Treatment drugs for immune and inflammatory diseases

Abstract

The present invention provides a therapeutic agent for immune / inflammatory disease in which an anti-human TIGIT antibody that activates an inhibitory immune checkpoint molecule (TIGIT) serves as an active ingredient. Furthermore, the present invention provides an antibody having the characteristics of a) and b): a) CDR3 of the heavy chain variable region includes the amino acid sequence of SEQ ID NO: 4; and b) CDR3 of the light chain variable region includes the amino acid sequence of SEQ ID NO: 6.

Description

[Technical Field]

[0001] This invention relates to therapeutic agents for immune and inflammatory diseases, including antibodies against human TIGIT. The invention also relates to anti-human TIGIT antibodies having a specific primary structure that may have therapeutic activity for immune and inflammatory diseases.

[0002] (Background of the invention) In recent years, there has been active development of drugs targeting immune checkpoint proteins, particularly anticancer drugs. Immune checkpoint proteins are factors that prevent the onset of immune and inflammatory diseases caused by abnormal activation of the immune system, and major molecules known to be involved include PD-1 (Programmed cell death protein 1, also known as CD279), PD-L1 (Programmed cell death ligand 1, also known as B7-H1 / CD274), PD-L2 (Programmed cell death ligand 2, also known as B7-DC / CD273), and CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4, also known as CD152). In addition to the above, TIGIT (T-cell immunoreceptor with immunoglobulin and ITIM domains, also known as zB7R1) is another example of an immune checkpoint protein. TIGIT is an immune checkpoint receptor found in natural killer (NK) cells, cytotoxic T cells (CTLs), memory T cells, regulatory T cells (Treg cells), and follicular helper T cells (Tfh cells). In CTLs and NK cells, immune activation is suppressed by the interaction between TIGIT and either of its two ligands, CD155 (PVR) or CD112 (Nectin2). On the other hand, TIGIT expression in Treg cells is known to enhance the ability to suppress the immune response through this interaction. The immunosuppressive effect of TIGIT binding to CD155 or CD112 arises from competitive inhibition of the immune activation receptor CD226, which is expressed in cytotoxic T cells and NK cells, against the same ligands (i.e., CD155 ligand and CD112 ligand).

[0003] Focusing on TIGIT-mediated signal transduction, methods have been reported for treating immune and inflammatory diseases by suppressing the function of TIGIT-positive T cells using agonists or anti-TIGIT agonistic antibodies against TIGIT (Patent Documents 1 and 2). However, to date, no approved pharmaceuticals containing anti-TIGIT agonistic antibodies exist, and there is a need for the creation of anti-TIGIT antibodies with superior therapeutic efficacy and safety. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2006 / 124667 [Patent Document 2] International Publication No. 2009 / 126688 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Accordingly, the object of the present invention is to provide an anti-human TIGIT agonist antibody that has the ability to bind to human TIGIT and enhance signaling mediated by said TIGIT, and to provide a novel therapeutic agent for immune and inflammatory diseases that includes an agonist antibody against human TIGIT. [Means for solving the problem]

[0006] While conducting research on drug target discovery based on elucidating the pathogenesis of immune diseases, the inventors discovered that TIGIT expression levels are elevated in patients with rheumatoid arthritis. In particular, they confirmed that Tfh cells, peripheral helper T cells (Tph cells), and Treg cells, which already have high TIGIT expression levels in healthy individuals, show significantly higher TIGIT expression levels in patients with the disease. Furthermore, they found that TIGIT expression levels in Tph cells in rheumatoid arthritis patients correlate with disease activity. Therefore, the inventors conceived the idea that targeting TIGIT could lead to the development of therapeutic drugs for immune and inflammatory diseases such as rheumatoid arthritis, and diligently investigated how to solve the above problem. First, they established multiple mouse anti-human TIGIT (anti-hTIGIT) antibodies, and from these established antibodies, they selected several particularly high agonistic antibodies. Further testing of the selected agonist antibodies in Tfh cells with high TIGIT expression levels revealed that a specific antibody (Clone M1-8) could suppress Tfh cell proliferation, thus completing the present invention.

[0007] In other words, the present invention provides the following: [1] An immunotherapy drug for treating immunological and inflammatory diseases, containing an anti-human TIGIT antibody as its active ingredient, which activates the inhibitory immune checkpoint molecule (TIGIT). [1'] A method for treating immune and inflammatory diseases in mammals, characterized by administering an effective amount of an anti-human TIGIT antibody that activates an inhibitory immune checkpoint molecule (TIGIT) to the mammal. [1”] Anti-human TIGIT antibodies that activate suppressive immune checkpoint molecules (TIGITs) for use in the treatment of immune and inflammatory diseases. [2] A therapeutic agent having Tfh cell inhibitory activity [1]. [2'] A Tfh cell inhibitor containing an anti-human TIGIT antibody that activates the inhibitory immune checkpoint molecule (TIGIT). [3] The therapeutic agent according to [1] or [2] having an effect of activating Treg cells. [3’] An activator of Treg cells, comprising an anti-human TIGIT antibody that activates an inhibitory immune checkpoint molecule (TIGIT). [4] The therapeutic agent according to any one of [1] to [3], wherein the immune and inflammatory disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, systemic sclerosis, polymyositis, dermatomyositis, IgG4-related disease, Takayasu arteritis, giant cell arteritis, polyarteritis nodosa, ANCA-associated vasculitis, mixed connective tissue disease, spondyloarthritis, Behçet's disease, adult Still's disease, multiple sclerosis, neuromyelitis optica, myasthenia gravis, primary biliary cirrhosis, non-alcoholic fatty liver disease, primary sclerosing cholangitis, autoimmune hepatitis, ulcerative colitis, Crohn's disease, psoriasis (psoriatic arthritis), vitiligo vulgaris, bullous pemphigoid, alopecia areata, idiopathic dilated cardiomyopathy, type 1 diabetes, Graves' disease, Hashimoto's disease, IgA nephropathy, membranous nephropathy, hemolytic anemia, and idiopathic thrombocytopenic purpura. [5] The therapeutic agent according to any one of [1] to [4], wherein the anti-human TIGIT antibody is an antibody that binds to human TIGIT (SEQ ID NO: 1). [6] The antibody a) The third CDR of the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 4, and b) The third CDR of the light chain variable region contains the amino acid sequence of SEQ ID NO: 6, The therapeutic agent according to [5]. [7] The antibody a) The heavy chain variable region containing the following: i) The first CDR containing the amino acid sequence of SEQ ID NO: 2; ii) The second CDR containing the amino acid sequence of SEQ ID NO: 3; and b) The light chain variable region containing the following: i) The first CDR containing the amino acid sequence of SEQ ID NO: 5; ii) The second CDR containing the amino acid sequence of tyrosine-alanine-serine; The therapeutic agent according to [5] or [6], comprising [8] The therapeutic agent according to any one of [5] to [7], further comprising an antibody containing an Fc domain. [9] The therapeutic agent according to [8], wherein the Fc domain is of human origin.

[10] The therapeutic agent according to [8] or [9], wherein the Fc domain is a mutant Fc domain.

[11] a) The third CDR of the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 4, and b) The third CDR of the light chain variable region contains the amino acid sequence of SEQ ID NO: 6, Antibody.

[12] The antibody according to

[11] , which binds to human TIGIT (SEQ ID NO: 1).

[13] a) A heavy chain variable region comprising: i) The first CDR containing the amino acid sequence of SEQ ID NO: 2; ii) The second CDR containing the amino acid sequence of SEQ ID NO: 3; and b) A light chain variable region comprising: i) The first CDR containing the amino acid sequence of SEQ ID NO: 5; ii) The second CDR containing the amino acid sequence of tyrosine-alanine-serine; The antibody according to

[11] or

[12] , comprising

[14] The antibody according to any one of

[11] to

[13] , further comprising an Fc domain.

[15] The antibody according to

[14] , wherein the Fc domain is of human origin.

[16] The antibody according to

[14] or

[15] , wherein the Fc domain is a mutant Fc domain.

[17] An isolated nucleic acid encoding the antibody according to any one of

[11] to

[16] .

[18] A host cell comprising the isolated nucleic acid according to

[17] .

[19] A method for producing an antibody according to any one of

[11] to

[16] , comprising the step of culturing a host cell according to

[18] under conditions under which the antibody is produced. [Effects of the Invention]

[0008] According to the present invention, an antibody that binds to human TIGIT is provided. Since this antibody can serve as a therapeutic agent for immune and inflammatory diseases, the present invention is useful for treating immune and inflammatory diseases using this therapeutic agent. [Brief explanation of the drawing]

[0009] [Figure 1] This graph shows the results of measuring antibody titers in antiserum from mice immunized with recombinant human TIGIT-His-mFc protein. The vertical axis of each graph represents the median fluorescence intensity. The horizontal axis of each graph represents log [plasma dilution]. [Figure 2] This shows the binding activity of the M1-8 antibody in the culture supernatant to doxycycline-expression-induced human TIGIT-expressing cells. [Figure 3] This shows the inhibitory activity of recombinant TIGIT, an M1-8 antibody in the culture supernatant, on the binding of PVR-expressing cells. [Figure 4] This shows the biological activity (agonist activity, antagonist activity, and cytotoxic activity) of the purified M1-8 antibody. [Figure 5] This shows the binding activity of the purified M1-8 antibody to human, cynomolgus monkey, and mouse TIGIT-expressing cells. [Figure 6]From top to bottom, the graphs show the inhibitory effect of TIGIT stimulation on the proliferation of CD8-positive cells (% inhibition of proliferation) and the inhibitory effect on the production of cytokines (IFNγ, Granzyme B, Perforin) (% suppression of cytokine production). The horizontal axis of each graph, from left to right, shows CD3 / PVR 0, CD3 / PVR 0.075, CD3 / PVR 0.025, CD3 / PVR 0.75, and CD3 / PVR 2.5. [Figure 7-1] a: This figure shows how TIGIT expression changes in cells with low TIGIT expression (CD4+ naive T cells) and cells with high TIGIT expression (Tfh cells) upon stimulation. b: This figure shows that stimulation with an anti-TIGIT agonistic antibody suppresses the proliferation of Tfh cells. [Figure 7-2] a: This figure shows the time course of TIGIT expression in Tfh cells, non-Tfh cells, and CD4+ naive T cells induced by activation stimulation. Non-Tfh cells showed TIGIT expression at an intermediate level between Tfh cells and naive T cells. b: This figure shows that stimulation with an anti-TIGIT agonistic antibody suppresses the proliferation of both Tfh cells and non-Tfh cells, but the inhibitory effect is more pronounced in Tfh cells. [Figure 8] This figure shows that stimulation of Tfh cells with an anti-TIGIT agonistic antibody suppresses the activation of B cells via Tfh cells. The stimulation of B cells from Tfh cells is evaluated by the proportion of plasma cells and the IgG concentration in the culture supernatant. The results are shown as absolute values ​​and relative values ​​with the isotype result set to 100. [Figure 9-1] This figure shows that stimulation of Treg cells with an anti-TIGIT agonistic antibody suppressed the proliferation of effector T cells. [Figure 9-2]This figure shows that stimulation of Treg cells (FrI and FrII) with anti-TIGIT agonistic antibodies suppresses the proliferation of non-Treg responder CD4+ T cells (CD25-), and that this suppressive effect is more pronounced in Treg cells (FrI). The vertical axis of the bar graph on the right shows the suppression rate (%) = [1 - (proliferation of co-cultured responder T cells) / (proliferation of isolated responder T cells)] × 100. *: p < 0.05 [Figure 10] This figure shows that an anti-TIGIT agonistic antibody improves splenomegaly and suppresses splenic lymphocyte proliferation in imiquimod-induced lupus model mice. Comparisons were made in three groups: untreated, anti-TIGIT, and isotype, with n=4, 3, and 4 respectively. [Figure 11-1] From left to right, the figures compare the proportion of CD69+ (activated) T cells in the spleen of imiquimod-induced lupus model mice, the proportion of CD4+ Effector memory T cells and Tfh cells in the spleen, the proportion of germinal center B cells and plasma cells in the B cells, and the amount of anti-dsDNA antibody in the mouse plasma, using anti-TIGIT agonistic antibody, in the same three groups as in Figure 10. Only in the isotype group for anti-dsDNA antibody, blood could not be collected from one mouse, resulting in n=3. The box plots for each data point, from left to right, show the uncoated group, the anti-TIGIT coated group, and the isotype coated group, respectively. *:p<0.05 [Figure 11-2]This figure shows the effect of administering anti-TIGIT agonistic antibodies on the immune response in imiquimod-induced lupus model mice. From the top left, the percentage of CD69+ (activated) T cells among CD4+ T cells in the spleen, the percentage of CD4+ Effector memory T(Tem) cells and Tfh cells among CD4+ T cells, and the percentage of germinal center (GC) B cells among B220+ cells are compared in three groups (isotype only, imiquimod application + anti-TIGIT, and imiquimod application + isotype, n=4, 5, and 5 respectively). Each bar graph, from left to right, shows the uncoated group, the isotype application group, and the anti-TIGIT application group, respectively. *: p<0.05 [Figure 12] This figure shows that anti-TIGIT agonistic antibodies improve clinical scores in EAE mice. The study involved 6 mice per group, and the clinical scores are shown over time for both the isotype antibody group and the anti-TIGIT agonistic antibody group. [Figure 13] This figure shows that administration of anti-TIGIT agonistic antibody promotes the proliferation of Treg cells in the brain, spinal cord, and spleen of EAE mice. The experiment was conducted with 3 mice per group, and cells in the brain, spinal cord, and spleen were stained and the proportion of Treg cells was compared between the isotype-treated group and the anti-TIGIT agonistic antibody-treated group. The box plots for each data point, from left to right, show the anti-TIGIT-treated group and the isotype-treated group, respectively.

[0010] (Detailed description of the invention) Antibodies or fragments thereof that bind to human TIGIT. The present invention provides an antibody or fragment thereof having a complementarity-determining region (CDR) containing a specific sequence and binding to human TIGIT. Hereinafter, the term "antibody of the present invention" will be used to encompass both the antibody and the fragment thereof. The antibody of the present invention may also be isolated. In this specification, "isolated" means a state in which a specific component (e.g., the antibody of the present invention, the nucleic acid encoding the antibody, etc.) has been identified, separated, or recovered from a component of its natural environment (e.g., a cell, etc.).

[0011] The antibody of the present invention comprises a third CDR (also referred to as CDR3) of the heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 4, and a third CDR of the light chain variable region containing the amino acid sequence shown in SEQ ID NO: 6. In one embodiment, the antibody of the present invention comprises i) a first CDR (also referred to as CDR1) of the heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2, ii) a second CDR (also referred to as CDR2) of the heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 3, iii) a first CDR of the light chain variable region containing the amino acid sequence shown in SEQ ID NO: 5, and iv) a second CDR of the light chain variable region containing the amino acid sequence shown in tyrosine-alanine-serine (YAS). An example of a heavy chain variable region containing CDR1 to 3 of the heavy chain variable regions shown in SEQ ID NOs: 2 to 4, respectively, is one containing the amino acid sequence shown in SEQ ID NO: 7, but the heavy chain variable region is not limited to containing this sequence. Furthermore, while one example of a light chain variable region containing CDR1-3 as shown in SEQ ID NO: 5, YAS, and SEQ ID NO: 6, respectively, is the amino acid sequence shown in SEQ ID NO: 8, the light chain variable region is not limited to those containing this sequence.

[0012] Immunological and inflammatory disease treatments including anti-human TIGIT agonist antibodies The present invention provides an immunotherapy for inflammatory diseases (hereinafter sometimes referred to as "the therapeutic agent of the present invention") comprising an anti-human TIGIT antibody as an active ingredient that activates human TIGIT, an inhibitory immune checkpoint molecule. One embodiment of the antibody that is the active ingredient of the therapeutic agent of the present invention is an antibody or fragment thereof that has a complementarity-determining region (CDR) containing a specific sequence and binds to human TIGIT. Hereinafter, the term "anti-human TIGIT agonist antibody" will be used to encompass the above antibody that is the active ingredient of the present invention and its fragment.

[0013] As shown in the examples below, anti-human TIGIT agonist antibodies have an inhibitory effect on follicular helper T (Tfh) cells (hereinafter sometimes referred to as "Tfh cell inhibitory effect"), and therefore have a therapeutic effect on immune and inflammatory diseases caused by Tfh cell activation. Furthermore, anti-human TIGIT agonist antibodies have an activating effect on Treg cells (hereinafter sometimes referred to as "Treg cell activating effect"). Accordingly, in another embodiment of the present invention, a Tfh cell inhibitor comprising an anti-human TIGIT agonist antibody, or a method for inhibiting Tfh cells comprising the step of contacting Tfh cells with an anti-human TIGIT agonist antibody, is provided. In yet another embodiment, a Treg cell activator comprising an anti-human TIGIT agonist antibody, or a method for activating Treg cells comprising the step of contacting Treg cells with an anti-human TIGIT agonist antibody, is also provided.

[0014] In this specification, "immunological and inflammatory diseases" means diseases that involve inflammation due to a breakdown of immune tolerance. Examples of immune and inflammatory diseases that can be treated with the therapeutic agent of the present invention include rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, systemic scleroderma, polymyositis, dermatomyositis, IgG4-related disease, Takayasu's arteritis, giant cell arteritis, polyarteritis nodosa, ANCA-associated vasculitis, mixed connective tissue disease, spondyloarthritis, Behçet's disease, adult-onset Still's disease, multiple sclerosis, neuromyelitis optica, myasthenia gravis, primary biliary cirrhosis, non-alcoholic fatty liver disease, primary sclerosing cholangitis, autoimmune hepatitis, ulcerative colitis, Crohn's disease, psoriasis (psoriatic arthritis), vitiligo, bullous pemphigoid, alopecia areata, idiopathic dilated cardiomyopathy, type 1 diabetes, Graves' disease, Hashimoto's disease, IgA nephropathy, membranous nephropathy, hemolytic anemia, and idiopathic thrombocytopenic purpura. Examples of animals to which the therapeutic agent of the present invention can be administered include primates, such as lemurs, lorises, Zweigera monkeys, monkeys (e.g., cynomolgus macaques), and humans. Humans are preferred. Therefore, a therapeutic method involving administering an effective amount of anti-human TIGIT agonist antibody or the therapeutic agent of the present invention to the aforementioned primates is also included in the present invention.

[0015] In this specification, "Tfh cell inhibitory effect" encompasses both the inhibitory effect on Tfh cell proliferation and the inhibitory effect on Tfh cell function. Examples of such Tfh cell functions include the maturation and activation of B cells and the promotion of antibody production. Furthermore, "Treg cell activating effect" encompasses both the inhibitory effect on Treg cell proliferation and the activation effect on Treg cell function. Examples of such Treg cell functions include the suppression of autoimmune responses through the inhibition of effector T cells and the suppression of excessive inflammation through the secretion of anti-inflammatory cytokines such as IL-10 and TGFβ.

[0016] (Anti-human TIGIT agonist antibody) In one embodiment, the anti-human TIGIT agonist antibody includes a third CDR (also called CDR3) of the heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 4, and a third CDR of the light chain variable region containing the amino acid sequence shown in SEQ ID NO: 6. Furthermore, the anti-human TIGIT agonist antibody may also include i) a first CDR (also called CDR1) of the heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 2, ii) a second CDR (also called CDR2) of the heavy chain variable region containing the amino acid sequence shown in SEQ ID NO: 3, iii) a first CDR of the light chain variable region containing the amino acid sequence shown in SEQ ID NO: 5, and iv) a second CDR of the light chain variable region containing the amino acid sequence shown as tyrosine-alanine-serine (YAS). An example of a heavy chain variable region containing CDR1-3 of the heavy chain variable regions shown in SEQ ID NOs: 2-4, respectively, is one containing the amino acid sequence shown in SEQ ID NO: 7, but the heavy chain variable region is not limited to containing this sequence. Furthermore, while one example of a light chain variable region containing CDR1-3 as shown in SEQ ID NO: 5, YAS, and SEQ ID NO: 6, respectively, is the amino acid sequence shown in SEQ ID NO: 8, the light chain variable region is not limited to those containing this sequence.

[0017] Anti-human TIGIT agonist antibodies can activate TIGIT (in other words, exert agonist activity against TIGIT) by binding to TIGIT. In this specification, "activating TIGIT" means that the TIGIT-mediated signal is activated by binding the antibody, compared to a control that does not have the antibody bound (including a group that has been contacted with an antibody that does not have a TIGIT-activating effect), or compared to before binding the antibody. Also in this specification, "anti-human TIGIT agonist antibody" means an antibody that activates the TIGIT-mediated signal by binding the antibody, compared to a control that does not have the antibody bound, or compared to before binding the antibody. Such activation of the TIGIT-mediated signal can be evaluated by any known method, such as a luciferase assay. Furthermore, such evaluation methods can also be used to screen for anti-human TIGIT agonist antibodies other than those containing the specific CDR1-3 described above.

[0018] In this specification, the term "to bind" means "having an ability to bind," and refers to the ability to form a non-covalent complex with one or more other molecules. An example of the complex of the present invention is a complex of an anti-human TIGIT agonist antibody with TIGIT. The anti-human TIGIT agonist antibody binds to the human TIGIT protein consisting of the amino acid sequence shown in SEQ ID NO: 1 (NCBI Reference Sequence: NP_776160.2). In one embodiment, the anti-human TIGIT agonist antibody binds not only to the human TIGIT protein but also to the TIGIT of non-human primates (e.g., cynomolgus monkeys), but not to the TIGIT of rodents (e.g., mice). Therefore, "human TIGIT" can be read as "primate TIGIT" below. The binding ability of such an antibody can be evaluated by any known method, for example, by contacting TIGIT with the antibody and detecting or measuring the antibody bound to TIGIT.

[0019] The anti-human TIGIT agonist antibody may be any antibody or fragment thereof, as long as it contains the CDR3 of the heavy chain variable region and the CDR3 of the light chain variable region. The class of the antibody is not particularly limited and may be any isotype such as IgG, IgM, IgA, IgD, or IgE. Preferably, it is IgG or IgM, and more preferably IgG considering the ease of purification, etc. The antibody fragment is not particularly limited as long as it contains the CDR3 of the heavy chain variable region and the CDR3 of the light chain variable region and binds to human TIGIT, and examples include antibody fragments such as Fab, Fab', and F(ab')2 that have the above-mentioned CDRs.

[0020] Furthermore, anti-human TIGIT agonist antibodies preferably have an Fc domain, such as full-body antibodies or antibodies in which the Fab region and the Fc domain are conjugated. Such Fc domains may be wild-type or mutant. As is known in the art, the Fc domain of an antibody interacts with numerous Fc receptors and ligands, conferring an important function known as effector function. Such Fc receptors include, but are not limited to, the following: FcγRI(CD64) including isoforms FcγRIa, FcγRIb, and FcγRIc (in humans); FcγRII(CD32) including isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII(CD16) including isoforms FcγRIIIa (including allotypes V158 and F158, associated with antibody-dependent cell-mediated cytotoxicity (ADCC)) and FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); FcRn (neonatal receptor), C1q (complement protein involved in complement-dependent cell-mediated cytotoxicity (CDC)); and FcRn (neonatal receptor involved in serum half-life). Suitable amino acid mutations can be made at one or more positions, as outlined below: for example, US Patent Application 11 / 841,654 and the references cited therein, US2004 / 013210, US2005 / 0054832, US2006 / 0024298, US2006 / 0121032, US2006 / 0235208, US2007 / 0148170.

[0021] Furthermore, the portion of the anti-human TIGIT agonist antibody other than the CDR (e.g., the variable region other than the CDR, the constant region, the Fc domain, etc.) may consist of any amino acid sequence as long as it has agonist activity against TIGIT, and may be derived from any animal. Examples of such animals include mammals such as mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, and humans, but it is preferably derived from humans.

[0022] Anti-human TIGIT agonist antibodies can be produced genetically engineered using nucleic acids or vectors encoding the antibody or its fragments. For example, nucleic acids encoding an anti-human TIGIT agonist antibody can be introduced into host cells to express the antibody or its fragments, and the antibody or its fragments can be isolated by known methods. Such isolation methods include affinity columns using protein A, other chromatography columns, filters, ultrafiltration, salting out, and dialysis, and these methods may be combined as appropriate. Antibody fragments can also be produced by treating the full-body antibody produced above with enzymes such as papain and pepsin.

[0023] (Nucleic acid encoding an anti-human TIGIT agonist antibody) The present invention provides isolated nucleic acids encoding the antibody of the present invention (hereinafter sometimes referred to as "the nucleic acid of the present invention"). The nucleic acid of the present invention includes nucleic acids encoding the heavy chain and light chain of the antibody of the present invention, nucleic acids encoding antibody fragments, etc. The nucleic acid encoding the antibody, anti-human TIGIT agonist antibody, or a fragment thereof may be DNA, RNA, or a DNA / RNA chimera, but is preferably DNA. Furthermore, the nucleic acid may be double-stranded or single-stranded. In the case of double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. Furthermore, the nucleic acid encoding the antibody or a fragment thereof may contain native nucleotides, modified nucleotides, nucleotide analogs, or mixtures thereof, as long as polypeptides can be expressed in vitro or in cells.

[0024] A nucleic acid encoding an antibody or a fragment thereof (for example, the nucleic acid of the present invention) can be constructed by a method known to the present day. For example, based on the amino acid sequence of an anti-human TIGIT agonist antibody listed in a sequence listing, a base sequence encoding the amino acids can be designed, and a DNA strand can be chemically synthesized. Alternatively, the synthesized partially overlapping oligoDNA short chains can be joined using PCR or Gibson Assembly to construct DNA encoding the full length or a portion of the anti-human TIGIT agonist antibody.

[0025] Furthermore, nucleic acids encoding antibodies or fragments thereof can be incorporated into expression vectors. Therefore, an expression vector containing either the antibody or the nucleic acid encoding a fragment thereof is provided. In this case, the nucleic acid encoding the heavy chain of the antibody and the nucleic acid encoding the light chain of the antibody may be incorporated into separate expression vectors or into a single expression vector. If incorporated into a single expression vector, these two types of nucleic acids may be incorporated via a sequence that enables polycistronic expression. Using a sequence that enables polycistronic expression makes it possible to express multiple genes incorporated into a single expression vector more efficiently. Examples of sequences that enable polycistronic expression include 2A sequences (e.g., 2A sequences derived from foot-and-mouth disease virus (FMDV) (F2A), 2A sequences derived from equine rhinitis A virus (ERAV) (E2A), 2A sequences derived from Porcine teschovirus (PTV-1) (P2A), and 2A sequences derived from Thosea asigna virus (TaV) (T2A sequences) (PLoS ONE 3, e2532, 2008, Stem Cells 25, 1707, 2007)), and internal ribosome entry sites (IRES) (US Patent No. 4,937,190).

[0026] Examples of promoters used in the above vectors include the EF1α promoter, CAG promoter, SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, TCR Vα gene promoter, and TCR Vβ gene promoter.

[0027] In addition to the promoter, the above vector may optionally include transcription and translation regulatory sequences, ribosome binding sites, enhancers, origins of replication, poly(A) addition signals, and selection marker genes. Examples of selection marker genes include dihydrofolate reductase genes, neomycin resistance genes, and puromycin resistance genes.

[0028] (Host cells containing nucleic acids encoding anti-human TIGIT agonistic antibodies) In one embodiment of the present invention, an expression vector containing a nucleic acid encoding the heavy chain and a nucleic acid encoding the light chain of the nucleic acid of the present invention described above can be introduced into a host cell to form an antibody within the cell. Accordingly, in one embodiment of the present invention, a host cell (hereinafter sometimes referred to as "the host cell of the present invention") containing the nucleic acid or vector of the present invention is provided. Furthermore, a method for producing the antibody of the present invention using the host cell (hereinafter sometimes referred to as "the method of the present invention") is provided. The method of the present invention includes the step of culturing the host cell of the present invention under conditions under which the antibody of the present invention is produced.

[0029] Examples of cells that can be used to express the antibodies of the present invention or anti-human TIGIT agonist antibodies include mammalian cells. Such mammalian cells include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), Manassas, and VA, including Chinese hamster ovary (CHO) cells, HEK 293 cells, NSO cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and numerous other cell lines. Recombinant antibodies can also be expressed using non-mammalian cells, including (but not limited to) bacteria (e.g., Escherichia coli), yeast, insects, and plants. Furthermore, anti-human TIGIT agonist antibodies can also be produced in transgenic animals such as cattle and chickens.

[0030] There are no particular limitations on the method for introducing nucleic acids encoding antibodies or fragments thereof (e.g., the nucleic acids of the present invention) or vectors into host cells, and known methods can be used. When introducing nucleic acids or plasmid vectors, methods such as calcium phosphate coprecipitation, PEG, electroporation, microinjection, and lipofection can be used. Nucleic acids encoding antibodies or fragments thereof may also be introduced directly into cells in RNA form to express antibodies within the cells. Known methods can be used for RNA introduction, and methods such as lipofection and electroporation are preferably used.

[0031] (Method for producing anti-human TIGIT agonistic antibodies) The resulting recombinant host cells can be maintained under conditions suitable for expression (e.g., in the presence of an inducer, in a suitable non-human animal, in a culture medium supplemented with suitable salts, growth factors, antibiotics, nutritional supplements, etc.), thereby producing one or more encoded polypeptides. In some cases, the heavy chain is produced in one cell and the light chain in another.

[0032] When mammalian cells are used as cells to express the antibodies or anti-human TIGIT agonist antibodies of the present invention, suitable culture media for these cells include, for example, minimal essential medium (MEM) containing approximately 5-20% fetal bovine serum [Science, 122, 501 (1952)], Dulbecco's modified Eagle medium (DMEM) [Virology, 8, 396 (1959)], RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], and 199 medium [Proceedings of the Society for the Biological Medicine, 73, 1 (1950)]. The pH of the medium is preferably approximately 6-8. Culturing is usually carried out at approximately 30°C-40°C. Aeration and stirring may be performed as needed.

[0033] Reagents or kits for the detection or measurement of human TIGIT As described above, the antibody of the present invention has the ability to bind to human TIGIT. Therefore, in another embodiment, a reagent for the detection or measurement of human TIGIT (or non-human TIGIT) containing the antibody of the present invention (hereinafter sometimes referred to as "the reagent of the present invention") is provided. The antibody of the present invention can be used as a reagent for the detection or measurement of human TIGIT by conjugating a labeling substance or the like to such antibody of the present invention, or it can be used to detect or measure human TIGIT in combination with other reagents. Therefore, a kit for the detection or measurement of human TIGIT containing the antibody of the present invention (hereinafter sometimes referred to as "the kit of the present invention") is also provided. The reagent or kit of the present invention can be used in methods using Western blotting, immunohistochemistry, EIA (Enzyme Immunoassay), or ELISA (Enzyme-Linked immunosorbent assay) (e.g., direct method, indirect method, competitive method, sandwich method, etc.).

[0034] The reagents or kits of the present invention may contain other antibodies (including fragments thereof, hereinafter the same) or reagents in addition to the antibody of the present invention. These antibodies or reagents may be pre-packaged with the antibody or stored in separate containers. Examples of antibodies or reagents include antibodies or fragments thereof different from the antibody of the present invention that binds to human TIGIT (hereinafter sometimes referred to as "anti-human TIGIT antibody"), secondary antibodies, labeling substances (e.g., fluorescent dyes (e.g., fluorescein), enzymes (e.g., horseradish peroxidase: HRP, alkaline phosphatase (AP)), radioactive materials, etc.), substrates (PNPP (p-nitrophenyl phosphate), a substrate of AP; ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt), OPD (o-phenylenediamine dihydrochloride), TMB), a substrate of HRP. Examples of reagents include (3,3',5,5'-tetramethylbenzidine, etc.), a stop solution to halt the enzyme reaction, a carrier, human TIGIT bound with a labeling substance (hereinafter sometimes referred to as "labeled human TIGIT"), a reaction vessel, buffer for diluting the processing solution and antibody, a standard substance of known concentration, a positive control (e.g., recombinant human TIGIT), a negative control, and instructions describing the protocol. These reagents can be pre-mixed as needed.

[0035] The antibody of the present invention contained in the reagent or kit described above may be immobilized on a carrier beforehand. The carrier used in the present invention is not particularly limited and can be an insoluble carrier such as a polymer such as polystyrene, glass beads, magnetic particles, a microplate, an immunochromatographic filter paper, or a glass filter. Preferably, it is a microplate used for ELISA. In addition, human TIGIT (including labeled human TIGIT, and human TIGIT to which other peptides such as BSA are bound) may be immobilized on the carrier (preferably a microplate). Accordingly, in one embodiment of the present invention, a reagent or kit for ELISA containing a microplate on which the antibody of the present invention is immobilized, or a reagent or kit for ELISA containing a microplate on which human TIGIT is immobilized and the antibody of the present invention is provided.

[0036] The reagent or kit of the present invention, which includes a secondary antibody, can be used, for example, in Western blotting, immunohistochemistry, EIA, or ELISA indirect methods. The secondary antibody is not particularly limited as long as it can bind to the antibody of the present invention, and can be appropriately selected. Furthermore, it is preferable that the secondary antibody is bound to the labeling substance described above, and the reagent or kit may also contain the substrate described above for the labeling substance. When using the reagent or kit of the present invention in an ELISA indirect method, for example, human TIGIT can be immobilized on a microplate, the antibody of the present invention, followed by the enzyme-labeled secondary antibody against that antibody, and after washing, the enzyme activity remaining on the microplate can be detected or measured to detect or measure human TIGIT or its amount.

[0037] The reagent or kit of the present invention, which includes labeled human TIGIT, can be used, for example, in competitive methods such as EIA or ELISA. The labeled human TIGIT is not particularly limited as long as it can bind to the antibody of the present invention. The labeling substance to be bound to human TIGIT is not particularly limited and includes, for example, the fluorescent dyes and enzymes mentioned above. The labeling substance may be bound to human TIGIT directly or indirectly. Direct binding is performed, for example, by a crosslinking reaction via a linker (e.g., NHS ester, maleimide, etc.), but is not limited to this method. Indirect binding can be performed, for example, by binding one or more tags (e.g., biotin, etc.) to biotin, and then binding the labeling substance to the tag via a substance that binds to the tag (e.g., (strept)avidin, antibody against the tag, etc.). Therefore, the labeled human TIGIT may be provided in a form in which the tagged human TIGIT and the labeling substance having a substance that binds to the tag are different substances. In addition to labeled human TIGIT, the reagent or kit may also contain the above-mentioned substrates for the labeling substance. When using the reagents or kits of the present invention in a competitive EIA or ELISA method, for example, the antibody of the present invention is immobilized on a microplate, and a sample containing human TIGIT and labeled human TIGIT of known concentration are reacted simultaneously in the same microplate. By detecting or measuring the enzyme activity remaining in the microplate after the reaction, human TIGIT or its quantity can be detected or measured.

[0038] The reagent or kit of the present invention, which contains anti-human TIGIT, can be used, for example, in the ELISA sandwich method. The antibody that binds to human TIGIT is not particularly limited as long as it can bind to human TIGIT, but it is preferable that a labeling substance is bound to it. Such a labeling substance is not particularly limited, and examples include the fluorescent dyes and enzymes mentioned above. In addition to the anti-human TIGIT antibody, the reagent or kit may also contain the substrates mentioned above for the labeling substance. When using the reagent or kit of the present invention in the ELISA sandwich method, for example, the antibody of the present invention can be immobilized on a microplate, human TIGIT can be reacted with it, then the labeled anti-human TIGIT antibody can be reacted with it, and after washing, the enzyme activity remaining on the microplate can be detected or measured to detect or measure human TIGIT.

[0039] Anti-human TIGIT antibodies can be produced using existing general manufacturing methods with human TIGIT, its partial peptides, or modified human TIGIT with added amino acids as immunogens. Such antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies (mAbs), and their fragments. Preferred antibodies are monoclonal antibodies or their fragments. Examples of antibodies or their fragments include, but are not limited to, genetically engineered conjugate molecules such as Fab, Fab', and F(ab')2 fragments that have the ability to bind to human TIGIT, or derivatives thereof modified with molecules that have protein-stabilizing effects, such as polyethylene glycol (PEG). In addition, commercially available antibodies may be used as the above-mentioned anti-human TIGIT antibodies.

[0040] Method for detecting or measuring human TIGIT In another embodiment, the present invention provides a method for detecting or measuring human TIGIT using the antibody, reagent, or kit of the present invention (hereinafter sometimes referred to as "the method of the present invention"). In one embodiment, the method of the present invention is (1) A step of contacting human TIGIT in a sample with the antibody of the present invention, and (2) A step of detecting or measuring the antibody of the present invention bound to the human TIGIT. This includes the following. The method can be applied, for example, to Western blotting, immunohistochemistry, EIA, or ELISA (e.g., direct method, indirect method, sandwich method, etc.).

[0041] In step (1) above, the method of contacting the human TIGIT in the sample with the antibody of the present invention is not particularly limited, but can be done, for example, by mixing the sample with a solution containing the antibody of the present invention. If the sample is a tissue section, the human TIGIT can also be contacted with the antibody of the present invention by adding a solution containing the antibody of the present invention to the tissue section. The conditions for contacting the human TIGIT with the antibody of the present invention are not particularly limited, but contact is usually made at a temperature of 0 to 45°C, preferably at a temperature of 0 to 40°C, more preferably at a temperature of 4 to 37°C, and even more preferably at a temperature of 25 to 37°C. There are also no particular limitations on the contact time, but it is usually 5 minutes to 6 hours, preferably 10 minutes to 2 hours, and more preferably 20 minutes to 1 hour.

[0042] Furthermore, step (1) above can be carried out by adding a sample to a carrier on which the antibody of the present invention is immobilized, or by immobilizing human TIGIT in the sample onto a carrier and then adding the antibody of the present invention to the carrier. The same carrier as described in 1. above can be used. There are no particular limitations on the method for immobilizing the antibody of the present invention or human TIGIT in the sample onto a carrier, but examples include methods using physical adsorption, electrostatic interaction, hydrophobic interaction, crosslinking agents, etc. The concentration of the antibody of the present invention in solution when immobilized can be appropriately adjusted according to the material, shape, and method of immobilization of the carrier, for example, a concentration of 5 to 50 μg / mL is possible, and preferably 10 to 40 μg / mL. When immobilizing human TIGIT, the concentration of human TIGIT in solution can also be appropriately adjusted. If a blocking operation is performed on the carrier after immobilization using a blocking agent, non-specific adsorption of the antibody of the present invention or human TIGIT is suppressed, and background noise during measurement is also suppressed. The blocking agent can be appropriately selected from agents commonly used in the field of antigen-antibody reactions such as EIA and ELISA, and collagen peptides are a preferred blocking agent.

[0043] After step (1), a washing step may be included to remove antibodies that did not bind to human TIGIT or human TIGIT that did not bind to the antibodies. Examples of washing solutions used in the washing step include buffer solutions (pH 6-8), and more specifically, Tris buffer, phosphate buffer, HEPES buffer, etc. These buffer solutions may also contain salts, surfactants, proteins, sugars, zwitterionic compounds, etc. as appropriate. Washing solutions included in commercially available immunoassay kits can also be suitably used.

[0044] The method for detecting or measuring the antibody of the present invention bound to human TIGIT in step (2) above is not particularly limited and can be carried out by methods known to the public. Examples of such methods include those described in "Methods in ENZYMOLOGY" Vol. 70 (Immunochemical Techniques (Part A)), Vol. 73 (Immunochemical Techniques (Part B)), Vol. 74 (Immunochemical Techniques (Part C)), Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)), Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), and Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology and Monoclonal Antibodies)) (all published by Academic Press).

[0045] Specifically, for example, if the antibody of the present invention to which a labeling substance has been bound in step (1) above is used, the antibody of the present invention bound to human TIGIT or its amount can be detected or measured by detecting or measuring the labeling substance or its amount. Alternatively, after step (2), the complex of the antibody of the present invention and human TIGIT may be brought into contact with a secondary antibody to which a labeling substance has been bound, or an anti-human TIGIT antibody to which a labeling substance has been bound, which binds to the antibody of the present invention, and the labeling substance or its amount may be detected or measured. Before detection or measurement, a washing step may be provided to remove secondary antibodies or anti-human TIGIT antibodies that could not bind to the antibody of the present invention or human TIGIT. The washing step can be performed in the same manner as the washing step optionally performed after step (1) described above. In addition, the secondary antibody and anti-human TIGIT antibody can be the same as those described in 3 above.

[0046] When a fluorescent substance is used as a labeling agent, the fluorescence it emits can be detected or measured using a plate reader or similar device. When an enzyme is used as a labeling agent, a substrate that decomposes and emits color or light due to the enzyme's action can be added, allowing the resulting color, light emission, and their levels to be detected or measured using a plate reader or similar device. Furthermore, when a radioactive substance is used as a labeling agent, the radiation emitted by the substance can be detected or measured using a scintillation counter or similar device. Based on the data detected or measured in this way, the amount of human TIGIT can also be quantified using image processing software (e.g., ImageJ).

[0047] Furthermore, in another embodiment of the method of the present invention, the method of the present invention is (1') A step of contacting labeled human TIGIT and human TIGIT in a sample with the antibody of the present invention, and (2') A step of detecting or measuring the antibody of the present invention conjugated to the labeled human TIGIT. This includes the above. The above method can be applied, for example, to competitive methods such as EIA or ELISA.

[0048] In step (1') described above, the method for contacting the labeled human TIGIT and the human TIGIT in the sample with the antibody of the present invention is not particularly limited, but can be carried out, for example, by mixing a solution containing the labeled human TIGIT, the sample, and a solution containing the antibody of the present invention. The conditions for contacting the labeled human TIGIT and the human TIGIT with the antibody of the present invention are the same conditions as those described in step (1) described above.

[0049] Furthermore, step (1') above can also be carried out by adding a sample and labeled human TIGIT of known concentration to a carrier on which the antibody of the present invention is immobilized, or by immobilizing human TIGIT in the sample onto a carrier and then adding the labeled human TIGIT and the antibody of the present invention to the carrier. The type of carrier, the method of immobilization, the blocking agent, etc., can be the same as those described in step (1) above.

[0050] After step (1'), a washing step may be provided to remove antibodies that did not bind to human TIGIT or labeled human TIGIT, or human TIGIT or labeled human TIGIT that did not bind to the antibodies. The washing solution used in the washing step may be the same as that described in step (1) above.

[0051] The method for detecting or measuring the antibody of the present invention bound to labeled human TIGIT in step (2') above involves detecting or measuring the labeled substance bound to the labeled human TIGIT or the amount thereof, thereby enabling the detection or measurement of the antibody of the present invention bound to human TIGIT or the amount thereof.

[0052] When a fluorescent substance is used as a labeling substance, the fluorescence it emits can be detected or measured using a plate reader or the like to determine the labeling substance or its quantity. When an enzyme is used as a labeling substance, by adding a substrate that decomposes and produces color or light emission through the action of the enzyme, the resulting color, light emission, or its level can be detected or measured using a plate reader or the like. When a radioactive substance is used as a labeling substance, the amount of radiation emitted by the substance can be detected or measured using a scintillation counter or the like. In the method of the present invention, if the amount of human TIGIT contained in the sample is large, the amount of labeled human TIGIT that can bind to the antibody of the present invention decreases, and the labeling level (e.g., color or fluorescence level) becomes weaker. On the other hand, if the amount of human TIGIT in the sample is small, the amount of labeled human TIGIT that can bind to the antibody of the present invention increases, and the labeling level (e.g., color or fluorescence level) becomes stronger.

[0053] Examples of samples used in the method of the present invention include samples collected from animals. These samples may be known to contain human TIGIT (for example, including the morphology of cells expressing human TIGIT), or they may be unknown to contain human TIGIT. Examples of such animals include mammals such as mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, and humans. Examples of animal-derived samples include blood, serum, plasma, saliva, urine, tears, sweat, milk, nasal secretions, semen, pleural fluid, gastrointestinal secretions, cerebrospinal fluid, interstitial fluid, and lymph, with serum and plasma being preferred. Cell populations obtained by cell culture are also preferred. These samples can be obtained by known methods; for example, serum and plasma can be prepared by collecting blood from a test animal according to conventional methods and separating the liquid components, and cerebrospinal fluid can be collected by known means such as spinal puncture.

[0054] Pharmaceuticals comprising the antibody of the present invention The present invention also provides a pharmaceutical product containing the antibody of the present invention as an active ingredient. The antibody of the present invention can suppress immune activation via TIGIT activation in cytotoxic T cells (CTLs) such as CD8-positive T cells or natural killer (NK) cells, and can enhance the ability to suppress immune responses via TIGIT activation in regulatory T cells (Treg cells). Therefore, a pharmaceutical product containing the antibody of the present invention can be used for the prevention or treatment of autoimmune diseases or diseases involved in the activation of CTLs or NK cells. Examples of such diseases include rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis (MS), inflammatory bowel disease (IBD), celiac disease, graft-versus-host disease (GVHD), and irritable bowel syndrome. Other examples of such diseases include Sjögren's syndrome, non-alcoholic steatohepatitis (NASH), and primary biliary cirrhosis. Examples of animals to which the pharmaceutical product of the present invention can be administered include primates, such as lemurs, lorises, Zweigera monkeys, monkeys (e.g., crab-eating macaques), and humans, preferably humans.

[0055] The antibodies and anti-human TIGIT agonist antibodies of the present invention are preferably prepared as pharmaceutical compositions according to conventional methods. Furthermore, the therapeutic agents of the present invention may contain pharmaceutically acceptable carriers and / or additives as needed. For example, they may contain surfactants (PEG, Tween, etc.), excipients, antioxidants (ascorbic acid, etc.), colorants, flavorings, preservatives, stabilizers, buffers (phosphoric acid, citric acid, other organic acids, etc.), chelating agents (EDTA, etc.), suspending agents, isotonic agents, binders, disintegrants, lubricants, flow enhancers, flavoring agents, etc. However, the pharmaceutical compositions are not limited to these and may contain other commonly used carriers as appropriate. Specifically, examples include light anhydrous silicic acid, lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium-chain triglyceride, polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, etc. It may also contain other low molecular weight polypeptides, proteins such as serum albumin, gelatin and immunoglobulins, and amino acids. When preparing an aqueous solution for injection, the anti-human TIGIT agonist antibody is dissolved in an isotonic solution containing, for example, physiological saline, glucose, or other adjuvants. Examples of adjuvants include D-sorbitol, D-mannose, D-mannitol, and sodium chloride. Furthermore, appropriate solubilizers, such as alcohol (ethanol, etc.), polyalcohols (propylene glycol, PEG, etc.), and nonionic surfactants (polysorbate 80, HCO-50), may be used in combination.

[0056] Furthermore, polypeptides can be encapsulated in microcapsules (such as hydroxymethylcellulose, gelatin, or poly[methylmethacrylate]) or incorporated into colloidal drug delivery systems (liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules, etc.) as needed (see Remington's Pharmaceutical Science 16th edition &, Oslo Ed. (1980), etc.).

[0057] The content of the anti-human TIGIT agonist antibody in the pharmaceutical composition is, for example, about 0.01 to 100% by weight of the total pharmaceutical composition, preferably 0.1 to 99.9% by weight.

[0058] The above-mentioned pharmaceutical composition can be administered orally or parenterally, but parenteral administration is preferred. Specifically, it is administered to patients by injection and transdermal administration. Examples of injectable formulations include, for example, intravenous injection, intramuscular injection, or subcutaneous injection, which can be administered systemically or locally. Local injection, particularly intramuscular injection, may be administered to or around the treatment site. Examples of transdermal formulations include, for example, ointments, gels, creams, patches, and transdermal patches, which can be administered systemically or locally. Furthermore, the method of administration can be appropriately selected depending on the patient's age and symptoms. As for the dosage, for example, it is possible to select a range of 0.5 mg to 10 mg of anti-human TIGIT agonist antibody per kg of body weight per dose. However, the pharmaceutical composition of the present invention is not limited to these dosages.

[0059] The sequence numbers in the sequence listing of this specification indicate the following sequences: (SEQ ID NO: 1) Amino acid sequence of human TIGIT (SEQ ID NO: 2) Amino acid sequence of Clone M1-8 H-CDR1 (Sequence ID 3) Amino acid sequence of Clone M1-8 H-CDR2 (SEQ ID NO: 4) Amino acid sequence of Clone M1-8 H-CDR3 (SEQ ID NO: 5) Amino acid sequence of Clone M1-8 L-CDR1 (SEQ ID NO: 6) Amino acid sequence of Clone M1-8 L-CDR3 (SEQ ID NO: 7) Clone M1-8 heavy chain variable region amino acid sequence (mouse-type heavy chain variable region) (Sequence ID 8) Clone M1-8 Light Chain Variable Region Amino Acid Sequence (Mouse-type Light Chain Variable Region)

[0060] The present invention will be described in more detail by the following examples, but the scope of the present invention is not limited to these examples. [Examples]

[0061] Example 1: Preparation of novel anti-human TIGIT agonist antibodies (M1-8) <Construction of recombinant human TIGIT-His-mFc protein> Recombinant human TIGIT-His-mFc protein was prepared using the Expi293 Expression System (Thermo). After culturing cells transfected with the human TIGIT-His-mFc expression plasmid, the recombinant human TIGIT-His-mFc protein was purified from the culture supernatant by gel filtration using a Ni-NTA cartridge (Fujifilm Wako Pure Chemical Industries) column and a Superdex 200 pg column.

[0062] <Immunization method> Female CD2F1 mice aged 6-8 weeks were subcutaneously immunized bilaterally in the popliteal fossa with recombinant human TIGIT-His-mFc protein along with TiterMax at a dose of 10 μg / m / m. Subsequently, the same amount of immunogen was administered 11 times at 3-4 days intervals along with CpG / Alum. As a final immunization, 100 μL of immunogen diluted to 1 mg / mL in phosphate-buffered saline (PBS, pH 7.4) was administered intraperitoneally to each mouse three days before lymph node dissection.

[0063] <Measurement of blood antibody titers> Blood was collected from the tail vein 36 days after the initial immunization, and the obtained blood was diluted 100-fold with physiological saline. Antiserum was obtained by centrifugation of the diluted blood at 4°C for 5 minutes and collection of the supernatant. Antibody titers were measured by the following method. Doxycycline-induced human TIGIT-expressing cells were cultured for 2 days under conditions with and without doxycycline. After washing the cells with PBS, cell dissociation buffer (GIBCO) was added, and the cells were allowed to stand at room temperature for 10 minutes to detach them from the flask. The obtained cells were washed twice with FACS buffer (PBS containing 1% FBS), and then 1 x 10⁶ 6 Each cell was suspended in FACS buffer to achieve a cell density of cells / mL. 100 μL of each suspension was dispensed into 96-well V-bottom plates, centrifuged, and the supernatant was removed. 100 μL of antiserum diluted in FACS buffer was then dispensed into these plates and allowed to stand at 4°C for 30 minutes. After washing three times with FACS buffer, 100 μL of AlexaFluoro647®-labeled anti-mouse IgG antibody was dispensed and allowed to stand at 4°C for 30 minutes. After washing twice with FACS buffer, the antibodies were suspended in 200 μL of FACS buffer, and the bound antibodies were measured using a Cytomics FC500MPL.

[0064] Figure 1 shows the obtained median fluorescence intensity. An increase in antibody titer against doxycycline-induced human TIGIT-expressing cells was observed in the antiserum of mice immunized with recombinant TIGIT protein.

[0065] <Production of monoclonal antibodies> Three days after the final immunization, lymph nodes were excised from mice No. 1, 2, 4, and 5, in which an increase in antibody titer was observed, compressed and filtered on a 40 μm strainer, and suspended in DMEM. P3-X63.Ag8.U1 (P3U1) was used as the fusion partner for the production of hybridomas. Lymphocytes and P3U1 were each washed three times with DMEM and mixed so that the cell number ratio was 1:1. The cells washed twice with fusion buffer (0.3M mannitol, 0.1 mM calcium chloride, 0.1 mM magnesium sulphate) were suspended in fusion buffer at 1.51 x 10 7 cells / mL. Cell fusion was carried out manually using BTX. The fused cells were washed once with ClonalCell TM -HY Medium C (STEMCELL), suspended in the same medium at 1.67 x 10 6 cells / mL, and then left standing overnight at 37°C under 5% CO2 conditions. The obtained fused cells were centrifuged, suspended in ClonaCell TM -HY Medium C at 1 x 10 6 cells / mL, mixed with ClonaCell TM -HY Medium D, and seeded on plates. After culturing for 10 days at 37°C under 5% CO2 conditions, hybridoma colonies were picked using Clone Pix and seeded on plates to which 200 μL / well of ClonaCell TM -HY Medium E was added. After culturing for 3 days at 37°C under 5% CO2 conditions, the culture supernatant was collected and used for screening.

[0066] <Preparation of transient expression cells> Transient expression cells were prepared manually using the Expi293F expression system (Thermo). Specifically, Expi293F cells in the logarithmic growth phase were seeded at 1x10 6After suspension to a cell density of cells / mL, it was dispensed into 125 mL Meyer flasks at 35 mL each. 37.5 μg of the TIGIT expression plasmid and 50 μL of 293Fectin were each diluted with OptiMEM I medium to a final volume of 1.25 mL and allowed to stand at room temperature for 5 minutes. The diluted DNA solution and 293Fectin solution were mixed and further allowed to stand at room temperature for 20 minutes. The mixture was added to the cells and cultured at 37°C, 8% CO2, and 120 rpm for 2 days, and the cells were collected.

[0067] <Primary screening (Mirrorball assay)> The primary screening was performed using transient expression cell lines and their parental lines. After collecting the cells, they were washed 3 times with FACS buffer and suspended in the same buffer at 3 x 10 5 cells / mL. Equal volumes of the cells were mixed with AlexaFluor® 647-labeled anti-mouse IgG (subclasses 1+2a+2b+3) antibody diluted to 1 μg / mL in the same buffer, and 20 μL each was dispensed into 384-well plates. To each plate, 10 μL of the culture supernatant of the hybridoma diluted 10-fold with FACS buffer was added, stirred with a plate mixer, and centrifuged for 1 minute. After standing at room temperature for 1 hour, the fluorescence intensity was measured using a plate imager (Mirrorball).

[0068] <Measurement of antibody concentration in culture supernatant by ELISA method> 50 μL of anti-mouse IgG (subclass 1+2a+2b+3) antibody, diluted to 5 μg / mL in PBS, was dispensed into 96 half-well ELISA plates and left to stand overnight at 4°C. Next, the plates were washed twice with wash buffer (PBS containing 0.05% tween 20), and then 100 μL of blocking buffer (PBS containing 20% ​​Immunoblock) was dispensed into each plate and left to stand at room temperature for 1 hour. To this plate, 50 μL of hybridoma culture supernatant or mouse IgG1 standard solution (0-300 ng / mL), diluted 100-fold or 1,000-fold in assay buffer (PBS containing 10% Immunoblock), was added and left to stand at room temperature for 1 hour. After washing the plates three times with wash buffer, 50 μL of HRP-labeled anti-mouse IgG (subclass 1+2a+2b+3) antibody was dispensed into each plate and left to stand at room temperature for 1 hour. After washing three times with washing buffer, 50 μL of Sure Blue / TMB Peroxidase Substrate (KPL) was dispensed into each sample and reacted at room temperature for 5 minutes. The reaction was stopped by dispensing 50 μL of 0.5 M sulfuric acid into each sample, and the absorbance at 450 nm was measured using a plate reader (SpectraMax). Softmax was used to create the calibration curve and calculate the concentrations.

[0069] Human, monkey, and mouse TIGIT-expressing cells were prepared using doxycycline-expressing CHO cell lines. Specifically, human, monkey, and mouse TIGIT expression plasmids were transfected into doxycycline-expressing CHO cell lines. After cloning the transfected cells using the limiting dilution method, TIGIT expression was induced by culturing them in doxycycline-containing medium, and cells showing confirmed expression were selected as TIGIT-expressing cells.

[0070] <Secondary Screening (FACS)> Secondary screening was performed using the same method as for measuring blood antibody titers. Specifically, doxycycline-induced human TIGIT-expressing cells were cultured for 2 days under conditions with and without doxycycline. After washing the cells with PBS, cell dissociation buffer (GIBCO) was added, and the cells were allowed to stand at room temperature for 10 minutes to detach them from the flask. The obtained cells were washed twice with FACS buffer (PBS containing 1% FBS), and then 1 x 10⁶ cells were used. 6 Each cell was suspended in FACS buffer to achieve a cell density of cells / mL. 100 μL of each suspension was dispensed into 96-well V-bottom plates, centrifuged, and the supernatant was removed. 100 μL of hybridoma culture supernatant, diluted to 1 μg / mL with FACS buffer, was added to each plate and allowed to stand at 4°C for 30 minutes. After washing three times with FACS buffer, 100 μL of AlexaFluoro647®-labeled anti-mouse IgG antibody was dispensed and allowed to stand at 4°C for 30 minutes. After washing twice with FACS buffer, the antibodies were suspended in 200 μL of FACS buffer, and the bound antibodies were measured using Cytomics FC500MPL.

[0071] Figure 2 shows the binding activity of the M1-8 antibody in the culture supernatant to doxycycline-induced human TIGIT-expressing cells.

[0072] <Third-stage screening (recombinant TIGIT, PVR-expressing cell binding inhibition assay)> Recombinant human TIGIT human IgG1Fc fusion protein (R&D systems, 7898-TG-050), diluted to 2 nM in FACS buffer, and hybridoma culture supernatant, diluted to 10-1,000 ng / mL, were mixed in a 1:1 ratio in 96-well V-bottom plates and allowed to stand at room temperature for 30 minutes. Doxycycline-induced human PVR-expressing cells cultured in the presence of doxycycline for 2 days were then mixed in 1 x 10⁶ FACS buffer. 6Suspended in cells / mL and dispensed 100 μL each into another 96-well V-bottom plate. The plate containing the cells was centrifuged, and after discarding the supernatant, the pre-mixed TIGIT and culture supernatant mixture was added and left standing at 4°C for 30 minutes. After washing three times with FACS buffer, 100 μL each of AlexaFluoro647 (registered trademark)-labeled anti-human IgG antibody was dispensed and left standing at 4°C for 30 minutes. After washing twice with FACS buffer, it was suspended in 200 μL of FACS buffer, and the bound TIGIT-Fc was measured using Cytomics FC500MPL.

[0073] Figure 3 shows the results of the recombinant TIGIT and PVR-expressing cell binding inhibition assay. The commercially available anti-human TIGIT antibody (clone MSBA43) inhibited the binding of TIGIT and PVR-expressing cells, but the M1-8 antibody in the culture supernatant did not.

[0074] <Antibody Isotyping by ELISA Method> 50 μL each of various anti-mouse isotype antibodies (ITM, BioLegend), diluted to 5 μg / mL in PBS, was dispensed into 96 half-well ELISA plates and left to stand overnight at 4°C. Next, the plates were washed twice with washing buffer (PBS containing 0.05% tween 20), then 100 μL of SuperBlock (PBS) Blocking Buffer (Thermo) was dispensed into each plate and left to stand at room temperature for 1 hour. 50 μL of hybridoma culture supernatant, diluted 10-fold with assay buffer (PBS containing 10% SuperBlock (PBS) Blocking Buffer), was added to each plate and left to stand at room temperature for 1 hour. After washing the plates three times with washing buffer, 50 μL of HRP-labeled anti-mouse IgG antibody was dispensed into each plate and left to stand at room temperature for 1 hour. After washing three times with washing buffer, 50 μL of Sure Blue / TMB Peroxidase Substrate (KPL) was dispensed into each plate and reacted at room temperature for 5 minutes. After stopping the reaction by dispensing 50 μL of 0.5 M sulfuric acid into each sample, the absorbance at 450 nm was measured using a plate reader (SpectraMax). Isotyping revealed that the M1-8 antibody was mouse IgG1 / k.

[0075] <Determination of the CDR region of the antibody> The CDR of the M1-8 antibody was defined according to the IMGT numbering system.

[0076] <Antibody purification> ClonaCell TM-Hybridomas cultured in HY Medium E and in the logarithmic growth phase were added to a 24-well plate containing 0.5 mL of Daigo's T medium (10% Ultra Low IgG FBS, 1% MEM NEAA, 1% Sodium Pyruvate, 1% L-Alanyl-L-Glutamine, 1% Penicillin-Streptomycin, 43% F-12 nutrient mixture, 43% Iscove's Modified Dulbecco's Medium) and cultured at 37°C under 5% CO2 conditions. After 2-3 days of culture, the cells were added to a 6-well plate containing 4 mL of Daigo's T medium and cultured for a further 2-3 days to acclimate them to Daigo's T medium. After confirming that the cells were sufficiently acclimatized, they were added to a T-75 flask containing 45 mL of Daigo's T medium and cultured at 37°C under 5% CO2 conditions. After 7-10 days, the culture medium was collected, and the culture supernatant was obtained by centrifugation. Monoclonal antibodies were purified from the obtained culture supernatant using protein A resin. 0.6 mL of 50% slurry protein A Sepharose resin, equilibrated with PBS, was added to the culture supernatant and shaken overnight at 4°C. After centrifugation at 4°C for 10 minutes, the supernatant was partially aspirated and the resin was suspended in the remaining medium and added to a 24-well filter plate (Whatman). The resin was washed three times with 5 mL of PBS, and the conjugated antibodies were eluted with 2.5 mL of elution buffer (0.1 M Glycine-HCl / 0.3 M NaCl, pH 3.0). The eluate was immediately neutralized with 0.3 mL of neutralization buffer (1 M Tris-HCl, pH 8.0). For the purification of mouse IgG1, the culture supernatant was mixed in equal volumes with mouse IgG1 conjugation buffer (1.5 M Glycine-HCl, 3 M NaCl, pH 9.5), and then protein A Sepharose resin was added. Furthermore, mouse IgG1 binding buffer was used instead of PBS for washing. The purified antibodies obtained were buffered in PBS using an ultrafiltration column (Amicon), and their concentrations were measured using a spectrophotometer (NanoDrop).

[0077] <Measurement of Biological Activity of Purified Antibodies> • Preparation of serially diluted antibody solutions (Day 0) The test antibody and PVR (Recombinant Human CD155 / PVR, R&D systems, 2530-CD-050) were used as a positive control, and IgG was used as a negative control, both serially diluted in culture medium (RPMI1640: Fujifilm Wako Pure Chemical Industries, 189-02025, 10% FBS: CORNING, 37-076-CVR, 1×PS: Penicillin-streptomycin solution (×100), Fujifilm Wako Pure Chemical Industries, 168-23191). Five serial dilutions were prepared for the test antibody and IgG from a final concentration of 10 μg / mL to 100 ng / mL using a 3-fold common ratio, and five serial dilutions were prepared for PVR from a final concentration of 33 μg / mL to 333 ng / mL using a 3-fold common ratio.

[0078] • Preparing the coating plate (Day 0) Anti-human CD3 (eBioscience, 16-0037) was diluted to 3 μg / mL with PBS (D-PBS(-), Fujifilm Wako Pure Chemical Industries, 045-29795) and added to 50 μL / well of a 96-well plate (black solid flat-bottom cell culture surface-treated polystyrene microplate, CORNING, 3916). The plates were sealed and left to stand overnight in a refrigerator at 4°C.

[0079] • Cell seeding and sample addition (agonist assay) (Day 0) Doxycycline-induced human TIGIT-expressing cells (hTIGIT / Jurkat) were treated with doxycycline to a concentration of 2 μg / mL and seeded at 60 μL / well in 96-well plates (clear, flat-bottom, cell culture surface-treated microplates, CORNING, 3598) that had been pre-filled with 48 μL / well of culture medium. Further, 12 μL / well each of the previously serially diluted test antibody, positive control, and negative control were added. 12 μL / well each of the 100% control wells was added to contain a final concentration of 3.3 μg / mL of PVR, and 12 μL / well each of the 0% control wells was added to contain culture medium. The cells were incubated overnight in a CO2 incubator (37°C, 5% CO2).

[0080] • Cell seeding and sample addition (antagonist assay) (Day 0) Doxycycline was added to doxycycline-inducible human TIGIT-expressing cells (hTIGIT / Jurkat) to a concentration of 2 μg / mL. These cells were then seeded at 60 μL / well in a 96-well plate (clear, flat-bottom, cell culture surface-treated microplate, CORNING, 3598) that had been pre-filled with 36 μL / well of culture medium in the sample, positive control, and negative control wells, and 48 μL / well in the 100% control and 0% control wells. The previously serially diluted test antibody, positive control, and negative control were added at 12 μL / well each. Culture medium was added to the 100% control well, and 12 μL / well of PVR at a final concentration of 3.3 μg / mL was added to the 0% control well. Finally, 12 μL / well of PVR at a final concentration of 3.3 μg / mL was added to the sample, positive control, and negative control wells. The cells were incubated overnight in a CO2 incubator (37°C, 5% CO2).

[0081] • Cell seeding and sample addition (Cytotoxicity / Growth inhibition assay) (Day 0) Doxycycline-induced human TIGIT-expressing cells (hTIGIT / Jurkat) were treated with doxycycline to a concentration of 2 μg / mL. These cells were then seeded into 96-well plates (clear, flat-bottom, cell culture surface-treated microplates, CORNING, 3598) that had been pre-filled with 48 μL / well of culture medium. 60 μL / well of culture medium was added to all but the 0% control well. 12 μL / well each of the previously serially diluted test antibody, positive control, and negative control were then added. 12 μL / well of PVR (Phase-Vitamin Rase) with a final concentration of 3.3 μg / mL was added to the 100% control well, and 12 μL / well of culture medium was added to the 0% control well. The cells were incubated overnight in a CO2 incubator (37°C, 5% CO2).

[0082] • Cellular stimulation (Day 1) After removing the solution from the anti-human CD3 coated plate, the plate was washed once with 200 μL / well PBS, and 90 μL / well of cell solution from each assay, to which the sample had been added on Day 0, was transferred. Anti-human CD28 (eBioscience, 16-0289) was diluted to a final concentration of 2 μg / mL and added to each plate at a rate of 10 μL / well. After mixing with a plate mixer (Titramax 100, Heidolph), the plates were incubated overnight in a CO2 incubator (37°C, 5% CO2).

[0083] • Measurement (Day 2) For the Agonist assay and Antagonist assay, the Nano-Glo® Luciferase Assay System (Promega, N1120) was used, and for the Cytotoxicity / Growth inhibition assay, the CellTiter-Glo® Luminescent Cell Viability Assay System (Promega, G7572) was used. Cells that had been returned to room temperature from a CO2 incubator were then treated with Nano-Glo. TMNano-Glo diluted 50-fold with Luciferase Assay Buffer TM Luciferase Assay Substrate solution was added at a rate of 100 μL / well, or CellTiter-Glo® Substrate solution dissolved in CellTiter-Glo® Buffer was added at a rate of 100 μL / well. The mixture was stirred using a plate mixer (Titramax 100, Heidolph), incubated for 3 minutes, and then the luminescence value for 1 second was measured using a multi-label counter Envision (2103 Envision, PerkinElmer).

[0084] ·analysis The sample values ​​were calculated using the following formula. Agonist assay: Sample agonist activity (%) = (1 - (measured value of compound - mean value of 100% control well) / (mean value of 0% control - mean value of 100% control well)) × 100 The calculated expression levels (%) were graphed using GraphPad Prism, and the EC50 value was calculated. Aliagonist assay: Antagonist activity (%) of the sample = (Measured value of the compound - Mean value of the 100% control well) / (Mean value of the 0% control - Mean value of the 100% control well) × 100 The calculated inhibition rate (%) was graphed using GraphPad Prism, and the IC50 value was calculated. Cytotoxicity / Growth inhibition assay: Cytotoxicity of the sample (%) = (1 - (measured value of compound - mean value of 0% control well) / (mean value of 100% control - mean value of 0% control well)) × 100 The calculated injury percentage was graphed using GraphPad Prism, and the IC50 value was calculated.

[0085] Figure 4 shows the results of the measurement of the biological activity of the M1-8 antibody. The M1-8 antibody showed agonist activity but not antagonist activity. Furthermore, under these conditions, the M1-8 antibody did not show cytotoxic activity against the human TIGIT-expressing cells used in this measurement, nor did it affect cell proliferation.

[0086] <Cross-reactivity test> Cross-reactivity testing of the obtained antibodies was performed by FACS using human, monkey, and mouse TIGIT-expressing cells. Specifically, doxycycline-inducing cells were cultured for 2 days under conditions with and without doxycycline. After washing the cells with PBS, Cell Dissociation Buffer (GIBCO) was added, and the cells were allowed to stand at room temperature for 10 minutes to detach them from the flask. The obtained cells were washed twice with FACS buffer (PBS containing 1% FBS), and then 1 x 10⁶ cells were separated. 6 Each suspension was prepared using FACS buffer to achieve a cell density of cells / mL. 100 μL of each suspension was dispensed into 96-well V-bottom plates, centrifuged, and the supernatant was removed. 100 μL of purified antibody, diluted to 1 μg / mL with FACS buffer, was added to each plate, and the plates were incubated at 4°C for 30 minutes. After washing three times with FACS buffer, 100 μL of AlexaFluoro647®-labeled anti-mouse IgG antibody was dispensed and incubated at 4°C for 30 minutes. After washing twice with FACS buffer, the antibodies were suspended in 200 μL of FACS buffer, and the bound antibodies were measured using a Cytomics FC500MPL.

[0087] Figure 5 shows the results of the cross-reactivity of the M1-8 antibody against human, monkey, and mouse TIGIT. It was found that the M1-8 antibody binds to human and monkey TIGIT, but not to mouse TIGIT.

[0088] In the following embodiments, the following antibodies and culture medium were used: Human; anti-CD3 (Hit3a), anti-CD4 (FITC, OKT4 or BV510, OKT4), anti-CD19(BV421, HIB19), anti-CD25 (PE, BC96), anti-CD27(BV510, O323), CD38(FITC, HIT2)anti-CD45RA (BV421, HI100), anti-CD127 (FITC, A019D5), anti-CD138(APC, MI15), anti-CXCR5(PerCP / Cy5.5, J252D4), anti-human TIGIT(PE-Cy7, A15153G) (いずれもBioLegend), 7-aminoactinomycin D(Bay Bioscience), agonistic anti-TIGIT antibody(Clone M1-8, antibody). Mouse; Specifically anti-CD44 (FITC, IM7) , anti-CD62L (PE, MEL-14) , anti-CD69 (APC,H.12F3), anti-CD95(APC, SA367H8), anti-CD138 (BV421, 281-2), anti-CD185(PE, L138D7), anti-GL7(PE-Cy7, GL7), (and BioLegend), anti-B220(PE, RA3-6B2), anti-CD3ε (PE-cy5, 145-2C11), and anti-CD4 (eFluor450 or APC (and Thermo Fisher Scientific). Anti-Foxp3 antibodies (PE, FJK-16s Thermo Fisher Scientific). Isotype control; mIgG1(MG1-45) (Biolegend). Complete RPMI (RPMI1640 with the following added: 2 mM L-glutamine, 1% non-essential amino acids, 55 μM 2-mercaptoethanol (Thermo Fisher Scientific), 1 mM sodium pyruvate, 100 U / ml penicillin, 100 U / ml streptomycin, 10% FBS (Thermo Fisher Scientific)).

[0089] Statistical analysis was performed using the Wilcoxon rank-sum test to examine differences between continuous variables. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using JMP 15 (SAS Institute).

[0090] Example 2: Verification of suppression of CD8-positive T cell activation via TIGIT activation Peripheral blood was collected from four healthy individuals using heparinized blood collection tubes (Terumo), and PBMCs were isolated using Ficoll-paque plus (GE Healthcare). CD8-positive T cells were isolated from the PBMCs using MACS with a CD8+ T cell isolation kit (Milteny). After staining these cells with CFSE (Invitrogen) reagent, they were suspended in RPMI1640 / 10% FBS / PSG medium and 1x10⁻¹⁶ cells were used. 5 Cells were seeded in a 96-well round-bottom plate at a rate of cells / well. The seeded cells were then treated with 1x10⁻¹⁶ cells each, which consisted of an anti-human CD3 antibody (Biolegend) and either human PVR-Fc (R&D systems) or human IgG1-Fc (R&D systems) conjugated to Dynabeads-M450 (Invitrogen). 5 The mixture was combined to form beads per well and incubated for 3 days under conditions of 5% CO2 and 37°C.

[0091] The method for binding antibodies to Dynabeads-M450 is as follows: Suspend Dynabeads-M450 in Binding buffer (0.1 M sodium phosphate pH 7.6), wash once using a magnet, then resuspend in Binding buffer, and add 2.5 μg / 10 of anti-CD3 antibody to it. 7 beads, PVR-Fc and IgG1-Fc, total amount of PVR-Fc and IgG1-Fc: 2.5 μg / 10 7 The mixture was mixed to form beads and stirred overnight at 4°C. The next day, it was washed twice with Wash buffer (PBS / 0.1% BSA / 2 mM EDTA) using a magnet, and then stirred with Blocking buffer (0.2 M Tris-HCl pH 8.5 / 0.1% BSA) at room temperature for 4 hours. It was washed once with Wash buffer and a magnet, and finally 4x10 units were added to Wash buffer. 7 The Dynabeads / antibody / Fc complex was suspended to a concentration of beads / ml. The prepared Dynabeads / antibody / Fc complex was stored at 4°C and used in subsequent experiments.

[0092] CD8-positive T cells were mixed with Dynabeads / anti-CD3 antibody / PVR-Fc and cultured for 3 days under 5% CO2 and 37°C conditions. The culture supernatant was then collected and the production levels of IFNγ (Biolegend), Granzyme B (Mabtech), and Perforin (Mabtech) were measured by ELISA. Cells were also collected, stained with anti-CD3 antibody-APCCy7, anti-CD8 antibody-BV421, and anti-CD4 antibody-BV510 (all Biolegend), and detected by FACS along with CFSE. The rate of cell proliferation was quantitatively analyzed based on the dilution ratio of CFSE. The results are shown in Figure 6. From Figure 6, it can be seen that the proliferation of CD8-positive T cells and the production of cytokines (IFNγ, Granzyme B, Perforin) were suppressed in a dose-dependent manner by PVR-Fc. These results suggest that PVR suppresses the activation of CD8-positive T cells through binding to its ligand, TIGIT, and that anti-TIGIT agonistic antibodies also suppress the activation of CD8-positive T cells.

[0093] Example 3: Effects on Tfh cells Blood was collected in heparinized blood collection tubes (TERUMO, Tokyo, Japan) and converted into PBMCs using Lymphoprep (Axis-Shield). Furthermore, CD4+ T cells were negatively selected using the CD4+ T Cell Isolation Kit (Miltenyi Biotec). The cell surface was stained with various antibodies, and the cells were fractionated into the following cell populations using a FACSAria III flow cytometer: Tfh; CD45RA-CXCR5+, Naive T; CD45RA+CXCR5-, non-Tfh; CD45RA-CXCR5-. The obtained CD4+ naive T cells and Tfh cells were stored in a 96-well U plate in a 1 × 10⁶ arrangement. 4 Cells were cultured in one well with the same amount of Dynabeads human T-Activator CD3 / CD28 (Thermo Fisher Scientific) added as the cells. Each cell was cultured in four wells under the same conditions, and TIGIT expression on the cells was analyzed using a FACSVerse flow cytometer at 24, 48, 72, and 96 hours. Cell proliferation was analyzed using FlowJo software version 10.4.2 (FlowJo, OR, USA).

[0094] As shown in Figure 7-1a, Tfh cells, which originally had high TIGIT expression, showed a further increase in TIGIT expression upon stimulation, whereas naive T cells, which had low TIGIT expression, did not show an increase in TIGIT expression upon stimulation.

[0095] Each cell was stained with 2 μM CellTrace Violet (Thermo Fisher Scientific) at 37°C for 7 minutes, washed, and cultured for 96 hours in a 96-well flat-bottom plate immobilized with 2 μg / ml anti-CD3 antibody, 1 μg / ml anti-CD28 antibody, and 10 μg / ml isotype (mouseIgG1) or anti-TIGIT agonistic antibody. Dead cells were removed by staining with 7-aminoactinomycin D, and viable cells were analyzed using a FACSVerse flow cytometer. Cell proliferation was analyzed using FlowJo software version 10.4.2 (FlowJo, OR, USA).

[0096] As shown in Figure 7-1b, the proliferation of Tfh cells was significantly suppressed by stimulation with anti-TIGIT agonistic antibodies, while no inhibitory effect was observed in naive T cells. In other words, the selective inhibitory effect of anti-TIGIT agonistic antibodies on the proliferation of cells with high TIGIT expression was demonstrated.

[0097] In addition to CD4+ naive T cells and Tfh cells, TIGIT expression on non-Tfh cells was also examined in the same manner as described above. As a result, non-Tfh cells showed TIGIT expression at a level intermediate between that of Tfh cells and naive T cells (Figure 7-2a). Furthermore, flow cytometry analysis using CellTrance violet, similar to the above, compared the proliferation inhibitory effect of the anti-TIGIT agonistic antibody on Tfh cells and non-Tfh cells. The results showed that the antibody also inhibits the proliferation of non-Tfh cells, but exhibits a more selective inhibitory effect on Tfh cells (Figure 7-2b).

[0098] Example 4: Effects of Tfh cells on B cells Blood was collected in heparinized blood collection tubes (TERUMO, Tokyo, Japan) and converted into PBMCs using Lymphoprep (Axis-Shield). Furthermore, CD19+ cells were positively selected using CD19 Microbeads. The cell surface was stained with various antibodies, and the cells were fractionated into the following cell populations using a FACSAria III flow cytometer: Memory B; CD19+CD27+, Effector T; CD25-CD45RA- (Tfh cells were isolated using the method described in Example 3).

[0099] Tfh cells were cultured at 37°C for 30 minutes in 96-well U plates immobilized with isotype (mouseIgG1) or anti-TIGIT agonistic antibody, 10 μg / ml. Memory B cells were added and cultured for 1 week in a medium mixed with 0.2 μg / ml staphylococcal enterotoxin B (Sigma). Dead cells were removed by staining with 7-aminoactinomycin D, and viable cells were analyzed using a FACSVerse flow cytometer. The percentage of plasma cells was analyzed using FlowJo software version 10.4.2 (FlowJo, OR, USA), and IgG in the culture supernatant was measured using a human IgG ELISA kit (Bethyl).

[0100] As shown in Figure 8, the group co-cultured with Tfh cells stimulated with anti-TIGIT agonistic antibodies suppressed plasma cell proliferation and also inhibited IgG production.

[0101] Example 5: Effects on Treg cells Blood was collected in heparinized blood collection tubes (TERUMO, Tokyo, Japan) and converted into PBMCs using Lymphoprep (Axis-Shield). Furthermore, CD4+ T cells were negatively selected using the CD4+ T Cell Isolation Kit (Miltenyi Biotec). The cell surface was stained with various antibodies, and the cells were fractionated into the following cell fractions using a FACSAria III flow cytometer (after 96 hours for Effector cells): Treg (FrI); CD25+CD45RA+, Treg (FrII); CD25+CD45RA-, Effector T; CD25-CD45RA-, Non-Treg responder CD4+ T cells; CD25-.

[0102] The obtained Treg cells (FrI) were cultured for 6 hours in a 96-well flat-bottom plate immobilized with 2 μg / ml anti-CD3 antibody, 1 μg / ml anti-CD28 antibody, and 10 μg / ml isotype (mouseIgG1) or anti-TIGIT agonistic antibody, in medium supplemented with 10 ng / ml IL-2 (R&D). Meanwhile, the CD4+ Effector T cells fractionated after 96 hours were stained with CellTrace violet (CTV; Thermo Fisher Scientific) using the same method as in Example 2, washed, and activated, then mixed with the Treg cells in a 1:1 ratio (1 x 10⁶ each). 4 Cells were cultured for 96 hours. Dead cells were stained and removed with 7-aminoactinomycin D, and viable cells were analyzed using a FACSVerse flow cytometer. Cell proliferation was analyzed using FlowJo software version 10.4.2 (FlowJo, OR, USA).

[0103] As shown in Figure 9-1, Treg cells stimulated with anti-TIGIT agonistic antibodies suppressed the proliferation of effector T cells. Stimulation of Treg cells with anti-TIGIT agonistic antibodies acted to activate Treg cells.

[0104] Similarly to the above, Treg cells (FrI) or Treg cells (FrII) were stimulated for 6 hours with anti-CD3 and anti-CD28 antibodies in the presence of isotype (mouseIgG1) or anti-TIGIT agonistic antibody. Non-Treg responder CD4+ T cells (CD25-) were labeled with CTV and mixed with each activated Treg cell fraction in a 1:1 ratio (1 x 10⁶ each). 4 The cells were co-cultured for 96 hours in the following proportions. Subsequently, the proliferation of responder T cells was analyzed using dilution of CTV fluorescence as an indicator, in the same manner as described above. As a result, all Treg cell fractions suppressed the proliferation of responder T cells, but activation of each Treg cell fraction in the presence of an anti-TIGIT agonistic antibody further enhanced Treg cell activation, and this effect was particularly pronounced in Treg(FrI), also known as naive Treg cells (Figure 9-2).

[0105] Example 6: Effects on imiquimod-induced lupus model mice Female C57BL / 6JJcl mice (CREA Japan) aged 6-8 weeks were purchased and crossbred with mice of the same strain that had been knocked in with the human TIGIT (hTIGIT) gene using conventional methods. Homozygous and wild-type hTIGIT knock-in mice of the same generation were used. All experiments were conducted at the Keio University School of Medicine Animal Laboratory and carried out in accordance with the regulations concerning animal experiments at the Keio University School of Medicine.

[0106] Imiquimod-induced lupus model mice were created by applying 5% imiquimod cream (Mochida Pharmaceutical) at a dose of 50 mg / kg three times a week to the right auricle of 10-12 week old female TIGIT-KI mice. The administration experiments were conducted as follows. All mice were administered twice a week. The mice were divided into three groups and analyzed on day 42 of the study. Non-topical administration: Imiquimod without topical application / Isotype (mIgG1) 300 μg / ml intraperitoneal administration Anti-TIGIT: Imiquimod topical application / 300 μg / ml intraperitoneal administration of anti-TIGIT agonistic antibody Isotype: Imiquimod topical application / Isotype (mIgG1) 300 μg / ml intraperitoneal administration

[0107] Mouse spleens were removed and weighed. The spleens were finely chopped, passed through a 40 μm cell strainer, and hemolyzed using HLB solution (IBL) to determine the total number of splenocytes. Mouse splenocytes were cultured at 4°C for 5 minutes using anti-CD16 / CD32 (BD Biosciences), and surface staining was performed for 20 minutes. The culture medium used for staining was PBS mixed with 0.5% BSA and 2 mM EDTA. Each cell type was defined as follows: Tem; CD44+CD62L-, Tfh; CXCR5+PD-1+, plasma cells; CD19-CD138+, GC B cells; CD95+GL7+. During analysis, blood was collected and the anti-dsDNA antibody titer in the blood was measured using the "Levis anti-dsDNA-mouse ELISA KIT" (Shibayagi Co., Ltd.).

[0108] As shown in Figure 10, the anti-TIGIT agonistic antibody suppressed splenomegaly and lymphocyte proliferation in the group administered the antibody. As shown in Figure 11-1, administration of the anti-TIGIT agonistic antibody significantly suppressed the increase in the proportion of plasma cells in B cells. It also showed a tendency to suppress the increase in the proportion of activated T cells in T cells, the proportion of CD4+ Effector memory T (Tem) cells and Tfh cells in CD4-positive T cells, the proportion of germinal center B cells in B cells, and the increase in anti-dsDNA antibody production. When the same experiment was performed with a larger number of mice, the anti-TIGIT agonistic antibody significantly suppressed the increase in the proportion of Tem cells and Tfh cells in CD4-positive T cells, the proportion of germinal center B cells in B cells, the proportion of plasma cells in splenocytes, and the increase in anti-dsRNA antibody production compared to the isotype (Figure 11-2). As described above, the anti-TIGIT agonistic antibody suppressed the proliferation of lymphocytes induced by the disease, particularly the proliferation of activated T cells. Furthermore, it also suppressed B cell activation mediated by Tfh cells, similar to the results obtained in culture experiments.

[0109] Example 7: Effects on mice with experimental autoimmune encephalomyelitis (EAE) 12-week-old male TIGIT-KI mice were given 200 μg of MOG. 35-55 A peptide (Synpeptide, Shanghai, China) and Freud's incomplete adjuvant, along with 400 μg of M. tuberculosis (Difco Laboratories), were administered subcutaneously. Additionally, 400 ng of pertussis toxin (List Biologicals) was administered intraperitoneally on the same day and on day 2. 100 μg of anti-TIGIT agonistic antibody or isotype (mIgG1) was administered intraperitoneally on days 0, 2, 4, 10, and 17 of the induction phase. Clinical scores were measured daily using the following method and analyzed on day 30 (0: no change; 1: tail muscle weakness; 2: tail paralysis; 3: hindlimb muscle weakness; 4: forelimb muscle weakness; 5: forelimb paralysis; 6: morbid or dead). Cells infiltrating the brain, spinal cord, and spleen were analyzed by flow cytometry using various antibodies.

[0110] As shown in Figure 12, the antibody-administered group showed a reduction in the clinical score of EAE. As shown in Figure 13, the antibody-administered group showed a tendency to promote Treg cell proliferation in the brain, spinal cord, and spleen.

[0111] Results / discussion The results above demonstrate that the anti-TIGIT agonistic antibody exhibits inhibitory effects on Tfh cells and Tfh cells-mediated inhibitory effects on B cells, both in vitro and in vivo. In vitro, the anti-TIGIT agonistic antibody showed an inhibitory effect on effector T cells against Treg cells, and in vivo, administration of this antibody suggested the promotion of Treg cell proliferation. Studies using disease model animals confirmed the effectiveness of this antibody. These effects were not observed with conventional antibodies. Suppressing activated T cells and activating Treg cells in immune and inflammatory diseases is logical for therapeutic use and could potentially become a widely used treatment for human immune and inflammatory diseases in the future. [Industrial applicability]

[0112] This invention enables the activation of human TIGIT-mediated signaling, thereby allowing for the suppression of Tfh cells and the activation of Treg cells, making it useful for the prevention or treatment of immune and inflammatory diseases.

[0113] This application is based on Japanese Patent Application No. 2020-171278, filed in Japan on 9 October 2020, the entirety of which is incorporated herein by reference.

Claims

1. An immunotherapy for treating immunological and inflammatory diseases, comprising an anti-human TIGIT antibody that activates an inhibitory immune checkpoint molecule (TIGIT) as an active ingredient, wherein the antibody: a) comprising a heavy chain variable region including a first CDR containing the amino acid sequence of SEQ ID NO: 2, a second CDR containing the amino acid sequence of SEQ ID NO: 3, and a third CDR containing the amino acid sequence of SEQ ID NO: 4; and b) A light chain variable region comprising a first CDR containing the amino acid sequence of SEQ ID NO: 5, a second CDR containing the amino acid sequence of tyrosine-alanine-serine, and a third CDR containing the amino acid sequence of SEQ ID NO:

6. A therapeutic drug.

2. The therapeutic agent according to claim 1, which has a Tfh cell inhibitory effect.

3. The therapeutic agent according to claim 1, which has a Treg cell activating effect.

4. The therapeutic agent according to claim 1, wherein the immune and inflammatory disease is selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, Sjögren's syndrome, systemic scleroderma, polymyositis, dermatomyositis, IgG4-related disease, Takayasu's arteritis, giant cell arteritis, polyarteritis nodosa, ANCA-associated vasculitis, mixed connective tissue disease, spondyloarthritis, Behçet's disease, adult Still's disease, multiple sclerosis, neuromyelitis optica, myasthenia gravis, primary biliary cirrhosis, non-alcoholic fatty liver disease, primary sclerosing cholangitis, autoimmune hepatitis, ulcerative colitis, Crohn's disease, psoriasis (psoriatic arthritis), vitiligo, bullous pemphigoid, alopecia areata, idiopathic dilated cardiomyopathy, type 1 diabetes mellitus, Graves' disease, Hashimoto's disease, IgA nephropathy, membranous nephropathy, hemolytic anemia, and idiopathic thrombocytopenic purpura.

5. The therapeutic agent according to claim 1, wherein the anti-human TIGIT antibody is an antibody that binds to human TIGIT (SEQ ID NO: 1).

6. The therapeutic agent according to claim 5, further comprising an antibody containing an Fc domain.

7. The therapeutic agent according to claim 6, wherein the Fc domain is of human origin.

8. The therapeutic agent according to claim 6, wherein the Fc domain is a mutated Fc domain.

9. An antibody that binds to human TIGIT (SEQ ID NO: 1), a) comprising a heavy chain variable region including a first CDR containing the amino acid sequence of SEQ ID NO: 2, a second CDR containing the amino acid sequence of SEQ ID NO: 3, and a third CDR containing the amino acid sequence of SEQ ID NO: 4; and, b) A light chain variable region comprising a first CDR containing the amino acid sequence of SEQ ID NO: 5, a second CDR containing the amino acid sequence of tyrosine-alanine-serine, and a third CDR containing the amino acid sequence of SEQ ID NO:

6. antibody.

10. The antibody according to claim 9, further comprising an Fc domain.

11. The antibody according to claim 10, wherein the Fc domain is of human origin.

12. The antibody according to claim 10, wherein the Fc domain is a mutant Fc domain.

13. An isolated nucleic acid encoding the antibody according to claim 9.

14. A host cell comprising the isolated nucleic acid described in claim 13.

15. A method for producing an antibody according to claim 9, comprising the step of culturing a host cell according to claim 14 under conditions under which the antibody is produced.

Images