Immunosuppressive agent

Abstract

To provide a novel immunosuppressive agent.SOLUTION: The present application provides an immunosuppressive agent containing a substance that is selected from anti-CD80 antibodies and anti-PD-L1 antibodies and that promotes binding between PD-L1 and PD-1.SELECTED DRAWING: Figure 47

Description

[Technical Field]

[0001] This patent application claims priority to Japanese Patent Application No. 2018-229774, the entire contents of which are incorporated herein by reference. The present application relates to an immunosuppressant comprising a substance that promotes the binding of PD-L1 selected from an anti-CD80 antibody and an anti-PD-L1 antibody to PD-1. [Background technology]

[0002] The immune system protects the body from disease by recognizing and destroying non-self substances such as pathogens and abnormal cells such as cancer cells. The immune system is precisely regulated to attack pathogens and abnormal cells and not to attack normal self substances, but failure of this control mechanism can cause various intractable diseases such as autoimmune diseases and chronic inflammatory diseases.

[0003] Various molecules are known to regulate the immune system. Programmed death-1 (PD-1) is an immune checkpoint receptor present on the surface of T cells and binds to two ligands: programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2). CD80, along with CD86, functions as a ligand for two structurally similar molecules expressed on T cells: CD28 and CTLA-4. CD28 activates T cells, whereas CTLA-4 inhibits them. Targeted inhibition of PD-1, PD-L1, and CTLA-4 can activate tumor-specific T cells and is known to be effective in treating tumors in human patients. Furthermore, an anti-CD80 antibody that blocks the binding of CD80 to CD28 has been reported to suppress T cell activation (Patent Document 1).

[0004] Interestingly, it has been reported that anti-PD-L1 antibodies that inhibit CD80 / PD-L1 interaction activate tumor immunity and are useful in cancer treatment (Non-Patent Document 1, Patent Document 2). On the other hand, it has also been reported that CD80 and PD-L1 are expressed on the same cells and bind to each other, and that the maintenance of T cell activity by CD80 is related to the inhibition of immunosuppression by the PD-1 / PD-L1 system in addition to its action via CD28 (Non-Patent Documents 2 and 3). Thus, the physiological function of CD80 / PD-L1 interaction has not been elucidated. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 1998 / 019706 [Patent Document 2] US Patent Application Publication No. 2011 / 0280877 [Non-patent literature]

[0006] [Non-Patent Document 1] The Journal of Immunology, 2011, 187: 1113-1119 [Non-patent document 2] Cancer Immunol Res. 2014 July; 2(7): 610-615 [Non-patent document 3] Cancer Immunology Research, 2018 June 5, 6(8), 921-929 Summary of the Invention [Problem to be solved by the invention]

[0007] One object of the present application is to provide an immunosuppressant. [Means for solving the problem]

[0008] In some embodiments, the present disclosure provides an immunosuppressant comprising a substance that promotes the binding of PD-L1 to PD-1, selected from an anti-CD80 antibody and an anti-PD-L1 antibody. In one embodiment, the disclosure provides an anti-CD80 antibody comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the disclosure provides an anti-CD80 antibody comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22. In certain embodiments, the present disclosure provides an anti-CD80 antibody that competes with any of the above-described antibodies for binding to CD80. In one aspect, the present disclosure provides an immunosuppressant comprising any of the above antibodies as an active ingredient. In certain aspects, the present disclosure provides an agent for the prophylaxis and / or treatment of autoimmune diseases, allergic diseases, or graft-versus-host disease, comprising any of the above-mentioned antibodies as an active ingredient. [Effects of the Invention]

[0009] The present disclosure provides immunosuppressants, or agents for the prevention and / or treatment of autoimmune diseases, allergic diseases, or graft-versus-host disease, or antibodies usable therefor. [Brief explanation of the drawings]

[0010] [Figure 1]The binding intensity of PD-1-EC (left) and the labeled antibodies indicated in the figure (right) to IIAdL1 cells expressing PD-L1 (top) or PD-L2 (bottom) and CD80 or CD86 is shown. [Figure 2] Figure 1 shows the binding intensity of PD-1-EC and anti-PD-L1 antibodies to IIAdL1-PD-L1 cells with or without co-culture with IIAdL1-mock or IIAdL1-CD80 cells. [Figure 3] Co-immunoprecipitation of CD80 and PD-L1 in IIAdL1 cells. WCL: uncrosslinked whole cell lysate. [Figure 4] IL-2 production from T cells was measured when antigen-presenting cells pulsed with the indicated amounts of antigen peptide were cocultured with PD-1-expressing DO11.10 T cells (PD-1(+)) (left) or PD-1-deficient DO11.10 T cells (PD-1KO) (right). The percentage of PD-1-mediated inhibition of IL-2 production is shown in the figure. [Figure 5] The binding intensity of PD-L1 antibody (left) and CD80 antibody (right) to each IIAdL1 cell line expressing PD-L1 and CD80 at various levels is shown. [Figure 6] The binding strength of PD-1-EC to each IIAdL1 cell line, which expresses the highest level of PD-L1 and various levels of CD80, is shown. [Figure 7] The relative binding strength of PD-1-EC to each of IIAdL1 cells expressing PD-L1 and CD80 at 25 different expression levels is shown. [Figure 8] This shows the percentage of PD-1-mediated inhibition of IL-2 production when IL-2 production from DO11.10 T cells was induced by each of IIAdL1 cells expressing PD-L1 and CD80 at 25 different expression levels.

[0011] [Figure 9] The binding intensities of PD-1-EC and the indicated labeled antibodies to LPS-activated splenic CD8α+ and CD11b+ DCs and TG-MΦs are shown. The dashed histograms represent isotype control staining. [Figure 10] The binding intensities of PD-1-EC and the antibodies indicated in the figure to LPS-activated splenic CD8α+ and CD11b+ DCs and TG-MΦ derived from wild-type or C57BL / 6-Cd80- / - mice are shown. [Figure 11] The figures show IL-2 production by T cells following stimulation of BW-OT-I cells (left) and BW-OT-II cells (right) with LPS-activated TG-MΦ (top), splenic CD8α+ (middle), and CD11b+ (bottom) DCs pulsed with the indicated amounts of antigen peptide. Function-blocking antibodies against PD-L1 and PD-L2, and isotype control IgG, were added as indicated. [Figure 12] Percentage of PD-1-mediated inhibition of IL-2 production is shown for the conditions indicated. One-way ANOVA with Dunnett's post-hoc test. **p < 0.01; ***p < 0.001.

[0012] [Figure 13] Schematic representation of chimeric molecules with swapped IgV and IgC domains of PD-L1, PD-L2, CD80, and CD86. [Figure 14] The PD-1-EC binding intensity of IIAdL1 cells expressing the molecules shown in the figure is shown. [Figure 15] Figure 1 shows the PD-1-EC binding ability of IIAdL1-CD80 cells expressing isolated PD-L1 mutants (top row) and PD-L1 with an amino acid substitution at Y56 (bottom row). The PD-1-EC binding intensity of the cells is compared to that of IIAdL1-PD-L1 cells without CD80. [Figure 16] The predicted 3D structures of mouse PD-L1 (left) and mouse CD80 (right) are shown. Amino acid residues that influence the cis-PD-L1 / CD80 interaction are indicated. [Figure 17] Figure 1 shows the binding of PD-1-EC to PD-L1Y56A in the presence of CD80. Figure 2 shows the binding intensity of PD-1-EC (left) and anti-PD-L1 antibodies (right) to IIAdL1 cells expressing the indicated molecules. [Figure 18]The hydrophobicity (gray) of human CD80 (left) and CD86 (right) is shown. The hydrophobicity of these molecules was analyzed using UCSF Chimera software. The circled area in the figure indicates the unique hydrophobic patch found on the DEB surface of CD80. [Figure 19] Figure 1 shows the binding of PD-1-EC to PD-L1 in the presence of CD80L107E. Figure 2 shows the binding intensity of PD-1-EC, anti-CD80 antibody, CD28-EC, and CTLA-4-EC to IIAdL1 cells expressing the molecules shown in the figure. [Figure 20] Figure 1 shows the PD-1-EC binding ability of IIAdL1-PD-L1 cells expressing CD80 with amino acid substitutions at hydrophobic residues in the hydrophobic patch. The PD-1-EC binding intensity of the cells is compared to that of IIAdL1-PD-L1 cells without CD80. [Figure 21] Lack of co-immunoprecipitation of PD-L1Y56A / CD80 and PD-L1 / CD80L107E is shown. WCL: non-crosslinked whole cell lysate. [Figure 22] This figure shows IL-2 production by T cells when antigen-presenting cells expressing the molecules indicated in the figure were co-cultured with PD-1-expressing DO11.10 T cells (PD-1(+)) (left) or PD-1-deficient DO11.10 T cells (PD-1KO) (right). The amounts of antigen peptide indicated in the figure were used. [Figure 23] The results of further analysis of the data shown in the upper panel of Figure 22 are shown, showing the percentage of PD-1-dependent inhibition mediated by PD-L1 and PDL1Y56A. [Figure 24] The results of further analysis of the data shown in the middle and lower panels of Figure 22 are shown. The percentage of PD-1-dependent inhibition mediated by the molecules shown in the figure (0.3 μM antigen peptide) is shown. One-way ANOVA with Dunnett's post-hoc test. ***p<0.001.

[0013] [Figure 25] The figures show the binding intensities of human PD-1-EC and the antibodies indicated in the figures to IIAdL1 cells expressing human PD-L1 (top row) or human PD-L2 (bottom row) and human CD80 or human CD86. [Figure 26] Figure 1 shows the binding intensity of human PD-1-EC to IIAdL1 cells expressing human PD-L1, with or without co-culture with IIAdL1 cells expressing human CD80. [Figure 27] Co-immunoprecipitation of human CD80 and human PD-L1 in IIAdL1 cells. WCL: uncrosslinked whole cell lysate. [Figure 28] The figures show IL-2 production from T cells when the antigen-presenting cells shown in the figure were pulsed with the indicated amount of antigen peptide and co-cultured with DO11.10 T cells containing human PD-1 (hPD-1(+)) (left) or not (PD-1KO) (right). The percentage of human PD-1-mediated inhibition of IL-2 production is shown in the figures. [Figure 29] PD-1-EC binding intensity to IIAdL1 cells expressing human PD-L1 N63D / G119S mutant (top row) and IIAdL1 cells expressing human CD80, CD80I92E, and CD80L104E (bottom row) is shown. [Figure 30] This figure shows IL-2 production from T cells when antigen-presenting cells expressing the indicated molecules were co-cultured with DO11.10 T cells expressing human PD-1 (hPD-1(+)) (left) or lacking human PD-1 (PD-1KO) (right). The amounts of antigen peptides shown in the figure were used.

[0014] [Figure 31] PD-1-EC binding intensity, PD-L1, PD-L2, and CD80 expression levels in LPS-activated BM-DCs and splenic CD8α+ and CD11b+ DCs from C57BL / 6N-Cd80L107E and C57BL / 6N-Cd274Y56A mice are shown. [Figure 32] PD-1-mediated inhibition of IL-2 production in BW-OT-I cells (left) and BW-OT-II cells (right) stimulated with BM-DCs derived from C57BL / 6N-Cd80L107E and C57BL / 6N-Cd274Y56A mice is shown as a function of IL-2 concentration. [Figure 33]The results in Figure 32 are shown as relative values. Data were obtained using antigen concentrations of 3 pM for BW-OT-I cells and 0.1 μM for BW-OT-II cells. One-way ANOVA with Dunnett's post-hoc test. ***p < 0.001. [Figure 34] Figure 1 shows PD-1-mediated inhibition of IL-2 production in BW-OT-I cells (left) and BW-OT-II cells (right) stimulated with splenic CD8α+ and CD11b+ DCs from C57BL / 6N-Cd80L107E and C57BL / 6N-Cd274Y56A mice. One-way ANOVA with Dunnett's post-hoc test. *p < 0.05; **p < 0.01. [Figure 35] Immune induction experiments in the absence of cis-PD-L1 / CD80 interaction show that OVA-specific T cells capable of producing IFN-γ (top row) and IL-2 (bottom row) are not induced (n=9). One-way ANOVA with Dunnett's post-hoc test. *p < 0.05; **p < 0.01; ***p < 0.001. [Figure 36] 1 shows the experimental design for vaccination with OVA and poly(I:C). [Figure 37] E.G7 tumor volumes in wild-type, Cd80L107E, and Cd274Y56A mice immunized with PBS (Ctrl) or OVA and poly(I:C) (Vac). Unpaired two-tailed Student's t-test. ***p < 0.001. [Figure 38] Relative E.G7 tumor volumes (day 15) in wild-type, Cd80L107E, and Cd274Y56A mice immunized with PBS (Ctrl) or OVA and poly(I:C) (Vac) are shown (n ≥ 8). One-way ANOVA with Dunnett's post-hoc test. *p < 0.05; ***p < 0.001. [Figure 39] 1 shows the experimental design for vaccination with OVA-pulsed BM-DCs. [Figure 40]Figure 1 shows the volume of E.G7 tumors in wild-type mice immunized with BM-DCs derived from wild-type, Cd274Y56A, and Cd80L107E mice. Unpaired two-tailed Student's t-test. *p < 0.05; **p < 0.01. [Figure 41] Relative E.G7 tumor volumes (day 14) in wild-type mice immunized with BM-DCs derived from wild-type, Cd274Y56A, and Cd80L107E mice are shown (n = 7). One-way ANOVA with Dunnett's post-hoc test. *p < 0.05; **p < 0.01. [Figure 42] Clinical scores of experimental autoimmune encephalomyelitis (EAE) in wild-type, Cd274Y56A, and Cd80L107E mice (n=12). Two-way repeated measures ANOVA with Tukey HSD post-hoc test. ***p < 0.001. [Figure 43] Figure 1: IL-17-producing cells are not induced in the absence of cis-PD-L1 / CD80 interaction upon EAE induction (n=8). One-way ANOVA with Dunnett's post-hoc test. *p < 0.05; **p < 0.01.

[0015] [Figure 44] We show that the anti-mouse CD80 antibody TKMG48 dissociates mPD-L1 from mCD80 and restores the mPD-1-binding ability of mPD-L1. [Figure 45] 1 shows that the anti-mouse CD80 antibody TKMG48 does not inhibit the binding of mCD80 to mCTLA-4, but weakly inhibits the binding of mCD80 to mCD28. [Figure 46] Addition of the anti-mouse CD80 antibody TKMG48 demonstrates that mPD-L1 binds to mPD-1 and suppresses T cell activation, even in the presence of mCD80. [Figure 47] 1 shows that administration of the anti-mouse CD80 antibody TKMG48 substantially alleviates the symptoms of EAE in wild-type mice. [Figure 48]This shows that administration of the anti-mouse CD80 antibody TKMG48 substantially alleviates the symptoms of EAE in wild-type mice, but not in PD-L1 knockout mice (PD-L1 KO in the figure). [Figure 49] Addition of the anti-human CD80 antibody TKMF5 demonstrates that hPD-L1 binds to hPD-1 and suppresses T cell activation, even in the presence of hCD80. [Figure 50] The anti-mouse CD80 antibody TKMG48 shows low binding to a CD80 mutant that lacks PD-L1 binding ability. 16-10A1 is a commercially available anti-mouse CD80 antibody. [Figure 51] The anti-human CD80 antibody TKMF5 shows low binding to a CD80 mutant that lacks PD-L1 binding ability. 2D10 is a commercially available anti-human CD80 antibody. [Figure 52] The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-human CD80 antibody TKMF5 are shown. Each CDR is indicated by a box. [Figure 53] The amino acid sequences of the heavy chain variable region and light chain variable region of the anti-mouse CD80 antibody TKMG48 are shown. Each CDR is indicated by a box. DETAILED DESCRIPTION OF THE INVENTION

[0016] In this disclosure, when a numerical value is accompanied by the term "about," it is intended to include a range of ±10% of that value. For example, "about 20" includes "18 to 22." A range of numerical values ​​includes all values ​​between and including the endpoints. When "about" refers to a range, it applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33."

[0017] In this disclosure, amino acid residues are represented by the following abbreviations: Ala or A: Alanine Arg or R: arginine Asn or N: Asparagine Asp or D: aspartic acid Cys or C: cysteine Gln or Q: glutamine Glu or E: glutamic acid Gly or G: glycine His or H: histidine Ile or I: Isoleucine Leu or L: leucine Lys or K: Lysine Met or M: methionine Phe or F: phenylalanine Pro or P: proline Ser or S: serine Thr or T: threonine Trp or W: Tryptophan Tyr or Y: Tyrosine Val or V: Valine

[0018] PD-L1 (also known as CD274) is a ligand for PD-1 and is expressed on a variety of cells, including dendritic cells and tumor cells. PD-1 is a representative immune checkpoint receptor on the surface of T cells, and binding of PD-L1 to PD-1 can suppress immune responses. Therefore, substances that promote the binding of PD-L1 to PD-1 can be used as immunosuppressants. The inventors of the present application have found that certain anti-CD80 antibodies and anti-PD-L1 antibodies promote the binding of PD-L1 to PD-1.

[0019] The binding of PD-L1 to PD-1 can be measured, for example, by contacting cells expressing PD-L1 with a soluble peptide comprising the extracellular domain of PD-1 that has a detectable label (e.g., a fluorescent label, a luminescent label, a radioactive label, a magnetic label, etc.) and measuring the amount of label bound to the cells. Unlabeled soluble peptides may also be used in combination with a labeled substance (e.g., a secondary antibody) that binds to the peptide. Specifically, the binding of PD-L1 to PD-1 can be measured by the methods described in the Examples of this application.

[0020] CD80 is primarily expressed on the surface of dendritic cells, activated B cells, and macrophages, and can regulate T cell activation and survival as a ligand for two different proteins (CD28 and CTLA-4) present on the surface of T cells. Without being limited by theory, the inventors of the present application have demonstrated that binding of CD80 to PD-L1 on the same cell (cis binding) inhibits the binding of PD-L1 to PD-1 on T cells. Therefore, inhibition of the cis binding of CD80 to PD-L1 can promote the binding of PD-L1 to PD-1. In one embodiment, the substance that promotes the binding of PD-L1 to PD-1 is a substance that promotes the binding of PD-L1 to PD-1 on the same cell as CD80. In another embodiment, the substance that promotes the binding of PD-L1 to PD-1 is a substance that inhibits the cis binding of CD80 to PD-L1. In another embodiment, the substance that promotes the binding of PD-L1 to PD-1 is an anti-CD80 antibody. In some embodiments, the agent that enhances the binding of PD-L1 to PD-1 is an anti-PD-L1 antibody.

[0021] As used herein, the term "cis" refers to the presence of two or more different proteins or molecules on the same cell, for example, cis-PD-L1 / CD80 refers to the presence of PD-L1 and CD80 on the same cell. As used in this disclosure, the term "trans" refers to the presence of two or more different proteins or molecules on different cells, for example, trans-PD-L1 / CD80 means that PD-L1 and CD80 are present on separate cells.

[0022] As used herein, "cis binding" refers to the binding, association, or interaction of two or more distinct membrane proteins expressed on the surface of a single cell. The cis binding of CD80 and PD-L1 can occur in any cell, for example, in cells of the immune system, particularly antigen-presenting cells, such as dendritic cells, macrophages, and B cells.

[0023] The cis binding of CD80 and PD-L1 can be confirmed by treating cells expressing CD80 and PD-L1 with a cross-linking agent (e.g., bis(sulfosuccinimidyl)suberate) that cross-links adjacent proteins, followed by detection or measurement of cross-linked CD80 and PD-L1. Cross-linked CD80 and PD-L1 can be detected or measured in assay systems such as immunoprecipitation, ELISA, and mass spectrometry, by capturing molecules with a substance (e.g., an antibody) that binds to either CD80 or PD-L1, and then detecting or measuring the other substance (e.g., an antibody) that binds to the other. Specifically, the cis binding of CD80 and PD-L1 can be measured using the methods described in the Examples herein. Substances that bind to CD80 include CD28, CTLA-4, anti-CD80 antibodies, and fragments thereof. Substances that bind to PD-L1 include PD-1, anti-PD-L1 antibodies, and fragments thereof. The substance for detection or measurement may have a detectable label (eg, a fluorescent label, a luminescent label, a radioactive label, a magnetic label, etc.).

[0024] As used herein, "inhibiting cis-binding" includes dissociating cis-bound CD80 and PD-L1 and / or preventing cis-binding of non-cis-bound CD80 and PD-L1. In one embodiment, a substance that promotes the binding of PD-L1 to PD-1 competitively inhibits the cis-binding of CD80 to PD-L1.

[0025] In one embodiment, a substance that enhances the binding of PD-L1 to PD-1 enhances the binding of PD-L1 to PD-1 by at least about 2-fold or more, for example, about 5-fold or more or about 10-fold or more, when cultured cells that highly express CD80 and PD-L1 (e.g., DO11.10 T cells in which the PD-1 and PD-L1 genes have been deleted and which express CD80 and PD-L1 under the LTR promoter (DOdKO cells)) are cultured in the presence of 10 μg / ml of the substance. For example, an example of measuring binding when the substance that enhances the binding of PD-L1 to PD-1 is an anti-CD80 antibody is described in the Examples section of this application.

[0026] In the present disclosure, CD80, PD-L1, and PD-1 may be from any species, typically mammals (e.g., human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.). Mouse or human is preferred, with human beings being particularly preferred. The amino acid sequences of CD80, PD-L1, and PD-1 from various species are readily available from publicly available databases. Representative amino acid sequences of human and mouse CD80 are registered in GenBank under accession numbers NP_005182 (SEQ ID NO: 1) and NP_033985 (SEQ ID NO: 2), respectively. Representative amino acid sequences of human and mouse PD-L1 are registered in GenBank under accession numbers NP_054862 (SEQ ID NO: 3) and NP_068693 (SEQ ID NO: 4), respectively. Representative amino acid sequences of human and mouse PD-1 are registered in GenBank under accession numbers NP_005009 (SEQ ID NO: 5) and NP_032824 (SEQ ID NO: 6), respectively. In this disclosure, CD80, PD-L1, and PD-1 include their naturally occurring allelic products.

[0027] In one embodiment, the cis binding between CD80 and PD-L1 occurs via at least a region containing amino acids equivalent to isoleucine at position 92 and / or leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1 (preferably a region containing amino acids equivalent to isoleucine at position 92 and leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1) and a region containing amino acids equivalent to asparagine at position 63 and / or glycine at position 119 of human PD-L1 having the amino acid sequence of SEQ ID NO: 3 (preferably a region containing amino acids equivalent to asparagine at position 63 and glycine at position 119 of human PD-L1 having the amino acid sequence of SEQ ID NO: 3). or via a region containing at least amino acids corresponding to leucine at position 96 and / or leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2 (preferably a region containing amino acids corresponding to leucine at position 96 and leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2) and a region containing amino acids corresponding to valine at position 54, tyrosine at position 56, and / or glutamic acid at position 58 of mouse PD-L1 having the amino acid sequence of SEQ ID NO: 4 (preferably a region containing amino acids corresponding to valine at position 54, tyrosine at position 56, and glutamic acid at position 58 of mouse PD-L1 having the amino acid sequence of SEQ ID NO: 4). The region containing the above amino acids may be composed of consecutive or discontinuous amino acid residues.

[0028] In one embodiment, the anti-CD80 antibody binds to a region containing amino acids corresponding to isoleucine at position 92 and / or leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1. In a preferred embodiment, the anti-CD80 antibody binds to a region containing amino acids corresponding to isoleucine at position 92 and leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1. The region containing these amino acids may consist of consecutive or non-consecutive amino acid residues.

[0029] In one embodiment, the anti-CD80 antibody binds to a region containing amino acids corresponding to leucine at position 96 and / or leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2. In a preferred embodiment, the anti-CD80 antibody binds to a region containing amino acids corresponding to leucine at position 96 and leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2. The region containing these amino acids may consist of consecutive amino acid residues or may consist of non-consecutive amino acid residues.

[0030] In some embodiments, the anti-PD-L1 antibody binds to a region containing amino acids corresponding to asparagine at position 63 and / or glycine at position 119 of human PD-L1, having the amino acid sequence of SEQ ID NO: 3. In a preferred embodiment, the anti-PD-L1 antibody binds to a region containing amino acids corresponding to asparagine at position 63 and glycine at position 119 of human PD-L1, having the amino acid sequence of SEQ ID NO: 3. The region containing these amino acids may be composed of consecutive or non-consecutive amino acid residues.

[0031] In some embodiments, the anti-PD-L1 antibody binds to a region containing amino acids corresponding to valine at position 54, tyrosine at position 56, and / or glutamic acid at position 58 of mouse PD-L1, which has the amino acid sequence of SEQ ID NO: 4. In a preferred embodiment, the anti-PD-L1 antibody binds to a region containing amino acids corresponding to valine at position 54, tyrosine at position 56, and glutamic acid at position 58 of mouse PD-L1, which has the amino acid sequence of SEQ ID NO: 4. The region containing these amino acids may be composed of consecutive or non-consecutive amino acid residues.

[0032] The term "an amino acid corresponding to isoleucine at position 92 of human CD80 having the amino acid sequence of SEQ ID NO: 1" refers to the amino acid in a CD80 that corresponds to isoleucine at position 92 of SEQ ID NO: 1 when the amino acid sequences of that CD80 and the amino acid sequence of SEQ ID NO: 1 are aligned optimally (maximum amino acid identity). The terms "an amino acid corresponding to leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1," "an amino acid corresponding to leucine at position 96 and / or leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2," "an amino acid corresponding to asparagine at position 63 and / or glycine at position 119 of human PD-L1 having the amino acid sequence of SEQ ID NO: 3," and "a valine at position 54, tyrosine at position 56, and / or glutamic acid at position 58 of mouse PD-L1 having the amino acid sequence of SEQ ID NO: 4" are similarly defined. For example, the leucine at position 96 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2 and the leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2 correspond to the isoleucine at position 92 of human CD80 having the amino acid sequence of SEQ ID NO: 1 and the leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1, respectively.

[0033] In the present disclosure, the term "antibody" is used to encompass various antibody structures, such as monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, and multispecific antibodies (e.g., bispecific antibodies). The species of the antibody is not particularly limited, and examples include antibodies derived from mouse, rat, rabbit, goat, and human. Preferred antibodies are humanized antibodies and human antibodies (fully human antibodies). Furthermore, preferred antibodies are monoclonal antibodies, and more preferably isolated monoclonal antibodies.

[0034] In the present disclosure, the term "isolated monoclonal antibody" refers to a monoclonal antibody that has been identified, separated, and / or purified from contaminants, including multiple or numerous components, extracted from host cells such as hybridomas or their culture supernatants, resulting in a substantially single, pure component.

[0035] In the present disclosure, the term "antibody" also encompasses molecules that contain a portion of an antibody as a component and retain antigen-binding ability. For example, but not limited to, antibody heavy and light chain variable regions (V H and V L ), F(ab')2, Fab', Fab, Fv, disulphide-linked FV (sdFv), single-chain FV (scFV), Fab3, diabody, triabody, tetrabody, minibody, Bis-scFv, (scFv)2-Fc, intact-IgG, and polymers thereof are included in the antibodies of the present application.

[0036] The immunoglobulin class of an antibody is determined based on the heavy chain constant region. Immunoglobulin classes include IgA, IgD, IgE, IgG, and IgM, and the corresponding heavy chains are called α chains, δ chains, ε chains, γ chains, and μ chains, respectively. Immunoglobulin classes can be further classified into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The immunoglobulin class and subclass of antibodies herein are not particularly limited. In one embodiment, the immunoglobulin class is IgG. The light chains of antibodies can be divided into κ chains and λ chains based on their constant regions, and the antibodies herein may have either κ chains or λ chains.

[0037] The variable region of an antibody is generally composed of three complementarity determining regions (CDRs) sandwiched between four framework regions (FRs). In this specification, the amino acid positions assigned to the CDRs and frameworks of an antibody variable region are defined according to Kabat (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., (1987) and (1991)).

[0038] Anti-CD80 antibodies and anti-PD-L1 antibodies can be obtained by conventional methods using peptides containing all or part of CD80 or PD-L1, for example, all or part of the extracellular domain of CD80 or PD-L1, as immunogens. Peptides containing all or part of CD80 or PD-L1 can be produced by conventional peptide synthesis methods, such as genetic engineering or chemical synthesis.

[0039] Polyclonal antibodies can be produced by standard methods such as those described in "Antibodies: A Laboratory Manual," Lane, HD et al. eds., Cold Spring Harbor Laboratory Press, New York, 1989. Specifically, anti-CD80 polyclonal antibodies and anti-PD-L1 polyclonal antibodies can be produced by immunizing mammals such as rats, mice, rabbits, goats, and horses with peptides containing all or a portion of CD80 or PD-L1, respectively.

[0040] Monoclonal antibodies can be obtained by known methods, such as producing hybridomas that produce antibodies, or using genetic engineering techniques to produce expression vectors containing antibody genes and expressing them in cells.

[0041] Hybridomas secreting monoclonal antibodies can be prepared according to the method described by Kohler et al., Nature 256:495, 1975. First, an immunogen is mixed with an appropriate substance for enhancing antigenicity (e.g., keyhole limpet hemocyanin or bovine serum albumin) and, if necessary, an immunostimulant (e.g., Freund's complete or incomplete adjuvant), and the mixture is used to immunize non-human mammals such as rats, mice, rabbits, goats, and horses. Typically, immunized animals are immunized multiple times at intervals of 3 to 10 days, and 1 to 100 μg of the immunogen peptide is administered. Next, immunocompetent cells (cells capable of producing antibodies in the immunized animal) are collected from the immunized animal after multiple immunizations and fused with myeloma cells (e.g., cells derived from mammals such as mice, rats, guinea pigs, hamsters, rabbits, or humans) that are not capable of producing autoantibodies. Cell fusion can be achieved using polyethylene glycol, electrofusion, or other methods. Furthermore, cells that have successfully undergone cell fusion are selected based on the selection marker possessed by the fused cells, and the reactivity of the antibodies produced by the selected cells to the immunogen is confirmed by ELISA, radioimmunoassay, fluorescent antibody technique, etc., to obtain hybridomas that produce the desired monoclonal antibody. Monoclonal antibodies can be isolated from the culture supernatant of the resulting hybridomas cultured in vitro. Alternatively, monoclonal antibodies can be isolated from the ascites fluid of mice, rats, guinea pigs, hamsters, rabbits, etc. after in vivo culture.

[0042] Monoclonal antibodies can also be obtained by cloning antibody genes from the resulting hybridomas, incorporating them into appropriate expression vectors, and expressing them in host cells, as described below (P.J. Delves., ANTIBODY PRODUCTION ESSENTIAL TECHNIQUES., 1997 WILEY; P. Shepherd and C. Dean., Monoclonal Antibodies., 2000 OXFORD UNIVERSITY PRESS; J.W. Goding., Monoclonal Antibodies: principles and practice., 1993 ACADEMIC PRESS). Furthermore, transgenic animal production techniques can be used to produce transgenic animals (e.g., cows, goats, sheep, or pigs) in which the gene for the antibody of interest has been incorporated into their endogenous genes, and monoclonal antibodies derived from the antibody gene can then be obtained, for example, from the milk of the transgenic animals.

[0043] The obtained monoclonal antibody can be purified by an appropriate combination of methods well known in the art, such as chromatography on a protein A column, ion exchange chromatography, hydrophobic chromatography, ammonium sulfate precipitation, gel filtration, affinity chromatography, etc.

[0044] A chimeric antibody is an antibody containing sequences that are derived from different sources, for example, an antibody in which variable and constant regions that are derived from different sources are linked. In one embodiment, a chimeric antibody is composed of a variable region of an antibody derived from a mammal other than human and a constant region derived from a human antibody. A chimeric antibody can be obtained, for example, by linking a polynucleotide encoding the variable region of an antibody derived from a mammal other than human with a polynucleotide encoding the constant region of a human antibody, incorporating the resulting polynucleotide into an expression vector, and introducing the expression vector into a host for expression.

[0045] CDRs are regions that essentially determine the binding specificity of an antibody, and their amino acid sequences are highly diverse. On the other hand, the amino acid sequences constituting FRs show high homology even among antibodies with different binding specificities. Therefore, CDR grafting can transfer the binding specificity of one antibody to another.

[0046] A humanized antibody generally consists of a CDR from an antibody derived from a nonhuman animal, a FR from a human antibody, and a constant region from a human antibody. Humanized antibodies can be obtained by grafting the CDR from an antibody derived from a nonhuman animal onto a human antibody. Humanized antibodies can be produced by various methods, including overlap extension PCR (Almagro and Fransson, Front. Biosci. 13:1619-1633(2008)). In this method, PCR is performed using oligonucleotides as primers that overlap the ends of the CDR from an antibody derived from a nonhuman animal (e.g., a mouse antibody) and the FR from a human antibody, synthesizing a polynucleotide in which the CDR from the nonhuman animal antibody and the FR from a human antibody are linked. The resulting polynucleotide is then ligated to a polynucleotide encoding the constant region of a human antibody, incorporated into an expression vector, and the expression vector is introduced into a host for expression, thereby obtaining a humanized antibody.

[0047] Methods for selecting FRs suitable for producing humanized antibodies are known. For example, FRs selected by the best-fit method (Sims et al. J. Immunol. 151:2296(1993)) or FRs derived from consensus sequences of specific subgroups of human antibody light or heavy chain variable regions (Carter et al. Proc. Natl. Acad. Sci. USA 89:4285(1992); Presta et al. J. Immunol. 151:2623(1993)) can be used.

[0048] Human antibodies can be obtained, for example, by sensitizing human lymphocytes in vitro with a desired antigen and then fusing the sensitized lymphocytes with human myeloma cells (Japanese Patent Publication No. 1-59878). U266 and other types of human myeloma cells can be used as the fusion partner. Human antibodies can also be obtained by immunizing transgenic animals carrying a full repertoire of human antibody genes with a desired antigen (Lonberg, Nat. Biotech. 23: 1117-1125, 2005). Furthermore, a technique for obtaining human antibodies by panning using a human antibody library is also known (Antibody Phage Display: Methods and Protocols, Methods in Molecular Biology 178, 2001). For example, the variable regions of human antibodies can be expressed on the surface of phages as single-chain fragments (scFvs) using phage display, phages that bind to the antigen are selected, and the DNA sequences encoding the variable regions of the antigen-binding human antibodies can be determined by analyzing the genes of the selected phages. Next, this variable region sequence is linked in frame to the sequence of a human antibody constant region, inserted into an appropriate expression vector, and this expression vector is introduced into a host for expression, thereby obtaining a human antibody.

[0049] Multispecific antibodies are antibodies that bind to at least two different sites. Examples of multispecific antibodies include bispecific antibodies and trispecific antibodies. In one embodiment, a multispecific antibody binds to CD80 or PD-L1 and one or more other antigens. Multispecific antibodies can be produced, for example, by genetic engineering techniques or by combining two or more antibodies or antibody fragments that recognize different antigens.

[0050] Antibody fragments can be obtained, for example, by digesting antibodies with proteases such as papain and pepsin. Alternatively, antibody fragments can be obtained by introducing an expression vector containing a polynucleotide encoding the antibody fragment into a host cell and expressing the polynucleotide (see, for example, Co, MS et al., J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, AH, Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A., Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol. (1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986) 121, 663-669; Bird, RE and Walker, BW, Trends Biotechnol. (1991) 9, 132-137; Hudson et al. al., Nat. Med., (2003)9, 129-134).

[0051] As described above, antibodies can be obtained by introducing an expression vector containing a polynucleotide encoding the antibody into cells and expressing it. Specifically, an expression vector is constructed so that a sequence encoding the antibody is expressed under the control of an expression control region such as an enhancer or promoter, and host cells are transformed with this expression vector to express the antibody.

[0052] Thus, the present disclosure also provides polynucleotides encoding anti-CD80 antibodies or anti-PD-L1 antibodies, expression vectors containing the polynucleotides, and transformed cells containing the polynucleotides or expression vectors.

[0053] Eukaryotic cells such as animal cells, plant cells, and fungal cells can be used as host cells. Animal cells include mammalian cells (e.g., CHO, COS, NIH3T3, myeloma, BHK (baby hamster kidney), HeLa, and Vero), amphibian cells (e.g., Xenopus oocytes), and insect cells (e.g., Sf9, Sf21, and Tn5). Fungal cells include yeast (e.g., Saccharomyces, e.g., Saccharomyces cerevisiae) and filamentous fungi (e.g., Aspergillus, e.g., Aspergillus niger). Prokaryotic cells such as Escherichia coli (e.g., JM109, DH5α, and HB101) and Bacillus subtilis can also be used as host cells. Vectors can be introduced into host cells by, for example, the calcium phosphate method, the DEAE-dextran method, electroporation, lipofection, or the like.

[0054] The binding of the obtained anti-CD80 or anti-PD-L1 antibodies to CD80 or PD-L1 was measured by ELISA, immunofluorescence, radioimmunoassay (RIA), and BIACORE. (登録商標) This can be confirmed by a surface plasmon resonance assay or the like.

[0055] The binding of the obtained anti-CD80 antibody or anti-PD-L1 antibody to CD80 or PD-L1 can also be confirmed by a competition assay. For example, FACS, ELISA, or the like can be used to determine whether the obtained anti-CD80 antibody competes with a known anti-CD80 antibody for binding to CD80, or whether the obtained anti-PD-L1 antibody competes with a known anti-PD-L1 antibody for binding to PD-L1. Examples of known anti-CD80 antibodies that can be used include anti-CD80 antibodies having the heavy chain variable region, light chain variable region, or CDR sequences described below.

[0056] In one embodiment, the anti-CD80 antibody or anti-PD-L1 antibody is administered at a dose of 10 -7 M or less or 10 -8 M or less, e.g. 10 -7 M~10 -15 M, 10 -7 M~10 -13 M, 10 -7 M~10 -9 M, 10 -8 M~10 -15 M, 10 -8 M~10 -13 M, 10 -8 M~10 -9 M, 10 -9 M~10 -12 M, or 10 -9 M~10 -11 It binds to CD80 or PD-L1 with an equilibrium dissociation constant (KD) of M. The equilibrium dissociation constant can be measured, for example, by biolayer interferometry. Specifically, the equilibrium dissociation constant can be measured by the methods described in the Examples of this application.

[0057] Preferably, the anti-CD80 antibody does not substantially inhibit the binding of CD80 to CTLA-4. The binding of CD80 to CTLA-4 can be measured, for example, by contacting cells expressing CD80 with a soluble peptide comprising the extracellular domain of CTLA-4 that has a detectable label (e.g., a fluorescent label, a luminescent label, a radioactive label, a magnetic label, etc.) and measuring the amount of label bound to the cells. An unlabeled soluble peptide may also be used in combination with a substance (e.g., a secondary antibody) that binds to the peptide and has a label. The amount of label bound to the cells can be compared in the presence and absence of the anti-CD80 antibody to determine whether or not, or the degree of, inhibition of the binding of CD80 to CTLA-4 is present. In this context, "does not substantially inhibit binding" means that in the presence of a sufficient amount (e.g., about 10 μg / ml) of anti-CD80 antibody to cell surface-expressed CD80, the amount of binding between CD80 and CTLA-4 is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% (and may exceed 100%) of the amount of binding in the absence of anti-CD80 antibody.

[0058] Alternatively, an anti-CD80 antibody that does not strongly inhibit the binding of CD80 to CD28 may be used. The binding of CD80 to CD28 can be measured, for example, by contacting cells expressing CD80 with a soluble peptide containing the extracellular domain of CD28 that has a detectable label (e.g., a fluorescent label, a luminescent label, a radioactive label, a magnetic label, etc.) and measuring the amount of label bound to the cells. An unlabeled soluble peptide may also be used in combination with a substance (e.g., a secondary antibody) that binds to the peptide and has a label. The amount of label bound to the cells can be compared in the presence and absence of the anti-CD80 antibody to determine whether or not, or the degree of, inhibition of the binding of CD80 to CD28 is present. In this context, "does not strongly inhibit binding" means that in the presence of a sufficient amount (e.g., about 10 μg / ml) of anti-CD80 antibody to CD80 expressed on the cell surface, the amount of binding of CD80 to CD28 is about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% or more (and may exceed 100%) of the amount of binding in the absence of anti-CD80 antibody.

[0059] In one embodiment, the anti-CD80 antibody a heavy chain variable region comprising CDR1, CDR2, and CDR3 in the amino acid sequence of SEQ ID NO: 7, or the amino acid sequence of SEQ ID NO: 7; and / or a light chain variable region comprising CDR1, CDR2, and CDR3 in the amino acid sequence of SEQ ID NO: 8, or the amino acid sequence of SEQ ID NO: 8; Includes.

[0060] In one embodiment, the anti-CD80 antibody a heavy chain variable region comprising CDR1, CDR2, and CDR3 in the amino acid sequence of SEQ ID NO: 15, or the amino acid sequence of SEQ ID NO: 15; and / or a light chain variable region comprising CDR1, CDR2, and CDR3 in the amino acid sequence of SEQ ID NO: 16, or the amino acid sequence of SEQ ID NO: 16; Includes.

[0061] In one embodiment, the anti-CD80 antibody CDR1 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 9; CDR2 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 10; and CDR3 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 11; a heavy chain variable region comprising: CDR1 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 12; CDR2 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 13; and CDR3 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 14; a light chain variable region comprising: Includes.

[0062] In one embodiment, the anti-CD80 antibody CDR1 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 17; CDR2 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 18; and CDR3 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 19; a heavy chain variable region comprising: CDR1 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 20; CDR2 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 21; and CDR3 comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the sequence of SEQ ID NO: 22; a light chain variable region comprising: Includes.

[0063] In one embodiment, the anti-CD80 antibody CDR1 comprising a sequence in which 0, 1 or 2 amino acids are deleted, substituted or added in the sequence of SEQ ID NO: 9; CDR2 comprising the sequence of SEQ ID NO: 10 in which 0, 1 or 2 amino acids are deleted, substituted or added; and CDR3 comprising a sequence in which 0, 1 or 2 amino acids are deleted, substituted or added in the sequence of SEQ ID NO: 11; a heavy chain variable region comprising: CDR1 comprising a sequence in which 0, 1 or 2 amino acids are deleted, substituted or added in the sequence of SEQ ID NO: 12; CDR2 comprising the sequence of SEQ ID NO: 13 in which 0, 1 or 2 amino acids are deleted, substituted or added; and CDR3 comprising a sequence in which 0, 1 or 2 amino acids are deleted, substituted or added in the sequence of SEQ ID NO: 14; a light chain variable region comprising: Includes.

[0064] In one embodiment, the anti-CD80 antibody CDR1 comprising a sequence in which 0, 1 or 2 amino acids are deleted, substituted or added in the sequence of SEQ ID NO: 17; CDR2 comprising the sequence of SEQ ID NO: 18 in which 0, 1 or 2 amino acids are deleted, substituted or added; and CDR3 comprising a sequence in which 0, 1 or 2 amino acids are deleted, substituted or added in the sequence of SEQ ID NO: 19; a heavy chain variable region comprising: CDR1 comprising a sequence in which 0, 1 or 2 amino acids are deleted, substituted or added in the sequence of SEQ ID NO: 20; CDR2 comprising the sequence of SEQ ID NO: 21 in which 0, 1 or 2 amino acids are deleted, substituted or added; and CDR3 comprising a sequence in which 0, 1 or 2 amino acids are deleted, substituted or added in the sequence of SEQ ID NO: 22; a light chain variable region comprising: Includes.

[0065] In one embodiment, the anti-CD80 antibody comprises a heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 9, CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and CDR3 comprising the amino acid sequence of SEQ ID NO: 11, and / or a light chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 12, CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR3 comprising the amino acid sequence of SEQ ID NO: 14. In one embodiment, the anti-CD80 antibody comprises a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 9, CDR2 consisting of the amino acid sequence of SEQ ID NO: 10, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 11, and / or a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 14.

[0066] In one embodiment, the anti-CD80 antibody comprises a heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 17, CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and CDR3 comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 20, CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and CDR3 comprising the amino acid sequence of SEQ ID NO: 22. In one embodiment, the anti-CD80 antibody comprises a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 17, CDR2 consisting of the amino acid sequence of SEQ ID NO: 18, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 19, and / or a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 20, CDR2 consisting of the amino acid sequence of SEQ ID NO: 21, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 22.

[0067] In one embodiment, the anti-CD80 antibody comprises a heavy chain variable region comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 7, and / or a light chain variable region comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 8. In a further embodiment, the anti-CD80 antibody comprises a heavy chain variable region comprising an amino acid sequence in which 0 to 5 amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 7, and / or a light chain variable region comprising an amino acid sequence in which 0 to 5 amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 8. These embodiments include anti-CD80 antibodies in which the CDRs of the heavy chain variable region and / or light chain variable region have not been altered, specifically anti-CD80 antibodies comprising a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 9, CDR2 having the amino acid sequence of SEQ ID NO: 10, and CDR3 having the amino acid sequence of SEQ ID NO: 11, and / or a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 12, CDR2 having the amino acid sequence of SEQ ID NO: 13, and CDR3 having the amino acid sequence of SEQ ID NO: 14.

[0068] In one embodiment, the anti-CD80 antibody comprises a heavy chain variable region comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 15, and / or a light chain variable region comprising a sequence having 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity to the amino acid sequence of SEQ ID NO: 16. In a further embodiment, the anti-CD80 antibody comprises a heavy chain variable region comprising an amino acid sequence in which 0 to 5 amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 15, and / or a light chain variable region comprising an amino acid sequence in which 0 to 5 amino acids have been deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 16. These embodiments include anti-CD80 antibodies in which the CDRs of the heavy chain variable region and / or light chain variable region have not been altered, specifically anti-CD80 antibodies comprising a heavy chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 17, CDR2 having the amino acid sequence of SEQ ID NO: 18, and CDR3 having the amino acid sequence of SEQ ID NO: 19, and / or a light chain variable region comprising CDR1 having the amino acid sequence of SEQ ID NO: 20, CDR2 having the amino acid sequence of SEQ ID NO: 21, and CDR3 having the amino acid sequence of SEQ ID NO: 22.

[0069] In one embodiment, the anti-CD80 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8. In a further embodiment, the anti-CD80 antibody comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 7 and / or a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 8.

[0070] In one embodiment, the anti-CD80 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16. In a further embodiment, the anti-CD80 antibody comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15 and / or a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 16.

[0071] In one embodiment, the anti-CD80 antibody comprises a heavy chain variable region comprising CDRs 1 to 3 of the above-mentioned light chain variable region and / or a light chain variable region comprising CDRs 1 to 3 of the above-mentioned heavy chain variable region.

[0072] In one embodiment, the anti-CD80 antibody is an antibody that competes with any of the anti-CD80 antibodies identified by the sequences above for binding to CD80. Competition can be determined, for example, by the competition assays described above.

[0073] "Sequence identity" is determined by comparing two sequences that are optimally aligned across the entire region of the sequences to be compared. The sequences to be compared may have additions or deletions (e.g., gaps) in the optimal alignment of the two sequences. Sequence identity can be calculated using programs such as FASTA, BLAST, and CLUSTAL W provided in public databases (e.g., DDBJ (http: / / www.ddbj.nig.ac.jp)). Alternatively, commercially available sequence analysis software (e.g., Vector NTI (登録商標) Software, GENETYX (登録商標) ver. 12).

[0074] Various methods are known for modifying amino acid sequences to obtain antibodies with desired properties. For example, mutants with improved binding affinity can be obtained by phage display-based methods. In this method, the site for mutation is determined by, for example, alanine scanning mutagenesis to identify amino acid residues that affect the antibody-antigen interaction, or by analyzing the crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Mutants with modified amino acids at these sites are generated by error-prone PCR or site-directed mutagenesis, and the resulting mutant library can be screened to obtain mutants with desired properties.

[0075] The anti-CD80 antibody or anti-PD-L1 antibody may have modified glycosylation in the Fc region. Examples of antibodies with modified glycosylation include antibodies lacking fucose attached to their glycosylation (U.S. Patent Publication No. 2003 / 0157108) and antibodies with glycosylation containing bisecting N-acetylglucosamine (GlcNAc) (WO 2003 / 011878).

[0076] The anti-CD80 antibody or anti-PD-L1 antibody may be conjugated to a polymer such as polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, or a copolymer of polyethylene glycol and polypropylene glycol, for example to increase the half-life or improve the stability of the antibody.

[0077] Substances that enhance the binding between PD-L1 and PD-1 can suppress immunity when administered to a subject in an effective amount. Thus, there is provided an immunosuppressant comprising, as an active ingredient, a substance that enhances the binding between PD-L1 and PD-1 selected from an anti-CD80 antibody and an anti-PD-L1 antibody.

[0078] The antibodies or immunosuppressants disclosed herein have low toxicity and can therefore be used safely as pharmaceuticals.

[0079] [Application to pharmaceuticals] The antibodies or immunosuppressants disclosed herein can be used for the prevention and / or treatment of diseases characterized by enhanced immunity. Accordingly, in one embodiment, there is provided an agent for the prevention and / or treatment of diseases characterized by enhanced immunity, comprising as an active ingredient a substance that promotes the binding of PD-L1 to PD-1, which is selected from an anti-CD80 antibody and an anti-PD-L1 antibody.

[0080] Diseases characterized by enhanced immunity include autoimmune diseases, allergic diseases, and graft-versus-host disease. Examples of autoimmune diseases include Behçet's disease, systemic lupus erythematosus, multiple sclerosis (systemic sclerosis, progressive systemic sclerosis), scleroderma, polymyositis, dermatomyositis, periarteritis nodosa (polyarteritis nodosa, microscopic polyangiitis), aortitis syndrome (Takayasu's arteritis), malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, Wegener's granulomatosis, mixed connective tissue disease, Sjögren's syndrome, adult-onset Still's disease, and allergies. Granulomatous vasculitis, hypersensitivity vasculitis, Cogan's syndrome, RS3PE, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid syndrome, eosinophilic fasciitis, IgG4-related disorders (e.g., primary sclerosing cholangitis, autoimmune pancreatitis), Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cirrhosis, aortitis syndrome, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastoma Neutropenic anemia, autoimmune hemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenic purpura, Graves' disease (hyperthyroidism), Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, idiopathic Addison's disease (chronic hypoadrenalism), type I diabetes, slowly progressive type I diabetes (adult latent autoimmune diabetes), chronic discoid lupus erythematosus, localized scleroderma, psoriasis, psoriatic arthritis, pemphigus, pemphigoid, herpes gestationis, Examples of autoimmune diseases include linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo, vitiligo vulgaris, atopic dermatitis, neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, sarcoidosis, bullous pemphigoid, giant cell arteritis, amyotrophic lateral sclerosis, eosinophilic granulomatosis with polyangiitis, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent abortion, inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease), and celiac disease. In some embodiments, the autoimmune disease is type 1 diabetes, multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis. In some embodiments, the autoimmune disease is multiple sclerosis. Allergic diseases include, for example, asthma, atopic dermatitis, rhinitis, conjunctivitis, and hay fever.

[0081] As used herein, "treating" or "treatment" means reducing or eliminating the cause of a disease, slowing or halting its progression, alleviating, ameliorating, or eliminating its symptoms, and / or inhibiting the worsening of its symptoms in a subject with the disease.

[0082] As used herein, "preventing" or "prevention" refers to preventing the onset of a disease or reducing the likelihood of developing a disease in a subject, particularly in a subject who is likely to develop the disease but has not yet done so, including preventing recurrence. Subjects who may develop an autoimmune disease or an allergic disease but have not yet done so include, for example, subjects with enhanced immune systems, subjects with a genetic predisposition to an autoimmune disease or an allergic disease, and subjects who have previously suffered from and recovered from an autoimmune disease or an allergic disease. Subjects who may develop graft-versus-host disease but have not yet done so include subjects who have received organ transplants.

[0083] Subjects to which immunosuppressants or agents for the prevention and / or treatment of diseases characterized by immune enhancement are administered include animals, typically mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, etc.), with humans being particularly preferred. Preferred subjects are those in need of immune suppression or the above-mentioned prevention and / or treatment, particularly those in need of the above-mentioned treatment.

[0084] The dosage of the active ingredient is appropriately selected depending on the administration method, the age, weight, health condition, etc. of the subject. For example, a daily dose of 10 μg / kg to 100 mg / kg, 100 μg / kg to 10 mg / kg, or 1 mg / kg to 10 mg / kg for an adult can be administered continuously for a period ranging from 30 minutes to 24 hours, or once to several times a day, or once to several times a day or every few days, or once a week or every few weeks, for example, once every 1 to 3 weeks, but is not limited thereto. The administration method is also appropriately selected depending on the age, weight, health condition, etc. of the subject. The administration method may be oral or parenteral, with parenteral administration being preferred. Examples of parenteral administration include subcutaneous administration, intradermal administration, intraperitoneal administration, intramuscular administration, and intravenous administration, with intravenous administration being preferred.

[0085] Immunosuppressants or agents for the prevention and / or treatment of diseases characterized by immune enhancement can be formulated using conventional methods. The formulations may contain various pharmaceutically acceptable formulation substances as needed. The formulation substances can be selected appropriately depending on the dosage form of the formulation, and examples include buffering agents, surfactants, stabilizers, preservatives, excipients, diluents, additives, disintegrants, binders, coating agents, lubricants, glidants, solubilizers, etc. For example, immunosuppressants can be formulated as injections or infusions. Injections or infusions can be in the form of sterile aqueous solutions, suspensions, or emulsions, or they can be in the form of solids or lyophilized preparations to be dissolved, suspended, or emulsified in a sterile liquid. The sterile liquid can be, for example, water for injection, physiological saline, glucose solution, or isotonic solution. Furthermore, immunosuppressants can be formulated to provide sustained or controlled release of the active ingredient. Methods for producing these formulations are well known in the art.

[0086] The formulation may contain a pharmaceutically acceptable carrier. In the present disclosure, "pharmaceutically acceptable carrier" includes any substance that, when combined with an active ingredient, can maintain the biological activity of the ingredient and is non-reactive with the subject's immune system. Examples include stabilizers, solubilizers, suspending agents, emulsifiers, soothing agents, buffers, preservatives, pH adjusters, and antioxidants. Stabilizers that can be used include, for example, various amino acids, albumin, globulin, gelatin, mannitol, glucose, dextran, ethylene glycol, propylene glycol, polyethylene glycol, ascorbic acid, sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, and dibutylhydroxytoluene. Solubilizers that can be used include, for example, alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 20). (登録商標) , Polysorbate 80 (登録商標), HCO-50, etc.) can be used. Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, sodium lauryl sulfate, etc. Examples of emulsifying agents that can be used include gum arabic, sodium alginate, tragacanth, etc. Examples of soothing agents that can be used include benzyl alcohol, chlorobutanol, sorbitol, etc. Examples of buffering agents that can be used include phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, epsilon aminocaproic acid buffer, etc. Examples of preservatives that can be used include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax, etc. Examples of preservatives that can be used include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol. Examples of pH adjusters that can be used include hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid. Examples of antioxidants that can be used include (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid, sorbitol, tartaric acid, and phosphoric acid.

[0087] Injections or infusion solutions for intravenous drip can be produced by sterilizing them in the final step or by an aseptic procedure, for example, by filtration, followed by filling into a sterile container. Alternatively, injections or infusion solutions for intravenous drip can be prepared by dissolving sterile powders (which may contain a powder of a pharmaceutically acceptable carrier) obtained by vacuum drying or freeze-drying in an appropriate solvent before use.

[0088] Immunosuppressants or agents for the prevention and / or treatment of diseases characterized by immune enhancement can be used alone or in combination with one or more additional active ingredients, particularly active ingredients for immune suppression. "Combined use" of ingredients refers not only to the use of a dosage form containing all ingredients or the use of a combination of dosage forms containing each ingredient separately, but also to the simultaneous administration of each ingredient or the administration of any ingredient with a delay, as long as they are used for the treatment and / or prevention of diseases characterized by immune suppression or immune enhancement. When any ingredient is administered with a delay, there may be a period during which the ingredients are administered simultaneously. Two or more additional active ingredients can also be used in combination. This combination can, for example, complement the preventive and / or therapeutic effects of other active ingredients, maintain and / or reduce the dosage or administration frequency of the other active ingredients, and / or reduce the dosage. Active ingredients suitable for combination use include, for example, anti-inflammatory agents, antibacterial agents, antifungal agents, antiviral agents, immunosuppressants, molecular targeted drugs, etc.

[0089] For example, when the immunosuppressant or the preventive and / or therapeutic agent for a disease characterized by immune enhancement of the present invention is applied to the prevention and / or treatment of type I diabetes, insulin preparations (e.g., human insulin, insulin glargine, insulin lispro, insulin detemir, insulin aspart, etc.), sulfonylureas (e.g., glibenclamide, gliclazide, glimepiride, etc.), rapid-acting insulin secretagogues (e.g., nateglinide, etc.), biguanide preparations (e.g., metformin, and the like), insulin sensitizers (e.g., pioglitazone, etc.), α-glucosidase inhibitors (e.g., acarbose, voglibose, etc.), diabetic neuropathy treatment drugs (e.g., epalrestat, mexiletine, imidapril, etc.), GLP-1 analogue preparations (e.g., liraglutide, exenatide, lixisenatide, etc.), and DPP-4 inhibitors (e.g., sitagliptin, vildagliptin, alogliptin, etc.).

[0090] Furthermore, for example, when the immunosuppressant or the agent for preventing and / or treating a disease characterized by immune enhancement of the present invention is applied to the prevention and / or treatment of multiple sclerosis, steroid drugs (e.g., cortisone acetate, hydrocortisone, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, fludrocortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone butylacetate, prednisolone sodium phosphate, halopredone acetate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, triamcinolone,

[0111] The present invention may be used in combination with any one or more drugs selected from the group consisting of benzodiazepine, benzodiazepine, benzocaine, benzodiazepine, benzocaine, benzodiazepine, benzocaine sodium phosphate, benzocaine palmitate, benzocaine sodium phosphate ...

[0091] Furthermore, for example, when the immunosuppressant or the prophylactic and / or therapeutic agent for a disease characterized by immune enhancement of the present invention is applied to the prophylaxis and / or treatment of systemic lupus erythematosus, it may be used in combination with any one or more drugs selected from steroid drugs (e.g., the steroid drugs described above), other immunosuppressants (e.g., cyclosporine, tacrolimus, fingolimod, etc.), and belimumab.

[0092] Furthermore, for example, when the immunosuppressant or the preventive and / or therapeutic agent for diseases characterized by immune enhancement of the present invention is applied to the prevention and / or treatment of rheumatoid arthritis, it may be used in combination with any one or more drugs selected from steroid drugs (e.g., the steroid drugs described above), antirheumatic drugs (e.g., methotrexate, sulfasalazine, bucillamine, leflunomide, mizoribine, tacrolimus, etc.), anticytokine drugs (e.g., infliximab, adalimumab, tocilizumab, etanercept, golimumab, certolizumab, etc.), abatacept, etc.

[0093] When applied to the prevention and / or treatment of other autoimmune diseases, allergic diseases, or graft-versus-host disease, the immunosuppressant or the preventive and / or therapeutic agent for diseases characterized by immune enhancement of the present invention may be used in combination with one or more of the other drugs described above.

[0094] In one aspect, a method for immune suppression is provided, comprising administering to a subject in need of immune suppression an effective amount of a substance that enhances the binding of PD-L1 to PD-1, selected from an anti-CD80 antibody and an anti-PD-L1 antibody, or an immunosuppressant comprising such a substance. As used herein, the term "effective amount" refers to an amount that can exert an immunosuppressive effect in a subject. In one aspect, there is provided a substance that promotes the binding of PD-L1 to PD-1, selected from an anti-CD80 antibody and an anti-PD-L1 antibody, or an immunosuppressant comprising such a substance, for use in immune suppression. In one aspect, there is provided use of a substance that promotes the binding of PD-L1 to PD-1, selected from an anti-CD80 antibody and an anti-PD-L1 antibody, or an immunosuppressant comprising such a substance, in the manufacture of a pharmaceutical composition for immunosuppression.

[0095] In one aspect, there is provided a method for preventing and / or treating a disease characterized by immune enhancement, the method comprising administering to a subject in need thereof an effective amount of a substance that enhances the binding of PD-L1 to PD-1, selected from an anti-CD80 antibody and an anti-PD-L1 antibody, or an immunosuppressant comprising such substance. In one aspect, there is provided a substance that enhances the binding of PD-L1 selected from an anti-CD80 antibody and an anti-PD-L1 antibody to PD-1, or an immunosuppressant comprising such a substance, for use in the prevention and / or treatment of a disease characterized by immune enhancement. In one aspect, there is provided use of a substance that enhances the binding of PD-L1 to PD-1, selected from an anti-CD80 antibody and an anti-PD-L1 antibody, in the manufacture of a medicament for the prevention and / or treatment of a disease characterized by enhanced immunity, or an immunosuppressant comprising such a substance.

[0096] The present application provides, for example, the following embodiments. [1-1] An immunosuppressant comprising a substance that promotes the binding of PD-L1 selected from an anti-CD80 antibody and an anti-PD-L1 antibody to PD-1. [1-2] The immunosuppressant according to [1-1] above, which promotes binding between PD-1 and PD-L1, which is present on the same cells as CD80. [1-3] The immunosuppressant according to [1-1] or [1-2] above, wherein the substance enhances the binding of PD-L1 to PD-1 by approximately 2-fold or more. [1-4] The immunosuppressant according to any one of [1-1] to [1-3] above, wherein PD-1 is present on T cells. [1-5] The immunosuppressant according to any one of the above [1-1] to [1-4], wherein the substance is an anti-CD80 antibody. [1-6] The anti-CD80 antibody is 10 -7 The immunosuppressant according to [1-5] above, which binds to CD80 with an equilibrium dissociation constant of M or less. [1-7] The immunosuppressant according to [1-5] or [1-6] above, wherein the anti-CD80 antibody inhibits the cis binding between CD80 and PD-L1. [1-8] The cis-linked domain is mediated by at least a region containing an amino acid sequence equivalent to isoleucine at position 92 and / or leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1, and a region containing an amino acid sequence equivalent to asparagine at position 63 and / or glycine at position 119 of human PD-L1 having the amino acid sequence of SEQ ID NO: 3; or Alternatively, the immunosuppressant according to the above item [1-7], wherein the cis-binding is via a region containing at least an amino acid corresponding to leucine at position 96 and / or leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2, and a region containing amino acids corresponding to valine at position 54, tyrosine at position 56, and / or glutamic acid at position 58 of mouse PD-L1 having the amino acid sequence of SEQ ID NO: 4. [1-9] The immunosuppressant according to any one of [1-5] to [1-8] above, wherein the anti-CD80 antibody binds to a region containing an amino acid corresponding to isoleucine at position 92 and / or leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1, or to a region containing an amino acid corresponding to leucine at position 96 and / or leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2. [1-10] The immunosuppressant according to any one of [1-5] to [1-9] above, wherein the anti-CD80 antibody does not substantially inhibit the binding of CD80 to CTLA-4. [1-11] The immunosuppressant according to any one of [1-5] to [1-10] above, wherein the anti-CD80 antibody does not strongly inhibit the binding between CD80 and CD28. [1-12] An immunosuppressant comprising an anti-CD80 antibody that promotes binding of PD-1 to PD-L1, which is present on the same cells as CD80, but does not strongly inhibit binding of CD80 to CD28.

[0097] [1-13] The anti-CD80 antibody (1) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; (2) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (3) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:11; or (4) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; The immunosuppressant according to any one of the above [1-5] to [1-12], comprising: [1-14] The anti-CD80 antibody a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:11; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14 The immunosuppressant according to any one of the above [1-5] to [1-13], comprising: [1-15] The anti-CD80 antibody a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and The immunosuppressant according to any one of [1-5] to [1-13] above, comprising a light chain variable region including a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22. [1-16] The immunosuppressant according to any one of [1-5] to [1-14] above, wherein the anti-CD80 antibody comprises a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8. [1-17] The immunosuppressant described in any one of [1-5] to [1-13] and [1-15] above, wherein the anti-CD80 antibody comprises a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 16. [1-18] The immunosuppressant according to any one of [1-5] to [1-12] above, wherein the anti-CD80 antibody competes with the antibody according to any one of [1-13] to [1-17] above for binding to CD80.

[0098] [1-19] The immunosuppressant according to any one of [1-1] to [1-4] above, wherein the substance is an anti-PD-L1 antibody. [1-20]The anti-PD-L1 antibody is 10 -7 The immunosuppressant according to [1-19] above, which binds to PD-L1 with an equilibrium dissociation constant of M or less. [1-21] The immunosuppressant according to [1-19] or [1-20] above, wherein the anti-PD-L1 antibody inhibits the cis binding of PD-L1 and CD80. [1-22] The cis bond is mediated by at least an amino acid corresponding to isoleucine at position 92 and / or leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1 and an amino acid corresponding to asparagine at position 63 and / or glycine at position 119 of human PD-L1 having the amino acid sequence of SEQ ID NO: 3; or Alternatively, the immunosuppressant according to the above [1-21], wherein the cis-linked bond is mediated by at least an amino acid corresponding to leucine at position 96 and / or leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2, and an amino acid corresponding to valine at position 54, tyrosine at position 56, and / or glutamic acid at position 58 of mouse PD-L1 having the amino acid sequence of SEQ ID NO: 4. [1-23] The immunosuppressant according to any one of [1-19] to [1-22] above, wherein the anti-PD-L1 antibody binds to a region containing amino acids corresponding to asparagine at position 63 and / or glycine at position 119 of human PD-L1 having the amino acid sequence of SEQ ID NO: 3, or to a region containing amino acids corresponding to valine at position 54, tyrosine at position 56, and / or glutamic acid at position 58 of mouse PD-L1 having the amino acid sequence of SEQ ID NO: 4.

[0099] [1-24] The immunosuppressant according to any one of the above [1-1] to [1-23], for the prevention and / or treatment of an autoimmune disease, an allergic disease, or graft-versus-host disease. [1-25] A method for preventing and / or treating an autoimmune disease, an allergic disease, or graft-versus-host disease, comprising administering an effective amount of the immunosuppressant described in any one of [1-1] to [1-23] to a subject in need thereof. [1-26] The immunosuppressant according to any one of [1-1] to [1-23] above, for use in the prevention and / or treatment of an autoimmune disease, an allergic disease, or graft-versus-host disease. [1-27] Use of the immunosuppressant according to any one of [1-1] to [1-23] above for the manufacture of an agent for the prevention and / or treatment of autoimmune diseases, allergic diseases, or graft-versus-host disease.

[0100] [2-1]An anti-CD80 antibody or anti-PD-L1 antibody that promotes the binding of PD-L1 to PD-1. [2-2] The anti-CD80 antibody or anti-PD-L1 antibody according to [2-1] above, which promotes binding between PD-1 and PD-L1 present on the same cells as CD80. [2-3] The anti-CD80 antibody or anti-PD-L1 antibody according to [2-1] or [2-2] above, which enhances the binding of PD-L1 to PD-1 by approximately 2-fold or more. [2-4] The anti-CD80 antibody or anti-PD-L1 antibody according to any one of [2-1] to [2-3] above, wherein PD-1 is present on T cells. [2-5] The antibody according to any one of [2-1] to [2-4] above, which is an anti-CD80 antibody. [2-6]10 -7 The antibody according to [2-5] above, which binds to CD80 with an equilibrium dissociation constant of M or less. [2-7] The antibody described in [2-5] or [2-6] above, which inhibits the cis binding of CD80 and PD-L1. [2-8] The cis-binding is via at least a region containing an amino acid corresponding to isoleucine at position 92 and / or leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1, and a region containing an amino acid corresponding to asparagine at position 63 and / or glycine at position 119 of human PD-L1 having the amino acid sequence of SEQ ID NO: 3; or Alternatively, the antibody of [2-7] above, wherein the cis-binding is via a region containing at least an amino acid corresponding to leucine at position 96 and / or leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2, and a region containing amino acids corresponding to valine at position 54, tyrosine at position 56, and / or glutamic acid at position 58 of mouse PD-L1 having the amino acid sequence of SEQ ID NO: 4. [2-9] The antibody according to any one of [2-5] to [2-8] above, which binds to a region containing amino acids corresponding to isoleucine at position 92 and / or leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1, or a region containing amino acids corresponding to leucine at position 96 and / or leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2. [2-10] The antibody according to any one of [2-5] to [2-9] above, which does not substantially inhibit the binding of CD80 to CTLA-4. [2-11] The antibody according to any one of [2-5] to [2-10] above, which does not strongly inhibit the binding between CD80 and CD28. [2-12] An anti-CD80 antibody that promotes binding of PD-1 to PD-L1, which is present on the same cells as CD80, but does not strongly inhibit binding of CD80 to CD28.

[0101] [2-13] (1) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; (2) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (3) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:11; or (4) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; The antibody according to any one of [2-5] to [2-12] above, comprising:

[0102] [2-14] a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14 The antibody according to any one of [2-5] to [2-13] above, comprising: [2-15] a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22 The antibody according to any one of [2-5] to [2-13] above, comprising: [2-16] The antibody according to any one of [2-5] to [2-14] above, wherein the anti-CD80 antibody comprises a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8. [2-17] The antibody according to any one of [2-5] to [2-13] and [2-15] above, wherein the anti-CD80 antibody comprises a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 16. [2-18] The antibody according to any one of [2-1] to [2-12] above, which competes with the antibody according to any one of [2-13] to [2-17] above for binding to CD80.

[0103] [2-19] The antibody according to any one of [2-1] to [2-4] above, which is an anti-PD-L1 antibody. [2-20]10 -7 The antibody of [2-19] above, which binds to PD-L1 with an equilibrium dissociation constant of M or less. [2-21] The antibody described in [2-19] or [2-20] above, which inhibits the cis binding of PD-L1 and CD80. [2-22] The cis-binding is via at least a region containing an amino acid corresponding to isoleucine at position 92 and / or leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1, and a region containing an amino acid corresponding to asparagine at position 63 and / or glycine at position 119 of human PD-L1 having the amino acid sequence of SEQ ID NO: 3; or Alternatively, the antibody of [2-21] above, wherein the cis-binding is via a region containing at least an amino acid corresponding to leucine at position 96 and / or leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2, and a region containing amino acids corresponding to valine at position 54, tyrosine at position 56, and / or glutamic acid at position 58 of mouse PD-L1 having the amino acid sequence of SEQ ID NO: 4. [2-23] The antibody according to any one of [2-19] to [2-22] above, which binds to a region containing amino acids corresponding to asparagine at position 63 and / or glycine at position 119 of human PD-L1 having the amino acid sequence of SEQ ID NO: 3, or to a region containing amino acids corresponding to valine at position 54, tyrosine at position 56, and / or glutamic acid at position 58 of mouse PD-L1 having the amino acid sequence of SEQ ID NO: 4.

[0104] [2-24] (1) a heavy chain variable region comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; (2) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22; (3) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:11; or (4) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; an anti-CD80 antibody comprising:

[0105] [2-25] a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14 The antibody described in [2-24] above, comprising: [2-26] a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 22 The antibody described in [2-24] above, comprising: [2-27] The antibody according to [2-24] or [2-25], wherein the anti-CD80 antibody comprises a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 8. [2-28] The antibody according to [2-24] or [2-26], wherein the anti-CD80 antibody comprises a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 16. [2-29] An anti-CD80 antibody that competes with the antibody according to any one of [2-24] to [2-28] above for binding to CD80. [2-30] An anti-CD80 antibody that binds to a region containing amino acids corresponding to isoleucine at position 92 and / or leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1, or a region containing amino acids corresponding to leucine at position 96 and / or leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO: 2. [2-31]10 -7 The antibody according to any one of [2-24] to [2-30] above, which binds to CD80 with an equilibrium dissociation constant of M or less. [2-32] An anti-PD-L1 antibody that binds to a region containing amino acids equivalent to asparagine at position 63 and / or glycine at position 119 of human PD-L1 and having the amino acid sequence of SEQ ID NO: 3, or a region containing amino acids equivalent to valine at position 54, tyrosine at position 56, and / or glutamic acid at position 58 of mouse PD-L1 and having the amino acid sequence of SEQ ID NO: 4. [2-33]10 -7 The antibody of [2-32] above, which binds to PD-L1 with an equilibrium dissociation constant of M or less. [2-34] The antibody according to any one of [2-1] to [2-33] above, which is a monoclonal antibody. [2-35] The antibody according to any one of [2-1] to [2-34] above, which is an isolated monoclonal antibody. [2-36] A pharmaceutical composition comprising the antibody according to any one of [2-1] to [2-35] above. [2-37] The pharmaceutical composition according to [2-36] above for the prevention and / or treatment of a disease characterized by immune suppression or immune enhancement.

[0106] [2-38] An immunosuppressant comprising, as an active ingredient, the antibody according to any one of [2-1] to [2-35] above. [2-39] The immunosuppressant according to [2-38] above, further comprising a pharmaceutically acceptable carrier. [2-40] A method for suppressing immunity, comprising administering an effective amount of the antibody described in any one of [2-1] to [2-35] to a subject in need thereof. [2-41] The antibody according to any one of [2-1] to [2-35] above, for use in immune suppression. [2-42] Use of the antibody according to any one of [2-1] to [2-35] above for the manufacture of an immunosuppressant.

[0107] [2-43] A preventive and / or therapeutic agent for a disease characterized by enhanced immunity, comprising the antibody according to any one of [2-1] to [2-35] above as an active ingredient. [2-44] The preventive and / or therapeutic agent according to [2-43] above, further comprising a pharmaceutically acceptable carrier. [2-45] A method for preventing and / or treating a disease characterized by enhanced immunity, the method comprising administering an effective amount of the antibody described in any one of [2-1] to [2-35] to a subject in need thereof. [2-46] The antibody according to any one of [2-1] to [2-35] above, for use in the prevention and / or treatment of a disease characterized by enhanced immunity. [2-47] Use of the antibody according to any one of [2-1] to [2-35] above for the manufacture of an agent for the prevention and / or treatment of a disease characterized by enhanced immunity. [2-48] The preventive and / or therapeutic agent according to [2-43] or [2-44], the method according to [2-45], the antibody according to [2-46], or the use according to [2-47], wherein the disease characterized by enhanced immunity is an autoimmune disease, an allergic disease, or graft-versus-host disease. [2-49] The preventive and / or therapeutic agent, method, antibody or use according to [2-48] above, wherein the disease characterized by enhanced immunity is an autoimmune disease. [2-50] Autoimmune diseases include Behçet's disease, systemic lupus erythematosus, multiple sclerosis, scleroderma, polymyositis, dermatomyositis, periarteritis nodosa, aortitis syndrome, malignant rheumatoid arthritis, rheumatoid arthritis, juvenile idiopathic arthritis, Wegener's granulomatosis, mixed connective tissue disease, Sjögren's syndrome, adult Still's disease, allergic granulomatous vasculitis, hypersensitivity vasculitis, Cogan's syndrome, RS3PE, temporal arteritis, polymyalgia rheumatica, fibromyalgia, antiphospholipid syndrome, eosinophilic fasciitis, IgG4-related disease, Guillain-Barré syndrome, myasthenia gravis, chronic atrophic gastritis, autoimmune hepatitis, primary biliary cirrhosis, aortitis syndrome, Goodpasture's syndrome, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune hemolytic anemia, and autoimmune neutropenia. , idiopathic thrombocytopenic purpura, Graves' disease, Hashimoto's disease, autoimmune adrenal insufficiency, primary hypothyroidism, idiopathic Addison's disease, type 1 diabetes, slowly progressive type 1 diabetes, chronic discoid lupus erythematosus, localized scleroderma, psoriasis, psoriatic arthritis, pemphigus, pemphigoid, herpes gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, alopecia areata, vitiligo, vitiligo vulgaris, atopic dermatitis , neuromyelitis optica, chronic inflammatory demyelinating polyneuropathy, sarcoidosis, bullous pemphigoid, giant cell arteritis, amyotrophic lateral sclerosis, eosinophilic granulomatosis with polyangiitis, Harada's disease, autoimmune optic neuropathy, idiopathic azoospermia, recurrent abortion, inflammatory bowel disease, and celiac disease. [2-51] The preventive and / or therapeutic agent, method, antibody or use according to [2-49] or [2-50] above, wherein the autoimmune disease is type I diabetes, multiple sclerosis, systemic lupus erythematosus or rheumatoid arthritis. [2-52] The preventive and / or therapeutic agent, method, antibody or use according to any one of the above [2-49] to [2-51], wherein the autoimmune disease is multiple sclerosis. [2-53] The preventive and / or therapeutic agent, method, antibody or use according to [2-52] above, wherein the multiple sclerosis is systemic sclerosis or progressive systemic sclerosis. [2-54] A polynucleotide encoding the antibody according to any one of [2-1] to [2-35] above. [2-55] A vector comprising the polynucleotide described in [2-54] above. [2-56] A host cell comprising the polynucleotide described in [2-54] or the vector described in [2-55].

[0108] All documents cited herein are hereby incorporated by reference. All of the above descriptions are non-limiting and can be modified without departing from the scope of the present invention as defined in the appended claims. Furthermore, all of the following examples are non-limiting and are provided solely to illustrate the present invention. [Example]

[0109] Materials and Methods cell culture DO11.10 cells, TCRα / β-deficient BW-1100.129.237 cells (White, J. et al., J. Immunol. 143, 1822-5 (1989)) (provided by Leszek Ignatowicz, Georgia Regents University), IIA1.6 cells, and E.G7 cells were maintained in RPMI 1640 medium (Gibco) supplemented with 10% (v / v) fetal bovine serum (FBS, Biowest), 0.5 mM monothioglycerol (Wako), 2 mM L-alanyl-L-glutamine dipeptide (Gibco), 100 U / mL penicillin (Nacalai Tesque), and 100 μg / mL streptomycin (Nacalai Tesque). Plat-E cells were maintained in Dulbecco's modified Eagle's medium (D'MEM, Invitrogen) supplemented with 10% (v / v) FBS, 100 U / mL penicillin (Nacalai Tesque), and 100 μg / mL streptomycin (Nacalai Tesque).

[0110] Plasmid and retroviral gene transfer cDNA fragments were amplified by PCR and cloned into a retroviral expression plasmid vector modified from pFB-ires-Neo (Agilent). To generate a plasmid library of mouse and human PD-L1 variants, the IgV domain of PD-L1 was amplified using Thermo-Start Taq DNA polymerase (Thermo Fisher Scientific) containing 400 μM MnCl2 and cloned into pFB-ires-Neo. PD-L1 and CD80 variants with site-directed mutations were generated by overhang PCR. To control expression levels, cDNA fragments were cloned into a retroviral expression plasmid vector modified from pSUPER.retro.puro (OligoEngine), in which the promoter regions were replaced with those of EF-1α (human elongation factor-1 alpha), CAG, CMV, and MC1 promoters. Plasmids were transfected into Plat-E cells cultured in D'MEM (high glucose) (Gibco) supplemented with 20% (v / v) FBS, 100 U / ml penicillin (Nacalai Tesque), and 100 μg / ml streptomycin (Nacalai Tesque) using FuGENE. (登録商標) Gene transduction was performed using HD (Promega), and the virus-containing supernatant was used to transduce the gene into target cells. Infected cells were selected using G418 (Wako), puromycin (Sigma-Aldrich), Zeocin (InvivoGen), or blasticidin (InvivoGen).

[0111] Generation of targeted gene knockout cell lines using CRISPR / Cas9 PD-1-deficient IIA1.6 cells, PD-1-, PD-L1-, and CD28-deficient DO11.10 cells, and PD-1-deficient BW-1100.129.237 cells were generated using the CRISPR / Cas9 system. The guide RNA sequences are shown in the table below. The guide RNA sequences were cloned into pEF-BOS-Cas9-U6-guide (which was modified from pEF-BOS (Mizushima, S. & Nagata, S. Nucleic Acids Res. 18, 5322 (1990)) to express humanized cas9 cDNA (Addgene) with or without the D10A mutation under the human EF-1α promoter and guide RNA under the reverse U6 promoter). The plasmids were then electroporated into the cells (Nucleofector II). (登録商標) Gene transduction was performed using a chromatographic microscope (Lonza). Cells lacking target gene expression were sorted using a cell sorter (MoFlo XDP, Beckman Coulter). Cell clones were obtained by limiting dilution, and loss-of-function mutations of the target gene and their lack of expression were confirmed by sequencing and flow cytometry, respectively.

[0112] [Table 1] * 1 :To increase efficiency, two guide RNAs were used simultaneously. * 2: Exons 2, 3 and 4 were targeted by introducing staggered nicks.

[0113] mouse C57BL / 6N mice were purchased from SLC and housed under specific pathogen-free conditions in an environmentally controlled clean room. Age- and sex-matched mice were used in each experiment. All mouse protocols were approved by the Animal Care and Use Committee of Tokushima University.

[0114] Generation of targeted gene knockout and knock-in mice Cas9 mRNA, gRNA, and single-stranded oligodeoxynucleotides (ssODN) were introduced into C57BL / 6N zygotes by electroporation as previously described (Hashimoto, M. & Takemoto, T., Sci. Rep. 5, 11315(2015)). - / - (PD-L1 knockout mice), C57BL / 6N-Cd80 - / - , C57BL / 6N-Cd274 Y56A and C57BL / 6N-Cd80 L107E The mice were generated as follows: C57BL / 6N-Cd80 - / - , C57BL / 6N-Cd274 Y56A and C57BL / 6N-Cd80 L107E The nucleotide sequences of the guide RNA and ssODN used to generate the C57BL / 6N-Cd274 mice are shown in the table below. - / - The mice have a single nucleotide insertion, which results in the creation of a premature stop codon at Y56. C57BL / 6N-Cd80 - / - The mice contained a single nucleotide insertion, resulting in a frameshift at G109, the addition of 20 unrelated amino acids, and the creation of a premature stop codon. The first-generation mosaic mice were crossed with C57BL / 6N wild-type mice to obtain heterozygous mice, and the heterozygous mice were crossed with each other to obtain homozygous mice. The primer set 5'-GAGACACTATCTCTAAAAAT-3' and 5'-TTAGTAGAGGTCTCCACCTT-3' for CD80 and 5'-GTTCATGTGATTCCCTAAAT-3' and 5'-CTGAAGTTGCTGTGCTGAGG-3' for PD-L1 were used to amplify genomic fragments. The amplified fragments were sequenced (ABI Prism (登録商標) 3700 DNA Analyzer, Thermo Fisher Scientific) or was used for restriction length polymorphism analysis.

[0115] [Table 2]

[0116] DO11.10 Stimulation of T cells and TCR-reconstituted cells DO11.10 T cells (5×10 4 Cells / well) were plated in a 96-well round-bottom plate (BD Biosciences) with a predetermined amount of OVA. 323-339 IIAdL1 cells (1 × 10 ) pulsed with the peptide (ISQAVHAAHAEINEAGR, >95% purity, Sigma-Aldrich Japan or Eurofins genomics) were cultured in 100 wells. 4 The PD-1 gene in BW-1100.129.237 cells was knocked out using the CRISPR / Cas9 system, resulting in BW-Pdcd1 cells. - / - Cells were prepared using BW-Pdcd1 - / - The cells were reconstituted with CD8α / CD8β / OT-I TCR or CD4 / OT-II TCR along with CD3δ, CD3ζ, CD28, and PD-1 to generate BW-OT-I or BW-OT-II cells, respectively. BW-OT-I and BW-OT-II cells (2.5 × 10 4 cells / well) in a 96-well round-bottom plate and a predetermined amount of MHCI-restricted OVA. 257-264 Peptide (SIINFEKL, >98% purity, MBL) or MHCII-restricted OVA 323-339 Peptide-pulsed BM-DCs or splenic DCs (5 × 10 3 Cells were stimulated with 100 μg / ml anti-PD-L1 antibody (1-111A), 5 μg / ml anti-PD-L2 antibody (TY25), or rat IgG2a isotype control (RTK2758, Biolegend) for 12–14 hours, where indicated. IL-2 concentrations in the culture supernatants were determined by ELISA (Biolegend). PD-1-mediated inhibition was calculated by comparing the amount of IL-2 in conditions with and without PD-1 activation.

[0117] Flow cytometry analysis Cultured and primary cells were stained with the indicated antibodies or soluble chimeric proteins. Splenocytes were stimulated with LPS (1 μg / ml, Escherichia coli O111:B4, Merck) or poly(I:C) (20 μg / ml, Merck) for 16–18 h before staining. Antibodies against mouse CD8α (5H10), CD28 (37.51), and PD-L1 (1-111A, MIH5) were purchased from Thermo Fisher Scientific. Antibodies against mouse PD-1 (RMP1-30), MHCII (M5 / 114.15.2), B220 (RA3-6B2), F4 / 80 (BM8), CD80 (16-10A1), CD86 (GL-1), DYKDDDDK tag (L5), CD19 (6D5), CD3e (17A2), CD4 (RM4-5), CD8α (53-6.7), CD11b (M1 / 70), CD11c (N418), and CD317 (927) were purchased from Biolegend. Streptavidin-phycoerythrin (PE) and streptavidin-allophycocyanin (APC) were purchased from Biolegend. Isotype control antibodies for rat IgG2a (RTK2758), rat IgG2b (RTK4530), and hamster IgG (HTK888) were purchased from Biolegend. Unless otherwise noted, 30 μg / ml biotinylated 1-111A was used for detection of mouse PD-L1. To prepare soluble chimeric proteins, cDNA fragments encoding the extracellular regions of mouse PD-1 (amino acids 1-167), human PD-1 (amino acids 1-167), mouse CD28 (amino acids 1-149), and mouse CTLA-4 (amino acids 1-162) were amplified by PCR. The five-stranded coiled-coil domain of cartilage oligomeric matrix protein (CMP) containing a DYKDDDDK tag (Terskikh, A. V et al., Proc. Natl. Acad. Sci. USA 94, 1663-8(1997)) was fused to the C-terminus of each protein, and the chimeric cDNA was cloned into an expression vector modified from pEBMulti-Neo (Wako).The plasmids were transfected into 293T or Plat-E cells using Avalanche-Omni transfection reagent (EZ Biosystems), and the culture supernatants were collected 48 and 96 hours later. The supernatants were diluted and used for staining. Binding of the chimeric protein to the cells was detected with an anti-DYKDDDDK tag antibody (L5). Data were acquired using Gallios (Beckman Coulter) and analyzed using FlowJo (Tree Star).

[0118] Co-immunoprecipitation The DYKDDDDK tag was fused to the C-terminus of PD-L1 and PD-L1Y56A. The SHSLQKYYITGEAEGFPATA tag (hER tag), recognized by a rabbit polyclonal antibody against human ERα protein (HC-20, Santa Cruz Biotechnology), was fused to the C-terminus of CD80, CD80L107E, and CD86. IIAdL1 cells expressing the tagged proteins in the desired combinations were washed extensively with PBS and then cross-linked with BS, a water-soluble, non-cleavable, membrane-impermeable cross-linker. 3 The cells were treated with 1 mM Tris-CoA (1 mM, Thermo Fisher Scientific) for 30 minutes. After the crosslinking reaction was stopped with 25 mM Tris, the cells were lysed in lysis buffer containing 1% NP-40. DYKDDDDK-tagged proteins were immunoprecipitated with anti-FLAG M2 agarose beads (Merk), separated by SDS-PAGE under reducing conditions, and transferred to a PVDF membrane. DYKDDDDK-tagged and hER-tagged proteins were detected with anti-DYKDDDDK (L5) and HC-20 antibodies, followed by IRDye800-anti-rat IgG (H+L) and IRDye680-anti-rabbit IgG (H+L) antibodies (LI-COR Biosciences). Fluorescent signals on the membrane were detected using an Odyssey imaging system (LI-COR Biosciences).

[0119] Generation of BM-DCs BM cells were collected from the femur and tibia of mice and cultured in RPMI 1640 medium supplemented with 10% (v / v) FBS, 0.5 mM monothioglycerol, 2 mM L-alanyl-L-glutamine dipeptide, 100 U / ml penicillin, 100 μg / ml streptomycin, and 20 ng / ml recombinant mouse GM-CSF (Biolegend). Two-thirds of the medium was replaced with fresh medium on day 4. Nonadherent cells were collected on day 6 and analyzed by CD11c immunofluorescence for mouse immunofluorescence experiments. + cells, CD86 for in vitro co-culture experiments + Cells were sorted using the BD iMag Cell Separation System (BD Biosciences). Isolated cells were stimulated with LPS (1 μg / ml). For dendritic cell vaccination of E.G7-bearing mice, 100 μg / ml of OVA protein (low endotoxin, Wako) was added. Nonadherent cells were collected after 16–18 h and used for further experiments.

[0120] Preparation of TG-MΦ Naive mice were intraperitoneally injected with 2 ml of 3% Brewer's thioglycollate medium (BD Biosciences). Four days later, peritoneal exudate cells were collected and seeded onto tissue culture plates at 37°C for 2 h. Floating cells were extensively washed away, and firmly adherent cells were stimulated with LPS (1 μg / ml) for 16–18 h and used for flow cytometry analysis. For in vitro coculture assays, F4 / 80 + Cells were purified by cell sorter (>95% purity).

[0121] Isolation of splenic DCs Spleens were treated with collagenase (1 mg / ml, Wako) for 20 minutes at 37°C, pulverized, and a single-cell suspension was prepared. After erythrocyte lysis, whole splenocytes were stimulated with LPS (1 μg / ml) for 16–18 hours. Cells were harvested and stained with bio-CD11c antibody followed by PE-streptavidin for CD11c immunoreactivity. + Cells were enriched with anti-PE magnetic particles and the BD iMag Cell Separation System. - F4 / 80- CD3 - CD11c + CD8α + CD11b - cells (CD8α + DC) or B220 - F4 / 80 - CD3 - CD11c + CD8α - CD11b + cells (CD11b + DCs) were sorted using a cell sorter (CD8α + DCs; >85% purity and CD11b + DCs; >90% purity), were used as antigen-presenting cells for in vitro coculture assays.

[0122] Protein structure The structures of mouse PD-L1 and CD80 were predicted by SWISS-MODEL (https: / / swissmodel.expasy.org / ) based on the published structures of human PD-L1 (PDB ID: 4Z18) and the IgV domain of mouse CD80 (PDB ID: 4RWH), respectively. The structures of human CD80 (PDB ID: 1DR9) and CD86 (PDB ID: 1NCN) were solved by UCSF Chimera software.

[0123] Induction of T cell responses to OVA OVA protein (100 μg) emulsified in Freund's complete adjuvant (BD Biosciences) was inoculated into the footpad of naive mice. One week after inoculation, 5 × 10 OVA-derived ... 5 The cells were treated with OVA protein (100 μg / ml), OVA 257-264 Peptide (100 nM) and OVA 323-339 The cells were stimulated with the peptide (3 μM) for 48 hours, and the concentrations of IL-2 and IFN-γ in the culture supernatant were determined by ELISA (Biolegend).

[0124] Tumor immunotherapy On day 0, mice were injected with 5 × 10 5 E.G7 lymphoma cells were subcutaneously administered. On days 5 and 12, OVA protein (100 μg) mixed with poly(I:C) (50 μg) in PBS was subcutaneously inoculated near the tumor. Alternatively, 4.5 × 10 OVA protein-pulsed cells were administered on days 3 and 10. 5 LPS-activated BM-DCs were subcutaneously inoculated near the tumor. Tumor size was measured every 3 days with a vernier caliper. Tumor volume was calculated using the following formula: 1 / 2 × (minor diameter) 2 ×(longitude).

[0125] Experimental autoimmune encephalomyelitis (EAE) EAE was induced according to a previously published protocol (Stromnes, IM & Goverman, Nat. Protoc. 1, 1810-1819 (2006)). Briefly, on day 0, mice were injected with MOG (Mouse Overgrowth Antibody) emulsified in Freund's incomplete adjuvant (BD Biosciences) supplemented with Mycobacterium tuberculosis H37RA (200 μg, BD Biosciences). 35-55 The peptide (200 μg, MEVGWYRSPFSRVVHLYRNGK, >95% purity, Eurofins) was administered subcutaneously as an antigen. On days 0 and 2, 200 ng of pertussis toxin (List Biological Laboratories) was administered intraperitoneally. Clinical scores were blindly assessed daily as follows: 0, no clinical signs; 1, limping; 2, hind limb weakness; 3, hind limb paralysis; 4, hind and forelimb paralysis; and 5, moribund. To assess the in vitro recall response, 1 × 10 mice were administered 7 days after antigen inoculation. 6 Splenocytes from 100 mice were cultured in 5 or 50 μg / ml of MOG. 35-55 The cells were stimulated with the peptide for 66 hours, and the IL-17A concentration in the culture supernatant was determined by ELISA (Thermo Fisher Scientific).

[0126] statistics An unpaired two-tailed Student's t-test was used for comparisons between two groups. One-way or two-way analysis of variance with post-hoc tests was used for multiple comparisons. p<0.05 was considered statistically significant. Error bars in the figures indicate s.e.m. Figures 1-12, 17, 19, 21-28, and 30-34 show representative data from three or more independent experiments.

[0127] [result] Study 1: cis-PD-L1 / CD80 interaction disrupts PD-L1 / PD-1 binding and subsequent PD-1-mediated inhibition We used the CRISPR / Cas9 system to knock out the PD-L1 gene in IIA1.6 cells, which express PD-L1 but not PD-L2, to generate IIAdL1 cells. We overexpressed PD-L1, PD-L2, CD80, and CD86 in various combinations in IIAdL1 cells and assessed their PD-1 binding ability by staining with soluble mouse PD-1 extracellular domain (PD-1-EC) protein (Figure 1). PD-1-EC binding to PD-L1 was strongly blocked by coexpression of CD80, but not by coexpression of CD86, in IIAdL1-PD-L1 cells (Figure 1, top panel). These results indicate that CD80 interacts with PD-L1 on the same antigen-presenting cells and that this cis-PD-L1 / CD80 interaction disrupts PD-L1 / PD-1 binding. In contrast, the binding of PD-1-EC to PD-L2 was not affected by coexpression of CD80 and CD86 in IIAdL1-PD-L2 cells (Figure 1, bottom panel). Addition of IIAdL1-CD80 cells to IIAdL1-PD-L1 cells (which allows trans-PD-L1 / CD80 interaction, but not cis-PD-L1 / CD80 interaction) did not affect the binding of PD-1-EC to IIAdL1-PD-L1 cells (Figure 2). This result indicates that CD80 must be expressed on the same cells as PD-L1 to disrupt PD-L1 / PD-1 binding. Notably, the cis-PD-L1 / CD80 interaction did not disrupt CD80 / CD28 binding or CD80 / CTLA-4 binding (data not shown). When adjacent proteins on the cell surface were crosslinked with a cell-impermeable crosslinker, CD80, but not CD86, coimmunoprecipitated with PD-L1 (Figure 3). This result further supports the cis interaction between PD-L1 and CD80.

[0128] To investigate the functional significance of this, we used IIAdL1 cells overexpressing various combinations of PD-L1, PD-L2, CD80, and CD86 as antigen-presenting cells to stimulate DO11.10 T cells. IL-2 production from antigen-stimulated DO11.10 T cells was strongly inhibited when PD-L1 or PD-L2 was expressed on the antigen-presenting cells (Figure 4, top panel). The suppression of IL-2 production was also observed in PD-1 knockout DO11.10 (DO11.10-Pdcd1). - / - ) This was not observed in T cells, indicating that PD-L1- or PD-L2-mediated inhibition was dependent on PD-1. Coexpression of CD80 strongly suppressed the PD-1-mediated inhibitory effect induced by IIAdL1-PD-L1 cells but not by IIAdL1-PD-L2 cells (Figure 4, middle panel). Coexpression of CD86 did not suppress the PD-1-mediated inhibitory effect induced by IIAdL1-PD-L1 cells or IIAdL1-PD-L2 cells (Figure 4, bottom panel). Thus, CD80 interacts with PD-L1 in cis and prevents PD-L1 from inducing PD-1 function during T cell activation.

[0129] Next, we investigated the dose-dependent effects of cis-PD-L1 / CD80 interaction. CD80 and PD-L1 were expressed alone or simultaneously on IIAdL1 cells at four different expression levels (25 combinations in total) (Figure 5). As expected, coexpression of significant amounts of CD80 with PD-L1 reduced the binding of PD-1-EC to PD-L1 in a manner consistent with the amount of CD80 expression (Figures 6 and 7). Antigen-presenting cells capable of binding PD-1-EC were able to induce PD-1 function and suppress IL-2 production upon activation of DO11.10 T cells (Figure 8). These results indicate that the relative amounts of PD-L1 and CD80 determine the ability of antigen-presenting cells to bind and induce PD-1 function on T cells.

[0130] Test 2: Primary DCs disrupt PD-1-mediated inhibition of T cell activation To investigate the cis-PD-L1 / CD80 interaction in vivo, activated CD8α + and CD11b+ We analyzed splenic DCs and thioglycollate-induced peritoneal MΦs (TG-MΦs) (Fig. 9). Interestingly, despite the common high levels of PD-L1 expression (Fig. 9, second row), the PD-1-EC binding strength of the three cell populations differed significantly. TG-MΦs bound strongly to PD-1-ECs, whereas CD8α + DCs bind weakly to PD-1-ECs and express CD11b + DCs hardly bound to PD-1-EC (Fig. 9, top panel). + and CD11b + The expression level of CD80 on DCs was much higher than that on TG-MΦ (Fig. 9, bottom panel).

[0131] Next, CD80 knockout (C57BL / 6N-Cd80 - / - ) mice and analyzed the same cell populations as above (Figure 10). - / - Mouse-derived CD8α + and CD11b + DCs bound to PD-1-EC with similar strength to that of TG-MΦ (Fig. 10, top panel). This result suggests that in wild-type mice, CD8α expression is suppressed due to the strong CD80 expression. + and CD11b + The expression level of CD80 on wild-type TG-MΦ was not high enough to interfere with PD-L1 / PD-1 binding, indicating that DCs were defective in binding to PD-1-ECs. CD80 deficiency did not alter the binding strength of TG-MΦ to PD-1-ECs.

[0132] Next, CD8α + and CD11b + DCs and TG-MΦ were used as antigen-presenting cells, and pOVA was 257-264 / H2-K b T lymphoma cells (BW-OT-I cells) or pOVA responding to 323-339 / IA bTG-MΦ stimulated T lymphoma cells (BW-OT-II cells) that respond to CD8α (Fig. 11). Consistent with its strong PD-1-EC binding ability, TG-MΦ induced the inhibitory function of PD-1 in both BW-OT-I and BW-OT-II cells (Fig. 11, top). On the other hand, CD8α + and CD11b + DCs failed to induce PD-1-mediated inhibitory effects on the activation of BW-OT-I and BW-OT-II cells (Figure 11, middle and bottom panels). Figure 12 shows the results of further analysis of the data shown in Figure 11. These results indicate that DCs express sufficient amounts of CD80 to interfere with PD-L1 / PD-1 binding and limit the function of PD-1 during T-DC interaction-mediated T cell activation.

[0133] Study 3: PD-L1 and CD80 mutants without cis-PD-L1 / CD80 interaction We attempted to isolate PD-L1 mutants that retained other functions but were unable to bind to CD80, and CD80 mutants that were unable to bind to PD-L1. By analyzing the binding characteristics of chimeric molecules in which the IgV and IgC domains of PD-L1, PD-L2, CD80, and CD86 were exchanged, we elucidated the involvement of the IgV domains of PD-L1 and CD80 in their interactions (Figures 13 and 14). Random mutations were introduced into the IgV domain of PD-L1 using error-prone PCR, and these mutants were overexpressed in IIAdL1-CD80 cells. Cell sorting identified cells that retained PD-1-EC binding ability. PD-L1 mutants isolated from these cells had high mutation rates at V54, Y56, and E58 (Figure 15, top panel). Prediction of the 3D structure of mouse PD-L1 indicated that these amino acid residues are located in the C chain of PD-L1. This C chain is close to the PD-1 interaction surface (Lin, DY-W. et al. Proc. Natl. Acad. Sci. USA 105, 3011-6(2008)) (Figure 16, left). These results suggest that the PD-L1 / CD80 interaction surface partially overlaps with the PD-L1 / PD-1 interaction surface. A series of point mutations at Y56 were examined (Figure 15, bottom). The Y56A mutant bound equally well to PD-1-EC in the presence and absence of CD80 (Figure 17, left), and expressed levels comparable to wild-type PD-L1 in IIAdL1 cells (Figure 17, right). Therefore, PD-L1Y56A was used for further analysis.

[0134] CD28 binds to the AGFCC'C' surface of CD80 (Ikemizu, S. et al., Immunity 12, 51-60(2000); Stamper, CC et al. Nature 410, 608-11(2001); Evans, EJ et al. Nat. Immunol. 6, 271-279(2005)), and CD80 can bind to CD28 in the presence of PD-L1 (data not shown). Therefore, we focused on the DEB surface of CD80. CD80 (Ikemizu, S. et al., previously cited) and CD86 (Zhang, X., Schwartz, J.-CD, Almo, SC & Nathenson, SG, Proc. Natl. Acad. Sci. USA 100, By comparing the hydrophobicity of CD80 with that of wild-type CD80 (2586-91 (2003)), we identified a unique hydrophobic patch on the DEB surface of CD80 (Figure 18). Therefore, we mutated amino acid residues in this hydrophobic patch. CD80 with the L96E or L107E mutation failed to interfere with PD-L1 / PD-1 binding (Figure 19, top panel and Figure 20). The CD80L107E mutant did not interfere with PD-L1 / PD-1 binding, bound CD28 and CTLA-4 to the same extent as wild-type CD80, and was expressed at similar levels to wild-type CD80 in IIAdL1 cells (Figure 19). Therefore, CD80L107E was used for further analysis. Furthermore, co-immunoprecipitation with CD80 using PD-L1Y56A was significantly reduced, and co-immunoprecipitation with CD80 using CD80L107E was almost abolished (Figure 21).

[0135] Next, we investigated the functions of these mutants. When DO11.10 T cells were stimulated with IIAdL1 cells expressing PD-L1Y56A as antigen-presenting cells, PD-L1Y56A induced PD-1-mediated inhibitory effects comparable to those of wild-type PD-L1 in the absence of CD80 (Figure 22, upper panel and Figure 23). Furthermore, PD-L1Y56A induced PD-1 function even in the presence of CD80 (Figure 22, middle panel and Figure 24). These results indicate that PD-L1Y56A can circumvent the effects of CD80 and induce PD-1 function. When CD80L107E was expressed on IIAdL1 cells in the absence of PD-L1, CD80L107E enhanced antigen-stimulated IL-2 production from DO11.10 T cells to a similar extent as wild-type CD80. Furthermore, PD-L1 was able to induce PD-1 function in the presence of CD80L107E (Figure 22, bottom panel and Figure 24), indicating that CD80L107E cannot inhibit PD-L1-mediated induction of PD-1 function.

[0136] Study 4: Attenuation of PD-1-mediated inhibition by cis-PD-L1 / CD80 interaction in humans To examine whether the attenuation of PD-1-mediated inhibitory effects by cis-PD-L1 / CD80 interaction also occurs in humans, we performed similar experiments using human orthologs. Similar to the murine molecule, human CD80 bound to human PD-L1 and attenuated human PD-L1 binding to human PD-1, whereas human CD86 did not (Figures 25 and 26). Human CD80, but not human CD86, co-immunoprecipitated with human PD-L1 (Figure 27). Furthermore, the cis interaction between human CD80 and human PD-L1 prevented human PD-L1 from inducing the inhibitory function of human PD-1 (Figure 28). We also successfully isolated human CD80 and human PD-L1 mutants that lacked the cis-PD-L1 / CD80 interaction and were unable to restrict PD-1 function (Figures 29 and 30). The human PD-L1 N63D / G119S mutant induced PD-1-mediated inhibition to a similar extent as wild-type human PD-L1 in the absence of human CD80 (Figure 30, top panel), and also induced PD-1-mediated inhibition in the presence of human CD80 (Figure 30, middle panel).Furthermore, the human CD80 L104E mutant was unable to prevent PD-L1 from inducing PD-1-mediated inhibition (Figure 30, bottom panel).

[0137] Study 5: Attenuation of T cell responses to immunogens, tumor cells, and autologous tissue in the absence of cis-PD-L1 / CD80 interactions To investigate the biological significance of cis-PD-L1 / CD80 interaction in vivo, we generated PD-L1Y56A and CD80L107E knock-in mice using the CRISPR / Cas9 system (C57BL / 6N-Cd274 Y56A Mice and C57BL / 6N-Cd80 L107E Mouse). C57BL / 6N-Cd274 Y56A Mice and C57BL / 6N-Cd80 L107E The mice were born normal and developed normally without any obvious abnormalities. + DC, CD11b +DCs and GM-CSF-induced bone marrow-derived dendritic cells (BM-DCs) showed much stronger PD-1-EC binding ability than those cells from wild-type mice, despite comparable expression levels of CD80, PD-L1, and PD-L2 (Figure 31). Expression levels of CD86 and MHCII on these cells were also comparable. Activated DCs from wild-type mice induced little inhibitory effect of PD-1 on the activation of BW-OT-I and BW-OT-II cells, whereas C57BL / 6N-Cd274 cells did not. Y56A Mice and C57BL / 6N-Cd80 L107E Activated DCs from mice induced an inhibitory effect (FIGS. 32-34).

[0138] Next, we investigated the effect of cis-PD-L1 / CD80 interaction on immune responses to exogenous antigens in vivo. Y56A Mice and C57BL / 6N-Cd80 L107E Mice were inoculated with OVA protein emulsified in complete Freund's adjuvant (CFA) as an antigen, and T cells in the draining lymph nodes were restimulated one week later. IFN-γ and IL-2 production from T cells stimulated with OVA protein and MHC1- and MHCII-restricted OVA peptides was significantly lower in both knockin mice than in wild-type mice (Figure 35). These results indicate that in wild-type mice, the function of PD-1 in inducing immune responses to foreign antigens is limited by cis-PD-L1 / CD80 interaction.

[0139] Next, we investigated the involvement of cis-PD-L1 / CD80 interaction in cancer immunotherapy. OVA-expressing E.G7 lymphoma cells (Moore, MW, Carbone, FR & Bevan, MJ, Cell 54, 777-85 (1988)) were transplanted into mice, and OVA protein and poly(I:C) were vaccinated (Figure 36). Tumor growth was strongly suppressed in wild-type mice by vaccination, but not in C57BL / 6N-Cd274 mice. Y56AMice and C57BL / 6N-Cd80 L107E No significant therapeutic effect was observed in C57BL / 6N-Cd274 mice (Figures 37 and 38). Furthermore, to directly examine the role of cis-PD-L1 / CD80 interactions on antigen-presenting cells, we used dendritic cell vaccines. BM-DCs derived from wild-type and knock-in mice were pulsed with OVA and administered to wild-type mice transplanted with E.G7 cells to induce an immune response (Figure 39). Consistent with the effects of protein vaccination, dendritic cell vaccine therapy using BM-DCs derived from wild-type mice induced a potent anti-tumor immune response, whereas C57BL / 6N-Cd274 mice induced a potent anti-tumor immune response (Figure 39). Y56A Mice and C57BL / 6N-Cd80 L107E When mouse-derived BM-DCs were used, they did not induce anti-tumor immune responses (Figures 40 and 41). These results indicate that the restriction of PD-1 function by cis-PD-L1 / CD80 interaction is crucial for the induction of anti-tumor immune responses.

[0140] Furthermore, the role of cis-PD-L1 / CD80 interaction in autoimmunity was investigated using experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (C57BL / 6N-Cd274). Y56A Mice and C57BL / 6N-Cd80 L107E In mice, the symptoms of EAE induced by immunization with the MOG peptide were significantly milder than those in wild-type mice (Figure 42). Similarly, upon restimulation with the MOG peptide, C57BL / 6N-Cd274 mice immunized with the MOG peptide showed significantly milder EAE symptoms than wild-type mice (Figure 42). Y56A Mice and C57BL / 6N-Cd80 L107E IL-17 production from splenocytes derived from C57BL / 6N-Cd274 mice was significantly reduced (Figure 43). These findings suggest that the inhibitory effect of PD-1 is prevented by cis-PD-L1 / CD80 interaction in wild-type mice during EAE development, whereas C57BL / 6N-Cd274 mice were significantly reduced in IL-17 production from splenocytes derived from C57BL / 6N-Cd274 mice. Y56A Mice and C57BL / 6N-Cd80 L107E In mice, PD-L1 is released from CD80, allowing it to bind to PD-1 and exert an inhibitory effect, suppressing the symptoms of EAE.

[0141] Tests using anti-CD80 antibodies Materials and Methods Soluble proteins A cDNA fragment encoding the extracellular region of mouse CD80 was amplified by PCR and fused to the Fc region of human IgG1 (hIgG1Fc). The chimeric cDNA was cloned into an expression vector modified from pEFBOSneo. The plasmid was transfected into Plat-E cells using Avalanche-Omni Transfection Reagent (EZ Biosystems), and the culture supernatant was collected 48 hours later. Mouse CD80-hIgG1Fc was purified using Protein G (GE Healthcare).

[0142] mouse C57BL / 6N-Cd80 - / - Mice were housed under specific pathogen-free conditions in an environmentally controlled clean room. All mouse protocols were approved by the Animal Care and Use Committee of Tokushima University.

[0143] Generation of anti-mouse CD80 monoclonal antibody (mAb) C57BL / 6N-Cd80 mice vaccinated with mouse CD80-hIgG1Fc protein as an antigen to induce an immune response - / - Mouse lymph node cells were fused with SP2 / o cells by electrical cell fusion (LF301, BEX). Culture supernatants of hybridoma clones were tested for their ability to dissociate the cis-PD-L1 / CD80 interaction.

[0144] Generation of anti-human CD80 mAb Lymph node cells from BALB / c mice vaccinated with human CD80-hIgG1Fc protein (R&D Systems) were fused with SP2 / o cells using Sendai virus envelope (GenomONE-CF, Ishihara Sangyo Kaisha). The culture supernatants of the hybridoma clones were tested for their ability to dissociate the cis-PD-L1 / CD80 interaction.

[0145] DO11.10 Stimulation of T cells and TCR-reconstituted cells DO11.10 T cells (5×10 4 IIAdL1 cells (1 × 10 cells / well) were pulsed with the indicated amount of OVA323-339 peptide (ISQAVHAAHAEINEAGR, >95% purity, Sigma-Aldrich Japan or eurofins genomics). 4 The cells were stimulated with 5 μg / ml of anti-mouse CD80 antibodies (TKMG48, 1G10 (BD Bioscience), RM80 (Biorad)), mouse IgG1 isotype control (MOPC-21, Biolegend), anti-human CD80 antibody (TKMF5), and mouse IgG2a isotype control (MOPC-173, Biolegend) as described below. IL-2 concentrations in the culture supernatants were measured by ELISA (Biolegend).

[0146] EAE MOG emulsified with Freund's incomplete adjuvant (BD Biosciences) containing Mycobacterium tuberculosis H37RA (200 μg, BD Biosciences) 35-55 Mice were immunized by subcutaneous inoculation with the peptide (200 μg, MEVGWYRSPFSRVVHLYRNGK, >95% purity, Eurofins) (day 0). Pertussis toxin (200 ng, List Biological Laboratories) was administered intraperitoneally on days 0 and 2. As shown in Figures 47 and 48, 500 μg of anti-mouse CD80 antibody (TKMG48) or isotype control mouse IgG1 (MOPC21, Bio X cell) was administered intraperitoneally. Clinical scores were blindly assessed daily as follows: 0, no clinical signs; 1, limp; 2, hind limb weakness; 3, hind limb paralysis; 4, hind limb and forelimb paralysis; and 5, moribund.

[0147] Binding of antibodies to the CD80 molecule The binding of TKMG48 and 16-10A1 to DOdKO cells transfected with mouse CD80, a chimeric molecule in which the IgV and IgC domains of CD80 and CD86 were swapped (see Figure 13), or a mouse CD80 mutant (harboring L96E and / or L107E mutations) that lacks the ability to bind to mouse PD-L1 was assessed by flow cytometry. The binding of TKMF5 and 2D10 to DOdKO cells transfected with human CD80 or a human CD80 mutant (harboring I92E and L104E mutations) that lacks the ability to bind to human PD-L1 was assessed by flow cytometry.

[0148] Binding evaluation of anti-mouse CD80 antibody (TKMG48) and anti-human CD80 antibody (TKMF5) The binding affinity of an anti-mouse CD80 antibody (TKMG48) to mouse CD80 soluble protein and an anti-human CD80 antibody (TKMF5) to human CD80 soluble protein was measured using biolayer interferometry. Briefly, cDNA fragments encoding the extracellular domains of mouse or human CD80 were amplified by PCR. A strep tag was added to the C-terminus of CD80. The chimeric cDNA was cloned into an expression vector modified from pEBMulti-Neo (Wako). The plasmid was transfected into Plat-E cells using Avalanche-Omni Transfection Reagent (EZ Biosystems), and the culture supernatant was collected 48 hours later. Monomeric CD80 (with a strep tag) was immobilized on a streptavidin-coated biosensor chip (Pall ForteBio), and binding of various concentrations of anti-CD80 antibody was monitored using BLItz (Pall ForteBio). The chip was washed with PBS, and the dissociation rate was analyzed. The association rate constant (ka), dissociation rate constant (kd) and dissociation constant (KD) were calculated using BLItz Pro software.

[0149] [result] Immunosuppression with anti-CD80 antibodies We investigated whether anti-mouse CD80 antibodies could enable mouse PD-1-ECs to bind to mouse PD-L1 when both mouse CD80 and mouse PD-L1 were present on the same antigen-presenting cells. Mouse PD-L1 and CD80 were introduced into DO11.10 T cells (DOdKO cells) lacking the PD-1 and PD-L1 genes to generate DOdKO-mPD-L1 / mCD80 cells. DOdKO-mPD-L1 / mCD80 cells were pretreated with 10 μg / ml of each anti-mouse CD80 antibody for 20 minutes at 37°C and then stained with mouse PD-1-ECs. Mouse PD-1-ECs bound to DOdKO-mPD-L1 / mCD80 cells pretreated with TKMG48, but not with other antibodies (Figure 44). The effect of TKMG48 pretreatment was evaluated using the mean fluorescence intensity of mouse PD-1-EC binding, calculated using the following formula: (TKMG48 treatment group - control group) / (control IgG group - control group) The binding of mouse PD-1-EC to DOdKO-mPD-L1 / mCD80 cells pretreated with TKMG48 was approximately 10.1-fold higher than that of cells pretreated with control IgG. These results indicate that TKMG48 mediated the binding of mouse PD-1-EC to mouse PD-L1 when both mouse CD80 and mouse PD-L1 were present on the same antigen-presenting cells.

[0150] The effects of anti-mouse CD80 antibodies on the interactions between CD80 and CD28, CD80 and CTLA-4, and CD80 and PD-L1 were investigated. DOdKO-CD80 cells were pretreated with 10 μg / ml of each anti-mouse CD80 antibody shown in Figure 45 for 20 minutes at 4°C, followed by staining with mouse CD28-EC, CTLA-4-EC, or PD-L1-EC. The results are shown in Figure 45. TKMG48 strongly inhibited the binding of PD-L1-EC to CD80. TKMG48 weakly inhibited the binding of CD28-EC to CD80, but not the binding of CTLA-4-EC to CD80. 16-10A1 strongly inhibited the interactions between CD80 and CD28-EC and CD80 and PD-L1-EC, and partially inhibited the interaction between CD80 and CTLA-4-EC. 1G10 strongly inhibited the interactions between CD80 and CD28-EC and CD80 and PD-L1-EC, but not between CD80 and CTLA-4-EC.RM80 did not inhibit the binding of CD28-EC, CTLA-4-EC, or PD-L1-EC to CD80.

[0151] We investigated whether TKMG48 could enable PD-L1 to induce the inhibitory effects of PD-1 when CD80 and PD-L1 are present on the same antigen-presenting cells. DO11.10 T cells expressing or lacking PD-1 were transfected with OVA. 323-339 Antibody to mouse CD80 or control IgG (5 μg / ml) was added as indicated in Figure 46. The results are shown in Figure 46. IL-2 production by antigen-stimulated DO11.10 T cells was inhibited by PD-1 when TKMG48 was added, but not when 16-10A1 or IG10 was added. These results indicate that TKMG48 dissociates the interaction between cis-PD-L1 and CD80, allowing PD-L1 to elicit the inhibitory effects of PD-1.

[0152] The therapeutic effect of TKMG48 on experimental autoimmune encephalomyelitis (EAE) was evaluated. C57BL / 6N mice were treated with MOG emulsified with CFA. 35-55 The peptide was inoculated as an antigen (day 0). 500 μg of TKMG48 or an isotype control (mouse IgG1) was intraperitoneally administered to mice on days 1, 3, 5, 7, 10, and 13. The results are shown in Figure 47. TKMG48 treatment significantly alleviated the symptoms of EAE. Similarly, C57BL / 6N mice and C57BL / 6N-Cd274 mice showed a significant improvement in EAE symptoms. - / - MOG emulsified in CFA was administered to mice (PD-L1 knockout mice). 35-55 The peptide was inoculated as an antigen (day 0). 500 μg of TKMG48 or an isotype control (mouse IgG1) was intraperitoneally administered to mice on days 1, 3, 5, 7, 10, 13, and 16. The results are shown in Figure 48. TKMG48 treatment significantly alleviated EAE symptoms in a PD-L1-dependent manner. These results indicate that activation of PD-1 signaling by dissociating the interaction between cis-PD-L1 and CD80 is effective in treating autoimmune diseases.

[0153] We investigated whether the anti-human CD80 antibody TKMF5 could mediate the binding of human PD-1-ECs to human PD-L1 when both human CD80 and human PD-L1 were present on the same antigen-presenting cells. DOdKO-hPD-L1 / hCD80 cells were pretreated with TKMF5 for 20 minutes at 37°C and then stained with human PD-1-ECs. Binding of PD-1-ECs to DOdKO-hPD-L1 / hCD80 cells increased in a TKMF5 concentration-dependent manner; for example, in the presence of 30 μg / mL TKMF5, the percentage of cells that bound PD-1-ECs was approximately 50%. These results demonstrate that TKMF5 mediates the binding of human PD-1-ECs to human PD-L1 when both human CD80 and human PD-L1 are present on the same antigen-presenting cells.

[0154] The effect of TKMF5 on the interactions between CD80 and CD28, CD80 and CTLA-4, and CD80 and PD-L1 was investigated. DO11.10 T cells lacking the PD-1 gene (DOdPD cells) were transfected with human CD80 to generate DOdPD-hCD80 cells. DOdPD-hCD80 cells were pretreated with 10 μg / ml TKMF5 for 20 minutes at 4°C and then stained with human CD28-EC, human CTLA-4-EC, or human PD-L1-EC. In TKMF5-pretreated DOdPD-hCD80 cells, the binding intensity of human CD80 to human CD28-EC and human CTLA-4-EC was over 80% of that in untreated controls, whereas the binding intensity of human CD80 to human PD-1-EC was less than 10% of that in untreated controls. These results indicate that TKMF5 inhibits the binding of human PD-L1-ECs to human CD80, but does not inhibit the binding of human CD28-ECs and human CTLA-4-ECs to human CD80.

[0155] We investigated whether TKMF5 could enable PD-L1 to elicit the inhibitory effects of PD-1 when CD80 and PD-L1 are present on the same antigen-presenting cells. OVA peptide-pulsed IIAdL1-hCD80 / hCD86 or IIAdL1-hCD80 / hCD86 / hPD-L1 cells were cultured with DO11.10 cells expressing or lacking PD-1 in the presence of TKMF5 or isotype control IgG (10 μg / ml). The results are shown in Figure 49. Addition of TKMF5 induced inhibition of TCR-induced IL-2 production in PD-1-expressing DO11.10 T cells, but not in PD-1-deficient cells. These results indicate that TKMF5 dissociates the interaction between cis-hPD-L1 and hCD80 on antigen-presenting cells, allowing PD-L1 to elicit the inhibitory effects of PD-1.

[0156] Figure 50 shows the results of flow cytometry analysis of the binding of TKMG48 (left) and 16-10A1 (right) to DOdKO cells transfected with mouse CD80, chimeric molecules in which the IgV and IgC domains of CD80 and CD86 were swapped (see Figure 13) (top), or mouse CD80 mutants (containing the L96E and / or L107E mutations) that lack the ability to bind to mouse PD-L1 (bottom). TKMG48 and 16-10A1 recognized the IgV region of CD80 (top). TKMG48 showed low binding to the CD80 mutant lacking the ability to bind to PD-L1 (left). In contrast, 16-10A1 showed binding to the CD80 mutant lacking the ability to bind to PD-L1 similar to that of wild-type CD80 (right).

[0157] Figure 51 shows the results of flow cytometry analysis of the binding of TKMF5 (left) and 2D10 (right) to DOdKO cells transfected with human CD80 or a human CD80 mutant (containing I92E and L104E mutations) that lacks the ability to bind to human PD-L1. The binding of TKMF5 to the human CD80 mutant that lacks the ability to bind to human PD-L1 was significantly reduced (left). In contrast, 2D10 showed binding to the human CD80 mutant that lacks the ability to bind to human PD-L1 similar to that of wild-type human CD80 (right).

[0158] The binding affinity of anti-mouse CD80 antibody (TKMG48) and anti-human CD80 antibody (TKMF5) to mouse CD80 and human CD80 was evaluated. The equilibrium dissociation constant (KD value) of TKMG48 was 1.232 nmol / L, and the equilibrium dissociation constant (KD value) of TKMF5 was 11.44 nmol / L. [Industrial Applicability]

[0159] A substance that promotes the binding of PD-L1 to PD-1, selected from anti-CD80 antibodies and anti-PD-L1 antibodies of the present invention, is useful as an immunosuppressant, or for the prevention and / or treatment of autoimmune diseases, allergic diseases, or graft-versus-host disease.

Claims

1. An immunosuppressant comprising a substance that promotes the binding of PD-L1 selected from an anti-CD80 antibody and an anti-PD-L1 antibody to PD-1.

2. The immunosuppressant of claim 1, which promotes binding between PD-1 and PD-L1, which is present on the same cells as CD80.

3. The immunosuppressant according to claim 1 or 2, wherein the substance is an anti-CD80 antibody.

4. The immunosuppressant of claim 3, wherein the anti-CD80 antibody inhibits the cis binding of CD80 and PD-L1.

5. The immunosuppressant described in claim 3 or 4, wherein the anti-CD80 antibody binds to a region containing amino acids corresponding to isoleucine at position 92 and / or leucine at position 104 of human CD80 having the amino acid sequence of SEQ ID NO: 1, or a region containing amino acids corresponding to leucine at position 96 and / or leucine at position 107 of mouse CD80 having the amino acid sequence of SEQ ID NO:

2.

6. The immunosuppressant according to any one of claims 3 to 5, wherein the anti-CD80 antibody does not substantially inhibit the binding of CD80 to CTLA-4.

7. the anti-CD80 antibody comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:11; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14 The immunosuppressant according to any one of claims 3 to 6, comprising:

8. the anti-CD80 antibody comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and The immunosuppressant of any one of claims 3 to 6, comprising a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

22.

9. The immunosuppressant of claim 7, wherein the anti-CD80 antibody comprises a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:

8.

10. The immunosuppressant of claim 8, wherein the anti-CD80 antibody comprises a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:

16.

11. The immunosuppressant of claim 3, wherein the anti-CD80 antibody competes with the antibody of any one of claims 7 to 10 for binding to CD80.

12. The immunosuppressant of claim 1 or 2, wherein the substance is an anti-PD-L1 antibody.

13. The immunosuppressant of claim 12, wherein the anti-PD-L1 antibody inhibits the cis binding of PD-L1 and CD80.

14. The immunosuppressant of claim 12 or 13, wherein the anti-PD-L1 antibody binds to a region containing amino acids corresponding to asparagine at position 63 and / or glycine at position 119 of human PD-L1, which has the amino acid sequence of SEQ ID NO: 3, or a region containing amino acids corresponding to valine at position 54, tyrosine at position 56, and / or glutamic acid at position 58 of mouse PD-L1, which has the amino acid sequence of SEQ ID NO:

4.

15. The immunosuppressant according to any one of claims 1 to 14 for use in the prevention and / or treatment of autoimmune diseases, allergic diseases or graft-versus-host disease.

16. A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; and An anti-CD80 antibody comprising a light chain variable region including a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

14.

17. A heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and An anti-CD80 antibody comprising a light chain variable region including a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 20, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 21, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

22.

18. 17. The anti-CD80 antibody of claim 16, comprising a heavy chain variable region comprising an amino acid sequence having 90% or greater identity to the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising an amino acid sequence having 90% or greater identity to the amino acid sequence of SEQ ID NO:

8.

19. 18. The anti-CD80 antibody of claim 17, comprising a heavy chain variable region comprising an amino acid sequence having 90% or greater identity to the amino acid sequence of SEQ ID NO: 15 and a light chain variable region comprising an amino acid sequence having 90% or greater identity to the amino acid sequence of SEQ ID NO:

16.

20. An anti-CD80 antibody that competes with the antibody of any one of claims 16 to 19 for binding to CD80.

21. An immunosuppressant comprising the antibody according to any one of claims 16 to 20 as an active ingredient.

22. 21. A preventive and / or therapeutic agent for autoimmune diseases, allergic diseases, or graft-versus-host disease, comprising the antibody according to any one of claims 16 to 20 as an active ingredient.

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