Fusion protein comprising antibody specifically binding to TIGIT and interleukin-15 and use thereof

Abstract

The present invention provides a fusion protein comprising an anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to TIGIT and interleukin-15 (IL-15), and also provides a pharmaceutical use of the fusion protein.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to Korean Patent Application No. 10-2022-0070914 filed on June 10, 2022, Korean Patent Application No. 10-2022-0112314 filed on September 5, 2022, and Korean Patent Application No. 10-2023-0007617 filed on January 18, 2023, which are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to a fusion protein comprising an anti-TIGIT antibody or an antigen-binding fragment thereof that specifically binds to TIGIT and interleukin-15 (IL-15), and pharmaceutical uses of the fusion protein. Background Art

[0004] Cancer immunotherapy is a method of treating cancer by utilizing the body's immune response, and is known as the fourth cancer treatment method after chemotherapy, surgery, and radiation therapy. Cancer immunotherapy requires a mechanism to enhance the recognition and response to tumor cells by activating the immune system. The activation of the immune system involves a complex mechanism that includes the functions of various cells, such as antigen-presenting cells, which are essential for initiating antigen-specific responses, and effector cells, which are responsible for destroying tumor cells.

[0005] A representative example of these effector cells is the cytotoxic T cell.

[0006] TIGIT (T cell immunoglobulin and ITIM (immunoreceptor tyrosine inhibitory motif) domain), also known as WUCAM, VSIG9 or Vstm3, is a co-inhibitory receptor that is mainly expressed in NK cells, CD8+T cells and CD4+T cells, and regulatory T cells (Treg). TIGIT is a transmembrane protein that contains an ITIM domain, a transmembrane domain, and an immunoglobulin variable domain.

[0007] In disease settings such as tumor infiltrating lymphocytes (TILs) and infections, the expression of TIGIT is increased. The expression of TIGIT can be a characteristic of exhausted T cells, which exhibit lower effector function compared to TIGIT-negative cells. In addition, Treg cells expressing TIGIT exhibit enhanced immunosuppressive activity compared to TIGIT-negative Treg populations.

[0008] Based on these properties, drugs that are antagonists to TIGIT (eg, antagonistic anti-TIGIT antibodies) are expected to induce activation of the immune system, enhance immune responses in disease states such as cancer, and thereby have a beneficial effect on disease treatment. Public Content

[0009] Technical issues

[0010] In one aspect, a fusion protein is provided, which comprises: (1) an anti-TIGIT antibody or an antigen-binding fragment thereof, which specifically recognizes TIGIT; and (2) IL-15.

[0011] In the fusion protein according to the embodiment, IL-15 can be directly linked to the C-terminus and / or N-terminus of the heavy chain (heavy chain variable region or heavy chain constant region) or light chain (light chain variable region or light chain constant region) of the anti-TIGIT antibody or its antigen-binding fragment through a linker or without a linker.

[0012] In another aspect, a nucleic acid molecule encoding a fusion protein is provided.

[0013] In another aspect, a recombinant vector carrying a nucleic acid molecule encoding a fusion protein is provided.

[0014] In another aspect, a recombinant cell having a nucleic acid molecule or a recombinant vector anchored thereat is provided.

[0015] On the other hand, an immunopotentiator or an immunopotentiating pharmaceutical composition is provided, which comprises as an active ingredient at least one substance selected from a fusion protein, a nucleic acid molecule encoding a fusion protein, a recombinant vector carrying a nucleic acid molecule, and a recombinant cell at which a nucleic acid molecule or a recombinant vector is anchored.

[0016] On the other hand, a pharmaceutical composition for preventing and / or treating immune-related diseases is provided, which comprises as an active ingredient at least one substance selected from a fusion protein, a nucleic acid molecule encoding a fusion protein, a recombinant vector carrying a nucleic acid molecule, and a recombinant cell at which a nucleic acid molecule or a recombinant vector is anchored.

[0017] On the other hand, an anticancer agent or pharmaceutical composition for preventing and / or treating cancer is provided, which comprises at least one substance selected from fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells at which nucleic acid molecules or recombinant vectors are anchored as an active ingredient.

[0018] On the other hand, a method for enhancing immunity is provided, which includes the step of administering a pharmaceutically effective amount of at least one substance to a subject in need of enhanced immunity, wherein the substance is selected from a fusion protein, a nucleic acid molecule encoding a fusion protein, a recombinant vector carrying a nucleic acid molecule, and a recombinant cell at which a nucleic acid molecule or a recombinant vector is anchored.

[0019] On the other hand, a method for preventing and / or treating immune-related diseases is provided, the method comprising the step of administering a pharmaceutically effective amount of one or more substances to a subject in need of prevention and / or treatment of immune-related diseases, the substances being selected from fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells containing nucleic acid molecules or recombinant vectors.

[0020] On the other hand, a method for preventing and / or treating cancer is provided, the method comprising the step of administering a pharmaceutically effective amount of at least one substance selected from a fusion protein, a nucleic acid molecule encoding a fusion protein, a recombinant vector carrying a nucleic acid molecule, and a recombinant cell at which a nucleic acid molecule or a recombinant vector is anchored to a subject in need of preventing and / or treating cancer.

[0021] On the other hand, provided is the use of at least one substance selected from fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells at which nucleic acid molecules or recombinant vectors are anchored for enhancing immunity or for preparing immunopotentiators.

[0022] On the other hand, provided is the use of at least one substance selected from a fusion protein, a nucleic acid molecule encoding a fusion protein, a recombinant vector carrying a nucleic acid molecule, and a recombinant cell at which a nucleic acid molecule or a recombinant vector is anchored for preventing and / or treating immune-related diseases or for preparing a drug for preventing and / or treating immune-related diseases.

[0023] On the other hand, provided is the use of at least one substance selected from fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells at which nucleic acid molecules or recombinant vectors are anchored for preventing and / or treating cancer or for preparing a drug for preventing and / or treating cancer.

[0024] Technical Solutions

[0025] Provided herein is a fusion protein comprising: an anti-TIGIT antibody or an antigen-binding fragment thereof that binds to TIGIT; and IL-15, and its pharmaceutical use. The fusion protein exhibits (1) the activity of blocking TIGIT function by an anti-TIGIT antibody or an antigen-binding fragment thereof, thereby activating the immune system (e.g., enhancing effector T cell function, regulating Treg activity, increasing cytokine secretion, etc.), and (2) due to the advantages of high binding affinity and low immunogenicity of IL-15 to receptors, it has been found to be used as various immune activators and / or immunotherapeutics.

[0026] Specific embodiments of the present disclosure will be given below.

[0027] Definition of terms

[0028] The phrases used in this article that a nucleic acid molecule (which can be used interchangeably with "polynucleotide" or "gene") or a polypeptide (which can be used interchangeably with "protein") "comprises a specific nucleic acid sequence or amino acid sequence", "consists of a specific nucleic acid sequence or amino acid sequence" or "is represented by a specific nucleic acid sequence or amino acid sequence" mean that the nucleic acid molecule or polypeptide essentially comprises the specific nucleic acid sequence or amino acid sequence, and can be interpreted as comprising a "substantially equivalent sequence" with changes (such as deletions, substitutions, modifications and / or additions) in the specific nucleic acid sequence or amino acid sequence (or does not exclude these changes in 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.

[0029] In an embodiment, the nucleic acid sequence or amino acid sequence provided herein may include mutants obtained by conventional mutagenesis methods, such as directed evolution or site-directed mutagenesis, as long as the mutant retains the original function or intended function of the sequence. In an embodiment, when a nucleic acid molecule or a polypeptide "comprises or consists of a specific nucleic acid sequence or an amino acid sequence", it means that the nucleic acid molecule or polypeptide (i) essentially comprises a specific nucleic acid sequence or an amino acid sequence, or (ii) essentially consists of or essentially comprises an amino acid sequence, which has 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, 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 function and / or intended function.

[0030] The nucleic acid sequences described herein may be modified in various ways within the coding region to reflect the codon preference of the microorganism in which the protein is expressed, as long as the amino acid sequence and / or function of the expressed protein remains unchanged due to the degeneracy of the codons.

[0031] The term "homology" as used herein refers to the degree of similarity between a given nucleic acid sequence or amino acid sequence and another sequence, which can be expressed as a percentage (%). The homology of nucleic acid sequences can be determined by 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).

[0032] Fusion Protein

[0033] According to one aspect thereof, the present disclosure provides a fusion protein comprising: (1) an anti-TIGIT antibody or an antigen-binding fragment thereof, which specifically recognizes TIGIT; and (2) IL-15.

[0034] In the fusion protein, the anti-TIGIT antibody or its antigen-binding fragment may have the activity of blocking TIGIT function to activate the immune system (e.g., enhancing effector T cell function, regulating Treg activity, increasing cytokine secretion, etc.). In addition, IL-15 is a safe polypeptide with high binding affinity to receptors and low immunogenicity, providing excellent immunoenhancing effects while minimizing the risk of the immune response recognizing it as an antigen when administered in vivo. Therefore, the fusion protein can be used as a variety of immunoenhancers and / or immunotherapeutics.

[0035] In the fusion protein according to the embodiment, IL-15 can be directly linked to the C-terminus and / or N-terminus of the heavy chain (heavy chain variable region or heavy chain constant region) or light chain (light chain variable region or light chain constant region) of the anti-TIGIT antibody or its antigen-binding fragment through a linker or without a linker.

[0036] More specifically, the fusion protein may comprise: (1) an anti-TIGIT antibody (e.g., an IgG-type antibody comprising two heavy chains and two light chains) and (2) an IL-15, wherein the IL-15 is directly linked to the C-terminus (C-terminus of the constant region) or N-terminus of one of the two heavy chains or one of the two light chains of the anti-TIGIT antibody with or without a peptide linker.

[0037] In another embodiment, the fusion protein may comprise: (1) an anti-TIGIT antibody (e.g., an IgG-type antibody comprising two heavy chains and two light chains); and (2) two IL-15 molecules, wherein each IL-15 is directly linked to the C-terminus or N-terminus of one of the two heavy chains or one of the two light chains of the anti-TIGIT antibody with or without a linker.

[0038] For example, in a fusion protein:

[0039] (i) one of the two IL-15 molecules may be directly linked 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 via a linker or without a linker, and the other IL-15 molecule may be directly linked 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 the other heavy chain of the anti-TIGIT antibody via a linker or without a linker, or

[0040] (ii) One of the two IL-15 molecules can be directly connected 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 through a linker or without a linker, and the other IL-15 molecule can be directly connected 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 through a linker or without a linker.

[0041] More specifically, in the fusion protein:

[0042] (i) one of the two IL-15 molecules may be directly linked to the C-terminus of one of the Fc regions of the two heavy chains of the anti-TIGIT antibody with or without a linker, and the other IL-15 molecule may be directly linked to the C-terminus of the other Fc region of the anti-TIGIT antibody with or without a linker, or

[0043] (ii) One of the two IL-15 molecules can be directly linked to the C-terminus of one of the two light chains of the anti-TIGIT antibody with or without a linker, and the other IL-15 molecule can be directly linked to the C-terminus of the other light chain of the anti-TIGIT antibody with or without a linker.

[0044] The linker can be, but is not limited to, a rigid peptide linker (e.g., [EAAAK]n, wherein n is an integer from 1 to 5), a flexible peptide linker (e.g., [(G)mS]l, wherein m is an integer from 1 to 5, and l is an integer from 1 to 5).

[0045] Anti-TIGIT antibodies or antigen-binding fragments thereof

[0046] The anti-TIGIT antibody or antigen-binding fragment thereof may comprise:

[0047] A polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 (CDR-H1),

[0048] A polypeptide comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 (CDR-H2),

[0049] A polypeptide comprising the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9 (CDR-H3),

[0050] A polypeptide comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 13 or SEQ ID NO: 14 (CDR-L1),

[0051] A polypeptide comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17 (CDR-L2), and

[0052] A polypeptide comprising the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20 (CDR-L3).

[0053] The polypeptide (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 10 may comprise the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.

[0054] Here, the complementarity determining regions (CDRs) are determined based on the CDR definition according to the Kabat system.

[0055] In an embodiment, six CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) that may be included in an anti-TIGIT antibody or antigen-binding fragment thereof are shown in Table 1 below:

[0056] [Table 1]

[0057]

[0058] (In Table 1, CDR-H1, CDR-H2, and CDR-H3 represent the complementarity determining regions on the heavy chain, and CDR-L1, CDR-L2, and CDR-L3 represent the complementarity determining regions on the light chain.)

[0059] In embodiments, the anti-TIGIT antibody or antigen-binding fragment thereof may comprise:

[0060] A heavy chain variable region comprising CDR-H1 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, CDR-H2 of SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, and CDR-H3 of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9; and

[0061] A light chain variable region comprising CDR-L1 of SEQ ID NO: 10 (e.g., SEQ ID NO: 11 or SEQ ID NO: 12), SEQ ID NO: 13 or SEQ ID NO: 14, CDR-L2 of SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, and CDR-L3 of SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

[0062] More specifically, the anti-TIGIT antibody or antigen-binding fragment thereof may comprise:

[0063] a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28; and

[0064] A light chain variable region comprising the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO: 36.

[0065] In certain cases (eg, during recombinant construction), the heavy chain variable region and / or the light chain variable region may also contain a suitable signal sequence at its N-terminus.

[0066] Table 2 below illustrates the amino acid sequences of the heavy chain variable region and the light chain variable region that may be included in the anti-TIGIT antibody or antigen-binding fragment thereof:

[0067] [Table 2]

[0068]

[0069]

[0070]

[0071] (In Table 2, the underlined regions represent CDR1, CDR2, and CDR3 of the heavy chain and light chain, respectively.)

[0072] In an embodiment, the anti-TIGIT antibodies or antigen-binding fragments thereof provided herein may bind to one or more amino acids selected from residues 51 to 70 of the human TIGIT protein (TAQVTQVNWEQQDQLLAICN; SEQ ID NO: 37), for example, but not limited to, the human TIGIT protein (SEQ ID NO: 64; NCBI reference sequence NP_776160.2; UniProtKB / SwissProt Q495A1-1).

[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

[0079] The term "antibody" as used herein refers to a protein that specifically binds to a particular antigen. Such a protein can be produced in the immune system in response to antigenic stimulation, or can be chemically synthesized or recombinantly produced, and there is no limitation on its type. The antibody can be a non-naturally produced antibody, such as a recombinant or synthetically produced antibody. The antibody can be an animal antibody (e.g., a mouse antibody), a chimeric antibody, a humanized antibody, or a human antibody. The antibody can be a monoclonal antibody or a polyclonal antibody.

[0080] In the anti-TIGIT antibodies or antigen-binding fragments thereof 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 may be derived from immunoglobulins of any subtype (e.g., IgA, IgD, IgE, IgG (IgG1, IgG2, IgG3, or IgG4), IgM, etc.). For example, the remaining regions may be derived from framework regions of any immunoglobulin subtype and / or from constant regions of light and / or heavy chains. In embodiments, the anti-TIGIT antibodies provided herein may be human IgG-type antibodies, such as, but not limited to, in the form of IgG1, IgG2, IgG3, or IgG4.

[0081] A complete antibody (such as IgG type) has a structure comprising two full-length light chains and two full-length heavy chains, wherein each light chain is connected to a heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region. The heavy chain constant region has gamma (γ) type, mu (μ) type, alpha (α) type, delta (δ) type and epsilon (ε) type, among which subtypes include gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1) and alpha2 (α2). The light chain constant region has kappa (κ) type and lambda (λ) type.

[0082] In an embodiment, the anti-TIGIT antibodies provided herein may include, but are not limited to, an IgG constant region as a heavy chain constant region and a kappa constant region as a light chain constant region.

[0083] In an embodiment, the constant region of IgG (such as human IgG1) can be wild type. In another embodiment, the constant region of IgG can be a variant, which comprises one or more mutations selected from 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 deletion.

[0089] The term "heavy chain" is understood to include full-length heavy chains and fragments thereof, which include variable region domains (V H ), and three constant region domains C H1 , C H2 and C H3 The term "light chain" is understood to include full-length light chains and fragments thereof, which contain variable region domains V and V containing amino acid sequences sufficient to confer antigen specificity. L and constant region domain C L .

[0090] The term "complementarity determining region" (CDR) refers to the region of an antibody that confers antigen binding specificity, which comprises the amino acid sequence of the heavy chain hypervariable region and the light chain hypervariable region of an immunoglobulin. The heavy chain and light chain may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). These CDRs can provide key contact residues for the binding of antibodies to antigens or epitopes. The terms "specific binding" or "specific recognition" used herein refer to the interaction between an antigen and an antibody that results in an immune response, which is well known in the art.

[0091] The complementarity determining regions (CDRs) described herein are defined based on the Kabat system.

[0092] Unless otherwise indicated, the term "antibody" as used herein is understood to include antigen-binding fragments of antibodies that retain antigen-binding ability.

[0093] The term "antigen-binding fragment" refers to any form of polypeptide comprising a portion that is capable of binding to an antigen (eg, the six CDRs as 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 light chain variable region and a heavy chain variable region, a light chain constant region and a heavy chain first constant region (C H1 ) structure, which has a single antigen binding site.

[0095] Fab' differs from Fab in that the former has a H1 The C-terminus of the domain includes a hinge region containing one or more than one cysteine ​​residues.

[0096] When the cysteine ​​residues in the hinge region of Fab' form a disulfide bond, an F(ab')2 antibody is generated. Fv is the smallest antibody fragment, which contains only the variable regions of the heavy chain and the light chain. Recombinant techniques for generating Fv fragments are well known in the art.

[0097] A double-chain Fv comprises a heavy chain variable region and a light chain variable region, which are non-covalently linked, while a single-chain Fv (scFv) comprises a light chain variable region and a heavy chain variable region, which are usually covalently linked via a peptide linker or directly covalently linked at the C-terminus to form a dimer-like structure similar to a double-chain Fv.

[0098] Antigen-binding fragments can be obtained using proteolytic enzymes (eg, Fab can be obtained by restriction digestion of whole antibodies with papain, and F(ab')2 fragments can be obtained by digestion with pepsin), or can be produced by recombinant DNA technology.

[0099] The term "hinge region" refers to the region of an antibody heavy chain located between the C H1 Domain and C H2 between domains, whose function is to provide flexibility to the antigen-binding site in antibodies.

[0100] The antibodies provided herein can be monoclonal antibodies. Monoclonal antibodies can be produced by methods well known in the art. For example, phage display technology can be used to produce monoclonal antibodies, or conventional methods can be used to generate monoclonal antibodies from animals (e.g., mice).

[0101] Standard ELISA (enzyme-linked immunosorbent assay) methods can be used to screen individual monoclonal antibodies based on their ability to bind to the TIGIT receptor binding domain. For example, competitive ELISA or cell-based functional assays can be used to assess the interaction and inhibitory activity of the molecules. The affinity (Kd value) of each monoclonal antibody to the TIGIT receptor binding domain can then be assessed based on the selected antibodies with strong inhibitory activity.

[0102] In the finally selected antibody, regions other than the antigen binding site may be humanized immunoglobulin antibodies or human immunoglobulin antibodies, and methods for producing humanized antibodies are well known in the art.

[0103] The antigen-binding fragment of the anti-TIGIT antibody provided herein refers to a fragment derived from an anti-TIGIT antibody that retains binding affinity to an antigen (TIGIT), which may be any polypeptide comprising the six CDRs of the above-mentioned anti-TIGIT antibody, such as but not limited to scFv, scFv-Fc, scFv-Cκ (kappa constant region), scFv-Cλ (lambda constant region), (scFv)2, Fab, Fab' or F(ab')2. In an embodiment, the antigen-binding fragment may be, but is not limited to, scFv or a fusion polypeptide, wherein the fusion polypeptide is fused with the Fc region (scFv-Fc) of an immunoglobulin (such as IgG1, IgG2, IgG3, IgG4, etc.) or with the constant region (scFv-Cκ or scFv-Cλ) of a light chain (such as kappa or lambda).

[0104] The phrase "comprising a specific amino acid sequence" or "comprising or consisting of a specific amino acid sequence" used in this article for an antibody (e.g., CDR, variable region or heavy chain / light chain, antigen-binding fragment, etc.) includes not only the case where the antibody necessarily comprises a specific amino acid sequence, but also includes the case where modifications (e.g., substitutions, deletions and / or additions) that do not significantly affect the activity of the antibody (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 have a binding affinity (K) to TIGIT (e.g., human TIGIT) of, e.g., 10 mM or less, 5 mM or less, 1 mM or less, 0.5 mM or less, 0.2 mM or less, 0.1 mM or less, 0.05 mM or less, 0.01 mM or less, 0.005 mM or less, 0.001 ... D ). Specifically, K DThe amount of the mixture may be, but is not limited to, 0.0001 nM to 10 mM, 0.0005 nM to 10 mM, 0.001 nM to 10 mM, 0.005 nM to 10 mM, 0.01 nM to 10 mM, 0.05 nM to 10 mM, 0.1 nM to 10 mM, 0.5 nM to 10 mM, 1 nM to 10 mM, 0.0001 nM to 5 mM, 0.0005 nM to 5 mM, 0.001 nM to 5 mM, 0.005 nM to 5 mM, 0.01 nM to 5 mM, 0.05 nM to 5 mM, 0.1 nM to 5 mM, 0. 5nM to 5mM, 1nM to 5mM, 0.0001nM to 1mM, 0.0005nM to 1mM, 0.001nM to 1mM, 0.005nM to 1mM, 0.01nM to 1mM, 0.05nM to 1mM, 0.1nM to 1mM, 0.5nM to 1mM, 1nM to 1mM, 0.0001nM to 0.5mM, 0.0005nM to 0.5mM, 0.001nM to 0.5mM, 0.005nM to 0.5mM, 0.01nM to 0.5mM, 0.05nM to 0.5mM, 0.1nM to 0.5mM, 0.5nM to 0.5mM, 1nM to 0.5mM, 0.0001nM to 0.2mM, 0.0005nM to 0.2mM, 0.001nM to 0.2mM, 0.005nM to 0.2mM, 0.01nM to 0.2mM, 0.05nM to 0.2mM, 0.1nM to 0.2mM, 0.5nM to 0.2mM, 1nM to 0.2mM, 0.0001nM to 0.1mM, 0.0005nM to 0.1mM, 0.001nM to 0.1mM, 0.00 In some embodiments, the present invention relates to a method for preparing an aqueous solution of at least one embodiment of the present invention that is preferably at least 0.5 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 0.5 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, or 1 nM to 0.05 mM.

[0106] Interleukin-15 (IL-15)

[0107] Interleukin-15 (IL-15) can be selected from:

[0108] (1) wild-type IL-15 polypeptide,

[0109] (2) 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 to polypeptide (1).

[0110] Interleukin-15 (IL-15) has a similar structure to interleukin-2 (IL-2), and binds to the IL-2 / IL-15 receptor beta chain (CD122) and common gamma chain (gamma-C, CD132) complex, through which it participates in signal transduction. In an embodiment, IL-15 can be derived from humans, such as, but not limited to, NCBI accession number NP_000576.1 (amino acid residues 49 to 162).

[0111] In an embodiment, IL-15 may have the following amino acid sequence (N-terminus → C-terminus):

[0112] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 38).

[0113] Polypeptide (1) may contain other oligopeptides for other purposes, such as but not limited to facilitating protein purification, such as a tag for labeling at the N-terminus (eg MAPRRARGCRTLGLPALLLLLLLRPPATRGDYKDDDDKIEGR (SEQ ID NO: 76)).

[0114] Due to its human origin, interleukin-15 is characterized by low immunogenicity when administered to humans.

[0115] IL-15 can exhibit binding activity to its receptor (e.g., binding activity to the IL-2 / IL-15 receptor beta chain (CD122) and / or the IL-2 / IL-15 receptor beta chain (CD122) and common gamma chain (gamma-C, CD132) complex), and / or cytokine production activity and / or secretion activity in immune cells (e.g., T cells (CD4+, CD8+), NK cells, etc.).

[0116] In an embodiment, IL-15 may be selected from:

[0117] (1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 38,

[0118] (2) A polypeptide having 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homology with polypeptide (1), and exhibiting binding activity to IL-2 / IL-15 receptors and / or cytokine production and / or secretion activity in immune cells. IL-15 may exhibit stronger binding activity to IL-2 / IL-15 receptors, and / or cytokine production and / or secretion activity in immune cells, and / or anti-cancer activity (e.g., inhibiting cancer cell proliferation, inducing cancer cell apoptosis).

[0119] Connectors

[0120] The linker can be a peptide linker, such as, but not limited to, [EAAAK]n (n is an integer from 1 to 5, representing the number of [EAAAK] (SEQ ID NO: 77)), [(G)mS]l (m is an integer from 1 to 5, representing the number of G residues, l is an integer from 1 to 5, representing the number of [(G)mS]), A EAAAK A (SEQ ID NO: 78), A EAAAK EAAAK A (SEQ ID NO: 79), A EAAAKEAAAK EAAAK A (SEQ ID NO: 80), A EAAAK EAAAK EAAAK EAAAK A (SEQ ID NO: 81), AEAAAK EAAAK EAAAK EAAAK A (SEQ ID NO: 82), AEAAAKEAAAKAG (SEQ ID NO: 83), AEAAAKEAAAKAGS (SEQ ID NO: 84), GGGGG (SEQ ID NO: 85), GGAGG (SEQ ID NO: 86), GGGGGGGG (SEQ ID NO: 87). NO: 87), GAGAGAGAGA (SEQ ID NO: 88), RPLSYRPPFPFGFPSVRP (SEQ ID NO: 89), YPRSIYIRRRHPSPSLTT (SEQ ID NO: 90), TPSHLSHILPSFGLPTFN (SEQ ID NO: 91), RPVSPFTFPRLSNSWLPA (SEQ ID NO: 92), SPAAHFPRSIPRPGPIRT (SEQ ID NO: 93), APGPSAPSHRSLPSRAFG (SEQ ID NO: 94), PRNSIHFLHPLLVAPLGA (SEQ ID NO: 95), MPSLSGVLQVRYLSPPDL (SEQ ID NO: 96), SPQYPSPLTLTLPPHPSL (SEQ ID NO: 97), NPSLNPPSYLHRAPSRIS (SEQ ID NO: 98), LPWRTSLLPSLPLRRRP (SEQ ID NO: 99), PPLFAKGPVGLLSRSFPP (SEQ ID NO: 100), VPPAPVVSLRSAHARPPY (SEQ ID NO: 101), LRPTPPRVRSYTCCPTP (SEQ ID NO: 102), PNVAHVLPLLTVPWDNLR (SEQ ID NO: 103), CNPLLPLCARSPAVRTFP (SEQ ID NO:104), LGTPTPTPTPTGEF (SEQ ID NO: 105), EDFTRGKL (SEQ ID NO: 106), L EAAAR EAAAR EAAAR EAAAR (SEQ ID NO: 107), L EAAAR EAAAREAAAR (SEQ ID NO: 108), L EAAAR (SEQ ID NO: 109), L EAAAR EAAAR (SEQ ID NO: 110), EAAAR EAAAR EAAAR EAAAR (SEQ ID NO: 111), EAAAR EAAAR EAAAR (SEQ ID NO: 112), EAAAR EAAAR (SEQ ID NO: 113), EAAAR (SEQ ID NO: 114), LTEEQQEGGG (SEQ ID NO: 115), TEEQQEGGG (SEQ ID NO: 116), LAKLKQKTEQLQDRIAGGG(SEQ ID NO: 117), LELKTPLGDT(SEQID NO: 118), THTCPRCPEP (SEQ ID NO: 119), KSCDTPPPCP (SEQ ID NO: 120), RCPEPKSCDT (SEQ ID NO: 121), PPPCPRCPEP (SEQ ID NO: 122), KSCDTPPPCP (SEQ ID NO: 123), RCPGG (SEQ ID NO: 124), and LEPKSSDKTHTSPPSPGG (SEQ ID NO: 125). In an embodiment, the peptide linker can be a rigid linker, such as but not limited to [EAAAK]n (wherein n is an integer from 1 to 5, representing the number of [EAAAK]), or a flexible linker, such as but not limited to [(G)mS]l (wherein 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]).

[0121] Nucleic acid molecules, recombinant vectors and recombinant cells

[0122] In another aspect, a nucleic acid molecule encoding a fusion protein is provided.

[0123] In another aspect, a recombinant vector carrying a nucleic acid molecule is provided. The vector can be used as an expression vector of the nucleic acid molecule.

[0124] In another aspect, a recombinant cell anchoring a nucleic acid molecule or a recombinant vector carrying a nucleic acid molecule is provided. The cell can be used to produce a fusion protein.

[0125] In another aspect, a method for producing a fusion protein is provided, the method comprising the step of expressing a nucleic acid molecule in a suitable host cell. The step of expressing a nucleic acid molecule in a suitable host cell may comprise culturing the above-mentioned recombinant cell under standard conditions.

[0126] The term "vector" refers to a means for expressing a target gene in a host cell. Examples of vectors include plasmid vectors, cosmid vectors, and viral vectors, such as phage vectors, adenoviral vectors, retroviral vectors, and adeno-associated viral 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, pGEX series, pET series, and pUC19), phages (e.g., λgt4λB, λ-Charon, λΔz1, and M13), or viruses (e.g., SV40).

[0127] In a recombinant vector, a nucleic acid molecule encoding a fusion protein can be operably linked to a promoter. The term "operably linked" refers to a functional connection between a nucleotide regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. When "operably linked," the regulatory sequence controls the transcription and / or translation of the other nucleotide sequence.

[0128] Recombinant vectors can generally be constructed as cloning vectors or expression vectors. Expression vectors can be any conventional vectors used in the art for expressing foreign proteins in plants, animals or microorganisms. Recombinant vectors can be constructed using various methods known in the art.

[0129] Recombinant vectors can be constructed using prokaryotic cells or eukaryotic cells as hosts. For example, when the vector is an expression vector and a prokaryotic cell is used as a host, the vector generally includes a strong promoter (e.g., pL λ The vector may include a promoter, a CMV promoter, a trp promoter, a lac promoter, a tac promoter, a T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When a eukaryotic cell is used as a host, the vector may include a replication origin derived from a eukaryotic cell, such as, but not limited to, a f1 origin, a SV40 origin, a pMB1 origin, an adenovirus origin, an AAV origin, and a BBV origin. In addition, the vector may include a promoter derived from a mammalian genome (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, a SV40 promoter, a cytomegalovirus promoter, and a HSVtk promoter), and generally includes a polyadenylation sequence as a transcription termination sequence.

[0130] Recombinant cells can be obtained by introducing the recombinant vector into a suitable host cell. As long as it is known in the art, any host cell that can stably and continuously clone or express the recombinant vector can be used. For example, prokaryotic host cells can include Escherichia coli (E. 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, and enterobacteria such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. For eukaryotic host cells, transformed host cells can include yeast (Saccharomyces cerevisiae), insect cells, plant cells, and animal cells, such as, but not limited to, Sp2 / 0 cell line, CHO (Chinese Hamster Ovary) K1 cell line, CHO cell line, DG44 cell line, PER.C6 cell line, W138 cell line, BHK cell line, COS-7 cell line, 293 cell line, HepG2 cell line, Huh7 cell line, 3T3 cell line, RIN cell line, MDCK cell line.

[0131] The delivery (introduction) of nucleic acid molecules or recombinant vectors into host cells can be carried out using commonly known methods. For example, when the host cell is a prokaryotic cell, a method such as, but not limited to, the CaCl2 method or electroporation method can be used; when the host cell is a eukaryotic cell, a method such as, but not limited to, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection or gene bombardment can be used.

[0132] The method for selecting transformed (introduced recombinant vector) host cells can be easily performed based on the phenotype expressed by selectable markers, using methods well known in the art. For example, if the selectable marker is a gene resistant to a specific antibiotic, the recombinant cells introduced into the recombinant vector can be easily selected by culturing them in a culture medium containing the antibiotic.

[0133] Drug Use

[0134] The anti-TIGIT antibody or its antigen-binding fragment contained in the fusion protein provided herein plays a role in blocking the effect of TIGIT (e.g., the interaction between TIGIT and its ligand CD155 (PVR)), thereby activating the immune system (e.g., enhancing effector T cell function, regulating Treg activity, increasing cytokine secretion), and it also exhibits good anti-cancer effects (e.g., cancer cell apoptosis, inhibiting cancer cell proliferation). In addition, IL-15 has a high binding affinity and low immunogenicity to its receptor (IL-2 / IL-15 receptor), so it can enhance the immune response when administered in vivo, such as increasing cytokine secretion, while maintaining safety by minimizing the risk of being identified as an immunogen. IL-15 also exhibits excellent anti-cancer effects (e.g., cancer cell apoptosis, inhibiting cancer cell proliferation). Therefore, the fusion peptide combines the advantages of anti-TIGIT antibodies or their antigen-binding fragments and IL-15, and can be effectively used in immune enhancement, prevention and / or treatment of immune-related diseases, particularly for the prevention and / or treatment of cancer.

[0135] On the one hand, an immunopotentiator or a pharmaceutical composition for immunopotentiation is provided, which comprises at least one substance selected from a fusion protein, a nucleic acid molecule encoding a fusion protein, a recombinant vector carrying a nucleic acid molecule, and a recombinant cell anchoring a nucleic acid molecule or a recombinant vector as an active ingredient.

[0136] On the other hand, a pharmaceutical composition for preventing and / or treating immune-related diseases is provided, which comprises at least one substance selected from fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells anchoring nucleic acid molecules or recombinant vectors as an active ingredient.

[0137] On the other hand, an anticancer agent or a pharmaceutical composition for preventing and / or treating cancer is provided, which comprises at least one substance selected from fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells anchoring nucleic acid molecules or recombinant vectors as an active ingredient.

[0138] On the other hand, a method for enhancing immunity is provided, the method comprising the step of administering a pharmaceutically effective amount of at least one substance to a subject in need of immune enhancement, the substance being selected from the group consisting of a fusion protein, a nucleic acid molecule encoding the fusion protein, a recombinant vector carrying the nucleic acid molecule, and a recombinant cell anchoring the nucleic acid molecule or the recombinant vector. The immune enhancement method may also include the step of identifying a subject in need of immune enhancement before the administering step.

[0139] On the other hand, a method for preventing and / or treating an immune-related disease is provided, the method comprising applying a pharmaceutically effective amount of at least one substance to an object that needs to prevent and / or treat an immune-related disease, the substance being selected from a fusion protein, a nucleic acid molecule encoding a fusion protein, a recombinant vector carrying a nucleic acid molecule, and a recombinant cell anchoring a nucleic acid molecule or a recombinant vector. The method for preventing and / or treating an immune-related disease may also include a step of identifying an object that needs to prevent and / or treat an immune-related disease before the administering step.

[0140] On the other hand, a method for preventing and / or treating cancer is provided, the method comprising the step of applying a pharmaceutically effective amount of at least one substance to a subject in need of preventing and / or treating cancer, the substance being selected from a fusion protein, a nucleic acid molecule encoding a fusion protein, a recombinant vector carrying a nucleic acid molecule, and a recombinant cell anchoring a nucleic acid molecule or a recombinant vector. The method for preventing and / or treating cancer may also include the step of identifying a subject in need of preventing and / or treating cancer before the administering step.

[0141] On the other hand, provided is the use of at least one agent selected from a fusion protein, a nucleic acid molecule encoding a fusion protein, a recombinant vector carrying a nucleic acid molecule, and a recombinant cell anchoring a nucleic acid molecule or a recombinant vector for use in immune enhancement, prevention and / or treatment of immune-related diseases, and / or prevention and / or treatment of cancer, or for the manufacture of a pharmaceutical composition for use in immune enhancement, prevention and / or treatment of immune-related diseases, and / or prevention and / or treatment of cancer.

[0142] The term "immune enhancement" as used herein refers to inducing a primary immune response to an antigen or increasing an existing immune response, and can be used interchangeably with terms such as immunopotentiation, immune activation, etc. More specifically, the "immune enhancement effect" of the fusion protein provided herein may include, but is not limited to, enhancing the function (activation) and / or proliferation of immune cells (e.g., NK cells, NKT cells, CD3+T cells, effector T cells (CD4+T cells), cytotoxic T cells (CD8+T cells), etc.), inhibiting and / or consuming the activity of regulatory T (Treg) cells, increasing the production and / or secretion of immune proteins (e.g., cytokines, such as IL-2, IFN-gamma, etc.), and activation of IL-15 receptors (located on the surface of immune cells (NK cells, etc.)) (e.g., confirmed as IL2RB / IL2RG dimerization, etc.).

[0143] As used herein, the term "immune-related disease" is intended to include all diseases caused by a disorder and / or insufficient activation of the immune system, including but not limited to cancer, infectious diseases, autoimmune diseases, and inflammatory diseases.

[0144] The cancer may be a solid tumor or a blood cancer, and may be, but is not limited to, at least one selected from squamous cell carcinoma, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma), peritoneal cancer, skin cancer, melanoma (e.g., cutaneous melanoma or ocular melanoma), rectal cancer, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic leukemia or acute leukemia, lymphocytic lymphoma, liver cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, head and neck cancer, brain cancer, and osteosarcoma.

[0145] The preventive and / or therapeutic effects on cancer include not only inhibiting the appearance and / or growth of cancer cells, but also inhibiting the progression of cancer caused by migration, invasion and metastasis.

[0146] Infectious diseases, autoimmune diseases and inflammatory diseases can be any diseases that can be treated, alleviated and / or prevented by the aforementioned immune enhancement (e.g., enhancing the function (activation) and / or proliferation of immune cells (e.g., NK cells, NKT cells, CD3+T cells, effector T cells (CD4+T cells), cytotoxic T cells (CD8+T cells), etc.), inhibiting and / or consuming the activity of regulatory T (Treg) cells, increasing the production and / or secretion of immune proteins (e.g., cytokines, such as IL-2, IFN-gamma, etc.), etc.). For example, infectious diseases are a general term for diseases caused by pathogens that spread and invade organisms (e.g., animals, including humans), such as viruses (e.g., influenza viruses, coronaviruses (e.g., severe acute respiratory syndrome (SARS) viruses (SARS-CoV, SARS-CoV-2), Middle East respiratory syndrome (MERS) viruses (MERS-CoV), cytomegalovirus (CMV), Epstein-Barr virus (EBV)), bacteria, fungi or parasites), which may include infections caused by at least one pathogen selected from viruses, bacteria, fungi and parasites and the diseases caused thereby. Autoimmune diseases may include, but are not limited to, 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, and cytokine storm.Inflammatory diseases are a general term for inflammation (chronic inflammation or acute inflammation) or diseases caused by inflammation, and may be selected from, but not limited to, cardiac inflammation (e.g., coronary artery disease, angina, myocardial infarction, pericarditis, myocarditis), vascular inflammation (e.g., 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), ), inflammation of the lower respiratory tract and / or lungs (e.g. bronchitis, bronchiectasis, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, interstitial lung disease, tuberculosis), inflammation of the upper digestive tract (e.g. gastritis, esophagitis), inflammation of the lower digestive tract (e.g. enteritis, ulcerative colitis, Crohn's disease, celiac disease, diverticulitis, irritable bowel syndrome, appendicitis, fistula), inflammation of the liver, bile ducts and / or pancreas (e.g. hepatitis, fatty liver, cholangitis, cholecystitis, pancreatitis, type 1 diabetes), inflammation of the kidneys (upper urinary tract) (e.g. pyelonephritis, nephrotic syndrome), glomerulonephritis, urinary tract infection), lower urinary tract inflammation (e.g., urinary tract infection, ureteritis, urethritis, cystitis, prostatitis / chronic pelvic pain syndrome), thyroid and / or parathyroid inflammation (e.g., thyroiditis, parathyroiditis), adrenal gland inflammation (e.g., adrenalitis), reproductive organ inflammation (e.g., pelvic inflammatory disease, oophoritis, orchitis, epididymitis), bone inflammation and / or joint inflammation (e.g., osteoarthritis, rheumatoid arthritis, osteomyelitis, synovitis), skin inflammation (e.g., cellulitis, erysipelas, tinea pedis, acne), muscle inflammation (e.g., At least one of the following: myositis, brain inflammation (e.g., encephalitis, major depressive disorder), neuroinflammation (e.g., neuritis in different parts of the eye or ear, complex regional pain syndrome, Guillain-Barré syndrome), eye inflammation (e.g., hordeolum, uveitis, conjunctivitis), ear inflammation (e.g., otitis media, mastoiditis), oral inflammation (e.g., stomatitis, periodontitis, gingivitis), systemic inflammation (e.g., systemic inflammatory response syndrome (SIRS), metabolic syndrome-related diseases), peritonitis, reperfusion injury, transplant rejection, and hypersensitivity reaction.

[0147] The object of administering the fusion protein and / or pharmaceutical composition provided herein can be any animal or any cell. For example, the object can be an animal selected from mammals, including humans, primates such as monkeys, rodents such as rats and mice, or cells, tissues, body fluids (such as serum) or cultures derived from (separated from) these animals, such as human cells, human tissues or human body fluids (such as serum) derived from humans.

[0148] In addition to the fusion protein, nucleic acid molecule, carrier and / or cell as active ingredient, the pharmaceutical composition may also include a pharmaceutically acceptable carrier. The carrier may be any carrier commonly used in the preparation of protein drugs, which may be, but is not limited to, selected from lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talcum, magnesium stearate and mineral oil. The pharmaceutical composition may also include at least one material selected from diluents, excipients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents and preservatives that are commonly used in the preparation of pharmaceutical compositions.

[0149] The content of the fusion protein, nucleic acid molecule, vector and / or cell as an active ingredient in the pharmaceutical composition may be, but is not limited to, 0.00001% to 99% 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, 10% to 99% by weight, 15% to 99% by weight, 20% to 99% by weight, 25% to 99% by weight, 30% to 99% by weight, 35% to 99% by weight, 40% to 99% by weight, 45% to 99% by weight, or 50% to 99% by weight, based on the total weight of the composition.

[0150] Fusion proteins, nucleic acid molecules, vectors, cells and / or pharmaceutical compositions can be administered by oral or parenteral routes. Parenteral administration can include intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal injection, topical application, nasal administration, pulmonary administration and rectal administration. For oral administration, since proteins or peptides are digested, oral compositions should be formulated to protect the active agent from degradation in the stomach.

[0151] In addition, the fusion protein, nucleic acid molecule, vector, cell and / or pharmaceutical composition can be formulated as a solution, suspension, syrup or emulsion in an oil medium or an aqueous medium, or as an extract, powder, granule, tablet, capsule or injection, and may also contain a dispersant or stabilizer for formulation.

[0152] The content of active ingredient in the pharmaceutical composition can vary according to formulation method, route of administration, patient's age, body weight, sex, disease state, diet, administration time, administration interval, route of administration, excretion rate and reaction sensitivity. Based on active ingredient (fusion protein, nucleic acid molecule, carrier, cell), the daily dose of the pharmaceutical composition can be, but is not limited to, 0.00001 mg / kg to 1000 mg / kg, 0.00001 mg / kg to 500 mg / kg, 0.00001 mg / kg to 100 mg / kg, 0.00001 mg / kg to 50 mg / kg, 0.0001 mg / kg to 1000 mg / kg, 0.0001 mg / kg to 500 mg / kg, 0.0001 mg / kg to 100 mg / kg, 0.0001 mg / kg to 50 mg / kg, 0.001 mg / kg to 10 ... The dosage of the present invention can be 0.1mg / kg to 1000mg / kg, 0.001mg / kg to 500mg / kg, 0.001mg / kg to 100mg / kg, 0.001mg / kg to 50mg / kg, 0.01mg / kg to 1000mg / kg, 0.01mg / kg to 500mg / kg, 0.01mg / kg to 100mg / kg, 0.01mg / kg to 50mg / kg, 0.1mg / kg to 1000mg / kg, 0.1mg / kg to 500mg / kg, 0.1mg / kg to 100mg / kg, or 0.1mg / kg to 50mg / kg. Daily dosage can be formulated as a single unit dose or suitable packaging, or made into a multi-dose container. The term "pharmaceutically effective amount" used herein refers to the amount of the active ingredient showing the desired pharmaceutical activity, which can be within the above dosage range.

[0153] On the other hand, a health functional food for enhancing immunity or promoting metabolism is provided, wherein the composition comprises at least one substance selected from fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells anchoring nucleic acid molecules or recombinant vectors as an active ingredient.

[0154] On the other hand, a health functional food for preventing and / or improving immune-related diseases is provided, wherein the food comprises at least one substance selected from fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells anchoring nucleic acid molecules or recombinant vectors as an active ingredient.

[0155] On the other hand, a health functional food for preventing and / or improving cancer is provided, wherein the food comprises at least one substance selected from fusion proteins, nucleic acid molecules encoding fusion proteins, recombinant vectors carrying nucleic acid molecules, and recombinant cells anchoring nucleic acid molecules or recombinant vectors as an active ingredient.

[0156] The term "health functional food" as used herein refers to food manufactured (processed) using nutrients that are easily lacking in the daily diet or raw materials or ingredients that are beneficial to the human body (hereinafter referred to as "functional raw materials"), which is intended to include all foods that are beneficial to maintaining health or preventing and / or alleviating certain diseases or symptoms. There is no specific restriction on the final product. For example, health functional food can be selected from, but not limited to, various foods, beverages and food additives.

[0157] The food composition may contain acceptable food additives, and may also contain suitable carriers, fillers and diluents commonly used in producing health functional foods.

[0158] When used as a food additive, the food composition may be added directly or used together with other foods or food ingredients, and may be appropriately used by conventional methods.

[0159] The content of the active ingredient in the food composition can be appropriately determined according to the form, expected effect and purpose of the food, and the active ingredient can be, but is not limited to, 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 weight of the food.

[0160] Beneficial Effects

[0161] The anti-TIGIT antibody or its antigen-binding fragment contained in the fusion protein provided herein blocks the action of TIGIT (e.g., the interaction between TIGIT and its ligand CD155 (PVR)), thereby activating the immune system (e.g., enhancing effector T cell function, regulating and / or consuming Treg cells, increasing cytokine secretion) and exhibiting good anti-cancer effects (e.g., cancer cell apoptosis, inhibiting cancer cell proliferation). In addition, IL-15 has high binding affinity and low immunogenicity to its receptor (IL-2 / IL-15 receptor), resulting in a strong immunoenhancing effect, such as increased cytokine secretion when administered in vivo, while maintaining safety by minimizing the risk of inducing an immune response as an immunogen. IL-15 also exhibits excellent anti-cancer effects (e.g., cancer cell apoptosis, inhibiting cancer cell proliferation). Therefore, the fusion peptide combines the benefits of anti-TIGIT antibodies or their antigen-binding fragments and IL-15, and can be effectively used for immunoenhancement, prevention and / or treatment of immune-related diseases, particularly for the prevention and / or treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0162] Figure 1 is a graph of the binding affinity of fusion proteins according to the embodiments to human TIGIT protein.

[0163] Figure 2a to Figure 2c 2 are diagrams showing that the fusion protein according to the example increases the production of cytokines in immune cells (2a: CD4+T cells, 2b: CD8+T cells, 2c: CD56+NK cells).

[0164] Figure 3 A graph showing that fusion proteins according to embodiments increase the expression of cytokines in PBMCs.

[0165] Figure 4a and Figure 4b is a diagram showing the induction of STAT5 in immune cells by the fusion protein according to the embodiment ( Figure 4a :CD8+T cells, Figure 4b : NK cells).

[0166] Figure 5 A diagram showing the effect of fusion proteins according to embodiments on long-term cytokine production in immune cells.

[0167] Figure 6 is a graph showing the depletion ability of regulatory T cells by fusion proteins according to the embodiment.

[0168] Figures 7a to 7c 7a is a diagram showing the ability of fusion proteins according to the embodiment to proliferate immune cells (7a: T cells, 7b: NKT cells, 7c: NK cells).

[0169] Figure 8 is a graph showing sustained cytokine production in NK cells by fusion proteins according to an embodiment.

[0170] Fig. 9 The cytotoxicity of the fusion protein according to the embodiments to lung cancer cells (A549 cells) is described.

[0171] Fig.10 The cytotoxicity of the fusion protein according to the embodiments to breast cancer cells (MCF-7 cells) is described. Mode for carrying out the invention

[0172] The present disclosure can be better understood through the following examples, which are intended to illustrate the present disclosure but are not meant to limit the present disclosure. For those skilled in the art, it is obvious that the following examples can be modified without departing from the spirit of the present disclosure.

[0173] Example 1. Generation of [anti-TIGIT monoclonal antibody]-[wild-type IL-15] fusion proteins (7A615, 2B715 and 3F815)

[0174] 1.1. Expression and purification of [TIGIT monoclonal antibody]-[wild-type IL-15] fusion protein

[0175] The fusion protein in which the anti-TIGIT antibody and IL-15 are fused to each other directly or via a linker is designed to have the structure shown in Table 3 below:

[0176] [Table 3]

[0177]

[0178]

[0179] (In Table 3, anti-TIGIT monoclonal antibodies (TIGIT Ab): 7A6 (7A6_VH3 / Vk5), 2B7, 3F8;

[0180] IL-15: wild-type IL-15;

[0181] LC variant: IL-15 fused to the C-terminus of the antibody light chain constant region (LC);

[0182] Fc variant: IL-15 is fused to the C-terminus of the antibody heavy chain constant region (Fc; including the ΔK478 mutation. )

[0183] To generate fusion proteins, genes encoding the heavy and light chain variable regions of TIGITmAb and genes encoding the heavy and light chain constant regions linked to [linker]-[wild-type IL-15] were synthesized and cloned into the pcDNA3.1 (Invitrogen) vector. CHO-K1 cells (ATCC) were seeded on plates and cultured to 70% to 90% confluence, after which 1 µg / µl of DNA of each gene and Lipofectamine reagent (Thermo) were diluted in Opti-MEM medium and mixed at a 1:1 ratio. After incubation for 5 minutes, the DNA lipid complexes were added to CHO-K1 cells and incubated at 37°C for transient expression. The culture supernatant was purified using Protein A and preparative SEC. The resulting protein was exchanged with PBS buffer, quantified by OD280nm measurement, and analyzed by SDS-PAGE.

[0184] [Table 4]

[0185] 7A615(7A6 VH3 / Vk5-IL-15)

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] [Table 5]

[0195] 2B715

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203] [Table 6]

[0204] 3F815

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214] Definitions related to the linker between the anti-TIGIT antibody and IL-15 in the fusion proteins described in Tables 4 to 6 and the Examples below are summarized as follows:

[0215] TL-1: Linker V-1 is attached to the C-terminus of the anti-TIGIT antibody light chain;

[0216] TL-2: Linker V-2 is attached to the C-terminus of the anti-TIGIT antibody light chain;

[0217] TL-3: linker V-1 attached to the C-terminus of the anti-TIGIT antibody heavy chain (Fc);

[0218] TL-4: linker V-2 attached to the C-terminus of the anti-TIGIT antibody heavy chain (Fc);

[0219] TL-5: There is no linker between the anti-TIGIT antibody heavy chain (Fc) and IL-15.

[0220] Example 2. Measurement of the binding affinity of the fusion protein to human TIGIT (ELISA)

[0221] ELISA plates were coated with recombinant human TIGIT protein (Acrobiosystems, China) at a concentration of 1 μg / ml in PBS (phosphate buffered saline) and incubated overnight at 4°C. The plates were blocked with blocking buffer (3 volume / volume % skim milk dissolved in PBS) for 1 hour, followed by serial dilutions of anti-TIGIT monoclonal antibodies (7A6) and fusion proteins ([7A6]-[TL-2]-[IL-15]; indicated as "7A615TL-2") (starting concentration: 61.097 nM; 1:3 serial dilutions) added to the coated plates. After incubation, anti-mouse / human IgG-HRP antibody (Abcam; 1:10000 dilution) was added to each well. Color was developed with TMB solution and stopped with 1N HCl. OD values ​​were read at 450 nm using GloMax® Explorer Fully Loaded.

[0222] The results are as follows Figure 1 As shown. As shown in the results, the binding affinity of the 7A615TL-2 fusion protein to human TIGIT was similar to that of the parent antibody (anti-TIGIT monoclonal antibody 7A6). This result indicates that the fusion of IL-15 with the anti-TIGIT antibody did not affect the antigen binding site of the antibody, and it maintained all the functional properties of the parent antibody.

[0223] Example 3. Fusion protein increases cytokine secretion in human PBMC

[0224] To measure cytokine production in human CD3+ T cells and CD56+ NK cells, 500,000 human PBMCs (Stemcell Technologies) were incubated with anti-CD3 monoclonal antibody (BD Biosciences, 0.2 µg / ml; or anti-CD4 antibody or anti-CD8 antibody) and anti-CD28 monoclonal antibody (BD Biosciences, 1 µg / ml) in the presence of anti-TIGIT 7A6 antibody (10 µg / ml) or fusion protein (7A615TL-2, 68.79 nM). Control groups consisted of cells not treated with anti-TIGIT antibody or 7A615TL-2. After incubation for 4 h at 37°C and 5% CO2, brefeldin A (10 µg / ml) was added to inhibit cytokine secretion, and cells were incubated for an additional 15 h, for a total incubation time of 19 h.

[0225] To analyze the proportion of cells producing cytokines, cells were washed twice by spinning at 400 × g for 10 minutes at 4 ° C, followed by staining as follows: cells were stained with Zombie Aqua Fixable Dead Cell Staining Solution (Biolegend) at room temperature for 20 minutes, followed by washing cells by spinning at 400 × g for 10 minutes. Cells were then labeled with antibodies conjugated to fluorophores for surface markers of CD4+T cells, CD8+T cells, or CD56+NK cells and incubated on ice for 30 minutes. All anti-CD3, anti-CD4, anti-CD8, and anti-CD56 antibodies were from Biolegend.

[0226] According to the manufacturer's instructions, cells were fixed and permeated using eBioscience™ Foxp3 / Transcription FactorFixation / Permeabilization Concentrate and Diluent kit (eBiosciences). Cells were then stained with fluorophore-bound antibodies for intracellular IFN-γ and TNFα (Biolegend) at room temperature for 30 minutes. After washing the cells, flow cytometry (CytoFLEX, Beckman Coulter) was used to detect the fluorescence of labeled surface markers and cytokines. Data were analyzed using FlowJo software (BD Biosciences) and visualized using GraphPadPrism 9 software (Dotmatics).

[0227] The results are as follows Figure 2a (CD4+T cells), Figure 2b (CD8+ T cells) and Figure 2c (CD56+NK cells). Figure 2a to Figure 2cAs shown, the fusion protein provided herein increases the production of Th1 / Tc1 cytokines (IFN-gamma) and TNF-alpha in T cells and NK cells, thereby enhancing the immune response. The results showed that compared with anti-TIGIT antibodies, 7A615 showed a stronger and dose-dependent effect in enhancing the function of effector T cells and NK cells.

[0228] Example 4. Fusion protein increases cytokine expression

[0229] Human peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured at 0.15×10 6 to 0.16×10 6 Cells were plated in a round-bottom 96-well plate and cultured at 37°C and 5% CO2 for 44 hours in the presence of 1 μg / ml anti-CD3 antibody (Biolegend). 7A615TL-2 fusion protein and parent antibody (7A6) were added to the culture medium at the same concentration (68.79 nM). The culture volume per well was 250 μl. After incubation, centrifugation was performed at 660×g for 10 minutes at 4°C. The supernatant was collected in a 1.5 ml microcentrifuge tube and stored at -80°C.

[0230] Cytokine levels were measured using the Bio-Plex cytokine assay (4-plex; #PPX-04, Invitrogen). A 4-plex cytokine analysis was performed for the following four human cytokines: IFN-γ, IL-2, TNF-α, and IL-9. All analyses were performed according to the manufacturer's instructions and the results were read using the Bio-Plex200 array reader (Bio-Rad).

[0231] The results obtained are as follows Figure 3 In an in vitro culture of human PBMCs activated by anti-CD3 monoclonal antibodies to stimulate T cells, co-stimulation of T cells with the fusion protein provided herein (7A615TL-2) resulted in higher levels of cytokine (IFN-γ, IL-2, TNF-α and IL-9) expression compared to co-stimulation with the parental antibody (7A6).

[0232] Example 5. Fusion protein induces STAT5 phosphorylation

[0233] Cells (human PBMC) were thawed and cultured at 10 6 The cells were resuspended in R10 medium (RPMI1640 medium (Gibco) containing 10 vol / vol % FBS (Gibco), penicillin-streptomycin (Sigma), and L-glutamine (Sigma)) at a concentration of 1×10 cells / mL. A total of 100 μl of the cell suspension (equivalent to 1×10 6cells / well). The 7A615TL-2 fusion protein was diluted to 68.7 nM and serially diluted 10-fold from this concentration to a minimum of 69 fM. The cells and fusion protein were mixed thoroughly by pipetting and incubated at 37°C for 15 minutes. After incubation, the cells were pelleted by centrifugation at 500×g for 5 minutes. The supernatant was discarded and the cell pellet was resuspended by vortexing. The cells were then resuspended in PBS containing a viability marker (eBioScience, 1:500), an anti-CD3 antibody (Biolegend, 1:50), and an anti-CD8 antibody (Biolegend, 1:50). The cells were stained at room temperature for 10 minutes, then centrifuged at 500×g for 5 minutes to obtain cells as pellets, and the supernatant was discarded. The cells were broken up by vortexing the pellets. The cells were then resuspended in 200 μL of Fixation Buffer (Biolegend) preheated to 37°C and incubated at room temperature for 15 minutes. The cell suspension was centrifuged at 500 × g for 5 minutes to agglomerate the cells, and the supernatant was discarded. The cell agglomerates were vortexed to break the cells, and the cells were then resuspended in 200 μL of ice-cold True-Nuclear 1X Perm Buffer (Biolegend). The cells were stored at -20°C for 1 hour, and then centrifuged at 500 × g for 5 minutes to obtain cells as a group. The supernatant was discarded, and the cells were vortexed to break the cells. The cells were then resuspended in a staining buffer containing an anti-pSTAT5 antibody (Biolegend, 1:25) and incubated at room temperature for 15 minutes. The cell suspension was centrifuged at 500 × g for 5 minutes, and the supernatant was discarded. The resulting group was vortexed and resuspended in PBS. The cells were then centrifuged again as described above, and finally resuspended in 100 μL PBS, and data were acquired using a flow cytometer (CytoFlex, Beckman Coulter). Data were analyzed using FlowJo (BD, USA).

[0234] The results are as follows Figure 4a (CD8+ T cells) and Figure 4b (NK cells) shown. IL-15 connected to anti-TIGIT antibodies induces STAT5 phosphorylation in CD8+T cells and NK cells. In primary cells, receptor heterodimerization must induce transmembrane signal transduction. For the signal transduction of the IL-15 receptor complex, its measurement is carried out by STAT5 phosphorylation. Stimulation of CD8+T cells and NK cells with the fusion protein provided herein induced high levels of STAT5 phosphorylation, which indicates that the IL-15 in the fusion protein maintains its ability to induce strong signal transduction.

[0235] Example 6. Fusion protein maintains long-term cytokine production

[0236] The fusion proteins provided herein were observed to stimulate immune cells over a long period of time, inducing sustained production of cytokines.

[0237] More specifically, the ability of the 7A615TL-2 fusion protein to induce long-term signaling was tested.

[0238] To this end, PBMCs from healthy human donors were seeded at a density of 250,000 cells per well in 96-well U-bottom plates containing R10 medium (RPMI1640 medium (Gibco) with 10 vol / vol % FBS (Gibco), penicillin-streptomycin (Sigma) and L-glutamine (Sigma). The cells were stimulated with IL-15 or 7A615TL-2 fusion protein at a concentration of 378.6 pM for 1 hour at 37° C. and 5% CO2, followed by centrifugation at 400×g for 3 minutes and washing 5 times with R10 medium. The cells were then placed at 37° C. and 5% CO2 for 24 hours.

[0239] After 24 hours of standing, cells were stimulated with anti-CD3 antibody (CytoStim, 1:500; Miltenyi) and incubated overnight at 37°C and 5% CO2. Two hours after adding CytoStim as a stimulator, Brefeldin A (Biolegend, 1:1000) and Monensin (Biolegend, 1:1000) were added. The cells were centrifuged at 400×g for 3 minutes and washed with PBS, and then resuspended in 50μl of PBS containing Fixable Viability dye eFluor 780 (Thermo Fisher, 1:500). The cells were incubated in the dark at room temperature for 10 minutes, then centrifuged at 400×g for 3 minutes and washed with 100μl PBS. After centrifugation at 400×g for 3 minutes again, the cells were resuspended in 50μl of Fixation Buffer (Biolegend) and incubated in the dark at room temperature for 30 minutes. The cells were then centrifuged again at 400×g for 3 minutes, washed with 100 μl of Perm Buffer (Biolegend, diluted in water to a 1× solution), and resuspended in 200 μl of Perm Buffer. The cells were incubated in Perm Buffer for 1 hour at room temperature in the dark, centrifuged at 400×g for 5 minutes, and the supernatant was discarded. The resulting cell pellet was resuspended in 50 μl of Perm buffer containing the following antibodies: anti-CD4-AlexaFluor700 (1:50), anti-CD8-PerCP (1:50), anti-CD56-BV421 (1:50), anti-IL-2-PE (1:50), and anti-IFN-γ-FITC (1:50). All antibodies were from Biolegend. The cells were centrifuged at 400×g, the supernatant was discarded, the cell pellet was resuspended in 100 μl of PBS, and analyzed using a CytoFlex flow cytometer.

[0240] FCS files were generated using CytExpert software (Beckman Coulter) and analyzed using FlowJo v10.8.1. Graphs were generated and statistical analyses were performed using GraphPad Prism 9. Statistical significance was assessed by one-way ANOVA corrected for multiple comparisons with Dunnett’s test (n = 8), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

[0241] The results obtained are as follows Figure 5 As shown. Figure 5As shown, stimulation with the fusion protein followed by 24 hours of incubation resulted in a significant increase in the production of cytokines (IL-2, IFN-γ) in both CD4+ and CD8+ T cells. However, this increase was not generally observed when the cells were stimulated with IL-15 alone. These results suggest that the anti-TIGIT antibody component of the fusion protein promotes sustained signaling on the surface of T cells and that the fusion protein remains active for a long time.

[0242] Example 7. Fusion protein-mediated depletion of Treg cells

[0243] Frozen human peripheral blood mononuclear cells (PBMCs) from Birmingham Hospital were thawed and allowed to stand at 37°C and 5% CO2 for 16 hours. The next day, Treg cells and NK cells were separated by magnetic bead separation (Miltenyi Biotec). The cells were counted and seeded into 96-well U-bottom plates at a ratio of 10 NK cells per 1 Treg cell. 7A615TL-2 fusion protein was added to the wells at a concentration of 10 µg / mL. The co-cultured cells were incubated at 37°C and 5% CO2 for 24 hours. After incubation, the co-cultured cells were centrifuged at 500×g for 5 minutes to pellet the cells. The supernatant was discarded and the pellet was vortexed to break the cells. The cells were resuspended in phosphate buffered saline (PBS) and then centrifuged at 500×g for 5 minutes to obtain the cells as pellets again. The cells were then resuspended in staining buffer containing viability markers (eBioScience, 1:500), anti-CD3 antibody (Biolegend, 1:50), anti-CD4 antibody (Biolegend, 1:50), anti-CD127 antibody (Biolegend, 1:50), anti-CD25 antibody (Biolegend, 1:50), and anti-CD56 antibody (Biolegend, 1:50) and incubated in the dark for 10 minutes. The cells were then washed with 200 µl of PBS. Finally, the cells were resuspended with 100 µl of PBS and data were acquired using a flow cytometer (CytoFlex, Beckman Coulter). Data were analyzed using FlowJo (BD, USA).

[0244] The results are as follows Figure 6 As shown. Figure 6 It can be seen that the fusion protein provided in the present application has a strong ability to consume Treg cells in co-culture with NK cells.

[0245] Example 8. Effect of fusion protein therapy on the proliferation of T cells, NKT cells and NK cells

[0246] PBMCs obtained from four healthy human donors were counted and centrifuged at 400 × g for 3 min. PBMCs were centrifuged at 5 × 10 6 The cells were resuspended in PBS containing CFSE dye (Biolegend, 1:2000 dilution) at a concentration of 10 cells / ml. After this step, the cells were incubated in a 37°C incubator for 20 minutes, avoiding direct light. R10 medium was then added to the test tube and incubated for another 10 minutes at 37°C. After centrifugation at 400×g for 3 minutes, the cells (PBMC) were washed three times with R10 medium, counted, and seeded in 100µl of R10 medium at 500,000 cells. The cells were then stimulated with 7A615TL-2 fusion protein or soluble IL-15 (Peprotech) at a concentration of 3876pM and incubated for 7 days at 37°C and 5% CO2. After incubation, cells were centrifuged at 400 × g for 3 min and subsequently stained with 50 µl of PBS solution containing an antibody cocktail (anti-CD3 APC (Biolegend; 1:50), anti-CD4-AF700 (Biolegend, 1:50), anti-CD56 BV421 (Biolegend, 1:50), and FixableViability Dye eFluor 780 (eBioscience; 65-0865-18)). Cells were incubated at room temperature for 10 min, centrifuged at 400 × g for 3 min, and washed with 100 µl of PBS. Cells were then resuspended in 100 µl of PBS and analyzed using a CytoFlex flow cytometer (Beckman Coulter). The resulting data were analyzed using FlowJo v10.8.1. The gating strategy defined T cells as CD3+CD56-, NKT cells as CD3+CD56+, and NK cells as CD3-CD56+. As cells divide, the level of CFSE dye decreases, so all cells with CFSE expression below the starting population are labeled as total proliferating. Unstimulated controls from each donor were used to set thresholds. Graphs were generated and statistical analyses performed using GraphPad Prism9. Statistical significance was assessed by one-way ANOVA corrected for multiple comparisons with Dunnett's test (n=8), *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.

[0247] The results are as follows Figure 7a (T cells), Figure 7b (NKT cells) and Figure 7c(NK cells). In this experiment, the proliferation of PBMCs was measured using CFSE dye. After stimulation with the 7A615TL-2 fusion protein or free IL-15, all three cell populations (T cells, NKT cells, NK cells) showed significant proliferation, with slightly higher proliferation of cells stimulated by the 7A615TL-2 fusion protein. This suggests that the 7A615TL-2 fusion protein is at least as effective as free IL-15 in stimulating the proliferation of T cells, NKT cells, and NK cells. These results indicate that the IL-15 component of the fusion protein still has the ability to stimulate the proliferation of immune cells.

[0248] Example 9. Effect of fusion protein on cytokine expression in NK cells

[0249] To observe whether the stimulation of immune cells by the fusion proteins provided herein can induce long-term signaling, an examination was performed.

[0250] To this end, PBMCs obtained from healthy human donors were seeded at a density of 500,000 cells per well in 96-well U-bottom plates containing R10 medium (RPMI1640 medium (Gibco) with 10 vol / vol % FBS (Gibco), penicillin-streptomycin (Sigma) and L-glutamine (Sigma). The cells were stimulated with IL-15 or 7A615TL-2 fusion protein at a concentration of 378.6 pM at 37° C. and 5% CO2 for 1 hour. The cells were then centrifuged at 400×g for 3 minutes and washed 5 times with R10 medium. After washing, the cells were placed at 37° C. and 5% CO2 for 24 hours.

[0251] After 24 hours, cells were stimulated with IL-12 (Peprotech) at a concentration of 50 ng / ml and incubated overnight at 37°C and 5% CO2. After 2 hours of stimulation with CytoStim (Miltenyi Biotec), Brefeldin A (Biolegend, 1:1000) and Monensin (Biolegend, 1:1000) were added. The cells were centrifuged at 400×g for 3 minutes, washed with PBS, and resuspended in 50μl of PBS containing Fixable Viability dye eFluor 780 (Thermo Fisher, 1:500). The cells were incubated at room temperature in the dark for 10 minutes, centrifuged at 400×g for 3 minutes, and washed with 100µl of PBS. After centrifugation at 400×g for 3 minutes, the cells were resuspended in 50μl of Fixation Buffer (Biolegend) and incubated at room temperature in the dark for 30 minutes. The cells were washed with 100 µl of Perm buffer (Biolegend; diluted 1× in water) and incubated with Perm buffer for 1 h at room temperature in the dark. After washing and centrifugation, the cells were resuspended in 50 µl of Perm buffer containing the following antibodies: CD3-APC (1:50), CD4-AlexaFluor700 (1:50), CD8-PerCP (1:50), CD56-BV421 (1:50), and IFN-γ-FITC (1:50). All antibodies were from Biolegend. The cells were then centrifuged at 400 × g, resuspended in 100 µl of PBS, and analyzed using a CytoFlex flow cytometer (Beckman Coulter).

[0252] Data were processed using CytExpert software (Beckman Coulter) to generate FCS files and analyzed using FlowJov10.8.1. Graphical analysis and statistical analysis were performed using GraphPad Prism9. Statistical significance was assessed by one-way ANOVA with Dunnett's test corrected for multiple comparisons (n ​​= 8), with significance levels of *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001, respectively.

[0253] The results are as follows Figure 8As shown. As shown in the figure, NK cells stimulated with 7A615TL-2 fusion protein and then stimulated again with IL-12 after 24 hours significantly induced high levels of IFN-γ expression. In contrast, the combined treatment of 7A6 antibody and IL-15 did not significantly increase the production of IFN-γ compared with the control group (no fusion protein or combined treatment) (ns = not significant). These results indicate that the fusion protein of the present application can provide sustained signaling to induce long-term IFN-γ production.

[0254] Example 10. Cytotoxicity of fusion protein (lung cancer cells; A549)

[0255] Frozen human PBMCs were thawed, washed and resuspended in complete medium (RPMI Merk, UK, plus 10 vol / vol % FCS Merk, UK). Cells were stimulated with IL-15 (Peprotech) or 7A615TL-2 fusion protein at a concentration of 68.7 nM for 1 hour. The stimulation density was 5×10 6 Cells were washed five times by centrifugation at 1500 rpm for 3 min in complete medium (2 ml each time). 6 The cells were resuspended at a density of 100,000 cells / ml (100,000 cells in 50 µl) and transferred into a 96-well U-bottom plate for 24 hours.

[0256] On the day of analysis, adherent A549-GFP target cells (A549-GFP (lung) MERK (ECACC) cat# 86012804-1VL) were washed with PBS (phosphate buffered saline) and incubated with trypsin / EDTA (0.05%, Gibco, Fisher UK) for 5 minutes at 37°C and 5% CO2. A549 cells were harvested, centrifuged at 500×g for 5 minutes and pelleted. The supernatant was discarded, and the A549-GFP cells were resuspended in complete medium (RPMI Merk, UK and 10% FCS Merk, UK) and plated at 5×10 3 The cells were seeded into 96-well plates at a density of 1×10 cells / well in 50 µl. The cells were allowed to adhere for 3 hours at 37°C and 5% CO2. The prepared PBMCs were then plated at a density of 1×10 5Cells were cultured at a density of 10 cells / ml at 50 µl / well onto adherent A549-GFP cells. Anti-CD3 antibody (Biolegend) was added at a concentration of 10 ng / ml. Cell death marker DRAQ7 (Biolegend) was diluted to 1 / 200, resulting in a final concentration of 1.5 µM, and added to each well. Co-culture was performed at 37°C and 5% CO2 in an IncuCyte (Sartorius, UK). Images were taken every 2 hours for 5 consecutive days and the results were analyzed using the IncuCyte's native software.

[0257] The results are as follows Fig. 9 [A) shows the log2-fold change of the number of A549 cells over time, and B) shows the area under the curve of A). Compared with untreated PBMCs, anti-CD3 antibody-treated PBMCs, or anti-CD3 antibody and IL-15-treated PBMCs, the PBMCs activated by the 7A615TL-2 fusion protein in the present application showed significantly higher levels of cytotoxicity against the A549-GFP target cell line. It is noteworthy that these PBMCs exhibited a longer duration of cytotoxicity (up to 72 hours) than the PBMCs treated with anti-CD3 antibodies and IL-15.

[0258] Example 12. Cytotoxicity of fusion protein (breast cancer cells; MCF-7)

[0259] Frozen human PBMCs were thawed. NK cells were isolated using magnetic bead separation (Miltney UK). Isolated cells were pre-activated by incubation for 24 h at 37°C and 5% CO2 in the presence or absence of 10 µg / ml of 7A615TL-2 fusion protein or pembrolizumab (Invivogen). On the day of analysis, adherent MCF-7-GFP target cells (MCF7-GFP (breast); MacroPhox Ltd) were washed with PBS and incubated with trypsin / EDTA (0.05%, Gibco, Fisher UK) for 5 min at 37°C and 5% CO2. MCF-7 cells were harvested and pelleted by centrifugation at 500 × g for 5 min. The supernatant was discarded and MCF-7-GFP cells were resuspended in complete medium (RPMI Merk, UK, plus 10 vol / vol % FCS Merk, UK) and subsequently cultured at 1 × 10 4 The cells were seeded in a 96-well flat-bottom plate at a density of 50 μl per well. The cells were incubated at 37°C and 5% CO2 for 3 hours to allow the cells to adhere. The pre-activated NK cells were then inoculated at a density of 1×10 5Adherent MCF-7-GFP cells were added at a density of 10 cells / ml, 50 µl per well. In addition, 10 µg / ml of 7A615TL-2 fusion protein or pembrolizumab (Invivogen) was added to each well. Co-culture was carried out in IncuCyte (Sartorius, UK) at 37°C and 5% CO2. Four images were taken every 2 hours for 6 days. Analysis was performed using the native software of IncuCyte.

[0260] The results are as follows Fig.10 Shown [A) shows the log2-fold change in the number of MCF-7 cells over time, and B) shows the area under the curve (AUC) of A). NK cells treated with 7A615TL-2 fusion protein had significantly higher levels of cytotoxicity against the MCF-7-GFP target cell line compared to the untreated group or the pembrolizumab-treated group.

Claims

1. A fusion protein comprising: An anti-TIGIT antibody or an antigen-binding fragment thereof; and Interleukin-15 (IL-15), The anti-TIGIT antibody or antigen-binding fragment thereof comprises A CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, A CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9, A CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 13 or SEQ ID NO: 14, A CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 17, and A CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:

20.

2. The fusion protein according to claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof comprises: a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 7, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 15, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18; a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 5, a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 8, a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 13, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 19; or CDR-H1 comprising the amino acid sequence of SEQ ID NO: 3, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 6, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 9, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 14, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

20.

3. The fusion protein according to claim 1, wherein the anti-TIGIT antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28; and A light chain variable region comprising the amino acid sequence of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 or SEQ ID NO:

36. 4 . The fusion protein according to claim 1 , wherein the anti-TIGIT antibody is an animal antibody, a chimeric antibody or a humanized antibody. 5 . The fusion protein of claim 1 , wherein the antigen binding fragment is scFv, scFv-Fc, (scFv) 2 , Fab, Fab′ or F(ab′) 2 of the anti-TIGIT antibody.

6. The fusion protein of claim 1, wherein the interleukin-15 comprises the amino acid sequence of SEQ ID NO:

38.

7. The fusion protein according to claim 1, wherein there is no peptide linker between the anti-TIGIT antibody or antigen-binding fragment thereof and interleukin-15.

8. The fusion protein according to claim 1, wherein a peptide linker is included between the anti-TIGIT antibody or antigen-binding fragment thereof and interleukin-15.

9. The fusion protein according to claim 8, wherein the peptide linker is [EAAAK]n (wherein n is an integer from 1 to 5, representing the number of [EAAAK]) or [(G)mS]l (wherein 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]).

10. A nucleic acid encoding the fusion protein according to any one of claims 1 to 9.

11. A recombinant vector comprising the nucleic acid according to claim 10.

12. A recombinant cell harboring the nucleic acid according to claim 10 or a recombinant vector comprising the nucleic acid.

13. A pharmaceutical composition for preventing or treating cancer, comprising the fusion protein according to any one of claims 1 to 9.

14. The pharmaceutical composition according to claim 13, wherein the cancer is a solid tumor or a hematological cancer.

15. A pharmaceutical composition for immune enhancement, comprising the fusion protein according to any one of claims 1 to 9. 16 . A pharmaceutical composition for preventing or treating immune-related diseases, comprising the fusion protein according to any one of claims 1 to 9.

17. The pharmaceutical composition according to claim 16, wherein the immune-related disease is an infectious disease, an inflammatory disease or an autoimmune disease.

Images