Antibodies that bind to the IL-18 receptor, IL-18 receptor activators, and their use

Abstract

This invention relates to the technology of antibodies, and more specifically, to antibodies that bind to the IL-18 receptor, IL-18 receptor activators, and their use. The invention provides antibodies that specifically bind to IL-18R1 and IL-18RAP with high affinity, and further provides bispecific antibodies that can efficiently bind both IL-18R1 and IL-18RAP simultaneously. These bispecific antibodies are novel IL-18 receptor activators that can activate the intracellular signaling pathway mediated by IL-18R1 / IL-18RAP, exert biological functions similar to IL-18 both in vitro and in vivo, and possess broad immunostimulatory effects and remarkable antitumor activity. Furthermore, they possess controllable activating activity, excellent molecular stability, and in vivo pharmacokinetic characteristics, which are advantageous for fully exhibiting antitumor efficacy in vivo, and offer prospects for a wide range of antitumor clinical applications.

Description

[Technical Field]

[0001] This invention relates to the technology of antibodies, and more particularly to antibodies that bind to the IL-18 receptor, IL-18 receptor activators, and the use of the same. [Background technology]

[0002] IL-18 is an important pro-inflammatory factor belonging to the IL-1 family (Okamura H, and et al. Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature. 1995 Nov 2;378(6552):88-91. doi:10.1038 / 378088a0. PMID:7477296.), and is mainly secreted by macrophages, dendritic cells, and epithelial cells. IL-18 exists as an inactive precursor within cells, and when inflammation occurs, the precursor peptide is cleaved by easpase-1 in the inflammasome, and its active form is rapidly released. Active IL-18 then binds to its heterodimer receptor (IL-18R1 / Rap), mediating the MyD88-NFκB signaling pathway. One of the main functions of IL-18 is to stimulate NK cells and antigen-paired lymphocytes to produce gamma interferon (Nakanishi k, Yoshimoto T, Tsutsui H, Okamura H. Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol. 2001; 19: 423-74. doi: 10.1146 / annurev.immunol. 19.1.423. PMID: 11244043).

[0003] IL-18, due to its immune-activating function, was once applied to tumor immunotherapy, but its Phase II clinical trial ended in failure (Tarhini AA, and et al. A phase 2, randomized study of SB-485232, rhIL-18, in patients with previously untreated metastatic melanoma. Cancer. 2009 Feb 15;115(4):859-68.doi:10.1002 / cncr.24100.PMID:19140204). Studies have shown that after multiple treatments with IL-18 in clinical patients, the drug's effectiveness gradually weakens, and large amounts of IL-18-binding protein (IL-18BP) are induced and produced in the body (Robertson MJ, and et al. A dose-escalation study of recombinant human interleukin-18 using two different schedules of administration in patients with cancer. Clin Cancer Res. 2008 Jun 1;14(11):3462-9. doi:10.1158 / 1078-0432.CCR-07-4740.PMID:18519778;PMCID:PMC8603216). IL-18BP is both a decoy receptor and a natural inhibitor of IL-18, and it inhibits its binding to the receptor by binding to IL-18 with extremely high affinity (1.1 pM). The presence of IL-18BP counteracts the immune-activating function of IL-18, leading to treatment failure.

[0004] On June 24, 2020, a research paper by the Aaron Ring research group at Yale University was published in the journal Nature (Zhou T, and et al. IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy. Nature. 2020 Jul;583(7817):609-614. doi:10.1038 / s41586-020-2422-6. Epub 2020 Jun 24. PMID:32581358;PMCID:PMC7381364.). It reported that by modifying IL-18 through directional evolution into DR-18 (decoy-resistant IL-18), which maintains signal transduction function without binding to the decoy receptor, it showed good antitumor effects. Preclinical trials and drug manufacturing for DR-18 have already been completed, and an Investigational New Drug (IND) application has been submitted. Currently, the project is conducting Phase 1 / 2 clinical trials. However, although DR-18 avoids antagonism by IL-18BP and shows clear antitumor effects and good safety characteristics, it is a cytokine analog, and therefore still has problems such as a short half-life in the body, difficulty in sustained activation activity, poor stability, and a complex manufacturing process. [Overview of the Initiative]

[0005] The present invention provides an antibody that binds to the IL-18 receptor, an IL-18 receptor activator, and the use of the same.

[0006] To develop antibodies that bind to the IL-18 receptor and IL-18 receptor activators, the present invention first obtained multiple anti-IL-18R1 and IL-18RAP single-domain antibodies possessing multiple unique sequences using camel immunotherapy library technology. These single-domain antibodies exhibit high affinity for IL-18R1 and IL-18RAP and can efficiently bind to the IL-18 receptor. Furthermore, by constructing a dual-target tandem-conjugated combinatorial library using the anti-IL-18R1 and IL-18RAP single-domain antibodies and combining ELISA with functional activity screening, multiple tandem-conjugated bispecific antibodies of aIL-18R1 and aIL-18RAP, which have activation activity similar to that of IL-18, were obtained. Based on this, the present invention humanizes the single-domain antibodies and bispecific antibodies, and the humanized aIL-18R1 and aIL-18RAP bispecific antibodies can still maintain good cell activation activity. The bispecific antibodies aIL-18R1 and aIL-18RAP have the biological function of stimulating and activating the proliferation of PBMC cells (human peripheral blood mononuclear cells) in vitro, thereby producing IFNγ. They can significantly promote immune reconstitution of PBMC immune cells in mice, exert antitumor efficacy in a dose-dependent manner, and, compared to IL-18, the bispecific antibodies have mild and sustained activating activity and possess superior safety potential.

[0007] Specifically, this invention proposes the following technical solutions. Firstly, the present invention provides an antibody or antigen-binding fragment that binds to the IL-18 receptor, wherein the antibody or antigen-binding fragment binds to IL-18R1 or IL-18RAP and includes a heavy chain variable region, the complementarity-determining region of the heavy chain variable region in the antibody or antigen-binding fragment that binds to IL-18R1 is one of the following (1) to (5), and the complementarity-determining region of the heavy chain variable region in the antibody or antigen-binding fragment that binds to IL-18RAP is one of the following (6) to (8). (1) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown by SEQ ID NO. 27, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 27. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is shown by SEQ ID NO. 28, or is a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 28. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is represented by SEQ ID NO. 29 or 51, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 29 or 51. (2) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown by SEQ ID NO. 30, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 30. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is the one shown by SEQ ID NO.31, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO.31. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is represented by SEQ ID NO. 32 or 52, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 32 or 52. (3) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown by SEQ ID NO.33, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO.33. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is represented by SEQ ID NO. 34 or 53, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 34 or 53. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is either the sequence shown by SEQ ID NO. 35, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 35. (4) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown by SEQ ID NO.36, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO.36. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is represented by SEQ ID NO. 37 or 54, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 37 or 54. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is represented by SEQ ID NO. 38 or 55, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 38 or 55. (5) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown by SEQ ID NO.39, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO.39. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is shown by SEQ ID NO. 40, or is a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 40. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is either the sequence shown by SEQ ID NO. 41, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 41. (6) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown by SEQ ID NO. 42, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 42. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is represented by SEQ ID NO. 43 or 56, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 43 or 56. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is either the sequence shown by SEQ ID NO. 44, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 44. (7) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown by SEQ ID NO. 45, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 45. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is represented by SEQ ID NO. 46 or 57, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 46 or 57. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is either the sequence shown by SEQ ID NO. 47, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 47. (8) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown by SEQ ID NO. 48, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 48. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is represented by SEQ ID NO. 49 or 58, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 49 or 58. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is either the sequence shown by SEQ ID NO. 50, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO. 50.

[0008] In the complementarity determination region of the heavy chain variable region described above, the sequences indicated by SEQ ID NO. 27-50 are the CDR sequences of the camel-derived antibody, and the sequences indicated by SEQ ID NO. 51-58 are the CDR sequences of the humanized antibody. In this invention, experiments have verified that the CDR sequences of the camel-derived antibody and the humanized antibody have high affinity for IL-18R1 and IL-18RAP, and can efficiently bind to IL-18R1 and IL-18RAP. Regarding the aforementioned sequence variants, the present invention obtained sequence variants that bind to IL-18R1 and IL-18RAP with high affinity by mutating the above CDR sequence using histidine scanning mutation. These sequence variants have at least 80% homology to the sequence before mutation (original sequence).

[0009] In (1) to (8) above, the sequence variant having at least 80% homology is obtained by substituting any one amino acid in the original sequence with histidine, and it is preferable that the original sequence is one of the sequences indicated by SEQ ID NO. 27 to 58. For example, in the case of the sequence variant corresponding to CDR1 described in (1) above, the original sequence is the sequence indicated by SEQ ID NO. 27. That is, the sequence variant is obtained by substituting one amino acid in the sequence indicated by SEQ ID NO. 27 with histidine. This variant still has a high affinity for IL-18R1. Preferably, in the amino acid sequences of CDR1, CDR2, and CDR3 of each antibody or its antigen-binding fragment, only one amino acid is substituted with histidine. For example, for the complementarity-determining regions (CDR1, CDR2, and CDR3) shown in (1), the histidine mutation in the sequence variant occurs at only one amino acid site in one amino acid sequence selected from SEQ ID NO. 27-29 and 51.

[0010] The sequence variants obtained by the above histidine scanning mutations are illustrative examples only. Those skilled in the art can modify the above CDR by "conservative sequence modifications" to obtain different sequence variants. "Conservative sequence modifications" refer to amino acid sequence modifications that do not significantly affect or change the binding properties of the antibody or its antigen-binding fragment, and include amino acid substitutions, additions, or deletions. For example, modifications can be introduced into the sequences shown in SEQ ID NO. 27-58 by common techniques in the art, such as site-directed mutagenesis or PCR-mediated mutagenesis. Here, a conservative amino acid substitution includes replacing an amino acid residue with an amino acid residue having a similar side chain or with other amino acid residues. Families of amino acid residues having similar side chains have already been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0011] Preferably, the amino acid sequence of the heavy chain variable region in the antibody or antigen-binding fragment that binds to IL-18R1 is one of the sequences indicated by SEQ ID NO. 7 to 11, one of the sequences indicated by SEQ ID NO. 20 to 23, or a sequence variant having at least 80% homology to the sequence indicated by either SEQ ID NO. 7 to 11 or SEQ ID NO. 20 to 23. Here, the sequences shown by SEQ ID NOs. 7 to 11 are the sequences of the heavy chain variable regions of the camel-derived antibody, and the sequences shown by SEQ ID NOs. 20 to 23 are the sequences of the heavy chain variable regions of the humanized antibody. In the present invention, it has been verified by experiments that the heavy chain variable regions of the above camel-derived antibody and the humanized antibody have a high affinity for IL-18R1 and can efficiently bind to IL-18R1.

[0012] The amino acid sequence of the heavy chain variable region in the antibody or its antigen-binding fragment that binds to the IL-18RAP is preferably any one shown by SEQ ID NOs. 12 to 14, or any one shown by SEQ ID NOs. 24 to 26, or a sequence variant having at least 80% homology with the sequence shown by any one of SEQ ID NOs. 12 to 14 and SEQ ID NOs. 24 to 26. Here, the sequences shown by SEQ ID NOs. 12 to 14 are the sequences of the heavy chain variable regions of the camel-derived antibody, and the sequences shown by SEQ ID NOs. 24 to 26 are the sequences of the heavy chain variable regions of the humanized antibody. In the present invention, it has been verified by experiments that the heavy chain variable regions of the above camel-derived antibody and the humanized antibody have a high affinity for IL-18RAP and can efficiently bind to IL-18RAP.

[0013] In the above heavy chain variable region, the sequence variant having at least 80% homology is obtained by substituting one amino acid in CDR1, CDR2, and CDR3 of the sequence shown by any one of SEQ ID NOs. 7 to 14 and SEQ ID NOs. 20 to 26 with histidine, that is, only one amino acid site in only one CDR of CDR1, CDR2, and CDR3 of each heavy chain variable region is mutated to histidine.

[0014] In the case of sequences indicated by SEQ ID NO. 7 or 20, it is preferable that sequence variants having at least 80% homology are obtained by substituting the amino acid at positions 27, 28, 29, 30, 31, 32, 34, 35, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, or 110 of the sequence indicated by SEQ ID NO. 7 or 20 with histidine. For sequences indicated by SEQ ID NO. 10 or 23, sequence variants having at least 80% homology are sequence variants obtained by substituting histidine at the amino acid positions 31, 32, 33, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 107, 108, 109, 110, 111, 112, or 29 of the sequence indicated by SEQ ID NO. 10 or 23, or SEQ ID It is preferable that the sequence variant is obtained by substituting the amino acids at positions 52 and 59, 52 and 105, 52 and 107, 59 and 99, 59 and 105, 59 and 107, 59 and 109, 99 and 107, 105 and 109, 52 and 112, 59 and 112, 101 and 112, or 105 and 112 of the sequence indicated by NO.10 or 23 with histidine. In the case of sequences indicated by SEQ ID NO. 13 or 25, it is preferable that sequence variants having at least 80% homology are sequence variants obtained by substituting the amino acid at positions 161, 162, 163, 164, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 228, 229, 230, 231, 232, 233, 234, or 235 of the sequence indicated by SEQ ID NO. 13 or 25 with histidine.

[0015] The array variants obtained by performing the above histidine scanning mutations are merely illustrative, and those skilled in the art can modify the above heavy chain variable region by techniques such as "conserved sequence modification" to obtain other different array variants.

[0016] In the present invention, the antibody or its antigen-binding fragment is a single-domain antibody, a mini-antibody, a bispecific antibody, a multispecific antibody, a monoclonal antibody, a single-chain antibody, Fab, Fab′, F(ab′)2, Fd, Fv, scFv, BsFv, dsFv or (dsFv)2. In some embodiments of the present invention, the antibody is a single-domain antibody. In some embodiments of the present invention, the antibody is a bispecific antibody containing the single-domain antibody. In the present invention, the antibody or its antigen-binding fragment is a camel-derived antibody, a humanized antibody, a mouse-derived antibody, a rabbit-derived antibody, or a chimeric antibody. In some embodiments of the present invention, the antibody is a camel-derived antibody. In some embodiments of the present invention, the antibody is a humanized antibody.

[0017] In the present invention, in addition to containing the above heavy chain variable region, the antibody or its antigen-binding fragment that binds to the IL-18 receptor can be linked with one or more selected from protein tags, enzyme cleavage sites, and linker peptides at the N-terminus and / or C-terminus of the heavy chain variable region, and the ligation of these sequences does not significantly affect the function of the antibody or its antigen-binding fragment. The sequences of the protein tag, enzyme cleavage site, and linker peptide are not particularly limited. Examples of the protein tag include, but are not limited to, tags such as His-tag, Gst, MBP, Strep, and Flag. The enzyme cleavage site may be an enzyme for cleaving the protein tag, and examples include, but are not limited to, TEV protease and sortase. The linker peptide may be a short-chain peptide containing a large amount of glycine and serine.

[0018] Secondly, the present invention provides a bispecific antibody comprising an antibody that binds to the IL-18 receptor or an antigen-binding fragment thereof. Specifically, the bispecific antibody may contain an antibody or its antigen-binding fragment that binds to IL-18R1, and an antibody or its antigen-binding fragment that binds to IL-18RAP, or an antibody or its antigen-binding fragment that binds to IL-18RAP not described in the present invention (for example, an antibody against IL-18RAP known in the prior art). Similarly, the bispecific antibody may contain an antibody or antigen-binding fragment thereof that binds to IL-18RAP, and an antibody or antigen-binding fragment thereof that binds to IL-18R1 not described in the present invention (for example, an antibody against IL-18R1 known in the prior art).

[0019] Furthermore, the bispecific antibody may contain an antibody that binds to IL-18R1 or IL-18RAP or its antigen-binding fragment, as well as an antibody having other functions (non-IL-18 receptor binding function). In some embodiments of the present invention, the bispecific antibody comprises one or more antibodies or antigen-binding fragments thereof that bind to the aforementioned IL-18R1, and one or more antibodies or antigen-binding fragments thereof that bind to the aforementioned IL-18RAP.

[0020] Preferably, in the bispecific antibody, The amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown by SEQ ID NO. 7, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown by SEQ ID NO. 12, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown by SEQ ID NO. 8, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown by SEQ ID NO. 14, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown by SEQ ID NO. 8, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown by SEQ ID NO. 13, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown by SEQ ID NO. 9, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown by SEQ ID NO. 14, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown by SEQ ID NO. 9, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown by SEQ ID NO. 13, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown by SEQ ID NO. 10, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown by SEQ ID NO. 13, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown by SEQ ID NO. 11, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown by SEQ ID NO. 12, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown as SEQ ID NO. 20, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown as SEQ ID NO. 24, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown by SEQ ID NO. 21, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown by SEQ ID NO. 26, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown by SEQ ID NO. 21, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown by SEQ ID NO. 25, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 22, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 26, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 22, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 25, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown by SEQ ID NO. 23, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown by SEQ ID NO. 25, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown by SEQ ID NO. 11, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown by SEQ ID NO. 24, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is obtained by substituting the amino acids at positions 27, 28, 29, 30, 31, 32, 34, 35, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, or 110 of the sequence shown in SEQ ID NO. 20 with histidine, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 24, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is obtained by substituting histidine at the 31st, 32nd, 33rd, 48th, 49th, 50th, 51st, 52nd, 53rd, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 107th, 108th, 109th, 110th, 111th, 112th, or 29th position of the sequence shown in SEQ ID NO. 23, or SEQ ID This is obtained by substituting the amino acids at positions 52 and 59, 52 and 105, 52 and 107, 59 and 99, 59 and 105, 59 and 107, 59 and 109, 99 and 107, 105 and 109, 52 and 112, 59 and 112, 101 and 112, or 105 and 112 with histidine, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 25, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 23, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 25, and obtained by substituting the amino acid at positions 161, 162, 163, 164, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 228, 229, 230, 231, 232, 233, 234, or 235 with histidine.

[0021] In the aforementioned bispecific antibody, it is preferable that the antibody or its antigen-binding fragment that binds to IL-18R1 is linked (tandem) to the antibody or its antigen-binding fragment that binds to IL-18RAP. The aforementioned linkage may be in a direction in which the antibody or antigen-binding fragment that binds to IL-18R1 is located at the N-terminus and the antibody or antigen-binding fragment that binds to IL-18RAP is located at the C-terminus, or in a direction in which the antibody or antigen-binding fragment that binds to IL-18RAP is located at the N-terminus and the antibody or antigen-binding fragment that binds to IL-18R1 is located at the C-terminus.

[0022] It is preferable that the antibody or antigen-binding fragment that binds to IL-18R1 and the antibody or antigen-binding fragment that binds to IL-18RAP are linked by a linker. The linker may be any flexible peptide or linker peptide, for example, a G4S or other similar linker peptide or flexible peptide consisting of amino acids of a certain length. In some embodiments of the present invention, a G4S flexible linker (SEQ ID NO. 15), a GS flexible linker, a (G4S)2 flexible linker (SEQ ID NO. 17), a (G4S)3 flexible linker (SEQ ID NO. 18), a (G4S)4 flexible linker (SEQ ID NO. 19), GGGGSG (SEQ ID NO. 66), GGGSGG (SEQ ID NO. 67), GGSGGG (SEQ ID NO. 68), GSGGGG (SEQ ID NO. 69), GGSGSG (SEQ ID NO. 70), or GSGSGG (SEQ ID NO. 71) is used to link an antibody or its antigen-binding fragment that binds to IL-18R1 with an antibody or its antigen-binding fragment that binds to IL-18RAP.

[0023] The bispecific antibody preferably contains one of the following structural domains (1) to (21): (1) The amino acid sequence is the sequence shown by SEQ ID NO. 7, the linker, and the sequence shown by SEQ ID NO. 12, in order from the N-terminus to the C-terminus. (2) The amino acid sequence is the sequence indicated by SEQ ID NO. 8, the linker, and the sequence indicated by SEQ ID NO. 14, in order from the N-terminus to the C-terminus. (3) The amino acid sequence is the sequence indicated by SEQ ID NO. 8, the linker, and the sequence indicated by SEQ ID NO. 13, in order from the N-terminus to the C-terminus. (4) The amino acid sequence is the sequence indicated by SEQ ID NO. 9, the linker, and the sequence indicated by SEQ ID NO. 14, in order from the N-terminus to the C-terminus. (5) The amino acid sequence is the sequence indicated by SEQ ID NO. 9, the linker, and the sequence indicated by SEQ ID NO. 13, in order from the N-terminus to the C-terminus. (6) The amino acid sequence is the sequence shown by SEQ ID NO. 10, the linker, and the sequence shown by SEQ ID NO. 13, in order from the N-terminus to the C-terminus. (7) The amino acid sequence is the sequence shown by SEQ ID NO. 12, the linker, and the sequence shown by SEQ ID NO. 7, in order from the N-terminus to the C-terminus. (8) The amino acid sequence is the sequence shown by SEQ ID NO. 13, the linker, and the sequence shown by SEQ ID NO. 9, in order from the N-terminus to the C-terminus. (9) The amino acid sequence is the sequence shown by SEQ ID NO. 13, the linker, and the sequence shown by SEQ ID NO. 10, in order from the N-terminus to the C-terminus. (10) The amino acid sequence is the sequence shown by SEQ ID NO. 12, the linker, and the sequence shown by SEQ ID NO. 11, in order from the N-terminus to the C-terminus. (11) The amino acid sequence is the sequence shown by SEQ ID NO. 20, the linker, and the sequence shown by SEQ ID NO. 24, in order from the N-terminus to the C-terminus. (12) The amino acid sequence is the sequence shown by SEQ ID NO. 21, the linker, and the sequence shown by SEQ ID NO. 26, in order from the N-terminus to the C-terminus. (13) The amino acid sequence is the sequence shown by SEQ ID NO. 21, the linker, and the sequence shown by SEQ ID NO. 25, in order from the N-terminus to the C-terminus. (14) The amino acid sequence is the sequence shown by SEQ ID NO. 22, the linker, and the sequence shown by SEQ ID NO. 26, in order from the N-terminus to the C-terminus. (15) The amino acid sequence is the sequence shown by SEQ ID NO. 22, the linker, and the sequence shown by SEQ ID NO. 25, in order from the N-terminus to the C-terminus. (16) The amino acid sequence is the sequence shown by SEQ ID NO. 23, the linker, and the sequence shown by SEQ ID NO. 25, in order from the N-terminus to the C-terminus. (17) The amino acid sequence is the sequence shown by SEQ ID NO. 24, the linker, and the sequence shown by SEQ ID NO. 20, in order from the N-terminus to the C-terminus. (18) The amino acid sequence is the sequence shown by SEQ ID NO. 25, the linker, and the sequence shown by SEQ ID NO. 22, in order from the N-terminus to the C-terminus. (19) The amino acid sequence is the sequence shown by SEQ ID NO. 25, the linker, and the sequence shown by SEQ ID NO. 23, in order from the N-terminus to the C-terminus. (20) The amino acid sequence is the sequence shown by SEQ ID NO. 24, the linker, and the sequence shown by SEQ ID NO. 11, in order from the N-terminus to the C-terminus. (21) The amino acid sequence is the sequence shown by SEQ ID NO. 20, the linker, and the sequence shown by SEQ ID NO. 24, in order from the N-terminus to the C-terminus, and is obtained by substituting the amino acid at positions 27, 28, 29, 30, 31, 32, 34, 35, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, or 110 with histidine. (22) A sequence obtained by substituting the amino acids at positions 31, 32, 33, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 107, 108, 109, 110, 111, 112 or 29 of the sequence shown by SEQ ID NO. 23 in order from the N-terminus to the C-terminus with histidine, or SEQ ID This is the linker sequence, indicated by SEQ ID NO. 25, obtained by substituting histidine amino acids at positions 52 and 59, 52 and 105, 52 and 107, 59 and 99, 59 and 105, 59 and 107, 59 and 109, 99 and 107, 105 and 109, 52 and 112, 59 and 112, 101 and 112, or 105 and 112 in the sequence indicated by NO. 23. (23) The amino acid sequence obtained by substituting histidine at the 161st, 162nd, 163rd, 164th, 179th, 180th, 181st, 182nd, 183rd, 184th, 185th, 186th, 187th, 188th, 189th, 190th, 191st, 192nd, 193rd, 194th, 195th, 228th, 229th, 230th, 231st, 232nd, 233rd, 234th, or 235th amino acid in the sequence indicated by SEQ ID NO. 23, the linker, and the sequence indicated by SEQ ID NO. 25, in order from the N-terminus to the C-terminus.

[0024] The above (1) to (10) are camel-derived bispecific antibodies, and (11) to (23) are humanized bispecific antibodies. Here, the humanized bispecific antibody (21) is a variety of mutants obtained by histidine scanning mutation in the humanized bispecific antibody (11). The humanized bispecific antibody (22) is a variety of mutants obtained by histidine scanning mutation in the humanized bispecific antibody (16). The humanized bispecific antibody (23) is a variety of mutants obtained by histidine scanning mutation in the humanized bispecific antibody (16). In this invention, experiments have verified that all of these mutants maintain good immune cell activation activity. In the present invention, there are no particular restrictions on the structure of the bispecific antibody, as long as it includes an antibody that binds to IL-18R1 or an antigen-binding fragment thereof, and an antibody that binds to IL-18RAP or an antigen-binding fragment thereof. The bispecific antibody can be designed to any currently known structure.

[0025] In some embodiments of the present invention, the bispecific antibody further comprises one or more selected from KappaL, CH1, CH2, CH3, hinge region, and KIH structure. The structures of the selectable bispecific antibodies are shown in Figure 4.

[0026] Third, the present invention provides a fusion protein comprising an antibody that binds to the IL-18 receptor or an antigen-binding fragment thereof, or the bispecific antibody. The fusion protein is preferably obtained by fusing an antibody that binds to the IL-18 receptor or an antigen-binding fragment thereof, or by fusing the bispecific antibody with another protein.

[0027] For example, to facilitate manufacturing, improve molecular stability, and enhance pharmacokinetic characteristics in the body, a fusion protein can be formed by fusing an antibody that binds to the IL-18 receptor, or its antigen-binding fragment, or the bispecific antibody with a protein such as Fab, Fc, or HAS. Furthermore, to further enhance the efficacy of the drug, a fusion protein can be formed by fusing an antibody that binds to the IL-18 receptor, or its antigen-binding fragment, or the bispecific antibody with other cytokines or antibodies (e.g., aPD-L1, aPD-1, etc.). The other proteins mentioned above preferably include one or more selected from protein tags, Fab, Fc, HSA, cytokines, and antibodies.

[0028] In some embodiments of the present invention, a fusion protein is obtained by fusing an antibody that binds to the IL-18 receptor or its antigen-binding fragment, or the bispecific antibody, with Fc. Fc may be a humanized Fc-tag (human Fc tag, hFc, SEQ ID NO.1), a mouse-derived Fc-tag (mouse Fc tag, mFc, SEQ ID NO.2), or a camel-derived Fc-tag (alpaca Fc tag, aFc, SEQ ID NO.3).

[0029] Fourth, the present invention provides a bifunctional protein that includes an antibody or its antigen-binding fragment that binds to the IL-18 receptor, or the bispecific antibody, and further includes a target protein, antibody, or cytokine. Preferably, the antibody is an immune checkpoint antibody and / or a tumor target antibody.

[0030] Fifth, the present invention provides nucleic acid molecules encoding an antibody that binds to the IL-18 receptor or an antigen-binding fragment thereof, or the bispecific antibody, or the fusion protein, or the bifunctional protein. Based on the amino acid sequences of antibodies or their antigen-binding fragments, bispecific antibodies, fusion proteins, and bifunctional proteins that bind to the IL-18 receptor, the nucleotide sequences of nucleic acid molecules encoding antibodies or their antigen-binding fragments, bispecific antibodies, fusion proteins, and bifunctional proteins that bind to the IL-18 receptor can be obtained. Due to codon degeneracy, the nucleotide sequences of nucleic acid molecules encoding the same protein are not unique, and all nucleic acid molecules that can generate antibodies or their antigen-binding fragments, bispecific antibodies, fusion proteins, and bifunctional proteins that bind to the IL-18 receptor by encoding are all included within the scope of protection of the present invention.

[0031] Sixth, the present invention provides a biomaterial comprising an expression cassette, vector, or host cell containing the nucleic acid molecule. The expression cassette is a recombinant nucleic acid molecule obtained by linking the nucleic acid molecule with a regulatory element used for transcription, translation, etc. Examples of the aforementioned vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, and artificial chromosome vectors. The host cells include microbial cells (e.g., E. coli, yeast, etc.), insect cells, or other animal cells.

[0032] Seventh, in the present invention, recombinant host cells are obtained by introducing a nucleic acid molecule encoding an antibody that binds to the IL-18 receptor or an antigen-binding fragment thereof, or the bispecific antibody, or the fusion protein, or the bifunctional protein into host cells, and The recombinant host cells are cultured and isolated to obtain an antibody that binds to the IL-18 receptor or its antigen-binding fragment, the bispecific antibody, or the fusion protein. A method for producing an antibody that binds to the IL-18 receptor, or an antigen-binding fragment thereof, or the bispecific antibody, or the fusion protein, or the bifunctional protein is provided. The host cells mentioned above include, but are not limited to, microbial cells (e.g., E. coli, yeast, etc.), insect cells, or other animal cells.

[0033] VIII. The present invention provides the use of an antibody or antigen-binding fragment thereof that binds to the IL-18 receptor, or the bispecific antibody, or the fusion protein, or the bifunctional protein, or the nucleic acid molecule, or the biological material, which is any of the following uses. (1) Use in the manufacture of antitumor drugs (2) Use in the manufacture of drugs that enhance the immune response of living organisms (3) Use in the manufacture of drugs to activate the IL-18 signaling pathway (4) Use in the manufacture of drugs to stimulate the secretion of INFγ and / or GZMB (5) Use in the manufacture of IL-18 receptor activators (6) Use in the manufacture of drugs to prevent or treat diseases associated with the IL-18 receptor (7) Use in the manufacture of drugs to measure the content of IL-18 receptors

[0034] In (1) above, the tumors include, but are not limited to, all tumors treatable by immunotherapy, including, gastrointestinal tumors (e.g., hepatobiliary cancer, gastric cancer, pancreatic cancer, intestinal cancer, etc.), respiratory tumors (e.g., squamous cell carcinoma of the lung, non-small cell lung cancer, small cell lung cancer, etc.), urinary tract tumors (e.g., bladder cancer, urothelial carcinoma, etc.), nervous system tumors, reproductive system tumors (e.g., ovarian cancer, breast cancer, cervical cancer, etc.), and melanoma.

[0035] Ninth, the present invention provides an IL-18 receptor activator comprising an antibody that binds to the IL-18 receptor or an antigen-binding fragment thereof, or the bispecific antibody, or the fusion protein, or the bifunctional protein.

[0036] Tenth, the present invention provides a pharmaceutical composition comprising an antibody that binds to the IL-18 receptor or an antigen-binding fragment thereof, or the bispecific antibody, or the fusion protein, or the bifunctional protein.

[0037] Eleventh, the present invention provides a detection reagent comprising an antibody that binds to the IL-18 receptor or an antigen-binding fragment thereof, or the bispecific antibody, or the fusion protein, or the bifunctional protein.

[0038] In the present invention, it is preferable that the IL-18 is human IL-18. [Effects of the Invention]

[0039] The beneficial effects of the present invention are as follows: The present invention provides antibodies that specifically bind to IL-18R1 and antibodies that specifically bind to IL-18RAP, and these antibodies have high affinity for IL-18R1 and IL-18RAP. Furthermore, by combining the above antibodies in a tandem manner, it is possible to form a bispecific antibody (IL-18Rα / β heterodimer complex) that can efficiently bind to both IL-18R1 and IL-18RAP simultaneously. These bispecific antibodies can activate the intracellular signaling pathway mediated by IL-18R1 / IL-18RAP, activate the IL-18 signaling pathway, exert biological functions similar to IL-18 both in vitro and in vivo, have a relatively broad immunoenhancing effect, stimulate the proliferation and activation of NK cells and T cells, and stimulate the secretion of cytokines such as IFNγ, and have significantly suppressed tumor growth in in vivo experiments with animal models, suggesting a wide range of potential anti-tumor clinical applications.

[0040] The bispecific antibody provided in this application is a completely novel IL-18 receptor activator that avoids the negative feedback phenomenon of IL-18 pathway activation due to the antagonistic effect of IL-18BP, and possesses controllable activating activity, superior molecular stability, and in vivo pharmacokinetic characteristics, making it advantageous for fully exerting the antitumor effect of the drug in vivo. [Brief explanation of the drawing]

[0041] To more clearly explain the technical concepts of the present invention or the prior art, the drawings necessary for describing the examples or the prior art will be briefly described below. Clearly, the drawings shown below are some examples of the present invention, and those skilled in the art can obtain other drawings based on these without requiring any creative work.

[0042] Figure 1 is a schematic diagram showing the construction of a prokaryotic expression vector for the forward / reverse bispecific antibody against human IL-18R1 / RAP in Example 3 of the present invention, where a is Anti-IL-18R1 VHH, B is Anti-IL-18RAP VHH, and L is GGGGS Linker. Figure 2 shows the results of the rhIL-18 activity detection using the reporter gene system in Example 5 of the present invention, indicating that rhIL-18-1 and rhIL-18-2 were obtained from two independent, repeated experiments. Figure 3 is a result diagram showing the effect of different linker lengths on the activity of Wio-02-01 in Example 6 of the present invention. Figure 4 is a schematic diagram showing the structural form of the anti-human IL-18R1 / RAP bispecific antibody in Example 7 of the present invention. Figure 5 is a schematic diagram showing the structure of the recombinant anti-human IL-18R1 or IL-18RAP camel-derived heavy chain single-domain antibody in Example 8 of the present invention. Figure 6 is a schematic diagram showing the structure of the anti-human IL-18R1 / RAP bispecific antibody Fc fusion protein in Example 9 of the present invention, where a is the forward-structured humanized bispecific antibody and B is the reverse-structured humanized bispecific antibody. Figure 7 shows the binding activity of the anti-human IL-18R1 / RAP bispecific antibody to IL-18R1 and IL-18RAP on the cell membrane surface in Example 12 of the present invention. Figure a shows the binding activity of the anti-human IL-18R1 / RAP bispecific antibody to IL-18R1 on the cell membrane surface, with antibody concentrations of 30, 10, 3.33, 1.11, 0.37, 0.12, 0.041, and 0.014 μg / ml. Figure b shows the binding activity of the anti-human IL-18R1 / RAP bispecific antibody to IL-18RAP on the cell membrane surface, with antibody concentrations of 30, 10, 3.33, 1.11, 0.37, 0.12, 0.041, and 0.014 μg / ml. Figure 8 shows the results of the activity evaluation of the humanized anti-IL-18R1 / RAP bispecific antibody by PBMC activation experiment in Example 12 of the present invention, where a is the result of the activity evaluation of the humanized anti-IL-18R1 / RAP bispecific antibody (with hFc linked) by PBMC activation experiment (+IL-12), and b is the result of the activity evaluation of the humanized anti-IL-18R1 / RAP bispecific antibody (with histidine tag linked) by PBMC activation experiment (+IL-12). Figure c is a result diagram (+IL-15) showing the activity evaluation of a humanized anti-IL-18R1 / RAP bispecific antibody (with hFc linked) by PBMC activation experiment, figure d is a result diagram (+IL-15) showing the activity evaluation of a humanized anti-IL-18R1 / RAP bispecific antibody (with histidine tag linked) by PBMC activation experiment, and figure e is a result diagram (+IL-15) showing the activity evaluation of a humanized anti-IL-18R1 / RAP bispecific antibody (mutant) by PBMC activation experiment. Figure 9 is a list of histidine scanning mutants of hzWA23 in Example 13 of the present invention, where a is the hzWA23 hzR1-F9 mutant and b is the hzWA23 hzRAP-C10 mutant. Figure 10 shows the results of the analysis of the binding activity of hzWA23Hm38 and hzWA23Hm47 to both IL-18R1 and IL-18RAP, performed by the ELISA method in Example 14 of the present invention, with concentrations of hzWA23Hm38, hzWA23Hm47 of 10, 3.33, 1.11, 0.370, 0.123, 4.11E-2, 1.37E-2, 4.57E-3, 1.52E-3, 5.08E-4, 1.69E-4, and 5.65E-05 ng / ml. Figure 11 shows the results of the FACS method for the binding activity of the hzWA23 mutant to IL-18R on the cell surface in Example 14 of the present invention. Figures a-d show the binding activity of the hzR1-F9 mutant to the human IL-18R1 antigen on the cell surface, with antibody concentrations of 3, 1, and 0.33 μg / ml. Figures e and f show the binding activity of the hzRAP-C10 mutant to the human IL-18RAP antigen on the cell surface, with antibody concentrations of 5, 1, and 0.2 μg / ml. The results indicate that, under current experimental conditions, after mutating most of the sites to histidine, all mutants can bind to antigens on the cell membrane surface. Figure 12 shows the results of the reporter gene system's activation activity of the hzWA23 mutant in Example 15 of the present invention. The antibody concentrations were 8, 1.6, and 0.32 ng / ml. Under current experimental conditions, after most of the sites were mutated to histidine, all mutants maintained good activation activity. Figure 13 shows the results of the PBMC activation experiment in Example 15 of the present invention, where the antibody concentrations were 200, 20, and 2 ng / ml. Under current experimental conditions, after most of the sites were mutated to histidine, all mutants maintained good activation activity against PBMCs. Figure 14 shows the results of the CD3+ T cell activation experiment in Example 15 of the present invention, where the antibody concentrations were 500, 100, 20, 4, 0.8, and 0.16 ng / ml. The EC50s for activating CD3+ T cells by hzWA23Hm38 and hzWA23Hm47 were 8.25 ng / ml and 3.78 ng / ml, respectively, which correspond to the activity of hzWAm23 (EC50 = 3.44 ng / ml). However, the maximum activation intensity (Top value) of hzWA23Hm47 was relatively low, and the activity of hzWAm23 was weak, so the EC50 could not be calculated. Figure 15 is a result figure showing the pharmacodynamic evaluation in the subcutaneous A375 cell CDX model of PBMC immune-reconstituted mice hzWio-02-01, hzWio-02-09, hzWio-02-11, and hzWio-02-44 in Example 16 of the present invention. Figure a shows the tumor growth curves representing the pharmacodynamic evaluation in the subcutaneous A375 cell CDX model of PBMC immune-reconstituted mice hzWio-02-01, hzWio-02-09, hzWio-02-11, and hzWio-02-44, with each curve representing one mouse. Figure b shows the tumor weights representing the pharmacodynamic evaluation in the subcutaneous A375 cell CDX model of PBMC immune-reconstituted mice hzWio-02-01, hzWio-02-09, hzWio-02-11, and hzWio-02-44. Figure 16 shows the results of the pharmacodynamic evaluation in the subcutaneous A375 cell CDX model of PBMC immune-reconstituted mice hzWio-02-03, hzWio-02-05, and hzWio-02-23 in Example 16 of the present invention. Figure a shows the tumor growth curve, which represents the pharmacodynamic evaluation in the subcutaneous A375 cell CDX model of PBMC immune-reconstituted mice hzWio-02-03, hzWio-02-05, and hzWio-02-23, with each curve representing one mouse. Figure b shows the tumor weight, which represents the pharmacodynamic evaluation in the subcutaneous A375 cell CDX model of PBMC immune-reconstituted mice hzWio-02-03, hzWio-02-05, and hzWio-02-23. Figure 17 shows the results of the pharmacodynamic evaluation in the subcutaneous A375 cell CDX model of hzWio-02-01a(G4) and hzWio-02-01h(G1, G2, G3) PBMC immune reconstituted mice in Example 16 of the present invention. Figure a shows the tumor growth curve, which represents the pharmacodynamic evaluation in the subcutaneous A375 cell CDX model of hzWio-02-01a(G4) and hzWio-02-01h(G1, G2, G3) PBMC immune reconstituted mice, with each curve representing one mouse. Figure b shows the tumor weight, which represents the pharmacodynamic evaluation in the subcutaneous A375 cell CDX model of hzWio-02-01a(G4) and hzWio-02-01h(G1, G2, G3) PBMC immune reconstituted mice. Figure 18 shows the results of the pharmacodynamic evaluation in the subcutaneous HuCC-T1 cell CDX model of hzWio-02-01-Hm20 PBMC immune reconstituted mice in Example 16 of the present invention. Figure a shows the tumor growth curve, which represents the pharmacodynamic evaluation in the subcutaneous HuCC-T1 cell CDX model of hzWio-02-01-Hm20 PBMC immune reconstituted mice, with each curve representing one mouse. Figure b shows the tumor weight, which represents the pharmacodynamic evaluation in the subcutaneous HuCC-T1 cell CDX model of hzWio-02-01-Hm20 PBMC immune reconstituted mice. Figure 19 shows the results of the pharmacodynamic evaluation of hzWA23 in a CDX model with mixed inoculation of human tumor cells (human colorectal adenocarcinoma epithelial cells DLD-1) and human PBMCs in Example 16 of the present invention. Figure a shows the average value of the tumor growth curve, which is the result of the pharmacodynamic evaluation in the CDX model with mixed inoculation of human colorectal adenocarcinoma epithelial cells DLD-1 and human PBMCs. Figure b shows the tumor weight, which is the result of the pharmacodynamic evaluation in the same model for hzWA23, with TGI = 34.9%. Figure 20 shows the results of detecting the activation activity of WAm17 and WAm21 by Luciferase HEK 293 Cells in Example 18 of the present invention, with working concentrations of WAm17 and WAm21 being 25, 5, 1, 0.2, 4.0E-2, 8.0E-3, 1.6E-3, and 3.2E-4 nM. Figure 21 shows the activity of WAm17 and WAm21 in an activation experiment of mouse spleen immune cells in Example 18 of the present invention, with working concentrations of 10, 2, 0.4, 8.0E-2, 1.6E-2, 3.2E-3, 6.4E-4, 1.28E-4, and 2.56E-5 nM. Figure 22 is a result diagram showing the evaluation of the in vivo activity of the activated camel-derived anti-mouse IL-18R antibody in the mouse MC38 tumor model in Example 19 of the present invention, where a is a result diagram showing the tumor growth curve, which is the result of the pharmacodynamic evaluation, and b is a result diagram showing the tumor weight, which is the result of the pharmacodynamic evaluation. [Modes for carrying out the invention]

[0043] To further clarify the object, technical proposal, and advantages of the present invention, the following description of the technical proposal will be accompanied by drawings of the present invention. Clearly, the embodiments shown below are some, but not all, embodiments of the present invention. To those skilled in the art, all other embodiments derived from the embodiments of the present invention without requiring any creative work are included within the scope of the present invention.

[0044] In the following examples, camel-derived single-domain antibodies targeting IL-18R1 or IL-18RAP are obtained using camel-derived single-domain immunoassay library technology. Bispecific antibodies are obtained by linking the IL-18R1 and IL-18RAP single-domain antibodies in tandem using a short-chain peptide linker. In vitro activity screening is performed to obtain bispecific antibodies that have biological functions similar to IL-18 and can activate the intracellular signaling pathway mediated by IL-18R1 / IL-18RAP. Humanization technology is used to obtain humanized single-domain antibodies hz-aIL-18R1 and hz-aIL-18RAP by humanizing the anti-IL-18R1 and anti-IL-18RAP single-domain antibodies, respectively, and these are further combined to form a humanized bispecific antibody. Cytological activity analysis is performed on the humanized bispecific antibodies to confirm that they have the activity to activate the IL-18R1 / IL-18RAP-mediated signaling pathway and PBMCs. In vivo antitumor drug efficacy experiments confirmed that the bispecific antibody possesses activity that can effectively suppress tumors.

[0045] Example 1: Expression of recombinant protein 1. Method for producing Fc-tag (or His-tag) recombinant proteins Using gene cloning technology, the target protein coding gene and the Fc-tag (or His-tag) coding gene were sequentially ligated, and then ligated to the correct expression cassette of a eukaryotic expression vector to construct a eukaryotic expression vector with a recombinant Fc-tag (or His-tag) protein fused to its C-terminus. After obtaining a sequenced expression vector plasmid, it was transfected into HEK293 cells for transient expression, the supernatant was collected, purified using a Protein A (or Ni-NTA) affinity chromatography column, desalted to PBS using a HiTrap desalting column, and finally, a recombinant protein with an Fc-tag (or His-tag) fused to its C-terminus was obtained. The Fc-tag types include human Fc-tag (hFc, SEQ ID NO.1), mouse-derived Fc-tag (mFc, SEQ ID NO.2), and camel-derived Fc-tag (aFc, SEQ ID NO.3), while the His-tag type is 6×His-tag (His-tag, His, SEQ ID NO.4). 2. Production of IL-18R1 / IL-18RAP recombinant protein Similar to the method described above, the coding gene of the N-terminal extracellular domain of human IL-18 receptor 1 (IL-18R1 ECD, SEQ ID NO. 5, NP_003846.1, Met1-Arg329) or the N-terminal extracellular domain of human IL-18-related receptor (IL-18RAP ECD, SEQ ID NO. 6; NP_003844.1, Met1-Gly357) is fusion expressed with hFc (SEQ ID NO. 1), mFc (mFc, SEQ ID NO. 2), aFc (aFc, SEQ ID NO. 3), or His-tag (SEQ ID NO. 4). Purification and desalting are performed using an affinity chromatography column, and finally, IL-18R1 ECD-hFc (the sequence indicated by SEQ ID NO. 5 and the sequence indicated by SEQ ID NO. 1, in order from the N-terminus to the C-terminus), IL-18RAP ECD-mFc (the sequence indicated by SEQ ID NO. 6 and the sequence indicated by SEQ ID NO. 2, sequentially from the N-terminus to the C-terminus), IL-18R1 ECD-aFc (the sequence indicated by SEQ ID NO. 5 and the sequence indicated by SEQ ID NO. 3, sequentially from the N-terminus to the C-terminus), IL-18R1 ECD-His (the sequence indicated by SEQ ID NO. 5 and the sequence indicated by SEQ ID NO. 4, sequentially from the N-terminus to the C-terminus), and IL-18RAP ECD-hFc (the sequence indicated by SEQ ID NO. 6 and the sequence indicated by SEQ ID NO. 1, sequentially from the N-terminus to the C-terminus), IL-18R1 ECD-mFc (the sequence indicated by SEQ ID NO. 5 and the sequence indicated by SEQ ID NO. 2, sequentially from the N-terminus to the C-terminus), IL-18RAP ECD-aFc (the sequence indicated by SEQ ID NO. 6 and the sequence indicated by SEQ ID We obtained recombinant proteins of IL-18RAP ECD-His (the sequence indicated by SEQ ID NO. 6 and the sequence indicated by SEQ ID NO. 4, respectively, from the N-terminus to the C-terminus).

[0046] Example 2: Screening of anti-human IL-18R1 or IL-18RAP camel-derived heavy chain single-domain antibodies Camels were immunized using both IL-18R1 ECD-aFc (the sequences indicated by SEQ ID NO. 5 and SEQ ID NO. 3, sequentially from the N-terminus to the C-terminus) and IL-18RAP ECD-aFc (the sequences indicated by SEQ ID NO. 6 and SEQ ID NO. 3, sequentially from the N-terminus to the C-terminus) as immunoantigens. The titers of the immunoserum were detected by ELISA using IL-18R1 ECD-His (the sequences indicated by SEQ ID NO. 5 and SEQ ID NO. 4, sequentially from the N-terminus to the C-terminus) and IL-18RAP ECD-His (the sequences indicated by SEQ ID NO. 6 and SEQ ID NO. 4, sequentially from the N-terminus to the C-terminus) as detection antigens. After the serum titer reaches the requirements for library construction, peripheral blood mononuclear cells (PBMCs) are isolated, total RNA is extracted and reverse transcribed, the reverse transcription product is amplified using a template, the variable domain of the heavy-chain of heavychain antibody (VHH) of the single variable domain antibody is isolated and cloned into a phage display vector, and introduced into E. coli TG1 competent cells by electroporation, resulting in a yield of 1.2 × 10⁶. 9A camel anti-human IL-18R1 / RAP immune antibody library was constructed. By displaying this antibody library, a phage display library was obtained. Using IL-18R1 ECD-His (the sequences indicated by SEQ ID NO. 5 and SEQ ID NO. 4, respectively, from the N-terminus to the C-terminus) and IL-18RAP ECD-His (the sequences indicated by SEQ ID NO. 6 and SEQ ID NO. 4, respectively, from the N-terminus to the C-terminus) as screening antigens and identification antigens, the constructed camel immune library was screened three times by solid-phase screening. After screening, single clones were selected and identified by ELISA after phage display (the identification results are shown in Tables 1 and 2), obtaining positive clones of specific phage-display single variable domain antibodies against IL-18R1 or IL-18RAP, respectively. Phage-display plasmids (phagemids) were extracted from the obtained single clones and used as spares.

[0047] Table 1: Identification results of anti-IL-18R1 phage antibodies by ELISA. [Table 1]

[0048] Table 2. Identification results of anti-IL-18RAP phage antibodies by ELISA. [Table 2]

[0049] Example 3: Construction of a forward / reverse bispecific antibody library of micro-anti-human IL-18R1 / RAP and screening of clones Fifteen coding gene fragments for an anti-IL-18R1 single-domain antibody and eleven coding gene fragments for an anti-IL-18RAP single-domain antibody were obtained by PCR amplification and conjugated. During conjugation, the coding gene for the GGGGS linker was introduced between the two gene fragments using appropriately designed primers. Next, the conjugated gene fragments were ligated to a pET32a vector (pET-Bis-R1 / RAP, as shown in Figure 1) using genetic engineering methods such as enzymatic cleavage, and transformed into BL-21 competent cells to construct a micro anti-human IL-18R1 / RAP bispecific antibody library with an amino acid sequence diversity of 165 (forward library). In addition to changing the ligation order of the IL-18R1 single-domain antibody and the anti-IL-18RAP single-domain antibody (pET-Bis-Rap / R1, as shown in Figure 1), a micro anti-human IL-18RAP / R1 bispecific antibody library with an amino acid sequence diversity of 165 (reverse library) was constructed in the same manner as above. Monoclones were randomly selected from the above micro-anti-human IL-18R1 / RAP (IL-18RAP / R1) bispecific antibody library and cultured overnight. Subsequently, they were subcultured in 300 μl of medium at a ratio of 1:10 and cultured in a 96-well plate until OD=0.6-0.8. Then, 0.1 mM IPTG was added, and induction expression was carried out overnight at 30°C. The supernatant was collected by centrifugation and sterilization was performed to obtain the soluble expression supernatant of bispecific antibody prokaryotic cells, which was then used as a spare.

[0050] Example 4: Analysis of the binding activity of prokaryotically expressed anti-human IL-18R1 / RAP bispecific antibody Human IL-18R1 ECD-His protein (the sequence indicated by SEQ ID NO. 5 and the sequence indicated by SEQ ID NO. 4, sequentially from the N-terminus to the C-terminus) was coated onto an ELISA plate overnight at 1 μg / ml, 50 μl / well. The ELISA plate was then blocked with a blocking agent, and soluble expression supernatant diluted 25-fold with the blocking agent was added. The mixture was incubated at 37°C for 1 hour, washed with PBST, and then IL-18RAP ECD-hFc (the sequence indicated by SEQ ID NO. 5 and the sequence indicated by SEQ ID NO. 1, sequentially from the N-terminus to the C-terminus) was added. The mixture was incubated at 37°C for 30 minutes, and finally, HRP-labeled anti-human Fc diantibody (Cat: 115-035-071 / lot: 144460) was added and incubated, washed, and colored. The colored clones are thought to be able to simultaneously bind to IL-18R1 and IL-18RAP. Forty-eight clones with high chromogenic values ​​were selected from both the forward and reverse libraries, and further identification was performed (the results of clone-binding activity identification are shown in Tables 3 and 4).

[0051] Table 3. Analysis of the binding activity of prokaryotically expressed anti-human IL-18R1 / RAP forward bispecific antibodies. [Table 3]

[0052] Table 4. Analysis of the binding activity of prokaryotically expressed anti-human IL-18RAP / R1 reverse bispecific antibody. [Table 4]

[0053] Example 5: Evaluation of the activity of prokaryotically expressed anti-human IL-18R1 / RAP bispecific antibody using a reporter gene system. 1. IL-18 Reporter HEK 293 Cells Reporter Gene System The IL-18 Reporter HEK 293 Cells were purchased from Beijing Kangyuan Boyuang Biotechnology (Beijing) Co., Ltd. (KC-2328). This reporter system was obtained by expressing an NF-κB / AP-1-induced secreted embryonic alkaline phosphatase (SEAP) reporter gene in HEK293 cells, and then stably transfecting them with genes encoding human IL-18R and IL-18RAP. The system exhibits a specific response to human IL-18; after human IL-18 binds to its cell surface receptor, it induces the expression of the SEAP reporter gene downstream of the NF-κB / AP-1 pathway, catalyzing a specific substrate and causing a color reaction, thus activating human IL-18. The results of activity detection of recombinant human IL-18 (rhIL-18, Novoprotein, CH29) using this system are shown in Figure 2, with an EC50 of rhIL-18 of 0.107 ± 0.020 ng / ml. Similarly, this system can detect the activating effect of activated bispecific antibodies targeting the IL-18 receptor on the NF-κB / AP-1 pathway. For activity measurement, cells were revived, subcultured to a sufficient number, collected, adjusted to an appropriate density, and 180 μl of cell suspension (approximately 50,000 cells) and 20 μl of diluted test sample (anti-human IL-18R1 / RAP bispecific antibody) were added to each well of a 96-well plate. A positive control (rhIL-18, Novoprotein, CH29) was established, and after homogeneous mixing, the cells were incubated at 37°C in a CO2 incubator for 20-24 hours. The following day, 180 μl of SEAP chromogenic substrate was added to each well of a new flat-bottom 96-well plate, followed by 20 μl of induced HEK-Blue. TMIL-18 cell supernatant was added and incubated in a 37°C incubator for 1-3 hours. The absorbance at 630 nm was measured using a spectrophotometer. The resulting data indicates activation activity by color development value (single concentration data) or EC50 (concentration gradient data) under specific concentration conditions. When EC50 is used, a concentration (log)-color development value variable slope curve (log(agonist) vs. response, Variable slope) is created, and EC50 is calculated using nonlinear regression 4-parameter curve fitting (four parameters), and the maximum activation signal value (Top 630nm The activation activity of the test sample was determined after binding it to the compound. 2. Activation evaluation and sequence measurement of prokaryotically expressed anti-human IL-18R1 / RAP bispecific antibodies. Following the method described above, the IL-18 Reporter HEK 293 Cells reporter gene system was used to detect the activity of prokaryotically expressed anti-human IL-18R1 / RAP bispecific antibodies in inducing the expression of NF-κB / AP-1 pathway genes. The results are shown in Table 5.

[0054] Table 5: Detection results of activation activity of prokaryotically expressed anti-human IL-18R1 / RAP bispecific antibodies using a reporter gene system. [Table 5]

[0055] Clones exhibiting relatively strong activation activity were selected, sequenced, and subjected to comparative analysis to remove duplicate sequences. As a result, single-domain antibodies targeting IL-18R1 mainly consist of R1-A12, R1-C7, R1-C10, R1-F9, and R1-E12 (sequences shown in SEQ ID NO. 7-11, respectively), while single-domain antibodies targeting IL-18RAP mainly consist of Rap-A10, Rap-C10, and Rap-F11 (sequences shown in SEQ ID NO. 12-14, respectively). By combining these sequences, the forward-structure bispecific antibodies with activation activity were obtained as follows: Wio-02-01(R1-A12-GGGGS-Rap-A10, SEQ ID NO.63), Wio-02-03(R1-C7-GGGGS-Rap-F11, SEQ ID NO.64), Wio-02-05(R1-C7-GGGGS-Rap-C10), Wio-02-09(R1-C10-GGGGS-Rap-F11), Wio-02-11(R1-C10-GGGGS-Rap-C10), Includes Wio-02-23(R1-F9-GGGGS-Rap-C10) Furthermore, the reverse-structure bispecific antibody having activating activity obtained as described above is Wio-02-29(Rap-A10-GGGGS-R1-A12), Wio-02-30(Rap-C10-GGGGS-R1-C10), Wio-02-31(Rap-C10-GGGGS-R1-F9), Includes Wio-02-44 (Rap-A10-GGGGS-R1-E12).

[0056] Example 6: Production of Wio-02-01-hFc fusion protein and study of the effects of different linker lengths and sequences on activity. Referring to the method for constructing the hFc fusion protein described in Example 1, the bispecific antibody Wio-02-01 and hFc (SEQ ID NO. 1) were fusion-expressed, and the anti-human IL-18R1 / IL-18RAP bispecific antibody-hFc fusion protein Wio-02-01-hFc (SEQ ID NO. 65) was obtained by affinity chromatography and desalting. The linker between the single-domain antibody R1-A12 and Rap-A10 in this fusion protein is GGGGS (named L0, SEQ ID NO. 15). The linker between the single-domain antibody R1-A12 and Rap-A10 in Wio-02-01-hFc was substituted. The substituted linker sequences included L1:GS, L2:GGGGSGGGGS (SEQ ID NO.17), L3:GGGGSGGGGSGGGGS (SEQ ID NO.18), and L4:GGGGSGGGGSGGGGSGGGGS (SEQ ID NO.19). Subsequently, referring to the method for constructing hFc fusion proteins described in Example 1, Wio-02-01-L1-hFc, Wio-02-01-L2-hFc, Wio-02-01-L3-hFc, and Wio-02-01-L4-hFc, which are fusion proteins of the bispecific antibody with the substituted linker and hFc (SEQ ID NO.1), were prepared. Finally, referring to the method of Example 5, the in vitro activity of the above anti-human IL-18R1 / RAP bispecific antibody was evaluated using the reporter gene system. The results are shown in Figure 3. The results indicate that when the linker length was increased to (G4S)3 and (G4S)4, the activation activity of the bispecific antibody decreased, but still maintained good activation activity. On the other hand, several other types of linkers had little effect on the activation activity of the bispecific antibody. The above results indicate that when GS flexibility linkers of different lengths are selected, good activation activity can be maintained for all anti-human IL-18R1 / RAP bispecific antibodies.

[0057] Furthermore, the Linker (L0:GGGGS (SEQ ID NO:15)) of the bispecific antibody hzWio-02-23 (SEQ ID NO.62) was substituted, and the Linker sequence to be substituted was: L5:GGGGSG (SEQ ID NO.66) L6:GGGSGG (SEQ ID NO.67) L7:GGSGGG (SEQ ID NO.68) L8:GSGGGG (SEQ ID NO.69) L9:GGSGSG (SEQ ID NO.70) L10: Includes GSGSGG (SEQ ID NO. 71). Then, referring to the method for constructing the hFc fusion protein described in Example 1, we prepared fusion proteins of a bispecific antibody with a substituted Linker and hFc as shown below. (Here, hzWio-02-23-L5-hFc is a fusion protein obtained by substituting L5 for the linker in hzWio-02-23-hFc, and the same applies to the others.) hzWio-02-23-hFc hzWio-02-23-L5-hFc(SEQ ID NO.72) hzWio-02-23-L6-hFc, hzWio-02-23-L7-hFc, hzWio-02-23-L8-hFc, hzWio-02-23-L9-hFc, hzWio-02-23-L10-hFc. Finally, referring to the method of Example 5, the in vitro activity of the above anti-human IL-18R1 / RAP bispecific antibodies was evaluated using a reporter gene system. The results are shown in Table 6. When the linker selects one of the six different amino acid combinations mentioned above, all of the anti-human IL-18R1 / RAP bispecific antibodies can maintain good activation activity. The above results further demonstrate that when different GS flexibility linkers are selected, all of the anti-human IL-18R1 / RAP bispecific antibodies can maintain good activation activity.

[0058] Table 6. Activity evaluation results of t-induced anti-IL-18R1 / RAP bispecific antibodies using reporter gene systems with different linkers (6 aa Linker) [Table 6]

[0059] Example 7: Production of human IL-18R1 / RAP bispecific antibodies with different structures, and analysis of their activation activity. Based on the R1-A12 / RAP-A10 single-domain antibody, bispecific antibodies with different structures were constructed, and their activity was then analyzed and validated. The antibody structures are shown in Figure 4, with a-h representing a total of eight different structures. Structure a is the parent form of Wio-02-01 (R1-A12-GGGGS-Rap-A10). The other structures were obtained by combining R1-A12 / Rap-A10 according to new structures using different ligation and tag fusion methods. Referring to the method of Example 5, the in vitro activity of each form of anti-human IL-18R1 / RAP bispecific antibody described above was evaluated using a reporter gene system. The results are shown in Table 7. These results indicate that all bispecific antibodies of each derived structure (e.g., a, c, d, g, h, etc.), including the AB tandem structure, possessed activating activity, and that the Fc region did not affect the activating activity of the bispecific antibodies. On the other hand, b, e, and f, which do not contain the tandem structure, showed only slight activating activity, indicating that the AB tandem structure is an important element for influencing activating activity.

[0060] Table 7 Results of activation activity of anti-human IL-18R1 / RAP bispecific antibodies with different structural forms. [Table 7]

[0061] Example 8: Humanization and eukaryotic expression of heavy chain single-domain antibodies against human IL-18R1 or IL-18RAP Humanization modifications were performed on specific single-domain antibodies against human IL-18R1 or IL-18RAP. First, the VHH sequences of camel-derived antibodies were comprehensively analyzed to measure the antigen complementarity-determining region (CDR) where the antibody and antigen bind, and the framework region that supports the conservative three-dimensional structure of the antibody. Then, based on the results of homology comparison, the sequence closest to the humanized antibody sequence was selected as the base template, and humanization of the framework region of the nano-antibody variable region (VHH) was achieved by combining the results of the full sequence blast and transplanting the CDR. Subsequently, site-directed mutagenesis was introduced into some important amino acids to maintain affinity equivalent to that of the camel-derived antibody. Ultimately, we obtained humanized sequences for anti-human IL-18R1 specific single-domain antibodies (the sequences of hzR1-A12, hzR1-C7, hzR1-C10, and hzR1-F9 are shown in SEQ ID NO. 20-23, respectively) and humanized sequences for anti-human IL-18RAP specific single-domain antibodies (the sequences of hzRap-A10, hzRap-C10, and hzRap-F11 are shown in SEQ ID NO. 24-26, respectively). Referring to the method for constructing the hFc fusion protein described in Example 1, the genes encoding the variable regions of the anti-human IL-18R1 (or IL-18RAP) single-domain antibody before and after humanization were fused with hFc (SEQ ID NO.1) using a GS linker. The anti-human IL-18R1 (or IL-18RAP) single-domain antibody-hFc fusion protein was obtained by affinity chromatography and desalting. The structure of the fusion protein is shown in Figure 5.

[0062] Example 9: Production of a humanized anti-human IL-18R1 / RAP bispecific antibody A tandem-type humanized bispecific antibody was constructed by linking the humanized anti-human IL-18R1 or IL-18RAP specific heavy chain single-domain antibody obtained in Example 8 to the camel-derived bispecific antibody with activating activity obtained in Example 5. Here, the forward-structured humanized bispecific antibody is hzWio-02-01(hzR1-A12-GGGGS-hzRap-A10, SEQ ID NO.59), hzWio-02-03(hzR1-C7-GGGGS-hzRap-F11, SEQ ID NO.60), hzWio-02-05(hzR1-C7-GGGGS-hzRap-C10, SEQ ID NO.61), hzWio-02-09(hzR1-C10-GGGGS-hzRap-F11), hzWio-02-11(hzR1-C10-GGGGS-hzRap-C10), Includes hzWio-02-23 (hzR1-F9-GGGGS-hzRap-C10, SEQ ID NO.62), Reverse-structured humanized bispecific antibodies are, hzWio-02-29(hzRap-A10-GGGGS-hzR1-A12), hzWio-02-30(hzRap-C10-GGGGS-hzR1-C10), hzWio-02-31(hzRap-C10-GGGGS-hzR1-F9), It includes hzWio-02-44 (hzRap-A10-GGGGS-R1-E12, where R1-E12 is a camel-derived sequence). Next, referring to the method for constructing the hFc fusion protein described in Example 1, the humanized bispecific antibody and hFc (SEQ ID NO.1) were fusion-expressed, and the humanized anti-human IL-18R1 / IL-18RAP bispecific antibody hFc fusion protein was obtained by affinity chromatography and desalting. The structure of the fusion protein is shown in Figure 6. The name and sequence of the fusion protein are as follows: hzWio-02-01-hFc, SEQ ID NO.59-SEQ ID NO.1, hzWio-02-03-hFc, SEQ ID NO.60-SEQ ID NO.1, hzWio-02-05-hFc, SEQ ID NO.61-SEQ ID NO.1, hzWio-02-09-hFc, SEQ ID NO.22-SEQ ID NO.26-SEQ ID NO.1, hzWio-02-11-hFc, SEQ ID NO.22-SEQ ID NO.25-SEQ ID NO.1, hzWio-02-23-hFc, SEQ ID NO.62-SEQ ID NO.1, hzWio-02-29-hFc, SEQ ID NO.24-SEQ ID NO.20-SEQ ID NO.1, hzWio-02-30-hFc, SEQ ID NO.25-SEQ ID NO.22-SEQ ID NO.1, hzWio-02-31-hFc, SEQ ID NO.25-SEQ ID NO.23-SEQ ID NO.1, hzWio-02-44-hFc, SEQ ID NO.24-SEQ ID NO.11-SEQ ID NO.1.

[0063] Example 10: Affinity measurement of anti-human IL-18R1 or IL-18RAP heavy chain single-domain antibodies The binding ability of anti-human IL-18R1 (or IL-18RAP) specific heavy chain single-domain antibodies (expressed fusion-expressed with the C-terminus and human Fc) to recombinant antigens was measured using the Octet® R4 system obtained from Fortebio, by capturing the antibody Fc region with an anti-human Fc region capture antibody biosensor probe. For measurement, the test sample was diluted to 5 μg / mL in PBS buffer and reacted with the AHC probe (Cat: 18-0015, PALL) to immobilize it on the surface of the chip for 180 seconds. Recombinant human IL-18R1 ECD-His (the sequence indicated by SEQ ID NO. 5 and the sequence indicated by SEQ ID NO. 4, sequentially from the N-terminus to the C-terminus) or IL-18RAP ECD-his (the sequence indicated by SEQ ID NO. 6 and the sequence indicated by SEQ ID NO. 4, sequentially from the N-terminus to the C-terminus) protein was used as the liquid phase and reacted with the antibody immobilized on the surface of the chip. The recombinant protein concentration was 100 nM. The binding time for each antigen was 300 s, and the dissociation time was 300 s. As a result (Table 8), under current experimental conditions, anti-human IL-18R1 and IL-18RAP antibodies were shown to have high affinity for their respective antigens. Before and after humanization, only the affinity for R1-C10 decreased significantly, and no clear changes were observed in the affinity of the other clones.

[0064] Table 8. Measurement results of affinity of anti-human IL-18R1 (or IL-18RAP) single-domain antibody (fusion expression of the C-terminus and human Fc). [Table 8]

[0065] Example 11: Histidine scanning mutation in the bispecific antibody hzWio-02-01 Using site-directed mutagenesis, the three CDR regions on the R1-A12 side of hzWio-02-01 were histidine-scanned. Specifically, site-directed mutagenesis primers were designed, and using the coding gene of hzWio-02-01 as a template, site-directed mutagenesis was introduced into the original gene sequence by PCR. Subsequently, recombinant mutant proteins were produced by eukaryotic expression and affinity purification, and were named hzWio-02-01-Hm1~35, respectively. These mutations were formed from SEQ ID NO. 59, with mutations occurring in T27H, A28H, Y29H, D30H, N31H, A32H, M34H, G35H, S50H, I51H, L53H, R54H, D55H, R56H, T57H, Y58H, Y59H, A60H, D61H, S62H, V63H, K64H, G65H, S98H, N99H, I100H, N102H, A103H, L104H, N105H, L106H, Q107H, G108H, N109H, and T110H, respectively. Here, the sequence of hzWio-02-01-Hm1 is shown as SEQ ID NO. 16.

[0066] Example 12: Activity analysis of a humanized anti-human IL-18R1 / RAP bispecific antibody 1. FACS analysis and binding activity to cell surface antigens. Eukaryotic transient expression vectors for the full-length genes IL-18R1 and IL-18RAP (IL-18R1, NP_003846.1; IL-18RAP, NP_003844.1) were constructed, transfected into HEK293 cells, cultured for 48 hours, then the cells were collected and 2 × 10⁶ were used. 5The sample was prepared in 100 μL in a cell / sample ratio. Humanized anti-human IL-18R1 / RAP bispecific antibodies, hzWio-02-01-hFc, hzWio-02-03-hFc, hzWio-02-05-hFc, hzWio-02-09-hFc, hzWio-02-11-hFc, and hzWio-02-23-hFc, were diluted and added to the cells. The mixture was then homogenized to the working concentration and incubated at 4°C for 60 minutes. The supernatant was removed by centrifugation, the cells were resuspended in cell medium, washed, and the supernatant was removed again by centrifugation. Subsequently, the flow cytometry antibody (Goat Anti human IgG1-FITC, sigma, F9512) was added and homogenized. The mixture was incubated at 4°C for 30 minutes, the supernatant was removed by centrifugation, and finally the cells were resuspended in cell medium. The mixture was then set in the analyzer and detection was performed. Based on the resulting data, a log(agonist) vs. response variable slope curve was created, and the EC50 was calculated using nonlinear regression 4-parameter curve fitting. 412nm Based on the above, the binding activity of the test samples was determined. As a result (Figure 7), the detected bispecific antibodies were shown to have good binding activity to cell surface antigens. 2. Activity evaluation of humanized anti-IL-18R1 / RAP bispecific antibody using a reporter gene system. Referring to the method of Example 5, the activity of anti-human IL-18R1 / RAP bispecific antibody in inducing the expression of NF-κB / AP-1 pathway genes was detected using the IL-18 Reporter HEK 293 Cells reporter gene system. As a result (Figure 9), it was shown that the detected humanized bispecific antibody could initiate the expression of NF-κB / AP-1 pathway genes even at extremely low concentrations (ng). However, compared to IL-18, the bispecific antibody showed relatively mild activation activity in the reporter gene system. The tandem His-tag fusion form showed superior activity compared to the bivalent Fc-tag form.

[0067] Table 9: Activity evaluation results of humanized anti-IL-18R1 / RAP bispecific antibodies using a reporter gene system. [Table 9]

[0068] The activation activity of the hzWio-02-01 mutant was detected using the same method. The results are shown in Table 10. The results indicate that mutating the amino acids of the single-domain antibody on the anti-IL-18R1 side had some effect on the binding activity of that side, and the activation activity of the bispecific antibody also changed to some extent, but all mutants still maintained good activation activity. Here, the activation activity of hzWio-02-01-Hm20 was clearly improved.

[0069] Table 10: Results table of activation activity of the histidine scanning mutant (His-tag fusion form) of hzWio-02-01* [Table 10] Note: *: Activity detection using a reporter gene system.

[0070] 3. Evaluate the activity of the humanized anti-IL-18R1 / RAP bispecific antibody by PBMC activation experiments. The in vitro activity of an anti-human IL-18R1 / RAP bispecific antibody was evaluated using primary cultured human peripheral blood mononuclear cells (PBMCs). Human PBMCs were extracted and cultured for 24 hours in RPMI-1640 medium containing FBS and recombinant human interleukin-2 (rhIL-2). Cells were collected and the cell concentration was increased to 3 × 10⁶ in a medium containing IL-12 (or IL-15). 6 The solution was adjusted to cells / ml, inoculated into cell culture plates, and the test sample was added. After 48 hours of incubation, the supernatant was collected, and the IFN-γ content in the supernatant was measured using a human IFN-γ detection kit (Biolegend human IFN-γ Kit). A concentration (log)-IFN-γ content curve was created, and the top value and EC50 value were calculated. The results are shown in Figure 8. Under current experimental conditions, in the presence of IL-12 (or IL-15), both bispecific antibodies can activate IFN-γ secretion by PBMCs, similar to IL-18. The bispecific antibodies exhibit activating activity whether fused with hFc or His-Tag. There are some differences in the activating activity of different bispecific antibodies; while the activity of hzWio-02-01-Hm20-hFc is almost equivalent to that of IL-18, the Emax values ​​for the activation activity of the other molecules are all significantly lower than those of IL-18, but the onset concentrations are all significantly lower than those of IL-18. The above results suggest that the activation of the IL-18R1 / RAP signaling pathway by bispecific antibodies has the characteristics of lower onset concentrations and milder activating activity compared to IL-18, and can be expected to have superior safety potential and a wider therapeutic window. Under current experimental conditions, bispecific antibodies such as hzWio-02-01-hFc, hzWio-02-03-hFc, hzWio-02-05-hFc, hzWio-02-11-hFc, and hzWio-02-23-hFc all play a role in stimulating PBMCs to produce IFNγ, exhibiting superior EC50 compared to rhIL-18 and significantly lower Emax values ​​than IL-18. Under current experimental conditions, bispecific antibodies such as hzWio-02-01-His, hzWio-02-03-His, hzWio-02-05-His, hzWio-02-11-His, and hzWio-02-23-His all play a role in stimulating PBMCs to produce IFNγ, exhibiting superior EC50 compared to rhIL-18 and significantly lower Emax values ​​than IL-18. Under current experimental conditions, bispecific antibodies such as hzWio-02-01-His, hzWio-02-03-His, hzWio-02-05-His, hzWio-02-11-His, and hzWio-02-23-His all play a role in stimulating PBMCs to produce IFNγ, exhibiting superior EC50 compared to rhIL-18 and significantly lower Emax values ​​than IL-18. Under current experimental conditions, the activity of the mutants hzWio-02-01-Hm11-hFc and hzWio-02-01-Hm20-hFc is superior to that of the parent strain hzWio-02-01-hFc. Compared to rhIL-18, the EC50 of the mutants is superior to that of rhIL-18, and the Top value increased to approach that of rhIL-18.

[0071] Example 13: Histidine scanning mutation of hzWio-02-23-L5-hFc Site-directed mutagenesis was used to perform histidine scanning on three CDR regions of each of the two single-domain antibodies (hzR1-F9, hzRAP-C10) contained in hzWio-02-23-L5-hFc (SEQ ID NO. 72, hereinafter abbreviated as hzWA23). Specifically, site-directed mutagenesis primers were designed, and using the hzWA23 coding gene as a template, site-directed mutations were introduced into the proto-gene sequence by PCR. In some cases, histidine mutations were introduced simultaneously at two sites (Figure 9). Recombinant proteins of the mutants were then produced by eukaryotic expression and affinity purification. The mutants on the hzR1-F9 side were named hzWA23Hm1~49 in order, and they are each named hzWA23 (SEQ ID NO. 72). Based on the HzR1-F9 side of NO.72), D31H, M32H, G33H, T48H, I49H, S50H, S51H, D52H, A53H, S54H, T55H, Y56H, Y57H, A58H, D59H, S60H, V61H, K62H, G63H, D96H, S97H, F98H, T99H, G100H, L101H, Q102H, L103H, A104H, E105H, K107H, A108H, D The mutations that resulted were 109H, F110H, G111H, Y112H, V29H, D52H / D59H, D52H / E105H, D52H / K107H, D59H / T99H, D59H / E105H, D59H / K107H, D59H / D109H, T99H / K107H, E105H / D109H, D52H / Y112H, D59H / Y112H, L101H / Y112H, and E105H / Y112H. The mutants on the hzRAP-C10 side were named hzWA23-2Hm1 to 29 in order, and they are based on the hzRAP-C10 side of hzWA23 (SEQ ID NO. 72), with the mutations A161H, Y162H, M163H, G164H, S179H, I180H, S181H, S182H, T183H, E184H, G185H, S186H, T187H, T188H, Y189H, A190H, D191H, S192H, V193H, K194H, G195H, Y228H, Q229H, F230H, G231H, A232H, F233H, R234H, and V235H occurring.

[0072] Example 14: Analysis of the binding activity of the HzWA23 mutant. 1. Analysis of binding activity to recombinant antigens using Fortebio Using the Octet® R4 system obtained from Fortebio, the binding ability of different mutants of hzWA23 to recombinant antigens was measured by capturing the antibody Fc region using an anti-human Fc region capture antibody biosensor probe. For measurement, the test sample was diluted to 5 μg / mL in PBS buffer and immobilized on the chip surface by reacting with the AHC probe (Cat: 18-0015, PALL). Human IL-18R1 ECD-His or IL-18RAP ECD-his recombinant protein was used as the liquid phase and reacted with the antibody immobilized on the chip surface. The recombinant proteins were dissolved in phosphate buffers at pH 7.4 and pH 6.0, respectively, to a concentration of 60 nM, and then reacted with the antibody coated on the chip surface. The binding time for each antigen was 300 s, and the dissociation time was 300 s. The results (Tables 11 and 12) show that, under current experimental conditions, good affinity can still be maintained even if most of the site is mutated to histidine. Furthermore, under pH 6.0 conditions, most mutants have been shown to have higher affinity.

[0073] Table 11. Affinity between the hzWA23 hzR1-F9 mutant and IL-18R1 ECD-His using Fortebio (pH 7.4 / 6.0) [Table 11]

[0074] Table 12 Affinity between the hzWA23 hzRAP-C10 mutant and IL-18Rap ECD-his using Fortebio (pH 7.4) [Table 12]

[0075] 2. ELISA analysis of the activity of hzWA23Hm38 and hzWA23Hm47 to simultaneously bind to both IL-18R1 and IL-18RAP. Human IL-18R1 ECD-His protein was coated onto ELISA plates overnight with 1 μg / ml, 50 μl / well. The ELISA plates were then blocked with a blocking agent. Diluted test samples hzWA23, hzWA23Hm38 (SEQ ID NO. 73), and hzWA23Hm47 (SEQ ID NO. 74) were added and incubated at 37°C for 1 hour. After washing the ELISA plates with PBST, 1 μg / ml of IL-18RAP ECD-mFc was added and incubated at 37°C for 30 minutes. Finally, HRP-labeled anti-mouse Fc compounds were added and incubated, washed, and colored to create OD-concentration curves. The results (Figure 10) show that hzWA23 and its mutants hzWA23Hm38 and hzWA23Hm47 can simultaneously bind to both IL-18R1 and IL-18RAP.

[0076] 3. Analysis of cell surface antigen binding activity by FACS Eukaryotic transient expression vectors for the full-length genes IL-18R1 and IL-18RAP (IL-18R1, NP_003846.1; IL-18RAP, NP_003844.1) were constructed, transfected into HEK293 cells, and cultured for 48 hours. After that, the cells were collected and 2 × 10⁶ were used. 5The sample was prepared in 100 μL cells / sample. The hzWA23 and its respective mutants were diluted and added to cells, then uniformly mixed to the working concentration and incubated at 4°C for 60 minutes. The supernatant was removed by centrifugation, the cells were resuspended in cell medium, washed, and the supernatant was removed again by centrifugation. Flow cytometry antibody (Goat Anti human IgG1-FITC, sigma, F9512) was added and uniformly mixed, followed by incubation at 4°C for 30 minutes. The supernatant was removed by centrifugation, and finally the cells were resuspended in cell medium. The sample was then placed in the analyzer for detection. The results (Figure 11) demonstrate that, under current experimental conditions, even with mutations to histidine in most parts of hzWA23, excellent binding activity to cell membrane surface antigens can be maintained.

[0077] Example 15: Analysis of the activating activity of the HzWA23 mutant (1) The IL-18 Reporter HEK 293 Cells reporter gene system was used to detect the activity of hzWA23 mutants in inducing NF-κB / AP-1 pathway activation, and the color value (OD) produced when the reporter gene SEAP catalyzes the chromogenic substrate was measured. As a result (Figure 12), most mutants maintained excellent activation activity, indicating that their activation characteristics are similar to those of their parental antibodies. (2) The in vitro activating activity of primary cultured human peripheral blood mononuclear cell (PBMC) mutants was used to measure the IFN-r content in the culture supernatant. The measurement results (Figure 13) show that most mutants maintained good activating activity, and that this activity is similar to the activating properties of the parent antibody. (3) The in vitro activation activities of hzWA23Hm23 (SEQ ID NO.75), hzWA23Hm38, and hzWA23Hm47 were evaluated using T cells (CD3+) isolated from primary cultured human PBMCs. Human PBMCs were extracted, and T cells were separated using CD3+ antibody magnetic beads and cultured in RPMI-1640 medium containing FBS and recombinant human interleukin 2 (rhIL-2) for 24 hours. The cells were collected, and the cell concentration was adjusted to 2×10 6 cells / ml in a medium containing IL12, inoculated into a cell culture plate, and the test sample was added. After culturing for 48 hours, the supernatant was collected, and the content of IFN-γ in the supernatant was detected using a human IFN-γ detection kit (Biolegend human IFN-γ Kit). A curve diagram of the concentration (log)-IFN-γ content was prepared, and the EC50 value was calculated. The results are shown in Figure 14.

[0078] Example 16: Evaluation of the in vivo activity of a humanized anti-human IL-18R1 / RAP bispecific antibody Human tumor cells (melanoma cell line A375) were inoculated subcutaneously into the right anterior rib region in the body of NSG mice reconstructed by human PBMC immunization, and when the tumor reached 60 - 100 mm 3The mice were divided into two groups, and an anti-human IL-18R1 / RAP bispecific antibody was administered via injection into the tail vein. A control group consisting of mice with the same type of IgG antibody was also established. Throughout the experiment, tumor volume and body weight were measured twice a week, and the relationship between changes in body weight and tumor volume and administration time was recorded. At the end of the experiment, the tumor mice were euthanized, the tumors were removed, weighed, and photographed. The relative tumor volume ratio (T / C) and tumor growth inhibition rate (1-T / C) for the treatment and control groups were calculated and statistically analyzed. The experimental results are shown in Figures 15, 16, 17, and 18. The results show that, under current experimental conditions, all of the bispecific antibodies (hFc structure) of the hzWio-02-01, hzWio-02-03, hzWio-02-05, hzWio-02-09, hzWio-02-11, hzWio-02-23, and the reversed hzWio-02-44 all exhibit clear tumor suppressor effects and maintain a dose-response relationship (Figures 15 and 16). Similarly, hzWio-02-01a, which lacks an Fc structure, and hzWio-02-01h, which has a complex structure, also exhibit clear tumor suppressor effects (Figure 17). When hzWio-02-01-m20, a highly active mutant of hzWio-02-01, was selected and evaluated in a cell tumor model of human intrahepatic cholangiocarcinoma cells (HuCC-T1), the results similarly showed clear tumor growth suppression activity (Figure 18). These results suggest that both bispecific antibodies with different structural forms that are cytologically activated have the potential to be developed as antitumor drugs. Human tumor cells (human colorectal adenocarcinoma epithelial cells DLD-1) and human PBMCs were mixed in a 10:1 ratio, and then inoculated subcutaneously into the right anterior rib region of NSG mice, when the tumor was 60-100 mm in size. 3The mice were divided into two groups, and hzWA23 was administered by injection into the tail vein. A control group was established containing the same type of IgG. Six mice were assigned to each group, and the dosage was 0.3 mg / kg, administered in two doses on day 1 and day 4 after group division. Tumor volume was measured twice a week during the experiment, and the relationship between tumor volume changes and administration time was recorded. At the end of the experiment, the tumor mice were euthanized, and the tumors were removed and weighed. The relative tumor volume ratio (T / C) and tumor growth inhibition rate (TGI=1-T / C) for the treatment and control groups were calculated and statistically analyzed, and the experimental results are shown in Figure 19. As a result, under the current experimental conditions, hzWA23 showed a clear tumor-suppressing effect, with a TGI of 34.9%.

[0079] Example 17: Production of activated camel-derived anti-mouse IL-18R antibody To further demonstrate that the structural form of the anti-IL-18R1 and IL-18RAP bispecific antibody possesses normal in vitro-in vivo activity, we constructed surrogate molecules of hzWA23 bound to mouse IL-18R1 and IL-18Rap, referring to the method for producing hzWA23. Specifically, a fusion protein (mIL-18R1-aFc) of the N-terminal extracellular domain of recombinant mouse IL-18 receptor 1 (mIL-18R1 ECD, NCBI:NP_032391.1) and camel Fc(aFc), and a fusion protein (mIL-18RAP-aFc) of the N-terminal extracellular domain of recombinant mouse IL-18-related receptor (IL-18RAP ECD NCBI:NP_034683.1) and camel Fc(aFc) were used as immunogens. These were mixed with Freund's adjuvant in a 1:1 volume ratio and thoroughly emulsified. Adult camels aged 3-5 years were selected, and immunization was performed by subcutaneous injection at a dose of 1 mg / dose / camel. The first immunization used a complete Freund's adjuvant, while the subsequent five immunizations used an incomplete Freund's adjuvant, with immunizations performed every two weeks. Ten days after the completion of the fifth immunization, peripheral blood was collected from camels, mononuclear cells (PBMCs) were isolated, RNA was extracted, and reverse transcription was performed to obtain a gene fragment library of the variable domain of heavy chain of heavy-chain antibody (VHH). Based on this, a VHH phage antibody library was constructed, and specific anti-mouse IL-18R1 and anti-mouse IL-18RAP single-domain antibodies were screened and obtained from it. By combining the anti-mouse IL-18R1 and anti-mouse IL-18RAP single-domain antibodies, the anti-mIL-18R1 / mIL-18Rap-hFc fusion proteins WAm17 and WAm21, which have the same molecular structure as hzWA23, were constructed.

[0080] Example 18 Evaluation of the in vitro activity of activated camel-derived anti-mouse IL-18R antibody (1) The activation activity of WAm17 and WAm21 was analyzed using Luciferase HEK 293 Cells. Luciferase HEK 293 Cells expressed a luciferase reporter gene induced by NF-κB / AP-1. After transient transfection of these cells with genes encoding mouse IL-18R1 and IL-18RAP, IL-18R1 and IL-18RAP expressed on the cell membrane bound to mouse IL-18, mediating the expression of a luciferase reporter gene downstream of the NF-κB / AP-1 pathway and catalyzing a specific substrate to induce a color reaction, thus demonstrating the activation of mouse IL-18. Similarly, this system can be used to detect the activation effect of anti-mouse IL-18R antibodies on the NF-κB / AP-1 pathway. The activation activity of WAm17 and WAm21 was measured using this system. Before measuring the activity, cells were revived and subcultured until a sufficient number were obtained. After collecting the cells and adjusting them to an appropriate density, they were transfected with transient expression vectors of mouse IL-18R1 and IL-18RAP. 24 hours after transfection, the cells were collected, adjusted to an appropriate density, and 80 μl / well of cell suspension (approximately 20,000 cells) and 20 μl / well of diluted test samples (WAm17, WAm21, mIL-18) were added to a 96-well plate and mixed uniformly. The cells were then cultured at 37°C in a CO2 incubator for 20-24 hours. The following day, 100 μl / well of luciferase chromogenic substrate was added to the plate, and after sufficient cell lysis, the fluorescence signal value was measured using a spectrophotometer. Based on the result data, a log(agonist) vs. response curve was created, and the EC50 was calculated using nonlinear regression liner curve fitting (four parameters), combined with the maximum activation signal value (Top 630nm). The measurement results show that both WAm17 and WAm21 can activate the expression of reporter genes in the mouse IL-18 downstream signaling pathway (Figure 20). (2) Using immune cells isolated from primary cultured mouse spleens, the activating activity of WAm17 and WAm21 was analyzed. Immune cells derived from mouse spleens were extracted and cultured for 24 hours in RPMI-1640 medium containing FBS and recombinant mouse interleukin 2 (rhIL-2). The cells were collected and the cell concentration was increased to 2 × 10⁶ in a medium containing mouse IL-12. 6 The solution was adjusted to the required concentration of cells / ml, inoculated into cell culture plates, and the test sample was added. After 48 hours of incubation, the supernatant was collected and tested using a mouse IFN-γ detection kit (ELISA MAX). TM The IFN-γ content in the supernatant was measured using Standard Set Mouse IFN-γ Cat. 430801 (Biolegend). The results are shown in Figure 21. Both WAm21 and WAm17 were able to stimulate mouse immune cells to secrete the IFN-γ toxic factor in a dose-dependent manner, and their EC50 values ​​were superior to those of mouse IL-18 (abbreviated as mIL-18), while the maximum activation capacity of WAm21 and WAm17 was weaker than that of mIL-18. The results indicate that the mechanisms and characteristics of WAm21 and WAm17 in activating mouse immune cells are similar to those of hzWA23, and therefore they can be used as alternative molecules to hzWA23 to evaluate the in vivo efficacy of mIL-18 activators.

[0081] Example 19 Evaluation of in vivo activity of activated camel-derived anti-mouse IL-18R antibody Mouse tumor cells (melanoma cell line MC38) were inoculated subcutaneously into the right anterior rib region of the mouse trunk, and the tumor was 60-100 mm in size. 3The mice were divided into groups, and each group was administered mouse IL-18R-activated antibodies WAm21 and WAm17 by intraperitoneal injection. A control group was also established containing the same type of IgG antibodies. Information for each group is shown in Table 13. During the experiment, tumor volume and body weight were measured twice a week, and the relationship between changes in body weight and tumor volume of the tumor mice and the administration time was recorded. At the end of the experiment, the tumor mice were euthanized, the tumors were removed, weighed, and photographed. The tumor growth and tumor weight suppression rates were calculated and statistically analyzed, and the experimental results are shown in Figure 22 and Table 14. These experimental results show that WAm17 and WAm21 clearly exhibit tumor growth inhibitory activity under the current experimental conditions.

[0082] Table 13. Classification information for evaluating the efficacy of WAm17 and WAm21 in the mouse colorectal adenocarcinoma (MC38 CDA) tumor model. [Table 13]

[0083] Table 14 Results obtained from evaluating the efficacy of WAm17 and WAm21 in a mouse colorectal adenocarcinoma (MC38 CDA) tumor model. [Table 14]

[0084] The above embodiments are for illustrative purposes only and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they may modify the above-described technologies or replace some of their technical features with equivalents, and that such modifications and replacements will not cause the corresponding technologies to deviate from the spirit and scope of the technologies relating to each embodiment of the present invention. [Industrial applicability]

[0085] The antibodies provided in this invention that specifically bind to IL-18R1 and IL-18RAP have high affinity for IL-18R1 and IL-18RAP, respectively. The bispecific antibodies provided in this invention can efficiently bind both IL-18R1 and IL-18RAP simultaneously, activate the IL-18 signaling pathway, exert biological functions similar to IL-18 both in vitro and in vivo, have broad immunoenhancing effects as novel IL-18 receptor activators, can stimulate the proliferation and activation of NK cells and T cells, and the secretion of cytokines such as IFNγ, can avoid the negative feedback phenomenon of IL-18 pathway activation caused by IL-18BP antagonism, and possess controllable activation activity, superior molecular stability, and in vivo pharmacokinetic characteristics, which are advantageous for fully exhibiting antitumor efficacy in vivo, significantly suppressing tumor growth in in vivo experiments in animal models, and have a wide range of potential antitumor clinical applications.

Claims

1. An antibody that binds to the IL-18 receptor or an antigen-binding fragment thereof, The aforementioned antibody or its antigen-binding fragment is bound to IL-18R1 or IL-18RAP and includes a heavy chain variable region. An antibody or antigen-binding fragment that binds to the IL-18 receptor, characterized in that the complementarity-determining region of the heavy chain variable region in the antibody or antigen-binding fragment that binds to IL-18R1 is one of the following (1) to (5), and the complementarity-determining region of the heavy chain variable region in the antibody or antigen-binding fragment that binds to IL-18RAP is one of the following (6) to (8). (1) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown in SEQ ID NO. 27, or a sequence variant having at least 80% homology to the sequence shown in SEQ ID NO.

27. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is the one shown in SEQ ID NO. 28, or a sequence variant having at least 80% homology to the sequence shown in SEQ ID NO.

28. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is represented by SEQ ID NO. 29 or 51, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 29 or 51. (2) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown in SEQ ID NO. 30, or a sequence variant having at least 80% homology to the sequence shown in SEQ ID NO.

30. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is the one shown in SEQ ID NO. 31, or a sequence variant having at least 80% homology to the sequence shown in SEQ ID NO.

31. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is represented by SEQ ID NO. 32 or 52, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 32 or 52. (3) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown in SEQ ID NO. 33, or a sequence variant having at least 80% homology to the sequence shown in SEQ ID NO.

33. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is represented by SEQ ID NO. 34 or 53, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 34 or 53. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is either the sequence shown in SEQ ID NO. 35, or a sequence variant having at least 80% homology to the sequence shown in SEQ ID NO.

35. (4) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown in SEQ ID NO. 36, or a sequence variant having at least 80% homology to the sequence shown in SEQ ID NO.

36. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is represented by SEQ ID NO. 37 or 54, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 37 or 54. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is represented by SEQ ID NO. 38 or 55, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 38 or 55. (5) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown in SEQ ID NO. 39, or a sequence variant having at least 80% homology to the sequence shown in SEQ ID NO.

39. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is represented by SEQ ID NO. 40, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO.

40. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is either the sequence shown in SEQ ID NO. 41, or a sequence variant having at least 80% homology to the sequence shown in SEQ ID NO.

41. (6) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown in SEQ ID NO. 42, or a sequence variant having at least 80% homology to the sequence shown in SEQ ID NO.

42. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is represented by SEQ ID NO. 43 or 56, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 43 or 56. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is either the sequence represented by SEQ ID NO. 44, or a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO.

44. (7) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown in SEQ ID NO. 45, or a sequence variant having at least 80% homology to the sequence shown in SEQ ID NO.

45. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is represented by SEQ ID NO. 46 or 57, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 46 or 57. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is either the sequence represented by SEQ ID NO. 47, or a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO.

47. (8) The amino acid sequence of the complementarity-determining region CDR1 of the heavy chain variable region is the one shown by SEQ ID NO. 48, or a sequence variant having at least 80% homology to the sequence shown by SEQ ID NO.

48. The amino acid sequence of the complementarity-determining region CDR2 of the heavy chain variable region is represented by SEQ ID NO. 49 or 58, or is a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO. 49 or 58. The amino acid sequence of the complementarity-determining region CDR3 of the heavy chain variable region is either the sequence represented by SEQ ID NO. 50, or a sequence variant having at least 80% homology to the sequence represented by SEQ ID NO.

50.

2. The sequence variant having at least 80% homology is obtained by substituting any one amino acid in the original sequence with histidine, wherein the original sequence is the sequence indicated by any of SEQ ID NO. 27 to 58. Preferably, only one amino acid in the amino acid sequences of CDR1, CDR2, and CDR3 of each antibody or its antigen-binding fragment is substituted with histidine. An antibody or antigen-binding fragment thereof that binds to the IL-18 receptor as described in feature 1.

3. The amino acid sequence of the heavy chain variable region of the antibody or antigen-binding fragment that binds to IL-18R1 is one of SEQ ID NO. 7 to 11, or one of SEQ ID NO. 20 to 23, or a sequence variant having at least 80% homology to the sequence shown in either SEQ ID NO. 7 to 11 or SEQ ID NO. 20 to 23. and / or, an antibody that binds to IL-18RAP or an antigen-binding fragment thereof, wherein the amino acid sequence of the heavy chain variable region is represented by any of SEQ ID NO. 12-14, or by any of SEQ ID NO. 24-26, or a sequence variant having at least 80% homology to the sequence represented by any of SEQ ID NO. 12-14 or SEQ ID NO. 24-26. Preferably, the sequence variant having at least 80% homology is obtained by substituting one amino acid of CDR1, CDR2, and CDR3 of the sequence represented by any of SEQ ID NO. 7-14 or SEQ ID NO. 20-26 with histidine. An antibody or antigen-binding fragment thereof that binds to the IL-18 receptor according to feature 1 or 2.

4. For sequences represented by SEQ ID NO. 7 or 20, sequence variants having at least 80% homology were obtained by substituting the amino acid at positions 27, 28, 29, 30, 31, 32, 34, 35, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, or 110 of the sequence represented by SEQ ID NO. 7 or 20 with histidine. and / or, For sequences indicated by SEQ ID NO. 10 or 23, sequence variants having at least 80% homology are obtained by substituting the amino acid at positions 31, 32, 33, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 107, 108, 109, 110, 111, 112, or 29 of the sequence indicated by SEQ ID NO. 10 or 23 with histidine, or SEQ ID NO. This is obtained by substituting the amino acids at positions 52 and 59, 52 and 105, 52 and 107, 59 and 99, 59 and 105, 59 and 107, 59 and 109, 99 and 107, 105 and 109, 52 and 112, 59 and 112, 101 and 112, or 105 and 112 of the sequence indicated by 10 or 23 with histidine. and / or, For sequences represented by SEQ ID NO. 13 or 25, sequence variants having at least 80% homology were obtained by substituting the amino acid at positions 161, 162, 163, 164, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 228, 229, 230, 231, 232, 233, 234, or 235 of the sequence represented by SEQ ID NO. 13 or 25 with histidine. An antibody or antigen-binding fragment thereof that binds to the IL-18 receptor as described in feature 3.

5. The aforementioned antibody or its antigen-binding fragment may be a single-domain antibody, mini-antibody, bispecific antibody, multispecific antibody, monoclonal antibody, single-chain antibody, Fab, Fab', F(ab') ２ , Fd, Fv, scFv, BsFv, dsFv, or (dsFv) ２ And, and / or, The aforementioned antibody or its antigen-binding fragment is a camel-derived antibody, a humanized antibody, a mouse-derived antibody, a rabbit-derived antibody, or a chimeric antibody. An antibody or antigen-binding fragment thereof that binds to the IL-18 receptor according to any one of claims 1 to 4.

6. A bispecific antibody comprising an antibody that binds to the IL-18 receptor as described in any one of claims 1 to 5, or an antigen-binding fragment thereof, Preferably, the bispecific antibody comprises one or more antibodies or antigen-binding fragments that bind to IL-18R1, and one or more antibodies or antigen-binding fragments that bind to IL-18RAP. A bispecific antibody characterized by the following features.

7. It is a bispecific antibody, In the aforementioned bispecific antibody, the amino acid sequence of the antibody or its antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 7, and the amino acid sequence of the antibody or its antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 12, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 8, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 14, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 8, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 13, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 9, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 14, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 9, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 13, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 10, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 13, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 11, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 12, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown as SEQ ID NO. 20, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown as SEQ ID NO. 24, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 21, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 26, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 21, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 25, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 22, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 26, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 22, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 25, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 23, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 25, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 11, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 24, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is obtained by substituting the amino acids at positions 27, 28, 29, 30, 31, 32, 34, 35, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, or 110 of the sequence shown in SEQ ID NO. 20 with histidine, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 24, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is obtained by substituting the amino acid at positions 31, 32, 33, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 107, 108, 109, 110, 111, 112, or 29 of the sequence shown in SEQ ID NO. 23 with histidine, or SEQ ID NO. This is obtained by substituting the amino acids at positions 52 and 59, 52 and 105, 52 and 107, 59 and 99, 59 and 105, 59 and 107, 59 and 109, 99 and 107, 105 and 109, 52 and 112, 59 and 112, 101 and 112, or 105 and 112 of the sequence shown in 23 with histidine, and the amino acid sequence of the antibody that binds to IL-18RAP or its antigen-binding fragment is shown in SEQ ID NO. 25, Alternatively, the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18R1 is shown in SEQ ID NO. 23, and the amino acid sequence of the antibody or antigen-binding fragment that binds to IL-18RAP is shown in SEQ ID NO. 25, and obtained by substituting the amino acid at positions 161, 162, 163, 164, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 228, 229, 230, 231, 232, 233, 234, or 235 with histidine. The bispecific antibody according to feature 6.

8. The bispecific antibody according to claim 7, characterized by comprising one of the structural domains described in any one of the following (1) to (21). (1) The amino acid sequence is the sequence shown in SEQ ID NO. 7, the linker, and the sequence shown in SEQ ID NO. 12, in order from the N-terminus to the C-terminus. (2) The amino acid sequence is the sequence shown in SEQ ID NO. 8, the linker, and the sequence shown in SEQ ID NO. 14, in order from the N-terminus to the C-terminus. (3) The amino acid sequence is the sequence shown in SEQ ID NO. 8, the linker, and the sequence shown in SEQ ID NO. 13, in order from the N-terminus to the C-terminus. (4) The amino acid sequence is the sequence shown in SEQ ID NO. 9, the linker, and the sequence shown in SEQ ID NO. 14, in order from the N-terminus to the C-terminus. (5) The amino acid sequence is the sequence shown in SEQ ID NO. 9, the linker, and the sequence shown in SEQ ID NO. 13, in order from the N-terminus to the C-terminus. (6) The amino acid sequence is the sequence shown in SEQ ID NO. 10, the linker, and the sequence shown in SEQ ID NO. 13, in order from the N-terminus to the C-terminus. (7) The amino acid sequence is the sequence shown in SEQ ID NO. 12, the linker, and the sequence shown in SEQ ID NO. 7, in order from the N-terminus to the C-terminus. (8) The amino acid sequence is the sequence shown in SEQ ID NO. 13, the linker, and the sequence shown in SEQ ID NO. 9, in order from the N-terminus to the C-terminus. (9) The amino acid sequence is the sequence shown in SEQ ID NO. 13, the linker, and the sequence shown in SEQ ID NO. 10, in order from the N-terminus to the C-terminus. (10) The amino acid sequence is the sequence shown in SEQ ID NO. 12, the linker, and the sequence shown in SEQ ID NO. 11, in order from the N-terminus to the C-terminus. (11) The amino acid sequence is the sequence shown in SEQ ID NO. 20, the linker, and the sequence shown in SEQ ID NO. 24, in order from the N-terminus to the C-terminus. (12) The amino acid sequence is the sequence shown in SEQ ID NO. 21, the linker, and the sequence shown in SEQ ID NO. 26, in order from the N-terminus to the C-terminus. (13) The amino acid sequence is the sequence shown in SEQ ID NO. 21, the linker, and the sequence shown in SEQ ID NO. 25, in order from the N-terminus to the C-terminus. (14) The amino acid sequence is the sequence shown in SEQ ID NO. 22, the linker, and the sequence shown in SEQ ID NO. 26, in order from the N-terminus to the C-terminus. (15) The amino acid sequence is the sequence shown in SEQ ID NO. 22, the linker, and the sequence shown in SEQ ID NO. 25, in order from the N-terminus to the C-terminus. (16) The amino acid sequence is the sequence shown in SEQ ID NO. 23, the linker, and the sequence shown in SEQ ID NO. 25, in order from the N-terminus to the C-terminus. (17) The amino acid sequence is the sequence shown in SEQ ID NO. 24, the linker, and the sequence shown in SEQ ID NO. 20, in order from the N-terminus to the C-terminus. (18) The amino acid sequence is the sequence shown in SEQ ID NO. 25, the linker, and the sequence shown in SEQ ID NO. 22, in order from the N-terminus to the C-terminus. (19) The amino acid sequence is the sequence shown in SEQ ID NO. 25, the linker, and the sequence shown in SEQ ID NO. 23, in order from the N-terminus to the C-terminus. (20) The amino acid sequence is the sequence shown in SEQ ID NO. 24, the linker, and the sequence shown in SEQ ID NO. 11, in order from the N-terminus to the C-terminus. (21) The amino acid sequence is the sequence shown in SEQ ID NO. 20, the linker, and the sequence shown in SEQ ID NO. 24, in order from the N-terminus to the C-terminus, and is obtained by substituting the amino acid at positions 27, 28, 29, 30, 31, 32, 34, 35, 50, 51, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 109, or 110 with histidine. (22) A sequence obtained by substituting the amino acid at positions 31, 32, 33, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 107, 108, 109, 110, 111, 112 or 29 of the sequence shown in SEQ ID NO. 23, in order from the N-terminus to the C-terminus, with histidine, or SEQ ID NO. The sequence obtained by substituting the amino acids at positions 52 and 59, 52 and 105, 52 and 107, 59 and 99, 59 and 105, 59 and 107, 59 and 109, 99 and 107, 105 and 109, 52 and 112, 59 and 112, 101 and 112, or 105 and 112 of the sequence shown in 23 with histidine is the linker, the sequence shown in SEQ ID NO.

25. (23) A sequence obtained by substituting histidine at the amino acids at positions 161, 162, 163, 164, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 228, 229, 230, 231, 232, 233, 234, or 235 of the sequence indicated by SEQ ID NO. 23, linker, or SEQ ID NO. 25, in order from the N-terminus to the C-terminus.

9. The bispecific antibody according to any one of claims 6 to 8, further comprising one or more selected from KappaL, CH1, CH2, CH3, hinge region, and KIH structure.

10. A fusion protein comprising an antibody that binds to the IL-18 receptor according to any one of claims 1 to 5 or an antigen-binding fragment thereof, or a bispecific antibody according to any one of claims 6 to 9. Preferably, the fusion protein is obtained by fusing an antibody that binds to the IL-18 receptor as described in any one of claims 1 to 5 or an antigen-binding fragment thereof, or a bispecific antibody as described in any one of claims 6 to 9, with another protein. More preferably, the fusion protein is characterized in that the other protein comprises one or more selected from protein tags, Fab, Fc, HSA, cytokines, and antibodies.

11. A bifunctional protein comprising an antibody or antigen-binding fragment thereof that binds to the IL-18 receptor as described in any one of claims 1 to 5, or a bispecific antibody as described in any one of claims 6 to 9, and further comprising a target protein, antibody, or cytokine. Preferably, the antibody is an immune checkpoint antibody and / or a tumor target antibody, making it a bifunctional protein.

12. A nucleic acid molecule characterized by encoding an antibody that binds to the IL-18 receptor according to any one of claims 1 to 5 or an antigen-binding fragment thereof, or a bispecific antibody according to any one of claims 6 to 9, or a fusion protein according to claim 10, or a bifunctional protein according to claim 11.

13. A biomaterial characterized by comprising an expression cassette, vector, or host cell containing the nucleic acid molecule described in claim 12.

14. Recombinant host cells are obtained by introducing a nucleic acid molecule encoding an antibody that binds to the IL-18 receptor according to any one of claims 1 to 5 or an antigen-binding fragment thereof, or a bispecific antibody according to any one of claims 6 to 9, or a fusion protein according to claim 10, or a bifunctional protein according to claim 11, into host cells. The recombinant host cells are cultured and isolated to obtain an antibody or its antigen-binding fragment that binds to the IL-18 receptor, the bispecific antibody, or the fusion protein. A method for producing an antibody that binds to an IL-18 receptor according to any one of claims 1 to 5, or an antigen-binding fragment thereof, or a bispecific antibody according to any one of claims 6 to 9, or a fusion protein according to claim 10, or a bifunctional protein according to claim 11.

15. The use of any of the following: an antibody or antigen-binding fragment thereof that binds to the IL-18 receptor as described in any one of claims 1 to 5; a bispecific antibody as described in any one of claims 6 to 9; a fusion protein as described in claim 10; a bifunctional protein as described in claim 11; a nucleic acid molecule as described in claim 12; or a biological material as described in claim 13. (1) Use in the manufacture of antitumor drugs (2) Use in the manufacture of drugs that enhance the body's immune response (3) Use in the manufacture of drugs to activate the IL-18 signaling pathway (4) Use in the manufacture of drugs to stimulate the secretion of INFγ and / or GZMB (5) Use in the manufacture of IL-18 receptor activators (6) Use in the manufacture of drugs to prevent or treat diseases associated with the IL-18 receptor (7) Use in the manufacture of drugs to measure the content of IL-18 receptors

16. An IL-18 receptor activator characterized by comprising an antibody that binds to the IL-18 receptor according to any one of claims 1 to 5 or an antigen-binding fragment thereof, or a bispecific antibody according to any one of claims 6 to 9, or a fusion protein according to claim 10, or a bifunctional protein according to claim 11.

17. A pharmaceutical composition characterized by comprising an antibody that binds to the IL-18 receptor according to any one of claims 1 to 5 or an antigen-binding fragment thereof, or a bispecific antibody according to any one of claims 6 to 9, or a fusion protein according to claim 10, or a bifunctional protein according to claim 11.

18. A detection reagent characterized by comprising an antibody that binds to the IL-18 receptor according to any one of claims 1 to 5 or an antigen-binding fragment thereof, or a bispecific antibody according to any one of claims 6 to 9, or a fusion protein according to claim 10, or a bifunctional protein according to claim 11.

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