New il-18 substitute and use thereof

Abstract

Provided are a new IL-18 substitute and the use thereof. Specifically, provided is a class of nanobodies targeting an IL-18 receptor. The IL-18Rb nanobody and the IL-18Ra nanobody specifically bind to an IL-18Rb chain and IL-18Ra chain of an IL-18 receptor, respectively, and a bispecific antibody based on the two monospecific nanobodies can effectively stimulate PBMC to release IFNγ, and exhibit a significant and effective tumor inhibitory effect. Therefore, the provided antibody can be used as a therapeutic long-acting substitute factor for IL-18, and provides a new strategy for clinical disease treatment.

Description

Novel IL-18 alternatives and their uses Technical Field

[0001] This invention relates to the field of antibody drugs, specifically to a novel IL-18 substitute, namely a nanobody targeting the IL-18 receptor and its application. Background Technology

[0002] IL-18 (interleukin-18), belonging to the IL-1 family, strongly induces the production of interferon-γ (IFN-γ) in cells. IL-18 exists intracellularly in an inactive precursor form (pro-IL-18), primarily produced by macrophages and T cells. During inflammation, it is rapidly secreted as active IL-18 by the recombinant NLRP3 inflammasome component caspase-1. IL-18 mediates downstream signaling pathways through receptor binding. The IL-18 receptor consists of IL-18Ra and IL-18Rb chains. Upon binding of IL-18 and IL-18Ra, IL-18Rb undergoes a conformational change to form a trimer, thereby activating the downstream MyD88-NFκB-mediated signaling pathway, promoting the activation and proliferation of immune cells, and the secretion of IFN-γ cytokines, thus possessing tumor-killing functions.

[0003] However, the anti-tumor efficacy of recombinant IL-18 in clinical trials was not ideal. Subsequent studies revealed that IL-18BP is highly expressed in the tumor microenvironment of patients. IL-18BP is a naturally occurring pseudoreceptor for IL-18 and has an extremely high affinity for IL-18 (KD < 1 nM). IL-18BP binds to IL-18 and blocks its interaction with cell surface receptors, thus blocking the activation of downstream IL-18 signaling pathways and rendering natural IL-18 ineffective. Therefore, how to bypass the strong binding of IL-18BP and exert a stronger immune cell activating effect is a pressing problem in IL-18 drug development.

[0004] Some studies have reported the use of antibodies targeting IL-18BP to block IL-18BP in the tumor microenvironment, thereby releasing the pro-inflammatory function of natural IL-18. However, such therapies have limited efficacy and are ineffective in patients with non-IL-18BP high expression. Other studies have reported IL-18 mutants, which also circumvent IL-18BP interference and exert potent effects, but their short in vivo half-life necessitates frequent injections, reducing patient adherence. Currently, no nanobodies targeting this target have been reported. Therefore, researching and developing novel therapeutic long-acting IL-18 substitutes based on nanobodies is of great significance and may provide new clinical treatment strategies.

[0005] Summary of the Invention

[0006] The purpose of this invention is to provide a nanobody that targets the IL-18 receptor, and the use of said nanobody in detection and disease treatment.

[0007] In a first aspect of the invention, an anti-IL-18Rb nanobody is provided, the nanobody specifically binding to IL-18Rb, wherein its VHH chain contains the following complementarity-determining region (CDR):

[0008] CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7, and CDR3 shown in SEQ ID NO:8.

[0009] In another preferred embodiment, the VHH chain of the anti-IL-18Rb nanobody further includes a framework region (FR), which includes human FR and camel-derived FR.

[0010] In another preferred embodiment, the VHH chain of the anti-IL-18Rb nanobody has an amino acid sequence as shown in SEQ ID NO:2.

[0011] In another preferred embodiment, the VHH chain of the anti-IL-18Rb nanobody has an amino acid sequence that has at least 75%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

[0012] In another preferred embodiment, the anti-IL-18Rb nanobody includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0013] In another preferred embodiment, the anti-IL-18Rb nanobody is capable of recognizing and binding to IL-18Rb in humans and cynomolgus monkeys.

[0014] In a second aspect of the invention, an anti-IL-18Rb antibody is provided, the antibody comprising a VHH chain of an anti-IL-18Rb nanobody as described in the first aspect of the invention.

[0015] In another preferred embodiment, the anti-IL-18Rb antibody comprises one or more VHH chains having an amino acid sequence as shown in SEQ ID NO:2.

[0016] In another preferred embodiment, the anti-IL-18Rb antibody comprises a monomer, a bivalent (bivalent antibody), a tetravalent (tetravalent antibody), and / or a multivalent (multivalent antibody).

[0017] In a third aspect of the invention, an anti-IL-18Rb nanobody Fc fusion protein is provided, the structure of the fusion protein from the N-terminus to the C-terminus being as shown in Formula Ia or Ib:

[0018] ALB(Ia);

[0019] BLA(Ib);

[0020] in,

[0021] "-" indicates a peptide bond;

[0022] A is one or more anti-IL-18Rb nanobodies as described in the first aspect of the present invention;

[0023] B is the Fc fragment of IgG; and

[0024] L represents no or flexible joints.

[0025] In another preferred embodiment, the flexible connector is a connecting peptide.

[0026] In another preferred embodiment, the Fc fragment of the IgG comprises the Fc fragment of human IgG.

[0027] In another preferred embodiment, the Fc fragment of the IgG is selected from the group consisting of the Fc fragments of IgG1, IgG2, IgG3, IgG4, or combinations thereof.

[0028] In another preferred embodiment, the linker peptide is selected from the following sequence: (G a S b ) x -(G m S n ) y , where a, b, m, n, x, y = 0 or 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 (preferably, a = 4 and b = 1, m = 3 and n = 1).

[0029] In a fourth aspect of the invention, a multispecific antibody is provided, the multispecific antibody comprising the VHH chain of the anti-IL-18Rb nanobody as described in the first aspect of the invention.

[0030] In a fifth aspect of the invention, a bispecific antibody is provided, the bispecific antibody comprising a VHH chain of an anti-IL-18Rb nanobody as described in the first aspect of the invention, and a VHH chain of an anti-IL-18Ra nanobody that specifically binds to IL-18Ra.

[0031] In another preferred embodiment, the VHH chain of the anti-IL-18Ra nanobody includes the following complementarity-determining region (CDR):

[0032] CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4, and CDR3 shown in SEQ ID NO:5.

[0033] In another preferred embodiment, the VHH chain of the anti-IL-18Ra nanobody further includes a framework region (FR), which includes human FR and camel-derived FR.

[0034] In another preferred embodiment, the VHH chain of the anti-IL-18Ra nanobody has an amino acid sequence as shown in SEQ ID NO:1.

[0035] In another preferred embodiment, the VHH chain of the anti-IL-18Ra nanobody has an amino acid sequence that has at least 75%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1.

[0036] In another preferred embodiment, the anti-IL-18Ra nanobody includes humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0037] In another preferred embodiment, the anti-IL-18Ra nanobody is capable of recognizing and binding to IL-18Ra in humans and cynomolgus monkeys.

[0038] In another preferred embodiment, the bispecific antibody further comprises an Fc segment.

[0039] In another preferred embodiment, the structure of the bispecific antibody from the N-terminus to the C-terminus is as shown in formula IIa or IIb:

[0040] in,

[0041] "-" represents a peptide bond. It is a disulfide bond;

[0042] N1 is the VHH chain of the anti-IL-18Ra nanobody;

[0043] N2 is the VHH chain of the anti-IL-18Rb nanobody;

[0044] P is either no or a connector;

[0045] H represents the hinge region of the antibody; and

[0046] Fc represents the Fc segment of the antibody.

[0047] In another preferred embodiment, the VHH chain of the anti-IL-18Ra nanobody includes the following complementarity-determining region (CDR):

[0048] CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4, and CDR3 shown in SEQ ID NO:5; and

[0049] The VHH chain of the anti-IL-18Rb nanobody contains the following complementarity-determining regions (CDRs):

[0050] CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7, and CDR3 shown in SEQ ID NO:8.

[0051] In another preferred embodiment, the VHH chain of the anti-IL-18Ra nanobody has the amino acid sequence shown in SEQ ID NO:1, and the VHH chain of the anti-IL-18Rb nanobody has the amino acid sequence shown in SEQ ID NO:2.

[0052] In another preferred embodiment, P is a linker, and the amino acid sequence of the linker is selected from the group consisting of: AA, AAA, GS, (GGGS). n (GGGGS) n , where n is a positive integer from 1 to 7.

[0053] In another preferred embodiment, the amino acid sequence of P is GS.

[0054] In another preferred embodiment, the amino acid sequence of H is as shown in SEQ ID NO:10 or 11.

[0055] In another preferred embodiment, the Fc is selected from Human IgG1 Fc, Human IgG2 Fc, Human IgG3 Fc, Human IgG4 Fc or variants thereof.

[0056] In another preferred embodiment, the amino acid sequence of the Fc is shown in SEQ ID NO:12.

[0057] In another preferred embodiment, the structure of the bispecific antibody from the N-terminus to the C-terminus is shown in Formula IIa; wherein the amino acid sequence of “N1-P-N2” is shown in SEQ ID NO:9.

[0058] In a sixth aspect of the invention, a recombinant protein is provided, the recombinant protein comprising:

[0059] (c1) the anti-IL-18Rb nanobody as described in the first aspect of the present invention, the anti-IL-18Rb antibody as described in the second aspect of the present invention, the anti-IL-18Rb nanobody Fc fusion protein as described in the third aspect of the present invention; and

[0060] (c2) Optional tag sequence for expression and / or purification.

[0061] In another preferred embodiment, the tag sequence includes (but is not limited to) the Flag tag, the HA tag, and the 6His tag.

[0062] In a seventh aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a polypeptide or protein selected from the group consisting of: an anti-IL-18Rb nanobody as described in the first aspect of the invention, an anti-IL-18Rb antibody as described in the second aspect of the invention, an anti-IL-18Rb nanobody Fc fusion protein as described in the third aspect of the invention, a multispecific antibody as described in the fourth aspect of the invention, a bispecific antibody as described in the fifth aspect of the invention, or a recombinant protein as described in the sixth aspect of the invention.

[0063] In another preferred embodiment, the polynucleotide includes DNA or RNA.

[0064] In an eighth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the seventh aspect of the invention.

[0065] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0066] In another preferred embodiment, the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0067] In a ninth aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the eighth aspect of the invention, or having a genome containing polynucleotides as described in the seventh aspect of the invention.

[0068] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0069] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0070] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0071] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Trichoderma, or combinations thereof.

[0072] In another preferred embodiment, the host cell is Pichia pastoris.

[0073] In a tenth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0074] (a) an anti-IL-18Rb nanobody as described in the first aspect of the present invention, an anti-IL-18Rb antibody as described in the second aspect of the present invention, an anti-IL-18Rb nanobody Fc fusion protein as described in the third aspect of the present invention, a multispecific antibody as described in the fourth aspect of the present invention, or a bispecific antibody as described in the fifth aspect of the present invention; and

[0075] (b) The coupling part selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

[0076] In an eleventh aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0077] (i) anti-IL-18Rb nanobody as described in the first aspect of the present invention, anti-IL-18Rb antibody as described in the second aspect of the present invention, anti-IL-18Rb nanobody Fc fusion protein as described in the third aspect of the present invention, multispecific antibody as described in the fourth aspect of the present invention, bispecific antibody as described in the fifth aspect of the present invention, or immunoconjugate as described in the tenth aspect of the present invention; and

[0078] (ii) Pharmaceutically acceptable carriers.

[0079] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating and / or preventing diseases or conditions associated with the IL-18 / IL-18R pathway.

[0080] In another preferred embodiment, the diseases or conditions associated with the IL-18 / IL-18R pathway include cancer or tumors, autoimmune diseases, and inflammatory diseases.

[0081] In another preferred embodiment, the diseases or conditions associated with the IL-18 / IL-18R pathway include (but are not limited to) solid tumors, pancreatic cancer, lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, triple-negative breast cancer, ovarian tumors, myeloma, lymphoma, leukemia, non-Hodgkin's lymphoma, multiple myeloma, rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, inflammatory bowel disease, Crohn's disease, Behcet's disease, pulmonary sarcoidosis, as well as hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), adult-onset Still's disease, systemic juvenile idiopathic arthritis (SJIA), cytokine release syndrome, XIAP deficiency, hidradenitis suppurativa, autoinflammation with infantile enterocolitis, and Behcet's disease.

[0082] In another preferred embodiment, the pharmaceutical composition further comprises other drugs for treating cancer or tumors, autoimmune diseases, or inflammatory diseases.

[0083] In another preferred embodiment, the pharmaceutical composition further comprises an immune checkpoint inhibitor, such as an anti-PD-1 / PD-L1 antibody or an anti-CTLA-4 antibody.

[0084] In another preferred embodiment, the pharmaceutical composition comprises: a bispecific antibody as described in the fifth aspect of the invention; an anti-PD-1 / PD-L1 antibody or an anti-CTLA-4 antibody; and a pharmaceutically acceptable carrier.

[0085] In another preferred embodiment, the anti-PD-1 antibody is Nivolumab.

[0086] In another preferred embodiment, the anti-CTLA-4 antibody is Ipilimumab.

[0087] In a twelfth aspect of the invention, an active ingredient combination is provided, the active ingredient combination comprising: (Z1) a bispecific antibody as described in the fifth aspect of the invention; and (Z2) an immune checkpoint inhibitor.

[0088] In another preferred embodiment, the immune checkpoint inhibitor is selected from: anti-PD-1 / PD-L1 antibody or anti-CTLA-4 antibody.

[0089] In another preferred embodiment, the anti-PD-1 antibody is Nivolumab.

[0090] In another preferred embodiment, the anti-CTLA-4 antibody is Ipilimumab.

[0091] In a thirteenth aspect of the present invention, a medicine box is provided, the medicine box comprising:

[0092] (C1) A first formulation, wherein the first formulation contains a bispecific antibody as described in the fifth aspect of the present invention and a pharmaceutically acceptable carrier;

[0093] (C2) A second formulation, wherein the second formulation contains an immune checkpoint inhibitor and a pharmaceutically acceptable carrier;

[0094] (C3) Specification, which describes a method of using a bispecific antibody and an immune checkpoint inhibitor as described in the fifth aspect of the invention in combination for the synergistic treatment of cancer or tumors.

[0095] In another preferred embodiment, the first and second formulations are independent of each other.

[0096] In another preferred embodiment, the first and second formulations are lyophilized or liquid formulations.

[0097] In another preferred embodiment, the first and second formulations are injectable formulations.

[0098] In another preferred embodiment, the first formulation is applied before, during, or after the application of the second formulation.

[0099] In another preferred embodiment, the immune checkpoint inhibitor is selected from: anti-PD-1 / PD-L1 antibody or anti-CTLA-4 antibody.

[0100] In another preferred embodiment, the anti-PD-1 antibody is Nivolumab.

[0101] In another preferred embodiment, the anti-CTLA-4 antibody is Ipilimumab.

[0102] In another preferred embodiment, the cancer or tumor includes: solid tumors, pancreatic cancer, lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, triple-negative breast cancer, ovarian tumors, myeloma, lymphoma, leukemia, non-Hodgkin lymphoma, and multiple myeloma.

[0103] In a fourteenth aspect of the invention, the use of the anti-IL-18Rb nanobody as described in the first aspect of the invention, the anti-IL-18Rb antibody as described in the second aspect of the invention, the anti-IL-18Rb nanobody Fc fusion protein as described in the third aspect of the invention, the recombinant protein as described in the sixth aspect of the invention, or the immunoconjugate as described in the tenth aspect of the invention is provided for the preparation of a reagent for detecting IL-18Rb protein in a sample.

[0104] In another preferred embodiment, the detection includes flow cytometry, ELISA, and immunofluorescence assay.

[0105] In another preferred embodiment, the detection is a non-diagnostic, non-therapeutic in vitro detection.

[0106] In a fifteenth aspect of the present invention, the use of an anti-IL-18Rb nanobody as described in the first aspect of the present invention, an anti-IL-18Rb antibody as described in the second aspect of the present invention, an anti-IL-18Rb nanobody Fc fusion protein as described in the third aspect of the present invention, a multispecific antibody as described in the fourth aspect of the present invention, a bispecific antibody as described in the fifth aspect of the present invention, or an immunoconjugate as described in the tenth aspect of the present invention is provided for the preparation of a medicament for the treatment and / or prevention of diseases or conditions related to the IL-18 / IL-18R pathway.

[0107] In another preferred embodiment, the diseases or conditions associated with the IL-18 / IL-18R pathway include cancer or tumors, autoimmune diseases, and inflammatory diseases.

[0108] In another preferred embodiment, the diseases or conditions associated with the IL-18 / IL-18R pathway include (but are not limited to) solid tumors, pancreatic cancer, lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, triple-negative breast cancer, ovarian tumors, myeloma, lymphoma, leukemia, non-Hodgkin's lymphoma, multiple myeloma, rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, inflammatory bowel disease, Crohn's disease, Behcet's disease, pulmonary sarcoidosis, as well as hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), adult-onset Still's disease, systemic juvenile idiopathic arthritis (SJIA), cytokine release syndrome, XIAP deficiency, hidradenitis suppurativa, autoinflammation with infantile enterocolitis, and Behcet's disease.

[0109] In a sixteenth aspect of the invention, a method for detecting IL-18Rb protein in a sample is provided, the method comprising the steps of:

[0110] (S1) Contact the sample with the anti-IL-18Rb nanobody as described in the first aspect of the present invention, or the anti-IL-18Rb antibody as described in the second aspect of the present invention, the anti-IL-18Rb nanobody Fc fusion protein as described in the third aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, or the immunoconjugate as described in the tenth aspect of the present invention.

[0111] (S2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of IL-18Rb protein in the sample.

[0112] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic in vitro method.

[0113] In a seventeenth aspect of the present invention, an IL-18R1 protein detection reagent is provided, the detection reagent comprising:

[0114] (D1) The anti-IL-18Rb nanobody as described in the first aspect of the present invention, or the anti-IL-18Rb antibody as described in the second aspect of the present invention, the anti-IL-18Rb nanobody Fc fusion protein as described in the third aspect of the present invention, the recombinant protein as described in the sixth aspect of the present invention, or the immunoconjugate as described in the tenth aspect of the present invention; and

[0115] (D2) A carrier that is scientifically acceptable for detection.

[0116] In another preferred embodiment, the conjugation portion of the immunoconjugate is a diagnostic isotope, a fluorescent marker, or a chemiluminescent marker.

[0117] In another preferred embodiment, the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0118] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0119] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0120] In another preferred embodiment, the test reagent is in liquid or powder form, such as aqueous solution, injection, lyophilized powder, tablet, lozenge, or inhaler.

[0121] In an eighteenth aspect of the invention, a kit for detecting IL-18Rb protein is provided, the kit comprising the detection reagents as described in the seventeenth aspect of the invention, and instructions.

[0122] In another preferred embodiment, the instruction manual states that the kit is used for non-invasive detection of IL-18Rb expression in a test subject.

[0123] In a nineteenth aspect of the invention, a method for treating and / or preventing diseases or conditions associated with the IL-18 / IL-18R pathway is provided, comprising the steps of administering to a subject in need an anti-IL-18Rb nanobody as described in a first aspect of the invention, an anti-IL-18Rb antibody as described in a second aspect of the invention, an anti-IL-18Rb nanobody Fc fusion protein as described in a third aspect of the invention, a multispecific antibody as described in a fourth aspect of the invention, a bispecific antibody as described in a fifth aspect of the invention, an immunoconjugate as described in a tenth aspect of the invention, or a pharmaceutical composition as described in an eleventh aspect of the invention.

[0124] In another preferred embodiment, the diseases or conditions associated with the IL-18 / IL-18R pathway include cancer or tumors, autoimmune diseases, and inflammatory diseases.

[0125] In another preferred embodiment, the diseases or conditions associated with the IL-18 / IL-18R pathway include (but are not limited to) solid tumors, pancreatic cancer, lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, triple-negative breast cancer, ovarian tumors, myeloma, lymphoma, leukemia, non-Hodgkin's lymphoma, multiple myeloma, rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, inflammatory bowel disease, Crohn's disease, Behcet's disease, pulmonary sarcoidosis, as well as hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), adult-onset Still's disease, systemic juvenile idiopathic arthritis (SJIA), cytokine release syndrome, XIAP deficiency, hidradenitis suppurativa, autoinflammation with infantile enterocolitis, and Behcet's disease.

[0126] In another preferred embodiment, the desired subject is a human or a non-human mammal (e.g., a cynomolgus monkey).

[0127] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0128] Figure 1 shows the results of ELISA detection of the binding activity between IL-18Ra nanobody and human IL-18Ra protein.

[0129] Figure 2 shows the results of ELISA detection of the binding activity between the IL-18Ra nanobody and monkey IL-18Ra protein.

[0130] Figure 3 shows the results of ELISA detection of the binding activity between IL-18Rb nanobody and human IL-18Rb protein.

[0131] Figure 4 shows the results of ELISA detection of the binding activity between IL-18Rb nanobody and human IL-18Rb protein.

[0132] Figure 5 shows the results of FACS detection of the binding activity of IL-18Ra nanobody with human IL-18Ra highly expressed cell line.

[0133] Figure 6 shows the results of FACS detection of the binding activity of IL-18Ra nanobody with cynomolgus monkey IL-18Ra highly expressed cell line.

[0134] Figure 7 shows the results of FACS detection of the binding activity of IL-18Rb nanobody with human IL-18Rb-overexpressing cell lines.

[0135] Figure 8 shows the results of FACS detection of the binding activity of IL-18Rb nanobody with cynomolgus IL-18Rb-overexpressing cell line.

[0136] Figure 9 is a schematic diagram of the structure of the IL-18Ra / IL-18Rb bispecific antibody.

[0137] Figure 10 shows the results of IL-18Ra / IL-18Rb bispecific antibody stimulating human PBMCs to release INFγ.

[0138] Figure 11 shows the results of human PBMCs releasing INFγ stimulated by the modified IL-18Ra / IL-18Rb bispecific antibody.

[0139] Figure 12 shows the sequencing results of RNA regulating gene expression in CD8+ T cells by bispecific antibodies.

[0140] Figure 13 shows the efficacy results of the bispecific antibody in the PK59 mouse tumor model.

[0141] Figure 14 shows the efficacy results of the bispecific antibody in the H23 mouse tumor model.

[0142] Figure 15 shows the efficacy results of bispecific antibody combined with PD1 and CTL1A4 inhibitors in the PK59 mouse tumor model. Detailed Implementation

[0143] Through extensive and in-depth research and screening, the inventors unexpectedly discovered for the first time a class of nanobodies targeting the IL-18 receptor, including nanobodies targeting IL-18Rb and IL-18Ra. The nanobodies of this invention exhibit good binding activity to IL-18Rb and IL-18Ra in humans and cynomolgus monkeys, respectively. Furthermore, based on the obtained monospecific nanobodies, the inventors constructed a bispecific antibody simultaneously targeting IL-18Rb and IL-18Ra. The constructed bispecific antibody effectively stimulates PBMCs to secrete IFNγ cytokines, serving as a long-acting IL-18 alternative, and demonstrated significant tumor-suppressive effects in a mouse tumor model. In addition, the inventors unexpectedly discovered that the bispecific antibody of this invention, when used in combination with immune checkpoint inhibitors such as anti-PD-1 antibodies or anti-CTL1A4 antibodies, has a synergistic tumor-suppressive effect.

[0144] Based on this, the present invention was completed.

[0145] the term

[0146] As used herein, the terms “anti-IL-18Ra nanobody of the present invention”, “IL-18Ra nanobody of the present invention”, “anti-IL-18Ra nanobody”, and “IL-18Ra nanobody” have the same meaning and can be used interchangeably, all referring to nanobodies that specifically recognize and bind to IL-18Ra (including human IL-18Ra and cynomolgus monkey IL-18Ra).

[0147] As used herein, the terms “anti-IL-18Rb nanobody of the present invention”, “IL-18Rb nanobody of the present invention”, “anti-IL-18Rb nanobody”, and “IL-18Rb nanobody” have the same meaning and can be used interchangeably, all referring to nanobodies that specifically recognize and bind to IL-18Rb (including human IL-18Rb and cynomolgus monkey IL-18Rb).

[0148] As used herein, the terms "bispecific antibody of the present invention" and "bispecific antibody of the present invention" have the same meaning and refer to bispecific antibodies that specifically recognize and bind to IL-18Ra and IL-18Rb.

[0149] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetragonal glycoprotein of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. There are two types of light chains, λ(l) and κ(k). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE, which can be further divided into different subclasses (isotypes), such as human IgG, which can be divided into IgG1, IgG2, IgG3, and IgG4; and IgA, which can be divided into IgA1 and IgA2. Subclasses of IgM, IgD, and IgE have not yet been identified. Each chain contains different sequence domains. The light chain comprises two domains or regions: a variable domain (VL) and a constant domain (CL). The heavy chain comprises four domains: a heavy chain variable region (VH) and three constant regions (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine antigen binding recognition and specificity. The constant domains (CL) of the light chain and the constant regions (CH) of the heavy chain confer important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding, and binding to the Fc receptor (FcR). The Fv fragment is the N-terminal portion of the immunoglobulin Fab fragment and consists of a variable portion of one light chain and one heavy chain. Antibody specificity depends on the structural complementarity between the antibody binding site and the antigen-determining region. The antibody binding site is composed primarily of residues from the highly variable region or complementarity-determining region (CDR). Occasionally, residues from non-highly variable regions or framework regions (FR) affect the overall domain structure and thus the binding site. Complementarity-determining regions (CDRs) are specific amino acid sequences that collectively define the binding affinity and the native Fv region of the immunoglobulin binding site. Each of the light and heavy chains of immunoglobulins has three CDRs, designated CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR3-H, respectively. Therefore, the conventional antibody-antigen binding site comprises six CDRs, containing a set of CDRs from the v region of each heavy and light chain.

[0150] As used herein, the terms "single domain antibody (sdAb)," "nanobody," and "heavy chain antibody" have the same meaning and are used interchangeably. They refer to the cloning of the variable region of an antibody heavy chain to construct a nanobody consisting of only one heavy chain variable region. This is the smallest antigen-binding fragment with complete function, the VHH chain. Typically, antibodies lacking both the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody consisting of only one heavy chain variable region.

[0151] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0152] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0153] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0154] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3.

[0155] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0156] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0157] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0158] The antibodies of the present invention also include variants of polypeptides containing the CDR region described in the present invention that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein.

[0159] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0160] The present invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the nanobodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0161] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0162] Table A

[0163] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0164] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0165] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0166] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0167] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0168] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0169] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0170] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0171] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0172] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0173] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0174] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0175] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0176] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes that can produce detectable products.

[0177] Therapeutic agents that can bind to or conjugate with the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0178] The IL-18Ra nanobody, IL-18Rb nanobody, and bispecific antibody of the present invention

[0179] Through extensive screening and verification, this invention has obtained multiple IL-18Ra nanobodies specifically targeting IL-18Ra and IL-18Rb nanobodies specifically targeting IL-18Rb. Based on the obtained monospecific antibodies, bispecific antibodies that simultaneously target IL-18Ra and IL-18Rb have been constructed.

[0180] In a preferred embodiment of the present invention, the amino acid sequence of the VHH chain of the anti-IL-18Ra nanobody is shown in SEQ ID NO:1, and it includes CDR1 as shown in SEQ ID NO:3, CDR2 as shown in SEQ ID NO:4 and CDR3 as shown in SEQ ID NO:5.

[0181] In a preferred embodiment of the present invention, the amino acid sequence of the VHH chain of the anti-IL-18Rb nanobody is shown in SEQ ID NO:2, which includes CDR1 as shown in SEQ ID NO:6, CDR2 as shown in SEQ ID NO:7 and CDR3 as shown in SEQ ID NO:8.

[0182] In a preferred embodiment of the present invention, the bispecific antibody comprises the anti-IL-18Ra nanobody and the anti-IL-18Rb nanobody as described above. In a specific embodiment, the structure of the bispecific antibody is shown in FIG9, which is formed by the polymerization of two polypeptide chains with the structure N1-P-N2-H-Fc or N2-P-N1-H-Fc; wherein, N1 is the VHH chain of the above-mentioned anti-IL-18Ra nanobody; N2 is the VHH chain of the above-mentioned anti-IL-18Rb nanobody; P is absent or linked; H is the hinge region of the antibody; and Fc is the Fc segment of the antibody.

[0183] Linkers that can be used to construct the bispecific antibodies of the present invention include adaptor peptides known in the art suitable for linking the variable region of antibodies, including but not limited to AA, AAA, GS, and (GGGS). n (GGGGS) n , where n is a positive integer from 1 to 7. In a preferred embodiment of the present invention, the connector used is GS.

[0184] As used in this article, the hinge region refers to the region located between CH1 and CH2 of the antibody. It is rich in proline, so it is easy to stretch and bend, which can change the distance between the two arms of the Y-shape, which is conducive to the simultaneous binding of antigen epitopes by both arms.

[0185] As used herein, the terms "Fc," "Fc segment," "Fc fragment," "Fc domain," and "Fc region" refer to crystallizable fragments that lack antigen-binding activity and are the sites where antibodies interact with effector molecules or cell surface Fc receptors (FcRs). Fc fragments bind to cells with corresponding Fc receptors on their surface, producing different biological effects. In ADCC (antibody-dependent cell-mediated cytotoxicity), the antibody's Fab segment binds to antigenic epitopes on virus-infected cells or tumor cells, while its Fc segment binds to FcRs on the surface of killer cells (NK cells, macrophages, etc.), mediating the direct killing of target cells by killer cells. The Fc fragment comprises the constant region polypeptide of the antibody excluding the CH1 constant region of the heavy chain, namely the two constant region domains CH2 and CH3 at the carboxyl terminus of the constant region of the heavy chain of human immunoglobulins IgA, IgD, and IgG, and the three constant region domains CH2, CH3, and CH4 at the carboxyl terminus of the constant region of the heavy chain of human immunoglobulins IgE and IgM. The Fc fragments of this invention include natural Fc fragments and modified Fc fragments. The Fc fragments are typically selected from Human IgG1 Fc, Human IgG2 Fc, Human IgG3 Fc, Human IgG4 Fc, or variants thereof, preferably from IgG1 Fc or Human IgG4 Fc or variants thereof. In some embodiments, the Fc region comprises the same or different subunits. In some embodiments, the Fc region of the human IgG heavy chain is defined as an amino acid residue extending from the Cys226 position or from Pro230 to its carboxyl terminus. Unless otherwise stated, the Fc region is numbered using the EU index. The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; or it can be a truncated C-terminus, for example, in which one or two C-terminal amino acid residues have been removed. In a preferred embodiment, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In some embodiments, the Fc segment of the present invention can be selectively mutated to eliminate immune effector function, including but not limited to combinations of the following mutations (according to EU index numbers): N297Q, N297A, L234A / L235A; L234A / L235A / P329G, L234F / L235E / P331S, etc. for IgG1; S228P / L235E, S228P / F234A / L235E, S228P / L235E / P329G, etc. for IgG4. In some embodiments, the Fc segment of the present invention includes a modification based on the knife-into-hole (KIH) technique, which involves introducing a protrusion (knob) at the interface of the first subunit and a hole (hole) at the interface of the second subunit.This allows the protrusion structure to be positioned within the pore structure, promoting heterodimer formation and inhibiting homodimer production. The protrusion structure is constructed by replacing the small amino acid side chain at the interface of the first subunit with a larger side chain (e.g., tyrosine or tryptophan). The pore structure is created at the interface of the second subunit by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). Both the protrusion and pore structures are prepared by altering the nucleic acid encoding the polypeptide. Optional KIH mutation combinations are shown in the table below.

[0186] KIH mutant combination

[0187] The active ingredient combination of the present invention

[0188] This invention also provides an active ingredient combination comprising the bispecific antibody of this invention and an immune checkpoint inhibitor (e.g., anti-PD-1 / PD-L1 antibody or anti-CTLA-4 antibody) as active components. Experiments have demonstrated that the bispecific antibody of this invention (e.g., A4B2-mdFc) combined with anti-PD-1 antibody or anti-CTLA-4 antibody exhibits a significantly enhanced tumor-suppressive effect, demonstrating a synergistic effect.

[0189] Furthermore, the present invention also provides pharmaceutical compositions and kits containing the above-described combination of active ingredients for the treatment and / or prevention of cancer or tumors, including but not limited to pancreatic cancer and lung cancer.

[0190] Pharmaceutical Composition

[0191] The present invention also provides a pharmaceutical composition comprising the aforementioned antibody or its active fragment or fusion protein or conjugate, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intraperitoneal, intravenous, or local administration.

[0192] The pharmaceutical compositions of the present invention can be directly used to bind to IL-18 receptor molecules and block the IL-18 / IL-18R pathway, and thus can be used to treat diseases related to the IL-18 / IL-18R pathway, including cancer or tumors, autoimmune or inflammatory diseases such as pancreatic cancer, lung cancer, rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, inflammatory bowel disease, and rare diseases such as hemophagocytic lymphohistiocytosis (HLH) and adult-onset Still disease.

[0193] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the antibody (or conjugate thereof) described above, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the pharmaceutical compositions of the present invention may also contain other therapeutic agents or be used in conjunction with other therapeutic agents.

[0194] When using a pharmaceutical composition, a safe and effective amount of the active ingredient is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0195] Detection methods

[0196] The present invention also relates to a method for detecting IL-18Rb protein. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of IL-18Rb protein in the dissolved samples using the antibody (or a fragment thereof) or its conjugate described in this invention.

[0197] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0198] Reagent test kit

[0199] The present invention also provides a kit containing the antibody (or fragment thereof) or conjugate thereof as a detection reagent. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0200] This invention also provides a detection kit for detecting IL-18Rb levels. The kit includes the antibody described in this invention that recognizes the IL-18Rb protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0201] application

[0202] The nanobody of this invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of diseases related to the IL-18 / IL-18R pathway, basic medical research, and biological research. A preferred application is for clinical diagnosis and targeted therapy against the IL-18 / IL-18R signaling pathway.

[0203] The main advantages of this invention are:

[0204] (1) The antibody of the present invention has good binding activity and can bind to human and cynomolgus monkey IL-18Ra and IL-18Rb at the same time.

[0205] (2) The antibody of the present invention has good functional activity and has a significantly stronger stimulatory effect on INFγ release than natural IL-18.

[0206] (3) The antibody of the present invention regulates the gene expression of cells without being interfered with by IL-18BP.

[0207] (4) The antibody of the present invention has a good tumor suppression effect and can be used for the treatment of cancers or tumors related to the IL-18 / IL-18R pathway.

[0208] (5) The antibodies of the present invention have a synergistic effect when used in combination with a variety of immune checkpoint inhibitors, providing a more effective treatment option for tumor immunotherapy.

[0209] The following specific embodiments further illustrate the present invention. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments without specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, the materials, reagents, instruments, etc., used in the embodiments are commercially available.

[0210] Example 1: Screening of nanobodies targeting the IL-18 receptor

[0211] The extracellular fragments of human IL-18Ra and human IL-18Rb genes were cloned and synthesized into the pFUSE vector according to the optimized human codon base sequences. The vector was then transfected into HEK293F cells, and the human IL-18Ra ECD-Fc and IL-18Rb ECD-Fc fusion proteins were purified by protein A affinity chromatography. The high-purity proteins were mixed with an immunoadjuvant and used to immunize three Bactrian camels from Xinjiang once a week. After seven immunizations, peripheral blood was collected from the camels, RNA was isolated, and the VHH gene fragment was transcribed and amplified. This fragment was then cloned into the pMECS vector and electroporated into TG1 competent cells to construct a high-quality phage display nanobody library. All libraries had a volume of 1×10⁻⁶. 8 CFU or higher, with fragment insertion rates all above 85%. Then, phage display technology was used to screen for IL-18Ra and IL-18Rb specific nanobodies. After 2-4 rounds of "adsorption-washing-enrichment" processes, significant phage enrichment of specific nanobodies was observed in all six libraries. Multiple clones were randomly selected from each library for initial screening using supernatant flow cytometry.

[0212] Stable 293T cells (293T-huIL-18Ra and 293T-huIL-18Rb) expressing high levels of human IL-18Ra and IL-18Rb were added to 96-well plates at a rate of 200,000 cells per well. 100 μL of lysed clonal supernatant was added and the cells were incubated at 4°C for 40 min. After washing the cells twice with PBS, diluted anti-HA-APC secondary antibody was added and the cells were incubated at 4°C for 30 min. After washing the cells twice with PBS, 200 μL of PBS was added to each well to resuspend the cells and the cells were analyzed by flow cytometry.

[0213] A total of 9 IL-18Ra nanobodies and 15 IL-18Rb nanobodies were obtained.

[0214] Example 2: Identification of the binding activity of IL-18Ra and IL18Rb nanobodies

[0215] ELISA was used to identify the binding activity of candidate antibodies: 100 μL of human and monkey IL-8R1 and human and monkey IL-8R2 antigens were coated in each well overnight; then blocked with 1% BSA at room temperature for 2 hours; serially diluted antibodies were added to the blocked antigen-coated plates and incubated at 37°C for 1 hour; after washing three times with PBST, diluted secondary antibody anti-HA-HRP was added and incubated at 37°C for 1 hour; after washing three times with PBST, TMB chromogenic buffer was added, and finally 2 mM H2SO4 was added. The plates were then read and detected using an OD450 microplate reader.

[0216] The results are shown in Figures 1-4, and all candidate antibodies exhibited good binding activity.

[0217] Further FACS was used to identify the binding activity of the candidate antibodies: 293T-human IL-18Ra, 293T-human IL-18Rb, 293T-cyno IL-18Ra, and 293T-cyno IL-18Rb high-expression cell lines were added to 96-well plates, serially diluted antibodies were added, and the plates were incubated at 4°C for 40 minutes; after washing twice with PBS, diluted secondary antibody anti-HA-APC was added, and the plates were incubated at 4°C for 30 minutes; after washing twice with PBS, 200 μL of PBS was added to each well to resuspend the cells, and flow cytometry was used for detection.

[0218] The results are shown in Figures 5-8, and all candidate antibodies exhibited good binding activity.

[0219] Example 3: Construction and expression of IL-18Ra / IL-18Rb bispecific nanobody

[0220] Nine IL-18Ra and 15 IL-18Rb were orthogonally combined to form heterobivalents, which were then fused with human Fc for expression. The antibody structure is shown in Figure 9. A total of 270 bivalent antibody combinations were formed and expressed using 293F cells. Briefly, the combined bivalent nanobodies were cloned into the pFUSE vector, mixed with PEI, and transfected into HEK 293F cells for 5 days to express bispecific antibodies. The bispecific antibodies were purified from the cell culture supernatant using protein A affinity chromatography.

[0221] Example 4: Detection of the activity of candidate bispecific nanobodies in stimulating the release of INFγ from human PBMCs

[0222] Human PBMCs were resuspended in 1640 complete medium and seeded at 200,000 cells per well in 96-well plates. 1 nM candidate antibody and 10 ng / mL IL-12 were added, and the plates were incubated overnight for 17-24 hours. The supernatant was then used for ELISA detection. Mouse anti-INFγ was coated onto the ELISA plate at a concentration of 1 μg / mL and incubated overnight at 4°C. The plates were blocked with 1% BSA at room temperature for 2 hours. Diluted cell supernatant was added and the plates were incubated at room temperature for 1 hour. After washing the plates three times with PBST, anti-INFγ-Biotin antibody was added and the plates were incubated at 37°C for 30 minutes. After washing the plates three times with PBST, SA-HRP was added, followed by TMB staining. Finally, 2 mM H2SO4 was added, and the plates were read using an OD450 microplate reader.

[0223] As shown in Figure 10, multiple candidate antibodies exhibited good stimulation of INFγ release.

[0224] Example 5: Modification of candidate bispecific nanobodies

[0225] To further enhance the functional activity of the combined antibodies, the linkers between different nanobodies were modified, and linkers of different lengths were designed for connecting different parts.

[0226] Candidate bispecific antibodies A4B2 (i.e., a combination of anti-IL-18Ra nanobody Nb4 (the amino acid sequence of the VHH chain is shown in SEQ ID NO:1) and anti-IL-18Rb nanobody Nb8 (the amino acid sequence of the VHH chain is shown in SEQ ID NO:2)) were selected. The two VHH chains were linked using different linkers and fused with the Fc segment through a hinge region to construct the bispecific antibody structure shown in Figure 9. The amino acid sequence of the hinge region used in this embodiment is shown in SEQ ID NO:10 or 11; the amino acid sequence of the Fc segment is shown in SEQ ID NO:12. The constructed bispecific antibodies are A4B2-Fc (Linker 1), A4B2-Fc (Linker 2), A4B2-Fc (Linker 3), and A4B2-Fc (Linker 4). The antigen-binding portion of A4B2-Fc (Linker 1) is shown in SEQ ID NO:9. The difference between A4B2-Fc (Linker 3) and A4B2-Fc (Linker 4) and A4B2-Fc (Linker 1) is that the linker sequence GS in the A4B2-Fc (Linker 1) sequence is replaced with GGS, GGGS and GGGGS, respectively.

[0227] After expressing the antibody using HEK293 cells, the activity was identified again, and the specific detection method was the same as in Example 4.

[0228] As shown in Figure 11, the linkers of different lengths between nanobodies have slightly different effects on antibody function, but all of them have a good effect on stimulating the release of INFγ.

[0229] Example 6: Regulation of gene expression in human CD8+ T cells by candidate bispecific nanobodies

[0230] The effect of the candidate bispecific nanobody prepared in Example 5 on gene expression regulation of human CD8+ T cells was detected.

[0231] CD8+ T cells were isolated from PBMCs of different healthy individuals and resuspended in 1640 medium containing FBS and human IL-12. The cells were then treated with the following methods: isotype control, IL-18, IL-18 + IL-18BP, A4B2-mdFc, and A4B2-mdFc + IL-18BP. After incubation at 37°C for 24 hours, cells were collected, RNA was extracted, and analyzed using TruSeq. TMRNA libraries were constructed using the RNA Sample Preparation Kit, and high-throughput sequencing was performed using the Illumina HiSeq 10 / NovaSeq 6000 sequencing platform. Differential expression was analyzed using DESeq2, and differentially expressed genes (DEGs) were identified by |log2FC|≥1 and FDR<0.05.

[0232] As shown in Figure 12, IL-18, A4B2-mdFc and A4B2-mdFc+IL-18BP have similar trends in regulating gene expression in cells, and the effect of A4B2-mdFc is not affected by IL-18BP.

[0233] Example 7: Antitumor effect of candidate bispecific nanobodies in a PK59 pancreatic cancer mouse model

[0234] The antitumor effect of the candidate bispecific nanobody prepared in Example 5 was tested in the PK59 pancreatic cancer mouse model.

[0235] PK-59 cells in logarithmic growth phase were resuspended in PBS and mixed with 50% Matrigel. 10 cells were seeded. 7 5 × 10⁵ cells were injected subcutaneously into the right rib area of ​​male NCG-severely immunodeficient mice. One week later, each mouse was injected via the tail vein. 6 PBMCs were used to reconstruct the immune system, and tumor volume was monitored in mice simultaneously. Tumor volume was increased to 100 mm². 3 At approximately 3:00 PM, tumor-bearing mice were randomly divided into 5 groups of 6 mice each. The mice in each group were treated with intraperitoneal injections of PBS, IL-18 (0.32 mg / kg), A4B2-mdFc (0.37 mg / kg), A4B2-mdFc (1.86 mg / kg), and A4B2-mdFc (9.32 mg / kg) (corresponding to Vehicle, IL-18, A4B2-mdFc L, A4B2-mdFc M, and A4B2-mdFc H in Figure 13, respectively), twice a week for 3 weeks. Tumor size and mouse condition were measured during the injection period.

[0236] As shown in Figure 13, the candidate antibody exhibited good tumor inhibition in this mouse model.

[0237] Example 8: Antitumor effect of candidate bispecific nanobodies in H23 lung cancer mouse model

[0238] The antitumor effect of the candidate bispecific nanobody prepared in Example 5 was detected in the H23 lung cancer mouse model.

[0239] H23 cells in logarithmic growth phase were resuspended in PBS and mixed with 50% Matrigel. 10 cells were seeded. 7 5 × 10⁵ cells were injected subcutaneously into the right rib area of ​​male NCG-severely immunodeficient mice. One week later, each mouse was injected via the tail vein. 6 PBMCs were used to reconstruct the immune system, and tumor volume was monitored in mice simultaneously. Tumor volume was increased to 100 mm². 3 At approximately 3:00 PM, tumor-bearing mice were randomly divided into 5 groups of 6 mice each. The mice in each group were treated with intraperitoneal injections of PBS, IL-18 (0.32 mg / kg), A4B2-mdFc (0.37 mg / kg), A4B2-mdFc (1.86 mg / kg), and A4B2-mdFc (9.32 mg / kg) (corresponding to Vehicle, IL-18, A4B2-mdFc L, A4B2-mdFc M, and A4B2-mdFc H in Figure 14, respectively), twice a week for 3 weeks. Tumor size and mouse condition were measured during the injection period.

[0240] As shown in Figure 14, the candidate antibody exhibited good tumor inhibition in this mouse model.

[0241] Example 9: Antitumor effect of candidate bispecific nanobody combined with PD1 and CTLA-4 inhibitors in PK59 pancreatic cancer mouse model

[0242] The antitumor effect of the candidate bispecific nanobody prepared in Example 5 in combination with PD1 and CTLA-4 inhibitors was tested in a PK59 pancreatic cancer mouse model.

[0243] PK-59 cells in logarithmic growth phase were resuspended in PBS and mixed with 50% Matrigel. 10 cells were seeded. 7 5 × 10⁵ cells were injected subcutaneously into the right rib area of ​​male NCG-severely immunodeficient mice. One week later, each mouse was injected via the tail vein. 6 PBMCs were used to reconstruct the immune system, and tumor volume was monitored in mice simultaneously. Tumor volume was increased to 100 mm². 3At approximately 3:00 PM, tumor-bearing mice were randomly divided into 6 groups, with 6 mice in each group. The 6 groups of mice were treated twice a week with intraperitoneal injections of PBS, A4B2-mdFc (1.86 mg / kg), Anti-PD-1 (Nivolumab, 5 mg / kg), A4B2-mdFc (1.86 mg / kg) combined with Anti-PD-1 (Nivolumab, 5 mg / kg), Anti-CTLA-4 (Ipilimumab, 5 mg / kg), and A4B2-mdFc (1.86 mg / kg) combined with Anti-CTLA-4 (Ipilimumab, 5 mg / kg), respectively, for 3 weeks. Tumor size and mouse condition were measured during the injection period.

[0244] As shown in Figure 15, the candidate antibody, together with PD1 inhibitor and CTLA-4 inhibitor, showed good inhibitory effects in this mouse model, and the combination had a synergistic effect.

Claims

1. An anti-IL-18Rb nanobody, said nanobody specifically binding to IL-18Rb, wherein its VHH chain contains the following complementarity-determining region (CDR): CDR1 shown in SEQ ID NO:6, CDR2 shown in SEQ ID NO:7, and CDR3 shown in SEQ ID NO:

8.

2. The anti-IL-18Rb nanobody as described in claim 1, characterized in that, The VHH chain of the anti-IL-18Rb nanobody has an amino acid sequence that has at least 75%, preferably at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

3. An anti-IL-18Rb antibody, characterized in that, The antibody comprises the VHH chain of the anti-IL-18Rb nanobody as described in claim 1.

4. An anti-IL-18Rb nanobody Fc fusion protein, wherein the structure of the fusion protein from the N-terminus to the C-terminus is as shown in Formula Ia or Ib: ALB(Ia); BLA(Ib); in, "-" represents a peptide bond; A is one or more anti-IL-18Rb nanobodies as described in claim 1; B is the Fc fragment of IgG; and L represents no or flexible joints.

5. A multispecific antibody, characterized in that, The multispecific antibody comprises the VHH chain of the anti-IL-18Rb nanobody as described in claim 1.

6. A bispecific antibody, characterized in that, The bispecific antibody comprises the VHH chain of the anti-IL-18Rb nanobody as described in claim 1, and the VHH chain of the anti-IL-18Ra nanobody that specifically binds to IL-18Ra.

7. The bispecific antibody as described in claim 6, characterized in that, The VHH chain of the anti-IL-18Ra nanobody contains the following complementarity-determining region (CDR): CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4, and CDR3 shown in SEQ ID NO:

5.

8. The bispecific antibody as described in claim 6, characterized in that, The structure of the bispecific antibody from the N-terminus to the C-terminus is shown in formula IIa or IIb: in, "-" represents a peptide bond, and "║" represents a disulfide bond; N1 is the VHH chain of the anti-IL-18Ra nanobody; N2 is the VHH chain of the anti-IL-18Rb nanobody; P is either no or a connector; H represents the hinge region of the antibody; and Fc represents the Fc segment of the antibody.

9. A recombinant protein, characterized in that, The recombinant protein comprises: (c1) the anti-IL-18Rb nanobody as described in claim 1, the anti-IL-18Rb antibody as described in claim 3, and the anti-IL-18Rb nanobody Fc fusion protein as described in claim 4; and (c2) Optional tag sequence for expression and / or purification.

10. A polynucleotide, characterized in that, The polynucleotide encodes a polypeptide or protein selected from the group consisting of: the anti-IL-18Rb nanobody as claimed in claim 1, the anti-IL-18Rb antibody as claimed in claim 3, the anti-IL-18Rb nanobody Fc fusion protein as claimed in claim 4, the multispecific antibody as claimed in claim 5, the bispecific antibody as claimed in claim 6, or the recombinant protein as claimed in claim 9.

11. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 10.

12. A host cell, characterized in that, The host cell contains the expression vector as described in claim 11, or its genome is integrated with the polynucleotide as described in claim 10.

13. An immunoconjugate, characterized in that, The immunoconjugate comprises: (a) the anti-IL-18Rb nanobody of claim 1, the anti-IL-18Rb antibody of claim 3, the anti-IL-18Rb nanobody Fc fusion protein of claim 4, the multispecific antibody of claim 5, or the bispecific antibody of claim 6; and (b) The coupling part selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, magnetic nanoparticles, viral capsid proteins or VLPs, or combinations thereof.

14. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the anti-IL-18Rb nanobody of claim 1, the anti-IL-18Rb antibody of claim 3, the anti-IL-18Rb nanobody Fc fusion protein of claim 4, the multispecific antibody of claim 5, the bispecific antibody of claim 6, or the immunoconjugate of claim 13; and (ii) Pharmaceutically acceptable carriers.

15. A combination of active ingredients, said combination of active ingredients comprising: (Z1) the bispecific antibody as described in claim 6; and (Z2) an immune checkpoint inhibitor.

16. A medicine box, the medicine box comprising: (C1) A first formulation, the first formulation comprising the bispecific antibody as described in claim 6 and a pharmaceutically acceptable carrier; (C2) A second formulation, wherein the second formulation contains an immune checkpoint inhibitor and a pharmaceutically acceptable carrier; (C3) Specification, which describes a method of combining the bispecific antibody and immune checkpoint inhibitor as described in claim 6 for the synergistic treatment of cancer or tumor.

17. The use of the anti-IL-18Rb nanobody as described in claim 1, the anti-IL-18Rb antibody as described in claim 3, the anti-IL-18Rb nanobody Fc fusion protein as described in claim 4, the recombinant protein as described in claim 9, or the immunoconjugate as described in claim 13, for the preparation of a reagent for detecting IL-18Rb protein in a sample.

18. Use of the anti-IL-18Rb nanobody of claim 1, the anti-IL-18Rb antibody of claim 3, the anti-IL-18Rb nanobody Fc fusion protein of claim 4, the multispecific antibody of claim 5, the bispecific antibody of claim 6, or the immunoconjugate of claim 13, for the preparation of a medicament for the treatment and / or prevention of diseases or conditions related to the IL-18 / IL-18R pathway.

19. The use as described in claim 18, characterized in that, The diseases or conditions associated with the IL-18 / IL-18R pathway include cancer or tumors, autoimmune diseases, and inflammatory diseases.

20. The use as described in claim 18, characterized in that, Diseases or conditions associated with the IL-18 / IL-18R pathway include solid tumors, pancreatic cancer, lung cancer, renal cell carcinoma, head and neck squamous cell carcinoma, triple-negative breast cancer, ovarian tumors, myeloma, lymphoma, leukemia, non-Hodgkin's lymphoma, multiple myeloma, rheumatoid arthritis (RA), systemic lupus erythematosus, type 1 diabetes, atopic dermatitis, psoriasis, inflammatory bowel disease, Crohn's disease, Behcet's disease, pulmonary sarcoidosis, as well as hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), adult-onset Still's disease, systemic juvenile idiopathic arthritis (SJIA), cytokine release syndrome, XIAP deficiency, hidradenitis suppurativa, autoinflammation with infantile enterocolitis, and Behcet's disease.

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