Heteromeric agonistic antibodies against il-18 receptor

Abstract

Provided herein are bispecific agonist antibodies specific for IL-18 receptor, as well as methods and compositions comprising the same.

Description

Related applications

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 545,278, filed October 23, 2023, the entire disclosure of which is hereby incorporated by reference. Background Technology

[0002] Interleukin-18 (IL-18) (also known as interferon-γ inducible factor, IGIF) is a pleiotropic pro-inflammatory cytokine that modulates both innate and adaptive immune system responses. IL-18 has been identified as playing an important role in regulating the inflammatory cascade, making it an ideal target for suppressing autoimmune diseases that include, but are not limited to, inflammatory diseases of the gut, heart, and lungs (Kaplanski G. Immunol. Rev. (2018); 281(1):138-153).

[0003] The IL-18 receptor (IL-18R) complex is a heterodimeric receptor (IL-18Rα / IL-Rβ) expressed on a variety of cells, including macrophages, neutrophils, natural killer (NK) cells, and antigen-experienced T cells (Gracie J.A. et al., J. Leukoc. Biol. (2003); 73(2): 213-24). Upon binding of IL-18 to the IL-18Rα subunit, IL-18Rβ is recruited to form a signaling pathway that induces a high-affinity complex shared with other members of the IL-1R family. These downstream signaling effector pathways are shared with other key immune regulatory molecules, such as Toll-like receptor cells (Gracie JA et al., J. Leukoc. Biol. (2003); 73(2): 213-24).

[0004] While early clinical trials are investigating the administration of therapeutic agents consisting solely of recombinant or engineered IL-18, several factors could lead to treatment failure. For example, IL-18 binds only to the extracellular signaling domain component IL-18Rα, but not to the adaptor molecule IL-18Rβ of the IL-18R receptor complex (Takei S. et al.; Arthritis Res. Ther. (2011); 13(2):R52). However, binding to both subunits of IL-18R is essential for signal transduction and therefore for the activation of inflammatory mediators. Recombinant IL-18 is rapidly neutralized by its endogenous inhibitor IL-18-binding protein (IL-18BP), which is induced by IL-18R signaling. Although IL-18 can be engineered to not bind to IL-18BP (Zhao T et al.; Nature; 583, 609-14 (2020)), mutant protein molecules carry a significant mutant load, making them potentially immunogenic and therefore readily neutralized by the host immune system. Recombinant or endogenous IL-18 is a high-affinity, short-half-life protein that is locally captured and rapidly eliminated at the application site, limiting its ability to induce signal transduction in tumors distal to the application site. Therefore, a stable IL-18R agonist that binds to both subunits with high affinity remains needed. Summary of the Invention

[0005] In one aspect, this document provides a multispecific binding protein comprising at least one first binding portion specifically binding to human interleukin-18 receptor α (IL-18Rα) and at least one second binding portion specifically binding to human interleukin-18 receptor β (IL-18Rβ), wherein the first binding portion comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17; and the second binding portion comprises the amino acid sequence of SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 32.

[0006] In some embodiments, the IL-18Rα binding moiety comprises: (a) an HCDR1 sequence containing the amino acid sequence of KHAMG (SEQ ID NO:10), The HCDR2 sequence containing the amino acid sequence AIDWSGGSTYYADSVKG (SEQ ID NO: 11), the HCDR3 sequence containing the amino acid sequence DSYTDYAQLWLPELESEYDY (SEQ ID NO: 12); or (b) the HCDR1 sequence containing the amino acid sequence SYTMG (SEQ ID NO: 13), the HCDR2 sequence containing the amino acid sequence AISWSAGRTYYADSVKG (SEQ ID NO: 14), the HCDR3 sequence containing the amino acid sequence EEAPDWAPIDCSGYGCLSLYDY (SEQ ID NO: 15).

[0007] In some embodiments, the IL-18Rβ binding moiety comprises: (a) an HCDR1 sequence comprising the amino acid sequence of SHNVMG (SEQ ID NO: 21), an HCDR2 sequence comprising the amino acid sequence of SIGSGGSTNYVDSVKG (SEQ ID NO: 22), and an HCDR3 sequence comprising the amino acid sequence of VVGVYRGS (SEQ ID NO: 23); or (b) an HCDR1 sequence comprising the amino acid sequence of RDTMG (SEQ ID NO: 18), an HCDR2 sequence comprising the amino acid sequence of VISSSGNTNYADSVLG (SEQ ID NO: 19), and an HCDR3 sequence comprising the amino acid sequence of HRTYGVDY (SEQ ID NO: 20).

[0008] In some embodiments, the first and second binding moieties are VHH domains. In some embodiments, the first and second binding moieties are located on the same polypeptide. In some embodiments, the first and second binding moieties are located on different polypeptides.

[0009] In some embodiments, the first and / or second binding moiety further comprises an amino acid substitution at the N-terminal position 14 of the binding moiety. In some embodiments, the amino acid at the N-terminal position 14 of the binding moiety is proline (P). In some embodiments, the substitution at the N-terminal position 14 of the binding moiety comprises alanine (A). In some embodiments, the substitution at the N-terminal position 14 of the binding moiety is alanine (A). In some embodiments, the substitution at the N-terminal position 14 of the binding moiety further stabilizes the binding moiety. In some embodiments, the substitution at the N-terminal position 14 of the binding moiety increases the agonist properties of the binding moiety.

[0010] In some implementations, the multispecific binding protein also includes one or more modified hinge regions.

[0011] In some embodiments, the one or more hinge regions include an upper hinge region of a length of up to 7 amino acids or the absence of an upper hinge region; and a middle hinge region and a lower hinge region, wherein the lower hinge region is connected to the N-terminus of the heavy chain constant region. In some embodiments, the upper hinge regions of the first modified hinge region and the second modified hinge region have the same sequence. In some embodiments, the upper hinge regions of the first modified hinge region and the second modified hinge region have different sequences.

[0012] In some embodiments, the upper hinge region comprises an amino acid sequence derived from the upper hinge region of a human IgG antibody. In some embodiments, the IgG antibody is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the IgG antibody is IgG1. In some embodiments, the upper hinge region comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the upper hinge region comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the IgG antibody is IgG4. In some embodiments, the upper hinge region comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, there is no upper hinge.

[0013] In some embodiments, the multispecific binding protein further comprises all or part of an immunoglobulin Fc domain or a variant thereof. In some embodiments, the Fc domain or a variant thereof comprises a first Fc heavy chain and a second Fc heavy chain.

[0014] In some embodiments, the multispecific binding protein further comprises a variant Fc domain having reduced effector function. In some embodiments, at least one Fc heavy chain comprises a substitution at amino acid position 234 according to EU numbering. In some embodiments, the substitution at amino acid position 234 is alanine (A). In some embodiments, at least one Fc heavy chain comprises a substitution at amino acid position 235 according to EU numbering. In some embodiments, the substitution at amino acid position 235 is alanine (A). In some embodiments, at least one Fc heavy chain comprises a substitution at amino acid position 237 according to EU numbering. In some embodiments, the substitution at amino acid position 237 is alanine (A).

[0015] In some embodiments, at least one Fc heavy chain contains one or more substitutions located at amino acid positions 234, 235, or 237 according to EU numbers. In some embodiments, the substitution at amino acid position 234 is alanine (A), the substitution at amino acid position 235 is alanine (A), and the substitution at amino acid position 237 is alanine (A).

[0016] In some embodiments, the Fc domain contains a heterodimerization mutation to promote heterodimerization of the first binding moiety with the second binding moiety. In some embodiments, the heterodimerization mutation is a kilometreotype (KIH) mutation. In some embodiments, the first Fc heavy chain contains a kilometreotype amino acid substitution at position 366, 368, or 407, and the second Fc heavy chain contains a kilometreotype amino acid substitution at position 366. In some embodiments, the first Fc heavy chain contains amino acid substitutions T366S, L368A, or Y407V, and the second Fc heavy chain contains amino acid substitution T366W.

[0017] In some embodiments, the heterodimerization mutation is a charge stabilization mutation. In some embodiments, the first Fc heavy chain contains an amino acid substituted for N297K, and the second Fc heavy chain contains an amino acid substituted for N297D.

[0018] In some embodiments, the first Fc heavy chain contains an amino acid substituted T299K, and the second Fc heavy chain contains an amino acid substituted T299D.

[0019] In some embodiments, the heterodimerization mutation comprises an engineered disulfide bond. In some embodiments, the engineered disulfide bond is formed by a first Fc heavy chain comprising an amino acid substituted for Y349C and a second Fc heavy chain comprising an amino acid substituted for S354C. In some embodiments, the engineered disulfide bond is formed by a C-terminal extension peptide fused to the C-terminus of each of the first and second Fc heavy chains. In some embodiments, the C-terminal extension of the first Fc heavy chain comprises the amino acid sequence GEC, and the C-terminal extension of the second Fc heavy chain comprises the amino acid sequence SCDKT.

[0020] In some embodiments, at least one Fc domain contains one or more mutations for promoting an increased half-life. In some embodiments, at least one Fc heavy chain contains one or more substitutions located at amino acid positions 252, 254, or 256 according to EU numbers. In some embodiments, the substitution at amino acid position 252 is tyrosine (Y), the substitution at amino acid position 254 is threonine (T), and the substitution at amino acid position 236 is glutamic acid (E).

[0021] In some embodiments, the first binding portion specifically binding to human IL-18Rα comprises the amino acid sequence shown in SEQ ID NO:26, and the second binding portion specifically binding to human IL-18Rβ comprises the amino acid sequence shown in any one of SEQ ID NO:27, 28, or 31.

[0022] In some embodiments, the binding protein comprises the amino acid sequence shown in SEQ ID NO: 29. In some embodiments, the binding protein further comprises an Fc region containing the amino acid sequence shown in SEQ ID NO: 30.

[0023] In some embodiments, the multispecific binding protein comprises a first polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO:26 (i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) and a second polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO:28 (i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0024] In some embodiments, the multispecific binding protein comprises a first polypeptide chain consisting of the amino acid sequence of SEQ ID NO:26 and a second polypeptide chain consisting of the amino acid sequence of SEQ ID NO:28.

[0025] In some embodiments, the multispecific binding protein comprises a first polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 29 (i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) and a second polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 30 (i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0026] In some embodiments, the multispecific binding protein comprises a first polypeptide chain consisting of the amino acid sequence of SEQ ID NO: 29 and a second polypeptide chain consisting of the amino acid sequence of SEQ ID NO: 30.

[0027] In some embodiments, the multispecific binding protein comprises a first polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 26 (i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) and a second polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 27 (i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0028] In some embodiments, the multispecific binding protein comprises a first polypeptide chain consisting of the amino acid sequence of SEQ ID NO:26 and a second polypeptide chain consisting of the amino acid sequence of SEQ ID NO:27.

[0029] In some embodiments, the multispecific binding protein comprises a first polypeptide chain having at least 80% identity (i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with the amino acid sequence of SEQ ID NO: 26 and a second polypeptide chain having at least 80% identity (i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) with the amino acid sequence of SEQ ID NO: 31.

[0030] In some embodiments, the multispecific binding protein comprises a first polypeptide chain consisting of the amino acid sequence of SEQ ID NO:26 and a second polypeptide chain consisting of the amino acid sequence of SEQ ID NO:31.

[0031] In one aspect, this document provides a pharmaceutical composition comprising the multispecific binding protein of any one of the preceding claims and a pharmaceutically acceptable carrier.

[0032] In another respect, this article provides isolated nucleic acid molecules encoding multispecific binding proteins as described herein.

[0033] In one respect, this article provides expression vectors containing nucleic acids as described herein.

[0034] In another respect, this article provides host cells containing expression vectors as described herein.

[0035] In one aspect, this disclosure provides a multispecific binding protein comprising a first binding portion specifically binding to human IL-18Rα and a second binding portion specifically binding to human IL-18Rβ, wherein the multispecific binding protein stimulates the production of antitumor cytokines but substantially does not stimulate the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers relative to the stimulation of IL-18 on the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers.

[0036] In some implementations, the antitumor cytokines are selected from the group consisting of IFN-γ, IL-2, IL-12, IL-15, CD40L and TNFα.

[0037] In some implementations, the markers for acute inflammation or Th2 response are selected from the group consisting of IL-6, IL-1β, IL-8, IL-4, IL-5 and IL-13.

[0038] In some implementations, antitumor cytokines and markers of acute inflammation or Th2 response are measured in peripheral blood mononuclear cell (PBMC) assays.

[0039] In some embodiments, the PBMC assay includes: 1) incubating a first PBMC population with a multispecific binding protein for at least 24 hours (e.g., 24, 36, 48, 60, 72, 84, or 96 hours); 2) incubating a second PBMC population with IL-18 for at least 24 hours (e.g., 24, 36, 48, 60, 72, 84, or 96 hours); and 3) measuring the production of antitumor cytokines and markers of acute inflammation or Th2 response from the first and second PBMC populations.

[0040] In some implementations, the multispecific binding protein stimulates the production of acute inflammatory or Th2 response markers at least 5, 10, 50, or 100 times less than IL-18.

[0041] In some implementations, the multispecific binding protein stimulates the production of acute inflammatory or Th2 response markers that are at least 5-fold, 10-fold, 50-fold, or 100-fold less than IL-18, as measured in a PBMC assay.

[0042] In some implementations, the production of GM-CSF stimulated by multispecific binding proteins is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than the production of IFN-γ.

[0043] In one aspect, this article provides a method for treating a disease or condition in a subject by administering to the subject a multispecific binding protein comprising a device for specifically binding to human IL-18Rα and a device for specifically binding to human IL-18Rβ.

[0044] In some aspects, this disclosure provides a method for inducing agonist activity of IL-18 receptor signaling in a subject, the method comprising administering to the subject a multispecific binding protein comprising a first binding portion specifically binding to human IL-18Rα and a second binding portion specifically binding to human IL-18Rβ, wherein the multispecific binding protein is capable of inducing IL-18 receptor signaling by inducing proximity between the IL-18Rα and IL-18Rβ subunits of human IL-18R.

[0045] In some aspects, this disclosure provides a method for inducing agonist activity of IL-18 receptor signaling in a subject, the method comprising administering to the subject a multispecific binding protein comprising a device for specifically binding to human IL-18Rα and a device for specifically binding to human IL-18Rβ.

[0046] In one aspect, this disclosure provides a method for stimulating IL-18-mediated IFN-γ expression in a subject, the method comprising administering to the subject a multispecific binding protein comprising a first binding portion specifically binding to human IL-18Rα and a second binding portion specifically binding to human IL-18Rβ, wherein the multispecific binding protein stimulates the production of antitumor cytokines in the subject but substantially does not stimulate the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers in the subject relative to the stimulation of IL-18 on the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers.

[0047] In some implementations, the antitumor cytokines are selected from the group consisting of IFN-γ, IL-2, IL-12, IL-15, CD40L and TNFα.

[0048] In some implementations, the markers for acute inflammation or Th2 response are selected from the group consisting of IL-6, IL-1β, IL-8, IL-4, IL-5 and IL-13.

[0049] In some implementations, the multispecific binding protein stimulates the production of acute inflammatory or Th2 response markers at least 5, 10, 50, or 100 times less than IL-18.

[0050] In some implementations, the production of GM-CSF in a subject stimulated by multispecific binding proteins is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than the production of IFN-γ in the subject.

[0051] In one aspect, this disclosure provides a method for treating a disease or condition in a subject, the method comprising administering the multispecific binding protein described herein to the subject in need.

[0052] In one aspect, this disclosure provides a method for stimulating IL-18R-mediated IFN-γ expression in a subject, the method comprising administering the multispecific binding protein described herein.

[0053] In some implementations, the multispecific binding protein stimulates the production of antitumor cytokines in the subject, but substantially does not stimulate the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers in the subject, relative to the stimulation of IL-18 on the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers.

[0054] In some implementations, the anti-tumor cytokines are selected from the group consisting of IFN-γ, IL-2, IL-12, IL-15, CD40L and TNFα.

[0055] In some implementations, the markers for acute inflammation or Th2 response are selected from the group consisting of IL-6, IL-1β, IL-8, IL-4, IL-5 and IL-13.

[0056] In some implementations, the multispecific binding protein stimulates the production of acute inflammatory or Th2 response markers at least 5, 10, 50, or 100 times less than IL-18.

[0057] In some implementations, the production of GM-CSF in subjects stimulated by multispecific binding proteins is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than the production of IFN-γ in subjects. Attached Figure Description

[0058] Figure 1 This is a graph illustrating the agonistic effects of DGL207, DGL333, and DGL620 as determined by HEK Blue.

[0059] Figures 2A-2H illustrate the effects of agonistic antibodies on various immune cell markers when cultured in the presence of PBMCs. The following markers are depicted: IFN-γ (Figure 2A), IL-5 (Figure 2B), IL-1β (Figure 2C), MCP-1 (Figure 2D), IL-6 (Figure 2E), IL-13 (Figure 2F), GM-CSF (Figure 2G), and IL-4 (Figure 2H).

[0060] Figure 3 These are gene expression data from PBMC samples cultured with IL-18R agonist antibodies. The data show that genes associated with IFNγ signaling, antiviral responses, NK and T cell activation, and lymphocyte signaling increased with increasing antibody levels, while neutrophil activation, monocyte activation, and pro-inflammatory signatures decreased.

[0061] Figures 4A and 4B depict the measurements of IFNγ and CD8 T cells in a graft-versus-host disease (GvHD) mouse model.

[0062] Figures 5A-5C The expression of CD69 on CD8 T cells over 21 days is shown for DGL336 (Fig. 5A), DGL346 (Fig. 5B), and DGL620 (Fig. 5C).

[0063] Figures 6A-6C The study demonstrates a 21-day reduction in CD159a receptors on CD56+ NK cells for DGL336 (Fig. 6A), DGL346 (Fig. 6B), and DGL620 (Fig. 6C).

[0064] Figures 7A-7C The expression of IFNγ over 21 days is shown for DGL336 (Fig. 7A), DGL346 (Fig. 7B), and DGL620 (Fig. 7C).

[0065] Figures 8A-8C The figure illustrates IL-2 expression over 21 days for DGL336 (Fig. 8A), DGL346 (Fig. 8B), and DGL620 (Fig. 8C).

[0066] Figures 9A-9C The figure shows the number of monocytes after exposure to DGL336 (Fig. 9A), DGL346 (Fig. 9B), or DGL620 (Fig. 9C) within 21 days.

[0067] Figures 10A-10C The figure illustrates IL-6 expression over 21 days for DGL336 (Fig. 10A), DGL346 (Fig. 10B), and DGL620 (Fig. 10C).

[0068] Figure 11A-11C The figure illustrates GM-CSF expression over 21 days for DGL336 (Fig. 11A), DGL346 (Fig. 11B), and DGL620 (Fig. 11C).

[0069] Figures 12A-12C The expression of IL-12 / 23 p40 over 21 days is shown for DGL336 (Fig. 12A), DGL346 (Fig. 12B), and DGL620 (Fig. 12C).

[0070] Figures 13A-13C The expression of TNFα over 21 days is shown for DGL336 (Fig. 13A), DGL346 (Fig. 13B), and DGL620 (Fig. 13C).

[0071] Figures 14A-14C The figure illustrates IL-15 expression over 21 days for DGL336 (Fig. 14A), DGL346 (Fig. 14B), and DGL620 (Fig. 14C).

[0072] Figures 15A-15C The figure illustrates sCD40L expression over 21 days for DGL336 (Fig. 15A), DGL346 (Fig. 15B), or DGL620 (Fig. 15C).

[0073] Figures 16A-16C The figure illustrates MCP-1 expression over 21 days for DGL336 (Fig. 16A), DGL346 (Fig. 16B), and DGL620 (Fig. 16C).

[0074] Figures 17A-17C The figure illustrates IL-4 expression over 21 days for DGL336 (Fig. 17A), DGL346 (Fig. 17B), and DGL620 (Fig. 17C).

[0075] Figures 18A-18C The figure illustrates IL-5 expression over 21 days for DGL336 (Fig. 18A), DGL346 (Fig. 18B), and DGL620 (Fig. 18C).

[0076] Figures 19A-19C The figure illustrates IL-13 expression over 21 days for DGL336 (Fig. 19A), DGL346 (Fig. 19B), and DGL620 (Fig. 19C). Detailed Implementation

[0077] Before describing this disclosure, it should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive, as the scope of this disclosure will be limited only by the appended claims.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0079] While any methods and materials similar to or equivalent to those described herein may be used in practice with this disclosure, exemplary methods and materials are described hereafter. All publications mentioned herein are incorporated by reference in order to provide a full description of them.

[0080] As used herein, the term "IL-18" or "interleukin-18" refers to a pro-inflammatory cytokine, also known as interferon-γ inducible factor (IGIF). In addition to its ability to induce interferon-γ, IL-18 has multiple functions, including activation of NF-κB, Fas ligand expression, and induction of CC and CXC chemokines. Because IL-18 can induce the production of interferon-γ in T cells and NK cells, it plays an important role in Th1 immune responses and participates in both innate and adaptive immunity. IL-18 is structurally and functionally related to the IL-1 family.

[0081] The biological activity of IL-18 is achieved through the binding of IL-18 to the heterodimeric IL-18 receptor (IL-18R), which consists of two subunits: the α subunit (a member of the IL-1R family, also known as IL-1R-associated protein-1 or IL-1Rrp1) and the β subunit (also known as IL-18R accessory protein, IL-18AP or AcPL). The IL-18R α subunit binds directly to IL-18 but does not participate in signal transduction. The β subunit itself does not bind to IL-18 but binds to the α subunit to form a high-affinity receptor (KD = ~0.3 nM) required for signal transduction (Sims, JE, (2002) Current Opin. Immunol. 14:117-122). IL-18 signal transduction via the IL-18R αβ complex is similar to that of the IL-1R and Toll-like receptor (TLR) systems. IL-18R signaling utilizes signal transduction molecules such as MyD88, IRAK, and TRAF6, and produces responses similar to IL-1 (e.g., activation of NIK, IκB kinase, NF-κB, INK, and p38 MAP kinase). The necessity of IL-18Rα and signal transduction molecules in mediating IL-18 biological activity has been confirmed by knockdown of the IL-18Rα subunit (Hoshino K., et al. (1999) J. Immunol. 162:5041-5044;), MyD88 (Adachi O., et al. (1998) Immunity 9:143-150), or IRAK (Kanakaraj P., (1999) J. Exp. Med. 189:1129-1138).

[0082] In some exemplary embodiments, the IL-18 cytokine is human IL-18 (Uniprot Q14116-1). In some exemplary embodiments, the IL-18 receptor is the human IL-18 receptor represented by sequences such as human IL-18Rα (Uniprot Q13478) and IL-18Rβ (Uniprot O95256-1).

[0083] As used herein, the terms “interleukin-18 receptor” or “IL-18R” or “IL18R” or “IL-18 receptor” refer to the receptor to which IL-18 binds. IL-18R is a member of the immunoglobulin superfamily. This receptor comprises two subunits: an α subunit (a member of the IL-1R family, also known as IL-1R-associated protein-1 or IL-1Rrp1) and a β subunit (also known as IL-18R accessory protein, IL-18AP, or AcPL). The IL-18R α subunit binds directly to IL-18 but does not participate in signal transduction. The β subunit itself does not bind to IL-18 but binds to the α subunit to form a high-affinity receptor (KD = ~0.3 nM) required for signal transduction (Sims, JE, (2002) Current Opin. Immunol. 14:117-122). In some embodiments, IL-18R is human IL-18R.

[0084] As used herein, the term "inducing proximity between the IL-18Rα and IL-18Rβ subunits of human IL-18R" refers to the aggregation of the IL-18Rα and IL-18Rβ subunits together, thereby stimulating human IL-18R activity. In some embodiments, the proximity induced by the multispecific binding protein of this disclosure is the same as or similar to the proximity induced when IL-18 aggregates the IL-18Rα and IL-18Rβ subunits of human IL-18R.

[0085] As used herein, the terms “antigen-binding moiety,” “binding domain,” or “binding specificity” refer to a molecule that specifically binds to an antigen, a binding that will be understood by those skilled in the art. For example, an antigen-binding moiety that specifically binds to an antigen typically binds to other molecules with a lower affinity (as determined by, for example, an immunoassay, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art). In some embodiments, the antigen-binding moiety that specifically binds to an antigen binds to that antigen with a Ka that is at least 2 log (e.g., a multiple of 10), 2.5 log, 3 log, 4 log, or higher than the Ka when the molecule nonspecifically binds to another antigen.

[0086] As used herein, the term “antibody” includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, heteroconjugate antibodies, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs (scFv), camel-derived antibodies (VHH), affinity antibodies, common light chain antibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In some embodiments, the antibodies described herein refer to a population of polyclonal antibodies. Antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecules. In some embodiments, the antibodies described herein are IgG antibodies, or their class (e.g., human IgG1 or IgG4) or subclass.

[0087] As used herein, the term “heavy chain” when used to refer to antibodies can refer to any different type of amino acid sequence based on a constant structural domain, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (µ), which produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including IgG subclasses such as IgG1, IgG2, IgG3, and IgG4.

[0088] As used in this article, the term "full-length antibody heavy chain" refers to an antibody heavy chain that includes VH, CH1 region, hinge region, CH2 domain and CH3 domain from the N-terminus to the C-terminus.

[0089] As used herein, the term "light chain" when used to refer to an antibody can refer to any different type of amino acid sequence based on a constant structural domain, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In a particular embodiment, the light chain is a human light chain. As used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids within the variable region of the antibody that confers antigen specificity and binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Frame region" or "FR," as known in the art, refers to the non-CDR portion of the variable regions of both the heavy and light chains. Generally, there are four FRs (FR-H1, FR-H2, FR-H3 and FR-H4) in each heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3 and FR-L4) in each light chain variable region.

[0090] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of many well-known schemes, including those described below: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD. (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., 2003 January; 27(1):55-77 (“IMGT” numbering scheme); and Honegger A. and Pluckthun A., “Yetanother numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J. Mol. Biol., 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme).

[0091] The boundaries of a given CDR or FR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on structure alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes number antibodies based on the length of the most common antibody region sequences, with insertions regulated by insert letters (e.g., "30a"), and deletions occurring in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, producing different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many ways.

[0092] As used herein, the term "single-chain variable fragment" (scFv) refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are sequentially linked via a short, flexible peptide linker and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise stated, as used herein, scFv may have VL and VH variable regions in either order; for example, with respect to the N-terminus and C-terminus of the polypeptide, scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0093] As used herein, the terms “VH” and “VL” refer to the variable domains of the antibody heavy and light chains, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety. As used herein, the term “VH / VL pair” refers to the combination of VH and VL that together form the antigen-binding site.

[0094] As used herein, the term “VHH domain” or “VHH antibody” refers to the heavy chain variable domain of camel-only heavy chain antibodies (HCAbs) and its humanized variants, as described in Hamers-Casterman C. et al., Nature (1993) 363:446–8.10.1038 / 363446a0, which is incorporated herein by reference in its entirety. The VHH domain or VHH antibody is also known as a “single-domain” antibody.

[0095] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain modified to have a high level of sequence homology with the human variable domain. This can be achieved by grafting six non-human antibody complementarity-determining regions (CDRs) that co-form the antigen-binding site onto a homologous human receptor frame region (FR). To fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to substitute frame residues from the parent antibody (i.e., the non-human antibody) into the human frame region (reversion mutation). Structural homology modeling may help identify amino acid residues in the frame region that are important for the antibody's binding properties. Therefore, a humanized antibody may contain a non-human CDR sequence (primarily a human frame region, which optionally contains one or more amino acid reversion mutations to a non-human amino acid sequence) and a fully human constant region. Optionally, additional amino acid modifications (not necessarily reversion mutations) may be applied to obtain humanized antibodies with preferred properties, such as affinity and biochemical properties.

[0096] As used herein, the term "multispecific antigen-binding molecule" refers to a bispecific, trispecific, or multispecific antigen-binding molecule and its antigen-binding fragments. A multispecific antigen-binding molecule may be specific to different epitopes of a target polypeptide, or may contain antigen-binding domains specific to epitopes of more than one target polypeptide. In some embodiments, the multispecific antigen-binding molecule of this disclosure comprises at least a first binding specificity against the IL-18Rα subunit and at least a second binding specificity against the IL-18Rβ subunit. A multispecific antigen-binding molecule may be a single multifunctional polypeptide, or it may be a multimeric complex of two or more polypeptides covalently or non-covalently associated with each other. The term "multispecific antigen-binding molecule" includes antibodies of this disclosure that can be linked to or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent association, or other means) to one or more other molecular entities, such as proteins or fragments thereof, to produce a bispecific or multispecific antigen-binding molecule having a second binding specificity. According to this disclosure, the term "multispecific antigen-binding molecule" also includes bispecific, trispecific, or multispecific antibodies or antigen-binding fragments thereof. In some embodiments, the antibody of this disclosure is functionally linked to another antibody or antigen-binding fragment thereof to produce a bispecific antibody having a second binding specificity.

[0097] Methods for preparing bispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537 (1983)). Due to the random allocation of the immunoglobulin heavy and light chains, hybridomas (tetramas) produce a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually accomplished by an affinity chromatography step. More modern techniques for producing bispecific antibodies employ heterodimerization domains that facilitate the desired pairing of the heavy chain from the antibody with the first specificity to the heavy chain from the antibody with the second specificity.

[0098] Antibody variable domain regions containing the desired binding specificity can be fused with immunoglobulin constant domain sequences. The fusion typically has an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge region, CH2 region, and CH3 region. It may also have a first heavy chain constant region (CH1) containing sites necessary for light chain binding, present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transformed into a suitable host organism. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Meth. Enzymol. 121:210 (1986).

[0099] As used herein, the term "Fc region" or "Fc domain" refers to a polypeptide comprising a CH2 domain and a CH3 domain, wherein the C-terminus of the CH2 domain is linked (directly or indirectly) to the N-terminus of the CH3 domain. In some embodiments, the Fc region is a variant Fc region. In some embodiments, the variant Fc region contains amino acid substitutions for improving stability. In some embodiments, the variant Fc region contains amino acid substitutions for promoting heterodimerization. In some embodiments, the variant Fc region contains amino acid substitutions for reducing or eliminating antibody effector function. The term "Fc polypeptide" includes an antibody heavy chain linked via disulfide bonds to an antibody light chain (e.g., to form a hapten).

[0100] As used herein, the term "CH1 domain" refers to the first constant domain of an antibody heavy chain (e.g., amino acid positions 118-215 of human IgG1 according to the EU index). The term includes naturally occurring CH1 domains and engineered variants of naturally occurring CH1 domains (e.g., CH1 domains containing one or more amino acid insertions, deletions, substitutions, or modifications relative to naturally occurring CH1 domains).

[0101] As used herein, the term "hinge region" refers to a portion of an antibody heavy chain containing cysteine ​​residues (e.g., cysteine ​​residues at amino acid positions 226 and 229 of human IgG1 according to the EU index) that mediate disulfide bonding between two heavy chains in a complete antibody. The term includes naturally occurring hinge regions and engineered variants of naturally occurring hinge regions (e.g., hinge regions containing one or more amino acid insertions, deletions, substitutions, or modifications relative to naturally occurring hinge regions). An exemplary full-length IgG1 hinge region contains amino acid positions 216-230 of human IgG1 according to the EU index. Hinge regions may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that result in flexible or semi-flexible bonding between adjacent variable regions and / or constant domains in a single polypeptide molecule. In some implementations, the immunoglobulin-like hinge region may be derived from or originate from any IgG1, IgG2, IgG3 or IgG4 subtype, or from IgA, IgE, IgD or IgM, including their chimeric forms.

[0102] In some embodiments, the hinge region may originate from a human IgG1 subtype extending from amino acid 216 to amino acid 230 according to the EU index numbering system or from amino acid 226 to amino acid 243 according to the Kabat numbering system. Those skilled in the art may have different understandings of the exact amino acids corresponding to the various domains of the IgG molecule. Therefore, the N-terminus or C-terminus of the domains outlined above may be extended or shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids.

[0103] As used herein, the term "upper hinge" generally refers to the last residue of the CH1 domain up to, but not including, the first heavy chain metacysteine. The upper hinge can sometimes be defined as the N-terminal sequence from position 216 to position 225 according to the Kabat EU numbering system for IgG1 antibodies (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institute of Health, Bethesda, Md., 1991). The term "middle hinge" refers to the region extending from the first heavy chain metacysteine ​​to the proline residue adjacent to the carboxyl terminus of the last middle hinge cysteine. The middle hinge can be the N-terminal sequence from position 226 to position 230 according to the Kabat EU numbering system. The term "lower hinge" refers to a highly conserved 7-8 amino acids. The lower hinge can be defined as the sequence from position 231 to 238 according to the Kabat EU numbering system for IgG1 antibodies. In some embodiments, the antibody according to the invention actually comprises an upper hinge, a middle hinge, and a lower hinge.

[0104] As used herein, the term "modified hinge region" refers to a hinge region in which one or more characteristics of the hinge (including, but not limited to, flexibility, length, conformation, charge, and hydrophobicity) are altered relative to the wild-type hinge. The modified hinge regions disclosed herein can be generated by methods well known in the art, such as, for example, introducing modifications into the wild-type hinge. In some embodiments, the hinge region may be modified with one or more amino acids. Modifications that can be used to generate modified hinge regions include, but are not limited to, amino acid insertions, deletions, substitutions, and rearrangements. The disclosed hinges and the modifications to the modified hinge regions are collectively referred to herein as "hinge modifications of the present invention," "one or more modified hinges of the present invention," or simply "hinge modifications" or "one or more modified hinges." The modified hinge regions disclosed herein may be incorporated into selected molecules (including, but not limited to, antibodies and fragments thereof). In some embodiments, the hinge region may be truncated and contain only a portion of the complete hinge region. As demonstrated herein, molecules containing modified hinges can exhibit altered (e.g., enhanced) agonistic activity when compared to molecules having the same amino acid sequence except for the modified hinge (such as, for example, molecules having the same amino acid sequence except for containing a wild-type hinge). In some embodiments, the antibody contains a modified hinge region, wherein the upper hinge region is at most 7 amino acids in length. In some embodiments, the upper hinge region is absent. In some embodiments, the modified hinge is a modified IgG1 linker. In some embodiments, the modified IgG1 hinge is derived from the sequence PLAPDKTHT (SEQ ID NO: 1). In some embodiments, the modified IgG1 hinge contains the sequence PLAP (SEQ ID NO: 2). In some embodiments, the modified IgG1 hinge contains the sequence DKTHT (SEQ ID NO: 5). In some embodiments, the modified hinge is a modified IgG4 hinge. In some embodiments, the modified IgG1 hinge contains the sequence EKSYGPP (SEQ ID NO: 4). In some embodiments, the modified hinge is a Gly / Ser hinge. In some implementations, the Gly / Ser hinge contains the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3).

[0105] Table 1. Hinge sequence.

[0106] As used herein, the term “EU index” refers to the EU numbering convention for constant regions of antibodies, as described in Edelman, GM. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, US Dept Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety. All amino acid positions of Fc peptides or fragments thereof used herein are numbered according to the EU index.

[0107] In some implementations, the term "linker" refers to 1-100 consecutive amino acid residues. Typically, linkers provide flexibility and spatial separation between two amino acids or between two polypeptide domains. Linkers can be inserted between VH, VL, CH, and / or CL domains to provide sufficient flexibility and mobility to the light and heavy chain domains, depending on the molecular format. At the amino sequence level, linkers are typically inserted at transitions between variable domains, between a variable domain and a knockout domain, or between a variable domain and a constant domain. Because the approximate size of immunoglobulin domains is well understood, transitions between domains can be identified. The precise location of a domain transition can be determined by locating peptides that do not form secondary structural elements (such as β-sheets or α-helices), as can be demonstrated by experimental data or determined by modeling or secondary structure prediction techniques.

[0108] As used herein, the terms “specifically binds,” “binding specificity,” or “specifically recognized” refer to an antigen-binding protein or antigen-binding fragment thereof that exhibits significant affinity for an antigen (e.g., IL-18R antigen) and does not exhibit significant cross-reactivity with targets other than IL-18R proteins. As used herein, the term “affinity” refers to the strength of the interaction between the antigen-binding site of an antigen-binding protein or antigen-binding fragment thereof and the epitope to which it binds. In some exemplary embodiments, affinity is measured by surface plasmon resonance (SPR), for example in a Biacore instrument. As will be readily understood by those skilled in the art, antigen-binding protein affinity can be reported as a dissociation constant (KD) in molar concentration (M). The antigen-binding proteins or antigen-binding fragments thereof disclosed herein have KD values ​​of about 10⁻⁵ M to about 10⁻¹² M (i.e., low micromolar to picomolar range), about 10⁻⁷ M to 10⁻¹¹ M, about 10⁻⁸ M to about 10⁻¹⁰ M, and about 10⁻⁹ M. In some embodiments, the antigen-binding protein or its antigen-binding fragment has a binding affinity of about 10⁻⁵ M, 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M. In some embodiments, the antigen-binding protein or its antigen-binding fragment has a binding affinity of about 10⁻⁷ M to about 10⁻⁹ M (nanomolar range).

[0109] Specific binding can be determined using any means recognized in the art for determining such binding. In some embodiments, specific binding is determined by a competitive binding assay (e.g., ELISA) or a Biacore assay. In some embodiments, the assay is performed at about 20°C, 25°C, 30°C, or 37°C.

[0110] As used herein, “administer / administration” means the act of physically delivering a substance present outside the body (e.g., the isolated binding peptides provided herein) to a patient via injection or other means, such as through, but not limited to, the lungs (e.g., inhalation), mucous membranes (e.g., intranasal), intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When managing or treating a disease or its symptoms, the administration of a substance typically occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the substance is typically administered before the onset of the disease or its symptoms and may be administered continuously over a long period to delay or reduce the onset or severity of disease-related symptoms.

[0111] As used herein, the term "composition" is intended to cover products containing a specified ingredient (e.g., the isolated binding polypeptide provided herein) in optional amounts, and any product produced directly or indirectly from a combination of specified ingredients in optional amounts.

[0112] "Effective amount" means the amount of an active pharmaceutical agent (e.g., the isolated bound polypeptide of this disclosure) sufficient to achieve the desired physiological outcome in an individual who requires the agent. Effective amounts can vary between individuals, depending on the health and physical condition of the individual being treated, the individual's taxonomy, the formulation of the composition, the evaluation of the individual's medical condition, and other relevant factors.

[0113] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject may be a mammal, such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, mice, etc.) or a primate (e.g., monkeys and humans). In some embodiments, as used herein, the term “subject” refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, wild animals, farm animals, sporting animals, and pets.

[0114] As used herein, the term "therapy" means any regimen, method, and / or agent that can be used to prevent, manage, treat, and / or improve a disease or related symptoms. In some embodiments, the term "therapy" means any regimen, method, and / or agent that can be used to modulate an immune response in a subject to an infection or related symptoms. In some embodiments, the term "therapies" means biological therapies, supportive therapies, and / or other therapies known to those skilled in the art (such as medical personnel) that can be used to prevent, manage, treat, and / or improve a disease or related symptoms. In other embodiments, the term "therapies" means biological therapies, supportive therapies, and / or other therapies known to those skilled in the art (such as medical personnel) that can be used to modulate an immune response in a subject to an infection or related symptoms.

[0115] As used herein, the term "treat" refers to a reduction or improvement in the progression, severity, and / or duration of disease or related symptoms resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as the isolated binding peptides provided herein). The term "treatment" as used herein may also refer to altering the disease course of a treated subject. Therapeutic effects of treatment include, but are not limited to: preventing the onset or recurrence of disease, relieving one or more symptoms, eliminating direct or indirect pathological consequences of disease, reducing the rate of disease progression, improving or alleviating disease status, and alleviating or improving prognosis.

[0116] The term “about” or “approximately” means about 20% of a given value or range, such as about 10%, about 5%, or about 1% or less.

[0117] IL-18R signal transduction path IL-18 was initially discovered to be a pro-inflammatory IFN-γ-inducing cytokine that shares some biological functions with IL-12 and acts synergistically with it. As a member of the IL-1 cytokine family, IL-18 is thought to play a role in early inflammatory responses and is constitutively synthesized by a range of hematopoietic and non-hematopoietic cells (e.g., macrophages, dendritic cells, Kupffer cells, keratinocytes, osteoblasts, astrocytes, adrenocortical cells, intestinal epithelial cells, microglia, and synovial fibroblasts) in response to lipopolysaccharide and other cytokines (such as TNF-α). It is post-translationally cleaved by cysteine ​​protease-1 to acquire the functional activity of its mature 18 kDa substance. Active IL-18 then targets cells expressing the IL-18 receptor, which is widely expressed in both hematopoietic and non-hematopoietic tissues.

[0118] The IL-18 receptor is a heterodimeric transmembrane protein composed of a ligand-binding IL-18Rα (IL-18Rα) subunit and a non-ligand-binding IL-18Rβ (IL-18Rβ) subunit, which is crucial for functional signal transduction. Ligand-induced receptor activation leads to the recruitment and activation of intracellular myeloid differentiation 88 (MyD88) and IL-1R-associated kinase (IRAK), simultaneously triggering at least two distinct phosphorylation cascades activating the PI3K and MAPK pathways (including activation of Akt, p38, and SAPK / JNK). Activation of these pathways ultimately results in the activation of NF-κB and the transcription of its downstream genes, including IFN-γ, chemokines, transcription factors, G proteins, and cell surface receptors. IL-18 shares signaling pathway elements with IL-1 but also carries some different elements.

[0119] IL-18 stimulation enhances T cell and NK cell maturation, cytokine secretion, cytotoxicity, and adhesion. IL-18-induced differentiation of naive T cells can independently induce Th1 or Th2 lineages, independent of IL-4 or IL-12. In differentiated Th1 clones, IL-18 can induce the production and secretion of IFN-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), or tumor necrosis factor (TNF); and it does so primarily in synergy with IL-12. In neutrophils, IL-18 has been shown to induce the expression and secretion of cytokines and chemokines, upregulate the expression of the cell surface adhesion molecule CD11b, and enhance neutrophil respiratory burst. Importantly, IL-12 can upregulate the IL-18 receptor itself on naive T cells, Th1 cells, and B cells, which partly explains the synergistic effect between these two cytokines. IL-18 also works synergistically with IL-2 to induce the expression of IL-13 (in an IFN-γ-dependent manner) and IL-10 (in an IFN-γ-independent manner). In summary, these results highlight the role of IL-18 in both innate and adaptive immune responses.

[0120] In non-hematopoietic cells such as endothelial and epithelial cells, synovial fibroblasts, and chondrocytes, IL-18 can upregulate the expression of adhesion molecules (such as E-selectin, ICAM, and VCAM), other cytokines, chemokines (CXCL8, CXCL5, CXCL1, CXCL12, CCL, CCL20), and angiogenesis mediators such as vascular endothelial growth factor (VEGF) and platelet-reactive protein. Overall, the effects of IL-18 inducing these effectors include increased leukocyte recruitment, cell adhesion, extravasation of immune cells, and promotion of cell migration and angiogenesis.

[0121] In the context of many inflammatory diseases, IL-18 has been shown to be upregulated, associated with disease, or a risk factor for disease development. Examples include Crohn's disease, rheumatoid arthritis, systemic lupus erythematosus, and cardiovascular disease. Elevated IL-18 levels have been observed in individuals at risk of developing type 1 diabetes (T1D) or type 2 diabetes (T2D). Elevated IL-18 levels have also been observed in the serum, urine, and pancreas of adolescents and adults with T1D and T2D, and this is associated with disease severity and the development of sequelae such as diabetic nephropathy. Studies in Alzheimer's disease patients have shown increased expression of IL-18 in the brain and are thought to contribute to immune and inflammatory processes, thereby enhancing oxidative stress and altering the expression of proteins that lead to the formation of amyloid-β (Aβ).

[0122] In summary, these studies suggest that inflammatory conditions may represent a class of pathologies for which blocking IL-18-mediated signaling using antagonistic anti-IL-18 agents can prove effective, with ample opportunity for well-defined preclinical investigations. However, the role of agonistic antibodies in upregulating the IL-18 signaling pathway remains unexplored. IL-18 is a potent immunostimulatory cytokine that selectively activates tumor-infiltrating NK cells and antigen-experienced T cells, but it induces its own inhibitor, the IL-18-binding protein, which acts as an immune checkpoint (Dinarello CA et al., Front. Immunol. 2013, Zhou T et al., Nature 2020). This biologically embedded constraint on pro-inflammatory signaling limits the utility of IL-18 as an oncology agent. Heteromery antibodies, acting as IL-18 receptor agonists, bypass this regulatory mechanism and can promote sustained pro-inflammatory signaling in tumors, thereby activating cytotoxic tumor-infiltrating lymphocytes. Importantly, this antibody will act through the NFkB signaling cascade, thereby working orthogonally and synergistically with the γc cytokine family (such as IL-2, IL-15) and IL-12, which act through the JAK / TYK / STAT signaling pathway.

[0123] Anti-IL-18Rα binding domain In some embodiments, the multispecific binding proteins described herein comprise a binding moiety, binding domain, or binding specificity of the IL-18Rα subunit that binds to IL-18R (e.g., human IL-18R). Any type of binding moiety that specifically binds to the IL-18Rα receptor subunit may be used in the multispecific binding proteins disclosed herein. In some embodiments, the binding moiety comprises an antibody variable domain. Exemplary binding moieties comprising antibody variable domains include, but are not limited to, VH, VL, VHH, VH / VL pairs, scFv, biantibodies, or Fab. Other suitable binding modalities include, but are not limited to, lipid carriers (see, for example, Gebauer M. et al., 2012, Method Enzymol. 503:157–188, which are incorporated herein by reference in their entirety), adnectins (see, for example, Lipovsek D., 2011, Protein Eng. Des. Sel. 24:3–9, which are incorporated herein by reference in their entirety), avimers (see, for example, Silverman J et al., 2005, Nat. Biotechnol. 23:1556–1561, which are incorporated herein by reference in their entirety), fynomers (see, for example, Schlatter D et al., 2012, mAb 4:497–508, which are incorporated herein by reference in their entirety), and kunitz domains (see, for example, Hosse RJ et al., 2006, Protein Sci.). 15:14–27, which is incorporated herein by reference in its entirety, knottin (see, for example, Kintzing JR et al., 2016, Curr. Opin. Chem. Biol. 34:143–150, which is incorporated herein by reference in its entirety), affibody (see, for example, Feldwisch J et al., 2010 J. Mol. Biol. 398:232–247, which is incorporated herein by reference in its entirety), and DARPin (see, for example, Pluckthun A., 2015, Annu. Rev. Pharmacol. Toxicol. 55:489–511, which is incorporated herein by reference in its entirety).

[0124] In some embodiments, the binding domain comprises a heavy chain and / or light chain variable region of a conventional antibody or its antigen-binding fragment (e.g., Fab or scFv), wherein the term "conventional antibody" is used herein to describe a heterotetrameric antibody containing heavy and light chain immunoglobulin chains arranged according to a "Y" conformation. Such conventional antibodies can be derived from any suitable species, including but not limited to antibodies derived from llamas, alpacas, camels, mice, rats, rabbits, goats, hamsters, chickens, monkeys, or humans. In some exemplary embodiments, the conventional antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and / or VL domains, or one or more of their complementarity-determining regions (CDRs), are derived from the same antibody. In some embodiments, the antigen-binding region of the conventional antibody may be referred to as "Fab" (antigen-binding fragment). Fab comprises a constant domain and a variable domain from each of the heavy and light chains. The variable heavy and light chains contain CDRs responsible for antigen binding.

[0125] In some embodiments, the IL-18Rα receptor subunit binding subunit contains at least the CDR or VHH domain of a VHH antibody or nanobody. VHH antibodies are heavy-chain antibodies derived from camels, consisting of two heavy chains and lacking a light chain (Hamers-Casterman et al., Nature. 1993; 363; 446-8). Each heavy chain of a VHH antibody has a variable domain at its N-terminus, and these variable domains are referred to in the art as “VHH” domains to distinguish them from the variable domains (i.e., VH domains) of the heavy chains of conventional antibodies. Similar to conventional antibodies, the VHH domain of this molecule contains HCDR1, HCDR2, and HCDR3 regions that confer antigen-binding specificity; therefore, VHH antibodies or fragments (such as isolated VHH domains) are suitable as components of the multispecific binding proteins of this disclosure.

[0126] Tables 2 and 3 below provide exemplary VHH CDRs or VHH domains that are specific to IL-18Rα.

[0127] Table 2. CDR region of IL-18Rα VHH heavy chain.

[0128] Table 3. IL-18Rα VHH domain sequence.

[0129] In some embodiments, the IL-18Rα binding domain comprises an amino acid sequence that is at least 75% identical (e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to at least one of the amino acid sequences in Table 2 or Table 3.

[0130] Anti-IL-18Rβ binding domain In some embodiments, the multispecific binding protein includes a binding domain or binding specificity for the IL-18Rβ subunit of IL-18R (e.g., human IL-18R).

[0131] In some embodiments, the binding domain comprises a heavy chain and / or light chain variable region of a conventional antibody or its antigen-binding fragment. In some embodiments, the binding domain is a Fab or scFv. In some embodiments, the IL-18Rβ binding domain is a Fab or scFv and pairs with the IL-18Rα binding domain, which is a Fab or scFv. In some embodiments, the IL-18Rβ binding domain is a Fab that shares a common light chain with the Fab of the IL-18Rα binding domain.

[0132] Exemplary binding portions containing antibody variable domains include, but are not limited to, VH, VL, VHH, VH / VL pairs, scFv, biantibodies, or Fab. Other suitable binding motifs include, but are not limited to, lipid carrier proteins (see, for example, Gebauer M. et al., 2012, Method Enzymol. 503:157–188, which are incorporated herein by reference in their entirety), attachment proteins (see, for example, Lipovsek D., 2011, Protein Eng. Des. Sel. 24:3–9, which are incorporated herein by reference in their entirety), affinity multimers (see, for example, Silverman J et al., 2005, Nat. Biotechnol. 23:1556–1561, which are incorporated herein by reference in their entirety), fynomers (see, for example, Schlatter D et al., 2012, mAb 4:497–508, which are incorporated herein by reference in their entirety), and kunitz domains (see, for example, Hosse RJ et al., 2006, Protein Sci). 15:14–27, which is incorporated herein by reference in its entirety, knottin (see, for example, Kintzing JR et al., 2016, Curr. Opin. Chem. Biol. 34:143–150, which is incorporated herein by reference in its entirety), affibody (see, for example, Feldwisch J et al., 2010 J. Mol. Biol. 398:232–247, which is incorporated herein by reference in its entirety), and DARPin (see, for example, Pluckthun A., 2015, Annu. Rev. Pharmacol. Toxicol. 55:489–511, which is incorporated herein by reference in its entirety).

[0133] In some embodiments, the IL-18Rβ receptor subunit binding domain includes at least the CDR or VHH domain of the VHH antibody or nanobody. In some embodiments, the IL-18Rβ VHH binding subunit is paired with the Fab or scFv IL-18Rα binding domain. In some embodiments, the IL-18Rβ VHH binding domain is paired with the IL-18Rα VHH binding domain.

[0134] Tables 4 and 5 below provide exemplary VHH CDRs and VHH domains that are specific to IL-18Rβ.

[0135] Table 4. CDR region of IL-18Rβ VHH heavy chain.

[0136] Table 5. IL-18Rβ VHH domain sequence.

[0137] Multispecific IL-18R binding protein In some embodiments, the IL-18Rα and IL-18Rβ binding domains disclosed herein may be paired together or operatively linked to generate a multispecific binding protein capable of crosslinking the IL-18Rα and IL-18Rβ subunits of an IL-18 receptor (e.g., human IL-18 receptor). In some embodiments, the IL-18Rα binding domain (e.g., VHH) is operatively linked (directly or indirectly) to a first Fc domain or the N and / or C-terminus of a peptide, and the IL-18Rβ binding domain is operatively linked to a second Fc domain or the N and / or C-terminus of a peptide, such that the first and second Fc domains promote heterodimerization of the IL-18Rα and IL-18Rβ binding domains.

[0138] In some embodiments, the multispecific binding proteins disclosed herein have agonistic effects on the IL-18R signaling pathway; that is, they do not antagonize the IL-18R pathway. In some embodiments, the agonistic effect can be measured using an IL-18 potency assay (e.g., the HEK-Blue™ IL-18 potency assay (InVivogen)). In this assay, HEK-Blue™ IL-18 cells are generated by stably transfecting HEK293-derived cells with genes encoding IL-18Rα and IL-18Rβ. In some embodiments, responses to human TNF-α and IL-1β are blocked, enabling cells to specifically respond to IL-18.

[0139] HEK-Blue™ IL-18 cells also express the NF-κB / AP-1-inducible secretory embryonic alkaline phosphatase (SEAP) reporter gene. The binding of bispecific antibodies to the heterodimeric IL-18 receptor on the surface of these cells triggers a signaling cascade that leads to NF-κB activation and subsequent quantifiable SEAP production.

[0140] The multispecific binding protein disclosed herein possesses agonist activity against IL-18R, stimulating IFN-γ expression while minimizing the induction of IL-5 and IL-1-β expression. This contrasts with IL-18R's natural ligand, IL-18, which potently stimulates IL-5 and IL-1-β expression. Therefore, the multispecific binding protein of this disclosure provides a unique and specific IL-18R agonist effect not found in IL-18.

[0141] Therefore, in one aspect, this disclosure provides a multispecific binding protein comprising a first binding portion specifically binding to human IL-18Rα and a second binding portion specifically binding to human IL-18Rβ, wherein the multispecific binding protein stimulates the production of antitumor cytokines but substantially does not stimulate the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers relative to the stimulation of IL-18 on the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers.

[0142] In some implementations, the antitumor cytokines are selected from the group consisting of IFN-γ, IL-2, IL-12, IL-15, CD40L and TNFα.

[0143] In some implementations, the markers for acute inflammation or Th2 response are selected from the group consisting of IL-6, IL-1β, IL-8, IL-4, IL-5 and IL-13.

[0144] In some implementations, antitumor cytokines and markers of acute inflammation or Th2 response are measured in peripheral blood mononuclear cell (PBMC) assays.

[0145] In some embodiments, the PBMC assay includes: 1) incubating a first PBMC population with a multispecific binding protein for at least 24 hours (e.g., 24, 36, 48, 60, 72, 84, or 96 hours); 2) incubating a second PBMC population with IL-18 for at least 24 hours (e.g., 24, 36, 48, 60, 72, 84, or 96 hours); and 3) measuring the production of antitumor cytokines and markers of acute inflammation or Th2 response from the first and second PBMC populations.

[0146] In some embodiments, approximately 10,000 to approximately 1,000,000 PBMCs are used in the first and second PBMC groups. In some embodiments, approximately 250,000 PBMCs are used in the first and second PBMC groups.

[0147] In some embodiments, the first PBMC population is incubated with IL-12 before or simultaneously with incubation with the multispecific binding protein. In some embodiments, the second PBMC population is incubated with IL-12 before or simultaneously with incubation with IL-18. In some embodiments, the first and second PBMC populations are incubated with IL-12 at a concentration of about 0.1 ng / mL to about 1 ng / mL. In some embodiments, the first and second PBMC populations are incubated with IL-12 at a concentration of about 0.5 ng / mL.

[0148] In some embodiments, the first PBMC population is incubated together with a multispecific binding protein at a concentration of about 1 nM to about 1,000 nM. In some embodiments, the first PBMC population is incubated together with a multispecific binding protein at a concentration of about 300 nM.

[0149] In some implementations, the multispecific binding protein stimulates the production of acute inflammatory or Th2 response markers at least 5, 10, 50, or 100 times less than IL-18.

[0150] In some implementations, the multispecific binding protein stimulates the production of acute inflammatory or Th2 response markers that are at least 5-fold, 10-fold, 50-fold, or 100-fold less than IL-18, as measured in a PBMC assay.

[0151] In some implementations, the production of GM-CSF stimulated by multispecific binding proteins is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than the production of IFN-γ.

[0152] In some embodiments, the multispecific binding protein stimulates at least 5-fold less IL-5 production than IL-18. In some embodiments, the multispecific binding protein stimulates at least 10-fold less IL-5 production than IL-18. In some embodiments, the multispecific binding protein stimulates at least 50-fold less IL-5 production than IL-18. In some embodiments, the multispecific binding protein stimulates at least 100-fold less IL-5 production than IL-18. In some embodiments, the multispecific binding protein stimulates at least 500-fold less IL-5 production than IL-18. In some embodiments, the multispecific binding protein stimulates at least 1,000-fold less IL-5 production than IL-18. In some embodiments, the multispecific binding protein stimulates at least about 5-fold and at least about 1,000-fold less IL-5 production than IL-18.

[0153] In some embodiments, the multispecific binding protein stimulates at least 5-fold less IL-1-β production than IL-18. In some embodiments, the multispecific binding protein stimulates at least 10-fold less IL-1-β production than IL-18. In some embodiments, the multispecific binding protein stimulates at least 50-fold less IL-1-β production than IL-18. In some embodiments, the multispecific binding protein stimulates at least 100-fold less IL-1-β production than IL-18. In some embodiments, the multispecific binding protein stimulates at least 500-fold less IL-1-β production than IL-18. In some embodiments, the multispecific binding protein stimulates at least 1,000-fold less IL-1-β production than IL-18. In some embodiments, the multispecific binding protein stimulates at least about 5-fold and at least about 1,000-fold less IL-1-β production than IL-18.

[0154] In some implementations, the fold change in the production of acute inflammatory or Th2 response markers of multispecific binding proteins compared to IL-18 is measured as in the PBMC assay described herein.

[0155] In some embodiments, the multispecific binding protein comprises at least one binding domain, such as VHH or Fab, located on each polypeptide. In some embodiments, the multispecific binding protein comprises a tandem binding domain bispecific construct, such as at least two VHH domains located on a single polypeptide. In some embodiments, the VHH domain targets two different targets, such as IL-18Rα and IL-18Rβ. In some embodiments, one or more of these binding domains are optimized. Optimization may include altering the primary amino acid sequence to reduce potentially adverse properties (e.g., immunogenicity in the human host; humanization).

[0156] In some embodiments, when measured using an agonist activity assay (e.g., the HEK-Blue assay), the agonist activity of the multispecific binding protein reaches or exceeds a specific threshold of background. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 2 times. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 3 times. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 4 times. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 5 times. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 6 times. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 7 times. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 8 times. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 9 times. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 10 times. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 11 times. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 12 times. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 13-fold. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 14-fold. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 15-fold. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 20-fold. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 30-fold. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 40-fold. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 50-fold. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 60-fold. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 70-fold. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 80-fold. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 90-fold. In some embodiments, the agonist activity of the multispecific binding protein exceeds the background by approximately 100-fold.

[0157] The Fc peptides used in the multispecific binding proteins of this disclosure typically comprise a CH2 domain and a CH3 domain, wherein the C-terminus of the CH2 domain is (directly or indirectly) linked to the N-terminus of the CH3 domain. Any naturally occurring or variant CH2 and / or CH3 domains can be used. For example, in some embodiments, the CH2 and / or CH3 domains are naturally occurring CH2 or CH3 domains from an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain). The CH2 and CH3 domains may originate from the same or different antibody heavy chains. In some embodiments, the Fc peptide comprises a portion from a single antibody heavy chain containing both the CH2 and CH3 domains. In some embodiments, the CH2 and / or CH3 domains are variants of either the naturally occurring CH2 or CH3 domains. In some embodiments, the CH2 domain and / or CH3 domain are variants comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to the naturally occurring CH2 domain or CH3 domain, respectively. In some embodiments, the CH2 domain and / or CH3 domain are chimeras of one or more CH2 domains or CH3 domains, respectively. In some embodiments, the CH2 domain comprises amino acid positions 231-340 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index. In some embodiments, the CH3 domain comprises amino acid positions 341-447 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index.

[0158] In some embodiments, the Fc peptide further comprises a hinge region, wherein the C-terminus of the hinge region is (directly or indirectly) connected to the N-terminus of the CH2 domain. For example, in some embodiments, the hinge region is a naturally occurring hinge region derived from an antibody heavy chain of IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain). The hinge region may be derived from an antibody heavy chain that is the same as or different from the CH2 and / or CH3 domains. In some embodiments, the hinge region is a variant comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to a naturally occurring hinge region. In some embodiments, the hinge region is a chimera of one or more hinge regions. In some embodiments, the hinge region comprises amino acid positions 226-229 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index. In some embodiments, the hinge region comprises amino acid positions 216-230 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index. In some embodiments, the hinge region comprises amino acid positions 216-230 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index. In some embodiments, the hinge region is a variant IgG4 hinge region comprising serine (S) located at amino acid position 228 according to the EU index.

[0159] In some embodiments, the Fc peptide further comprises a CH1 domain, wherein the C-terminus of the CH1 domain is (directly or indirectly) connected to the N-terminus of the hinge region. For example, in some embodiments, the CH1 domain is a naturally occurring CH1 domain from an antibody heavy chain of IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain). The CH1 domain may be derived from the same or different antibody heavy chain as the hinge region, CH2 domain, and / or CH3 domain. In some embodiments, the CH1 domain is a variant comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to the naturally occurring CH1 domain. In some embodiments, the CH1 domain is a chimera of one or more CH1 domains. In some embodiments, the CH1 domain comprises amino acid positions 118-215 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index.

[0160] In some embodiments, the Fc peptide lacks the CH1 domain, or contains a mutation located in the CH1 domain or a variable domain of the heavy chain that prevents the heavy chain from binding to the antibody light chain. In some embodiments, the antibody heavy chain lacks a portion of the hinge region.

[0161] Heterodimerization motif In some implementations, the first and second Fc domains are further engineered to enhance the heterodimerization of the IL-18Rα and IL-18Rβ binding domains and minimize the effects of incorrect chain pairing (i.e., pairing of the IL-18Rα binding domain or the same IL-18Rβ domain).

[0162] The production of desired multispecific antibodies can be improved using any method recognized in the art for addressing incorrect chain pairing. For example, US2010 / 0254989 A1 describes the construction of a bispecific cMet-ErbB1 antibody in which the VH and VL of a single antibody are fused via a GlySer linker. For bispecific antibodies containing an Fc domain, mutations can be introduced into the Fc to promote proper heterodimerization of the Fc moiety. Klein et al. (mAb (2012) 4:6, 1-11) reviewed several such methods, the contents of which are incorporated herein by reference in their entirety.

[0163] In some implementations, the IL-18Rα and IL-18Rβ binding specifics of multispecific binding proteins are heterodimerized via mortise-and-tenon (KiH) pairing of the Fc domain. This dimerization technique utilizes "protrusions" or "mortises" engineered into the interface of the CH3 domain with "cavities" or "mortises". When a appropriately positioned and sized mortise-and-tenon is present at the interface of the first or second CH3 domain, engineering of the corresponding mortise-and-tenon at adjacent interfaces is required, thereby promoting and enhancing Fc domain pairing at the CH3 / CH3 domain interface. The IgG Fc domain fused with VHH has a mortise-and-tenon, and the IgG Fc domain of a conventional antibody has a mortise-and-tenon designed to accommodate the mortise-and-tenon, and vice versa. A "mortise-and-tenon" refers to at least one amino acid side chain, typically a larger side chain, protruding from the interface of the CH3 portion of the first Fc domain. The protrusion produces a mortise-and-tenon that is complementary to and accommodated by a mortise-and-tenon in the CH3 portion of the second Fc domain. A “mortar” is at least one amino acid side chain, typically a smaller one, that is removed from the interface of the CH3 portion of the second Fc domain. This technique is described, for example, in U.S. Patent Nos. 5,821,333, 5,731,168, and 8,216,805; Ridgway et al., Protein Engineering (1996) 9:617-621; and Carter PJ Immunol. Methods (2001) 248: 7-15, which are incorporated herein by reference.

[0164] Exemplary amino acid residues that can act as pestles include arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). Existing amino acid residues in the CH3 domain may be replaced or substituted by pestle amino acid residues. Preferred substituted amino acids may include any amino acid with a small side chain, such as alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), or valine (V).

[0165] Exemplary amino acid residues that can serve as mordane include alanine (A), serine (S), threonine (T), or valine (V). Existing amino acid residues in the CH3 domain may be replaced or substituted by mordane amino acid residues. Preferred substituted amino acids may include any amino acid with a large side chain, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W).

[0166] The CH3 domain is preferably derived from human IgG1 antibody. Exemplary amino acid substitutions for the CH3 domain include Y349C, S354C, T366S, T366Y, T366W, F405A, F405W, Y407T, Y407A, Y407V, T394S, or combinations thereof. Preferred exemplary combinations are S354C, T366Y, or T366W for vallea mutations in the first CH3 domain and Y349C, T366S, L368A, Y407T, or Y407V for mortis mutations in the second CH3 domain.

[0167] In some implementations, the two Fc domains of the antigen-binding construct are heterodimerized via Fab arm exchange (FAE). Human IgG1 with a P228S hinge mutation may contain either an F405L or K409R CH3 domain mutation. These two antibodies are mixed with a reducing agent to generate the FAE. This technique is described in U.S. Patents 9,212,230 and Labrijn AF PNAS (2013) 110(13):5145-5150, which are incorporated herein by reference.

[0168] In some embodiments, the two Fc domains of the antigen-binding construct are heterodimerized via an electrostatic reversal effect. This dimerization technique utilizes electrostatic reversal to promote and enhance Fc domain pairing at the CH3 / CH3 domain interface. The charge complementarity between the two CH3 domains is altered to favor heterodimerization (opposite charge pairing) rather than homodimerization (same charge pairing). In this method, electrostatic repulsion prevents homodimerization. Certain exemplary amino acid residue substitutions conferring the electrostatic reversal effect include K409D, K392D, and / or K370D in the first CH3 domain and D399K, E356K, and / or E357K in the second CH3 domain. This technique is described in U.S. Patent Publication No. 2014 / 0154254 A1 and Gunasekaran K. JBC (2010) 285(25):19637-19646, which are incorporated herein by reference.

[0169] In some implementations, charge complementarity is formed by a first Fc domain containing N297K and / or T299K mutations and a second Fc domain containing N297D and / or T99D mutations.

[0170] In some embodiments, the two Fc domains of the antigen-binding construct are heterodimerized through hydrophobic interactions. This dimerization technique utilizes hydrophobic interactions, rather than electrostatic interactions, to promote and enhance Fc domain pairing at the CH3 / CH3 domain interface. Exemplary amino acid residue substitutions may include K409W, K360E, Q347E, Y349S, and / or S354C in the first CH3 domain and D399V, F405T, Q347R, E357W, and / or Y349C in the second CH3 domain. Preferred amino acid residue substitution pairs between the first and second CH3 domains include K409W:D399V, K409W:F405T, K360E:Q347R, Y349S:E357W, and S354C:Y349C. This technique is described in U.S. Patent Publication No. 2015 / 0307628 A1.

[0171] In some implementations, heterodimerization can be mediated by using leucine zipper fusion. Leucine zipper domains fused to the C-terminus of each CH3 domain of the antibody chain force heterodimerization. This technique is described in Wranik B. JBC (2012) 287(52):43331-43339.

[0172] In some implementations, heterodimerization can be mediated using a chain-exchange engineered domain (SEED) body. The CH3 domain, derived from IgG and IgA formats, forces heterodimerization. This technique is described in Muda M. PEDS (2011) 24(5): 447-454.

[0173] In some embodiments, the heterodimerization motif may comprise a non-natural disulfide bond formed from engineered cysteine ​​residues. In some embodiments, the first set of disulfide bonds may comprise a Y349C mutation in a first Fc domain and an S354C mutation in a second Fc domain. In other embodiments, engineered disulfide bonds may be introduced by fusing a C-terminal extension peptide having engineered cysteine ​​residues to the C-terminus of each of the two Fc domains. In some embodiments, the first Fc domain may comprise a substitution of "PGK" at the carboxyl terminus with "GEC", and the second Fc domain may comprise a substitution of "PGK" at the carboxyl terminus with "KSCDKT".

[0174] In some implementations, multispecific binding proteins can employ the CrossMab principle (as reviewed by Klein et al.), which involves domain exchange between the heavy and light chains to facilitate proper pairing. Another approach involves engineering the interface between the paired VH-VL domains or paired CH1-CL domains of the heavy and light chains to increase the affinity between the heavy chain and its homologous light chain (Lewis et al., Nature Biotechnology (2014) 32: 191-198).

[0175] An alternative approach for producing multispecific binding protein formulations with correct antigen specificity is to develop methods for enriching antibodies with correct heavy-light chain pairings. For example, Spiess et al. (Nature Biotechnology (2013) 31: 753-758) described a method for producing MET-EGFR bispecific antibodies from bacterial co-cultures expressing two different half-antibodies.

[0176] Methods for altering the binding affinity of at least one heavy chain of a multispecific binding protein to a specific region by mutating its constant region to a binding affinity for an affinity agent (e.g., protein a) have also been described. This allows for the separation of correctly paired heavy chain heterodimers based on purification techniques utilizing the differential binding of the two heavy chains to the affinity agent (see US2010 / 0331527, WO2013 / 136186).

[0177] International patent application number PCT / EP2012 / 071866 (WO2013 / 064701) addresses the problem of mispairing of chains by employing a multispecific antibody separation method based on the use of anti-idiotype binders (particularly anti-idiotype antibodies). The anti-idiotype binder is used in a two-step selection process, where a first agent is used to capture antibodies with VH-VL domains specific to a first antigen, and subsequently a second agent is used to capture antibodies with a second VH-VL domain specific to a second antigen.

[0178] In some embodiments, the multispecific binding protein employs a first binding specificity having a single-domain antibody (VHH) binding region and a second binding specificity containing the VHH binding region. In some embodiments, the first and second binding moieties are located on the same polypeptide. In some embodiments, the first and second binding moieties are located on different polypeptides.

[0179] In some embodiments, multispecific binding proteins as described herein also include a common light chain. As used herein, the term "common light chain" refers to a light chain capable of pairing with a first heavy chain of an antibody bound to a first antigen to form a binding site specifically binding to the first antigen, and also capable of pairing with a second heavy chain of an antibody bound to a second antigen to form a binding site specifically binding to the second antigen. The common light chain is a polypeptide comprising a variable domain (VL) and a constant domain (CL) of the antibody light chain from the N-terminus to the C-terminus, and is also abbreviated herein as "VL-CL". Multispecific binding proteins having a common light chain require heterodimerization of the different heavy chains. In some embodiments, the heterodimerization methods listed above can be used in conjunction with the common light chain. In some exemplary embodiments, the heterodimerization motif may comprise a non-natural disulfide bond formed from engineered cysteine ​​residues. Adding a disulfide bond between the heavy and light chains of the antibody has been shown to improve stability. In addition, disulfide bonds have also been used as a solution to improve light chain pairing within bispecific antibodies (Geddie ML et al., mAB (2022) 14(1)).

[0180] Unless otherwise stated, all antibody constant region numbers used in this paper correspond to the EU numbering scheme, as described by Edelman et al. (Proc. Natl. Acad. Sci. 63(1): 78-85. 1969).

[0181] Other methods for the heterodimerization of heavy and / or light chains, as well as the generation and purification of asymmetric antibodies, are known in the art. See, for example, Klein C. mAb (2012) 4(6): 653-663 and U.S. Patent 9,499,634, each of which is incorporated herein by reference.

[0182] Effector functional mutation In some implementations, multispecific binding proteins, as disclosed herein, can be provided in various isotypes and can be provided with different constant region variants. The multispecific binding Fc region primarily determines its effector functions in Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent phagocytosis (ADCP) activity. These “cellular effector functions,” distinct from effector T cell functions, involve recruiting cells carrying Fc receptors to target cell sites to kill antibody-bound cells.

[0183] In some embodiments, the multispecific binding protein as described herein may be a multispecific binding protein exhibiting reduced effector function. In some embodiments, the one or more mutations reduce one or more of antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). In some embodiments, the binding protein as described herein may lack ADCC, ADCP, and / or CDC activity. In any case, the antibody according to the invention may contain or optionally lack an Fc region binding to one or more types of Fc receptors. The use of different antibody formats and the presence or absence of FcR binding and cellular effector function allow antibodies to be tailored for specific therapeutic purposes, as discussed elsewhere herein.

[0184] In some embodiments, the first and second Fc domains contain one or more mutations that reduce the function of the Fc effector. In some embodiments, the first and second Fc domains each contain L234A and L235A mutations. These IgG1 mutations are also known as “LALA” mutations and are described in more detail by Xu et al. (Cell Immunol. 2000; 200:16–26). In some embodiments, the first and second Fc domains each contain L234A, L235A, G237A, and / or P329G mutations. The amino acid positions of the Fc domains mentioned herein are based on EU antibody numbers. Alternatively, the antibody may have a constant region that is ineffective for the effector. The antibody may have a heavy chain constant region that does not bind to the Fcγ receptor, for example, this constant region may contain an L235E mutation. Another optional mutation in the heavy chain constant region is S228P, which increases stability. The heavy chain constant region may be IgG4 containing both the L235E and S228P mutations. This “IgG4-PE” heavy chain constant region is effector-invalid. The disabled IgG1 heavy chain constant region is also effector-invalid. The disabled IgG1 heavy chain constant region may contain alanine residues at positions 234, 235, and / or 237 (EU index number), for example, it can be an IgG1 sequence containing mutations in L234A, L235A, and / or G237A (“LALAGA”).

[0185] Human IgG1 constant regions containing specific mutations or altered glycosylations (e.g., N297Q, N297D, and N297K, EU index numbers) located on residue Asn297 have been shown to reduce binding to the Fc receptor.

[0186] In other embodiments, it may be necessary to enhance the binding of the Fc region of a multispecific antibody to human Fc γ receptor IIIA (FcgRIIIA) relative to the binding of the corresponding naturally occurring antibody's Fc region. In some embodiments, the constant region can be engineered to enhance ADCC and / or CDC and / or ADCP. The potency of Fc-mediated effects can be enhanced by engineering the Fc domain using various established techniques. Such approaches increase affinity for certain Fc receptors, resulting in a potentially diverse spectrum of activation enhancements. This can be achieved by modifying one or more amino acid residues. Exemplary mutations are one or more residues selected from 239, 332, and 330 (or equivalent positions in other IgG isotypes) of the human IgG1 constant region. Thus, the antibody may comprise a human IgG1 constant region having one or more mutations independently selected from S239D, I332E, and A330L (EU index number).

[0187] Affinity to the Fc receptor can also be increased by altering the native glycosylation profile of the Fc domain (e.g., by generating hypofucosylated or defucosylated variants). Non-fucosylated antibodies contain a trimannose core structure of an Fc complex N-glycan lacking fucoose residues. These glycoengineered antibodies, lacking the core fucoose residues from the Fc N-glycan, may exhibit stronger ADCC than fucosylated equivalents due to enhanced FcγRIIIA binding. For example, to increase ADCC, residues in the hinge region can be altered to increase binding to FcγRIIIA. Therefore, the antibody may contain a variant of the human IgG heavy chain constant region as a wild-type human IgG heavy chain constant region. In some embodiments, the variant human IgG heavy chain constant region binds to the human Fcγ receptor selected from the group consisting of FcγRIIB and FcγRIIA with a higher affinity than the wild-type human IgG heavy chain constant region binding to human FcγRIIIA. The antibody may comprise a variant of the human IgG heavy chain constant region as a wild-type human IgG heavy chain constant region, wherein the variant human IgG heavy chain constant region binds to human FcγRIIB with a higher affinity than the wild-type human IgG heavy chain constant region. The variant human IgG heavy chain constant region may be a variant human IgG1, variant human IgG2, or variant human IgG4 heavy chain constant region. In one embodiment, the variant human IgG heavy chain constant region comprises one or more amino acid mutations selected from G236D, P238D, S239D, S267E, L328F, and L328E (EU index numbering system). In another embodiment, the variable human IgG heavy chain constant region comprises a group of amino acid mutations selected from the group consisting of: S267E and L328F; P238D and L328E; P238D and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G and A330R: P238D, E233D, G237D, H268D, P271G and A330R; G236D and S267E; S239D and S267E; V262E, S267E and L328F; and V264E, S267E and L328F (EU index numbering system).

[0188] Enhancement of CDC can be achieved through amino acid changes that increase affinity for C1q (the first component of the classical complement activation cascade). Another approach is to create a chimeric Fc domain created from human IgG1 and human IgG3 segments, which utilizes the higher affinity of IgG3 for C1q. The antibodies of the present invention may contain mutant amino acids located at residues 329, 331, and / or 322 to alter C1q binding and / or reduce or eliminate CDC activity. In another embodiment, the antibodies or antibody fragments disclosed herein may contain an Fc region with modifications located at residues 231 and 239, thereby replacing amino acids to alter the antibody's ability to fix complement. In one embodiment, the antibody or fragment has a constant region containing one or more mutations selected from E345K, E430G, R344D, and D356R, particularly containing a double mutation of R344D and D356R (EU index numbering system).

[0189] The functional properties of multispecific binding proteins can be further modulated by combining amino acid substitutions that alter Fc binding affinity with those that affect binding to FcRn. Binding proteins with amino acid substitutions that affect binding to FcRn (also referred to herein as "FcRn variants") may, in some cases, increase serum half-life in vivo compared to unmodified binding proteins. It should be understood that any combination of Fc and FcRn variants can be used to modulate the clearance of antigen-antibody complexes. Suitable FcRn variants that can be combined with any of the Fc variants described herein include, but are not limited to, N434A, N434S, M428L, V308F, V259I, M428L / N434S, V259I / V308F, Y436I / M428L, Y436I / N434S, Y436V / N434S, Y436V / M428L, M252Y, M252Y / S254T / T256E, and V259I / V308F / M428L.

[0190] Expression of antigen-binding proteins In one aspect, polynucleotides encoding binding proteins disclosed herein (e.g., antigen-binding proteins and their antigen-binding fragments) are provided. A method for preparing binding proteins, comprising expressing these polynucleotides, is also provided.

[0191] Polynucleotides encoding the binding proteins disclosed herein are typically inserted into expression vectors for introduction into host cells that can be used to produce the desired amount of the binding protein. Therefore, in some aspects, this disclosure provides expression vectors comprising the polynucleotides disclosed herein, and host cells comprising these vectors and the polynucleotides.

[0192] The terms "vector" or "expression vector" are used herein to refer to a vector used according to this disclosure as a means of introducing a desired gene into a cell and expressing that gene in the cell. As known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, bacteriophages, viruses, and retroviruses. Generally, vectors compatible with this disclosure will contain selection markers, appropriate restriction sites that promote the cloning and entry into eukaryotic or prokaryotic cells and / or replication within eukaryotic or prokaryotic cells.

[0193] Many expression vector systems are available for the purposes of this disclosure. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Other vectors involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells that have integrated DNA into their chromosomes can be selected by introducing one or more markers that allow selective transfection of host cells. These markers can provide protrophic status against auxotrophic hosts, resistance to biocides (e.g., antibiotics), or resistance to heavy metals such as copper. The selection marker gene can be directly linked to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Optimal synthesis of mRNA may also require additional elements. These elements may include signal sequences, splicing signals, and transcription promoters, enhancers, and termination signals. In some embodiments, a cloned variable region gene is inserted into the expression vector along with heavy and light chain constant region genes (e.g., human constant region genes) synthesized as discussed above.

[0194] In other embodiments, the binding protein can be expressed using a polycistronic construct. In such expression systems, a variety of gene products of interest, such as the heavy and light chains of antibodies, can be generated from a single polycistronic construct. These systems advantageously utilize internal ribosome entry sites (IRES) to provide relatively high levels of the peptide in eukaryotic host cells. A compatible IRES sequence is disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those skilled in the art will understand that such expression systems can be used to efficiently generate the full range of peptides disclosed in this application.

[0195] More generally, once a vector or DNA sequence encoding a binding protein (e.g., an antibody or fragment thereof) has been prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Plasmids can be introduced into host cells using a variety of techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with an intact virus. See Ridgway, AAG, “Mammalian Expression Vectors,” Chapter 24.2, pp. 470–472, Vectors, edited by Rodriguez and Denhardt (Butterworths, Boston, Mass. 1988). Plasmids can be introduced into the host via electroporation. Transformed cells are then grown under conditions suitable for the production of light and heavy chains, and the synthesis of heavy and / or light chain proteins in the cells is measured. Exemplary assays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence activated cell sorting (FACS), immunohistochemistry, etc.

[0196] As used in this article, the term "transformation" should be used in a broad sense to refer to the introduction of DNA into recipient host cells, thereby altering the genotype.

[0197] Similarly, "host cell" refers to a cell that has been transformed with a vector constructed using recombinant DNA technology and encoding at least one heterologous gene. Unless otherwise explicitly stated, the terms "cell" and "cell culture" are used interchangeably to refer to the source of the antibody when describing methods for isolating peptides from a recombinant host. In other words, recovering peptides from "cells" can mean recovering them from intact cells after centrifugation, from the supernatant of a lysed cell culture, or from a cell culture containing both culture medium and suspended cells.

[0198] In one embodiment, the host cell line used for antibody expression is of mammalian origin. Those skilled in the art can determine the specific host cell line most suitable for expressing the desired gene product therein. Exemplary host cell lines include, but are not limited to, GS-CHO and CHO-K1 (Chinese hamster ovary cell lines), DG44 and DUXB11 (Chinese hamster ovary cell lines, DHFR-), HELA (human cervical cancer), CV-1 (monkey kidney cell line), COS (CV-1 derivative with SV40 T antigen), R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HEK (human kidney cell line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). In one embodiment, the cell line provides altered glycosylation of the antibody expressed therein, such as fucosylation-free glycosylation (e.g., PER.C6® (Crucell) or FUT8 knockout CHO cell lines (POTELLIGENT® cells) (Biowa, Princeton, NJ)). In one embodiment, NSO cells may be used. CHO cells are particularly useful. Host cell lines are generally available from commercial services, such as the U.S. Tissue Culture Collection, or from authors of published literature.

[0199] In vitro production allows for scaling up to obtain large quantities of the desired peptides. Techniques for culturing mammalian cells under tissue culture conditions are known in the art, including homogeneous suspension culture, such as in airlift reactors or continuous stirred reactors, or immobilized or embedded cell cultures, such as in hollow fibers, microcapsules, agarose beads, or ceramic cylinders. If necessary and / or required, peptide solutions can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, DEAE-cellulose chromatography, and / or (immuno)affinity chromatography.

[0200] The gene encoding the binding protein specifically described in this disclosure can also be expressed in non-mammalian cells such as bacteria, yeast, or plant cells. In this regard, it should be understood that various single-celled non-mammalian microorganisms such as bacteria can also be transformed, i.e., those capable of growing in cultures or fermentations. Transformation-sensitive bacteria include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; members of the genus Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. It should also be understood that when expressed in bacteria, the binding protein can become part of an inclusion body. In some embodiments, the binding protein is then isolated, purified, and assembled into a functional molecule. In some embodiments, the binding protein of this disclosure is expressed in bacterial host cells. In some implementations, bacterial host cells are transformed with an expression vector containing a nucleic acid molecule encoding the binding protein disclosed herein.

[0201] Besides prokaryotes, eukaryotic microorganisms can also be used. *Saccharomyces cerevisiae* or *Bacillus bakerella* are the most commonly used eukaryotic microorganisms, but many other strains are also readily available. For expression in the genus *Saccharomyces*, the plasmid YRp7 is typically used, for example (Stinchcomb et al., *Nature*, 282:39 (1979); Kingsman et al., *Gene*, 7:141 (1979); Tschemper et al., *Gene*, 10:157 (1980)). This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow in tryptophan, such as ATCC 44076 or PEP4-1 (Jones, *Genetics*, 85:12 (1977)). The presence of *trpl* damage, a characteristic of the yeast host cell genome, provides an efficient environment for transformation by growth detection in the absence of tryptophan.

[0202] Formulations / Pharmaceutical Compositions In some embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the antigen-binding protein described herein. Some embodiments include a pharmaceutical composition comprising a therapeutically effective amount of any of the binding proteins described herein or a binding protein-drug conjugate, and mixed with a pharmaceutically or physiologically acceptable formulation selected for suitability to the manner of administration.

[0203] Acceptable formulations are generally non-toxic to recipients at the dosage and concentration used.

[0204] In some embodiments, the pharmaceutical composition may contain formulation materials for altering, maintaining, or retaining properties of the composition such as pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), swelling agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin), proteins (such as serum albumin, gelatin, or immunoglobulins), colorants, flavorings and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight peptides, salt-forming counterions (such as sodium), and antimicrobial agents. Preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants, or wetting agents (such as pluronics; PEG; sorbitol esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; glycerol; lecithin; cholesterol, or terosapane), stability enhancers (such as sucrose or sorbitol), tonicotinic agents (such as alkali metal halides, such as sodium chloride or potassium chloride, or mannitol and sorbitol), delivery media, diluents, excipients, and / or pharmaceutical adjuvants (see, for example, REMINGTON'S PHARMACEUTICAL). SCIENCES (18th edition, edited by AR Gennaro, Mack Publishing Company 1990) and subsequent editions thereof, which are incorporated herein by reference for any purpose.

[0205] In some embodiments, the optimal pharmaceutical composition will be determined by a technician based on, for example, the intended route of administration, delivery format, and required dosage. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[0206] In some embodiments, the primary medium or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable medium or carrier for injection may be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials commonly found in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary media. Other exemplary pharmaceutical compositions comprise a Tris buffer with a pH of about 7.0-8.5 or an acetate buffer with a pH of about 4.0-5.5, which may also contain sorbitol or a suitable alternative. In one embodiment of this disclosure, a binding protein composition for storage can be prepared by mixing a selected composition having the desired purity with an optional formulation in the form of a lyophilized cake or aqueous solution. Furthermore, the binding protein can be formulated as a lyophilized product using a suitable excipient (such as sucrose).

[0207] In some embodiments, the pharmaceutical compositions disclosed herein may be selected for parenteral or subcutaneous delivery. Alternatively, the compositions may be selected for inhalation or delivery via the digestive tract, such as oral delivery. The preparation of such pharmaceutically acceptable compositions is within the scope of the art.

[0208] In some embodiments, the components are formulated at concentrations acceptable for the application site. For example, a buffer solution is used to maintain the composition at a physiological pH or slightly lower, typically in the pH range of about 5 to about 8.

[0209] When considering parenteral administration, the therapeutic composition for use can be in the form of a pyrogen-free, parenteral-acceptable aqueous solution containing the desired binding protein in a pharmaceutically acceptable medium. A particularly suitable medium for parenteral injection is sterile distilled water in which the binding protein is formulated into a suitably preserved sterile isotonic solution. Another preparation may involve formulating the desired molecule with an agent (such as injectable microspheres, biodegradable particles, polymeric compounds such as polylactic acid or polyglycolic acid), beads, or liposomes) to provide a controlled or sustained release of the product, which can then be delivered via reservoir injection. Hyaluronic acid can also be used, and this can have a prolonged effect in promoting circulation. Other suitable means for introducing the desired molecule include implantable drug delivery devices.

[0210] In one embodiment, the pharmaceutical composition may be formulated for inhalation. For example, the binding protein may be formulated as a dry powder for inhalation. The binding protein inhalation solution may also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution may be nebulized.

[0211] Certain formulations are also envisioned for oral administration. In one embodiment of this disclosure, the multispecific binding protein administered in this manner may or may not be formulated with carriers conventionally used for compounding solid dosage forms such as tablets and capsules. For example, capsules may be designed to release the active portion of the formulation at a point in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be incorporated to promote the absorption of the binding protein. Diluents, flavoring agents, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0212] Another pharmaceutical composition may involve an effective amount of a multispecific binding protein mixed with a nontoxic excipient suitable for manufacturing tablets. A solution in unit dose form can be prepared by dissolving the tablet in sterile water or another suitable medium. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, or sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or gum arabic; or lubricants such as magnesium stearate, stearic acid, or talc.

[0213] Additional pharmaceutical compositions disclosed herein will be apparent to those skilled in the art, including formulations involving binding proteins in sustained or controlled delivery formulations. Techniques for formulating various other sustained or controlled delivery devices, such as liposome carriers, biocorrosive microparticles or porous beads, and reservoir injections, are also known to those skilled in the art. Additional examples of sustained-release formulations include semi-permeable polymer matrices in the form of shaped articles (e.g., membranes or microcapsules). Sustained-release matrices may include polyesters, hydrogels, polylactic acid, copolymers of L-glutamic acid and L-glutamic acid γ-ethyl ester, poly(2-hydroxyethyl methacrylate), ethylene-vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art.

[0214] In some embodiments, pharmaceutical compositions intended for intravenous administration must generally be sterile. This can be achieved by filtration through a sterile filter membrane. In the case of lyophilization of the composition, sterilization using this method can be performed before or after lyophilization and reconstitution. Compositions intended for parenteral administration can be stored in lyophilized or solution form. Additionally, parenteral compositions are typically placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be punctured by a hypodermic needle.

[0215] Once a pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored in ready-to-use form or in a form that requires reconstitution before application (e.g., lyophilized form).

[0216] This disclosure also covers kits for producing single-dose administration units. The kits may each contain a first container with a dried multispecific binding protein and a second container with an aqueous formulation. Kits containing single-compartment and multi-compartment pre-filled syringes (e.g., liquid syringes and lyophilized syringes) are also included within the scope of this disclosure.

[0217] The effective amount of the binding protein drug composition to be used in treatment will depend, for example, on the treatment context and purpose. Those skilled in the art will understand that the appropriate dose level for treatment will therefore vary in part depending on the molecule delivered, the indication for use of the binding protein, the route of administration, and the patient's size (weight, body surface or organ size) and condition (age and overall health). Therefore, clinicians can titrate the dose and modify the route of administration to achieve the best therapeutic effect.

[0218] The frequency of administration will depend on the pharmacokinetic parameters of the binding protein in the formulation used. Typically, clinicians will administer the composition until a dose is reached to achieve the desired effect. Therefore, the composition can be administered as a single dose, as two or more doses over time (which may or may not contain the same amount of the desired molecule), or as a continuous infusion via an implanted device or catheter. Those skilled in the art will typically refine the appropriate dosage further, and this is within the scope of their daily practice. The appropriate dosage can be determined by using appropriate dose-response data.

[0219] The drug composition can be administered via known methods, such as oral administration; injection via intravenous, intraperitoneal, intracerebral (brain parenchyma), intraventricular, intramuscular, intraocular, intraarterial, portal vein, or intralesional routes; via a continuous release system; or via an implantable device. If necessary, the composition can be administered by bolus injection, continuous infusion, or via an implantable device.

[0220] In some embodiments, the composition can also be applied topically via implantation of a membrane, sponge, or other suitable material that has absorbed or encapsulated the desired molecules. When an implantable device is used, it can be implanted into any suitable tissue or organ, and the delivery of the desired molecules can be achieved via diffusion, timed release bolus, or continuous administration.

[0221] The multispecific binding proteins disclosed herein can be formulated into aerosols for external application, such as aerosols administered by inhalation (see, for example, U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivering steroids that can be used to treat inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entirety). These formulations for administration to the respiratory tract can be, alone or in combination with an inert carrier (such as lactose), in the form of an aerosol or solution for use in a nebulizer or in the form of a fine powder for inhalation. In this case, the particles of the formulation will have a diameter of less than 50 micrometers in one embodiment and less than 10 micrometers in another embodiment.

[0222] The multispecific binding proteins disclosed herein can be formulated for topical or external application, such as for application to the skin and mucous membranes in the form of gels, creams, and lotions, such as for application to the eyes, and for application to the eyes or for intracranial or intraspinal application. Topical application is envisioned for transdermal delivery and is also envisioned for application to the eyes or mucous membranes, or for inhalation therapy. Nasal solutions of the heterodimeric proteins, alone or in combination with other pharmaceutically acceptable excipients, can also be administered.

[0223] Transdermal patches (including iontophoresis and electrophoresis devices) are well known to those skilled in the art and can be used to administer heterodimeric proteins. Such patches are disclosed, for example, in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference in their entirety.

[0224] In some embodiments, the pharmaceutical composition comprising the multispecific binding protein described herein is a lyophilized powder that can be reconstituted for administration as a solution, emulsion, and other mixture. It can also be reconstituted and formulated into a solid or gel. The lyophilized powder is prepared by dissolving the heterodimeric protein described herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability of the powder or a reconstituted solution prepared from the powder, or other pharmacological components. Excipients that can be used include, but are not limited to, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium phosphate, or potassium phosphate, or other such buffers known to those skilled in the art, wherein in one embodiment the buffer is at approximately neutral pH. The solution is then sterilely filtered and subsequently lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In one embodiment, the resulting solution is aliquoted into vials for lyophilization. Each vial will contain a single dose or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, such as from about 4°C to room temperature. This lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends on the compound chosen. This amount can be determined empirically. The multispecific binding proteins provided herein can also be formulated to target specific tissues, receptors, or other areas of the body of the subject to be treated. Many such targeting methods are well known to those skilled in the art. This document contemplates the use of all such targeting methods in the compositions of the present invention. For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are incorporated herein by reference in their entirety. In a specific embodiment, the heterodimeric protein described herein is targeted to a tumor.

[0225] Treatment / Usage One aspect of this disclosure is a multispecific binding protein as described herein, which is used as a drug.

[0226] In some implementations, a method for treating a disease or condition by activating IL-18R is provided, the method comprising administering an effective amount of the multispecific binding protein as described herein to a subject in need.

[0227] In one aspect, this disclosure provides a method for treating a disease or condition in a subject, the method comprising administering the multispecific binding protein described herein to the subject in need.

[0228] In one aspect, this disclosure provides a method for stimulating IL-18R-mediated IFN-γ expression in a subject, the method comprising administering the multispecific binding protein described herein.

[0229] In some implementations, the multispecific binding protein stimulates the production of antitumor cytokines in the subject, but substantially does not stimulate the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers in the subject, relative to the stimulation of IL-18 on the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers.

[0230] In some implementations, the anti-tumor cytokines are selected from the group consisting of IFN-γ, IL-2, IL-12, IL-15, CD40L and TNFα.

[0231] In some implementations, the markers for acute inflammation or Th2 response are selected from the group consisting of IL-6, IL-1β, IL-8, IL-4, IL-5 and IL-13.

[0232] In some implementations, the multispecific binding protein stimulates the production of acute inflammatory or Th2 response markers at least 5, 10, 50, or 100 times less than IL-18.

[0233] In some implementations, the production of GM-CSF in subjects stimulated by multispecific binding proteins is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than the production of IFN-γ in subjects.

[0234] In some implementations, the multispecific binding protein described herein can be used to detect and quantify one or more target antigens in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. The binding protein can bind to the one or more target antigens with an affinity suitable for the assay method employed.

[0235] For diagnostic applications, in some implementations, multispecific binding proteins, as described herein, can be labeled with a detectable moiety. This detectable moiety can be any substance capable of generating a detectable signal directly or indirectly. For example, the detectable moiety can be, but is not limited to, a radioactive isotope, such as... 3 H, 14 C 32 P, 35 S, 125 I, 99 Tc, 111 In or 67 Ga; fluorescent or chemiluminescent compounds, such as fluorescein isothiocyanate, rhodamine, or luciferin; or enzymes, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0236] The binding protein described herein can be used for in vivo imaging. The binding protein, labeled with a detectable portion, can be administered to an animal, for example, into the bloodstream, and the presence and location of the labeled antibody in the host can be determined. The binding protein can be labeled with any portion that is detectable in animals (whether by nuclear magnetic resonance (NMR), radiology, or other detection methods known in the art).

[0237] This disclosure also relates to a kit comprising binding proteins as described herein and other reagents for detecting target antigen levels in biological samples. Such reagents may include detectable markers, blocking sera, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition containing any binding protein, polynucleotide, vector, vector system, and / or host cell as described herein.

[0238] In some embodiments, the kit includes a container and a label or packaging insert located on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container contains a composition that, on its own or in combination with another composition, is effective in treating, preventing, and / or diagnosing a condition and may have a sterile inlet (e.g., the container may be an IV solution bag or vial with a stopper that can be punctured by a hypodermic needle). In some embodiments, the label or packaging insert indicates that the composition is used for the prevention, diagnosis, and / or treatment of the selected condition. Alternatively or additionally, the article or kit may also include a second (or third) container containing pharmaceutically acceptable buffers, such as sterile water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may also include other materials deemed necessary commercially and by the user, including additional buffers, diluents, filters, needles, and syringes.

[0239] In some embodiments, this disclosure relates to a method for preventing and / or treating a disease or condition (e.g., cancer). In some embodiments, the method includes administering to a patient a therapeutically effective amount of at least one of the binding proteins described herein or pharmaceutical compositions associated therewith. In some embodiments, the patient is a person.

[0240] The contents of any articles, patents and patent applications, and all other documents and electronic information mentioned or cited herein are hereby incorporated in their entirety by reference as if each separate publication were specifically and individually indicated to be incorporated by reference. The applicant reserves the right to incorporate any and all materials and information from any such articles, patents, patent applications or other physical and electronic documents into this application in their entirety.

[0241] While this disclosure has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of this disclosure. It will be apparent to those skilled in the art that suitable equivalents can be used to make other appropriate modifications and alterations to the methods described herein without departing from the scope of the embodiments disclosed herein. Furthermore, numerous modifications can be made to adapt particular circumstances, materials, composition, processes, process steps, or steps to the purpose, spirit, and scope of this disclosure. All such modifications are intended to be within the scope of the appended claims. Certain embodiments have been described in detail and will be more clearly understood by referring to the following examples, which are for illustrative purposes only and are not intended to be limiting.

[0242] Example The following embodiments are provided to provide a complete disclosure and description to those skilled in the art of how to manufacture and use the methods and compositions particularly described in this invention, and are not intended to limit the scope of what the inventors consider their invention. Efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should also be taken into account. Unless otherwise stated, parts are parts by weight, molecular weights are average molecular weights, temperatures are degrees Celsius, and pressures are atmospheric pressure or close to atmospheric pressure.

[0243] Example 1. Humanization of IL-18R conjugates.

[0244] The conjugates previously identified with high activity levels using HEK Blue assays were humanized and optimized for therapeutic use. VHH conjugates targeting IL-18Rα and IL-18Rβ were computationally modeled using the antigen on the DIAGONAL platform. Unnecessary residues for epitope recognition were replaced with human sequences. Reversion mutations were added solely to maintain antigen binding and stability. Affinity of the constructs was measured using Carterra, and activity was measured using HEK Blue assays. Furthermore, in some cases, proline at position 14 of the VHH-binding domain was replaced with alanine to improve the stability and agonistic activity of the conjugates.

[0245] When antibody DGL207 was humanized, decreased potency and affinity for IL-18Rβ (DGL333) were observed. Using the DIAGONAL platform, it was observed that proline at position 14 (P14) may destabilize the molecule in the context of a rigid hinge (hinge 1). Restoring this mutation to alanine, present in camel lines, improved the affinity and activity of the bispecific compound in the HEK Blue assay. Figure 1 ).

[0246] In addition to humanizing VHH, Fc was engineered for optimal therapeutic use (e.g., LALAGA, mortis, and YTE mutations).

[0247] Table 6. Optimized IL-18R antibody.

[0248] Table 7. Optimized sequences of agonist antibodies against IL-18R.

[0249] Example 2. Measurement results of the affinity of the humanized conjugate for IL-18R.

[0250] SPR assays were performed using Carterra LSA equipped with an HC30M core (Carterra-Bio). Binding was determined at 25°C using HBSTE (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20) and 0.5 g / L BSA. The density of the anti-human IgG-Fc capture lawn was established on the HC30M chip using amine coupling. To prepare the lawn, the chip was activated with 133 mM EDC and 33.3 mM S-NHS in 100 mM MES pH 5.5. Goat anti-human IgG (H+L) multispecies SP ads-UNLB (Southern Biotech, 2087-01) was coupled with a standard fixative in 10 mM sodium acetate pH 4.5 for 15 min and quenched with 1 M ethanolamine HCl pH 8.5. A band of antibodies at 20 μg / mL was then captured using the anti-human IgG-Fc capture surface. The monomeric antigens of human IL-18Rα, human IL-18Rβ, cynomolgus monkey IL-18Rα, or rhesus monkey IL-18Rβ targeted by these antibodies were injected into the captured antibody array at six concentrations in a 4-fold dilution series starting from 500 nM. The binding data were then globally fitted to a 1:1 Langmuir binding model using the injected buffer as a reference and blank control, and ka, kd, and KD were estimated using Carterra Kinetics software. Table 8 shows the KD values ​​for DGL336, DGL346, and DGL620.

[0251] Table 8. Measured Affinities of Non-humanized Leads

[0252] Table 9. Measured affinity of the lead compound for the target

[0253] Example 3. Evaluation of agonistic activity using HEK cells overexpressing IL-18Rα and IL-18Rβ.

[0254] HEK Blue™ IL-18 cells were purchased from Invivogen (hbk-hmIL-18). These cell lines overexpress IL-18Rα and IL-18Rβ while blocking responses to TNFα and IL-1β. Reporter cells were resuscitated and cultured according to the supplier's recommendations. Cells were washed with PBS and added to 96-well plates at a density of approximately 50,000 cells / well. Control or bispecific antibody was added to the wells at the final agonist concentrations listed in Table 10. The plates were incubated at 37°C and 5% CO2 for 20–24 hours. QUANTI-Blue™ solution (Invivogen) was prepared according to the manufacturer's instructions, and 180 µL of this solution was added to each well in a new 96-well plate, followed by 20 µL of supernatant from antibody-induced HEK-Blue IL-18 cells. Cell culture plates were incubated at 37°C and 5% CO2 for 3 hours, and then read at 630 nm on a spectrophotometer (Varioskan Lux, Thermo). All optimized antibodies exhibited similar activity to their parental dehumanized antibodies in the HEK Blue assay. To calculate EC50, bispecificities were evaluated using 10-point, 5-fold titrations starting at 100 nM. Human IL-18 (Invivogen, rcyec-hiI18) was titrated using 8-point, 4-fold titrations starting at 1 ng / ml. All data were fitted in PRISM using logarithmic (agonist) and response-variable slope (four parameters) analyses.

[0255] Table 10. Results using HEK-Blue with optimized antibody.

[0256] Example 4. IFNγ induction in human PBMCs.

[0257] To assess the activity of agonist antibodies in immune cells, frozen peripheral blood mononuclear cells (PBMCs) were obtained from six donors. These PBMCs were thawed and seeded at a concentration of 250,000 cells / well in 10% RPMI (Gibco, A10491). For the assay, IL-12 (R&D Systems, 10018-IL) was added to each well at a final concentration of 0.5 ng / mL. Bispecific antibody was added to a final concentration of 300 nM and diluted to generate a titration profile. Cells were incubated for 24 h, then the plates were centrifuged at 500 g for 5 min and the supernatant was collected. Supernatants from all samples were run on a Human IFNγ DUoSet ELISA Kit (DY285B) from R&D Systems. IL-18 (R&D Systems, 9124-IL) was used as a positive control. Plates were run on Luminex according to the manufacturer's instructions. Data were analyzed in PRISM using four-parameter least-squares fitting. The EC50 for each bispecific antibody tested was calculated and then averaged across six donors (Table 11). Table 12 shows the calculated maximum induction data for IFNγ after 24 hours.

[0258] The results from this PBMC assay are shown in Figures 2A-2H. After 24 hours, the IL-18R bispecific antibody was able to induce IFNγ, a marker of tumor effector lymphocyte activation. Unlike the ligand IL-18, which can activate a variety of immune cells, the IL-18R bispecific antibody did not induce IL-5 and other Th2 cytokines (e.g., IL-4 and IL-13, data not shown). Furthermore, this bispecific antibody did not activate IL-1β and other cytokines and chemokines involved in acute inflammation, such as IL-6, IL-8, MCP-1, and GM-CSF (Figures 2B-2H). These data suggest that heteromeric IL-18R bispecific agonists preferentially stimulate anti-tumor effector lymphocytes.

[0259] Table 11. EC50 under different human donor conditions.

[0260] Table 12. Calculated maximum induction of IFNγ after 24 hours

[0261] Example 5. Gene expression profiling analysis in PBMC.

[0262] Human PBMCs were treated with recombinant human IL-12 alone (R&D Systems, 10018-IL), IL-12 plus recombinant IL-18 (rhIL-18, R&D Systems, 9124-IL), or IL-12 plus 10 nM DIAGONAL IL-18R antibody agonist for 24 hours. Total RNA was isolated and analyzed using the NanoString platform. Compared with rhIL-18, all tested DIAGONAL IL-18R agonists showed selective gene expression profiles in human PBMCs. Specifically, DIAGONAL IL-18R agonists induced the expression of genes involved in antiviral responses, NK and T cell activation, and lymphocyte signaling. Unlike IL-18, IL-18R agonists did not affect genes involved in neutrophil and monocyte activation or genes driving pro-inflammatory responses. Figure 3 ).

[0263] Example 6. In vivo characterization of agonists in a mouse GvHD model.

[0264] The molecular profile of IL-18R agonists on the immune system was evaluated using a human PBMC transplanted mouse model of graft-versus-host disease (GvHD). Human PBMCs isolated from healthy adult donors were thawed on ice with cryopreservation stock solution, washed, and then processed at 2 x 10⁻⁶ ppm. 8 Cells / mL suspended in PBS. 2x10 cells / mL were implanted into 7- to 9-week-old female NOD / SCID / IL-2Rγnull immunodeficient mice on day 0. 7 Three PBMCs were used. Three mouse cohorts were established using three donors. Each agonist was administered at 3 mpk to a group of nine mice (representing three mice per donor) on days 0, 4, and 10. Cellularity and cell activation status were assessed using whole blood flow cytometry, and cytokine analysis was performed using plasma CBA analysis. Figure 4A shows IFNγ induction at two time points (days 7 and 12). All agonists showed robust IFNγ induction in the GVHD model. Figure 4B shows the increased number of CD8 T cells following agonist administration.

[0265] Example 7. Characterization of agonists in cynomolgus monkeys.

[0266] Male cynomolgus macaques (2–3 monkeys per agonist) received a single dose of a bispecific IL-18R agonist on day 0. Blood samples were collected at various time points before and after administration to evaluate PK and PD profiles. Blood samples were analyzed either as fresh whole blood or processed into plasma. Data are shown in Figures 5A–19C. The data show that a single dose of the IL-18R agonist resulted in activation of T cells and NK cells, such as CD69 expression on CD8 T cells (…). Figures 5A-5C ) and a decrease in inhibitory CD159a receptors on CD56+ NK cells ( Figures 6A-6C The signals indicated by IFNγ (a neurotransmitter) are consistent with observed cell activation, particularly in T cells and NK cells. Figures 7A-7C ) and IL-2 ( Figures 8A-8C The number of monocytes ( ) has increased. This agonist does not amplify monocytes ( ). Figures 9A-9C It does not induce acute inflammatory mediators such as IL-6 ( ), and it does not induce acute inflammatory mediators such as IL-6 ( ). Figures 10A-10C ), GM-CSF ( Figure 11A-11C It has no effect on other cytokines and chemokines involved in acute inflammation, myeloid-derived suppressor cells, or the Th2 response. This bispecific agonist is well tolerated. Due to its poor cross-reactivity with cynomolgus monkey IL-18Rβ, the activity of DGL620 in cynomolgus monkeys may not be representative of its activity in humans. DGL620 has complete cross-reactivity with IL-18Rα.

[0267] Another factor measured was IL-12 / 23 p40 ( Figures 12A-12C ), TNFα ( Figures 13A-13C ), IL-15 Figures 14A-14C ), sCD40L ( Figures 15A-15C MCP-1 Figures 16A-16C ), IL-4 Figures 17A-17C ), IL-5 Figures 18A-18C ) and IL-13 ( Figures 19A-19C ).

[0268] Example 8. Expression of agonist IL-18R antibody.

[0269] DGL336, DGL346, and DGL620 were transiently transfected into 10 L CHO-K1 cells using the WuXian Express transfection platform (WuXi Biologics). Antibodies were purified using MabSelect SuRe (Cytiva) and further refined using POROS XS (Thermo) and / or CaptoMMC ImpRes (Cytiva) according to purity. Purity at each step was analyzed using SEC-HPLC and SDS-PAGE, and the purity was confirmed by mass spectrometry. The final product yields for each product were as follows: DGL336 – 1 g / L; DGL346 – 1.3 g / L; DGL620 – 0.8 g / L (Table 13).

[0270] Table 13. Quantification of IL-18R antibody expression from CHO K1 cells.

Claims

1. A multispecific binding protein comprising at least one first binding portion specifically binding to human interleukin-18 receptor α (IL-18Rα) and at least one second binding portion specifically binding to human interleukin-18 receptor β (IL-18Rβ), wherein: a. The first binding portion comprises the amino acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17; and b. The second binding portion comprises the amino acid sequence of SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:

32.

2. The multispecific binding protein of claim 1, wherein the first binding portion and the second binding portion are VHH domains.

3. The multispecific binding protein of claim 1 or 2, wherein the first and second binding moieties are located on the same polypeptide.

4. The multispecific binding protein of claim 1 or 2, wherein the first and second binding moieties are located on different peptides.

5. The multispecific binding protein as described in any of the preceding claims, further comprising one or more modified hinge regions.

6. The multispecific binding protein of claim 5, wherein one or more hinges comprise: i) An upper hinge region, wherein the length of the upper hinge region is at most 7 amino acids or the upper hinge region is absent; and ii) The middle hinge region and the lower hinge region, wherein the lower hinge region is connected to the N end of the heavy chain constant region.

7. The multispecific binding protein of claim 5 or 6, wherein the upper hinge region of the first modified hinge region and the second modified hinge region are identical sequences.

8. The multispecific binding protein of claim 5 or 6, wherein the upper hinge region of the first modified hinge region and the second modified hinge region are different sequences.

9. The multispecific binding protein of any one of claims 1-8, wherein the upper hinge region comprises an amino acid sequence derived from the upper hinge region of a human IgG antibody.

10. The multispecific binding protein of claim 9, wherein the IgG antibody is selected from IgG1, IgG2, IgG3 and IgG4.

11. The multispecific binding protein of claim 10, wherein the IgG antibody is IgG1.

12. The multispecific binding protein of any one of claims 1-11, wherein the upper hinge region comprises the amino acid sequence of SEQ ID NO:

2.

13. The multispecific binding protein of any one of claims 1-11, wherein the upper hinge region comprises the amino acid sequence of SEQ ID NO:

5.

14. The multispecific binding protein of claim 13, wherein the IgG antibody is IgG4.

15. The multispecific binding protein of claim 14, wherein the upper hinge region comprises the amino acid sequence of SEQ ID NO:

4.

16. The multispecific binding protein of any one of claims 1-11, wherein the upper hinge is absent.

17. The multispecific binding protein as described in any of the preceding claims, further comprising all or part of an immunoglobulin Fc domain or a variant thereof.

18. The multispecific binding protein of claim 17, wherein the Fc domain or a variant thereof comprises a first Fc heavy chain and a second Fc heavy chain.

19. The multispecific binding protein of claim 17 or 18, further comprising a variant Fc domain having reduced effector function.

20. The multispecific binding protein of claim 19, wherein at least one Fc heavy chain comprises a substitution at amino acid position 234 according to EU number.

21. The multispecific binding protein of claim 20, wherein the substitution at amino acid position 234 is alanine (A).

22. The multispecific binding protein of claim 21, wherein at least one Fc heavy chain comprises a substitution located at amino acid position 235 according to EU number.

23. The multispecific binding protein of claim 22, wherein the substitution at amino acid position 235 is alanine (A).

24. The multispecific binding protein of claim 19, wherein at least one Fc heavy chain comprises a substitution at amino acid position 237 according to EU number.

25. The multispecific binding protein of claim 24, wherein the substitution at amino acid position 237 is alanine (A).

26. The multispecific binding protein of claim 18, wherein at least one Fc heavy chain comprises one or more substitutions located at amino acid positions 234, 235 or 237 according to EU number.

27. The multispecific binding protein as described in claim 26, The substitution located at amino acid position 234 is alanine (A). The substitution located at amino acid position 235 is alanine (A), and The substitution located at amino acid position 237 is alanine (A).

28. The multispecific binding protein as claimed in any of the preceding claims, wherein the Fc domain comprises a heterodimerization mutation for promoting heterodimerization of the first binding moiety and the second binding moiety.

29. The multispecific binding protein of claim 28, wherein the heterodimerization mutation is a kilometreotype (KIH) mutation.

30. The multispecific binding protein of claim 29, wherein the first Fc heavy chain comprises an amino acid substitution at position 366, 368, or 407 that produces a mortar, and the second Fc heavy chain comprises an amino acid substitution at position 366 that produces a pestle.

31. The multispecific binding protein of claim 30, wherein the first Fc heavy chain comprises amino acid substitutions of T366S, L368A, or Y407V, and the second Fc heavy chain comprises amino acid substitutions of T366W.

32. The multispecific binding protein of claim 30, wherein the heterodimerization mutation is a charge stabilization mutation.

33. The multispecific binding protein of claim 32, wherein the first Fc heavy chain comprises an amino acid substituted N297K, and the second Fc heavy chain comprises an amino acid substituted N297D.

34. The multispecific binding protein of claim 32, wherein the first Fc heavy chain comprises an amino acid substituted T299K, and the second Fc heavy chain comprises an amino acid substituted T299D.

35. The multispecific binding protein of claim 28, wherein the heterodimerization mutation comprises engineered disulfide bonds.

36. The multispecific binding protein of claim 35, wherein the engineered disulfide bond is formed by a first Fc heavy chain containing an amino acid substituted Y349C and a second Fc heavy chain containing an amino acid substituted S354C.

37. The multispecific binding protein of claim 35, wherein the engineered disulfide bond is formed by a C-terminal extension peptide fused to the C-terminus of each of the first Fc heavy chain and the second Fc heavy chain.

38. The multispecific binding protein of claim 37, wherein the first Fc heavy chain C-terminal extension comprises the amino acid sequence GEC, and the second Fc heavy chain C-terminal extension comprises the amino acid sequence SCDKT.

39. The multispecific binding protein of any one of claims 17-38, wherein at least one Fc domain contains one or more mutations for promoting an increased half-life.

40. The multispecific binding protein of claim 39, wherein at least one Fc heavy chain comprises one or more substitutions located at amino acid positions 252, 254 or 256 according to EU numbering.

41. The multispecific binding protein as described in claim 40, The substitution located at amino acid position 252 is tyrosine (Y). The substitution located at amino acid position 254 is threonine (T), and The substitution located at amino acid position 236 is glutamic acid (E).

42. The multispecific binding protein of any of the preceding claims, wherein the first binding portion specifically binding to human IL-18Rα comprises the amino acid sequence shown in SEQ ID NO: 26, and the second binding portion specifically binding to human IL-18Rβ comprises the amino acid sequence shown in any one of SEQ ID NO: 27, 28, or 31.

43. The multispecific binding protein as claimed in any of the preceding claims, wherein the binding protein comprises the amino acid sequence shown in SEQ ID NO:

29.

44. The multispecific binding protein as described in any of the preceding claims, further comprising an Fc region containing the amino acid sequence shown in SEQ ID NO:

30.

45. The multispecific binding protein according to any one of claims 1-4, comprising a first polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 26, and a second polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO:

28.

46. ​​The multispecific binding protein according to any one of claims 1-4, comprising a first polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 29, and a second polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO:

30.

47. The multispecific binding protein according to any one of claims 1-4, comprising a first polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 26, and a second polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO:

27.

48. The multispecific binding protein according to any one of claims 1-4, comprising a first polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO: 26, and a second polypeptide chain having at least 80% identity with the amino acid sequence of SEQ ID NO:

31.

49. A pharmaceutical composition comprising the multispecific binding protein of any one of the preceding claims and a pharmaceutically acceptable carrier.

50. An isolated nucleic acid molecule encoding a multispecific binding protein as described in any of the preceding claims.

51. An expression vector comprising the nucleic acid molecule of claim 50.

52. A host cell comprising the expression vector of claim 51.

53. A method for treating a disease or ailment of a subject, comprising administering to a subject in need the multispecific binding protein according to any one of the preceding claims.

54. The multispecific binding protein according to any one of the preceding claims, used as a drug.

55. A method for stimulating IL-18R-mediated IFN-γ expression in a subject, the method comprising administering a multispecific binding protein according to any one of the preceding claims.

56. The method of claim 55, wherein the multispecific binding protein stimulates the production of antitumor cytokines in the subject but substantially does not stimulate the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers in the subject relative to the stimulation of IL-18 on the production of MCP-1, GM-CSF, or acute inflammatory or Th2 response markers.

57. The method of claim 55 or 56, wherein the antitumor cytokine is selected from the group consisting of IFN-γ, IL-2, IL-12, IL-15, CD40L and TNFα.

58. The method of any one of claims 55-57, wherein the acute inflammatory or Th2 response marker is selected from the group consisting of IL-6, IL-1β, IL-8, IL-4, IL-5 and IL-13.

59. The method of any one of claims 55-58, wherein the multispecific binding protein stimulates the production of acute inflammatory or Th2 response markers at least 5 times, at least 10 times, at least 50 times, or at least 100 times less than IL-18.

60. The method of any one of claims 55-59, wherein the production of GM-CSF in the subject stimulated by the multispecific binding protein is at least 5 times, at least 10 times, at least 50 times, or at least 100 times less than the production of IFN-γ in the subject.

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