Heteromeric agonist antibodies against IL-18 receptor

Abstract

Bispecific agonist antibodies that bind to the IL-18 receptor and methods of using same are provided.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application Nos. 63 / 539,902, filed September 22, 2023, 63 / 458,042, filed April 7, 2023, and 63 / 418,333, filed October 21, 2022, the entire disclosures of which are incorporated herein by reference. [Background technology]

[0002] Interleukin-18 (IL-18) (also known as interferon-γ-inducing factor, IGIF) is a pleiotropic proinflammatory cytokine that modulates both innate and adaptive immune system responses. IL-18 has been shown to play a key role in modulating the inflammatory cascade, making it an ideal target for inhibiting autoimmune diseases, including but not limited to inflammatory diseases of the gut, heart, and lung (Kaplanski G. Immunol. Rev. (2018);281(1):138-153). IL-18 has also been shown to have antitumor activity in preclinical models. IL-18 therapy in clinical trials is focused on the use of recombinant IL-18 variants to agonize the IL-18 receptor complex (IL-18R). These therapies include SB-485232 (tadequinig alfa), a recombinant IL-18 being investigated by GSK for the treatment of melanoma, and ST-067, an engineered variant of IL-18 in clinical development by Simcha Therapeutic for the treatment of solid tumors.

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

[0004] Although early clinical trials have investigated the administration of therapeutic agents containing only recombinant or engineered IL-18, several factors may contribute to the failure of such treatments. For example, IL-18 binds solely to IL-18Rα, an extracellular signaling domain component, but not to IL-18Rβ, an adaptor molecule of the IL-18R receptor complex (Takei S. et al.; Arthritis Res. Ther. (2011); 13(2):R52). However, binding to both IL-18R subunits is required for signal transduction and, ultimately, activation of inflammatory mediators. In contrast, agonistic antibodies can be engineered to bind independently to both receptor subunits. Recombinant IL-18 is rapidly neutralized by its endogenous inhibitor, IL-18-binding protein (IL-18BP), which is induced by IL-18R signaling. Because the scaffold of the agonistic antibody does not bind to IL-18BP, it can sustainably activate IL-18R. Although IL-18 can be engineered to not bind to IL-18BP (Zhao T et al.; Nature; 583, 609-14 (2020)), this mutant protein molecule has a significant mutational burden, which may render it immunogenic and susceptible to rapid neutralization by the host immune system. Finally, recombinant or endogenous IL-18 is a high-affinity, short-half-life protein that is locally trapped at the administration site and rapidly eliminated, limiting its ability to induce signaling in tumors distant from the administration site. Antibody agonists can be engineered to have a longer elimination half-life and reduced affinity for IL-18R-expressing cells, thereby enhancing their pharmacokinetic and pharmacodynamic properties in target tissues compared to recombinant or engineered IL-18. Summary of the Invention

[0005] The present disclosure improves upon the prior art by providing heteromeric antibodies that can activate IL-18R-mediated cell signaling by effectively cross-linking the IL18Rα and IL18Rβ subunits of the IL-18 receptor.

[0006] In certain embodiments, the present disclosure provides a multispecific binding protein comprising a first binding moiety that specifically binds human IL-18Rα and a second binding moiety that specifically binds 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.

[0007] In one aspect, the disclosure provides a multispecific binding protein comprising a first binding moiety that specifically binds human IL-18Rα and a second binding moiety that specifically binds human IL-18Rβ, wherein the multispecific binding protein stimulates anti-tumor cytokine production without substantially stimulating MCP-1 production, GM-CSF production, or the production of markers of acute inflammatory or Th2 responses compared to IL-18 stimulation of MCP-1 production, GM-CSF production, or the production of markers of acute inflammatory or Th2 responses.

[0008] In certain embodiments, the anti-tumor cytokine is selected from the group consisting of IFN-γ, IL-2, IL-12, IL-15, CD40L, and TNFα.

[0009] In certain embodiments, the marker of an acute inflammatory response or a Th2 response is selected from the group consisting of IL-6, IL-1β, IL-8, IL-4, IL-5, and IL-13.

[0010] In certain embodiments, anti-tumor cytokines and markers of acute inflammatory or Th2 responses are determined in peripheral blood mononuclear cell (PBMC) assays.

[0011] In certain embodiments, the PBMC assay comprises: 1) incubating a first PBMC population with the 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 anti-tumor cytokines and markers of an acute inflammatory or Th2 response from the first and second PBMC populations.

[0012] In certain embodiments, the multispecific binding protein stimulates production of a marker of an acute inflammatory response or a Th2 response that is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than IL-18.

[0013] In specific embodiments, the multispecific binding protein stimulates production of a marker of an acute inflammatory response or a Th2 response that is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than IL-18 as measured in a PBMC assay.

[0014] In specific embodiments, the multispecific binding protein stimulates GM-CSF production that is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold lower than IFN-γ production.

[0015] In certain embodiments, the present disclosure provides a multispecific binding protein comprising a first binding moiety that specifically binds human IL-18Rα and a second binding moiety that specifically binds human IL-18Rβ, wherein the multispecific binding protein is capable of agonist activity of IL-18 receptor signaling.

[0016] In certain embodiments, the present disclosure provides a multispecific binding protein comprising a means for specifically binding to human IL-18Rα and a means for specifically binding to human IL-18Rβ. In certain embodiments, 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. In certain embodiments, the multispecific binding protein is capable of agonistic activity of IL-18 receptor signaling.

[0017] In certain embodiments, the first binding moiety comprises an IL-18Rα VHH domain and the second binding moiety comprises an IL-18Rβ VHH domain.

[0018] In certain embodiments, the IL-18Rα VHH domain and the IL-18Rβ VHH domain are in separate polypeptides.

[0019] In certain embodiments, the IL-18Rα VHH domain and the IL-18Rβ VHH domain are in the same polypeptide.

[0020] In certain embodiments, the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of SYDMG (SEQ ID NO: 1), an HCDR2 sequence comprising the amino acid sequence of ALRWSGGSTSYADSVKG (SEQ ID NO: 2), and an HCDR3 sequence comprising the amino acid sequence of TLETDSGTYWADY (SEQ ID NO: 3).

[0021] In certain embodiments, the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of ATGMG (SEQ ID NO: 4), an HCDR2 sequence comprising the amino acid sequence of RISSTGSPNYVDFVKG (SEQ ID NO: 5), and an HCDR3 sequence comprising the amino acid sequence of VGTTLFA (SEQ ID NO: 6).

[0022] In certain embodiments, the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of TKGLG (SEQ ID NO: 7), an HCDR2 sequence comprising the amino acid sequence of GISSAGWIFYTQSVKG (SEQ ID NO: 8), and an HCDR3 sequence comprising the amino acid sequence of AQSGVPLRS (SEQ ID NO: 9).

[0023] In certain embodiments, the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of INIMD (SEQ ID NO: 10), an HCDR2 sequence comprising the amino acid sequence of RISPGDIITYANDVKG (SEQ ID NO: 11), and an HCDR3 sequence comprising the amino acid sequence of RQGAGDY (SEQ ID NO: 12).

[0024] In certain embodiments, the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of DYVLG (SEQ ID NO: 13), It comprises an HCDR2 sequence comprising the amino acid sequence of CISSRGRYLNYAETVKG (SEQ ID NO: 14) and an HCDR3 sequence comprising the amino acid sequence of VRRVSEVCKLAEDDFAS (SEQ ID NO: 15).

[0025] In certain embodiments, the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of KHAMG (SEQ ID NO: 16).

[0026] In certain embodiments, the multispecific binding protein comprises an HCDR2 sequence comprising the amino acid sequence of AIDWSGGSTYYADSVKG (SEQ ID NO: 17), an HCDR3 sequence comprising the amino acid sequence of DSYTDYAQLWLPELESEYDY (SEQ ID NO: 18).

[0027] In certain embodiments, the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of SYTMG (SEQ ID NO: 19), an HCDR2 sequence comprising the amino acid sequence of AISWSAGRTYYADSVKG (SEQ ID NO: 20), and an HCDR3 sequence comprising the amino acid sequence of EEAPDWAPIDCSGYGCLSLYDY (SEQ ID NO: 21).

[0028] In certain embodiments, the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of IDFMG (SEQ ID NO: 22), an HCDR2 sequence comprising the amino acid sequence of TITTGGSTNYADSVKD (SEQ ID NO: 23), and an HCDR3 sequence comprising the amino acid sequence of VVHTTSRPPVLY (SEQ ID NO: 24).

[0029] In certain embodiments, the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of NYDMG (SEQ ID NO: 25), an HCDR2 sequence comprising the amino acid sequence of VISGPGGIAFYGDSVKG (SEQ ID NO: 26), and an HCDR3 sequence comprising the amino acid sequence of APRGSYYRRTNSYDY (SEQ ID NO: 27).

[0030] In certain embodiments, the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of RYG (SEQ ID NO: 28), an HCDR2 sequence comprising the amino acid sequence of DIYWNGGNTYYTDSVKG (SEQ ID NO: 29), and an HCDR3 sequence comprising the amino acid sequence of ATSYYAVTDPLKVAY (SEQ ID NO: 30).

[0031] In certain embodiments, the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of NWYMR (SEQ ID NO: 31), an HCDR2 sequence comprising the amino acid sequence of SINSGGDDTDYADSVKG (SEQ ID NO: 32), and an HCDR3 sequence comprising the amino acid sequence of GADRV (SEQ ID NO: 33).

[0032] In certain embodiments, the second binding portion of the multispecific binding protein comprises an IL-18Rβ VHH domain.

[0033] In certain embodiments, the IL-18Rβ VHH of the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of SYTMG (SEQ ID NO: 19), an HCDR2 sequence comprising the amino acid sequence of ALSWWNGGISTAYADSVKG (SEQ ID NO: 34), and an HCDR3 sequence comprising the amino acid sequence of ARDRMPRADEYDY (SEQ ID NO: 35).

[0034] In certain embodiments, the IL-18Rβ VHH of the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of RNSMA (SEQ ID NO: 36), an HCDR2 sequence comprising the amino acid sequence of AISSISSGGRTDYADFVKG (SEQ ID NO: 37), and an HCDR3 sequence comprising the amino acid sequence of PIRVASLAYDD (SEQ ID NO: 38).

[0035] In certain embodiments, the IL-18Rβ VHH of the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of NYHMG (SEQ ID NO: 39), an HCDR2 sequence comprising the amino acid sequence of AISSSGGKTSYPDSVNG (SEQ ID NO: 40), and an HCDR3 sequence comprising the amino acid sequence of DPRYWVAAGGSEPENVEV (SEQ ID NO: 41).

[0036] In certain embodiments, the IL-18Rβ VHH of the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of VNSMA (SEQ ID NO: 42), an HCDR2 sequence comprising the amino acid sequence of VISSGGSAVYADSVKG (SEQ ID NO: 43), and an HCDR3 sequence comprising the amino acid sequence of GSAAYRDY (SEQ ID NO: 44).

[0037] In certain embodiments, the IL-18Rβ VHH of the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of RNTMG (SEQ ID NO: 45), an HCDR2 sequence comprising the amino acid sequence of HFLWTGGETDYADAVKG (SEQ ID NO: 46), and an HCDR3 sequence comprising the amino acid sequence of NYAGYRIDGYQY (SEQ ID NO: 47).

[0038] In certain embodiments, the IL-18Rβ VHH of the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of IHVMG (SEQ ID NO: 48), an HCDR2 sequence comprising the amino acid sequence of FIINNGGTRYADSVKG (SEQ ID NO: 49), and an HCDR3 sequence comprising the amino acid sequence of EGTYRGRYSTDN (SEQ ID NO: 50).

[0039] In certain embodiments, the IL-18Rβ VHH of the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of ENDVR (SEQ ID NO: 51), an HCDR2 sequence comprising the amino acid sequence of AITSGITGYADSVRI (SEQ ID NO: 52), and an HCDR3 sequence comprising the amino acid sequence of TDQY (SEQ ID NO: 53).

[0040] In certain embodiments, the IL-18Rβ VHH of the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of LNTMG (SEQ ID NO: 54), an HCDR2 sequence comprising the amino acid sequence of VESSSGITNYADSVKG (SEQ ID NO: 55), and an HCDR3 sequence comprising the amino acid sequence of KLFGRDF (SEQ ID NO: 56).

[0041] In certain embodiments, the IL-18Rβ VHH of the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of SHNVMG (SEQ ID NO: 57), an HCDR2 sequence comprising the amino acid sequence of SIGSGGSTNYVDSVKG (SEQ ID NO: 58), and an HCDR3 sequence comprising the amino acid sequence of VVGVYRGS (SEQ ID NO: 59).

[0042] In certain embodiments, the IL-18Rβ VHH of the multispecific binding protein comprises an HCDR1 sequence comprising the amino acid sequence of RDTMG (SEQ ID NO: 116), an HCDR2 sequence comprising the amino acid sequence of VISSSGNTNYADSVLG (SEQ ID NO: 117), and an HCDR3 sequence comprising the amino acid sequence of HRTYGVDY (SEQ ID NO: 118).

[0043] In certain embodiments, the IL-18Rα VHH of the multispecific binding protein is at least about 90% identical, at least about 95% identical, or at least 98% identical to the amino acid sequence of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70.

[0044] In certain embodiments, the IL-18Rα VHH of the multispecific binding protein is at least about 90% identical, at least about 95% identical, or at least 98% identical to the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:98.

[0045] In certain embodiments, the IL-18Rα VHH of the multispecific binding protein comprises the amino acid sequence of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:70.

[0046] In certain embodiments, the IL-18Rβ VHH of the multispecific binding protein comprises the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:98.

[0047] In some embodiments, the multispecific binding protein has agonist activity that meets or exceeds a particular threshold over background as measured in an agonist activity assay, eg, a HEK-Blue assay.

[0048] In some embodiments, the agonist activity of the multispecific binding protein is about 2-fold over background.

[0049] In some embodiments, the agonist activity of the multispecific binding protein is about 3-fold over background.

[0050] In some embodiments, the agonist activity of the multispecific binding protein is about 4-fold over background.

[0051] In some embodiments, the agonist activity of the multispecific binding protein is about 5-fold over background.

[0052] In some embodiments, the agonist activity of the multispecific binding protein is about 6-fold over background.

[0053] In some embodiments, the agonist activity of the multispecific binding protein is about 7-fold over background.

[0054] In some embodiments, the agonist activity of the multispecific binding protein is about 8-fold over background.

[0055] In some embodiments, the agonist activity of the multispecific binding protein is about 9-fold over background.

[0056] In some embodiments, the agonist activity of the multispecific binding protein is about 10-fold over background.

[0057] In some embodiments, the agonist activity of the multispecific binding protein is about 11-fold over background.

[0058] In some embodiments, the agonist activity of the multispecific binding protein is about 12-fold over background.

[0059] In some embodiments, the agonist activity of the multispecific binding protein is about 13-fold over background.

[0060] In some embodiments, the agonist activity of the multispecific binding protein is about 14-fold over background.

[0061] In some embodiments, the agonist activity of the multispecific binding protein is about 15-fold over background.

[0062] In some embodiments, the first binding moiety binds to one or more of amino acids Ser24, Arg25, Pro26, Thr126, Ser127, Lys128, and Ile129 of human IL-18Rα (SEQ ID NO: 287).

[0063] In some embodiments, the first binding moiety binds to at least amino acids Ser24, Arg25, Pro26, Thr126, Ser127, Lys128, and Ile129 of human IL-18Rα (SEQ ID NO: 287).

[0064] In some embodiments, the first binding moiety binds to amino acids Ser24, Arg25, Pro26, Thr126, Ser127, Lys128, Ile129, Phe135, Phe136, Gln137, Ile138, Thr139, Cys140, Glu141, Asn142, Ser143, Lys200, Thr201, and Phe202 of human IL-18Rα (SEQ ID NO: 287).

[0065] In some embodiments, the first binding moiety binds to amino acids Ser24, Arg25, Pro26, His27, Ile28, Thr29, Glu122, Arg123, Gln124, Val125, Thr126, Ser127, Lys128, Ile129, and Val130 of human IL-18Rα (sequence number 287).

[0066] In some embodiments, the second binding moiety binds to one or more of amino acids Gln216, Gly217, Thr218, Gln239, Val240, Arg241, Thr242, Ile243, Lys309, Ser310, Thr311, and Leu312 of human IL-18Rβ (SEQ ID NO: 288).

[0067] In some embodiments, the second binding moiety binds to at least amino acids Gln216, Gly217, Thr218, Gln239, Val240, Arg241, Thr242, Ile243, Lys309, Ser310, Thr311, and Leu312 of human IL-18Rβ (SEQ ID NO: 288).

[0068] In some embodiments, the second binding moiety binds to at least amino acids Asp213, Tyr214, His215, Gln216, Gly217, Thr218, Gln239, Val240, Arg241, Thr242, Ile243, Lys306, Ser307, Ile308, Lys309, Ser310, Thr311, and Leu312 of human IL-18Rβ (SEQ ID NO: 288).

[0069] In some embodiments, the second binding moiety binds to at least amino acids Glu39, Glu40, Glu41, His112, Phe113, Leu114, Thr115, Pro116, Gln216, Gly217, Thr218, Gln239, Val240, Arg241, Thr242, Ile243, Phe279, Glu280, Arg281, Val282, Phe283, Asn284, Lys309, Ser310, Thr311, Leu312, Lys313, Asp314, and Glu315 of human IL-18Rβ (SEQ ID NO: 288).

[0070] In some embodiments, the IL-18Rα binding moiety and / or the IL-18Rβ binding moiety are optimized. In some embodiments, the optimized IL-18Rα binding moiety and / or the IL-18Rβ binding moiety are humanized. In some embodiments, the IL-18Rα binding moiety comprises the amino acid sequence of SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, or SEQ ID NO:123. In some embodiments, the IL-18Rβ binding moiety comprises the amino acid sequence of SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, or SEQ ID NO:128.

[0071] In some embodiments, the multispecific binding protein further comprises one or more modified hinge regions. In some embodiments, the one or more modified hinges comprise an upper hinge region that is up to 7 amino acids in length or absent, and a middle hinge region and a lower hinge region, wherein the lower hinge region is linked 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.

[0072] 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: 274. In some embodiments, the upper hinge region comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the IgG antibody is IgG4. In some embodiments, the upper hinge region comprises the amino acid sequence of SEQ ID NO: 276. In some embodiments, the upper hinge is absent.

[0073] In certain embodiments, the multispecific binding protein further comprises all or a portion of an immunoglobulin Fc domain or a variant thereof.

[0074] In certain embodiments, the Fc domain of the multispecific binding protein or variant thereof comprises a first Fc heavy chain and a second Fc heavy chain.

[0075] In certain embodiments, the multispecific binding protein further comprises a variant Fc domain with reduced effector function.

[0076] In certain embodiments, the multispecific binding protein comprises at least one Fc heavy chain comprising a substitution at amino acid position 234 according to EU numbering.

[0077] In certain embodiments, the multispecific binding protein comprises at least one Fc heavy chain comprising a substitution at amino acid position 234 according to EU numbering, wherein the substitution at amino acid position 234 is an alanine (A).

[0078] In certain embodiments, the multispecific binding protein comprises at least one Fc heavy chain comprising a substitution at amino acid position 235 according to EU numbering.

[0079] In certain embodiments, the multispecific binding protein comprises at least one Fc heavy chain comprising a substitution at amino acid position 235 according to EU numbering, wherein the substitution at amino acid position 235 is an alanine (A).

[0080] In certain embodiments, the multispecific binding protein comprises at least one Fc heavy chain comprising a substitution at amino acid position 237 according to EU numbering.

[0081] In certain embodiments, the multispecific binding protein comprises at least one Fc heavy chain comprising a substitution at amino acid position 237 according to EU numbering, wherein the substitution at amino acid position 237 is an alanine (A).

[0082] In certain embodiments, the multispecific binding protein comprises at least one Fc heavy chain comprising one or more substitutions at amino acid positions 234, 235, or 237 according to EU numbering.

[0083] In certain embodiments, the multispecific binding protein comprises at least one Fc heavy chain comprising one or more substitutions at amino acid position 234, 235, or 237 according to EU numbering, wherein the substitution at amino acid position 234 is an alanine (A), the substitution at amino acid position 235 is an alanine (A), and the substitution at amino acid position 237 is an alanine (A).

[0084] In certain embodiments, the Fc domain of the multispecific binding protein comprises a heterodimerization mutation that promotes heterodimerization of the first binding moiety with the second binding moiety.

[0085] In certain embodiments, the Fc domain of the multispecific binding protein comprises a heterodimerization mutation that promotes heterodimerization of the first binding moiety with the second binding moiety, and the heterodimerization mutation is a knob-in-hole (KIH) mutation.

[0086] In certain embodiments, the first Fc heavy chain domain of the multispecific binding protein comprises an amino acid substitution at position 366, 368, or 407 that creates a knob, and the second Fc heavy chain comprises an amino acid substitution at position 366 that creates a hole.

[0087] In certain embodiments, the first Fc heavy chain of the multispecific binding protein comprises the amino acid substitution T366S, L368A, or Y407V, and the second Fc heavy chain comprises the amino acid substitution T366W.

[0088] In certain embodiments, the Fc domain of the multispecific binding protein comprises a heterodimerization mutation that promotes heterodimerization of the first binding moiety with the second binding moiety, and the heterodimerization mutation is a charge-stabilizing mutation.

[0089] In certain embodiments, the Fc domain of the multispecific binding protein comprises a heterodimerization mutation that promotes heterodimerization of the first binding moiety and the second binding moiety, the heterodimerization mutation is a charge-stabilizing mutation, the first Fc heavy chain comprises the amino acid substitution N297K, and the second Fc heavy chain comprises the amino acid substitution N297D.

[0090] In certain embodiments, the Fc domain of the multispecific binding protein comprises a heterodimerization mutation that promotes heterodimerization of the first binding moiety and the second binding moiety, the heterodimerization mutation is a charge-stabilizing mutation, the first Fc heavy chain comprises the amino acid substitution T299K, and the second Fc heavy chain comprises the amino acid substitution T299D.

[0091] In certain embodiments, the Fc domain of the multispecific binding protein comprises a heterodimerization mutation that promotes heterodimerization between the first binding moiety and the second binding moiety, wherein the heterodimerization mutation comprises an engineered disulfide bond.

[0092] In certain embodiments, the Fc domain of the multispecific binding protein comprises a heterodimerization mutation that promotes heterodimerization between a first binding moiety and a second binding moiety, wherein the heterodimerization mutation comprises an engineered disulfide bond, and wherein the engineered disulfide bond is formed by a first Fc heavy chain that comprises the amino acid substitution Y349C and a second Fc heavy chain that comprises the amino acid substitution S354C.

[0093] In certain embodiments, the Fc domain of the multispecific binding protein comprises a heterodimerization mutation that promotes heterodimerization between the first binding moiety and the second binding moiety, wherein the heterodimerization mutation comprises an engineered disulfide bond.

[0094] In certain embodiments, the Fc domain of the multispecific binding protein comprises a heterodimerization mutation that promotes heterodimerization between the first binding moiety and the second binding moiety, wherein the heterodimerization mutation comprises an engineered disulfide bond, and 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.

[0095] In a particular embodiment, the Fc domain of the multispecific binding protein comprises a heterodimerization mutation that promotes heterodimerization of a first binding moiety and a second binding moiety, wherein the heterodimerization mutation comprises an engineered disulfide bond, and 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, 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.

[0096] In certain embodiments, at least one Fc domain of the multispecific binding protein comprises one or more mutations that promote increased half-life.

[0097] In certain embodiments, at least one Fc heavy chain of the multispecific binding protein comprises one or more substitutions at amino acid positions 252, 254, or 256 according to EU numbering.

[0098] In certain embodiments, at least one Fc heavy chain of the multispecific binding protein comprises one or more substitutions at amino acid position 252, 254, or 256 according to EU numbering, wherein 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).

[0099] In a specific embodiment, the first binding moiety that specifically binds to human IL-18Rα comprises the amino acid sequence set forth in any one of SEQ ID NOs: 240-251, and the second binding moiety that specifically binds to human IL-18Rβ comprises the amino acid sequence set forth in any one of SEQ ID NOs: 252-260.

[0100] In some embodiments, the first binding moiety and / or the second binding moiety further comprises an amino acid substitution at position 14 from the N-terminus of the binding moiety.

[0101] In some embodiments, the amino acid at position 14 from the N-terminus of the binding moiety is a proline (P).

[0102] In some embodiments, the substitution at position 14 from the N-terminus of the binding moiety comprises an alanine (A).

[0103] In some embodiments, the substitution at position 14 from the N-terminus of the binding moiety is an alanine (A).

[0104] In some embodiments, a substitution at position 14 from the N-terminus of the binding moiety further stabilizes the binding moiety.

[0105] In some embodiments, the substitution at position 14 from the N-terminus of the binding moiety increases the agonist properties of the binding moiety.

[0106] In certain embodiments, the multispecific binding protein is comprised in a pharmaceutical composition and a pharmaceutically acceptable carrier.

[0107] In certain embodiments, the multispecific binding protein is encoded by an isolated nucleic acid molecule.

[0108] In certain embodiments, the multispecific binding protein is encoded by an expression vector.

[0109] In certain embodiments, the multispecific binding protein is encoded by an expression vector contained within a host cell.

[0110] In certain embodiments, the multispecific binding protein is administered to a subject in need thereof in a method for treating a disease or disorder in the subject.

[0111] In certain embodiments, the multispecific binding proteins are used as pharmaceuticals.

[0112] In certain embodiments, the present disclosure provides methods for treating a disease or disorder in a subject by administering to the subject a multispecific binding protein comprising a means for specifically binding to human IL-18Rα and a means for specifically binding to human IL-18Rβ.

[0113] In certain aspects, the 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 moiety that specifically binds human IL-18Rα and a second binding moiety that specifically binds 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.

[0114] In certain embodiments, the present disclosure provides methods for inducing agonist activity of IL-18 receptor signaling in a subject, comprising administering to the subject a multispecific binding protein comprising a means for specifically binding to human IL-18Rα and a means for specifically binding to human IL-18Rβ.

[0115] In one aspect, the disclosure provides a method of stimulating IL-18R-mediated IFN-γ expression in a subject comprising administering to the subject a multispecific binding protein comprising a first binding moiety that specifically binds human IL-18Rα and a second binding moiety that specifically binds human IL-18Rβ, wherein the multispecific binding protein stimulates anti-tumor cytokine production in the subject without substantially stimulating MCP-1 production, GM-CSF production, or production of markers of acute inflammatory or Th2 responses in the subject compared to IL-18 stimulation of MCP-1 production, GM-CSF production, or production of markers of acute inflammatory or Th2 responses.

[0116] In certain embodiments, the anti-tumor cytokine is selected from the group consisting of IFN-γ, IL-2, IL-12, IL-15, CD40L, and TNFα.

[0117] In certain embodiments, the marker of an acute inflammatory response or a Th2 response is selected from the group consisting of IL-6, IL-1β, IL-8, IL-4, IL-5, and IL-13.

[0118] In certain embodiments, the multispecific binding protein stimulates production of a marker of an acute inflammatory response or a Th2 response that is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than IL-18.

[0119] In specific embodiments, the multispecific binding protein stimulates at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less GM-CSF production in the subject than IFN-γ production in the subject. [Brief explanation of the drawings]

[0120] [Figure 1] FIG. 1 depicts certain exemplary embodiments of the formats of the multispecific binding proteins described herein. [Figure 2] FIG. 1 is a schematic diagram showing a workflow for characterization of multispecific binding proteins of the present disclosure. [Figure 3] 1 shows a cluster plot used in the design of multispecific binding proteins of the present disclosure. [Figure 4] 1 shows the results of screening for IL-18 agonist activity using exemplary embodiments of heteromeric antibodies derived from prior art sequences described herein. [Figure 5A] 1 shows the results of an agonist assay using exemplary heteromeric IL-18R antibodies designed by the DIAGONAL platform. [Figure 5B] 1 shows the results of an agonist assay using exemplary heteromeric IL-18R antibodies designed by the DIAGONAL platform. [Figure 6] 1 shows the results of a PBMC tumor killing assay using exemplary heteromeric IL-18R agonist antibodies in the presence or absence of human IL-12. [Figure 7] 1 is a graph showing the agonism of DGL207, DGL333, and DGL620 in a HEK Blue assay. [Figure 8A]The effect of agonist antibodies on immune cell markers when cultured in the presence of PBMCs is shown. IFN-γ is indicated. [Figure 8B] Figure 1 shows the effect of agonist antibodies on immune cell markers when cultured in the presence of PBMCs. IL-5 is shown. [Figure 8C] The effect of agonist antibodies on immune cell markers when cultured in the presence of PBMCs is shown. IL-1β is shown. [Figure 8D] Figure 1 shows the effect of agonist antibodies on immune cell markers when cultured in the presence of PBMCs, with MCP-1 shown. [Figure 8E] Figure 1 shows the effect of agonist antibodies on immune cell markers when cultured in the presence of PBMCs. IL-6 is shown. [Figure 8F] Figure 1 shows the effect of agonist antibodies on immune cell markers when cultured in the presence of PBMCs, with IL-13 shown. [Figure 8G] The effect of agonist antibodies on immune cell markers when cultured in the presence of PBMCs is shown. GM-CSF is indicated. [Figure 8H] The effect of agonist antibodies on immune cell markers when cultured in the presence of PBMCs is shown. IL-4 is indicated. [Figure 9] Gene expression data from PBMC samples cultured with an IL-18R agonist antibody. The data show that genes related to IFNγ signaling, antiviral responses, NK cell and T cell activation, and lymphocyte signaling are increased by the antibody, while neutrophil activation, monocyte activation, and pro-inflammatory signatures are decreased. [Figure 10] A and B show measurements of IFNγ and CD8 T cells in a mouse model of graft-versus-host disease (GvHD). [Figure 11] AC show CD69 expression in CD8 T cells over 21 days with DGL336 (A), DGL346 (B), and DGL620 (C). [Figure 12]AC show the reduction of CD159a receptors on CD56+ NK cells by DGL336 (A), DGL346 (B), and DGL620 (C) over 21 days. [Figure 13] AC show IFNγ expression by DGL336 (A), DGL346 (B), and DGL620 (C) over 21 days. [Figure 14] AC show IL-2 expression by DGL336 (A), DGL346 (B), and DGL620 (C) over a 21-day period. [Figure 15] AC show monocyte counts after 21 days of exposure to DGL336 (A), DGL346 (B), or DGL620 (C). [Figure 16] A to C show IL-6 expression by DGL336 (A), DGL346 (B), and DGL620 (C) over 21 days. [Figure 17A] GM-CSF expression by DGL336 for 21 days is shown. [Figure 17B] GM-CSF expression by DGL346 for 21 days is shown. [Figure 17C] GM-CSF expression by DGL620 for 21 days is shown. [Figure 18A] IL-12 / 23 p40 expression by DGL336 for 21 days is shown. [Figure 18B] IL-12 / 23 p40 expression by DGL346 for 21 days is shown. [Figure 18C] IL-12 / 23 p40 expression by DGL620 for 21 days is shown. [Figure 19A] 21 days of TNFα expression by DGL336. [Figure 19B] 21 days of TNFα expression by DGL346. [Figure 19C] 21 days of TNFα expression by DGL620. [Figure 20A] IL-15 expression by DGL336 for 21 days is shown. [Figure 20B] IL-15 expression by DGL346 for 21 days is shown. [Figure 20C]IL-15 expression by DGL620 for 21 days is shown. [Figure 21A] sCD40L expression by DGL336 for 21 days is shown. [Figure 21B] sCD40L expression by DGL346 for 21 days is shown. [Figure 21C] sCD40L expression by DGL620 for 21 days is shown. [Figure 22A] 1 shows MCP-1 expression by DGL336 for 21 days. [Figure 22B] 1 shows MCP-1 expression by DGL346 for 21 days. [Figure 22C] 1 shows MCP-1 expression by DGL620 for 21 days. [Figure 23A] IL-4 expression by DGL336 for 21 days is shown. [Figure 23B] IL-4 expression by DGL346 for 21 days is shown. [Figure 23C] IL-4 expression by DGL620 for 21 days is shown. [Figure 24A] IL-5 expression by DGL336 for 21 days is shown. [Figure 24B] IL-5 expression by DGL346 for 21 days is shown. [Figure 24C] IL-5 expression by DGL620 for 21 days is shown. [Figure 25A] IL-13 expression by DGL336 for 21 days is shown. [Figure 25B] IL-13 expression by DGL346 for 21 days is shown. [Figure 25C] IL-13 expression by DGL620 for 21 days is shown. DETAILED DESCRIPTION OF THE INVENTION

[0121] Before describing the present disclosure, it is to be understood that this disclosure is not limited to the particular methods and experimental conditions described, as methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0122] 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 belongs.

[0123] Although any methods and materials similar or equivalent to those described herein can be used in the practice of this disclosure, exemplary methods and materials are described below. All publications mentioned herein are incorporated by reference as if set forth in their entirety.

[0124] As used herein, the term "IL-18" refers to a cytokine also known as interferon-γ-inducing factor (IGIF), a proinflammatory cytokine with various functions in addition to its ability to induce interferon-γ. These various functions include activation of NF-κB, Fas ligand expression, induction of both CC and CXC chemokines, and interferon-γ. IL-18 plays an important role in Th1-type immune responses due to its ability to induce interferon-γ production in T cells and NK cells, and is involved in both innate and adaptive immunity. IL-18 is related to the IL-1 family both structurally and functionally.

[0125] The biological activity of IL-18 is mediated by binding 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 (IL-18R accessory protein, also known as IL-18AP or AcPL). The IL-18Rα subunit directly binds IL-18 but is unable to signal. The β-subunit alone does not bind IL-18, but when combined with the α-subunit, it forms a high-affinity receptor (KD = approximately 0.3 nM) required for signal transduction (Sims, JE, (2002) Current Opin. Immunol. 14:117-122). IL-18 signaling via the IL-18Rαβ complex is similar to that of the IL-1R and Toll-like receptor (TLR) systems. IL-18R signaling uses signaling molecules such as MyD88, IRAK, and TRAF6, resulting in responses similar to those of IL-1 (e.g., activation of NIK, IκB kinase, NF-κB, INK, and p38 MAP kinase). The requirement for IL-18Rα and signaling molecules in mediating IL-18 biological activity was confirmed using knockouts 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), respectively.

[0126] In certain exemplary embodiments, the IL-18 cytokine is human IL-18 (Uniprot Q14116-1). In certain exemplary embodiments, the IL-18 receptor is a human IL-18 receptor represented by the human IL-18Rα (Uniprot Q13478) and IL-18Rβ sequences (Uniprot O95256-1).

[0127] As used herein, the term "inducing proximity between the IL-18Rα and IL-18Rβ subunits of human IL-18R" refers to bringing the IL-18Rα and IL-18Rβ subunits together such that human IL-18R activity is stimulated. In certain embodiments, the proximity induced by a multispecific binding protein of the disclosure is the same as or similar to the proximity induced when IL-18 brings together the IL-18Rα and IL-18Rβ subunits of human IL-18R.

[0128] As used herein, the terms "antigen-binding moiety" or "binding domain" or "binding specificity" refer to a molecule that specifically binds to an antigen, and such binding is understood by those of skill in the art. For example, an antigen-binding moiety that specifically binds to an antigen typically binds to other molecules with lower affinity, as determined by, for example, immunoassays, BIAcore®, KinExA 3000 instruments (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, an antigen-binding moiety that specifically binds to an antigen binds to that antigen with a Ka that is at least 2 logs (e.g., a factor of 10), 2.5 logs, 3 logs, 4 logs, or more greater than the Ka when the molecule nonspecifically binds to another antigen.

[0129] As used herein, the terms "antibody" (singular and plural) include full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising the CDRs, VH regions, and / or VL regions of an antibody. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules 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, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fvs (scFv), camelized antibodies, affibodies, 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 certain embodiments, the antibody described herein refers to a polyclonal antibody population. An antibody can be an immunoglobulin molecule of 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). In certain embodiments, the antibodies described herein are IgG antibodies, or a class (e.g., human IgG1 or IgG4) or subclass thereof. 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.

[0130] As used herein, the term "VHH" refers to the heavy chain variable domain of a camelid heavy chain-only antibody (HCAb) and humanized variants thereof, 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.

[0131] As used herein, the term "VH / VL pair" refers to the combination of a VH and a VL that together form a binding site for an antigen.

[0132] As used herein, the term "heavy chain" when used in reference to an antibody can refer to any of the specific types, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence of the constant domain, which give rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4.

[0133] As used herein, the term "full-length antibody heavy chain" refers to an antibody heavy chain comprising, from N-terminus to C-terminus, a VH, a CH1 region, a hinge region, a CH2 domain, and a CH3 domain.

[0134] As used herein, the term "light chain" when used with respect to an antibody can refer to either specific type, such as kappa (κ) or lambda (λ), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In certain embodiments, 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 in an antibody variable region that confers antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The term "framework region" or "FR" is known in the art to refer to the portions of heavy and light chain variable regions other than the CDRs. Generally, each heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).

[0135] The precise amino acid sequence boundaries of a particular CDR or FR can be determined according to the scheme described in 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 site topography," 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" domains,” Dev. Comp. Immunol., 2003 January;27(1):55-77 (the “IMGT” numbering scheme), and the scheme described in Honegger A. and Pluckthun A., “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J. Mol. Biol., 2001 June;309(3):657-70 (the AHo numbering scheme).

[0136] The boundaries of a particular CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on sequence alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, and some antibodies have insertions and deletions, designated by an inserted letter, e.g., "30a." The two schemes place certain insertions and deletions ("indels") at different positions, resulting in numbering differences. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0137] As used herein, the term "single-chain variable fragment" (scFv) refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguously linked by a short, flexible polypeptide linker and are expressible as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, an scFv as used herein may have the VL and VH variable regions in any order, e.g., relative to the N- and C-termini of the polypeptide, and may comprise VL·linker·VH or VH·linker·VL.

[0138] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, particularly CDR3. However, the term "human antibody" as used herein is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. This term includes antibodies recombinantly produced in a non-human mammal or in the cells of a non-human mammal. This term is not intended to include antibodies isolated from or generated in a human subject.

[0139] As used herein, the term "multispecific antigen-binding molecule" refers to bispecific, trispecific, or multispecific antigen-binding molecules and antigen-binding fragments thereof. Multispecific antigen-binding molecules may be specific for different epitopes on a single target polypeptide or may contain antigen-binding domains specific for epitopes on multiple target polypeptides. In certain embodiments, a multispecific antigen-binding molecule of the present disclosure comprises at least a first binding specificity for the IL-18Rα subunit and at least a second binding specificity for the IL-18Rβ subunit. A multispecific antigen-binding molecule may be a single multifunctional polypeptide or a multimeric complex of two or more polypeptides covalently or noncovalently linked to each other. The term "multispecific antigen-binding molecule" includes antibodies of the present disclosure that may be linked to or coexpressed with another functional molecule, such as another peptide or protein. For example, an antibody or fragment thereof can be operatively linked (e.g., by chemical bonding, genetic fusion, non-covalent bonding, or otherwise) 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 the present disclosure, the term "multispecific antigen-binding molecule" also includes bispecific, trispecific, or multispecific antibodies or antigen-binding fragments thereof. In certain exemplary embodiments, an antibody of the present disclosure is operatively linked to another antibody or antigen-binding fragment thereof to produce a bispecific antibody having a second binding specificity.

[0140] In an exemplary embodiment, the heteromeric antibody of the present disclosure is a bispecific antibody. A bispecific antibody may be a monoclonal antibody, such as a human or humanized antibody, that has binding specificities for at least two different antigens. In a specific embodiment, a bispecific antibody of the present disclosure comprises at least a first binding domain for the IL-18Rα subunit and at least a second binding domain for the IL-18Rβ subunit.

[0141] Methods for generating bispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies was based on the coexpression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537 (1983)). Because immunoglobulin heavy and light chains are randomly assorted, a hybridoma (quadroma) can produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually achieved by an affinity chromatography step. More modern techniques for generating bispecific antibodies use heterodimerization domains that favor the desired pairing of an antibody heavy chain having one specificity with an antibody heavy chain having a second specificity.

[0142] Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences. The fusion is typically with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. This may involve the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding being present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details regarding the generation of bispecific antibodies, see, e.g., Suresh et al., Meth. Enzymol. 121:210 (1986).

[0143] As used herein, the term "Fc" 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. The term "Fc polypeptide" includes an antibody heavy chain linked by a disulfide bond to an antibody light chain (e.g., forming a half antibody).

[0144] As used herein, the term "CH1 domain" refers to the first constant domain of an antibody heavy chain (e.g., amino acids 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 that contain one or more amino acid insertions, deletions, substitutions, or modifications compared to the naturally occurring CH1 domain).

[0145] As used herein, the term "CH2 domain" refers to the second constant domain of an antibody heavy chain (e.g., amino acids 231-340 of human IgG1 according to the EU index). The term includes naturally occurring CH2 domains and engineered variants of naturally occurring CH2 domains (e.g., CH2 domains that contain one or more amino acid insertions, deletions, substitutions, or modifications compared to the naturally occurring CH2 domain).

[0146] As used herein, the term "CH3 domain" refers to the third constant domain of an antibody heavy chain (e.g., amino acids 341-447 of human IgG1 according to the EU index). The term includes naturally occurring CH3 domains and engineered variants of naturally occurring CH3 domains (e.g., CH3 domains that contain one or more amino acid insertions, deletions, substitutions, or modifications compared to a naturally occurring CH3 domain).

[0147] As used herein, the term "hinge region" refers to the portion of an antibody heavy chain that contains the cysteine ​​residues that mediate disulfide bonds between the two heavy chains in an intact antibody (e.g., the cysteine ​​residues at amino acid positions 226 and 229 of human IgG1 according to the EU index). The term includes naturally occurring hinge regions and engineered variants of naturally occurring hinge regions (e.g., hinge regions that contain one or more amino acid insertions, deletions, substitutions, or modifications compared to a naturally occurring hinge region). An exemplary full-length IgG1 hinge region contains amino acids 216-230 of human IgG1 according to the EU index. A hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible bond between adjacent variable and / or constant domains in a single polypeptide molecule. In some embodiments, the immunoglobulin-like hinge region may be of or derived from any IgG1, IgG2, IgG3, or IgG4 subtype, or IgA, IgE, IgD, or IgM (including chimeric forms thereof).

[0148] In some embodiments, the hinge region may be of the 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 differ in their understanding of the exact amino acids that correspond to the various domains of an IgG molecule. Thus, the N- or C-termini 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.

[0149] As used herein, the term "upper hinge" typically refers to the last residue of the CH1 domain up to, but not including, the first inter-heavy chain cysteine. The upper hinge may be defined as the N-terminal sequence from positions 216 to 225 according to the Kabat EU numbering system for IgG1 antibodies (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). The term "middle hinge" refers to the region extending from the first inter-heavy chain cysteine ​​to the proline residue adjacent to the carboxyl terminus of the last middle hinge cysteine. The middle hinge may be the N-terminal sequence from positions 226 to 230 according to the Kabat EU numbering system. The term "lower hinge" refers to the highly conserved 7-8 amino acids. The lower hinge may be defined as the sequence from positions 231 to 238 according to the Kabat EU numbering system for IgG1 antibodies. In some embodiments, an antibody of the invention substantially comprises the upper, middle, and lower hinge.

[0150] As used herein, the term "modified hinge region" refers to a hinge region that has been altered in one or more of the hinge's characteristics, including, but not limited to, flexibility, length, conformation, charge, and hydrophobicity, compared to a wild-type hinge. The modified hinge regions disclosed herein can be generated by methods well known in the art, such as introducing modifications into a wild-type hinge. In some embodiments, the hinge region can be modified by one or more amino acids. Modifications that can be utilized to generate the modified hinge region include, but are not limited to, amino acid insertions, deletions, substitutions, and rearrangements. The above modifications of the disclosed hinges and modified hinge regions are collectively referred to herein as "hinge modifications of the invention," "modified hinge(s) of the invention," or simply "hinge modifications" or "modified hinge(s)." The modified hinge regions disclosed herein can be incorporated into molecules of choice, including, but not limited to, antibodies and fragments thereof. In some embodiments, the hinge region may be truncated and comprise only a portion of an intact hinge region. In some embodiments, the hinge region may be, and as shown herein, molecules comprising a modified hinge may exhibit altered (e.g., enhanced) agonist activity compared to a molecule having the same amino acid sequence except for the modified hinge, such as, for example, a molecule having the same amino acid sequence except for the wild-type hinge. In some embodiments, an antibody comprises a modified hinge region, and the upper hinge region is up to 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: 273). In some embodiments, the modified IgG1 hinge comprises the sequence PLAP (SEQ ID NO: 274). In some embodiments, the modified IgG1 hinge comprises the sequence DKTHT (SEQ ID NO: 275). In some embodiments, the modified hinge is a modified IgG4 hinge. In some embodiments, the modified IgG1 hinge comprises the sequence EKSYGPP (SEQ ID NO: 276).In some embodiments, the modified hinge is a Gly / Ser hinge. In some embodiments, the Gly / Ser hinge comprises the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 275).

[0151] As used herein, the term "EU index" refers to the EU numbering convention for antibody constant regions 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, USDept. Health and Human Services, 5th edition, 1991 (each of which is incorporated herein by reference in its entirety). All numbering of amino acid positions in an Fc polypeptide or fragment thereof used herein follows the EU index. As used herein, the term "linker" refers to 0 to 100 consecutive amino acid residues. Linkers may be present or absent, identical or different. Linkers contained in a protein or polypeptide may all have the same amino acid sequence or different amino acid sequences.

[0152] In some embodiments, the term "linker" refers to 1 to 100 consecutive amino acid residues. Typically, a linker provides flexibility and spatial separation between two amino acids or two polypeptide domains. Depending on the format of the molecule, a linker can be inserted between the VH, VL, CH, and / or CL domains to provide sufficient flexibility and flexibility to the light and heavy chain domains. Linkers are typically inserted at the amino acid sequence level at the transitions between variable domains, between variable and knockout domains, or between variable and constant domains, respectively. The approximate sizes of immunoglobulin domains are well understood, allowing the identification of transitions between domains. The precise locations of domain transitions can be identified by localizing peptide segments that do not form secondary structure elements, such as beta sheets or alpha helices, as demonstrated by experimental data or as determined by modeling or secondary structure prediction techniques.

[0153] As used herein, the terms "specifically bind," "specific binding," "binding specificity," or "specifically recognized" refer to an antigen-binding protein or antigen-binding fragment thereof that exhibits appreciable affinity for an antigen (e.g., an IL-18R antigen) and does not exhibit significant cross-reactivity to targets that are not 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 certain exemplary embodiments, affinity is measured by surface plasmon resonance (SPR), for example, on a Biacore instrument. As will be readily understood by one of skill in the art, the affinity of an antigen-binding protein may be reported as a dissociation constant (KD) in molar units (M). The antigen-binding proteins or antigen-binding fragments thereof of the present disclosure have a dissociation constant of about 10 -5 M ~ about 10 -12 M (i.e., low micromolar to picomolar range), approximately 10 -7 M~10 -11 M, about 10 -8 M ~ about 10 -10M, about 10 -9 In certain embodiments, the antigen binding protein or antigen binding fragment thereof has a KD value within the range 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 In certain embodiments, the antigen binding protein or antigen binding fragment thereof has a binding affinity of about 10 -7 M ~ about 10 -9 It has a binding affinity of M (nanomolar range).

[0154] Specific binding may be determined according to any art-recognized means for determining such binding. In some embodiments, specific binding is determined by competitive binding assays (e.g., ELISA) or Biacore assays. In certain embodiments, the assay is performed at about 20°C, 25°C, 30°C, or 37°C.

[0155] As used herein, "administering" or "administration" refers to the act of injecting or otherwise physically delivering an exogenous substance (e.g., an isolated binding polypeptide provided herein) to a patient, for example, but not limited to, pulmonary delivery (e.g., inhalation), mucosal (e.g., intranasal), intradermal, intravenous, intramuscular, and / or any other physical delivery method described herein or known in the art. When managing or treating a disease or its symptoms, administration of the substance typically occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, administration of the substance typically occurs before the onset of the disease or its symptoms and may be continued chronically to delay the onset of or reduce the severity of disease-related symptoms.

[0156] As used herein, the term "composition" is intended to include not only a product containing the specified components (e.g., the isolated binding polypeptides provided herein), optionally in the specified amounts, but also any product that results directly or indirectly from combining the specified components, optionally in the specified amounts.

[0157] By "effective amount" is meant an amount of an active pharmaceutical agent (e.g., an isolated binding polypeptide of the present disclosure) that is sufficient to bring about a desired physiological result in an individual in need of the agent. An effective amount may vary from individual to individual depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, an evaluation of the individual's medical condition, and other relevant factors.

[0158] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject can be a mammal, such as a non-primate animal (e.g., cows, pigs, horses, cats, dogs, rats, mice, etc.) or a primate (e.g., monkeys and humans). In certain embodiments, the term "subject" as used herein refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, feral animals, farm animals, sport animals, and pets.

[0159] As used herein, the term "therapy" refers to any protocol, method, and / or agent that can be used in the prevention, management, treatment, and / or amelioration of a disease or its associated symptoms. In some embodiments, the term "therapy" refers to any protocol, method, and / or agent that can be used in modulating an immune response to an infection or its associated symptoms in a subject. In some embodiments, the terms "therapy" and "singular" and "plural" refer to biological therapies, supportive therapies, and / or other therapies known to those of skill in the art, such as healthcare professionals, that are useful in the prevention, management, treatment, and / or amelioration of a disease or its associated symptoms. In other embodiments, the terms "therapy" and "plural" refer to biological therapies, supportive therapies, and / or other therapies known to those of skill in the art, such as healthcare professionals, that are useful in modulating an immune response to an infection or its associated symptoms in a subject.

[0160] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease or its associated 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 polypeptides provided herein). The term "treating," as used herein, can also refer to altering the disease course in the subject being treated. The therapeutic effect of treatment includes, but is not limited to, preventing the onset or recurrence of the disease, alleviating a symptom(s), reducing the direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or mitigating the disease state, and remission or improved prognosis.

[0161] The term "about" or "approximately" means within about 20%, eg, within about 10%, within about 5%, or within about 1% of a given value or range.

[0162] IL-18R signaling pathway IL-18 was initially discovered as a proinflammatory, IFN-γ-inducing cytokine that shares biological functions with IL-12 and acts synergistically with it. As a member of the IL-1 family of cytokines, IL-18 is thought to be involved in early inflammatory responses. It is synthesized constitutively by a wide range of hematopoietic and non-hematopoietic cells (e.g., macrophages, dendritic cells, Kupffer cells, keratinocytes, osteoblasts, astrocytes, adrenal cortical cells, intestinal epithelial cells, microglial cells, and synovial fibroblasts) and in response to other cytokines such as lipopolysaccharide and TNF-α. It is post-translationally cleaved by caspase-1 to reveal the functional activity of the mature 18-kDa species. Active IL-18 then targets cells expressing the IL-18 receptor, which is widely expressed in both hematopoietic and non-hematopoietic tissues.

[0163] The IL-18 receptor is a heterodimeric transmembrane protein composed of a ligand-binding IL-18R alpha (IL-18Rα) subunit and an unliganded IL-18R beta (IL-18Rβ) subunit, which is essential for functional signaling. Ligand activation of this receptor leads to the recruitment and activation of intracellular myeloid differentiation factor 88 (MyD88) and IL-1R-associated kinase (IRAK), which simultaneously initiates at least two distinct phosphorylation cascades that activate the PI3K and MAPK pathways, including activation of Akt, p38, and SAPK / JNK. Activation of these pathways leads to activation of NF-κB and the transcription of downstream genes, including IFN-γ, chemokines, transcription factors, G proteins, and cell surface receptors. IL-18 shares some common signaling pathways with IL-1, but also differs from them.

[0164] IL-18 stimulation can enhance the maturation, cytokine secretion, cytotoxicity, and adhesion of T cells and NK cells. IL-18-induced differentiation of naive T cells can induce either Th1 or Th2 lineages, independent of either 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), 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 the respiratory burst of neutrophils. Importantly, the IL-18 receptor itself can be upregulated by IL-12 on naive T cells, Th1 cells, and B cells, which may explain the synergistic effect between these two cytokines. IL-18 also acts synergistically with IL-2 to induce the expression of IL-13 (IFN-γ-dependent) and IL-10 (IFN-γ-independent). Together, these results clearly demonstrate a role for IL-18 in both innate and adaptive immune responses.

[0165] 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, and CCL20), and angiogenic mediators such as vascular endothelial growth factor (VEGF) and thrombospondin. Overall, the IL-18-induced effects of these effectors are to enhance leukocyte recruitment, cell adhesion, immune cell extravasation, and cell migration and promote neovascularization.

[0166] In the context of numerous inflammatory diseases, IL-18 has been shown to be upregulated, correlate with disease, or be 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 for developing either type 1 diabetes (T1D) or type 2 diabetes (T2D). High levels of IL-18 have also been observed in the serum, urine, and islet cells of young and adult T1D and T2D patients and correlate with disease severity and the development of sequelae such as diabetic nephropathy. Studies on Alzheimer's disease patients have revealed increased expression of IL-18 in the brain, where it is thought to contribute to immune and inflammatory processes that increase oxidative stress and alter the expression of proteins that contribute to amyloid beta (Aβ) formation.

[0167] Taken together, these studies suggest that inflammatory disorders may represent a group of pathologies in which blockade of IL-18-mediated signaling by anti-IL-18 antagonists may be effective, and there are ample opportunities for well-defined preclinical studies in this area. However, the role of agonist antibodies in upregulating the IL-18 signaling pathway has not yet been explored. IL-18 is a potent immunostimulatory cytokine that selectively activates tumor-infiltrating NK and antigen-experienced T cells, but also induces unique inhibitory IL-18-binding proteins that act as immune checkpoints (Dinarello CA et al., Front. Immunol. 2013; Zhou T. et al., Nature 2020). These biologically built-in constraints on proinflammatory signaling limit the utility of IL-18 as an oncological agent. Heteromeric antibodies that act as agonists of the IL-18 receptor can bypass this regulatory mechanism and promote persistent proinflammatory signaling in tumors that activate cytotoxic tumor-infiltrating lymphocytes. Importantly, such antibodies act through the NFkB signaling cascade, acting orthogonally and synergistically with the γc cytokine family (e.g., IL-2, IL-15) and IL-12, which act through the JAK / TYK / STAT signaling pathway.

[0168] Anti-IL18Rα binding domain One component of the multispecific binding proteins of the present disclosure is a binding moiety, binding domain, or binding specificity that binds to the IL-18Rα receptor subunit of an IL-18 receptor (e.g., human IL-18R). Any type of binding moiety that specifically binds to the IL-18Rα receptor subunit can be used in the multispecific binding proteins disclosed herein. In certain embodiments, the binding moiety comprises an antibody variable domain. Exemplary binding moieties that comprise an antibody variable domain include, but are not limited to, a VH, a VL, a VHH, a VH / VL pair, an scFv, a diabody, or a Fab. Other suitable binding moiety formats include lipocalins (see, e.g., Gebauer M. et al., 2012, Method Enzymol. 503:157-188, which is incorporated herein by reference in its entirety), adnectins (see, e.g., Lipovsek D., 2011, Protein Eng. Des. Sel. 24:3-9, which is incorporated herein by reference in its entirety), avimers (see, e.g., Silverman J, et al., 2005, Nat. Biotechnol. 23:1556-1561, which is incorporated herein by reference in its entirety), finomers (see, e.g., Schlatter D, et al., 2012, mAbs 4:497-508, which is incorporated herein by reference in its entirety), kunitz domains (see, e.g., Hosse R, et al., 2012, mAbs 4:497-508, which is incorporated herein by reference in its entirety), and adnectins. al., 2006, Protein Sci. 15:14-27, which is incorporated herein by reference in its entirety), knottins (see, e.g., Kintzing JR et al., 2016, Curr. Opin. Chem. Biol. 34:143-150, which is incorporated herein by reference in its entirety), affibodies (see, e.g., Feldwisch J. et al., 2010 J. Mol. Biol. 398:232-247).which is incorporated herein by reference in its entirety), and DARPins (see, e.g., Pluckthun A., 2015, Annu. Rev. Pharmacol. Toxicol. 55:489-511, which is incorporated herein by reference in its entirety).

[0169] In certain embodiments, the binding domain comprises the variable regions of the heavy and / or light chains of a conventional antibody or its antigen-binding fragment (e.g., Fab or scFv), and the term "conventional antibody" is used herein to refer to a heterotetrameric antibody containing immunoglobulin heavy and light chains arranged according to a "Y" configuration. Such conventional antibodies may be derived from any suitable species, including, but not limited to, antibodies of llama, alpaca, camel, mouse, rat, rabbit, goat, hamster, chicken, monkey, or human origin. In certain exemplary embodiments, a conventional antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), and the VH domain and / or VL domain, or one or more complementarity-determining regions (CDRs) thereof, are derived from the same antibody. In certain embodiments, the antigen-binding region of a conventional antibody may be referred to as "Fab" (Fragment antigen-binding). Fab comprises one constant domain and one variable domain from each of the heavy and light chains. The variable heavy and light chains contain the CDRs responsible for antigen binding.

[0170] In other embodiments, the IL-18Rα receptor subunit-binding subunit comprises at least the CDRs or VHH domains of a VHH antibody or nanobody. VHH antibodies, which are Camelidae-derived heavy-chain antibodies, are composed of two heavy chains and lack light chains (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 of the heavy chains of conventional antibodies, i.e., VH domains. Like conventional antibodies, the VHH domains of these molecules contain 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 the present disclosure.

[0171] Exemplary VHH CDRs or VHH domains with specificity for IL-18Rα and IL-18Rβ are shown in Tables 1 and 2 below, respectively. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0172] In certain embodiments, an IL-18Rα binding domain of the present disclosure 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 1 or Table 2.

[0173] In certain embodiments, an IL18Rβ binding domain of the present disclosure 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 1 or Table 2.

[0174] Anti-IL18Rα VHH binding domain epitope An epitope, also known as an antigenic determinant, is the specific portion of an antigen that is recognized and bound by an antibody. In some embodiments, the binding domain that binds to the IL-18Rα receptor subunit of the IL-18 receptor binds to a specific conformational epitope that includes amino acid residues listed in Table 3, according to the amino acid numbering scheme defined by Unitprot reference number Q13478.

[0175] Amino acid sequence of IL-18Rα: (SEQ ID NO: 287) [Uniprot Q13478 IL-18Ra] [Table 3]

[0176] Anti-IL18Rβ binding domain Another component of the multispecific binding proteins of the present disclosure is a binding domain or binding specificity that binds to the IL-18Rβ receptor subunit of the IL-18 receptor (eg, human IL-18R).

[0177] In certain embodiments, the binding domain comprises a heavy chain variable region and / or a light chain variable region of a conventional antibody or antigen-binding fragment thereof. In certain embodiments, the binding domain is a Fab or scFv. In certain embodiments, the IL-18Rβ binding domain is a Fab or scFv and is paired with an IL-18Rα binding domain that is a Fab or scFv. In certain embodiments, the IL-18Rβ binding domain is a Fab that shares a common light chain with the Fab of the IL-18Rα binding domain.

[0178] Exemplary binding moieties comprising antibody variable domains include, but are not limited to, a VH, a VL, a VHH, a VH / VL pair, an scFv, a diabody, or a Fab. Other suitable binding moiety formats include lipocalins (see, e.g., Gebauer M. et al., 2012, Method Enzymol. 503:157-188, which is incorporated herein by reference in its entirety), adnectins (see, e.g., Lipovsek D., 2011, Protein Eng. Des. Sel. 24:3-9, which is incorporated herein by reference in its entirety), avimers (see, e.g., Silverman J, et al., 2005, Nat. Biotechnol. 23:1556-1561, which is incorporated herein by reference in its entirety), finomers (see, e.g., Schlatter D, et al., 2012, mAbs 4:497-508, which is incorporated herein by reference in its entirety), kunitz domains (see, e.g., Hosse R, et al., 2012, mAbs 4:497-508, which is incorporated herein by reference in its entirety), and adnectins. al., 2006, Protein Sci. 15:14-27, which is incorporated herein by reference in its entirety), knottins (see, e.g., Kintzing JR et al., 2016, Curr. Opin. Chem. Biol. 34:143-150, which is incorporated herein by reference in its entirety), affibodies (see, e.g., Feldwisch J. et al., 2010 J. Mol. Biol. 398:232-247, which is incorporated herein by reference in its entirety), and DARPins (see, e.g., Pluckthun A., 2015, Annu. Rev. Pharmacol. Toxicol. 55:489-511, which is incorporated herein by reference in its entirety).

[0179] In other embodiments, the IL-18Rβ receptor subunit binding domain comprises at least the CDRs or VHH domain of a VHH antibody or nanobody. In certain embodiments, an IL-18Rβ VHH binding subunit is paired with a Fab or scFv IL-18Rα binding domain. In other embodiments, an IL-18Rβ VHH binding domain is paired with an IL-18Rα VHH binding domain.

[0180] Exemplary VHH CDRs or VHH domains with specificity for IL-18Rβ are shown in Tables 1 and 2.

[0181] Anti-IL18Rβ VHH binding domain epitope In some embodiments, the binding domain that binds to the IL-18Rβ receptor subunit of the IL-18 receptor binds to a specific conformational epitope that includes specific amino acid residues listed in Table 4 according to the amino acid numbering scheme defined by Unitprot reference number O95256-1. [Table 4]

[0182] Amino acid sequence of IL-18Rβ: MLCLGWIFLWLVAGERIKGFNISGCSTKKLLWTYSTRSEEEFVLFCDLPEPQKSHFCHRNRLSPKQVPEHLPFMGSNDLSDVQWYQQPSNGDPLEDIRKSYPHIIQDKCTLHFLTPGVNNSGSYICRPKMIKSPYDVACCVKMILEVKPQTNASCEYSASHKQDLLLGSTGSISCPSLSCQSDAQSPAVTWYKNGKLLSVERSNRIVVDEVYDYHQGTYVCDYTQSDTVSSWTVRAVVQVRTIVGDTKLKPDILDPVEDTLEVELGKPLTISCKARFGFERVFNPVIKWYIKDSDLEWEVSVPEAKSIKSTLKDEIIERNIILEKVTQRDLRRKFVCFVQNSIGNTTQSVQLKEKRGVVLLYILLGTIGTLVAVLAASALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEW (SEQ ID NO: 288) [Uniprot O95256-1 IL-18Rb]

[0183] Multi-specific IL-18R binding protein In certain embodiments, the IL-18Rα-binding domain and IL-18Rβ-binding domain disclosed herein can be paired or operably linked together to generate a multispecific binding protein capable of cross-linking the IL-18Rα and IL-18Rβ subunits of an IL-18 receptor (e.g., a human IL-18 receptor). In some embodiments, the IL-18Rα-binding domain (e.g., a VHH) is operably linked (directly or indirectly) to the N-terminus and / or C-terminus of a first Fc domain or polypeptide, and the IL-18Rβ-binding domain is operably linked to the N-terminus and / or C-terminus of a second Fc domain or polypeptide, such that the first and second Fc domains facilitate heterodimerization of the IL-18Rα-binding domain and the IL-18Rβ-binding domain.

[0184] In certain exemplary embodiments, the multispecific binding proteins of the present disclosure exhibit agonism toward the IL-18R signaling pathway, i.e., do not exhibit antagonism toward the IL-18R pathway. In some embodiments, agonism 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, thereby allowing the cells to respond specifically to IL-18.

[0185] HEK-Blue™ IL-18 cells also express the NF-κB / AP-1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene. Binding of the bispecific antibody to the heterodimeric IL-18 receptor on the surface of these cells initiates a signaling cascade that leads to the activation of NF-κB and the subsequent production of quantifiable SEAP.

[0186] The multispecific binding proteins of the present disclosure have agonist activity for IL-18R, stimulating expression of IFN-γ but barely inducing expression of IL-5 and IL-1-β. This is in contrast to IL-18, the natural ligand for IL-18R, which potently stimulates expression of IL-5 and IL-1-β. Thus, the multispecific binding proteins of the present disclosure provide a uniquely specific agonism of IL-18R that is not found in IL-18.

[0187] Accordingly, in one embodiment, the disclosure provides a multispecific binding protein comprising a first binding moiety that specifically binds human IL-18Rα and a second binding moiety that specifically binds human IL-18Rβ, wherein the multispecific binding protein stimulates anti-tumor cytokine production without substantially stimulating MCP-1 production, GM-CSF production, or the production of markers of acute inflammatory or Th2 responses compared to IL-18 stimulation of MCP-1 production, GM-CSF production, or the production of markers of acute inflammatory or Th2 responses.

[0188] In certain embodiments, the anti-tumor cytokine is selected from the group consisting of IFN-γ, IL-2, IL-12, IL-15, CD40L, and TNFα.

[0189] In certain embodiments, the marker of an acute inflammatory response or a Th2 response is selected from the group consisting of IL-6, IL-1β, IL-8, IL-4, IL-5, and IL-13.

[0190] In certain embodiments, anti-tumor cytokines and markers of acute inflammatory or Th2 responses are determined in peripheral blood mononuclear cell (PBMC) assays.

[0191] In certain embodiments, the PBMC assay comprises: 1) incubating a first PBMC population with the 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 anti-tumor cytokines and markers of an acute inflammatory or Th2 response from the first and second PBMC populations.

[0192] In certain embodiments, about 10,000 to about 1,000,000 PBMCs are used in the first and second PBMC populations, hi certain embodiments, about 250,000 PBMCs are used in the first and second PBMC populations.

[0193] In certain embodiments, the first PBMC population is incubated with IL-12 before or simultaneously with the multispecific binding protein. In certain embodiments, the second PBMC population is incubated with IL-12 before or simultaneously with IL-18. In certain 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 certain embodiments, the first and second PBMC populations are incubated with IL-12 at a concentration of about 0.5 ng / mL.

[0194] In certain embodiments, the first PBMC population is incubated with the multispecific binding protein at a concentration of about 1 nM to about 1,000 nM, hi certain embodiments, the first PBMC population is incubated with the multispecific binding protein at a concentration of about 300 nM.

[0195] In certain embodiments, the multispecific binding protein stimulates production of a marker of an acute inflammatory response or a Th2 response that is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than IL-18.

[0196] In specific embodiments, the multispecific binding protein stimulates production of a marker of an acute inflammatory response or a Th2 response that is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than IL-18 as measured in a PBMC assay.

[0197] In specific embodiments, the multispecific binding protein stimulates GM-CSF production that is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold lower than IFN-γ production.

[0198] In certain embodiments, the multispecific binding protein stimulates IL-5 production at least 5-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-5 production at least 10-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-5 production at least 50-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-5 production at least 100-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-5 production at least 500-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-5 production at least 1,000-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-5 production about 5-fold to about 1,000-fold less than IL-18.

[0199] In certain embodiments, the multispecific binding protein stimulates IL-1-β production at least 5-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-1-β production at least 10-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-1-β production at least 50-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-1-β production at least 100-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-1-β production at least 500-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-1-β production at least 1,000-fold less than IL-18. In certain embodiments, the multispecific binding protein stimulates IL-1-β production about 5-fold to about 1,000-fold less than IL-18.

[0200] In certain embodiments, the fold change in production of a marker of an acute inflammatory response or a Th2 response of the multispecific binding protein compared to IL-18 is as measured in a PBMC assay described herein. In some embodiments, the multispecific binding protein comprises at least one binding domain in each polypeptide, e.g., a VHH or Fab. In some embodiments, the multispecific binding protein comprises a tandem binding domain bispecific construct, e.g., at least two VHH domains in one polypeptide. In some embodiments, the VHH domains target two different targets, e.g., IL-18Rα and IL-18Rβ.

[0201] In some embodiments, the agonist activity of the multispecific binding protein meets or exceeds a certain threshold over background as measured in an agonist activity assay, e.g., a HEK-Blue assay. In some embodiments, the agonist activity of the multispecific binding protein is about 2-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 3-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 4-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 5-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 6-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 7-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 8-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 9-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 10-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 11-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 12-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 13-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 14-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 15-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 20-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 30-fold over background.In some embodiments, the agonist activity of the multispecific binding protein is about 40-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 50-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 60-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 70-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 80-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 90-fold over background. In some embodiments, the agonist activity of the multispecific binding protein is about 100-fold over background.

[0202] The Fc polypeptides used in the multispecific binding proteins of the present disclosure generally comprise a CH2 domain and a CH3 domain, with the C-terminus of the CH2 domain linked (directly or indirectly) to the N-terminus of the CH3 domain. Any naturally occurring or variant CH2 and / or CH3 domain may be used. For example, in certain embodiments, the CH2 and / or CH3 domain is a naturally occurring CH2 or CH3 domain derived from an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain, e.g., a human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain. The CH2 and CH3 domains may be derived from the same or different antibody heavy chains. In certain embodiments, the Fc polypeptide comprises a CH2- and CH3-domain-containing portion from a single antibody heavy chain. In certain embodiments, the CH2 and / or CH3 domain is a variant of a naturally occurring CH2 or CH3 domain, respectively. In certain embodiments, the CH2 and / or CH3 domains are variants comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to a naturally occurring CH2 or CH3 domain, respectively. In certain embodiments, the CH2 and / or CH3 domains are chimeras of one or more CH2 or CH3 domains, respectively. In certain embodiments, the CH2 domain comprises amino acids 231-340 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index. In certain embodiments, the CH3 domain comprises amino acids 341-447 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index.

[0203] In certain embodiments, the Fc polypeptide further comprises a hinge region, wherein the C-terminus of the hinge region is linked (directly or indirectly) to the N-terminus of the CH2 domain. For example, in certain embodiments, the hinge region is a naturally occurring hinge region derived from an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain, e.g., a human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain. The hinge region may be derived from the same or a different antibody heavy chain as the CH2 and / or CH3 domain. In certain embodiments, the hinge region is a variant comprising one or more amino acid insertions, deletions, substitutions, or modifications compared to a naturally occurring hinge region. In certain embodiments, the hinge region is a chimera of one or more hinge regions. In certain embodiments, the hinge region comprises amino acids 226 to 229 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index. In certain embodiments, the hinge region comprises amino acids 216-230 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index. In certain embodiments, the hinge region comprises amino acids 216-230 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index. In certain embodiments, the hinge region is a variant IgG4 hinge region comprising a serine (S) at amino acid position 228 according to the EU index.

[0204] In certain embodiments, the Fc polypeptide further comprises a CH1 domain, the C-terminus of which is linked (directly or indirectly) to the N-terminus of the hinge region. For example, in certain embodiments, the CH1 domain is a naturally occurring CH1 domain derived from an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain, e.g., a human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain. The CH1 domain may be derived from the same or a different antibody heavy chain as the hinge region, CH2 domain, and / or CH3 domain. In certain embodiments, the CH1 domain is a variant comprising one or more amino acid insertions, deletions, substitutions, or modifications compared to a naturally occurring CH1 domain. In certain embodiments, the CH1 domain is a chimera of one or more CH1 domains. In certain embodiments, the CH1 domain comprises amino acids 118 to 215 of a naturally occurring hinge region (e.g., human IgG1) according to the EU index.

[0205] In certain embodiments, the Fc polypeptide lacks a CH1 domain or contains a mutation in the CH1 domain or heavy chain variable domain that prevents association of the antibody light and heavy chains, hi certain embodiments, the antibody heavy chain lacks a portion of the hinge region.

[0206] Heterodimerization motif In certain exemplary embodiments, the first and second Fc domains are further engineered to enhance heterodimerization of the IL-18Rα-binding domain and the IL-18Rβ-binding domain and minimize the effects of incorrect chain pairing (i.e., pairing of IL-18Rα-binding domains or identical IL-18Rβ domains).

[0207] Any art-recognized approach to addressing the problem of incorrect chain pairing can be used to improve the production of desired multispecific antibodies. For example, US2010 / 0254989A1 describes the construction of a bispecific cMet-ErbB1 antibody in which the VH and VL of each antibody are genetically fused via a GlySer linker. In the case of bispecific antibodies containing an Fc domain, mutations can be introduced into the Fc to promote proper heterodimerization of the Fc portion. Some such approaches are reviewed in Klein et al. (mAbs (2012) 4:6, 1-11), the contents of which are incorporated herein by reference in their entirety.

[0208] In certain embodiments, the IL18Rα and IL18Rβ binding specificities of a multispecific antibody heterodimerize via knob-into-hole (KiH) pairing of the Fc domains. This dimerization technique utilizes engineered "protrusions" or "knobs" and "cavities" or "holes" at the interface of the CH3 domains. If a knob or hole with a suitable positioning and size is present at the interface of either the first or second CH3 domain, it is sufficient to simply engineer a corresponding hole or knob, respectively, at the adjacent interface, thereby promoting and strengthening Fc domain pairing at the CH3 / CH3 domain interface. The IgG Fc domain fused to the VHH comprises a knob, and the IgG Fc domain of a conventional antibody comprises a hole designed to accommodate the knob, or vice versa. The "knob" refers to at least one amino acid side chain, typically a large side chain, protruding from the interface of the CH3 portion of the first Fc domain. This protrusion is complementary to the "hole" in the CH3 portion of the second Fc domain, forming a "knob" that is received by the "hole." The "hole" is at least one amino acid side chain, typically a small side chain, that is recessed 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.

[0209] Exemplary amino acid residues that can function as the knob include arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). The amino acid residues present in the CH3 domain can be exchanged or substituted with the amino acid residues of the knob. Preferred amino acids for substitution can 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).

[0210] Exemplary amino acid residues that can function as holes include alanine (A), serine (S), threonine (T), or valine (V). Amino acid residues present in the CH3 domain can be exchanged or substituted with hole amino acid residues. Preferred amino acids for substitution can include any amino acid with a large side chain, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W).

[0211] The CH3 domain is preferably derived from a 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 the knob mutation of the first CH3 domain, and Y349C, T366S, L368A, Y407T, or Y407V for the hole mutation of the second CH3 domain.

[0212] In certain embodiments, the two Fc domains of the antigen-binding construct are heterodimerized by Fab arm exchange (FAE). A human IgG1 carrying a P228S hinge mutation may contain a F405L or K409R CH3 domain mutation. Mixing the two antibodies with a reducing agent results in FAE. This technique is described in U.S. Pat. No. 9,212,230 and Labrijn AFPNAS (2013) 110(13):5145-5150, which are incorporated herein by reference.

[0213] In another embodiment, the two Fc domains of the antigen-binding construct are heterodimerized by electrostatic steering. This dimerization technique utilizes electrostatic steering to promote and strengthen Fc domain pairing at the CH3 / CH3 domain interface. The charge complementarity between the two CH3 domains is altered to favor heterodimerization (pairing of opposite charges) over homodimerization (pairing of like charges). In this method, electrostatic repulsion prevents homodimerization. Specific exemplary amino acid residue substitutions that result in electrostatic steering 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 US Patent Publication No. 2014 / 0154254 A1 and Gunasekaran K. JBC (2010) 285(25):19637-19646, which are incorporated herein by reference.

[0214] In other embodiments, the charge complementarity is formed by a first Fc domain comprising an N297K and / or T299K mutation and a second Fc domain comprising an N297D and / or T299D mutation.

[0215] In one embodiment of the present invention, the two Fc domains of the antigen-binding construct are heterodimerized by hydrophobic interaction. This dimerization technique utilizes hydrophobic interactions instead of electrostatic interactions to promote and strengthen the pairing of the Fc domains 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 pairs of amino acid residue substitutions between the first and second CH3 domains include K409W:D399V, K409W:F405T, K360E:Q347R, Y349S:E357W, and S354C:Y349C. This technique is described in US Patent Publication No. 2015 / 0307628 A1.

[0216] In one embodiment of the present invention, heterodimerization can be mediated by the use of leucine zipper fusion. Heterodimerization is forced by leucine zipper domains fused to the C-terminus of each CH3 domain of the antibody chain. This technique is described in Wranik B. JBC (2012) 287(52):43331-43339.

[0217] In one embodiment of the present invention, heterodimerization can be mediated by the use of Strand Exchange Engineered Domain (SEED) bodies. CH3 domains from IgG and IgA formats force heterodimerization. This technique is described in Muda M. PEDS (2011) 24(5):447-454.

[0218] In other embodiments, the heterodimerization motif may comprise a non-native disulfide bond formed by engineered cysteine ​​residues. In certain embodiments, the first set of disulfides may comprise a Y349C mutation in the first Fc domain and a S354C mutation in the second Fc domain. In other embodiments, the engineered disulfide bond may be introduced by fusing a C-terminal extension peptide containing an engineered cysteine ​​residue to the C-terminus of each of the two Fc domains. In certain embodiments, the first Fc domain may comprise a substitution of the carboxyl-terminal "GEC" as "PGK" and the second Fc domain may comprise a substitution of the carboxyl-terminal amino acid "PGK" with "KSCDKT."

[0219] In yet another approach, multispecific antibodies can utilize the CrossMab principle (reviewed in Klein et al.), which involves domain swapping between heavy and light chains to promote proper pairing. Yet another approach involves engineering the interface between heavy and light chain VH-VL domain pairs or CH1-CL domain pairs to increase the affinity between a heavy chain and its cognate light chain (Lewis et al. Nature Biotechnology (2014) 32:191-198).

[0220] An alternative approach to producing multispecific antibody preparations with the correct antigen specificity is to develop methods to enrich for antibodies with the correct heavy-light chain pairing. For example, Spiess et al. (Nature Biotechnology (2013) 31:753-758) reported a method for producing MET-EGFR bispecific antibodies from the co-culture of bacteria expressing two different half-antibodies.

[0221] A method has also been reported in which the constant region of at least one of the heavy chains of a bispecific antibody is mutated to alter its binding affinity to an affinity factor, such as protein A. This allows for the isolation of properly paired heavy chain heterodimers based on purification techniques that exploit the difference in binding of the two heavy chains to the affinity factor (see US2010 / 0331527, WO2013 / 136186).

[0222] International Patent Application No. PCT / EP2012 / 071866 (WO2013 / 064701) addresses the problem of incorrect chain pairing using a method for isolating multispecific antibodies based on the use of anti-idiotypic binders, particularly anti-idiotypic antibodies. The anti-idiotypic binders are used in a two-step selection method in which a first agent is used to capture antibodies with a VH-VL domain pairing specific for a first antigen, followed by a second agent to capture antibodies with a second VH-VL domain pairing specific for a second antigen.

[0223] In yet another embodiment, the multispecific antibody uses a first binding specificity with a conventional Fab binding region and a second binding specificity comprising a single-domain antibody (VHH) binding region. The heterodimerization method used forces the heavy chain region of the Fab to associate only with the complete heavy chain of the VHH. Because the VHH chain does not associate with a light chain, the light chain region of the Fab portion associates only with its corresponding heavy chain.

[0224] In certain other embodiments, the multispecific binding proteins described herein further comprise a common light chain. As used herein, the term "common light chain" refers to a light chain that can pair with a first light chain of an antibody that binds to a first antigen to form a binding site that specifically binds to the first antigen, and can also pair with a second heavy chain of an antibody that binds to a second antigen to form a binding site that specifically binds to the second antigen. A common light chain is a polypeptide comprising, from N- to C-terminal, an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), and is also abbreviated herein as "VL-CL." Multispecific binding proteins with a common light chain require heterodimerization of different heavy chains. In certain embodiments, the heterodimerization methods described above can be used with a common light chain. In certain exemplary embodiments, the heterodimerization motif can include a non-native disulfide bond formed by engineered cysteine ​​residues. Adding disulfide bonds between both the heavy and light chains of an antibody has been shown to improve stability. Furthermore, disulfide bonds have also been used as a solution to improve light chain pairing in bispecific antibodies (Geddie MLet et al, mABs (2022) 14 (1)).

[0225] Unless otherwise stated, all numbering of antibody constant regions used herein corresponds to the EU numbering scheme described in Edelman et al. (Proc. Natl. Acad. Sci. 63(1):78-85. 1969).

[0226] Additional methods for heterodimerization of heavy and / or light chains and for producing and purifying asymmetric antibodies are known in the art. See, e.g., Klein C. mAbs (2012) 4(6):653-663 and U.S. Patent No. 9,499,634, each of which is incorporated herein by reference.

[0227] Effector function mutations As noted above, the multispecific binding proteins of the present disclosure can be prepared with various isotypes and with different constant regions. The Fc region of a multispecific binding protein primarily determines its effector function with respect to Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cellular phagocytosis (ADCP) activity. These "cellular effector functions," distinct from effector T-cell functions, involve recruiting Fc receptor-bearing cells to the site of target cells, resulting in killing of the antibody-bound cells.

[0228] Antibodies of the present invention may exhibit reduced effector function. In certain embodiments, one or more mutations reduce one or more of antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC). In certain embodiments, antibodies of the present invention may lack ADCC, ADCP, and / or CDC activity. In either case, antibodies of the present invention may comprise, or optionally lack, an Fc region that binds to one or more types of Fc receptor. The use of different antibody formats, and the presence or absence of FcR binding and cellular effector function, allows for the tailoring of antibodies for use for specific therapeutic purposes, as described elsewhere herein.

[0229] In certain embodiments, the first and second Fc domains contain one or more mutations that reduce Fc effector function. In certain embodiments, the first and second Fc domains each contain the L234A and L235A mutations. These IgG1 mutations are also known as "LALA" mutations and are described in further detail in Xu et al. (Cell Immunol. 2000;200:16-26). In certain embodiments, the first and second Fc domains each contain the L234A, L235A, G237A, and / or P329G mutations. The amino acid positions of the Fc domains referred to herein are based on EU antibody numbering. Alternatively, the antibody may have an effector-null constant region. The antibody may have a heavy chain constant region that does not bind to Fcγ receptors; for example, the constant region may contain the L235E mutation. Another optional mutation in the heavy chain constant region is S228P, which increases stability. The heavy chain constant region may be an IgG4 containing both the L235E and S228P mutations. This "IgG4-PE" heavy chain constant region is effector-null. A disabled IgG1 heavy chain constant region is also effector-null. The disabled IgG1 heavy chain constant region may contain alanine at positions 234, 235, and / or 237 (EU index numbering), for example, an IgG1 sequence containing the L234A, L235A, and / or G237A mutations ("LALAGA").

[0230] Human IgG1 constant regions containing specific mutations or altered glycosylation at residue Asn297 (e.g., N297Q, N297D, and N297K, EU index numbering) have been shown to reduce binding to Fc receptors.

[0231] In other embodiments, it may be desirable to enhance binding of the Fc region of a multispecific antibody to human Fc gamma receptor IIIA (FcgRIIIA) compared to that of the corresponding naturally occurring antibody Fc region. In certain 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 by a variety of established techniques. Such methods increase affinity for specific Fc receptors, thus creating a potentially diverse profile of enhanced activation. This can be achieved by modifying one or more amino acid residues. Exemplary mutations are one or more of residues selected from 239, 332, and 330 for the human IgG1 constant region (or equivalent positions in other IgG isotypes). Thus, an antibody can comprise a human IgG1 constant region with one or more mutations independently selected from S239D, I332E, and A330L (EU index numbering).

[0232] Increased affinity for Fc receptors can also be achieved by altering the native glycosylation profile of the Fc domain, for example, by generating hypofucosylated or defucosylated variants. Nonfucosylated antibodies contain the tri-mannosyl core structure of the complex N-glycan of Fc, which lacks fucose residues. These glycoengineered antibodies, lacking the core fucose residue from the Fc N-glycan, may exhibit stronger ADCC than their fucosylated counterparts due to enhanced FcγRIIIA binding ability. For example, to increase ADCC, residues in the hinge region can be altered to increase binding to FcγRIIIA. Thus, an antibody can comprise a human IgG heavy chain constant region that is a variant of the wild-type human IgG heavy chain constant region. In a specific embodiment, the variant human IgG heavy chain constant region binds to a human Fcγ receptor selected from the group consisting of FcγRIIB and FcγRIIA with higher affinity than the wild-type human IgG heavy chain constant region binds to human FcγRIIIA. The antibody may comprise a human IgG heavy chain constant region that is a variant of a wild-type human IgG heavy chain constant region, and that binds to human FcγRIIB with higher affinity than the wild-type human IgG heavy chain constant region binds to human FcγRIIB. The variant human IgG heavy chain constant region may be the heavy chain constant region of a variant human IgG1, a variant human IgG2, or a variant human IgG4. 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 variant human IgG heavy chain constant region comprises a set 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).

[0233] Enhanced CDC can be achieved by 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 made from a human IgG1 segment and a human IgG3 segment that takes advantage of the higher affinity of IgG3 for C1q. The antibodies of the invention can contain amino acid mutations at residues 329, 331, and / or 322 to alter C1q binding and / or reduced or eliminated CDC activity. In another embodiment, the antibodies or antibody fragments disclosed herein can contain an Fc region with modifications at residues 231 and 239 that replace 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 a double mutation comprising R344D and D356R (EU index numbering system).

[0234] The functional properties of multispecific binding proteins can be further adjusted by combining amino acid substitutions that alter Fc binding affinity with amino acid substitutions 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 certain circumstances, extend the in vivo serum half-life compared to the unmodified binding protein. Of course, any combination of Fc variants and FcRn variants can be used to adjust 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.

[0235] Expression of antigen-binding proteins In one aspect, polynucleotides encoding the binding proteins (e.g., antigen-binding proteins and antigen-binding fragments thereof) disclosed herein are provided. Methods of making the binding proteins, including expressing these polynucleotides, are also provided.

[0236] Polynucleotides encoding the binding proteins disclosed herein are typically inserted into expression vectors for introduction into host cells that can be used to produce desired quantities of the binding proteins. Thus, in certain aspects, the present disclosure provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0237] The term "vector" or "expression vector" is used herein to mean a vector used in accordance with the present disclosure as a vehicle for introducing and expressing a desired gene in a cell. As known to those skilled in the art, such vectors may be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors compatible with the present disclosure will contain a selectable marker, appropriate restriction sites to facilitate cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0238] For the purposes of this disclosure, numerous expression vector systems may be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV, or MOMLV), or SV40 virus. Others involve the use of polycistronic systems containing internal ribosome binding sites. Furthermore, cells into which the DNA has integrated into the chromosome can be selected by introducing one or more markers that allow for selection of transfected host cells. Markers may confer prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by cotransformation. Additional elements may be required for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In some embodiments, the cloned variable region genes are inserted into an expression vector along with heavy and light chain constant region genes (eg, human constant region genes) synthesized as described above.

[0239] In other embodiments, binding proteins may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as antibody heavy and light chains, may be produced from a single polycistronic construct. Such systems advantageously use internal ribosome entry sites (IRES) to provide relatively high levels of polypeptides in eukaryotic host cells. Suitable IRES sequences are disclosed in U.S. Pat. No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those skilled in the art will appreciate that such expression systems can be used to effectively produce any of the polypeptides disclosed in the present application.

[0240] More generally, once a vector or DNA sequence encoding a binding protein, such as 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. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those skilled in the art. These 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 intact virus. See Ridgway, AAG "Mammalian Expression Vectors," Chapter 24.2, pp. 470-472, Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Introduction of the plasmid into the host can be by electroporation. Transformed cells are grown under conditions appropriate for the production of light and heavy chains and assayed for the synthesis of heavy and / or light chain proteins. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and the like.

[0241] As used herein, the term "transformation" is intended to be used broadly to refer to the introduction of DNA into a recipient host cell, resulting in a change in the genotype.

[0242] In a similar vein, a "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. In describing processes for isolating a polypeptide from a recombinant host, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the antibody, unless otherwise specified. In other words, recovery of polypeptide from "cells" can mean recovery from spun-down whole cells, recovery from the supernatant of a lysed cell culture, or recovery from a cell culture containing both medium and suspension cells.

[0243] In one embodiment, the host cell line used for antibody expression is of mammalian origin. One skilled in the art can determine the particular host cell line most suitable for the desired gene product to be expressed. Exemplary host cell lines include, but are not limited to, GS-CHO and CHO-K1 (Chinese hamster ovary lines), DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA (human cervical carcinoma), CV-1 (monkey kidney line), COS (a derivative of CV-1 containing the SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HEK (human kidney 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, e.g., defucosylation, of antibodies expressed from the cell line (e.g., PER.C6® (Crucell) or a FUT8 knockout CHO cell line (POTELLIGENT® cells) (Biowa, Princeton, NJ)). In one embodiment, NSO cells may be used. CHO cells are particularly useful. Host cell lines are typically available from commercial services, e.g., the American Tissue Culture Collection, or from authors in published literature.

[0244] In vitro production allows for scale-up to obtain large amounts of the desired polypeptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art and include, for example, homogeneous suspension culture in airlift reactors or continuous stirred reactors, or immobilized or entrapped cell culture in, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE cellulose, and / or (immuno)affinity chromatography.

[0245] Genes encoding the binding proteins featured in this disclosure can also be expressed in non-mammalian cells, such as bacteria or yeast, or plant cells. In this regard, it will be understood that various unicellular non-mammalian microorganisms, such as bacteria, i.e., microorganisms capable of growth in culture or under fermentation, can also be transformed. Bacteria susceptible to transformation include members of the Enterobacteriaceae family, e.g., strains of Escherichia coli or Salmonella; Bacillaceae, e.g., Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. It will further be understood that, when expressed in bacteria, the binding proteins can become part of inclusion bodies. In some embodiments, the binding proteins are then isolated, purified, and assembled into functional molecules. In some embodiments, the binding proteins of the present disclosure are expressed in bacterial host cells. In some embodiments, the bacterial host cells are transformed with an expression vector comprising a nucleic acid molecule encoding the binding proteins of the present disclosure.

[0246] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, although several other strains are commonly available. For expression in Saccharomyces, for example, the plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used. 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 No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). Therefore, the presence of the trpl lesion, characteristic of the yeast host cell genome, provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0247] Formulations / pharmaceutical compositions In certain embodiments, pharmaceutical compositions are provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antigen binding protein described herein. Some embodiments include pharmaceutical compositions comprising a therapeutically effective amount of any one of the binding proteins described herein, or a binding protein-drug conjugate, admixed with a pharmaceutically or physiologically acceptable formulating agent selected for suitability with the mode of administration.

[0248] Acceptable formulation materials are typically nontoxic to recipients at the dosages and concentrations employed.

[0249] In some embodiments, pharmaceutical compositions may contain formulation materials to modify, maintain, or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or penetration of the composition.Suitable formulation materials include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (e.g., boric acid, bicarbonate, Tris-HCl, citric acid, phosphoric acid, or other organic acids), bulking agents (e.g., mannitol or glycine), chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), bulking agents, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin), proteins (e.g., serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), preservatives (e.g., benzalkonium chloride), and the like. benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvent (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohol (e.g., mannitol or sorbitol), suspending agent, surfactant or wetting agent (e.g., Pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancer (e.g., sucrose or sorbitol), tonicity enhancer (e.g., alkali metal halides such as sodium chloride or potassium chloride, or mannitol sorbitol), delivery vehicle, diluent, excipient, and / or pharmaceutical adjuvant (e.g., REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., AR Gennaro, ed., Mack Publishing Company 1990) and subsequent editions thereof).

[0250] In some embodiments, the optimal pharmaceutical composition will be determined by one of skill in the art depending, for example, on the intended route of administration, delivery format, and desired dosage. Such composition can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the binding protein.

[0251] In some embodiments, the primary vehicle or carrier in a pharmaceutical composition can be either aqueous or non-aqueous in nature. For example, a vehicle or carrier suitable for injection can be water, saline solution, or artificial cerebrospinal fluid, optionally supplemented with other ingredients common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Other exemplary pharmaceutical compositions include Tris buffer at a pH of about 7.0-8.5 or acetate buffer at a pH of about 4.0-5.5, and may further include sorbitol or a suitable substitute. In one embodiment of the present disclosure, a binding protein composition can be prepared for storage by combining a selected composition having the desired purity with optional formulating agents in the form of a lyophilized cake or aqueous solution. Additionally, the binding protein can be formulated as a lyophilizate using appropriate excipients, such as sucrose.

[0252] In some embodiments, the pharmaceutical compositions of the present disclosure can be selected for parenteral or subcutaneous delivery. Alternatively, the compositions can be selected for inhalation or delivery via the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of those skilled in the art.

[0253] In some embodiments, the components of the formulation are present in concentrations that are acceptable to the site of administration. For example, a buffer is used to maintain the composition at physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0254] When parenteral administration is intended, the therapeutic composition for use can be in the form of a pyrogen-free, parenterally 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 as a sterile, isotonic solution and properly preserved.Another preparation can include formulating the desired molecule with agents such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, which provide controlled or sustained release of the product, and then delivering it by depot injection.Hyaluronic acid can also be used, which can have the effect of promoting the duration of the product in the circulatory system.Other suitable means for introducing the desired molecule include implantable drug delivery devices.

[0255] In one embodiment, the pharmaceutical composition can be formulated for inhalation. For example, the binding protein can be formulated as a dry powder for inhalation. The binding protein can also be formulated as an inhalable solution using a propellant for aerosol delivery. In yet another embodiment, the solution can be nebulized.

[0256] It is also contemplated that certain formulations may be administered orally. In one embodiment of the present disclosure, multispecific binding proteins administered in this manner may be formulated with or without carriers traditionally used in the preparation of solid dosage forms such as tablets and capsules. For example, capsules can be designed to release the active portion of the formulation at a time when bioavailability is maximized in the gastrointestinal tract and pre-systemic degradation is minimized. Additional agents can be included to facilitate absorption of the binding protein. Diluents, flavorings, low-melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders can also be used.

[0257] Another pharmaceutical composition may contain an effective amount of the multispecific binding protein in a mixture with non-toxic excipients suitable for the manufacture of tablets. By dissolving the tablets in sterile water or another appropriate vehicle, a solution can be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate, or binding agents such as starch, gelatin, or acacia, or lubricating agents such as magnesium stearate, stearic acid, or talc.

[0258] Further pharmaceutical compositions of the present disclosure will be apparent to those skilled in the art, including formulations comprising the binding protein in sustained- or controlled-delivery formulations. Techniques for formulating various other sustained- or controlled-delivery means, such as liposome carriers, bioerodible microparticles or porous beads, and depot injections, are also known to those skilled in the art. Further examples of sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, such as films or microcapsules. Controlled-release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamic acid, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions can also include liposomes, which can be prepared by any of several methods known in the art.

[0259] In some embodiments, pharmaceutical compositions used for in vivo administration must typically be sterile. This can be achieved by filtration through a sterile filtration membrane. If the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. In addition, parenteral compositions are generally placed into a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0260] Once formulated, the pharmaceutical compositions may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or dehydrated or lyophilized powder. Such formulations may be stored either in a ready-to-use form or in a form (e.g., lyophilized) requiring reconstitution prior to administration.

[0261] The present disclosure also encompasses kits for producing single-dose administration units. Each kit may contain both a first container containing a dried multispecific binding protein and a second container containing an aqueous formulation. Kits containing single-chamber and multi-chamber pre-filled syringes (e.g., liquid syringes and lyosyringes) are also within the scope of the present disclosure.

[0262] The effective amount of a binding protein pharmaceutical composition used therapeutically will depend, for example, on the context and purpose of the treatment. Therefore, those skilled in the art will understand that appropriate dosage levels for treatment will depend, in part, on the molecule being delivered, the indication for which the binding protein is being used, the route of administration, and the size (weight, body surface, or organ size) and condition (age and general health) of the patient. Accordingly, clinicians can titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[0263] The dosing frequency is determined by the pharmacokinetic parameters of the binding protein in the formulation used. Typically, the clinician will administer the composition until a dosage that achieves the desired effect is reached. Thus, 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 continuous infusion via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of the tasks routinely performed by those skilled in the art. The appropriate dosage can be confirmed using appropriate dose-response data.

[0264] The route of administration of the pharmaceutical composition is in accordance with known methods, for example, orally, or via injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes, by sustained release system, or by implanted device. If desired, the composition can be administered by bolus injection or continuously by infusion, or by implanted device.

[0265] In some embodiments, the compositions may also be administered locally via implantation of a membrane, sponge, or other suitable material that has absorbed or encapsulated the desired molecule. When an implanted device is used, the device can be implanted in any suitable tissue or organ, and delivery of the desired molecule can be by diffusion, slow release bolus, or continuous administration.

[0266] The multispecific binding proteins disclosed herein can be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for the delivery of steroids useful in the treatment of inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entireties). These formulations for administration to the respiratory tract may be in the form of an aerosol or solution for nebulization, or an ultrafine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulation will have a diameter of less than 50 microns in one embodiment, and less than 10 microns in one embodiment.

[0267] The multispecific binding proteins disclosed herein can be formulated for topical or local application, e.g., topical application to the skin and mucous membranes, e.g., the eyes, in the form of gels, creams, and lotions, and for ocular application, or for intracapsular or intrathecal application. Topical administration is contemplated for transdermal delivery, and for administration to the eye 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.

[0268] Transdermal patches, including iontophoretic and electrophoretic devices, are well known to those skilled in the art and can be used to administer heterodimeric proteins. For example, such patches are disclosed in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference.

[0269] In certain embodiments, pharmaceutical compositions comprising the multispecific binding proteins described herein are lyophilized powders that can be reconstituted for administration as solutions, emulsions, and other mixtures. They can also be reconstituted and formulated as solids or gels. The lyophilized powder is prepared by dissolving the heterodimeric protein or a pharmaceutically acceptable derivative thereof described herein in a suitable solvent. In certain embodiments, the lyophilized powder is sterile. The solvent may contain excipients to improve stability or other pharmacological components of the powder or a reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer such as citric acid, sodium phosphate, or potassium phosphate, or other such buffers known to those of skill in the art, in one embodiment at about neutral pH. Sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial contains a single or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for parenteral administration. When reconstituted, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. Such 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. All such targeting methods are contemplated herein for use in the present compositions.For non-limiting examples of targeting methods, see, e.g., 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, 6,0 See, for example, Nos. 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 entireties. In certain embodiments, the heterodimeric proteins described herein target tumors.

[0270] Method of Treatment / Use Another aspect of the present disclosure is a multispecific antibody and / or antigen-binding protein described herein for use as a medicament.

[0271] In certain embodiments, a method of treating a disorder due to activation of IL-18R is provided, the method comprising administering to a subject in need thereof an effective amount of an antigen binding protein described herein.

[0272] The binding proteins can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays, for the detection and quantification of one or more target antigens. The binding proteins bind to one or more target antigens with an affinity appropriate for the assay method being used.

[0273] For diagnostic applications, in some embodiments, the binding protein can be labeled with a detectable moiety. The detectable moiety can be any moiety capable of producing a detectable signal, whether directly or indirectly. For example, the detectable moiety can be: 3 H, 14 C. 32 P, 35 S, 125 I, 99Tc, 111 In, or 67 It can be a radioisotope such as Ga; a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0274] Binding proteins are also useful for in vivo imaging. Binding proteins labeled with a detectable moiety can be administered to an animal (e.g., into the bloodstream) to assay for the presence and location of the labeled antibody in the host. Binding proteins can be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

[0275] The present disclosure also relates to kits comprising the binding protein and other reagents useful for detecting levels of a target antigen in a biological sample. Such reagents can include a detectable label, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any of the binding proteins, polynucleotides, vectors, vector systems, and / or host cells described herein. In some embodiments, the kit comprises a container and a label or package insert associated with or attached to the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds the composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing a condition and may have a sterile access port (e.g., the container can be an IV solution bag or vial with a stopper pierceable by a hypodermic needle). In some embodiments, the label or package insert indicates that the composition is used for preventing, diagnosing, and / or treating a selected condition. Alternatively, or additionally, the article of manufacture or kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0276] In some embodiments, the present disclosure relates to methods for preventing and / or treating a disease or disorder (e.g., cancer). In some embodiments, the methods comprise administering to a patient a therapeutically effective amount of at least one of the binding proteins described herein, or a pharmaceutical composition related thereto. In some embodiments, the patient is human.

[0277] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein are incorporated by reference in their entirety to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference. Applicant reserves the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

[0278] While the present disclosure has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure. Those skilled in the art will readily recognize that other suitable modifications and adaptations of the methods described herein, using suitable equivalents, may be made without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition, process, treatment step(s) to the objective, spirit, and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto. While certain embodiments have been described in detail above, the embodiments will be more clearly understood by reference to the following examples. The examples are included for illustrative purposes only and are not intended to be limiting. [Example]

[0279] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions featured in this invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.

[0280] Example 1. Computational prioritization of agonist molecules Recapitulating the desired pharmacological signal of a natural ligand using an exogenous protein scaffold is a challenging task. Receptor-ligand assemblies often coevolve over millions of years to possess complementary interfaces with elements of induced fit, positioning the intracellular domains in an active juxtaposition that promotes downstream signaling. In the case of the IL-18 receptor, the IL-18 ligand first binds to the IL-18Rα subunit to form a binary complex, which subsequently recruits the IL-18Rβ subunit to form the IL-18 receptor signaling complex (Tsutsumi et al. The structural basis for receptor recognition of human interleukin-18. Nat Commun. 2014, 5, 5340).

[0281] Recapitulation of IL-18 by cross-linking agonist antibodies requires that the positioning of IL-18Rα / IL-18Rβ, at least in their membrane-proximal portions, be precisely maintained in the absence of IL-18. This requirement reduces the epitope space available to antibody modules and imposes strict paratope composition requirements. 6 Considering that this would result in more than 10 antibody binding sites, exhaustive screening methods could be used to screen 10 of the heteromeric molecules. 12 Finding a productive agonist configuration from the many possibilities is unrealistic.

[0282] To address this challenge, we used a five-step approach to design approximately 100 agonistic bispecific molecules starting from approximately 500,000 unique antibody sequences obtained from a next-generation sequencing (NGS) antibody discovery campaign against each IL-18 receptor.

[0283] First, we used in silico tools to predict three-dimensional structures from NGS sequences. For VHH binders, we used a convolutional neural network (CNN) encompassing two blocks of a 1D residual neural network (ResNet) trained on thousands of antibody structures from the Protein Data Bank to predict the overall 3D structure of each nanobody. For Fab or scFv binders, we predicted the CDR structure using an equivariant graph neural network trained to reconstruct antibody loops. These loops were then grafted onto a typical Fab or scFv framework.

[0284] In the second step, we used a deep graph neural network (GNN) combined with a bidirectional long short-term memory (BiLSTM) network to extract low-level features for each paratope, allowing the construction of a pairwise distance matrix for all sequences. The metric used to compare each paratope reflected a sampling of the paratope surface space. A reduction analysis of this matrix using hierarchical clustering (Figure 3) revealed a prioritized set of 10,000 representative sequences. Each representative cluster is the structure with the smallest distance to other cluster members according to the surface metric.

[0285] In a third step, these structures were aligned to the target receptor subunits IL-18Rα and IL-18Rβ (respectively antigen-derived) using a GPU-accelerated version of a proprietary fast Fourier transform docking program augmented by an antibody-parameterized AlphaFold2-based machine learning refinement step.

[0286] The fourth step consisted of identifying epitopes of interest on the surface of each receptor as regions to which multiple paratopes dock in a similar manner. Hundreds of candidates were selected for each receptor based on the best poses for each epitope, as assessed by the docking energy function score.

[0287] The final step entailed assembling the best combination of binders by combining two modules for IL-18R alpha and beta (respectively) that aligned with the geometric constraints of the bispecific framework based on the knob-and-hole Fc construct. A deep learning algorithm based on the encoder-decoder architecture of a recurrent neural network complemented by reinforcement learning was used to design the linkers for each arm of the bispecific to the core Fc.

[0288] Example 2. Identification and generation of heteromeric antibodies To establish a screening triage, a set of prior art VHHs (WO2010040736A2) or Fabs (US20210130478A1) were selected for assembly into heteromeric molecules. The binding moieties were fused to Fc containing mutations that promote heterodimerization (Y349C, S354C, T366S, L368A, Y470V, T366W) and mutations that reduce effector function (L234A and L235A). This assembly was performed combinatorially without using the computational methods described in Example 1. Forty-four heteromeric molecules were assembled. Forty-one of the 44 showed no activity. Three Fab-VHH heteromeric antibodies with activity greater than two-fold above background are listed in Table 5. This activity was essential for establishing a screening cascade for the newly discovered heteromeric molecules.

[0289] Antibodies were transiently transfected into HEK293 cells using rPEx® technology. Cells were harvested 6 days post-transfection and purified using a HiTrap Fibro PrismA column. Protein-containing pools were concentrated using a 30 kDa spin filter (Amicon) and further purified using a Superdex 200 column. Protein-containing fractions were analyzed using LabChip® capillary electrophoresis (Perkin Elmer). The purity of the final product was assessed using analytical gel filtration using LabChip® and a Superdex™ 200 10 / 30 column. [Table 5] [Table 6-1] [Table 6-2]

[0290] Example 3. Screening for agonist activity HEK-Blue™ IL-18 cells were purchased from Invivogen (hbk-hmil18). These cell lines overexpress IL-18Rα and IL-18Rβ and block responses to TNFα and IL-1β. Reporter cells were revived and cultured according to the supplier's recommendations. Cells were rinsed with PBS and plated into 96-well plates at a density of approximately 50,000 cells / well. 20 μl of either control or heteromeric antibody was added to each well to achieve the final agonist concentrations listed in Table 5. Plates were incubated at 37°C and 5% CO2 for 20–24 hours. QUANTI-Blue™ (Invivogen) solution was prepared according to the manufacturer's instructions, and 180 μl was added per well to a new 96-well plate, followed by 20 μl of supernatant from antibody-induced HEK-Blue IL-18 cells. The cell culture plates were incubated at 37°C and 5% CO2 for 3 hours and then read at 630 nm by a spectrophotometer (Clariostar).

[0291] Most agonists (41) showed no activity. Three agonists from the prior art that showed minimal activity (more than 2-fold over background) are shown in Table 7 below and in Figure 4. No robust agonist activity was detected. [Table 7]

[0292] Example 4. Generation of anti-IL18R antibodies from an immunized VHH library Two llamas per target were injected with five weekly boosts of recombinant protein (human IL-18Rα-Fc or human IL-18Rβ-Fc, Acro Biosystems). Following the final protein immunization, blood was collected and PBMCs were isolated. RNA was then extracted and stored. RNA purity and integrity were analyzed by microcapillary electrophoresis using a 2100 Bioanalyzer (Agilent). RNA was converted to cDNA using reverse transcriptase and random primers. VHHs were amplified using multiplex primers and cloned into a phagemid vector. Phages were prepared, and libraries were generated and analyzed from each llama. Each library had a size greater than 1.8E+09 and contained more than 90% VHH inserts.

[0293] Recombinant proteins (human IL-18Rα-his, cynomolgus IL-18Rα-his, human IL-18Rβ-his, and rhesus IL-18Rβ-his, all from Sino Biological) were biotinylated using standard protocols and used to probe the library. Biotinylated antigens were incubated with phage at various concentrations during multiple rounds of panning. After each selection round, E. coli was infected with the output phage for subsequent selection or characterization of individual clones by panning.

[0294] Example 4. Generation of anti-IL18R antibodies from a fully human library Antibodies were isolated using a fully human antibody phage library. Recombinant proteins (human IL-18Rα-his, cynomolgus monkey IL-18Rα-his, and human IL-18Rβ-his, all from Sino Biological) were biotinylated using standard protocols and used to probe the library. Biotinylated antigen was incubated with phage at various concentrations during multiple rounds of panning. After each selection round, E. coli was infected with output phage for subsequent selection or characterization of individual clones. Periplasmic extracts of individual clones were sequenced and profiled for binding to the recombinant proteins using a FRET-based assay essentially as previously described (Rossant DG et al. PMID: 25381254). Selected clones were also sequenced using NGS, and binders were analyzed using the DIAGONAL platform. Positive binding clones with calculated epitope diversity were generated as bispecific antibodies.

[0295] Example 5. Generation of heteromeric antibodies After identifying binders for each receptor, antibodies with unique CDR-H3s and predicted epitope diversity (identified using the DIAGONAL platform described in Example 1) were selected for further characterization as heteromeric bispecific IL-18 receptor-binding molecules. Each VHH for IL-18Rα and IL-18Rβ was fused to an Fc containing the mutations shown below and linked using a DKTHT linker. The VHH pairings for IL-18Rα and IL-18Rβ are listed in Table 8 below, and the sequences of the VHHs for IL-18Rα and IL-18Rβ are listed in Tables 9 and 11, respectively. In some cases, the VHHs were fused in tandem to a single Fc domain and paired with the Fc domain via a hinge. Fabs were combined with VHHs using a common light chain or tested with further light chain engineering to ensure bispecific pairing.

[0296] Knob-into-hole mutations (T366S, L368A, Y470V, T366W) and bridging disulfides (Y349C, S354C) were used to design VHH-Fc. Reduced effector function mutations (L234A and L235A) were added. Additional Fc mutations included G237A, T299K / T299D to reduce effector function, and M252Y / S254T / T256E or M428L / N434S to extend half-life.

[0297] As an example, DGL097 (VHH3_VHH385_bsAb) and DGL093 (VHH285_VHH505_bsAb), shown in the HEK-IL-18 data from the two agonists shown in Figure 5, were assembled with the following sequence mutations: VHH3 or VHH285 against IL-18Rα was combined with the linker DKTHT, followed by L234A and L235A, knob-into-hole hole mutations (T366S, L368A, Y470V), and the bridging disulfide Y349C. VHH385 or VHH505 against IL-18Rβ was combined with the linker DKTHT, followed by L234A and L235A, knob-into-hole knob mutation (T366W), and the bridging disulfide S354C. [Table 8]

[0298] Antibodies were transiently transfected into HEK293 cells using rPEx® technology. Cells were harvested 6 days post-transfection and purified using a HiTrap Fibro PrismA column. Protein-containing pools were concentrated using a 30 kDa spin filter (Amicon) and further purified using a Superdex 200 column. Protein-containing fractions were analyzed using LabChip® capillary electrophoresis (Perkin Elmer). The purity of the final product was assessed using analytical gel filtration using LabChip® and a Superdex™ 200 10 / 30 column. [Table 9-1] [Table 9-2] [Table 10-1] [Table 10-2] [Table 10-3] [Table 11] [Table 12]

[0299] A tandem VHH construct containing two Fc domains was constructed for testing. [Table 13] [Table 14-1] [Table 14-2] [Table 15-1] [Table 15-2] [Table 15-3] [Table 15-4] [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4] [Table 17-5] [Table 17-6]

[0300] Example 6. Screening for agonist activity HEK-Blue™ IL-18 cells were purchased from Invivogen (hbk-hmil18). These cell lines overexpress IL-18Rα and IL-18Rβ and block responses to TNFα and IL-1β. Reporter cells were revived and cultured according to the supplier's recommendations. Cells were washed with PBS and added to a 96-well plate at approximately 50,000 cells per well. 20 μl of either control or heteromeric antibody was added to the wells at the listed final assay conditions. The plates were incubated for 20–24 hours in a 37°C, 5% CO2 incubator. QUANTI-Blue™ (Invivogen) solution was prepared according to the manufacturer's instructions, and 180 μl was added to a new plate, followed by 20 μl of supernatant from antibody-induced HEK-Blue IL-18 cells. The cell culture plate was incubated at 37°C for 3 hours and then read at 630 nm on a Clariostar spectrophotometer.

[0301] In the initial screen, antibodies were screened using a 4-point 20-fold titration starting at 100 nM. Constructs with greater than 2-fold activity at any concentration were considered for further characterization. Data reported are the average of two replicates at the highest reading.

[0302] To calculate EC50, bispecifics were evaluated in a 10-point, 5-fold titration starting at 100 nM. Human IL-18 (Invivogen, rcyec-hiI18) was titrated in an 8-point, 4-fold titration starting at 1 ng / ml. All data were fitted in PRISM using a log (agonist) vs. response-variable slope (4-parameter) analysis.

[0303] The agonist activity of the heteromeric antibodies listed in Table 17 was tested in the HEK-Blue™ assay. All antibodies exhibited agonist activity, as indicated by an increase in absorbance at 630 nm. Figure 5 shows an exemplary graph of the binding activity exhibited by DGL097 compared to PBS, IL-18, and IL-1. Titration curves were generated to determine the Emax and EC50 of these heteromeric antibodies. These values ​​are listed in Table 18. [Table 18] [Table 19] [Table 20] [Table 21-1] [Table 21-2]

[0304] Example 7: Engineering a hinge-containing bispecific antibody. The agonistic activity of heteromeric antibodies with modified hinges was also tested. A panel of DGL093 variants, DGL207-DGL222, was designed, expressed, and purified as described. All hinge variants contained fully human framework 4. The bispecifics were tested using the HEK Blue assay as described. As shown in Table 22, DGL207 and DGL209 outperformed the parent DGL093 and other hinge variations of other heteromeric antibodies. [Table 22] [Table 23-1] [Table 23-2] [Table 23-3] [Table 23-4] [Table 24-1] [Table 24-2] [Table 25]

[0305] The IL-18R bispecific DGL207 agonist with hinge variant 1 (hinge 1; no upper hinge) performed best among the hinge variants. Hinge 2 contains the upper hinge sequence of PLAPDKTHT (SEQ ID NO: 273). Hinge 3 contains the upper hinge sequence of PLAP (SEQ ID NO: 274). Hinge 4 contains the upper hinge sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 275). Hinge 5 contains the upper hinge sequence of EKSYGPP (SEQ ID NO: 276). Hinge 6 contains the upper hinge sequence of DKTHT (SEQ ID NO: 277) and is similar to DGL093 except that it contains a fully human framework 4.

[0306] Example 8: IL18R bispecific agonist antibodies induce effector cell-mediated cytotoxicity of tumor cells in vitro. The assay was initiated after 2 weeks of growth of Incucyte Nuclight Green A549 target cells, stably expressing a green indicator dye. Nuclight Green A549 target cells were seeded at 5,000 cells / well in a 96-well plate and incubated overnight at 37°C and 5% CO2. The following day, PBMCs from three healthy human donors were washed and resuspended in RPMITot (containing 10% FBS, β-mercaptoethanol, L-glutamine, NEAA, sodium pyruvate, and gentamicin), and 200,000 PBMCs were added to the Nuclight Green A549 target cells at a 40:1 effector (PBMC):target cell ratio. To determine the effect of IL-18R agonists on tumor killing, 30 nM, 3 nM, or 1 nM of the IL-18R agonist DGL093 was added to cell cocultures in the presence or absence of 10 ng / ml human IL-12 (Miltenyi Biotec). Human IL-18 (Biolegend) at 200 ng / mL, 20 ng / mL, or 2 ng / mL plus 10 ng / mL human IL-12 served as a positive control and comparator for the DGL093 agonist, and human IL-2 (3.3 ng / mL) plus cetuximab (5 μg / mL) served as a positive assay control optimized by CRO ImmunXperts. Cells were then incubated in an Incucyte chamber at 37°C and 5% CO2, and tumor cell growth was assessed over 5 days by monitoring the confluence of Incucyte Nuclight Green A549 target cells over time. Death of Incucyte® Nuclight Green A549 target cells over 5 days can be assessed using Cytotox Red dye reagent (250 nM) on the Incucyte® Analysis System, but as this does not distinguish between PBMC death and tumor cell death, data are presented as green object counts, representing only the number of Incucyte® Nuclight Green A549 target cells.

[0307] Only DGL093 showed similar agonist activity over time compared to IL-18 in the three donors. DGL093 showed similar tumor-killing activity over time compared to IL-18 in IL-12-primed PBMCs from the three donors (Figure 6).

[0308] Example 9. Humanization of IL-18R binders. Binders previously identified as having high levels of activity using the HEK Blue assay were humanized and optimized for therapeutic use. VHH binders to IL-18Rα and IL-18Rβ were computationally modeled using the antigens using the DIAGONAL platform. Residues not essential for epitope recognition were replaced with human sequences. Back mutations were added only to maintain antigen binding and stability. Construct affinity was measured using Carterra, and activity was measured using the HEK Blue assay. Additionally, in some cases, alanine was substituted for proline at position 14 in the VHH-binding domain to improve binder stability and agonism.

[0309] When DGL207 was humanized, it was observed that the potency and affinity for IL-18Rβ were reduced (DGL333). Using the DIAGONAL platform, it was observed that the proline at position 14 (P14) in the context of the rigidified hinge (hinge 1) could destabilize the molecule. Reverting this mutation to an alanine found in the llama germline improved both the affinity and activity of the bispecific in the HEK Blue assay (Figure 7).

[0310] In addition to humanizing the VHHs, the Fc was engineered for optimal therapeutic use (e.g., LALAGA, knob-in-hole, and YTE mutations). [Table 26] [Table 27-1] [Table 27-2]

[0311] Example 10. Determination of affinity for IL-18R by humanized binders. SPR experiments were performed using a Carterra LSA equipped with an HC30M chip (Carterra-Bio). Binding assays were performed 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. Amine coupling was used to determine the density of the anti-human IgG-Fc capture layer (lawn) on the HC30M chip. To prepare the capture layer, the chip was activated with 100 mM MES (pH 5.5) containing 133 mM EDC and 33.3 mM S-NHS. Standard immobilization coupling of goat anti-human IgG (H+L) multi-species SP ads-UNLB (Southern Biotech, 2087-01) was performed in 10 mM sodium acetate (pH 4.5) for 15 minutes and quenched using 1 M ethanolamine HCl (pH 8.5). Next, a panel of antibodies was captured at 20 μg / mL using an anti-human IgG-Fc capture surface. Monomeric antigens against antibody targets, human IL-18Rα, human IL-18Rβ, cynomolgus IL-18Rα, or rhesus IL-18Rβ, were injected over the captured antibody array at six concentrations in a 4-fold dilution series starting at 500 nM. After referencing the binding data and blanking from the injected buffer, global fitting to a 1:1 Langmuir binding model was performed using Carterra Kinetics software to estimate k, kd, and KD. Table 27 shows the KD values ​​for DGL336, DGL346, and DGL620. [Table 28] [Table 29]

[0312] Example 11. Evaluation of agonist activity using HEK cells overexpressing IL-18Rα and IL-18Rβ. HEK Blue™ IL-18 cells were purchased from Invivogen (hbk-hmIL-18). These cell lines overexpress IL-18Rα and IL-18Rβ and block responses to TNFα and IL-1β. Reporter cells were revived and cultured according to the supplier's recommendations. Cells were rinsed with PBS and added to 96-well plates at a density of approximately 50,000 cells / well. Either control or bispecific antibodies were added to the wells at a final agonist concentration of 100 nM (Table 29). 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 was added per well to 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 before reading at 630 nm on a spectrophotometer (Varioskan Lux, Thermo). All optimized antibodies had similar activity to their parent nonhumanized antibodies in the HEK Blue assay. To calculate EC50, bispecific antibodies were evaluated in a 10-point, 5-fold titration starting at 100 nM. Human IL-18 (Invivogen, rcyec-hiI18) was titrated in an 8-point, 4-fold titration starting at 1 ng / ml. All data were fitted using log (agonist) vs. response-variable slope (4-parameter) analysis in PRISM. [Table 30]

[0313] Example 12. Induction of IFNγ in human PBMCs and immune cell profiling. To evaluate the activity of agonist antibodies on immune cells, frozen peripheral blood mononuclear cells (PBMCs) were obtained from six donors. These PBMCs were thawed and plated at a concentration of 250,000 cells per well in 10% RPMI (Gibco, A10491). To perform the assay, IL-12 (R&D Systems, 10018-IL) was added to each well at a final concentration of 0.5 ng / mL. Bispecific antibodies were added to a final concentration of 300 nM and diluted to generate a titration curve. After incubating the cells for 24 hours, the plates were spun at 500 g for 5 minutes and the supernatants were collected. Supernatants from all samples were run using the R&D Systems Human IFNγ DUoSet ELISA Kit (DY285B). IL-18 (R&D Systems, 9124-IL) was used as a positive control. Plates were run using Luminex according to the manufacturer's instructions. Data were analyzed in PRISM using a four-parameter, least-squares fit. EC50 values ​​were calculated for each bispecific antibody tested and then averaged across the six donors (Table 30). Table 25 shows the data for calculated maximal induction of IFNγ after 24 hours.

[0314] The results of the PBMC assay are shown in Figures 8A–8H. After 24 h, the IL-18R bispecific antibodies were able to induce IFNγ, a marker of tumor effector lymphocyte activation. Unlike IL-18, a ligand that can activate multiple immune cells, the IL-18R bispecific antibodies did not induce IL-5 or other Th2 cytokines (e.g., IL-4 and IL-13, data not shown). Furthermore, these bispecific antibodies did not activate IL-1β or other cytokines and chemokines involved in acute inflammation, such as IL-6, IL-8, MCP-1, and GM-CSF (Figures 8B–8H). These data demonstrate that heteromeric IL-18R bispecific agonists can preferentially stimulate anti-tumor effector lymphocytes. [Table 31] [Table 32]

[0315] Example 13. Gene expression profiling in PBMCs. Human PBMCs were treated for 24 hours with recombinant human IL-12 alone (R&D Systems, 10018-IL), IL-12 and recombinant IL-18 (rhIL-18, R&D Systems, 9124-IL), or IL-12 and 10 nM of a DIAGONAL IL-18R antibody agonist. Total RNA was isolated and analyzed using the NanoString platform. All tested DIAGONAL IL-18R agonists exhibited selective gene expression profiles compared with rhIL-18 in human PBMCs. Specifically, DIAGONAL IL-18R agonists induced the expression of genes involved in antiviral responses, NK cell 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 proinflammatory responses (Figure 9).

[0316] Example 14. In vivo characterization of agonists in a mouse GvHD model. A human PBMC-transplanted mouse model of graft-versus-host disease (GvHD) was used to evaluate the molecular profile of IL-18R agonists in the immune system. Human PBMCs isolated from healthy adult donors were thawed on ice from frozen stocks, washed, and incubated at 2 x 10 cells. 8 On day 0, 2 x 10 cells were injected into 7-9 week-old female NOD / SCID / IL-2Rγ null immunodeficient mice. 7PBMCs were transplanted. Three mouse cohorts were established using three donors. Groups of nine mice, equivalent to three mice per donor, were administered 3 mpk of each agonist on days 0, 4, and 10. Flow cytometry analysis of whole blood was used to assess cellularity and activation status, and cytokine analysis was performed by CBA analysis using plasma. Figure 10A shows the induction of IFNγ at two time points (days 7 and 12). All agonists demonstrated robust induction of IFNγ in the GVHD model. Figure 10B shows the amount of CD8 T cells increased after agonist administration.

[0317] Example 15. In vivo characterization of agonists in cynomolgus monkeys. Male cynomolgus monkeys (two to three monkeys per agonist) received a single dose of IL-18R bispecific agonist on day 0. Blood was collected the day before dosing and at various time points after dosing to assess PK and PD profiles. Blood was analyzed as fresh whole blood or processed to plasma. Data are shown in Figures 11A-14C. The data indicate that a single dose of IL-18R agonist resulted in activation of both T and NK cells, as indicated by a decrease in CD69 expression on CD8+ T cells (Figures 11A-11C) and the inhibitory CD159a receptor on CD56+ NK cells (Figures 12A-12C), respectively. Consistent with the observed cell activation was an increase in the T and NK cell mediators IFNγ (Figures 13A-13C) and IL-2 (Figures 14A-14C). These agonists did not induce monocyte proliferation (Figures 15A-15C) or induction of acute inflammatory mediators such as IL-6 (Figures 16A-16C) or GM-CSF (Figures 17A-17C). Other cytokines and chemokines involved in acute inflammation, myeloid-derived suppressor cells, and Th2 responses were unaffected. The bispecific agonists were well tolerated. The activity of DGL620 in cynomolgus monkeys may not be representative of its activity in humans due to its limited cross-reactivity with nonhuman primate IL-18Rβ. DGL620 is fully cross-reactive with nonhuman primate IL-18Rα.

[0318] Example 16. Expression of agonistic IL-18R antibodies. DGL336, DGL346, and DGL620 were transiently transfected into 10 L of CHO-K1 cells using the WuXian Express Transfection Platform (WuXi Biologics). Antibodies were purified using MabSelect SuRe (Cytiva) and refined using POROS XS (Thermo) and / or CaptoMMC ImpRes (Cytiva) depending on purity. Purity at each step was analyzed using SEC-HPLC and SDS-PAGE and confirmed using mass spectrometry. The final product yields for each were 1 g / L for DGL336, 1.3 g / L for DGL346, and 0.8 g / L for DGL620 (Table 32). [Table 33]

Claims

1. A multispecific binding protein comprising a first binding moiety that specifically binds human IL-18Rα and a second binding moiety that specifically binds 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.

2. 1. A multispecific binding protein comprising a first binding moiety that specifically binds human IL-18Rα and a second binding moiety that specifically binds human IL-18Rβ, wherein the multispecific binding protein stimulates anti-tumor cytokine production without substantially stimulating MCP-1 production, GM-CSF production, or the production of markers of acute inflammatory or Th2 responses, as compared to IL-18 stimulation of MCP-1 production, GM-CSF production, or the production of markers of acute inflammatory or Th2 responses.

3. 3. The multispecific binding protein of claim 2, wherein the anti-tumor cytokine is selected from the group consisting of IFN-γ, IL-2, IL-12, IL-15, CD40L, and TNFα.

4. 4. The multispecific binding protein of claim 2 or 3, wherein the marker of an acute inflammatory response or a Th2 response is selected from the group consisting of IL-6, IL-1β, IL-8, IL-4, IL-5, and IL-13.

5. The multispecific binding protein of any one of claims 2 to 4, wherein the anti-tumor cytokines and markers of acute inflammatory or Th2 responses are determined in a peripheral blood mononuclear cell (PBMC) assay.

6. 6. The multispecific binding protein of claim 5, wherein the PBMC assay comprises: 1) incubating a first population of PBMCs with the multispecific binding protein for at least 24 hours; 2) incubating the second population of PBMCs with IL-18 for at least 24 hours; and 3) measuring the production of the anti-tumor cytokines and markers of an acute inflammatory response or a Th2 response from the first and second PBMC populations. The method comprising:

7. 7. The multispecific binding protein of any one of claims 2 to 6, wherein the multispecific binding protein stimulates production of a marker of an acute inflammatory response or a Th2 response at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than IL-18.

8. 8. The multispecific binding protein of any one of claims 2 to 7, wherein the multispecific binding protein stimulates the production of a marker of an acute inflammatory response or a Th2 response at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than IL-18 as measured in the PBMC assay.

9. 9. The multispecific binding protein of any one of claims 2-8, wherein the multispecific binding protein stimulates GM-CSF production at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than IFN-γ production.

10. 10. The multispecific binding protein of any one of claims 1 to 9, The multispecific binding protein, wherein the first binding moiety comprises an IL-18Rα VHH domain and the second binding moiety comprises an IL-18Rβ VHH domain.

11. 11. The multispecific binding protein of claim 10, wherein the IL-18Rα VHH domain and the IL-18Rβ VHH domain are on separate polypeptides.

12. The multispecific binding protein of claim 10, wherein the IL-18Rα VHH domain and the IL-18Rβ VHH domain are in the same polypeptide.

13. 13. The multispecific binding protein of any one of claims 1 to 12, wherein the IL-18Rα binding portion is: (a) an HCDR1 sequence comprising the amino acid sequence of SYDMG (SEQ ID NO: 1); an HCDR2 sequence comprising the amino acid sequence of ALRWSGGGSTSYADSVKG (SEQ ID NO: 2); an HCDR3 sequence comprising the amino acid sequence of TLETDSGTYWADY (SEQ ID NO: 3); (b) an HCDR1 sequence comprising the amino acid sequence of ATGMG (SEQ ID NO: 4), an HCDR2 sequence comprising the amino acid sequence of RISSTGSPNYVDFVKG (SEQ ID NO: 5), and an HCDR3 sequence comprising the amino acid sequence of VGTTLFA (SEQ ID NO: 6); (c) an HCDR1 sequence comprising the amino acid sequence of TKGLG (SEQ ID NO: 7), an HCDR2 sequence comprising the amino acid sequence of GISSAGWIFYTQSVKG (SEQ ID NO: 8), and an HCDR3 sequence comprising the amino acid sequence of AQSGVPLRS (SEQ ID NO: 9); (d) an HCDR1 sequence comprising the amino acid sequence of INIMD (SEQ ID NO: 10), an HCDR2 sequence comprising the amino acid sequence of RISPGDIITYANDVKG (SEQ ID NO: 11), and an HCDR3 sequence comprising the amino acid sequence of RQGAGDY (SEQ ID NO: 12); (e) an HCDR1 sequence comprising the amino acid sequence of DYVLG (SEQ ID NO: 13); an HCDR2 sequence comprising the amino acid sequence of CISSRGRYLNYAETVKG (SEQ ID NO: 14); an HCDR3 sequence comprising the amino acid sequence of VRRVSEVCKLAEDDFAS (SEQ ID NO: 15); (f) an HCDR1 sequence comprising the amino acid sequence of KHAMG (SEQ ID NO: 16); A HCDR2 sequence comprising the amino acid sequence of AIDWSGGSTYYADSVKG (SEQ ID NO: 17), a HCDR3 sequence comprising the amino acid sequence of DSYTDYAQLWLPELESEYDY (SEQ ID NO: 18), (g) an HCDR1 sequence comprising the amino acid sequence of SYTMG (SEQ ID NO: 19), an HCDR2 sequence comprising the amino acid sequence of AISWSAGRTYYADSVKG (SEQ ID NO: 20), and an HCDR3 sequence comprising the amino acid sequence of EEPDWAPIDCSGYGCLSLYDY (SEQ ID NO: 21); (h) an HCDR1 sequence comprising the amino acid sequence of IDFMG (SEQ ID NO: 22), an HCDR2 sequence comprising the amino acid sequence of TITTGGSTNYADSVKD (SEQ ID NO: 23), and an HCDR3 sequence comprising the amino acid sequence of VVHTTSRPPVLY (SEQ ID NO: 24); (i) an HCDR1 sequence comprising the amino acid sequence of NYDMG (SEQ ID NO: 25); an HCDR2 sequence comprising the amino acid sequence of VISGPGGIAFYGDSVKG (SEQ ID NO: 26); an HCDR3 sequence comprising the amino acid sequence of APRGSYYRRTNSYDY (SEQ ID NO: 27); (j) an HCDR1 sequence comprising the amino acid sequence of RYG (SEQ ID NO: 28), an HCDR2 sequence comprising the amino acid sequence of DIYWNGGNTYYTDSVKG (SEQ ID NO: 29), and an HCDR3 sequence comprising the amino acid sequence of ATSYYAVTDPLKVAY (SEQ ID NO: 30); or (k) an HCDR1 sequence comprising the amino acid sequence of NWYMR (SEQ ID NO: 31), an HCDR2 sequence comprising the amino acid sequence of SINSGGDDTDYADSVKG (SEQ ID NO: 32), and an HCDR3 sequence comprising the amino acid sequence of GADRV (SEQ ID NO: 33) The multispecific binding protein comprising:

14. 10. The multispecific binding protein of any one of the preceding claims, wherein the second binding moiety comprises an IL-18Rβ VHH domain.

15. 15. The multispecific binding protein of any one of claims 1 to 14, wherein the IL-18Rβ binding portion is: (a) an HCDR1 sequence comprising the amino acid sequence of SYTMG (SEQ ID NO: 19); A HCDR2 sequence comprising the amino acid sequence of ALSWWNGGISTAYADSVKG (SEQ ID NO: 34), a HCDR3 sequence comprising the amino acid sequence of ARDRMPRADEYDY (SEQ ID NO: 35), (b) an HCDR1 sequence comprising the amino acid sequence of RNSMA (SEQ ID NO: 36); an HCDR2 sequence comprising the amino acid sequence of AISSISSGGRTDYADFVKG (SEQ ID NO: 37); an HCDR3 sequence comprising the amino acid sequence of PIRVASLAYDD (SEQ ID NO: 38); (c) an HCDR1 sequence comprising the amino acid sequence of NYHMG (SEQ ID NO: 39); A HCDR2 sequence comprising the amino acid sequence of AISSSGGKTSYPDSVNG (SEQ ID NO: 40), a HCDR3 sequence comprising the amino acid sequence of DPRYWVAAGGSEPENVEV (SEQ ID NO: 41), (d) an HCDR1 sequence comprising the amino acid sequence of VNSMA (SEQ ID NO: 42); an HCDR2 sequence comprising the amino acid sequence of VISSGGSAVYADSVKG (SEQ ID NO: 43); an HCDR3 sequence comprising the amino acid sequence of GSAAYRDY (SEQ ID NO: 44); (e) an HCDR1 sequence comprising the amino acid sequence of RNTMG (SEQ ID NO: 45); an HCDR2 sequence comprising the amino acid sequence of HFLWTGGETDYADAVKG (SEQ ID NO: 46); an HCDR3 sequence comprising the amino acid sequence of NYAGYRIDGYQY (SEQ ID NO: 47); (f) an HCDR1 sequence comprising the amino acid sequence of IHVMG (SEQ ID NO: 48); an HCDR2 sequence comprising the amino acid sequence of FIINNGGTRYADSVKG (SEQ ID NO: 49); an HCDR3 sequence comprising the amino acid sequence of EGTYRGRYSTDN (SEQ ID NO: 50); (g) an HCDR1 sequence comprising the amino acid sequence of ENDVR (SEQ ID NO: 51); an HCDR2 sequence comprising the amino acid sequence of AITSSGITGYADSVRI (SEQ ID NO: 52); an HCDR3 sequence comprising the amino acid sequence of TDQY (SEQ ID NO: 53); (h) an HCDR1 sequence comprising the amino acid sequence of LNTMG (SEQ ID NO: 54), an HCDR2 sequence comprising the amino acid sequence of VESSSGITNYADSVKG (SEQ ID NO: 55), and an HCDR3 sequence comprising the amino acid sequence of KLFGRDF (SEQ ID NO: 56); (i) an HCDR1 sequence comprising the amino acid sequence of SHNVMG (SEQ ID NO: 57); an HCDR2 sequence comprising the amino acid sequence of SIGSGGSTNYVDSVKG (SEQ ID NO: 58), an HCDR3 sequence comprising the amino acid sequence of VVGVYRGS (SEQ ID NO: 59), or (j) an HCDR1 sequence comprising the amino acid sequence of RDTMG (SEQ ID NO: 116), an HCDR2 sequence comprising the amino acid sequence of VISSSGNTNYADSVLG (SEQ ID NO: 117), and an HCDR3 sequence comprising the amino acid sequence of HRTYGVDY (SEQ ID NO: 118) The multispecific binding protein comprising:

16. 10. The multispecific binding protein of any one of the preceding claims, wherein the IL-18Rα VHH is at least about 90% identical, at least about 95% identical, or at least 98% identical to the amino acid sequence of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:

70.

17. 10. The multispecific binding protein of any one of the preceding claims, wherein the IL-18Rβ VHH is at least about 90% identical, at least about 95% identical, or at least 98% identical to the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, or SEQ ID NO:

98.

18. 10. The multispecific binding protein of any one of the preceding claims, wherein the IL-18Rα VHH comprises the amino acid sequence of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, or SEQ ID NO:

70.

19. 10. The multispecific binding protein of any one of the preceding claims, wherein the IL-18Rβ VHH comprises the amino acid sequence of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79 or SEQ ID NO:

98.

20. 10. The multispecific binding protein of any one of the preceding claims, wherein the multispecific binding protein has agonist activity that meets or exceeds a certain threshold over background when measured in an agonist activity assay, such as a HEK-Blue assay.

21. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 2-fold over background.

22. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 3-fold over background.

23. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 4-fold over background.

24. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 5-fold over background.

25. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 6-fold over background.

26. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 7-fold over background.

27. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 8-fold over background.

28. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 9-fold over background.

29. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 10-fold over background.

30. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 11-fold over background.

31. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 12-fold over background.

32. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 13-fold over background.

33. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 14-fold over background.

34. 21. The multispecific binding protein of claim 20, wherein the agonist activity of the multispecific binding protein is about 15-fold over background.

35. 10. The multispecific binding protein of any one of the preceding claims, wherein the first binding moiety binds to one or more of amino acids Ser24, Arg25, Pro26, Thr126, Ser127, Lys128, and Ile129 of human IL-18Rα (SEQ ID NO: 287).

36. 10. The multispecific binding protein of any one of the preceding claims, wherein the first binding moiety binds to at least amino acids Ser24, Arg25, Pro26, Thr126, Ser127, Lys128, and Ile129 of human IL-18Rα (SEQ ID NO: 287).

37. 10. The multispecific binding protein of any one of the preceding claims, wherein the first binding moiety binds to amino acids Ser24, Arg25, Pro26, Thr126, Ser127, Lys128, Ile129, Phe135, Phe136, Gln137, Ile138, Thr139, Cys140, Glu141, Asn142, Ser143, Lys200, Thr201, and Phe202 of human IL-18Rα (SEQ ID NO: 287).

38. 10. The multispecific binding protein of any one of the preceding claims, wherein the first binding moiety binds to amino acids Ser24, Arg25, Pro26, His27, Ile28, Thr29, Glu122, Arg123, Gln124, Val125, Thr126, Ser127, Lys128, Ile129, and Val130 of human IL-18Rα (SEQ ID NO: 287).

39. 10. The multispecific binding protein of any one of the preceding claims, wherein the second binding moiety binds to one or more of amino acids Gln216, Gly217, Thr218, Gln239, Val240, Arg241, Thr242, Ile243, Lys309, Ser310, Thr311, and Leu312 of human IL-18Rβ (SEQ ID NO: 288).

40. 10. The multispecific binding protein of any one of the preceding claims, wherein the second binding moiety binds to at least amino acids Gln216, Gly217, Thr218, Gln239, Val240, Arg241, Thr242, Ile243, Lys309, Ser310, Thr311, and Leu312 of human IL-18Rβ (SEQ ID NO: 288).

41. 10. The multispecific binding protein of any one of the preceding claims, wherein the second binding moiety binds to at least amino acids Asp213, Tyr214, His215, Gln216, Gly217, Thr218, Gln239, Val240, Arg241, Thr242, Ile243, Lys306, Ser307, Ile308, Lys309, Ser310, Thr311, and Leu312 of human IL-18Rβ (SEQ ID NO: 288).

42. 10. The multispecific binding protein of any one of the preceding claims, wherein the second binding moiety binds to at least amino acids Glu39, Glu40, Glu41, His112, Phe113, Leu114, Thr115, Pro116, Gln216, Gly217, Thr218, Gln239, Val240, Arg241, Thr242, Ile243, Phe279, Glu280, Arg281, Val282, Phe283, Asn284, Lys309, Ser310, Thr311, Leu312, Lys313, Asp314, and Glu315 of human IL-18Rβ (SEQ ID NO: 288).

43. 10. The multispecific binding protein of any one of the preceding claims, further comprising one or more modified hinge regions.

44. 44. The multispecific binding protein of claim 43, wherein the one or more modified hinges comprise: i) an upper hinge region that is at most 7 amino acids in length or is absent; and ii) comprising a middle hinge region and a lower hinge region, wherein the lower hinge region is linked to the N-terminus of the heavy chain constant region; The multispecific binding protein.

45. 45. The multispecific binding protein of claim 43 or 44, wherein the upper hinge regions of the first modified hinge region and the second modified hinge region have the same sequence.

46. 45. The multispecific binding protein of claim 43 or 44, wherein the upper hinge regions of the first modified hinge region and the second modified hinge region are different sequences.

47. 10. The multispecific binding protein of claim 1, wherein the upper hinge region comprises an amino acid sequence derived from the upper hinge region of a human IgG antibody.

48. 48. The multispecific binding protein of claim 47, wherein the IgG antibody is selected from IgG1, IgG2, IgG3, and IgG4.

49. 49. The multispecific binding protein of claim 48, wherein the IgG antibody is IgG1.

50. 2. The multispecific binding protein of any preceding claim, wherein the upper hinge region comprises the amino acid sequence of SEQ ID NO:

274.

51. 10. The multispecific binding protein of any one of the preceding claims, wherein the upper hinge region comprises the amino acid sequence of SEQ ID NO:

277.

52. 48. The multispecific binding protein of claim 47, wherein the IgG antibody is IgG4.

53. 53. The multispecific binding protein of claim 52, wherein the upper hinge region comprises the amino acid sequence of SEQ ID NO:

276.

54. 54. The multispecific binding protein of any one of claims 1 to 53, wherein the upper hinge is absent.

55. 10. The multispecific binding protein of any one of the preceding claims, further comprising all or part of an immunoglobulin Fc domain or a variant thereof.

56. 56. The multispecific binding protein of claim 55, wherein the Fc domain or variant thereof comprises a first Fc heavy chain and a second Fc heavy chain.

57. 57. The multispecific binding protein of claim 55 or 56, further comprising a variant Fc domain with reduced effector function.

58. 58. The multispecific binding protein of claim 57, wherein at least one Fc heavy chain comprises a substitution at amino acid position 234 according to EU numbering.

59. 59. The multispecific binding protein of claim 58, wherein the substitution at amino acid position 234 is an alanine (A).

60. 60. The multispecific binding protein of any one of claims 57 to 59, wherein at least one Fc heavy chain comprises a substitution at amino acid position 235 according to EU numbering.

61. 61. The multispecific binding protein of claim 60, wherein the substitution at amino acid position 235 is an alanine (A).

62. 62. The multispecific binding protein of any one of claims 57 to 61, wherein at least one Fc heavy chain comprises a substitution at amino acid position 237 according to EU numbering.

63. 63. The multispecific binding protein of claim 62, wherein the substitution at amino acid position 237 is an alanine (A).

64. 64. The multispecific binding protein of any one of claims 57 to 63, wherein at least one Fc heavy chain comprises one or more substitutions at amino acid positions 234, 235, or 237 according to EU numbering.

65. 65. The multispecific binding protein of claim 64, 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); The multispecific binding protein.

66. 10. The multispecific binding protein of any one of the preceding claims, wherein the Fc domain comprises a heterodimerization mutation that promotes heterodimerization of the first binding moiety with the second binding moiety.

67. 64. The multispecific binding protein of claim 63, wherein the heterodimerization mutation is a knob-in-hole (KIH) mutation.

68. 68. The multispecific binding protein of claim 67, wherein the first Fc heavy chain comprises an amino acid substitution at position 366, 368, or 407 that creates a hole, and the second Fc heavy chain comprises an amino acid substitution at position 366 that creates a knob.

69. 69. The multispecific binding protein of claim 67 or 68, wherein the first Fc heavy chain comprises the amino acid substitution T366S, L368A, or Y407V and the second Fc heavy chain comprises the amino acid substitution T366W.

70. 68. The multispecific binding protein of claim 67, wherein the heterodimerization mutation is a charge-stabilizing mutation.

71. 71. The multispecific binding protein of claim 70, wherein the first Fc heavy chain comprises the amino acid substitution N297K and the second Fc heavy chain comprises the amino acid substitution N297D.

72. 72. The multispecific binding protein of claim 70 or 71, wherein the first Fc heavy chain comprises the amino acid substitution T299K and the second Fc heavy chain comprises the amino acid substitution T299D.

73. 68. The multispecific binding protein of claim 67, wherein the heterodimerization mutation comprises an engineered disulfide bond.

74. 74. The multispecific binding protein of claim 73, wherein the engineered disulfide bond is formed by a first Fc heavy chain comprising the amino acid substitution Y349C and a second Fc heavy chain comprising the amino acid substitution S354C.

75. 75. The multispecific binding protein of claim 73 or 74, 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.

76. 76. The multispecific binding protein of any one of claims 73 to 75, 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.

77. 10. The multispecific binding protein of any one of the preceding claims, wherein at least one Fc domain comprises one or more mutations that promote an increased half-life.

78. 78. The multispecific binding protein of claim 77, wherein at least one Fc heavy chain comprises one or more substitutions at amino acid positions 252, 254, or 256 according to EU numbering.

79. 79. The multispecific binding protein of claim 78, 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 256 is glutamic acid (E); The multispecific binding protein.

80. 10. The multispecific binding protein of any one of the preceding claims, wherein the first binding moiety that specifically binds human IL-18Rα comprises the amino acid sequence set forth in any one of SEQ ID NOs:240-251, and the second binding moiety that specifically binds human IL-18Rβ comprises the amino acid sequence set forth in any one of SEQ ID NOs:252-260.

81. 10. The multispecific binding protein of claim 1, wherein the first binding moiety and / or the second binding moiety further comprises an amino acid substitution at position 14 from the N-terminus of the binding moiety.

82. 82. The multispecific binding protein of claim 81, wherein the amino acid at position 14 from the N-terminus of the binding moiety is proline (P).

83. 83. The multispecific binding protein of claim 81 or 82, wherein the substitution at position 14 from the N-terminus of the binding moiety comprises an alanine (A).

84. 84. The multispecific binding protein of any one of claims 81 to 83, wherein the substitution at position 14 from the N-terminus of the binding moiety is an alanine.

85. 85. The multispecific binding protein of any one of claims 81 to 84, wherein the substitution at position 14 from the N-terminus of the binding moiety further stabilizes the binding moiety.

86. 86. The multispecific binding protein of any one of claims 81 to 85, wherein said substitution at position 14 from the N-terminus of said binding moiety increases the agonistic properties of said binding moiety.

87. 10. A pharmaceutical composition comprising a multispecific binding protein according to any one of the preceding claims and a pharmaceutically acceptable carrier.

88. 10. An isolated nucleic acid molecule encoding a multispecific binding protein according to any one of the preceding claims.

89. 89. An expression vector comprising the nucleic acid molecule of claim 88.

90. 90. A host cell comprising the expression vector of claim 89.

91. 10. A method for treating a disease or disorder in a subject, the method comprising administering to a subject in need thereof a multispecific binding protein of any one of the preceding claims.

92. 10. A multispecific binding protein according to any one of the preceding claims for use as a medicament.

93. 1. A method of stimulating IL-18R-mediated IFN-γ expression in a subject, comprising administering to the subject a multispecific binding protein comprising a first binding moiety that specifically binds human IL-18Rα and a second binding moiety that specifically binds human IL-18Rβ, wherein the multispecific binding protein stimulates anti-tumor cytokine production in the subject without substantially stimulating MCP-1 production, GM-CSF production, or the production of markers of acute inflammatory or Th2 responses in the subject compared to IL-18 stimulation of MCP-1 production, GM-CSF production, or the production of markers of acute inflammatory or Th2 responses.

94. 94. The method of claim 93, wherein the anti-tumor cytokine is selected from the group consisting of IFN-γ, IL-2, IL-12, IL-15, CD40L, and TNFα.

95. 95. The method of claim 93 or 94, wherein the marker of an acute inflammatory response or a Th2 response is selected from the group consisting of IL-6, IL-1β, IL-8, IL-4, IL-5, and IL-13.

96. 96. The method of any one of claims 93-95, wherein the multispecific binding protein stimulates production of a marker of an acute inflammatory response or a Th2 response that is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than IL-18.

97. 97. The method of any one of claims 93-96, wherein the multispecific binding protein stimulates production of GM-CSF in the subject that is at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold less than IFN-γ production in the subject.

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