising at least one selected from the group consisting of: an antibody or an antigen-binding fragment thereof, a fusion protein, a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof or a fusion protein, a recombinant vector comprising a nucleic acid molecule, and a recombinant cell comprising a nucleic acid molecule or a recombinant vector.

[0032] On the other hand, a method for suppressing immunity is provided, comprising the step of administering to a subject requiring immunosuppression a pharmaceutically effective amount selected from at least one of the following: an antibody or an antigen-binding fragment thereof, a fusion protein, a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof or a fusion protein, a recombinant vector containing a nucleic acid molecule, and a recombinant cell containing a nucleic acid molecule or a recombinant vector.

[0033] On the other hand, a method for preventing and / or treating immune-related diseases is provided, the method comprising administering to a subject requiring prevention and / or treatment of an immune-related disease a pharmaceutically effective amount selected from at least one of the following: an antibody or an antigen-binding fragment thereof, a fusion protein, a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof or a fusion protein, a recombinant vector comprising a nucleic acid molecule, and a recombinant cell comprising a nucleic acid molecule or a recombinant vector.

[0034] On the other hand, a method for preventing and / or treating autoimmune diseases and / or inflammatory diseases is provided, the method comprising administering to a subject requiring prevention and / or treatment of an autoimmune disease and / or inflammatory disease a pharmaceutically effective amount selected from at least one of the following: an antibody or an antigen-binding fragment thereof, a fusion protein, a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof or a fusion protein, a recombinant vector comprising a nucleic acid molecule, and a recombinant cell comprising a nucleic acid molecule or a recombinant vector.

[0035] On the other hand, it provides the use of at least one selected from the group consisting of an antibody or an antigen-binding fragment thereof, a fusion protein, a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof or a fusion protein thereof, a recombinant vector containing a nucleic acid molecule, and a recombinant cell containing a nucleic acid molecule or a recombinant vector for the purpose of suppressing immunity or for the purpose of manufacturing an immunosuppressant.

[0036] On the other hand, it provides the use of at least one selected from the group consisting of an antibody or an antigen-binding fragment thereof, a fusion protein, a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof or a fusion protein thereof, a recombinant vector containing a nucleic acid molecule, and a recombinant cell containing a nucleic acid molecule or a recombinant vector for the prevention and / or treatment of immune-related diseases or for the manufacture of a medicament for the prevention and / or treatment of immune-related diseases.

[0037] On the other hand, it provides the use of at least one selected from the group consisting of an antibody or an antigen-binding fragment thereof, a fusion protein, a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof or a fusion protein thereof, a recombinant vector comprising a nucleic acid molecule, and a recombinant cell comprising a nucleic acid molecule or a recombinant vector for the prevention and / or treatment of autoimmune diseases and / or inflammatory diseases, or for the manufacture of a medicament for the prevention and / or treatment of autoimmune diseases and / or inflammatory diseases.

[0038] [Technical Solution]

[0039] This application provides an anti-TIGIT antibody that binds to TIGIT or its antigen-binding fragment; a fusion protein comprising (1) an anti-TIGIT antibody or its antigen-binding fragment and (2) IL-10; and its pharmaceutical use. The anti-TIGIT antibody or its antigen-binding fragment has excellent binding affinity to TIGIT, and therefore it can have the activity of delivering other useful substances (e.g., IL-10) to sites where TIGIT is present, such as cells that express (overexpress) TIGIT on their surfaces. The fusion protein has the advantage of being usable as a variety of immunosuppressants and / or immunotherapeutic agents because (1) it suppresses immunity by targeting T cells and NK cells that are activated by reasons such as increased TIGIT expression via the anti-TIGIT antibody or its antigen-binding fragment, and (2) it has excellent binding affinity to the receptor and low immunogenicity via IL-10.

[0040] The following is a detailed description of this disclosure.

[0041] Definition of terminology

[0042] As used herein, the phrases “contain a specific nucleic acid sequence or amino acid sequence,” “compose of a specific nucleic acid sequence or amino acid sequence,” or “represented by a specific nucleic acid sequence or amino acid sequence” mean that the nucleic acid molecule or polypeptide substantially comprises a specific nucleic acid sequence or amino acid sequence and can be interpreted as containing “substantially equivalent sequences” (or not excluding such variations of the specific nucleic acid sequence or amino acid sequence) with variations (e.g., deletions, substitutions, modifications, and / or additions) within the specific nucleic acid sequence or amino acid sequence, as long as the original function and / or intended function of the nucleic acid molecule or polypeptide is maintained.

[0043] In embodiments, the nucleic acid sequences or amino acid sequences provided herein may comprise mutants obtained through conventional mutagenesis methods (e.g., directed evolution or site-directed mutagenesis), provided that the mutants retain the original or desired function of the sequence. In embodiments, when a nucleic acid molecule or polypeptide “contains or is composed of a specific nucleic acid sequence or amino acid sequence”, this means that the nucleic acid molecule or polypeptide (i) substantially contains the specific nucleic acid sequence or amino acid sequence, or (ii) substantially consists of or substantially contains the following amino acid sequences that are amino acid sequences having 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 91% or higher, 92% or higher, 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.5% or higher, or 99.9% or higher homology to the specific nucleic acid sequence or amino acid sequence, while retaining its original and / or desired function.

[0044] The nucleic acid sequences described herein can be modified in various ways within the coding region to reflect the codon preferences of the microorganisms used for protein expression, as long as the amino acid sequence and / or function of the expressed protein remain unchanged due to codon degeneracy.

[0045] As used herein, the term "homology" refers to the degree of similarity between a given nucleic acid sequence or amino acid sequence and another sequence, and can be expressed as a percentage (%). The homology of nucleic acid sequences can be determined using well-known algorithms such as BLAST or FASTA, and programs such as BLASTN or BLASTX have been developed based on the BLAST algorithm (see: http: / / www.ncbi.nlm.nih.gov).

[0046] Anti-TIGIT antibody or its antigen-binding fragment

[0047] One embodiment of this application provides an anti-TIGIT antibody or its antigen-binding fragment, which has the activity of specifically recognizing TIGIT and / or specifically binding TIGIT.

[0048] Anti-TIGIT antibodies or their antigen-binding fragments may include:

[0049] A polypeptide (CDR-H1) containing the amino acid sequence of SEQ ID NO:1.

[0050] A polypeptide (CDR-H2) containing the amino acid sequence of SEQ ID NO:2.

[0051] A polypeptide (CDR-H3) containing the amino acid sequence of SEQ ID NO:3.

[0052] A polypeptide (CDR-L1) containing the amino acid sequence of SEQ ID NO:4.

[0053] A polypeptide (CDR-L2) containing the amino acid sequence of SEQ ID NO:5, and

[0054] A polypeptide (CDR-L3) containing the amino acid sequence of SEQ ID NO:6.

[0055] In this paper, the complementarity determination region (CDR) is determined based on the CDR definition of the Kabat system.

[0056] In this implementation, the six CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) that may be included in the anti-TIGIT antibody or its antigen-binding fragment are summarized in Table 1 below:

[0057] [Table 1]

[0058]

[0059] (In Table 1, CDR-H1, CDR-H2, and CDR-H3 represent complementarity-determining regions on the heavy chain, while CDR-L1, CDR-L2, and CDR-L3 represent complementarity-determining regions on the light chain.)

[0060] In this embodiment, the anti-TIGIT antibody or its antigen-binding fragment may include:

[0061] The heavy chain variable region includes CDR-H1 of SEQ ID NO: 1, CDR-H2 of SEQ ID NO: 2, and CDR-H3 of SEQ ID NO: 3; and

[0062] The light chain variable region includes CDR-L1 of SEQ ID NO: 4, CDR-L2 of SEQ ID NO: 5, and CDR-L3 of SEQ ID NO: 6.

[0063] More specifically, an anti-TIGIT antibody or its antigen-binding fragment may include:

[0064] The heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:29 to 34; and

[0065] The light chain variable region contains an amino acid sequence selected from SEQ ID NO:8 and SEQ ID NO:35 to 41.

[0066] In some cases (e.g., when recombinant construction), the heavy chain variable region and / or the light chain variable region may also contain a suitable signal sequence at their N-terminus.

[0067] Table 2 below shows the amino acid sequences of the heavy chain variable region and light chain variable region that can be included in anti-TIGIT antibodies or their antigen-binding fragments:

[0068] [Table 2]

[0069]

[0070]

[0071] (In Table 2, the underlined areas represent CDR1, CDR2, and CDR3 for the heavy and light chains, respectively.)

[0072] In one instance, the anti-TIGIT antibody or its antigen-binding fragment provided herein is used to (bind / recognize) TIGIT protein, such as human TIGIT protein (NCBI reference sequence NP_776160.2; UniProtKB / SwissProt Q495A1-1), and can bind one or more amino acids (e.g., in consecutive positions) within the protein, but is not limited thereto.

[0073] [Human TIGIT protein (UniProtKB / SwissProt Q495A1-1)]

[0074] 1 MRWCLLLIWA QGLRQAPLAS GMMTGTIETT GNISAEKGGS IILQCHLSST TAQVTQVNWE

[0075] 61 QQDQLLAICN ADLGWHISPS FKDRVAPGPG LGLTLQSLTV NDTGEYFCIY HTYPDGTYTG

[0076] 121 RIFLEVLESS VAEHGARFQI PLLGAMAATL VVICTAVIVV VALTRKKKAL RIHSVEGDLR

[0077] 181 RKSAGQEEWS PSAPSPPGSC VQAEAAPAGL CGEQRGEDCA ELHDYFNVLS YRSLGNCSFF

[0078] 241 TETG (SEQ ID NO: 42)

[0079] As used herein, the term "antibody" refers to a protein that specifically binds to a particular antigen. This protein may be produced in response to antigenic stimulation within the immune system, or it may be chemically synthesized or recombinantly produced; there are no restrictions on its type. Antibodies can be non-naturally occurring, such as recombinant or synthetically produced antibodies. Antibodies can be animal antibodies (e.g., mouse antibodies), chimeric antibodies, humanized antibodies, or human antibodies. Antibodies can be monoclonal or polyclonal antibodies.

[0080] In the anti-TIGIT antibody or its antigen-binding fragment provided herein, regions other than the defined heavy chain CDR regions and light chain CDR regions, or heavy chain variable regions and light chain variable regions, can be derived from any subtype of immunoglobulin (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, etc.). For example, the remaining regions can be derived from the framework region of any immunoglobulin subtype and / or from the constant regions of the light chain and / or heavy chain. In embodiments, the anti-TIGIT antibody provided herein can be a human IgG-type antibody, for example, in the form of IgG1, IgG2, IgG3, or IgG4, but is not limited thereto.

[0081] Intact antibodies (e.g., IgG type) have a structure consisting of two full-length light chains and two full-length heavy chains, each light chain linked to the heavy chain by a disulfide bond. The constant regions of an antibody are divided into heavy chain constant regions and light chain constant regions. Heavy chain constant regions have gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses including gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha1 (α1), and alpha 2 (α2). Light chain constant regions have kappa (κ) and lambda (λ) types.

[0082] In the implementation of this invention, the anti-TIGIT antibody may include, but is not limited to, the IgG constant region as the heavy chain constant region and the κ constant region as the light chain constant region.

[0083] In one embodiment, the constant region of IgG (e.g., human IgG1) can be wild-type. In another embodiment, the constant region of IgG can be a variant comprising one or more mutations selected from the group consisting of S240D (serine at position 240 is replaced by aspartic acid), A331L, I333E, N298A, S299A, E334A, K335A, L235A, L236A, P330G, and K478 deletion (ΔK478), and can include, for example, the following mutations:

[0084] (1) S240D, A331L and I333E;

[0085] (2) N298A;

[0086] (3) S299A, E334A and K335A;

[0087] (4) L235A, L236A and P330G; or

[0088] (5) K478 is missing.

[0089] The term "heavy chain" is defined as including the full-length heavy chain and its fragments, which contains a variable region domain (VH) containing an amino acid sequence sufficient to confer antigen specificity, three constant region domains CH1, CH2, and CH3, and a hinge. The term "light chain" is defined as including the full-length light chain and its fragments, which contains a variable region domain VL and a constant region domain CL containing an amino acid sequence sufficient to confer antigen specificity.

[0090] The term "complementarity-determining region" (CDR) refers to an antibody region that confers antigen-binding specificity, comprising the amino acid sequences of both the heavy and light chain hypervariable regions of an immunoglobulin. Each heavy and light chain may contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). These CDRs provide key contact residues for antibody-antigen or epitope binding. As used herein, the terms "specific binding" or "specific recognition" refer to the interaction between an antigen and antibody that elicits an immune response, as is well known in the art.

[0091] The Complementarity Determinant Region (CDR) described in this paper is defined based on the Kabat system.

[0092] Unless otherwise stated, the term “antibody” as used herein may be understood to include antigen-binding fragments of antibodies that retain the ability to bind antigens.

[0093] The term “antigen-binding fragment” refers to any form of polypeptide containing a portion capable of binding an antigen (e.g., the six CDRs defined herein). Examples include, but are not limited to, scFv, scFv-Fc, (scFv)2, Fab, Fab', or F(ab')2.

[0094] In the antigen-binding fragment, Fab has a structure comprising a variable region of light and heavy chains, a constant region of light chains, and a first constant region (CH1) of heavy chains, and has a single antigen-binding site.

[0095] The difference between Fab' and Fab is that the former includes a hinge region containing one or more cysteine ​​residues at the C-terminus of the CH1 domain of the heavy chain.

[0096] When cysteine ​​residues in the hinge region of Fab' form disulfide bonds, F(ab')2 antibody is generated. Fv is the smallest antibody fragment, containing only the variable regions of the heavy and light chains, and recombinant techniques for generating Fv fragments are well known in the art.

[0097] Double-chain Fv contains variable regions of the heavy chain and the light chain, which are non-covalently bonded together. Single-chain Fv (scFv) contains variable regions of the light chain and the heavy chain. The two can usually be covalently bonded together through peptide linkers or directly at the C-terminus to form a dimer structure similar to double-chain Fv.

[0098] Antigen-binding fragments can be obtained using proteolytic enzymes (e.g., Fab can be obtained by restrictive digestion of the whole antibody with papain, while the F(ab')2 fragment can be obtained by digestion with pepsin), or can be produced using recombinant DNA technology.

[0099] The term "hinge region" refers to the region located between the CH1 and CH2 domains within the heavy chain of an antibody, which functions to provide flexibility for the antigen-binding site within the antibody.

[0100] The antibodies described herein may be monoclonal antibodies. Monoclonal antibodies can be produced using methods well known in the art. For example, they can be produced using phage display technology, or they can be generated using conventional methods as monoclonal antibodies derived from animals (e.g., mice).

[0101] Individual monoclonal antibodies can be screened using standard ELISA (enzyme-linked immunosorbent assay) based on their ability to bind to the receptor-binding domain of TIGIT. Functional assays such as competitive ELISA or cell-based assays can be used to assess molecular interactions and inhibitory activity. The affinity (Kd value) of each monoclonal antibody for the TIGIT receptor-binding domain can then be evaluated based on the selected antibody with strong inhibitory activity.

[0102] The ultimately selected antibody can be used not only to manufacture and serve as an antibody whose region, excluding the antigen-binding site, has been converted into a human immunoglobulin antibody, but also as a humanized antibody. Methods for preparing humanized antibodies are well known in the art.

[0103] The antigen-binding fragment of the anti-TIGIT antibody described herein refers to a fragment derived from the anti-TIGIT antibody that maintains its affinity for binding to the antigen (TIGIT). It can be any polypeptide containing one of the six CDRs of the aforementioned anti-TIGIT antibody, such as scFv, scFv-Fc, scFv-Ck (κ constant region), scFv-Cλ (λ constant region), (scFv)2, Fab, Fab', or F(ab')2, but is not limited thereto. In embodiments, the antigen-binding fragment can be scFv or a fusion polypeptide, wherein the scFv is fused to the Fc region (scFv-Fc) of an immunoglobulin (e.g., IgG1, IgG2, IgG3, IgG4, etc.) or the constant region (scFv-Ck or scFv-Cλ) of a light chain (e.g., κ or λ), but is not limited thereto.

[0104] As used herein, the phrase “contains a specific amino acid sequence” or “represents a specific amino acid sequence or is composed of a specific amino acid sequence” for antibodies (e.g., CDRs, variable regions or heavy / light chains, antigen-binding fragments, etc.) covers not only cases where antibodies must include a specific amino acid sequence, but also cases where modifications (e.g., substitution, deletion, and / or addition of amino acid residues) that do not significantly affect antibody activity (e.g., antigen affinity, pharmacological activity, etc.) are introduced into the amino acid sequence.

[0105] The anti-TIGIT antibodies or antigen-binding fragments provided herein can have binding affinity (KD) for TIGIT (e.g., human TIGIT) as measured by surface plasmon resonance (SPR), such as 10 mM or lower, 5 mM or lower, 1 mM or lower, 0.5 mM or lower, 0.2 mM or lower, 0.1 mM or lower, 0.05 mM or lower, 0.01 mM or lower, 0.005 mM or lower, or 0.001 mM or lower. Specifically, KD can be in the following ranges: 0.0001nM to 10mM, 0.0005nM to 10mM, 0.001nM to 10mM, 0.005nM to 10mM, 0.01nM to 10mM, 0.05nM to 10mM, 0.1nM to 10mM, 0.5nM to 10mM, 1nM to 10mM, 0.0001nM to 5mM, 0.0005nM to 5mM, 0.001nM to 5mM, 0.005nM to 5mM, 0.01nM to 5mM, 0.05nM to 5mM, 0.05nM to 5mM, 0.05nM to 5mM, 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 0.05 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, 0.01 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 0.0.05mM, 0.0001nM to 0.01mM, 0.0005nM to 0.01mM, 0.001nM to 0.01mM, 0.001nM to 0.01mM, 0.005nM to 0.01mM, 0.005nM to 0.01mM, which can be 0.01mM, 0.01nM to 0.01mM, 0.05nM to 0.01mM, 0.1nM to 0.01mM, 0.5nM to 0.01mM, or 1nM to 0.01mM, but are not limited to these values.

[0106] Another embodiment of this application provides a nucleic acid molecule encoding an anti-TIGIT antibody or an antigen-binding fragment thereof.

[0107] Another implementation provides a recombinant vector comprising a nucleic acid molecule encoding an anti-TIGIT antibody or an antigen-binding fragment thereof.

[0108] Another implementation provides a recombinant cell containing nucleic acid molecules or a recombinant vector.

[0109] Anti-TIGIT antibodies or their antigen-binding fragments possess excellent binding affinity for TIGIT and do not inhibit interaction (binding) with proteins that interact with TIGIT (e.g., CD155). Due to this characteristic, anti-TIGIT antibodies or their antigen-binding fragments can bind (fusion / conjugate) to bioactive substances (e.g., IL-10) with desired effects and have the activity of efficiently delivering the bioactive substance to sites where TIGIT is present, such as cells (e.g., immune cells, such as T cells or NK (natural killer) cells) that express (overexpress) TIGIT on their surface, without affecting the function / activity / effect of TIGIT.

[0110] Therefore, another embodiment of this application provides a composition for delivering a bioactive substance (a delivery carrier for a bioactive substance or a delivery system for a bioactive substance), comprising one or more selected from the group consisting of antibodies or their antigen-binding fragments, nucleic acid molecules, recombinant vectors, and recombinant cells. The composition for delivering a bioactive substance may further comprise the bioactive substance to be delivered. In this case, the bioactive substance may be linked to an antibody or antigen-binding fragment (in the form of an antibody-bioactive substance conjugate; for example, an antibody-drug conjugate (ADC), etc.), but is not limited thereto.

[0111] Another embodiment provides a method for preparing a composition for delivering a bioactive substance, comprising the step of binding the bioactive substance to an antibody or antigen-binding fragment. This binding may be recombinant, chemical, or physical, but is not limited thereto.

[0112] Another embodiment provides a method for delivering a bioactive substance, comprising the step of administering the above-described composition for delivering the bioactive substance to a subject who requires delivery of the bioactive substance.

[0113] Other embodiments provide a use for delivering one or more bioactive 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 bioactive substances.

[0114] Bioactive substances can be one or more selected from the group consisting of: all biocompatible substances, such as small molecule compounds, proteins, peptides, nucleic acid molecules (e.g., antisense oligonucleotides, siRNA, shRNA, microRNA, aptamers, etc.); and labeling substances, such as radioactive isotopes, wherein they can exhibit beneficial or desired effects, such as immunosuppression, immune activation, visualization, etc., at the site where the anti-TIGIT antibody or its antigen-binding fragment is delivered, such as on cells that express (overexpress) TIGIT on their surface (e.g., immune cells, such as T cells, NK (natural killer) cells).

[0115] For example, a bioactive substance may be one or more selected from the group consisting of: chemical drugs that have immunosuppressive or immunoactivating effects in immune cells; proteins (e.g., cytokines, chemokines, etc.) that have immunosuppressive or immunoactivating effects in immune cells; ligands that bind to receptors on the surface of immune cells; antibodies that bind to immune-related proteins (receptors on the surface of immune cells, ligands that bind to them, etc.), but are not limited thereto.

[0116] Other embodiments provide a composition for detecting TIGIT, comprising the aforementioned antibody or antigen-binding fragment. Another embodiment provides a method for detecting TIGIT, comprising the steps of: treating a biological sample with the aforementioned anti-TIGIT antibody or its antigen-binding fragment; and identifying whether an antigen-antibody reaction (binding) occurs. In the detection method, if an antigen-antibody reaction is detected, the presence of TIGIT in the biological sample can be determined. Another embodiment provides the use of the aforementioned anti-TIGIT antibody or its antigen-binding fragment for detecting TIGIT. TIGIT detection refers to identifying the presence or expression of TIGIT in a biological sample, or the degree of presence or expression of TIGIT.

[0117] Anti-TIGIT antibodies or antigen-binding fragments thereof may be conjugated to a detectable marker. If the detectable marker is visible, the anti-TIGIT antibody or antigen-binding fragment thereof can be used to visualize TIGIT in a biological sample. In one instance, the detectable marker may be one or more selected from the group consisting of: radioactive isotopes (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 1251, 1311, 177Lu, 166Ho, 153Sm, etc.), enzymes, fluorescent markers, luminescent markers (e.g., bioluminescent markers), magnetic markers, and chemicals such as, but not limited to, biotin.

[0118] Biological samples can be selected from groups of cells (e.g., immune cells, such as T cells and NK cells), tissues, and body fluids (e.g., blood, serum, etc.) obtained from mammals (such as humans, monkeys, primates, mice, rats, etc.) and can be isolated from living organisms.

[0119] Other embodiments provide a composition for diagnosing diseases associated with TIGIT expression or overexpression, comprising the above-described anti-TIGIT antibody or its antigen-binding fragment.

[0120] Another implementation provides a method for diagnosing diseases associated with TIGIT expression or overexpression, or a method for providing information for diagnosing diseases, comprising the steps of: contacting an anti-TIGIT antibody or an antigen-binding fragment thereof with a biological sample and identifying whether an antigen-antibody reaction (binding) occurs within the sample.

[0121] Other embodiments provide the use of an anti-TIGIT antibody or an antigen-binding fragment thereof for the diagnosis of diseases associated with TIGIT expression or overexpression.

[0122] Diseases associated with TIGIT expression or overexpression can be any disease caused by TIGIT expression or overexpression, such as autoimmune diseases. Autoimmune diseases include, but are not limited to, rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, Behcet's syndrome, lupus (systemic lupus erythematosus; SLE), and Sjögren's syndrome, and can be, but are 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.

[0123] In diagnostic methods, biological samples can be selected from groups of cells (e.g., immune cells, such as T cells, NK cells), tissues, and body fluids (e.g., blood, serum, etc.) obtained from mammals (such as primates (including humans and monkeys)) and rodents (including mice and rats) that require diagnosis of diseases associated with TIGIT expression or overexpression, and can be isolated from living organisms.

[0124] The steps for identifying whether an antigen-antibody reaction has occurred can be performed by a variety of methods known in the art. For example, it can be measured by conventional enzyme reactions, fluorescence, luminescence, and / or radiation detection. Specifically, it can be measured by methods selected from, but not limited to, the group consisting of immunochromatography, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay (FIA), luminescent immunoassay (LIA), Western blotting, and microarrays.

[0125] Fusion protein

[0126] In this disclosure, one embodiment 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).

[0127] Anti-TIGIT antibodies or their antigen-binding fragments are as described above.

[0128] Fusion proteins can have the functions of anti-TIGIT antibodies or their antigen-binding fragments (TIGIT recognition / binding, targeting, activation, etc.) and IL-10 functions (immunosuppression, IL-10 receptor (IL-10R) recognition / binding, etc.).

[0129] Interleukin-10 (IL-10) can be selected from:

[0130] (a) A peptide containing IL-10, and

[0131] (b) A polypeptide having 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 homology with polypeptide (a).

[0132] 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 two IL-10Rα (also known as IL-10R1) and two IL-10Rβ (also known as IL-10R2). For example, interleukin-10 can be human, such as NCBI accession number NP_000563.1 (amino acid residues 1-18 are the signal sequence; amino acid residues 19-178 are the maturation region), but is not limited to this.

[0133] In one embodiment, IL-10 can be represented by the following amino acid sequence (from N-terminus to C-terminus):

[0134] SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (wild type: SEQ ID NO: 9)

[0135] The polypeptide (a) including IL-10 can be in the form of a monomer containing one IL-10, or in the form of a polymer in which two or more IL-10s are linked (e.g., a dimer of two IL-10s). When IL-10 is in the form of a polymer (e.g., a dimer), two or more (e.g., two) IL-10 monomers can be linked by a linker or directly linked without a linker. When IL-10 is linked by a linker, the linker (second linker) can be selected from, but is not limited to, universal peptide linkers such as GGGGSGGG (SEQ ID NO: 16).

[0136] The polypeptide (a) may include, but is not limited to, additional oligopeptides for other purposes, such as oligopeptides that facilitate protein purification, such as tags for labeling at the N-terminus (e.g., MHSSALLCCLVLLTGVRA (SEQ ID No: 45) etc.).

[0137] Interleukin-10 is characterized by low immunogenicity when administered to humans due to its human origin.

[0138] IL-10 can inhibit the binding activity of its receptors, such as the IL-10 receptor α chain (IL10RA, CD210, CD210a, CDW210A, HIL-10R, IL-10R1, IL10R, interleukin-10 receptor, α subunit, interleukin-10 receptor α subunit) and / or the IL-10 receptor β chain (IL10RB, CDW210B, CRF2-4, CRFB4, D21S58, D21S66, IL-10R2, interleukin-10 receptor, β subunit, interleukin-10 receptor β subunit) and / or inhibit the production and / or secretion of cytokines in immune cells (e.g., T cells (CD4+, CD8+), NK cells, etc.).

[0139] In one implementation, IL-10 can be selected from the group consisting of:

[0140] (a) (a-1) A polypeptide (monomer) containing one amino acid sequence of SEQ ID NO:9;

[0141] (a-2) A polypeptide (dimer) comprising the two amino acid sequences of SEQ ID NO:9; and

[0142] (b) A polypeptide having 93% or higher, 94% or higher, 95% or higher, 96% or higher, 97% or higher, 98% or higher or 99% or higher homology with polypeptide (a) and inhibiting / inducing binding activity with the IL-10 receptor and / or cytokine production and / or secretion activity in immune cells.

[0143] The polypeptide (a-2) can be, but is not limited to, the two amino acid sequences of SEQ ID NO:9 linked together with or without a peptide linker (e.g., SEQ ID NO:16, etc.).

[0144] IL-10 may have activities such as binding to the 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).

[0145] In fusion proteins, anti-TIGIT antibodies or their antigen-binding fragments can be functionally capable of delivering IL-10 to effector cells (e.g., effector T cells and / or Treg cells) and / or NK cells. Additionally, by maintaining the antibody's native sequence, it is able to bind to Fc receptors. Therefore, anti-TIGIT antibodies can also target TIGIT-negative effector cells important for autoimmunity and / or inflammation, including monocytes and macrophages. IL-10 is a safe peptide with good receptor-binding affinity and low immunogenicity; when administered in vivo, it exhibits excellent immunosuppressive effects with a low risk of being recognized as an immunogen and inducing an immune response. Therefore, fusion proteins can be used as various immunosuppressants and immunotherapeutic agents.

[0146] In the fusion protein according to the embodiments, IL-10 can be a monomer or a dimer, linked via a linker or directly 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 its antigen-binding fragment, either directly or via a linker.

[0147] More specifically, the fusion protein may include: (1) an anti-TIGIT antibody (e.g., an IgG-type antibody comprising two heavy chains and two light chains) and (2) an IL-10, wherein the IL-10 is attached to the C-terminus (constant region C-terminus) or N-terminus of either of the two heavy chains or either of the two light chains of the anti-TIGIT antibody via a peptide linker or, in the absence of a peptide linker, directly.

[0148] In another embodiment, the fusion protein may include: (1) an anti-TIGIT antibody (e.g., an IgG-type antibody comprising two heavy chains and two light chains); and (2) two IL-10 molecules, wherein each IL-10 is attached to the C-terminus or N-terminus of either of the two heavy chains or either of the two light chains of the anti-TIGIT antibody via a linker or directly therewithout a linker.

[0149] For example, a fusion protein may contain:

[0150] (i) One of the two IL-10 molecules can be linked, either via a linker or directly without a linker, to the C-terminus (i.e., the C-terminus of the constant region (e.g., Fc)) or N-terminus (i.e., the N-terminus of the variable region) of one of the two heavy chains of the anti-TIGIT antibody, or via a linker or directly without a linker, or

[0151] (ii) One of the two IL-10 molecules may be linked, either via a linker or directly without a linker, to the C-terminus (i.e., the C-terminus of the constant region) or N-terminus (i.e., the N-terminus of the variable region) of one of the two light chains of the anti-TIGIT antibody, and the other IL-10 molecule may be linked, either via a linker or directly without a linker, to the C-terminus (i.e., the C-terminus of the constant region) or N-terminus (i.e., the N-terminus of the variable region) of the other light chain of the anti-TIGIT antibody.

[0152] More specifically, in fusion proteins:

[0153] (i) One of the two IL-10 molecules can be linked to the C-terminus of one Fc region of the two heavy chains of the anti-TIGIT antibody via a linker or directly without a linker, while the other can be linked to the C-terminus of the other Fc region of the anti-TIGIT antibody via a linker or directly without a linker, or

[0154] (ii) One of the two IL-10 molecules can be linked to the C-terminus of one of the two light chains by a linker or directly without a linker, and the other can be linked to the C-terminus of the other light chain of the anti-TIGIT antibody by a linker or directly without a linker.

[0155] The linker used to connect the antibody and IL-10 can be a rigid peptide linker (e.g., [EAAAK]n, where n is an integer from 1 to 5 representing the number of [EAAAK]), a flexible peptide linker (e.g., [(G)mS]l, where m is an integer from 1 to 5 representing the number of G, and l is an integer from 1 to 5 representing the number of [(G)mS]), but is not limited thereto.

[0156] connector

[0157] The linker (first linker) used to connect the antibody and IL-10 can be a peptide linker, for example, selected from at least one of the following: [EAAAK]n (n is an integer of 1 to 5 representing the number of [EAAAK](SEQ ID NO:77), [(G)mS]l (m is an integer of 1 to 5 representing the number of G residues, and l is an integer of 1 to 5 representing the number of [(G)mS]), A EAAAKA, A EAAAK EAAAK A, A EAAAK EAAAK EAAAK EAAAK A, A EAAAK EAAAK EAAAK EAAAK EAAAKA, A EAAAK EAAAK EAAAK EAAAK A.AEAAAAKEAAAAKAG,AEAAAAKEAAAAKAG,GGGG,GGGGG,GGGGGGGG,GAGAGAGAGA,RPLSYRPPPPPFFPFGFPSVRP,YPRSYIRRRHHPSSLTT,TPSHLSHILPSFGLPTFWN,RPVSPFTFPPRLSNSWLPA,SPAAHPRSIPRPPGPIRT,APGPSAPS HRSLPSRAAFG, PRNSHFLHPLLVAPLGA, MPSLSGVLQVRYLSPPDL, SPQYPSPLTLTLPPHPSL, NPSLNPPSYLHRAPSRIS, LPWRTSLPLPRRPP, PPLFAKGPVGLLSRSSFPP, VPPAPVVSSLRSAHARPPY, LRPTPPRVRSYTCCPP, PNVAHVLPLL TVPWDNLR, CNPLLPLCARSPAVRTFP, LGTPTPTPTTTGEF, EDFTRGKL, L EAAAR EAAAR EAAAR EAAAR EAAAR EAAAR EAAAR, L EAAAR EAAAR EAAAR EAAREAAR, L EAAR, L EAAR, EAAAR EAAAR EAAAR EAAAR, EAAAR EAAAR EAAAR, EAAAR, LTEEQQEGGG, TEEQQEGGG, LAKLKQKTEQLQDRIAGGG, LELKTPLGDT THTCPRCPEP KSCDTPPPCPRCPEPKSCDT PPPCPRCPEP KSCDTPPPCP RCPGG, and LEPKSSDKTHTSPPSPGG, but not limited thereto.For example, the peptide linker can be a rigid linker, such as [EAAAK]n (where n is an integer from 1 to 5 representing the number of [EAAAK], such as SEQ ID No: 14, 46 to 49, etc.), or a flexible linker, such as [(G)mS]l (where m is an integer from 1 to 5 representing the number of G residues, and l is an integer from 1 to 5 representing the number of [(G)mS], such as SEQ ID No: 15, 98 to 101, etc.), but is not limited thereto.

[0158] Nucleic acid molecules, recombinant vectors and recombinant cells

[0159] Another implementation provides a nucleic acid molecule encoding an antibody, antigen-binding fragment, or fusion protein.

[0160] Another implementation provides a recombinant vector carrying nucleic acid molecules. This vector can be used as an expression vector for nucleic acid molecules.

[0161] Another implementation provides a recombinant cell anchored to a nucleic acid molecule or a recombinant vector containing the nucleic acid molecule. The cell can be used to produce fusion proteins.

[0162] Another embodiment provides a method for producing a fusion protein, the method comprising the step of expressing a nucleic acid molecule in a suitable host cell. The step of expressing the nucleic acid molecule in a suitable host cell may include culturing the recombinant cells under standard conditions.

[0163] The term "vector" refers to a tool used to express a target gene in a host cell. Examples of vectors include plasmid vectors, granular vectors, and viral vectors, such as phage vectors, adenovirus vectors, retroviral vectors, and adeno-associated virus vectors. Vectors that can be used as recombinant vectors include plasmids commonly used in the art (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGE series, pET series, and pUC19), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13), or viruses (e.g., SV40).

[0164] In recombinant vectors, nucleic acid molecules encoding fusion proteins can be operatively linked to promoters. The term "operatively linked" refers to a functional connection between a nucleotide regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. When "operatively linked," the regulatory sequence controls the transcription and / or translation of the other nucleotide sequence.

[0165] Recombinant vectors can typically be constructed as cloning vectors or expression vectors. Expression vectors can be any conventional vector used in the art for expressing exogenous proteins in plants, animals, or microorganisms. Recombinant vectors can be constructed using a variety of methods known in the art.

[0166] Recombinant vectors can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector is an expression vector and a prokaryotic cell is used as the host, the vector typically includes a strong promoter capable of driving transcription (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When a eukaryotic cell is used as the host, the vector may include, but is not limited to, eukaryotic origins of replication such as f1 origin, SV40 origin, pMB1 origin, adenovirus origin, AAV origin, and BBV origin. Additionally, the vector may include promoters derived from mammalian genomes (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSVtk promoter), and typically includes a polyadenylated sequence as a transcription termination sequence.

[0167] Recombinant cells can be obtained by introducing a recombinant vector into a suitable host cell. Any host cell known in the art that can stably and continuously clone or express the recombinant vector is available. Prokaryotic host cells may include, for example, *Escherichia coli* JM109, *Escherichia coli* BL21, *Escherichia coli* RR1, *Escherichia coli* LE392, *Escherichia coli* B, *Escherichia coli* X1776, *Escherichia coli* W3110, *Bacillus* spp. such as *Bacillus subtilis* and *Bacillus thuringiensis*, as well as enteric bacteria such as *Salmonella typhimurium*, *Serratia marcescens*, and various *Pseudomonas* species. For eukaryotic host cells, the transformed host cells may include yeast (Saccharomyces 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, MDCK cell lines, but are not limited to these.

[0168] Nucleic acid molecules or recombinant vectors can be delivered (introduced) into host cells using commonly known methods. For example, when the host cell is a prokaryote, methods such as the CaCl2 method or electroporation can be used; and when the host cell is a eukaryote, methods such as microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, or gene bombardment can be used, but are not limited to these.

[0169] Methods for selecting transformed (recombinant vector-introduced) host cells can be readily performed using techniques known in the art based on a phenotype expressed by a selection marker. For example, if the selection marker is a gene conferring resistance to a specific antibiotic, recombinant cells that have been introduced with the recombinant vector can be readily selected by culturing them in a culture medium containing the antibiotic.

[0170] Medical use

[0171] The fusion protein provided herein contains an anti-TIGIT antibody or its antigen-binding fragment that has the ability to deliver IL-10 to effector immune cells to suppress immune cell activity (e.g., inhibit effector T cell function, promote Treg activity, reduce inflammatory cytokine secretion, etc.). Furthermore, IL-10 has a high binding affinity and low immunogenicity to its receptor (IL-10 receptor), resulting in immunosuppressive effects, such as reduced cytokine (inflammatory cytokine) secretion when administered in vivo, while maintaining safety by minimizing the risk of being recognized as an immunogen. IL-10 also exhibits excellent autoimmune and anti-inflammatory effects (e.g., inhibition of pro-inflammatory cytokine production and / or secretion, inhibition of effector immune cell proliferation, etc.). Therefore, the fusion peptide combines the advantages of an anti-TIGIT antibody or its antigen-binding fragment with IL-10 and can be effectively used for immunosuppression, prevention, and / or treatment of immune-related diseases, particularly autoimmune and / or inflammatory diseases.

[0172] One embodiment provides an immunosuppressant or pharmaceutical composition for immunosuppression, comprising at least one active ingredient selected from the group consisting of: an antibody or an antigen-binding fragment thereof, or a fusion protein, a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof or a fusion protein, a recombinant vector carrying the nucleic acid molecule, and a recombinant cell anchored to the nucleic acid molecule or the recombinant vector.

[0173] Another embodiment provides a pharmaceutical composition for the prevention and / or treatment of immune-related diseases, the composition comprising at least one active ingredient selected from the group consisting of: an antibody or an antigen-binding fragment thereof, or a fusion protein, a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof or a fusion protein, a recombinant vector carrying a nucleic acid molecule, and recombinant cells anchoring a nucleic acid molecule or a recombinant vector. The aforementioned immune-related diseases may be selected from the group consisting of autoimmune diseases, inflammation, and inflammatory diseases.

[0174] Other embodiments provide a pharmaceutical composition (or anti-autoimmune agent and / or anti-inflammatory agent) for the prevention and / or treatment of autoimmunity (or autoimmune diseases) and / or inflammation (or inflammatory diseases), comprising one or more of the following as active ingredients: the antibody or antigen-binding fragment, the fusion protein, the nucleic acid molecule encoding the antibody or antigen-binding fragment or fusion protein, the recombinant vector comprising the nucleic acid molecule, and the recombinant cell comprising the nucleic acid molecule or recombinant vector.

[0175] Another implementation provides a method for suppressing immunity, comprising administering to a subject requiring immunosuppression a pharmaceutically effective amount of at least one selected from the group consisting of: antibodies or antigen-binding fragments or fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells anchored to nucleic acid molecules or recombinant vectors. The immunosuppression method may further include a step of identifying the subject requiring immunosuppression prior to the administration step.

[0176] Another embodiment provides a method for preventing and / or treating immune-related diseases, the method comprising administering to a subject requiring prevention and / or treatment of an immune-related disease a pharmaceutically effective amount selected from at least one of the following: antibodies or antigen-binding fragments or fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells anchoring nucleic acid molecules or recombinant vectors. The method for preventing and / or treating immune-related diseases may further include identifying the subject requiring prevention and / or treatment of the immune-related disease prior to the administration step.

[0177] Other embodiments provide a method for preventing and / or treating autoimmunity (or autoimmune diseases) and / or inflammation (or inflammatory diseases), comprising administering to a subject requiring prevention and / or treatment of autoimmunity (or autoimmune diseases) and / or inflammation (or inflammatory diseases) a pharmaceutically effective amount of one or more steps selected from the group consisting of: antibodies or antigen-binding fragments, fusion proteins, nucleic acid molecules encoding antibodies or antigen-binding fragments or fusion proteins, recombinant vectors containing nucleic acid molecules, and recombinant cells containing nucleic acid molecules or recombinant vectors. The method for preventing and / or treating cancer may further include, prior to the administration step, identifying a subject requiring prevention and / or treatment of autoimmunity (or autoimmune diseases) and / or inflammation (or inflammatory diseases).

[0178] Other embodiments provide a use for preventing and / or treating immunosuppression, immune-related diseases, and / or for preventing and / or treating autoimmunity (or autoimmune diseases) and / or inflammation (or inflammatory diseases), or a use for preparing a pharmaceutical composition for preventing and / or treating immunosuppression, immune-related diseases, and / or for preventing and / or treating autoimmunity (or autoimmune diseases) and / or inflammation (or inflammatory diseases), selected from at least one of the following: antibody or antigen-binding fragments, fusion proteins, nucleic acid molecules encoding antibody or antigen-binding fragments or fusion proteins, recombinant vectors containing nucleic acid molecules, and recombinant cells containing nucleic acid molecules or recombinant vectors.

[0179] As used herein, the term "immunosuppression" refers to a reduction in the initial immune response to an antigen or a weakening of an existing immune response, and is used interchangeably with terms such as "immunosuppression" or "immunoinactivation."

[0180] As used herein, the term "immune-related disease" is intended to encompass all diseases caused by disorders and / or insufficient activation of the immune system, including but not limited to autoimmune diseases, inflammatory diseases, etc. Inflammation can be inflammatory (e.g., chronic or acute inflammation) and can include, but is not limited to, inflammation caused by infection (e.g., local inflammation, systemic inflammation, etc.).

[0181] Autoimmune diseases refer to any symptoms and / or diseases caused by autoimmunity, including but not limited to rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, Behçet's syndrome, lupus, and Sjögren's syndrome. They may also be selected from, but are not limited to, one or more of the following groups: myasthenia gravis (e.g., myasthenia gravis), scleroderma, hypothyroidism, hyperthyroidism, psoriasis, vitiligo, multiple sclerosis, autoimmune hepatitis, autoimmune nephritis, autoimmune pancreatitis, autoimmune encephalitis, and cytokine storm.

[0182] Inflammatory diseases refer to inflammation (e.g., chronic or acute inflammation) or diseases caused by inflammation, such as cardiac inflammation (e.g., coronary artery disease, angina pectoris, myocardial infarction, pericarditis, myocarditis, etc.), vascular inflammation (e.g., a general term for atherosclerosis, arteriosclerosis, myocarditis, etc.), atherosclerosis, vasculitis, disseminated intravascular coagulation (DIC), immune thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), anemia), upper respiratory tract inflammation (e.g., acute nasopharyngitis, allergic rhinitis, sinusitis, pharyngitis, tonsillitis, laryngitis) and lower respiratory tract inflammation. Inflammation of the lungs (e.g., bronchitis, bronchiectasis, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, interstitial lung disease, tuberculosis), upper gastrointestinal tract (e.g., gastritis, esophagitis), lower gastrointestinal tract (e.g., small bowel disease, ulcerative colitis, Crohn's disease, celiac disease, diverticulitis, irritable bowel syndrome, appendicitis, fistula, etc.), liver, gallbladder and / or pancreas (e.g., hepatitis, fatty liver, cholangitis, cholecystitis, pancreatitis, type 1 diabetes, etc.), kidney (upper urinary tract) (e.g., pyelonephritis, glomerulonephritis, urinary tract infection), lower urinary tract Inflammation of the reproductive system (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, tinea pedis, acne, etc.), inflammation of the muscles (e.g., myositis, etc.), inflammation of the brain (e.g., encephalitis, severe...). The treatment may include, but is not limited to, the prevention and / or treatment of autoimmune diseases and / or inflammatory diseases. This includes, but is not limited to, the inhibition of the occurrence and / or increase of inflammation, and the inhibition of the exacerbation of autoimmune and / or inflammatory diseases caused by migration, invasion, and spread. Other treatment options include, but are not limited to, the prevention and / or treatment of autoimmune diseases and / or inflammatory diseases. Neuroinflammatory diseases (e.g., neuritis in various parts of the body, such as the eyes and ears, complex regional pain syndromes, which may include one or more of the groups selected from Guillain-Barré syndrome), ocular inflammation (e.g., blepharitis, uveitis, conjunctivitis, etc.), ear inflammation (otitis media, mastoiditis, etc.), oral inflammation (stomatitis, periodontitis, gingivitis, etc.), systemic inflammation (systemic inflammatory response syndromes (e.g., sepsis, metabolic syndrome-related diseases, etc.), peritonitis, reperfusion injury, transplant rejection, hypersensitivity reactions, etc.).

[0183] Systemic inflammation caused by infection can be selected from any infectious disease that can be treated, mitigated, and / or prevented by the immunosuppression described above, which includes the suppression (e.g., inactivation) and / or reduction of the function and / or proliferation of immune cells (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 cells (Tregs), and reduction of the production and / or secretion of inflammatory proteins (e.g., cytokines (TNF-α, IFN-γ, etc.)). For example, the infectious disease can be an infection selected from one or more pathogens selected from the group consisting of viruses (e.g., influenza virus, coronaviruses such as severe acute respiratory syndrome (SARS) virus (SARS-CoV, SARS-CoV-2, etc.), Middle East respiratory syndrome (MERS) virus (MERS-CoV), bacteria, fungi, parasites, etc., or diseases (infectious diseases) caused by such infections).

[0184] Subjects who may be administered the fusion protein and / or pharmaceutical composition provided herein may be any animal or cell. For example, a subject may be an animal selected from mammals, including primates (e.g., humans and monkeys) and rodents (e.g., rats and mice), or cells, tissues, body fluids (e.g., serum) or cultures derived from (isolated from) such animal (e.g., human), or isolated cells, tissues or body fluids (e.g., serum) derived from humans.

[0185] In addition to fusion proteins, nucleic acid molecules, carriers, and / or cells as active ingredients, pharmaceutical compositions may additionally comprise pharmaceutically acceptable carriers. Carriers may be any commonly used in the formulation of protein drugs and may be selected from, but are not limited to, lactose, dextran, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. Pharmaceutical compositions may also comprise at least one of the following commonly used in the preparation of pharmaceutical compositions: diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives.

[0186] Based on the total weight of the composition, the content of the fusion protein, nucleic acid molecule, carrier and / or cell as the active ingredient in the pharmaceutical composition may be 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%, but is not limited thereto.

[0187] Fusion proteins, nucleic acid molecules, carriers, cells, and / or pharmaceutical compositions can be administered via oral or non-oral routes. Non-oral administration may include intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal injection, topical application, nasal application, pulmonary application, and rectal application. For oral administration, because proteins or peptides are digested, oral compositions should be formulated to protect the active agent from degradation in the stomach.

[0188] In addition, fusion proteins, nucleic acid molecules, carriers, cells and / or pharmaceutical compositions can be formulated as solutions, suspensions, syrups or emulsions in oily or aqueous media, or as extracts, powders, granules, tablets, capsules or injections, and may also contain dispersants or stabilizers for formulation.

[0189] The content of the active ingredient in a pharmaceutical composition can vary depending on the formulation method, route of administration, patient's age, weight, sex, disease state, diet, administration time, administration interval, route of administration, excretion rate, and response sensitivity. Based on the active ingredient (fusion protein, nucleic acid molecule, carrier, cell), the daily dose of the pharmaceutical composition can be 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 100 mg / kg, 0.0001 to 50 mg / kg, 0.0001 to 50 mg / kg, 0. The dosage ranges from 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 500 mg / kg, 0.01 to 100 mg / kg, 0.01 to 50 mg / kg, 0.1 to 100 mg / kg, or 0.1 to 50 mg / kg, but are not limited to these. Daily doses may be formulated as a single unit dose or suitably aliquoted or manufactured in multi-dose containers. As used herein, the term "pharmaceuticalally effective amount" refers to the amount of active ingredient that exhibits the desired pharmaceutical activity, which falls within the stated dosage range.

[0190] Another embodiment provides a health functional food for immunosuppression, comprising at least one active ingredient selected from the group consisting of: fusion proteins, nucleic acid molecules encoding antibodies or antibody-binding fragments or fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells anchoring nucleic acid molecules or recombinant vectors.

[0191] Another embodiment provides a health functional food for the prevention and / or improvement of immune-related diseases, the food comprising at least one active ingredient selected from the group consisting of: fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells anchored to nucleic acid molecules or recombinant vectors.

[0192] As used herein, the term "health functional food" refers to food products made (processed) using nutrients that are easily lacking in the daily diet or ingredients or raw materials or components that have beneficial functions for the human body (hereinafter referred to as "functional ingredients"), and is intended to encompass all foods that are beneficial to maintaining health or preventing and / or alleviating certain diseases or symptoms. There are no particular limitations on the final product. For example, health functional foods can be selected from, but are not limited to, the group consisting of various foods, beverages, and food additives.

[0193] Food compositions may contain acceptable food additives and may further include suitable carriers, fillers and diluents commonly used in the production of health functional foods.

[0194] When used as a food additive, the food composition can be added directly or in combination with other foods or food ingredients, and can be used appropriately by conventional methods.

[0195] The content of active ingredients in a food composition may be appropriately determined according to the form of the food, the desired effect and the purpose, and may be 0.00001 to 99% by weight, 0.0001 to 99% by weight, 0.001 to 99% by weight, 0.01 to 99% by weight, 0.1 to 99% by weight, 1 to 99% by weight or 10 to 99% by weight of the total food weight, but is not limited thereto.

[0196] [Beneficial Effects]

[0197] The fusion protein provided in this article has the advantages of both the aforementioned anti-TIGIT antibody or its antigen-binding fragment and IL-10, and can be effectively used for the prevention and / or treatment of immunosuppression or immune-related diseases. Attached Figure Description

[0198] Figure 1 This is a graph showing the results of measuring the degree of binding between human TIGIT and the anti-TIGIT antibody MMB0 and fusion protein (MMB5) provided according to one embodiment using an ELISA according to one embodiment.

[0199] Figure 2 This is a graph showing the results of ELISA measurements of the binding degree of anti-TIGIT antibody (MMB0) and fusion protein (MMB5) to IL-10Ra.

[0200] Figures 3a to 3c This is a graph showing the results of measuring the binding affinity of the fusion protein provided according to one embodiment to effector T cells and NK cells expressing TIMIT using FACS. Figure 3a CD4+ T cells; Figure 3b CD8+ T cells; Figure 3c NK cells).

[0201] Figure 4 This is a graph showing the results of FACS measurements to determine whether the TIGIT / CD155 interaction of the fusion protein provided according to one embodiment is blocked.

[0202] Figure 5a and Figure 5b This is a graph showing the results of confirming the STAT3 signaling level of the fusion protein provided according to one embodiment by STAT3 phosphorylation. Figure 5a CD4+ T cells; Figure 5b (CD8+ T cells).

[0203] Figure 6 This is a diagram showing the transport effect of IL-10 of the fusion protein provided according to one embodiment on immune cells (effector T cells, NK cells).

[0204] Figure 7 This is a graph showing the inhibitory effect of a fusion protein provided according to one embodiment on the production of inflammatory cytokines (TNF-α, granzyme B).

[0205] Figures 8a to 8c This is a graph showing the inhibitory effect of a fusion protein according to one embodiment on T cell proliferation. Figure 8a CD4+ T cells Figure 8b CD8+ T cells Figure 8c NK cells).

[0206] Figures 9a to 9c This is a graph showing the inhibitory effect of a fusion protein provided according to one embodiment on the expression of inflammatory cytokines (TNF-α) in PBMCs derived from patients with autoimmune diseases.

[0207] Figure 10 This is a graph showing the anti-inflammatory effect of the fusion protein provided according to one embodiment, based on the results of measuring the DAI score (Disease Activity Index score) in a DSS (dextran sulfate)-induced colitis model. Detailed Implementation

[0208] The invention will be described in more detail below by way of examples, but these are merely illustrative and not intended to limit the scope of the invention. It will be apparent to those skilled in the art that modifications can be made to the examples described below without departing from the spirit of the invention.

[0209] Example 1: Production of monoclonal anti-TIGIT antibody

[0210] 1.1. Production of mouse monoclonal anti-TIGIT antibody

[0211] Four BALB / c mice were immunized with His-labeled human TIGIT protein (R&D systems, UniProtKB / SwissProt Q495A1-1) via four consecutive cross-injections over 14 days. Lymphocytes were collected from the four mice, purified, and then fused with SP2 / 0 myeloma cells. The fused cells were proliferated in HAT selective single-step cloning medium, and the resulting 2 × 920 hybridoma clones were selected using ClonePix2 (Molecular Devices), transferred to 96-well plates, and cultured.

[0212] Supernatants from hybridoma tissue cultures effective against recombinant human TIGIT (His) (R / D system) were screened using indirect ELISA. For positive clones, Ig secretion and specificity were tested by additional indirect ELISA of the screening antigen (recombinant human TIGIT Fc chimeric protein (R&D system)) and cells. The CHO-K1 (ATCC, CCL-61) cell line was used as a negative control.

[0213] ELISA is performed under the following conditions;

[0214] - Coat ELISA plates at 4°C with a coating solution containing 0.1 μg / well of recombinant human TIGIT (rhTIGIT) protein at a concentration of 100 μL / well in carbonate coating buffer (pH 9.6) at room temperature.

[0215] - Block with PBS containing 3% skim milk powder at room temperature for 1 hour.

[0216] - Treat with PBS-Tween containing 1:10000 goat anti-mouse IgG / M(H+L)-HRP (100uL / well) for 1 hour (37°C, w / shake).

[0217] - All washing steps were performed with PBS-Tween for 3 minutes.

[0218] Add TMB substrate at a concentration of 50 μL / well, place in the dark (5-10 minutes), and terminate with an equal volume of 1 M HCl.

[0219] Clones expressing IgM were isolated and deexpressed by typing, and clones expressing IgG were collected.

[0220] Collected positive clones were subcloned to identify stably expressing clones. Elution was performed by passing culture supernatant containing the selected clones through a protein A column, with the buffer replaced with PBS. The purified antibodies were tested by indirect ELISA against the screening antigen (recombinant human TIGIT(T103) Fc chimeric protein; R&D systems) and the negative control antigen (His peptide).

[0221] Among the collected positive clones, the MMB0 clone (based on mouse skeleton (mIgG1) mouse antibody) was selected as the primary antibody.

[0222] The CDR and variable region of the antibody are listed in Table 3 below.

[0223] [Table 3]

[0224]

[0225] 1.2. Humanization of mouse anti-TIGIT antibody

[0226] To identify key amino acid residues for antibody structure and binding, a protein structure model of the monoclonal antibody variable region (mAb V region) was analyzed. Using this information, combined with computer simulations of human antibody structures, a large number of preliminary sequence fragments suitable for producing humanized variants of MMB0 were selected, and peptides binding to human MHC class II alleles were analyzed. Where possible, sequence fragments identified as significant non-human germline binders of human MHC class II were also included. This reduced the fragment set, and these combinations were reanalyzed using the methods described above to identify connections between fragments that 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 or had reduced significant T-cell epitopes, thus designing a human variable region that evaded recognition by potential T-cell epitopes (deimmunization).

[0227] Six heavy chain variable regions (VH1, VH6, VH7, VH5, VH8, VH9) and seven light chain variable regions (Vk1, Vk2, Vk7, Vk11, Vk5, Vk10, Vk14) sequences were selected for the preparation of humanized antibodies, and were used for gene synthesis and expression in mammalian cells.

[0228] The sequences of the six heavy chain variable regions and seven light chain variable regions used to prepare humanized antibodies, as well as the heavy chain variable region (VH0) and light chain variable region (Vk0) of mouse antibody MMB0, are summarized in Table 4 below.

[0229] [Table 4]

[0230]

[0231]

[0232] In addition, the human IgG-derived constant regions used to prepare humanized antibodies are summarized in Table 5 below:

[0233] [Table 5]

[0234]

[0235] Based on the variable region sequences listed in Tables 4 and 5 above, various combinations of humanized antibodies were constructed using the sequences of 6 heavy chain variable regions (VH1, VH6, VH7, VH5, VH8, VH9), 7 light chain variable regions (Vk1, Vk2, Vk7, Vk11, Vk5, Vk10, Vk14), and the human constant region sequences in Table 5. Furthermore, chimeric antibodies were constructed using the VH0 / VK0 combination (which is a variable region of a mouse antibody) and the human constant regions in Table 5. Among these, the humanized antibody constructed using the 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 application examples.

[0236] Example 2. Production of the [anti-TIGIT monoclonal antibody]-[wild-type IL-10] fusion protein

[0237] By fusing the humanized anti-TIGIT antibody and IL-10 prepared in Example 1.2 with or without a linker, the fusion protein was designed to have the structure shown in Table 6 below.

[0238] [Table 6]

[0239] Light and heavy chain TIGIT mAb-wild-type IL-10 fusion protein

[0240]

[0241] (In Table 6 above,

[0242] Anti-TIGIT monoclonal antibody (TIGIT mAb): MMB0_VH9 / Vk11 (hereinafter referred to as 'MMB0');

[0243] IL-10: Wild-type IL-10;

[0244] Two connectors or no connector: LC variant (fused to the C end of LC (light chain) with rigid and flexible connectors) & Fc variant (ΔK478) (no connector and fused to the C end of Fc with rigid and flexible connectors)

[0245] The gene encoding TIGIT mAb was synthesized (see Table 7) and cloned into a modified pDNA3.4 vector (Biointron) containing a "linker-wild-type IL-10 element". CHO-K1 cells (ATCC) were seeded onto culture plates and cultured to a density of 70–90%, then the gene DNA was transfected into CHO-K1 cells by electrophoresis. Cells were subsequently cultured at 37°C to induce transient expression. The culture supernatant was purified by protein A affinity chromatography and preparative SEC. The produced protein was buffered in PBS, quantified by OD280 nm, and analyzed by SDS-PAGE.

[0246] [Table 7]

[0247] Sequences of anti-TIGIT mAb and fusion protein MMB0-IL-10

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261] (In Table 7, the IL-10 dimer is formed by linking the IL-10 of SEQ ID NO:9 to the D-1 connector (GGGSGGG; SEQ ID NO:16))

[0262] In the following text, the nomenclature of the above-mentioned anti-TIGIT antibody fusion proteins with MMB0 and IL-10 (see Table 6) is shown in Table 8:

[0263] [Table 8]

[0264]

[0265] In the following text, the names of the anti-TIGIT antibody MMB0 and IL-10 fusion proteins prepared in this embodiment are listed in Table 8 above.

[0266] Example 3. Protein binding activity of the MMB0-IL-10 fusion protein

[0267] 3.1. TIGIT combines affinity

[0268] Recombinant human TIGIT protein (Acro Biosystems-Cat# TIT-H52H5) was diluted to a concentration of 1 mg / ml in coating buffer and coated onto ELISA plates (using an ELISA buffer kit (Invitrogen)). TM The 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 1:3 from 62.5 nM using assay buffer. The serially diluted drug was placed on the TIGT-coated plate and incubated. Subsequently, anti-human IgG-HRP antibody (Promega, Cat#W4031) diluted 1:10000 was added to each well. The plate was induced with TMB (tetramethylbenzidine) solution and the reaction was terminated using ELISA stop solution (Invitrogen, Cat#SS04). OD values ​​were measured at 450 nM using GloMax® Explorer.

[0269] The binding affinity of anti-TIGIT antibody (MMB0) and MMB0-IL-10 fusion protein (MMB5) to TIGIT is shown in... Figure 1 middle. Figure 1 The results showed that the MMB0-IL-10 fusion protein (MMB5) exhibits a similar affinity for human TIGIT to the parent antibody (MMB0). These results indicate 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 parent antibody (MMB0) or inhibit its binding ability, and that the fusion protein retains the functional characteristics of the parent antibody.

[0270] 3.2. Binding affinity to IL-10 receptor

[0271] Recombinant human IL-10RA His-tag protein (SinoBiological, 10419-H08H) was diluted to a concentration of 0.1 μg / mL using coating buffer B (Invitrogen, CB01100) and coated onto an ELISA plate. 200 μl of 1X assay buffer (Invitrogen, CNB0011) was added to each well, and the plate was blocked by incubation at room temperature (RT) for 1 hour. 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 the plate was incubated at 37°C with shaking (200 rpm) for 1 hour. Each of the above samples was then diluted to a concentration of 200 nM with assay buffer. The detection antibody (HRP-conjugated anti-human IgG (H+L) antibody (Promega, Cat#W4031)) was diluted 1:10000 in assay buffer, and 100 μl was added to each well. The plate was then incubated at 37°C with shaking (200 rpm) for 1 hour. Subsequently, 100 μl of TMB solution was added to each well. After addition and luminescence induction, 100 μl of ELISA stop solution (Invitrogen, Cat#SS04) was added to terminate the reaction. OD values ​​were measured at 450 nM using GloMax® Explorer.

[0272] The binding affinity of the anti-TIGIT antibody (MMB0) and fusion protein (MMB5) to IL-10Ra is shown in... Figure 2 In the middle. For example Figure 2 As shown, the parental antibody MMB0 does not bind to IL-10RA, while the fusion protein (MMB5) in which IL-10 is linked to the anti-TIGIT antibody shows binding affinity for IL-10RA and exhibits a stronger binding affinity for IL-10 than for IL-10-Fc.

[0273] Example 4. Cell-binding activity of the fusion protein

[0274] 4.1. Binding affinity of TIGIT to CD4+ T cells, CD8+ T cells, and CD56+ NK cells

[0275] In this embodiment, the binding ability of the fusion protein (MMB5) to each cell population of TIGIT-expressing immune cells (CD4+ T cells, CD8+ T cells, and CD56+ NK cells) was tested using a PE / Cy7-labeled anti-TIGIT antibody. Because the fusion protein (MMB5) competitively binds to TIGIT expressed on the surface of immune cells (including the PE / Cy7-labeled anti-TIGIT antibody), the percentage of binding of the PE / Cy7-labeled anti-TIGIT antibody to TIGIT on the surface of immune cells decreases as the fusion protein (MMB5) binds to the TIGIT expressed on the surface of immune cells. Therefore, the binding ability of the fusion protein (MMB5) to immune cells is tested by measuring the degree of reduction in the percentage of binding of the PE / Cy7-labeled anti-TIGIT antibody.

[0276] Peripheral blood mononuclear cells (PBMCs; STEMCELL Technologies) from healthy human donors were counted and resuspended at a rate of 5 million cells per 1 ml PBS (in SPL (catalog number 34296) 96-well plates (round bottom). The prepared cells (PBMCs) were bound to the MMB0-IL-10 fusion proteins MMB1, MMB3, and MMB5 (68.7 nM each) or their parental antibody MMB0 (68.7 nM) in a 37°C incubator for 15 min. The plates were centrifuged at 1500 rpm for 3 min at room temperature to remove unbound antibodies, and then stained with the antibody mixture. The antibody mixture described above consisted 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 staining in the dark at room temperature for 10 minutes, 100 μl of PBS was added, and the cells were centrifuged at 1500 rpm for 3 minutes at room temperature for washing. The supernatant was then removed, and the cells were resuspended in 100 μl of PBS. Flow cytometry was then performed using a CytoFLEX (Beckman Coulter) at a rate of 60 μl / min (discard rate ≤5%). Therefore, the level of TIGIT binding to the aforementioned anti-hTIGIT PE / Cy7 antibody (PE / Cy7-labeled anti-hTIGIT antibody) could be measured. For comparison, the same test was performed on the unstimulated group that was not treated with the fusion protein or parental antibody.

[0277] The proportion of TIGIT on the surface of immune cells labeled with anti-hTIGIT PE / Cy7 antibody (“expressed as %TIGIT expression”) is shown in Figures 3a to 3c ( Figure 3a CD4+ T cells; Figure 3b CD8+ T cells; Figure 3c NK cells).

[0278] As previously explained, the lower the level of TIGIT labeled by the binding of the anti-hTIGIT PE / Cy7 antibody, the higher the cell-binding affinity of the treated sample via TIGIT; therefore, Figures 3a to 3c The results showed that the anti-TIGIT antibody-IL-10 fusion protein or parent antibody (anti-TIGIT antibody) provided in this disclosure binds to effector T cells and NK cells with excellent binding affinity.

[0279] Example 5. Maintaining TIGIT / CD155 interaction / signal transduction function

[0280] In this embodiment, a TIGIT / CD155 blocking assay was performed according to Promega's TIGIT / CD155 blocking bioassay instructions to determine whether the MMB0-IL-10 fusion protein provided in this disclosure maintains TIGIT / CD155 interaction / signaling function. More specifically, TIGIT effector cells (Jurkat cell line expressing human TIGIT; Promega) were thawed, transferred to assay buffer (Promega), seeded in white 96-well plates, and incubated for one day. On the second day, the MMB0-IL-10 fusion proteins MMB1, MMB3, MMB5, and their parental antibody MMB0 were used in... Figure 4 The concentrations shown are used for treatment. Subsequently, CD155-CHO-K1 (Promega) was added to all experimental groups, including the negative control group (the group not treated with this drug (fusion protein or antibody)). The samples were then incubated at 37°C for 6 hours and then left at room temperature for 10–15 minutes. Finally, Bio-Glo... TM Reagents were added to all experimental groups, including the negative control, and after incubation at room temperature, luminescence was measured three times using a luminescence plate reader (using a BHS LumiStar luminescence plate reader).

[0281] For comparison, antagonistic anti-TIGIT antibodies were used (in... Figure 4 The same experiment was performed using "other anti-TIGIT mAb" as a control antibody. The sequence information of the control used is as follows:

[0282] Heavy chain:

[0283] QVQLQESGPGLVKPSQTLSLTCTVTGYSITSDYAWNWIRQPPGKGLEWMGYISYSGSARYNPSLKSRVTISRDTSKNQFSLKLSSVTAEDTATYYCARKGYPAYFAYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 43)

[0284] Light chain:

[0285] DIQMTQSPSSSLSASVGDRVSITCKASQDVSTAVAWYQQKPGQAPRLLIYSASYRYTGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYSTPYTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 44)

[0286] The results obtained above are shown in Figure 4 In the middle. For example Figure 4 As shown, the MMB0-IL-10 fusion proteins MMB1, MMB3, MMB5, and their parent antibody MMB0 do not block the interaction (binding) between TIGIT and CD155. This indicates that the fusion proteins provided in this application can utilize the benefits of CD155 signaling naturally present in the immune microenvironment of immune cells, and can also utilize the advantages of the TIMIT-stimulated signaling of the included anti-TIMIT monoclonal antibody.

[0287] Example 6. Induction of IL-10R (IL-10 receptor) signaling (measured by STAT3 phosphorylation)

[0288] The immunostimulatory effect of the MMB0-IL-10 fusion protein on human CD3+ T cells (CD4+ and CD8+ T cells) was tested using STAT3 phosphorylation assay.

[0289] PBMCs (STEMCELL Technologies) from healthy human donors were used at 5×10 5Cells were seeded into plates at the specified concentrations per well. Cells were treated with 68.7 nM of either the drug (MMB0-IL-10 fusion proteins MMB1, MMB2, MMB3, MMB4, MMB5, MMB6) or a human IL-10 control (Miltenyi Biotec, 130-093-948) and stimulated at 37°C for 15 min. After centrifugation (1500 rpm, 3 min, 4°C), the supernatant was removed, and the cells were stained with 50 μl of surface-labeled antibody at 4°C for 10 min. Surface-labeled antibodies were Live / Dead-AV750 (eBioscience, 65-0865-12, 1:500), anti-CD3-BV150 (Biolegend, 344828, 1:50), anti-CD4-A700 (Biolegend, 344622, 1:50), and anti-CD8-FITC (Biolegend, 980908, 1:50), and these markers were used after being diluted in PBS to the listed ratios. Subsequently, after removing the supernatant by centrifugation (1500 rpm, 3 min, 4 °C), 100 μl of Fix buffer (Biolegend, 420801) was added to the cells, and the cells were allowed to react at room temperature for 15 min. After the reaction was complete, the cells were centrifuged (1500 rpm, 3 min, room temperature), and the supernatant was removed. Then, 100 μl of True-Phos perm buffer (Biolegend, 425401) was added to the cells and the reaction was carried out at -20°C for 1 hour. Subsequently, the cells were centrifuged (1500 rpm, 3 min) to remove the supernatant and stained with pSTAT3 antibody. For staining, the pSTAT3-APC (Biolegend, 651008, 1:50) antibody was diluted in PBS and the process was performed in the dark. After staining, the cells were centrifuged (1500 rpm, 3 min) to remove the supernatant, and then 100 μl of PBS was added to resuspend the cells. The antibody detection surface marker labeling and fluorescence of phosphorylated STAT3 (pSTAT3) were detected in each cell using a cell analyzer (CytoFLEX, Beckman Coulter). Data were analyzed using Flowjo software (BD Biosciences) and plotted using GraphPad Prism 9 software (Dotmatics).

[0290] The results obtained above (signal transduction) are shown in Figure 5a and Figure 5b middle( Figure 5a CD4+ cells; Figure 5bCD8+ cells; obtained by normalizing the signal at each concentration relative to the signal at a drug concentration of 68.7 nM (set to 100%).

[0291] like Figure 5a and Figure 5b As shown, for all tested fusion proteins, phosphorylation of STAT3 was observed up to 68.7 nM in CD4+ T cells and CD8+ T cells, and in the presence of IL-10 (human IL-10 in its free form but not in the fusion protein; control drug), it was found that IL-10 began to lose its binding efficiency with IL-10R at 0.687 nM. This experiment demonstrates that, compared with free hIL-10, the MMB fusion antibody can induce pSTAT3 signaling at a lower concentration, and that antibody-bound IL-10 can maintain signal transduction.

[0292] Example 7. IL-10 Delivery

[0293] In this embodiment, the effect of the MMB0-IL-10 fusion protein provided in this disclosure in transporting IL-10 to immune cells (effector T cells, NK cells) was tested.

[0294] PBMCs (STEMCELL Technologies) obtained from healthy human donors will be used at a rate of 2 x 10 5Cells were seeded at a concentration of [specific concentration not specified] into 96-well plates, then the MMB0-IL-10 fusion protein MMB1 was injected and incubated at 37°C for 15 minutes. The cells were then centrifuged (1500 rpm, 3 minutes), the supernatant was removed, 50 μl of the antibody master mixture was added to the cells to resuspend them, and the reaction was carried out in the dark. The master mixture of the above antibodies was prepared by diluting 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), and Live / Dead-A750 (ebioscience, 1:50) in PBS buffer. After centrifugation (1500 rpm, 3 min), the supernatant was removed, and the solution was resuspended in 100 μl of PBS. Fluorescence values ​​were measured using a cell analyzer (CytoFLEX, Beckman Coulter), and the percentage of cells displaying the PE (phycoerythrin) color was also measured. The percentage of cells displaying the PE color indicates the percentage of cells that have bound (transported) IL-10. Data were analyzed using Flowjo software (BDBiosciences), and plotted using GraphPad Prism 9 software (Dotmatics).

[0295] The results obtained above (the percentage of cells that bind (transport) to IL-10) are shown in Figure 6 In the middle. For example Figure 6 As shown, the concentration of the MMB0-IL-10 fusion protein (MMB1) was as high as 6.87 nM, with increased IL-10 binding, followed by a slight decrease at 68.7 nM. This indicates that the MMB0-IL-10 fusion protein provided in this disclosure can deliver IL-10 to effector T cells and NK cells in a concentration-proportional manner.

[0296] Example 8. Inhibition of Function (In Vitro)

[0297] 8.1. Inhibition of pro-inflammatory cytokine production

[0298] In this embodiment, the inhibitory effect of the MMB0-IL-10 fusion protein provided in this disclosure on the production of pro-inflammatory cytokines was tested.

[0299] PBMCs from healthy human donors were stimulated using anti-CD3 antibody (BD, 1 μg / ml), anti-CD28 antibody (BD, 0.00032 μg / ml), and drugs (STEMCELL technology; 2 × 10⁻⁶). 5 Cells / well) for 72 hours. The drugs mentioned in this article are the MMB0-IL-10 fusion proteins MMB3 and MMB5, and their parental antibody MMB0. Each drug was used for stimulation at a concentration of 68.7 nM at 37°C. Subsequently, the supernatant was subjected to LegendPlex assays to measure the pro-inflammatory cytokines TNF-α and granzyme B. For comparison, the same test was performed using anti-CD3 antibody / anti-CD28 antibody (denoted as "anti-CD3 / CD28") without the above drug treatment.

[0300] According to the manufacturer's manual, use LEGENDplex TM Buffer group A (Biolegend, 740368), LEGENDplex TM Human CD8 / NK Panel standard (Biolegend, 740281), LEGENDplex TM Human CD8 / NK Panel for antibody detection (Biolegend, 740282), LEGENDplex TM Human TNF-α capturing microbeads A10 (Biolegend, 740273) and LEGENDplex TM Human granzyme B captures microbeads B6 (Biolegend, 740278) for LegendPlex.

[0301] More specifically, the supernatant was diluted 1:1 with assay buffer (Invitrogen) and mixed with microbeads. The mixture was then incubated at 8000 rpm for 2 hours at room temperature and in the dark using a shaker. After centrifugation (1050 rpm, 5 min), the supernatant was completely removed, wash buffer was added, and the mixture was incubated at room temperature (RT) for 1 min. Subsequently, the supernatant was removed by centrifugation (1050 rpm, 5 min). Then, the detection antibody was added, and the mixture was incubated at room temperature and in the dark for 1 hour. Then, SA-PE (streptavidin-phycoerythrin conjugate) was added, and the mixture was incubated at room temperature and in the dark for 30 min. Finally, the mixture was centrifuged (1050 rpm, 5 min), and the supernatant was removed. 150 μl of wash buffer was added for resuspending, and TNF-α and granzyme B were quantified by measuring fluorescence using LegendPlex.

[0302] The results obtained above are shown in Figure 7 In the middle. For example Figure 7As shown, all tested MMB0-IL-10 fusion proteins reduced the levels of pro-inflammatory cytokines TNF-α and granzyme (GZMB) that are increased by anti-CD3 / CD28 stimulation. These results indicate that the MMB0-IL-10 fusion protein provided in this disclosure maintains the anti-inflammatory efficacy of IL-10.

[0303] 8.2. Inhibition of T cell proliferation

[0304] In this embodiment, the inhibitory effect of the MMB0-IL-10 fusion protein provided in this disclosure on T cell proliferation was tested.

[0305] Anti-CD3 antibody (BD, 0.1 μg / ml), anti-CD28 antibody (BD, 0.00032 μg / ml), and a concentration of 2×10 were used. 6Cell / ml drug stimulation was performed on healthy human donor PBMCs (STEMCELL Technologies) labeled with CellTrace Violet (Invitrogen, C34557) for 72 hours. Here, the drugs refer to the MMB0-IL-10 fusion proteins MMB3 and MMB5, and their parental antibody MMB0, each at a concentration of 68.7 nM. Subsequently, the plates were centrifuged (400 g, 3 min, room temperature) to remove the supernatant. Cells were then cultured in the dark for 10 min at room temperature in Viability Dye eFluor 780 (1 / 1000) diluted with PBS. After centrifugation (400 g, 3 min, room temperature), the supernatant was removed. The resulting cells were placed in FOXP3Perm buffer (eBioscience) at room temperature in the dark for 15 min to allow them to become permeable. After centrifugation (400 g, 3 min, room temperature), the supernatant was removed, and the cells were stained with an internal staining antibody. Internal staining antibodies included 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), and PerCP anti-CD8 (Biolegend, 980916, 1 / 50). After centrifugation and removal of supernatant, 100 μl of PBS was added, and the cells were resuspended. Fluorescence values ​​were measured using an analyzer (CytoFLEX, Beckman Coulter). Data were analyzed using Flowjo software (BD Biosciences), and plots were generated 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 (in... Figures 8a-8c (This is represented as "anti-CD3 / CD28" in Chinese).

[0306] The results obtained above are shown in Figure 8a (CD4+ T cells) Figure 8b (CD8+ T cells) and Figure 8c In (NK cells). For example... Figures 8a-8cAs shown, all tested MMB0-IL-10 fusion proteins inhibited the proliferation of CD4+ T cells, CD8+ T cells, and NK cells induced by stimulation of anti-CD3 / CD28 antibodies. On the other hand, the parental antibody (MMB0) showed a weaker or lower statistical significance in this inhibitory effect. Therefore, it can be concluded that the inhibitory effect on T cell and NK cell proliferation is due to IL-10 signaling rather than the TIGIT signaling of the MMB0-IL-10 fusion protein. In summary, the MMB0-IL-10 fusion proteins provided in this disclosure demonstrate that they can inhibit the proliferative effects of anti-CD3 / CD28 on CD4+ T cells, CD8+ T cells, and NK cells.

[0307] 8.3. Inhibition of the production (expression) of pro-inflammatory cytokines in PBMCs derived from patients with autoimmune diseases.

[0308] PBMCs (STEMCELL technology) from patients with autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and ulcerative colitis (UC) will be processed at a rate of 5 × 10 5Cell / well seeding concentration. Cells were stimulated with anti-CD3 antibody (BD, 555336, 1 μg / ml) and anti-CD28 (BD, 555725, 1 μg / ml), and the drugs were added at 68.7 nM. Here, the drugs refer to the MMB0-IL-10 fusion proteins MMB1, MMB3, and MMB5, their parental antibody MMB0, and the control anti-TIGIT antibodies A15153G (Biolegend, 372702) and 22G2 (BPS, 71340). After treatment with the drugs and incubation at 37°C in a 5% CO2 incubator for 4 hours, brefeldin A (Brefeldin A, Sigma-Aldrich, B7651) was added at a concentration of 10 μg / ml to prevent cytokine release, and the cells were incubated at 37°C for 15 hours. In summary, cells (derived from PBMCs of patients with autoimmune diseases) were reacted with the drug and stimulant (anti-CD3 / CD28) for 19 hours. After the reaction, the supernatant was removed by centrifugation (1500 rpm, 5 min, 4 °C), and Zombie Aqua Fixable dead cell dye solution (Biolegend, 77143) was added for staining of dead / or cells (Live / Dead). The cells were then incubated at room temperature in the dark for 20 min. After the reaction, the supernatant was removed by centrifugation (1500 rpm, 5 min, 4 °C), 150 μl of the surface-labeled antibody master mixture was added, and the mixture was stained in the dark at 4 °C for 30 min. The master mixture of surface-labeled antibodies consisted of anti-CD3-BV421 (Biolegend, 300434, 1:100), anti-CD4-PerCP-Cy5.5 (Biolegend, 3446081:100), anti-CD8-AF700 (Biolegend, 301028, 1:200), and anti-CD56-APC (Biolegend, 362504, 1:100) diluted in FACS buffer (PBS containing 0.1% FBS). After staining, the cells were centrifuged (1500 rpm, 5 min, 4°C) to remove the supernatant, and 100 μl of Fix / perm buffer (eBioscience, 00-5523-00) was added. The cells were then fixed at room temperature in the dark for 40 min. Next, the cells were centrifuged (1500 rpm, 5 min, 4°C) to remove the supernatant, and stained with intracellular staining antibodies at room temperature in the dark for 30 min. The intracellular staining antibody was 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 minutes, 4°C), and 200 μl of PBS was added to resuspend the sample.Fluorescence values ​​were measured using a cell analyzer (CytoFLEX, Beckman Coulter). Data were analyzed using Flowjo software (BD Biosciences) and plotted using GraphPad Prism 9 software (Dotmatics). For comparison, the same test was performed using anti-CD3 antibody / anti-CD28 antibody without the aforementioned drug treatment. Figures 9a-9c (represented as "Stim" in Chinese).

[0309] The results obtained above (the ratio of CD4+ T cells expressing TNF-α) are shown in... Figure 9a (derived from PBMCs of SLE patients) Figure 9b (from PBMCs of RA patients) and Figure 9c (From PBMCs of UC patients). For example... Figures 9a-9c As shown, stimulation with anti-CD3 / CD28 antibodies increased the ratio of CD4+ T cells expressing TNF-α, a pro-inflammatory cytokine involved in the pathological symptoms of autoimmune diseases; conversely, this increased ratio of TNF-α-expressing CD4+ T cells was found to decrease upon treatment with the MMB0-IL-10 fusion protein tested. This effect of reducing TNF-α-expressing CD4+ T cells via 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)). Furthermore, the effect of reducing TNF-α-expressing CD4+ T cells via the MMB0-IL-10 fusion protein was superior to that of antibody A15153G, a control drug known to have anti-TIGIT agonist function. Using 22G2, a control drug with anti-TIGIT antagonist function, an actual increase in TNF-α-expressing CD4+ T cells was observed. Therefore, it can be seen that the MMB0-IL-10 fusion protein provided in this disclosure has excellent anti-inflammatory effects by reducing the production of the pro-inflammatory cytokine TNF-α by CD4+ T cells in patients with various autoimmune diseases.

[0310] Example 9. Anti-inflammatory effect (in vivo)

[0311] In this embodiment, the anti-inflammatory effect of the MMB0-IL-10 fusion protein was tested by measuring the DAI score in a DSS-induced colitis model.

[0312] More specifically, enteritis was induced in human TIGIT KI balb / c mice using 3% DSS (sodium dextran sulfate), and the mice were observed for 11 days after intraperitoneal or oral administration of the drugs. The drugs mentioned in this study were 5 mg / kg of the MMB0-IL-10 fusion protein MMB5, 40 mg / kg of cyclosporine A (MCE, 34526) (a drug primarily 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, while the other drugs were administered intraperitoneally. The DAI score (Disease Activity Index) was measured daily during the experiment and calculated using the following formula: (DAI score = (weight loss score + fecal characteristics score + fecal occult blood score) / 3).

[0313] The results obtained above are shown in Figure 10 In the middle. For example Figure 10 As shown, in a DSS-induced colitis animal model experiment, the group administered soluble human IL-10 did not show anti-inflammatory effects (decreased DAI score), while 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 higher anti-inflammatory efficacy than cyclosporine A at up to 8 days after administration. These results indicate that the MMB0-IL-10 fusion protein provided in this disclosure has a potent anti-inflammatory effect against inflammatory diseases.

Claims

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

6.

2. The anti-TIGIT antibody or its antigen-binding fragment according to claim 1, comprising: The heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:29 to 34; and The light chain variable region contains an amino acid sequence selected from SEQ ID NO:8 and SEQ ID NO:35 to 41.

3. The anti-TIGIT antibody or its antigen-binding fragment according to claim 1, wherein, The anti-TIGIT antibody is an animal antibody, a chimeric antibody, or a humanized antibody.

4. The anti-TIGIT antibody or its antigen-binding fragment according to claim 1, wherein, The antigen-binding fragment is scFv, scFv-Fc, (scFv)2, Fab, Fab', or F(ab')2 of the anti-TIGIT antibody.

5. A fusion protein comprising: (1) Anti-TIGIT antibody or its antigen-binding fragment; and (2) Interleukin-10 (IL-10).

6. The fusion protein according to claim 5, wherein, The anti-TIGIT antibody or its antigen-binding fragment comprises: A polypeptide (CDR-H1) containing the amino acid sequence of SEQ ID NO:1; A polypeptide (CDR-H2) containing the amino acid sequence of SEQ ID NO:2; A polypeptide (CDR-H3) containing the amino acid sequence of SEQ ID NO:3; A polypeptide (CDR-L1) containing the amino acid sequence of SEQ ID NO:4; A polypeptide (CDR-L2) containing the amino acid sequence of SEQ ID NO:5; and A polypeptide (CDR-L3) containing the amino acid sequence of SEQ ID NO:

6.

7. The fusion protein according to claim 5, wherein, The anti-TIGIT antibody or its antigen-binding fragment comprises: The heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:7 and SEQ ID NO:29 to 34; and The light chain variable region contains an amino acid sequence selected from SEQ ID NO:8 and SEQ ID NO:35 to 41.

8. The fusion protein according to claim 5, wherein, The IL-10 is a monomer or a dimer.

9. The fusion protein according to claim 5, wherein, The fusion protein is not contained in the peptide linker between the anti-TIGIT antibody or its antigen-binding fragment and interleukin-10.

10. The fusion protein according to claim 5, wherein, The fusion protein is contained in a peptide linker between the anti-TIGIT antibody or its antigen-binding fragment and interleukin-10.

11. The fusion protein according to claim 10, wherein, The peptide linker is represented by [EAAAK]n or [(G)mS]l, where n is an integer from 1 to 5 representing the quantity of [EAAAK], m is an integer from 1 to 5 representing the quantity of G, and l is an integer from 1 to 5 representing the quantity of [(G)mS].

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 encoding a fusion protein according to any one of claims 5 to 11.

13. A recombinant vector comprising the nucleic acid molecule according to claim 12.

14. A recombinant cell comprising the nucleic acid molecule according to claim 12 or a recombinant vector comprising said nucleic acid molecule.

15. A pharmaceutical composition for the prevention or treatment of immune-related diseases, 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. The pharmaceutical composition for the prevention or treatment of immune-related diseases according to claim 15, wherein, The immune-related diseases mentioned are autoimmune diseases or inflammatory diseases.

17. The pharmaceutical composition for the prevention or treatment of immune-related diseases according to claim 16, wherein, The autoimmune diseases mentioned are rheumatoid arthritis, type 1 diabetes, Crohn's disease, ulcerative colitis, Behcet's syndrome, lupus, Sjögren's syndrome, myasthenia gravis, scleroderma, hypothyroidism, hyperthyroidism, psoriasis, vitiligo, multiple sclerosis, autoimmune hepatitis, autoimmune nephritis, autoimmune pancreatitis, autoimmune encephalitis, or cytokine storm.

18. The pharmaceutical composition for the prevention or treatment of immune-related diseases according to claim 16, wherein, The inflammatory disease is inflammation or a disease caused by inflammation.

19. A pharmaceutical composition for immunosuppression, 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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