Hinge-modified bispecific antibodies

Abstract

This specification provides a bispecific agonist antibody having a modified hinge region, and a method for using the same.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 458,045 filed on 7 April 2023 and U.S. Provisional Patent Application No. 63 / 596,905 filed on 7 November 2023, the entirety of the disclosures of these documents being incorporated herein by reference. [Background technology]

[0002] Antibodies consist of two Fab regions connected to a crystallizable (Fc) fragment by a flexible hinge region. The hinge region is a flexible amino acid extension of the central heavy chain of the IgG and IgA immunoglobulin classes, linking these two chains by a disulfide bond.

[0003] The antibody hinge can be divided into three regions: the upper hinge, the core hinge, and the lower hinge, each with a different functional role. At the N-terminal end, the upper hinge allows for the movement and rotation of the fragment antigen-binding domain (Fab). The central core hinge contains a variable number of cysteine ​​residues, depending on the IgG subtype, which form disulfide bonds to stabilize HC association. At the C-terminal end is the lower hinge, which allows for the movement of Fc compared to Fab, and its amino acid residues may be involved in Fc gamma receptor (FcγR) binding.

[0004] The hinges of human IgG subtypes differ significantly in the number of residues and the number of possible disulfide crosslinks between the two heavy chains. This contributes to the overall stability of the antibody. For example, of all IgGs, IgG4 is the only subtype that produces a bispecific antibody molecule through natural Fab-arm exchange. Furthermore, this diversity, including differences in amino acid sequences, partially contributes to the strength of the interaction between IgG and FcγR. [Overview of the project]

[0005] This disclosure provides a heteromeric antibody having a modified hinge region, thereby improving the prior art by enhancing the antibody's agonist activity.

[0006] In one embodiment, the Specified herein provides a bispecific antibody exhibiting agonist activity comprising at least a first antigen-binding domain, a first modified hinge region, and a first heavy chain Fc domain, and at least a second antigen-binding domain and a second heavy chain Fc domain, wherein the first modified hinge region comprises an upper hinge region, a central hinge region, and a lower hinge region, which are up to 7 amino acids long or absent, and the lower hinge region is ligated to the N-terminus of the first heavy chain Fc domain. In some embodiments, the first antigen-binding domain binds to a first receptor subunit, and the second antigen-binding domain binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits. In some embodiments, the first and second receptor subunits are different subunits that form a heterodimer. In some embodiments, the first and second receptor subunits are the same subunit that forms a heterodimer. In some embodiments, the first and second receptor subunits are selected from tumor necrosis factor superfamily (TNFSF) receptors, interleukin type I receptors, interleukin type II receptors, Ig superfamily (IGSF) receptors, receptor tyrosine kinases (RTKs), growth hormone receptors, transforming growth factor beta (TGFβ) receptor superfamily, type C lectin-like receptors, interferon receptors, phosphatase receptors (i.e., receptor protein tyrosine phosphatases), and integrin receptors.

[0007] In some embodiments, the bispecific antibody further includes a second modified hinge region ligated to the N-terminus of the second heavy chain Fc domain. In some embodiments, the second modified hinge region includes an upper hinge region which is up to 7 amino acids long or absent, as well as a central hinge region and a lower hinge region, the lower hinge region being ligated to the N-terminus of the second heavy chain Fc domain. In some embodiments, the upper hinge region is ligated to the N-terminus of the second heavy chain Fc domain.

[0008] In some embodiments, the upper hinge regions of the first and second modified hinge regions are the same. In some embodiments, the upper hinge regions of the first and second modified hinge regions are different. In some embodiments, the upper hinge region includes 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 includes the amino acid sequence of SEQ ID NO: 1. In some embodiments, the upper hinge region includes the amino acid sequence of SEQ ID NO: 5. In some embodiments, the IgG antibody is IgG4. In some embodiments, the upper hinge region includes the amino acid sequence of SEQ ID NO: 4. In some embodiments, the upper hinge region is absent. In some embodiments, the central and lower hinge regions include the amino acid sequence of SEQ ID NO: 6.

[0009] In some embodiments, the first modified hinge region and / or the second modified hinge region include the amino acid sequence of SEQ ID NO: 7. In some embodiments, the first modified hinge region and / or the second modified hinge region include the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first modified hinge region and / or the second modified hinge region include the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first modified hinge region and / or the second modified hinge region include the amino acid sequence of SEQ ID NO: 9.

[0010] In some embodiments, the first heavy chain Fc domain and / or the second heavy chain Fc domain contains human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the first heavy chain Fc domain and / or the second heavy chain Fc domain contains the amino acid sequence of SEQ ID NO: 10.

[0011] In some embodiments, the first heavy chain Fc domain and / or the second heavy chain Fc domain contains one or more amino acid substitutions. In some embodiments, at least one heavy chain Fc domain contains a substitution at position 234 of amino acid according to EU numbering. In some embodiments, the substitution at position 234 of amino acid is alanine (A). In some embodiments, at least one heavy chain Fc domain contains a substitution at position 235 of amino acid according to EU numbering. In some embodiments, the substitution at position 235 of amino acid is alanine (A). In some embodiments, at least one heavy chain Fc domain contains a substitution at position 237 of amino acid according to EU numbering. In some embodiments, the substitution at position 237 of amino acid is alanine (A). In some embodiments, at least one heavy chain Fc domain contains one or more substitutions at position 234, 235, or 237 of amino acid according to EU numbering. In some embodiments, the substitution at amino acid position 234 is alanine(A), the substitution at amino acid position 235 is alanine(A), and the substitution at amino acid position 237 is alanine(A).

[0012] In some embodiments, the heavy chain Fc domain includes a heterodimerizing mutation that promotes heterodimerization between the first and second binding sites. In some embodiments, the heterodimerizing mutation is a knob-in-hole (KIH) mutation. In some embodiments, the first heavy chain Fc domain includes an amino acid substitution that creates a hole at position 366, 368, or 407, and the second heavy chain Fc domain includes an amino acid substitution that creates a knob at position 366. In some embodiments, the first heavy chain Fc domain includes the amino acid substitution T366S, L368A, or Y407V, and the second heavy chain Fc domain includes the amino acid substitution T366W.

[0013] In some embodiments, the heterodimerizing mutation is a charge-stabilizing mutation. In some embodiments, the first heavy chain Fc domain contains the amino acid substitution N297K, and the second heavy chain Fc domain contains the amino acid substitution N297D. In some embodiments, the first heavy chain Fc domain contains the amino acid substitution T299K, and the second heavy chain Fc domain contains the amino acid substitution T299D.

[0014] In some embodiments, the heterodimerization mutation includes an engineered disulfide bond. In some embodiments, the engineered disulfide bond is formed by a first heavy chain Fc domain containing the amino acid substitution Y349C and a second heavy chain Fc domain containing the amino acid substitution S354C. In some embodiments, the engineered disulfide bond is formed by a C-terminal elongation peptide fused to the C-terminus of each of the first and second heavy chain Fc domains. In some embodiments, the C-terminal elongation of the first heavy chain Fc domain contains the amino acid sequence GEC, and the C-terminal elongation of the second heavy chain Fc domain contains the amino acid sequence SCDKT.

[0015] In some embodiments, at least one heavy chain Fc domain contains one or more mutations that promote an increase in half-life. In some embodiments, at least one heavy chain Fc domain contains one or more substitutions at amino acid positions 252, 254, or 256, according to EU numbering. In some embodiments, the substitution at amino acid position 252 is tyrosine (Y), the substitution at amino acid position 254 is threonine (T), and the substitution at amino acid position 256 is glutamic acid (E).

[0016] In some embodiments, the first modified hinge region and the first heavy chain Fc domain are set forth in the amino acid sequence of SEQ ID NO: 11. In some embodiments, the first modified hinge region and the first heavy chain Fc domain are set forth in the amino acid sequence of SEQ ID NO: 12. In some embodiments, the first modified hinge region and the first heavy chain Fc domain are set forth in the amino acid sequence of SEQ ID NO: 13. In some embodiments, the first modified hinge region and the first heavy chain Fc domain are set forth in the amino acid sequence of SEQ ID NO: 14.

[0017] In some embodiments, the first antigen-binding domain is selected from a single-chain Fv (scFv), VHH, or Fab. In some embodiments, the second antigen-binding domain is selected from a single-chain Fv (scFv), VHH, or Fab.

[0018] In some embodiments, the bispecific antibody comprises, from the N-terminus to the C-terminus, ai) a first polypeptide chain comprising a first antigen-binding domain, a first modified hinge region, and a first Fc domain, and bi) a second polypeptide chain comprising a second antigen-binding domain, a second modified hinge region, and a second Fc domain; aii) a first polypeptide chain comprising a second antigen-binding domain, a first antigen-binding domain, a first modified hinge region, and a first Fc domain, and bii) a second polypeptide chain comprising a second modified hinge region, and a second Fc domain; or aiii) a first polypeptide chain comprising a first modified hinge region, and a first Fc domain, and biii) a second polypeptide chain comprising a second antigen-binding domain, a first antigen-binding domain, a second modified hinge region, and a second Fc domain; or aiv) a first polypeptide chain comprising a first antigen-binding domain, a second antigen-binding domain, a first modified hinge region, and a first constant region, and biv) a second polypeptide chain comprising a third antigen-binding domain, a fourth antigen-binding domain, a second modified hinge region, and a second constant region.

[0019] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, and the fourth antigen-binding domain comprise scFv.

[0020] In some embodiments, (a) the first antigen-binding domain comprises a VHH domain and the second antigen-binding domain comprises a VHH domain; (b) the first antigen-binding domain comprises a Fab domain and the second antigen-binding domain comprises a VHH domain; (c) the first antigen-binding domain comprises a VHH domain and the second antigen-binding domain comprises a Fab domain; (d) the first antigen-binding domain comprises a Fab domain and the second antigen-binding domain comprises a Fab domain; (e) the first antigen-binding domain comprises a Fab domain and the second antigen-binding domain comprises an scFv; (f) the first antigen-binding domain comprises an scFv and the second antigen-binding domain comprises a Fab domain; (g) the first antigen-binding domain comprises an scFv and the second antigen-binding domain comprises an scFv; (h) the first antigen-binding domain comprises an scFv and the second antigen-binding domain comprises a VHH domain; or (i) the first antigen-binding domain comprises a VHH domain and the second antigen-binding domain comprises an scFv.

[0021] In some embodiments, the first and / or second antibody-binding domains are cleaved at the C-terminus adjacent to the upper hinge domain. In some embodiments, the C-terminus adjacent to the upper hinge domain is cleaved by at least one residue. In some embodiments, the C-terminus adjacent to the upper hinge domain is cleaved by at least two residues.

[0022] In some embodiments, the bispecific antibody comprises a first and a second polypeptide chain, wherein the first polypeptide chain is VH1-(HX1)n-VH2-C-(HX2)n, where, VH1 is the first heavy chain variable domain, VH2 is the second heavy chain variable domain, C is a heavy chain constant domain, HX1 is a linker, HX2 is the Fc region, and n is independently either 0 or 1. The second polypeptide chain is Includes VL1-(LX1)n-VL2-C-(LX2)n, Here, VL1 is the first light chain variable domain, VL2 is the second light chain variable domain, C is a light chain constant domain, LX1 is a linker, LX2 does not include the Fc region, and n is independently either 0 or 1.

[0023] In some embodiments, linker HX1 includes the amino acid sequence of PLAP or PAPNLLGGP. In some embodiments, linker LX1 includes the amino acid sequence of PLAP or PAPNLLGGP. In some embodiments, linker HX1 includes the amino acid sequence of PLAP, and linker LX1 includes the amino acid sequence of PLAP or PAPNLLGGP.

[0024] In some embodiments, the first receptor subunit comprises an IL18Rα subunit, and the second receptor subunit is an IL18Rβ subunit. In some embodiments, the first receptor subunit comprises an ALK1 receptor subunit, and the second receptor subunit is selected from BMPRII, ActRIIA, and ActRIIB.

[0025] In another embodiment, as used herein, a) a first antigen-binding domain, a first modified hinge region, and a first heavy chain Fc domain, b) A bispecific antibody is provided that exhibits agonist activity comprising a second antigen-binding domain and a second heavy chain Fc domain, where, The first modified hinge region is, i) Upper hinge regions that are or do not exist and are at most 7 amino acids long, ii) Including a lower hinge region, the lower hinge region is ligated to the N-terminus of the first heavy chain Fc domain, The first antigen-binding domain binds to the first receptor subunit, and the second antigen-binding domain binds to the second receptor subunit, thereby inducing signal transduction by increasing proximity between the first and second receptor subunits.

[0026] In some embodiments, (a) the first antigen-binding domain includes a VHH domain, and the second antigen-binding domain includes a VHH domain; (b) The first antigen-binding domain includes a Fab domain, and the second antigen-binding domain includes a VHH domain; (c) The first antigen-binding domain includes a VHH domain, and the second antigen-binding domain includes a Fab domain; (d) The first antigen-binding domain includes a Fab domain, and the second antigen-binding domain includes a Fab domain; (e) The first antigen-binding domain includes the Fab domain, and the second antigen-binding domain includes the scFv; (f) The first antigen-binding domain includes scFv, and the second antigen-binding domain includes Fab domain; (g) The first antigen-binding domain contains scFv, and the second antigen-binding domain contains scFv; (h) The first antigen-binding domain includes scFv, and the second antigen-binding domain includes a VHH domain; or (i) The first antigen-binding domain includes a VHH domain, and the second antigen-binding domain includes an scFv domain.

[0027] In some embodiments, the first receptor subunit comprises an IL18Rα subunit, and the second receptor subunit is an IL18Rβ subunit. In some embodiments, the first receptor subunit comprises an ALK1 receptor subunit, and the second receptor subunit is selected from BMPRII, ActRIIA, and ActRIIB.

[0028] In another embodiment, as used herein, a) a first antigen-binding domain, a first modified hinge region, and a first heavy chain Fc domain, b) A bispecific antibody is provided that exhibits agonist activity comprising a second antigen-binding domain and a second heavy chain Fc domain, where, The first modified hinge region is, i) Upper hinge regions that are or do not exist and are at most 7 amino acids long, ii) Including a lower hinge region, the lower hinge region is ligated to the N-terminus of the first heavy chain Fc domain, The first antigen-binding domain binds to the first receptor subunit, and the second antigen-binding domain binds to the second receptor subunit, thereby inducing signal transduction by increasing proximity between the first and second receptor subunits.

[0029] In another embodiment, this specification provides a multispecific binding protein comprising at least a first polypeptide chain, where, The first polypeptide chain comprises a first variable heavy chain domain (VH1) linked to a second variable heavy chain domain (VH2) via at least one modified hinge region.

[0030] In some embodiments, one or both of VH1 and VH2 are VH domains or VHH domains.

[0031] In some embodiments, the multispecificity binding protein further comprises a second polypeptide chain, the second polypeptide chain comprising a first variable light chain domain (VL1) linked to a second variable light chain domain (VL2) via at least one modified hinge region.

[0032] In some embodiments, one or both of VH1 and VH2 are cleaved at the C-terminus.

[0033] In some embodiments, the C-terminus is cleaved by at least one residue.

[0034] In some embodiments, the C-terminus is cleaved by at least two residues.

[0035] In some embodiments, the C-terminal SS amino acid residue is deleted.

[0036] In some embodiments, the multispecific binding protein is a first polypeptide chain of VH1-HX1-VH2-C-Fc, where, VH1 is the first heavy chain variable domain, VH2 is the second heavy chain variable domain, C is a heavy chain constant domain, HX1 is a linker in the modified hinge region, and Fc is the Fc region, which is the first polypeptide chain, The second polypeptide chain of VL1-LX1-VL2-C, Here, VL1 is the first light chain variable domain, VL2 is the second light chain variable domain, C is a light chain constant domain, and LX1 comprises a second polypeptide chain, which is a linker in the modified hinge region.

[0037] In some embodiments, the modified hinge region includes or consists of the amino acid sequence PLAP or PAPNLLGGP.

[0038] In some embodiments, VH1 binds to a first receptor subunit and VH2 binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits.

[0039] In some embodiments, a first antigen-binding domain formed from VH1 and VL1 binds to a first receptor subunit, and a second antigen-binding domain formed from VH2 and VL2 binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits.

[0040] In some embodiments, the first receptor subunit and the second receptor subunit are different subunits that form a heterodimer.

[0041] In some embodiments, the first receptor subunit and the second receptor subunit are the same subunit that forms a heterodimer.

[0042] In some embodiments, the first and second receptor subunits are selected from tumor necrosis factor superfamily (TNFSF) receptors, interleukin type I receptors, interleukin type II receptors, Ig superfamily (IGSF) receptors, receptor tyrosine kinases (RTKs), growth hormone receptors, transforming growth factor beta (TGFβ) receptor superfamily, type C lectin-like receptors, interferon receptors, phosphatase receptors (i.e., receptor protein tyrosine phosphatases), and integrin receptors.

[0043] In some embodiments, the antigen-binding domain is a VHH containing the P14A amino acid substitution according to Kabat numbering.

[0044] In one embodiment, the disclosure provides a multispecific binding protein comprising at least a first binding domain and a second binding domain, wherein the first binding domain is linked to the second binding domain via at least one modified hinge region.

[0045] In some embodiments, the first binding domain is a first variable heavy chain domain (VH1), and the second binding domain is a second variable heavy chain domain (VH2).

[0046] In some embodiments, one or both of VH1 and VH2 are VH domains or VHH domains.

[0047] In some embodiments, the multispecificity binding protein further comprises a first variable light chain domain (VL1) linked to a second variable light chain domain (VL2) via at least one modified hinge region.

[0048] In some embodiments, the first binding domain is the first scFv, and the second binding domain is the second scFv.

[0049] In one embodiment, the disclosure provides a multispecificity binding protein comprising at least a first polypeptide chain, the first polypeptide chain comprising a first variable heavy chain domain (VH1) linked to a second variable heavy chain domain (VH2) via at least one modified hinge region.

[0050] In some embodiments, one or both of VH1 and VH2 are VH domains or VHH domains.

[0051] In some embodiments, the multispecificity binding protein further comprises a second polypeptide chain, the second polypeptide chain comprising a first variable light chain domain (VL1) linked to a second variable light chain domain (VL2) via at least one modified hinge region.

[0052] In some embodiments, one or both of VH1 and VH2 are cleaved at the C-terminus.

[0053] In some embodiments, the C-terminus is cleaved by at least one residue.

[0054] In some embodiments, the C-terminus is cleaved by at least two residues.

[0055] In some embodiments, the C-terminal SS amino acid residue is deleted.

[0056] In some embodiments, the multispecific binding protein is a first polypeptide chain of VH1-HX1-VH2-C-Fc, where, VH1 is the first heavy chain variable domain, VH2 is the second heavy chain variable domain, C is a heavy chain constant domain, HX1 is a linker in the modified hinge region, and Fc is the Fc region, which is the first polypeptide chain, The second polypeptide chain of VL1-LX1-VL2-C, Here, VL1 is the first light chain variable domain, VL2 is the second light chain variable domain, C is a light chain constant domain, and LX1 comprises a second polypeptide chain, which is a linker in the modified hinge region.

[0057] In some embodiments, the modified hinge region includes i) an upper hinge region that is up to 7 amino acids long or absent, and ii) a lower hinge region.

[0058] In some embodiments, the modified hinge region includes or consists of the amino acid sequence PLAP or PAPNLLGGP.

[0059] In some embodiments, the first binding domain binds to the first receptor subunit, and the second binding domain binds to the second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits.

[0060] In some embodiments, VH1 binds to a first receptor subunit and VH2 binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits.

[0061] In some embodiments, a first antigen-binding domain formed from VH1 and VL1 binds to a first receptor subunit, and a second antigen-binding domain formed from VH2 and VL2 binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits.

[0062] In some embodiments, a first scFv binds to a first receptor subunit, and a second scFv binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits.

[0063] In some embodiments, the first receptor subunit and the second receptor subunit are different subunits that form a heterodimer.

[0064] In some embodiments, the first receptor subunit and the second receptor subunit are the same subunit that forms a heterodimer.

[0065] In some embodiments, the first and second receptor subunits are selected from tumor necrosis factor superfamily (TNFSF) receptors, interleukin type I receptors, interleukin type II receptors, Ig superfamily (IGSF) receptors, receptor tyrosine kinases (RTKs), growth hormone receptors, transforming growth factor beta (TGFβ) receptor superfamily, type C lectin-like receptors, interferon receptors, phosphatase receptors (i.e., receptor protein tyrosine phosphatases), and integrin receptors.

[0066] In some embodiments, the antigen-binding domain is a VHH containing the P14A amino acid substitution according to Kabat numbering.

[0067] In some embodiments, the P14A amino acid substitution further stabilizes the multispecific binding protein.

[0068] In some embodiments, the P14A amino acid substitution increases the agonist properties of the multispecific binding protein.

[0069] In one embodiment, the present disclosure provides a multispecific binding protein comprising a first polypeptide chain and a second polypeptide chain, each comprising a first scFv ligated from the N-terminus to the C-terminus of a second single-chain variable fragment (scFv), wherein the first scFv is ligated to the second scFv via at least one modified hinge region.

[0070] In some embodiments, the modified hinge region includes i) an upper hinge region that is up to 7 amino acids long or absent, and ii) a lower hinge region.

[0071] In some embodiments, the modified hinge region includes or consists of the amino acid sequence PLAP or PAPNLLGGP.

[0072] In some embodiments, a first scFv binds to a first receptor subunit, and a second scFv binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits.

[0073] In some embodiments, the first receptor subunit and the second receptor subunit are different subunits that form a heterodimer.

[0074] In some embodiments, the first receptor subunit and the second receptor subunit are the same subunit that forms a heterodimer.

[0075] In some embodiments, the first and second receptor subunits are selected from tumor necrosis factor superfamily (TNFSF) receptors, interleukin type I receptors, interleukin type II receptors, Ig superfamily (IGSF) receptors, receptor tyrosine kinases (RTKs), growth hormone receptors, transforming growth factor beta (TGFβ) receptor superfamily, type C lectin-like receptors, interferon receptors, phosphatase receptors (i.e., receptor protein tyrosine phosphatases), and integrin receptors.

[0076] In some embodiments, the multispecific binding protein further includes a heavy chain constant region.

[0077] In some embodiments, the heavy chain constant region includes a substitution at amino acid position 234, according to EU numbering.

[0078] In some embodiments, the amino acid substitution at position 234 is alanine (A).

[0079] In some embodiments, the heavy chain constant region includes a substitution at amino acid position 235, according to EU numbering.

[0080] In some embodiments, the amino acid substitution at position 235 is alanine (A).

[0081] In some embodiments, the heavy chain constant region includes a substitution at the 237th amino acid position according to EU numbering.

[0082] In some embodiments, the substitution at amino acid position 237 is alanine (A).

[0083] In some embodiments, the heavy chain constant region includes one or more substitutions at amino acid positions 234, 235, or 237, according to EU numbering.

[0084] In some embodiments, the substitution at amino acid position 234 is alanine(A), the substitution at amino acid position 235 is alanine(A), and the substitution at amino acid position 237 is alanine(A).

[0085] In some embodiments, the heavy chain constant region includes a heterodimerization mutation that promotes heterodimerization between the first binding site and the second binding site.

[0086] In some embodiments, the heterodimerizing mutation is a knob-in-hole (KIH) mutation.

[0087] In some embodiments, the first heavy chain constant region includes an amino acid substitution that creates a hole at position 366, 368, or 407, and the second heavy chain constant region includes an amino acid substitution that creates a knob at position 366.

[0088] In some embodiments, the first heavy chain constant region includes the amino acid substitution T366S, L368A, or Y407V, and the second heavy chain constant region includes the amino acid substitution T366W.

[0089] In some embodiments, heterodimerizing mutations are charge-stabilizing mutations.

[0090] In some embodiments, the first heavy chain constant region includes the amino acid substitution N297K, and the second heavy chain constant region includes the amino acid substitution N297D.

[0091] In some embodiments, the first heavy chain constant region includes the amino acid substitution T299K, and the second heavy chain constant region includes the amino acid substitution T299D.

[0092] In some embodiments, the heterodimerization mutation includes an engineered disulfide bond.

[0093] In some embodiments, the manipulated disulfide bond is formed by a first heavy chain constant region containing the amino acid substitution Y349C and a second heavy chain constant region containing the amino acid substitution S354C.

[0094] In some embodiments, the manipulated disulfide bond is formed by a C-terminal extension peptide fused to the C-terminus of the first and second heavy chain constant regions, respectively.

[0095] In some embodiments, the C-terminal extension of the first heavy chain constant region includes the amino acid sequence GEC, and the C-terminal extension of the second heavy chain constant region includes the amino acid sequence SCDKT.

[0096] In some embodiments, at least one heavy chain constant region contains one or more mutations that promote an increase in half-life.

[0097] In some embodiments, at least one heavy chain constant region includes one or more substitutions at amino acid positions 252, 254, or 256, according to EU numbering.

[0098] In some embodiments, the substitution at amino acid position 252 is tyrosine (Y), the substitution at amino acid position 254 is threonine (T), and the substitution at amino acid position 256 is glutamic acid (E).

[0099] In some embodiments, at least one heavy chain constant region includes one or more substitutions at amino acid positions 428 or 434, according to EU numbering.

[0100] In some embodiments, at least one heavy chain constant region includes substitutions of M428L and N434S according to EU numbering.

[0101] In another embodiment, this specification provides a pharmaceutical composition comprising a bispecific antibody and a pharmaceutically acceptable carrier, as provided herein.

[0102] In one embodiment, the Specified herein provides an isolated nucleic acid molecule encoding a bispecific antibody as provided herein. In some embodiments, the expression vector comprises the nucleic acid molecule provided herein. In some embodiments, the host cell comprises the expression vector.

[0103] In another embodiment, this specification provides a method for treating a disease or disorder in a subject, comprising administering a bispecific antibody, such as those provided herein, to a subject in need thereof.

[0104] In another embodiment, bispecific antibodies as provided herein are used as pharmaceuticals. In yet another embodiment, bispecific antibodies as provided herein are used for diagnostic purposes.

[0105] In another aspect, the present disclosure provides a method for inducing signaling between a first receptor subunit and a second receptor subunit in a subject, comprising administering a multispecific binding protein provided herein to the subject.

[0106] In some embodiments, the multispecific binding protein can induce signal transduction by inducing proximity between a first receptor subunit and a second receptor subunit.

[0107] In some embodiments, multispecific binding proteins have higher agonist activity compared to multispecific binding proteins lacking at least one modified hinge region.

[0108] In some embodiments, the multispecific binding protein induces agonist activity that is at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the activity of the native ligand to the first and second receptor subunits. [Brief explanation of the drawing]

[0109] [Figure 1] This figure shows a specific exemplary embodiment of the bispecific antibody format described herein. [Figure 2] This is a schematic diagram illustrating the workflow for characterizing the bispecific antibodies disclosed herein. [Figure 3] This graph shows arteriovenous malformations (AVMs) in the retina of HHT mouse models. A shows mice treated with control (no bispecific antibody) compared to mice treated with DGL288 (15 mg / kg / day). Mice treated with DGL288 did not develop detectable AVMs compared to the control. B shows that mice treated with DGL292 at a dose of 1 mg / kg / day did not develop AVMs compared to mice treated with control. C demonstrates that DGL288 administered at a dose of 1 mg / kg / day also did not develop AVMs compared to mice treated with control. [Figure 4] This graph shows arteriovenous malformations (AVMs) in the retina of an HHT mouse model. Mice were treated with DGL292, DGL945, and DGL947 (1 mg / kg / day) compared with a control (no bispecific antibody). Mice treated with DGL292, DGL945, and DGL947 did not develop detectable AVMs compared to the control. [Figure 5] This graph shows the agonism of DGL207, DGL333, and DGL620 in the HEK Blue assay. [Modes for carrying out the invention]

[0110] Before describing this disclosure, it should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as methods and conditions can vary. Furthermore, since the scope of this disclosure is limited only by the appended claims, it should be understood that the terminology used herein is for illustrative purposes only and not intended to limit any particular embodiment.

[0111] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains.

[0112] In carrying out this disclosure, any methods and materials similar to or equivalent to those described herein may be used, but exemplary methods and materials are described below. All published documents referenced herein are incorporated herein by reference to their entirety.

[0113] As used herein, the term “antibody” (singular and plural) includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing the CDR, VH, and / or VL regions of an antibody. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies containing 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, aphibodies, common light chain antibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fvs(sdFv), anti-idiotype (anti-Id) antibodies (e.g., anti-anti-Id antibodies), bivariate variable regions (DVDs), and any of the antigen-binding fragments described above. In certain embodiments, the antibodies described herein refer to a population of polyclonal antibodies. Antibodies can be immunoglobulin molecules 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 their class (e.g., human IgG1, or IgG4), or subclasses. As used herein, the terms "VH" and "VL" refer to the variable domains of the heavy and light chains of an antibody, 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).

[0114] As used herein, the terms “antigen-binding moiety,” “binding domain,” or “binding specificity” refer to a molecule that specifically binds to an antigen, and such binding is understood by those skilled in the art. For example, an antigen-binding moiety that specifically binds to one antigen will typically bind to other molecules with lower affinity, as determined by, for example, immunoassays, BIAcore®, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID), or other assays known in the art. In certain embodiments, an antigen-binding moiety that specifically binds to one antigen will bind to that antigen with a Ka that is at least 2 log-logs (e.g., a coefficient of 10), 2.5 log-logs, 3 log-logs, 4 log-logs, or greater than the Ka when the molecule nonspecifically binds to another antigen.

[0115] As used herein, the term "VHH" refers to the heavy chain variable domain of camelid heavy chain-only antibodies (HCAbs) and their humanized variants, as described in Hamers-Casterman C. et al., Nature (1993) 363:446-8.10.1038 / 363446a0 (which is incorporated herein by reference in its entirety).

[0116] As used herein, the term "VH / VL pair" refers to a combination of VH and VL that, together, form a binding site for an antigen.

[0117] As used herein, the term “heavy chain” as used in relation to antibodies may 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 that gives rise to the IgA, IgD, IgE, IgG, and IgM classes of antibodies, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4.

[0118] As used herein, the term "full-length antibody heavy chain" refers to an antibody heavy chain comprising the VH, CH1 region, hinge region, CH2 domain, and CH3 domain from the N-terminus to the C-terminus.

[0119] As used herein, the term “light chain” as used in reference to an antibody may refer to one of a 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 within the antibody variable region that confer 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 “framework region” or “FR” is known in the art to refer to the portion of the heavy chain 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).

[0120] The precise amino acid sequence boundaries of a particular CDR or FR can be easily determined using one of several well-known schemes, including Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5 thEd. 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," Dev. Comp. Immunol., 2003 January; 27(1):55-77 ("IMGT" numbering scheme), and Honegger A. and Pluckthun Examples include the one described in A., “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J.Mol.Biol., 2001 Jun.8;309(3):657-70, ("Aho" numbering scheme).

[0121] The boundaries of specific CDRs or FRs can differ 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 length, and some antibodies have insertions and deletions given by insertion letters, e.g., "30a". The difference in numbering arises because these two schemes place specific insertions and deletions ("indels") in different positions. The Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.

[0122] As used herein, the term “single-chain variable fragment” (scFv) refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light and heavy chain variable regions are adjacently linked by a short, flexible polypeptide linker, and the scFv is expressible as a single-chain polypeptide, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, the scFv as used herein may have the VL and VH variable regions in any order, for example, with respect to the N-terminus and C-terminus of the polypeptide, and the scFv may contain VL-linker-VH or VH-linker-VL.

[0123] As used herein, the term “human antibody” is intended to include antibodies having variable domains and Fc domains derived from human germline immunoglobulin sequences. The human mAbs of this disclosure may include, for example, amino acid residues in the CDR, particularly in CDR3, that are not encoded by the human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutations in vivo). However, as used herein, the term “human antibody” is not intended to include mAbs in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) is grafted onto a human FR sequence. The term includes antibodies recombinantly produced in or from non-human mammals. The term is not intended to include antibodies isolated from or produced in human subjects.

[0124] As used herein, the term “multispecific antigen-binding molecule” refers to a bispecific, tripspecific, or multispecific antigen-binding molecule and its antigen-binding fragments. A multispecific antigen-binding molecule may be specific to a different epitope in one target polypeptide, or it may contain antigen-binding domains specific to epitopes in multiple target polypeptides. In certain embodiments, the multispecific antigen-binding molecules of this disclosure include at least a first binding specificity to a receptor subunit and at least a second binding specificity to the subunit. A multispecific antigen-binding molecule may be a single polyfunctional polypeptide, or it may be a polymeric complex of two or more polypeptides covalently or noncovalently bonded to each other. The term “multispecific antigen-binding molecule” includes the antibodies of this disclosure that may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or a fragment thereof may be functionally linked to one or more other molecular entities, such as a protein or a fragment thereof, to produce a bispecific or multispecific antigen-binding molecule having a second binding specificity (e.g., by chemical bonding, gene fusion, non-covalent bonding, or otherwise). According to this disclosure, the term “multispecific antigen-binding molecule” also includes bispecific, tripspecific, or multispecific antibodies or their antigen-binding fragments. In certain exemplary embodiments, an antibody of this disclosure is functionally linked to another antibody or its antigen-binding fragment to produce a bispecific antibody having a second binding specificity.

[0125] In exemplary embodiments, the heteromeric antibodies of the Disclosure are bispecific antibodies. A bispecific antibody may be a monoclonal antibody, such as a human antibody or a humanized antibody, having binding specificity to at least two different antigens. In certain embodiments, the bispecific antibody of the Disclosure comprises at least a first binding domain to a receptor subunit and at least a second binding domain to another receptor subunit.

[0126] Methods for producing bispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy / light chain pairs, each with two heavy chains having different specificities (Milstein et al., Nature 305:537 (1983)). Because the heavy and light chains of immunoglobulins are combined randomly, 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 affinity chromatography. More modern techniques for generating bispecific antibodies utilize heterodimerizing domains that favor the desired pairing of the heavy chain of an antibody with primary specificity with the heavy chain of an antibody with secondary specificity.

[0127] An antibody variable domain with desired binding specificity can be fused to an immunoglobulin constant domain sequence. This fusion is typically with an immunoglobulin heavy chain constant domain containing at least a portion of the hinge, CH2, and CH3 regions. The first heavy chain Fc domain, which contains the site required for light chain binding, may have (CH1) present in at least one of the fusions. DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain, is inserted into a separate expression vector and co-transformed into a suitable host organism. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Meth. Enzymol. 121:210 (1986).

[0128] 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 (directly or indirectly) linked to the N-terminus of the CH3 domain. The term "Fc polypeptide" includes antibody heavy chains (e.g., those forming half-antibodies) linked to antibody light chains by disulfide bonds.

[0129] In certain embodiments, the Fc chain begins in a hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc chain includes at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc chain includes at least one of the hinge (e.g., upper, middle, and / or lower hinge regions) domains, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, part, or fragment thereof. In certain embodiments, the Fc domain includes a complete Fc chain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, the Fc chain includes a hinge domain (or part thereof) fused to a CH3 domain (or part thereof). In certain embodiments, the Fc chain includes a CH2 domain (or part thereof) fused to a CH3 domain (or part thereof). In certain embodiments, the Fc chain consists of a CH3 domain or part thereof. In certain embodiments, the Fc chain consists of a hinge domain (or part thereof) and a CH3 domain (or part thereof). In certain embodiments, the Fc chain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In certain embodiments, the Fc chain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In certain embodiments, the Fc chain lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). As used herein, the Fc chain generally refers to a polypeptide comprising all or a portion of the Fc chain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides comprising only, for example, the hinge, CH2, and CH3 domains. The Fc chain may originate from any species and / or any subtype of immunoglobulin, including but not limited to human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. The Fc domain encompasses native Fc and Fc variant molecules. Similar to Fc variants and natural Fc, the term Fc chain includes molecules in monomeric or polymeric form, whether digested from the whole antibody or produced by other means.In some embodiments, the Fc chain includes the carboxyl termini of both heavy chains held together by a disulfide. In certain embodiments, the Fc chain consists of a CH2 domain and a CH3 domain.

[0130] In some embodiments, the Fc polypeptide contains some or all of the wild-type hinge sequence (typically at its N-terminus). In some embodiments, the Fc polypeptide does not contain any functional or wild-type hinge sequence.

[0131] As used herein, the term “CH1 domain” refers to the first constant domain of the antibody heavy chain (e.g., amino acid positions 118–215 of human IgG1, according to the EU index). This term includes the naturally occurring CH1 domain and engineered variants of the naturally occurring CH1 domain (e.g., a CH1 domain that includes one or more amino acid insertions, deletions, substitutions, or modifications compared to the naturally occurring CH1 domain).

[0132] As used herein, the term “CH2 domain” refers to the second constant domain of the antibody heavy chain (e.g., amino acid positions 231–340 of human IgG1, according to the EU index). This term includes the naturally occurring CH2 domain and engineered variants of the naturally occurring CH2 domain (e.g., a CH2 domain that includes one or more amino acid insertions, deletions, substitutions, or modifications compared to the naturally occurring CH2 domain).

[0133] As used herein, the term “CH3 domain” refers to the third constant domain of the antibody heavy chain (e.g., amino acid positions 341–447 of human IgG1, according to the EU index). This term includes both the naturally occurring CH3 domain and engineered variants of the naturally occurring CH3 domain (e.g., a CH3 domain that includes one or more amino acid insertions, deletions, substitutions, or modifications compared to the naturally occurring CH3 domain).

[0134] As used herein, the term “hinge region” refers to the portion of an antibody heavy chain that contains cysteine ​​residues mediating the disulfide bond between two heavy chains in an intact antibody (e.g., the cysteine ​​residues at positions 226 and 229 of human IgG1, according to the EU index). The hinge region may be divided into three peptide regions, namely the upper, middle, and lower hinges. This term includes both the naturally occurring hinge region and manipulated variants of the naturally occurring hinge region (e.g., a hinge region involving one or more amino acid insertions, deletions, substitutions, or modifications compared to the naturally occurring hinge region). An exemplary hinge region of full-length IgG1 contains amino acids 216–230 of human IgG1, according to the EU index. The hinge region may consist of at least two (e.g., five, ten, fifteen, twenty, forty, sixty, or more) amino acids that result in a flexible or semi-flexible bond between adjacent variable regions 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 their chimeric forms).

[0135] In some embodiments, the hinge region may be of a human IgG1 subtype, extending from amino acid 216 to amino acid 230 according to the EU Index numbering system, or from amino acid 226 to amino acid 243 according to the Kabat numbering system. Those skilled in the art may have different understandings of the exact amino acids corresponding to various domains of the IgG molecule. Therefore, the N-terminus or C-terminus of the domains outlined above may be extended or shortened by one, two, three, four, five, six, seven, eight, nine, or even ten amino acids.

[0136] As used herein, the term “upper hinge” typically refers to the last residue of the CH1 domain up to, but not including, the first interchain cysteine. The upper hinge is sometimes defined as the N-terminal sequence from position 216 to 225 according to the Kabat EU numbering system for IgG1 antibodies (Kabat et al., Sequences of Proteins of Immunological Interest, 5 th (Ed. Public Health Service, National Institute of Health, Bethesda, Md., 1991). The term “central hinge” refers to the region extending from the first interchain cysteine ​​to the proline residue adjacent to the carboxyl terminus of the last central hinge cysteine. The central hinge may be the N-terminal sequence from position 226 to 230 according to the Kabat EU numbering system. The term “lower hinge” refers to a highly conserved sequence of 7-8 amino acids. The lower hinge may be defined as the sequence from position 231 to 238 according to the Kabat EU numbering system for IgG1 antibodies. In some embodiments, the antibody according to the present invention substantially comprises upper, central, and lower hinges.

[0137] As used herein, the term “modified hinge region” refers to a hinge region that has been altered in one or more of the hinge characteristics, including but not limited to, flexibility, length, conformation, charge, and hydrophobicity, compared to a wild-type hinge. Modified hinge regions disclosed herein can be generated by methods well known in the art, such as introducing modifications to a wild-type hinge. In some embodiments, the hinge region may be modified by one or more amino acids. Modifications that may be used to generate a modified hinge region include, but are not limited to, insertions, deletions, substitutions, and rearrangements of amino acids. The modifications of the disclosed hinges and modified hinge regions are collectively referred herein to as “hinge modifications of the present invention,” “modified hinges of the present invention,” or simply “hinge modifications” or “modified hinges.” Modified hinge regions disclosed herein can be incorporated into optimal molecules, including, but not limited to, antibodies and fragments thereof. In some embodiments, the hinge region may be cleaved, and only a portion of the complete hinge region may be included. As shown herein, a molecule containing a modified hinge may exhibit altered (e.g., enhanced) agonist activity compared to a molecule having the same amino acid sequence except for the presence of a wild-type hinge, for example, a molecule having the same amino acid sequence except for the presence of a wild-type hinge. In some embodiments, the antibody contains a modified hinge region, and the upper hinge region is up to 7 amino acids long. In some embodiments, the upper hinge region is absent. In some embodiments, the modified hinge is a modified IgG1 linker. In some embodiments, the modified IgG1 hinge is derived from the sequence PLAPDKTHT (SEQ ID NO: 1). In some embodiments, the modified IgG1 hinge contains the sequence PLAP (SEQ ID NO: 2). In some embodiments, the modified IgG1 hinge contains the sequence DKTHT (SEQ ID NO: 5). In some embodiments, the modified hinge is a modified IgG4 hinge. In some embodiments, the modified IgG1 hinge includes the sequence EKSYGPP (sequence number 4). In some embodiments, the modified hinge is a Gly / Ser hinge.In some embodiments, the Gly / Ser hinge includes the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3). In some embodiments, the C-terminal residue of the variable domain adjacent to the upper hinge is cleaved. In some embodiments, at least one residue of the variable domain adjacent to the upper hinge is cleaved. In some embodiments, at least two residues of the variable domain adjacent to the upper hinge are cleaved. In some embodiments, the C-terminal SS amino acid of the heavy chain variable domain is deleted.

[0138] The modified hinge regions of this disclosure may be used as linkers for linking one or more antigen-binding domains of this disclosure. In certain embodiments, a first variable heavy chain domain (VH1) is linked to a second variable heavy chain domain (VH2) via at least one modified hinge region. In certain embodiments, a first variable light chain domain (VL1) is linked to a second variable light chain domain (VL2) via at least one modified hinge region. VH1 and VL1 associate to form a first antigen-binding domain, and VH2 and VL2 associate to form a second antigen-binding domain. In other embodiments, a first scFv is linked to a second scFv via at least one modified hinge region.

[0139] In certain embodiments, the multispecific binding proteins of this disclosure (i.e., multispecific binding proteins having at least a first antigen-binding protein and a second antigen-binding protein) have higher agonist activity compared to multispecific binding proteins lacking at least one modified hinge region. For example, a multispecific binding protein having VH1 linked to VH2 via at least one modified hinge region and / or VL1 linked to VL2 via at least one modified hinge region may have higher agonist activity of a target receptor pair (e.g., VH1 / VL1 binds to the first receptor subunit and VH2 / VL2 binds to the second receptor subunit) compared to the same multispecific binding protein lacking at least one modified hinge region.

[0140] As used herein, the term "EU Index" refers to 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, 5 th This refers to the EU numbering rules for antibody Fc domains, as described in edition, 1991, each of which is incorporated herein by reference in its entirety. All numbering of amino acid positions of Fc polypeptides or fragments used herein follows the EU index.

[0141] In some embodiments, the term "linker" refers to 1 to 100 contiguous amino acid residues. Typically, a linker provides flexibility and spatial separation between two amino acids or between two polypeptide domains. Depending on the format of the molecule, the linker can be inserted between the VH, VL, CH, and / or CL domains to provide sufficient flexibility and mobility to the domains of the light and heavy chains. Linkers are typically inserted at the amino acid sequence level at the transition between variable domains, between the variable domain and the knockout domain, or between the variable domain and the constant domain, respectively. Since the approximate sizes of immunoglobulin domains are well understood, the transition regions between domains can be identified. The exact position of the domain transition region can be identified by the positioning of a peptide extension that does not form secondary structure elements such as beta sheets or alpha helices, as demonstrated by experimental data or as determined by techniques of modeling or secondary structure prediction.

[0142] As used herein, the terms "specifically binds", "specific binding", "binding specificity", or "specifically recognized" refer to an antigen-binding protein or an antigen-binding fragment thereof that exhibits a detectable affinity for an antigen (e.g., an IL-18R antigen) and does not exhibit significant cross-reactivity for different target 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 an antigen-binding fragment thereof and the epitope to which it binds. In certain exemplary embodiments, affinity is measured, for example, by surface plasmon resonance (SPR) on a Biacore instrument. As will be readily understood by those skilled in the art, the affinity of an antigen-binding protein can be reported in molar concentration units (M) as a dissociation constant (KD). The antigen-binding proteins or antigen-binding fragments thereof of the present disclosure have a dissociation constant of about 10 -5 M to about 10 -12 M (i.e., in the range of low micromolar to picomolar), about 10 -7 M to 10 -11 M, about 10 -8 M to about 10 -10 M, about 10-9 It has a KD value within the range of M. In a particular embodiment, the antigen-binding protein or its antigen-binding fragment is 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 It has a binding affinity of M. In a particular embodiment, the antigen-binding protein or its antigen-binding fragment is about 10 -7 M~about 10 -9 It has binding affinity in the M (nanomolar range).

[0143] Specific binding may be determined by any means recognized in the art for determining such binding. In some embodiments, specific binding is determined by a competitive binding assay (e.g., ELISA) or a Biacore assay. In certain embodiments, the assay is performed at approximately 20°C, 25°C, 30°C, or 37°C.

[0144] As used herein, “administer” or “dosage” means the act of physically delivering an extracorporeal substance (e.g., an isolated bound polypeptide provided herein) to a patient by injection or other means, for example, by pulmonary delivery (e.g., inhalation), mucosal delivery (e.g., intranasal delivery), intradermal delivery, intravenous delivery, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When managing or treating a disease or its symptoms, administration of the substance is generally performed after the onset of the disease or its symptoms. When preventing a disease or its symptoms, administration of the substance is generally performed before the onset of the disease or its symptoms and may be continued for a long period to delay the appearance or reduce the severity of disease-related symptoms.

[0145] As used herein, the term “composition” is intended to include not only products that optionally contain a given component (e.g., an isolated bound polypeptide provided herein) in a predetermined amount, but also any products that result directly or indirectly from the combination of a given component in a predetermined amount, either optionally.

[0146] "Effective amount" means the amount of active pharmaceutical agent (e.g., the isolated bound polypeptide of this disclosure) that is sufficient to produce the desired physiological outcome in an individual requiring the drug. The effective amount may vary between individuals depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the method of preparing the composition, the assessment of the individual's medical condition, and other relevant factors.

[0147] As used herein, the terms “subject” and “patient” are used synonymously. As used herein, the subject may be an animal other than a primate (e.g., cattle, pigs, horses, cats, dogs, rats, mice, etc.) or a mammal such as a primate (e.g., monkeys and humans). In certain embodiments, as used herein, the term “subject” refers to a vertebrate such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, feral animals, livestock, sports animals, and pets.

[0148] As used herein, the term “therapy” refers to any protocol, method and / or agent that may be used in the prevention, management, treatment and / or improvement of a disease or its associated symptoms. In some embodiments, the term “therapy” refers to any protocol, method and / or agent that may be used in the modulation of the immune response to an infection or its associated symptoms in a subject. In some embodiments, the term “therapy” (singular and plural) refers to biological therapies, supportive therapies and / or other therapies known to those skilled in the art, such as healthcare workers, that are useful in the prevention, management, treatment and / or improvement of a disease or its associated symptoms. In other embodiments, the term “therapy” (singular and plural) refers to biological therapies, supportive therapies and / or other therapies known to those skilled in the art, such as healthcare workers, that are useful in the modulation of the immune response to an infection or its associated symptoms in a subject.

[0149] As used herein, the terms “to treat,” “treatment,” and “to treat” mean a reduction or improvement in 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 isolated conjugated polypeptides provided herein). The term “to treat” may also mean, as used herein, an alteration of the disease process of the subject being treated. The therapeutic effects of treatment include, but are not limited to, prevention of the onset or recurrence of the disease, relief of symptoms, reduction of direct or indirect pathological consequences of the disease, slowing of the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis.

[0150] The terms "about" or "approximately" mean within about 20% of a given value or range, for example, within about 10%, within about 5%, or within about 1%.

[0151] Joint domain One component of the bispecific antibody of this disclosure is one or more antigen-binding domains, or binding specificity that binds to a first cell surface target and a second cell surface target. In certain embodiments, the first cell surface target is a first receptor subunit, and the second cell surface target is a second receptor subunit.

[0152] The bispecific antibodies disclosed herein may utilize any type of binding moiety that specifically binds to a particular receptor subunit. In certain embodiments, the binding moiety includes an antibody-variable domain. Exemplary binding moieties containing an antibody-variable domain include, but are not limited to, VH, VL, VHH, VH / VL pairs, scFv, diabody, or Fab. Other preferred binding moiety formats include lipocalin (see, e.g., Gebauer M. et al., 2012, Method Enzymol. 503:157-188, which is incorporated herein by reference in its entirety), adonectin (see, e.g., Lipovsek D., 2011, Protein Eng. Des. Sel. 24:3-9, which is incorporated herein by reference in its entirety), avimer (see, e.g., Silverman J, et al., 2005, Nat. Biotechnol. 23:1556-1561, which is incorporated herein by reference in its entirety), finomer (see, e.g., Schlatter D, et al., 2012, mAbs 4:497-508, which is incorporated herein by reference in its entirety), and kunitz domain (see, e.g., Hosse RJet). See al., 2006, Protein Sci. 15:14-27 (this document is incorporated herein by reference in its entirety), knottin (see, for example, Kintzing JR et al., 2016, Curr. Opin. Chem. Biol. 34:143-150 (this document is incorporated herein by reference in its entirety)), afibody (see, for example, Feldwisch J. et al., 2010 J. Mol. Biol. 398:232-247 (this document is incorporated herein by reference in its entirety)), and DARPin (see, for example, Pluckthun A., 2015, Annu. Rev. Pharmacol. Toxicol. 55:489-511 (this document is incorporated herein by reference in its entirety)).

[0153] In certain embodiments, the binding domain comprises variable regions of the heavy and / or light chains of a conventional antibody or its antigen-binding fragment (e.g., Fab or scFv). The term “conventional antibody” is used herein to refer to a heterotetrameric antibody containing heavy and light chains of immunoglobulin arranged according to a “Y” configuration. Such conventional antibodies may originate 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, the conventional antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), where the VH domain and / or VL domain, or one or more complementarity-determining regions (CDRs) thereof, originate 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 CDRs involved in antigen binding.

[0154] In other embodiments, a specific receptor subunit-binding subunit comprises at least a CDR or VHH domain of a VHH antibody or nanobody. The VHH antibody, a heavy-chain antibody derived from camelids, consists of two heavy chains and lacks a light chain (Hamers-Casterman, et al. Nature. 1993;363;446-8). Each heavy chain of the VHH antibody has a variable domain at its N-terminus, which is referred to in the art as the "VHH" domain to distinguish it from the variable domain of the heavy chain of conventional antibodies, i.e., the VH domain. Similar to conventional antibodies, the VHH domain of this molecule contains HCDR1, HCDR2, and HCDR3 regions that confer antigen-binding specificity, and therefore fragments such as the VHH antibody or isolated VHH domain are suitable as components of the multispecific binding protein of this disclosure.

[0155] Multiple specific binding proteins In certain embodiments, the first and second binding domains disclosed herein may be paired together or operably ligated to generate a multispecific binding protein capable of crosslinking the first and second subunits of a particular receptor (e.g., the human IL-18 receptor). In some embodiments, the first specific binding domain (e.g., VHH or scFv) is operably ligated (directly or indirectly) to the N-terminus and / or C-terminus of the first Fc domain or polypeptide, and the second specific binding domain is operably ligated to the N-terminus and / or C-terminus of the second Fc domain or polypeptide, so as to facilitate heterodimerization of the first and second specific binding domains.

[0156] As used herein, the terms “dual variable domain” or “DVD” refer to a binding protein containing two or more antigen-binding sites, which may be a tetravalent or polyvalent binding protein. A DVD may be monospecific, i.e., capable of binding to one antigen, or multispecific, i.e., capable of binding to two or more antigens. A DVD-binding protein containing two heavy-chain DVD polypeptides and two light-chain DVD polypeptides is called “DVD immunoglobulin” or “DVD-Ig.” Each half of DVD-Ig contains a heavy-chain DVD polypeptide and a light-chain DVD polypeptide, as well as two or more antigen-binding sites. Each binding site contains a heavy-chain variable domain and a light-chain variable domain, with a total of six CDRs involved in antigen binding for each antigen-binding site.

[0157] Descriptions of the design, expression, and characterization of the DVD-Ig molecule are provided in PCT Publication No. WO2007 / 024715; U.S. Patent No. 7,612,181; and Wu et al., Nature Biotechnol., 25:1290-1297 (2007). A preferred example of such a DVD-Ig molecule includes a heavy chain comprising the structural formula VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker provided it is not CH1, X2 is an Fc region, and n is 0 or 1, but preferably 1; and a light chain comprising the structural formula VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker provided it is not CH1, X2 does not contain an Fc region, and n is 0 or 1, but preferably 1. Such DVD-Ig may comprise two such heavy chains and two such light chains, each chain comprising variable domains linked in series without a constant region interposing between the variable domains, the heavy chain and light chain may associate to form a series-type functional antigen-binding site, and the heavy chain and light chain pair may associate with another heavy chain and light chain pair to form a tetramer-binding protein having four functional antigen-binding sites. In another example, the DVD-Ig molecule may comprise a heavy chain and a light chain, each containing three variable domains (VD1, VD2, VD3) linked in series without a constant region interposing between the variable domains, the heavy chain and light chain pair may associate to form three antigen-binding sites, and the heavy chain and light chain pair may associate with another heavy chain and light chain pair to form a tetramer-binding protein having six antigen-binding sites.

[0158] In one embodiment, the Disclosure provides a binding protein comprising first and second polypeptide chains, wherein the first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first heavy chain variable domain, VD2 is a second heavy chain variable domain, C is a heavy chain constant domain, X1 is a linker subject to not being CH1, X2 is an Fc region, and n is independently 0 or 1; and the second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, where VD1 is a first light chain variable domain, VD2 is a second light chain variable domain, C is a light chain constant domain, X1 is a linker subject to not being CH1, X2 does not contain an Fc region, and n is independently 0 or 1.

[0159] In relation to the construction of DVD-Ig or other binding protein molecules, the term "linker" is used to indicate a polypeptide ("linker polypeptide") containing a single amino acid or two or more amino acid residues linked by a peptide bond, and is also used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), Poljak, RJ, Structure, 2:1121-1123 (1994)). Generally, flexible linkers composed of small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids can be used. Exemplary flexible linkers include, but are not limited to, GGGGSG(sequence ##), GGSGG(sequence ##), GGGGSGGGGS(sequence ##), GGSGGGGSG(sequence ##), GGSGGGGSGGGGS(sequence ##), GGGGSGGGGSGGGG(sequence ##), GGGGSGGGGSGGGGS(sequence ##), and RADAAAAGGGGSGGGGSGGGGSGGGGS(sequence ##).

[0160] Alternatively, a rigid linker may be used to link one or more antigen-binding proteins. This rigid linker allows for the maintenance of a certain distance between linked antigen-binding proteins, thereby potentially enhancing the activity of individual proteins. The rigid linker may use one or more proline amino acids to impart rigidity. Examples of flexible linkers include ASTKGP (SEQ ID NO: ##), ASTKGPSVFPLAP (SEQ ID NO: ##), TVAAP (SEQ ID NO: ##), RTVAAP (SEQ ID NO: ##), TVAAPSVFIFPP (SEQ ID NO: ##), RTVAAPSVFIFPP (SEQ ID NO: ##), AKTTPKLEEGEFSEAR (SEQ ID NO: ##), AKTTPKLEEGEFSEARV (SEQ ID NO: ##), AKTTPKLGG (SEQ ID NO: ##), SAKTTPKLGG (SEQ ID NO: ##), SAKTTP (SEQ ID NO: ##), RADAAP (SEQ ID NO: ##), RADAAPTVS (SEQ ID NO: ##), and RADAAAAGGPGS. Examples include, but are not limited to, (sequence number ##), SAKTTPKLEEGEFSEARV(sequence number ##), ADAAP(sequence number ##), ADAAPTVSIFPP(sequence number ##), QPKAAP(sequence number ##), QPKAAPSVTLFPP(sequence number ##), AKTTPP(sequence number ##), AKTTPPSVTPLAP(sequence number ##), AKTTAP(sequence number ##), AKTTAPSVYPLAP(sequence number ##), GENKVEYAPALMALS(sequence number ##), GPAKELTPLKEAKVS(sequence number ##), and GHEAAAVMQVQYPAS(sequence number ##).

[0161] In certain embodiments, the linker includes a modified hinge region as described herein.

[0162] In certain embodiments, the linker includes or consists of PLAP, PAPNLLGGP, PLAPDKTHT, EKSYGPP, or DKTHT.

[0163] In certain embodiments, the multispecificity binding protein comprises a first and a second polypeptide chain, where, The first polypeptide chain comprises VH1-(HX1)n-VH2-C-(HX2)n, VH1 is the first heavy chain variable domain, VH2 is the second heavy chain variable domain, C is the heavy chain constant domain, HX1 is the linker, HX2 is the Fc region, n is independently 0 or 1, and The second polypeptide chain comprises VL1-(LX1)n-VL2-C-(LX2)n, VL1 is the first light chain variable domain, VL2 is the second light chain variable domain, C is the light chain constant domain, LX1 is the linker, LX2 does not contain the Fc region, and n is independently 0 or 1.

[0164] In certain embodiments, the linker HX1 comprises the amino acid sequence PLAP or PAPNLLGGP.

[0165] In certain embodiments, the linker LX1 comprises the amino acid sequence PLAP or PAPNLLGGP.

[0166] In certain embodiments, linker HX1 comprises the amino acid sequence of PLAP, and linker LX1 comprises the amino acid sequence of PLAP or PAPNLLGGP.

[0167] In certain embodiments, the multispecific binding protein comprises two polypeptide chains: VH1-(HX1)n-VH2-C-(HX2)n and two polypeptide chains: VL1-(LX1)n-VL2-C-(LX2)n.

[0168] In a particular embodiment, if (HX1)n, then n is 1, and if (HX2)n, then n is 1.

[0169] In a particular embodiment, if (LX1)n, n is 1, and if (LX2)n, n is 0.

[0170] In certain embodiments, the multispecificity binding protein comprises a first and a second polypeptide chain, where, The first polypeptide chain comprises VH1-(HX1)n-VH2-C-Fc, VH1 is the first heavy chain variable domain, VH2 is the second heavy chain variable domain, C is the heavy chain constant domain, HX1 is the linker, Fc is the Fc region, n is independently 0 or 1, and The second polypeptide chain comprises VL1-(LX1)n-VL2-C, VL1 is the first light chain variable domain, VL2 is the second light chain variable domain, C is the light chain constant domain, LX1 is the linker, and n is independently 0 or 1.

[0171] Non-DVD-IG format In another aspect of this disclosure, the bispecific antibody is defined as having a length from the N-terminus to the C-terminus. ai) A first polypeptide chain comprising a first antigen-binding domain, a first linker (e.g., a modified hinge region), and a first constant region, and bi) A second polypeptide chain comprising a second antigen-binding domain, a second linker (e.g., a modified hinge region), and a second constant region, aii) A first polypeptide chain comprising a second antigen-binding domain, a first antigen-binding domain, a first linker (e.g., a modified hinge region), and a first constant region, and bii) A second linker (e.g., a modified hinge region) or the absence of a linker, and a second polypeptide chain including a second constant region, aiii) a first linker (e.g., a modified hinge region) or absence of a linker, and a first polypeptide chain including a first constant region, biii) A second polypeptide chain comprising a second antigen-binding domain, a first antigen-binding domain, a second linker (e.g., a modified hinge region), and a second constant region, or aiv) A first polypeptide chain comprising a first antigen-binding domain, an optional first linker (e.g., a modified hinge region), a second antigen-binding domain, an optional second linker (e.g., a modified hinge region), and a first constant region, and biv) comprising a second polypeptide chain including a third antigen-binding domain, an optional third linker (e.g., a modified hinge region), a fourth antigen-binding domain, an optional fourth linker (e.g., a modified hinge region), and a second constant region.

[0172] In certain embodiments, the first antigen-binding domain includes scFv, VHH, Fab, F(ab')2, or a single-domain antibody.

[0173] In certain embodiments, the second antigen-binding domain includes scFv, VHH, Fab, F(ab')2, or a single-domain antibody.

[0174] In certain embodiments, the third antigen-binding domain includes scFv, VHH, Fab, F(ab')2, or a single-domain antibody.

[0175] In certain embodiments, the fourth antigen-binding domain includes scFv, VHH, Fab, F(ab')2, or a single-domain antibody.

[0176] In certain embodiments, one or more of the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, and the fourth antigen-binding domain include scFv, VHH, Fab, F(ab')2, or a single-domain antibody.

[0177] In certain embodiments, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, and the fourth antigen-binding domain each comprise an scFv. In certain exemplary embodiments, the multispecific binding proteins of this disclosure exhibit agonism to any signaling pathway, i.e., they do not exhibit antagonism to any pathway. In some embodiments, agonism can be measured using a specific receptor efficacy assay (e.g., HEK-Blue® Efficacy Assay (InVivogen)). The efficacy assay (e.g., HEK-Blue) comprises a cell line expressing the target receptor of interest (e.g., HEK293). Binding of the bispecific antibody to the receptor triggers a signaling cascade that results in the expression of a quantifiable reporter gene. For example, HEK-Blue® IL-18 cells are generated by stably transfecting HEK293 cells with genes encoding IL-18Rα and IL-18Rβ to measure receptor binding and subsequent signaling.

[0178] As used herein, the term “inducing proximity” between a first subunit and a second subunit of a given receptor refers to binding the first subunit and the second subunit in such a way that a subsequent signaling cascade is stimulated. In certain embodiments, the proximity induced by the multispecific binding proteins of this disclosure is the same as or similar to the proximity induced when a native ligand attracts the first subunit and the second subunit of a target receptor.

[0179] The bispecific antibodies of this disclosure may utilize at least one modified hinge region. The modified hinge region functions as a linker for connecting different domains of the bispecific antibody. In certain embodiments, the modified hinge region links a first variable heavy chain domain (VH1) to a second variable heavy chain domain (VH2), and / or links the first variable light chain domain (VL1) to a second variable light chain domain (VL2). In another embodiment, the modified hinge region links a first scFv to a second scFv. In certain embodiments, the modified hinge region includes i) an upper hinge region that is up to 7 amino acids long or absent, and ii) a lower hinge region. In certain embodiments, the modified hinge region includes or consists of the amino acid sequence PLAP or PAPNLLGGP.

[0180] The bispecific antibodies (e.g., multispecific binding proteins) of this disclosure have higher agonist activity compared to bispecific antibodies lacking at least one modified hinge region. Agonist activity can be measured using specific receptor efficacy assays (e.g., Pathhunter U2OS dimerization assay (DiscoverX)) or efficacy assays (e.g., Pathhunter) that include cell lines expressing the target receptor of interest (e.g., U2OS). Binding of the bispecific antibody to the receptor triggers a signaling cascade that results in the expression of a quantifiable reporter gene.

[0181] In some embodiments, the multispecific binding protein induces agonist activity that is at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, or at least about 80% of the activity of the native ligand to the first and second receptor subunits.

[0182] The bispecific antibodies (e.g., multispecific binding proteins) of this disclosure induce agonist activity that is at least about 35% of the activity of the native ligand to the first and second receptor subunits. In certain embodiments, the bispecific antibodies (e.g., multispecific binding proteins) of this disclosure induce at least about 40% of the activity of the native ligand to the first and second receptor subunits. In certain embodiments, the bispecific antibodies (e.g., multispecific binding proteins) of this disclosure induce at least about 40% of the activity of the native ligand to the first and second receptor subunits. In certain embodiments, the bispecific antibodies (e.g., multispecific binding proteins) of this disclosure induce at least about 45% of the activity of the native ligand to the first and second receptor subunits. In certain embodiments, the bispecific antibodies (e.g., multispecific binding proteins) of this disclosure induce at least about 50% of the activity of the native ligand to the first and second receptor subunits. In certain embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 55% of the activity of BMP9. In certain embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 60% of the activity of the native ligand to the first and second receptor subunits. In certain embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 65% of the activity of the native ligand to the first and second receptor subunits. In certain embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 70% of the activity of the native ligand to the first and second receptor subunits. In certain embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 75% of the activity of the native ligand to the first and second receptor subunits.In certain embodiments, the bispecific antibodies (e.g., multispecific binding proteins) of this disclosure induce at least about 80% of the activity of the intrinsic ligand to a first receptor subunit and a second receptor subunit.

[0183] In certain embodiments, the activity of a native ligand is determined by measuring the activation of a protein activated by the target receptor. Activation may be measured in cells incubated with a multispecific binding protein and / or in cells incubated with the native ligand. Activation of the protein activated by the target receptor may be detected using an enzyme-linked immunosorbent assay (ELISA).

[0184] In certain embodiments, the activity of a native ligand is determined by measuring changes in gene expression of a gene known to be expressed upon activation of the target receptor. Detection of gene expression of a gene known to be expressed upon activation of the target receptor can be achieved using standard molecular biology techniques and PCR. Briefly, a first population of cells is incubated with the bispecific antibody of this disclosure, and a second population of cells is incubated with the native ligand. Following a certain incubation time, mRNA is isolated from the cells, cDNA is generated, and PCR is performed to detect the gene level compared to a control gene such as GAPDH. The gene level in the first population of cells is then compared to the gene level in the second population of cells.

[0185] FC Domain The heavy chain Fc domains used in the bispecific antibodies of this disclosure generally include a CH2 domain and a CH3 domain, with the C-terminus of the CH2 domain being (directly or indirectly) ligated to the N-terminus of the CH3 domain. Any naturally occurring or variant CH2 and / or CH3 domains may be used. For example, in certain embodiments, the CH2 and / or CH3 domains are naturally occurring CH2 or CH3 domains 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 includes CH2 and CH3 domain-containing portions from a single antibody heavy chain. In certain embodiments, the CH2 and / or CH3 domains are variants of the 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 compared to the naturally occurring CH2 or CH3 domain. In certain embodiments, the CH2 and / or CH3 domains are chimeras of one or more CH2 or CH3 domains. In certain embodiments, the CH2 domain comprises amino acids 231-340 of the 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 the naturally occurring hinge region (e.g., human IgG1) according to the EU index.

[0186] In certain embodiments, the Fc polypeptide further comprises a hinge region, the C-terminus of which is (directly or indirectly) linked to the N-terminus of the CH2 domain. For example, in certain embodiments, the hinge region is a spontaneously 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 domain and / or the 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 spontaneously 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 acid positions 226-229 of a spontaneously occurring hinge region (e.g., human IgG1) according to the EU index. In certain embodiments, the hinge region includes amino acids 216-230 of the naturally occurring hinge region (e.g., human IgG1) according to the EU index. In certain embodiments, the hinge region includes amino acids 216-230 of the 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 containing serine (S) at amino acid position 228 according to the EU index.

[0187] In certain embodiments, the Fc polypeptide further comprises a CH1 domain, the C-terminus of which is (directly or indirectly) linked to the N-terminus of the hinge region. For example, in certain embodiments, the CH1 domain is a spontaneously 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, the CH2 domain, and / or the 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 spontaneously 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 acid positions 118-215 of a spontaneously occurring hinge region (e.g., human IgG1) according to the EU index.

[0188] 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 between the antibody light chain and heavy chain. In certain embodiments, the antibody heavy chain lacks a portion of the hinge region.

[0189] Heterodimizing motif In certain exemplary embodiments, further manipulation of the first and second Fc domains enhances heterodimerization of the first and second specificity-binding domains, minimizing the effects of improper chain pairing (i.e., pairing of the IL-18Rα-binding domain or the same IL-18Rβ domain).

[0190] The production of desired multispecific antibodies can be improved by using any known technique in the art to address the problem of improper chain pairing. For example, US2010 / 0254989A1 describes the construction of a bispecific cMet-ErbB1 antibody in which the VH and VL of individual antibodies 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. Several such techniques are outlined in Klein et al. (mAbs(2012)4:6,1-11), the contents of which are incorporated herein by reference in their entirety.

[0191] In certain embodiments, the first and second specificity binding specificities of a multispecific antibody are heterodimerized via knob-into-hole (KiH) pairing of the Fc domain. This dimerization technique utilizes manipulated “protrusions” or “knobs” and “cavitations” or “holes” at the interface of the CH3 domain. If a knob or hole of suitable positioning and size exists at the interface of either the first or second CH3 domain, only the corresponding hole or knob at the adjacent interface needs to be manipulated, thereby promoting and enhancing the pairing of Fc domains at the CH3 / CH3 domain interface. The IgG Fc domain fused to VHH has a knob, and the IgG Fc domain of a conventional antibody has a hole designed to accommodate a 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” at the CH3 portion of the second Fc domain, forming a “knob” that is received by the “hole”. A "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. Patents 5,821,333, 5,731,168, and 8,216,805, Ridgway et al. Protein Engineering (1996) 9:617-621), and Carter PJImmunol. Methods (2001) 248:7-15, which are incorporated herein by reference.

[0192] Exemplary amino acid residues that can function as knobs include arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). Amino acid residues present in the CH3 domain may be exchanged or substituted with amino acid residues of the knob. Preferred amino acids for substitution may include any amino acid with a small side chain, such as alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), or valine (V).

[0193] 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 for the hole amino acid residue. Preferred amino acids for substitution may include any amino acid with a large side chain, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W).

[0194] 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.

[0195] In certain embodiments, the two Fc domains of an antigen-binding construct are heterodimerized by Fab-arm exchange (FAE). Human IgG1 carrying the P228S hinge mutation may contain the F405L or K409R CH3 domain mutation. Mixing the two antibodies with a reducing agent yields the FAE. This technique is described in U.S. Patent No. 9,212,230 and Labrijn AFPNAS (2013) 110(13):5145-5150, which are incorporated herein by reference.

[0196] In other embodiments, the two Fc domains of the antigen-binding construct are heterodimerized by an electrostatic steering effect. This dimerization technique utilizes electrostatic steering to promote and enhance the pairing of Fc domains at the CH3 / CH3 domain interface. The charge complementarity between the two CH3 domains is modified so that heterodimerization (pairing of opposite charges) is preferred over homodimerization (pairing of same charges). In this method, homodimerization is prevented by electrostatic repulsion. Specific exemplary amino acid residue substitutions that result in the electrostatic steering effect include K409D, K392D, and / or K370D in the first CH3 domain, and D399K, E356K, and / or E357K in the second CH3 domain. This technology is described in U.S. Patent Publication No. 2014 / 0154254A1 and Gunasekaran K. JBC (2010) 285(25):19637–19646, which are incorporated herein by reference.

[0197] In other embodiments, charge complementarity is formed by a first Fc domain containing mutations N297K and / or T299K, and a second Fc domain containing mutations N297D and / or T299D.

[0198] In one embodiment of the present invention, the two Fc domains of the antigen-binding construct are heterodimerized by hydrophobic interaction effects. This dimerization technique utilizes hydrophobic interactions instead of electrostatic interactions to promote and enhance the pairing of 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 technology is described in U.S. Patent Publication No. 2015 / 0307628A1.

[0199] In one embodiment of the present invention, heterodimerization can be mediated by the use of a leucine zipper fusion. Heterodimerization is forced by a leucine zipper domain fused to the C-terminus of each CH3 domain in the antibody chain. This technique is described in Wranik B. JBC (2012) 287(52):43331-43339.

[0200] In one embodiment of the present invention, heterodimerization can be mediated by the use of a Strand Exchange Engineered Domain (SEED) body. Heterodimerization is forced by CH3 domains derived from IgG and IgA formats. This technique is described in Muda M. PEDS (2011) 24(5):447-454.

[0201] In other embodiments, the heterodimerizing motif may include a non-native disulfide bond formed by the engineered cysteine ​​residue. In certain embodiments, the first set of disulfides may include a Y349C mutation in the first Fc domain and an 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 the engineered cysteine ​​residue to the C-terminus of each of the two Fc domains. In certain embodiments, the first Fc domain may include a substitution of the carboxyl terminus with "GEC" as "PGK", and the second Fc domain may include a substitution of the carboxyl terminal amino acid "PGK" with "KSCDKT".

[0202] Another approach involves using the CrossMab principle (outlined in Klein et al.), which includes domain exchange between the heavy and light chains, to facilitate the formation of proper pairings. Yet another approach involves manipulating the interface between the VH-VL domain pair or CH1-CL domain pair of the heavy and light chains to increase the affinity between the heavy chain and its homologous light chain (Lewis et al. Nature Biotechnology (2014) 32:191-198).

[0203] An alternative to producing multispecific antibody preparations with appropriate antigen specificity is the development of methods for enriching antibodies with appropriate heavy-light chain pairings. For example, Spiess et al. (Nature Biotechnology (2013) 31:753-758) reported a method for producing MET-EGFR bispecific antibodies from co-cultures of bacteria expressing two different half-antibodies.

[0204] Methods have also been reported to alter the binding affinity to affinity factors, such as protein A, by mutating at least one Fc domain in the heavy chain of a bispecific antibody. This makes it possible to isolate properly paired heavy chain heterodimers based on purification techniques that utilize the difference in binding between the two heavy chains to affinity factors (see US2010 / 0331527, WO2013 / 136186).

[0205] International Patent Application PCT / EP2012 / 071866 (WO2013 / 064701) addresses the problem of improper chain pairing using a method for isolating multispecific antibodies based on the use of anti-idiotype conjugates, particularly anti-idiotype antibodies. The anti-idiotype conjugate is used in a two-step selection method in which a first agent is used to capture an antibody having a VH-VL domain pairing specific to a first antigen, and then a second agent is used to capture an antibody having a second VH-VL domain pairing specific to a second antigen.

[0206] In yet another embodiment, the multispecific antibody employs a first binding specificity having a conventional Fab binding region and a second binding specificity including a single-domain antibody (VHH) binding region. The heterodimerization method used forces binding of the Fab heavy chain region to only the complete heavy chain of the VHH. Since the VHH chain does not associate with the light chain, the light chain region of the Fab portion associates only with its corresponding heavy chain.

[0207] 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, by pairing with a first heavy chain of an antibody that binds to a first antigen, can form a binding site that specifically binds to the first antigen, and by pairing with a second heavy chain of an antibody that binds to a second antigen, can form a binding site that specifically binds to the second antigen. The common light chain is a polypeptide comprising an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL) in the direction from the N-terminus to the C-terminus, and is also abbreviated herein as “VL-CL”. Multispecific binding proteins having a common light chain require heterodimerization of different heavy chains. In certain embodiments, the heterodimerization method described above may be used with a common light chain. In certain exemplary embodiments, the heterodimerization motif may comprise a non-natural disulfide bond formed by an engineered cysteine ​​residue. It has been shown that adding a disulfide bond between both the heavy and light chains of the antibody improves stability. Furthermore, disulfide bonds have also been used as a solution to improve light chain pairing in bispecific antibodies (Geddie M. Let al, mABs(2022)14(1)).

[0208] Unless otherwise stated, all antibody Fc domain numbering used herein corresponds to the EU numbering scheme described in Edelman et al. (Proc.Natl.Acad.Sci.63(1):78-85.1969).

[0209] Further methods for heterodimerization of heavy and / or light chains, and for the production and purification of asymmetric antibodies, are known in the art. See, for example, Klein C. mAbs (2012) 4(6):653-663 and U.S. Patent No. 9,499,634, which are incorporated herein by reference.

[0210] Effector function variation As described above, the multispecific binding proteins of this disclosure may be provided in various isotypes and using different Fc domains. The Fc domain of the multispecific binding 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 phagocytic activity (ADCP). These "cellular effector functions," unlike effector T cell functions, involve recruiting cells possessing Fc receptors to the site of target cells, resulting in the killing of antibody-bound cells.

[0211] The antibodies according to the present invention may exhibit reduced effector function. In certain embodiments, one or more mutations reduce one or more of the following: antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), or complement-dependent cell-mediated cytotoxicity (CDC). In certain embodiments, the antibodies according to the present invention may lack ADCC, ADCP, and / or CDC activity. In any case, the antibodies according to the present invention may contain or optionally lack an Fc region that binds to one or more types of Fc receptors. The use of different antibody formats, as well as the presence or absence of FcR binding and cell effector function, allows for the modification of the antibodies to suit use for specific therapeutic purposes as described elsewhere in this specification.

[0212] 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 Fc domain. The antibody may have a heavy-chain Fc domain that does not bind to the Fcγ receptor, for example, this Fc domain may contain the L235E mutation. Another optional mutation in the heavy chain Fc domain is S228P, which increases stability. The heavy chain Fc domain may be IgG4 containing both the L235E and S228P mutations. This “IgG4-PE” heavy chain Fc domain is effector null. The invalidated IgG1 heavy chain Fc domain is also effector null. The invalidated IgG1 heavy chain Fc domain may contain alanine at positions 234, 235, and / or 237 (EU index numbering), and may be an IgG1 sequence containing, for example, the L234A, L235A, and / or G237A mutations ("LALAGA").

[0213] Human IgG1Fc domains containing specific mutations or altered glycosylations at residue Asn297 (e.g., N297Q, N297D, and N297K, EU index numbering) have been shown to reduce binding to the Fc receptor.

[0214] In other embodiments, it may be desirable to enhance the binding of the Fc domain of a multispecific antibody to human Fc gamma receptor IIIA (FcγRIIIA) compared to that of the Fc domain of the corresponding naturally occurring antibody. In certain embodiments, the Fc domain may be manipulated for the enhancement of ADCC and / or CDC and / or ADCP. The potency of the Fc-mediated effect may be enhanced by manipulating the Fc domain by various established techniques. Such methods increase affinity to specific Fc receptors and thus create a potentially diverse profile of enhanced activation. This may be achieved by modification of one or more amino acid residues. Examples of mutations are one or more residues (or equivalent positions in other IgG isotypes) selected from 239, 332, and 330 for the human IgG1Fc domain. Thus, an antibody may contain a human IgG1Fc domain with one or more mutations independently selected from S239D, I332E, and A330L (EU index numbering).

[0215] Increased affinity for Fc receptors can also be achieved by altering the innate glycosylation profile of the Fc domain, for example, by generating low-fucosylated or defucosylated variants. Non-fucosylated antibodies encapsulate a tri-mannosyl core structure of a complex N-glycan of Fc that does not contain fucose residues. These glycoengineered antibodies, lacking core fucose residues derived 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, antibodies may contain human IgG heavy chain Fc domains that are variants of the wild-type human IgG heavy chain Fc domain. In certain embodiments, the variant human IgG heavy chain Fc domain 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 Fc domain binds to human FcγRIIIA. This antibody may contain a variant human IgG heavy chain Fc domain, which is a variant of the wild-type human IgG heavy chain Fc domain, and which binds to human FcγRIIB with higher affinity than the wild-type human IgG heavy chain Fc domain binds to human FcγRIIB. The variant human IgG heavy chain Fc domain may be the heavy chain Fc domain of variant human IgG1, variant human IgG2, or variant human IgG4. In one embodiment, the variant human IgG heavy chain Fc domain contains 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 Fc domain 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).

[0216] Enhancement of CDC can be achieved by amino acid changes that increase affinity to C1q, the first component of the classical complement activation cascade. Another approach is to create a chimeric Fc domain created from human IgG1 and human IgG3 segments that takes advantage of IgG3's higher affinity for C1q. The antibodies of the present invention may contain amino acids mutated at residues 329, 331, and / or 322 to alter C1q binding and / or reduced or lost CDC activity. In another embodiment, the antibodies or antibody fragments disclosed herein may contain an Fc region having modifications at residues 231 and 239 that replace amino acids to alter the antibody's ability to immobilize complement. In one embodiment, the antibody or fragment has an Fc domain containing one or more mutations selected from E345K, E430G, R344D, and D356R, in particular a double mutation including R344D and D356R (EU index numbering system).

[0217] The functional properties of multispecific binding proteins can be further modified 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 FcRn binding (also referred to herein as "FcRn variants") may, under certain circumstances, extend the in vivo serum half-life compared to unmodified binding proteins. Naturally, any combination of Fc variants and FcRn variants can be used to modulate antigen-antibody complex clearance. Preferred 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.

[0218] Expression of antigen-binding proteins In one embodiment, polynucleotides encoding binding proteins (e.g., antigen-binding proteins and antigen-binding fragments thereof) disclosed herein are provided. Methods for producing binding proteins, including expressing these polynucleotides, are also provided.

[0219] The polynucleotides encoding the binding proteins disclosed herein are typically inserted into expression vectors for introduction into host cells that can be used to produce a desired amount of the binding protein. Thus, in certain embodiments, this disclosure provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0220] The terms “vector” or “expression vector” are used herein to mean a vector used in accordance with this disclosure as a medium for introducing and expressing a desired gene in a cell. As is known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses, and retroviruses. Generally, vectors suitable for this disclosure include a selection marker, appropriate restriction sites to facilitate the cloning of the desired gene, and the ability to enter and / or replicate within eukaryotic or prokaryotic cells.

[0221] 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, vacciniavirus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others involve the use of polycistrone systems including an internal ribosome binding site. Furthermore, cells into which the DNA has been integrated into the chromosome may be selected by introducing one or more markers that enable the selection of transfected host cells. The markers may confer prototrophicity to a nutrient-demanding host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selection marker gene may be directly ligated to the DNA sequence to be expressed, or it may be introduced into the same cell by co-transformation. Additional elements may be required for optimal mRNA synthesis. These elements may include signal sequences, splice signals, and further, transcription promoters, enhancers, and termination signals. In some embodiments, the cloned variable region gene is inserted into an expression vector along with the heavy and light chain Fc domain genes (e.g., human Fc domain genes) synthesized as described above.

[0222] In other embodiments, the binding protein may be expressed using a polycistronic construct. In such an expression system, multiple gene products of interest, such as the heavy and light chains of an antibody, may be produced from a single polycistronic construct. Such a system advantageously utilizes intra-sequence ribosome entry sites (IRESs) to provide relatively high levels of polypeptides in eukaryotic host cells. A suitable IRES sequence is disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those skilled in the art will understand that such an expression system can be used to effectively produce the entire range of polypeptides disclosed in this application.

[0223] More generally, once a vector or DNA sequence encoding a binding protein, such as an antibody or a fragment thereof, is prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. The introduction of plasmids into host cells can be achieved by various 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 envelope DNA, microinjection, and infection with intact viruses. See Ridgway, AAG “Mammalian Expression Vectors” Chapter 24.2, pp.470-472; Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). The introduction of plasmids into the host may be by electroporation. Transformed cells are grown under conditions suitable for light and heavy chain production and assayed for heavy chain and / or light chain protein synthesis. Examples of assay techniques include enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), fluorescence-activated cell sequencing (FACS), and immunohistochemistry.

[0224] As used herein, the term “transformation” shall be used in a broad sense to refer to the introduction of DNA into recipient host cells that alters their genotype.

[0225] In the same sense, “host cells” refer to cells constructed using recombinant DNA technology and transformed with a vector encoding at least one heterologous gene. In describing the process for isolating polypeptides from recombinant hosts, the terms “cells” and “cell culture” are used interchangeably to indicate the source of antibodies unless otherwise specified. In other words, the recovery of polypeptides from “cells” may mean recovery from centrifugated whole cells, recovery from the supernatant of a lysed cell culture, or recovery from a cell culture containing both culture medium and suspension cells.

[0226] In one embodiment, the host cell line used for antibody expression is of mammalian origin. Those skilled in the art can determine the specific host cell line best suited to expressing the desired gene product. Exemplary host cell lines include, but are not limited to, GS-CHO and CHO-K1 (Chinese hamster ovary cell line), DG44 and DUXB11 (Chinese hamster ovary cell line, DHFR-negative), HELA (human cervical cancer), CV-1 (monkey kidney cell line), COS (a derivative of CV-1 containing the SV40 T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HEK (human kidney cell line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), and 293 (human kidney). In one embodiment, the cell line undergoes altered glycosylation of the antibodies expressed from the cell line, e.g., defucosylation (e.g., PER.C6® (Crucell) or FUT8 knockout CHO cell line (POTELLIGENT® cells) (Biowa, Princeton, NJ)). In one embodiment, NS0 cells may be used. CHO cells are particularly useful. Host cell lines are typically available through commercial services, e.g., the American Tissue Culture Collection, or from authors of published literature.

[0227] In vitro production allows for scale-up to obtain large quantities 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 agitation reactors, or immobilized or encapsulated cell culture in, for example, hollow fibers, microcapsules, agarose microbeads, or ceramic cartridges. If necessary and / or desired, polypeptide solutions may be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography in DEAE cellulose, and / or (immuno) affinity chromatography.

[0228] The genes encoding the binding proteins discussed in this disclosure can also be expressed in non-mammalian cells such as bacteria or yeast, or in plant cells. In this regard, it will be understood that various single-celled non-mammalian microorganisms, such as bacteria, i.e., microorganisms that can grow under culture or fermentation, can also be transformed. Easily transformable bacteria include members of the Enterobacteriaceae, e.g., strains of Escherichia coli or Salmonella; Bacillaceae, e.g., Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. When expressed in bacteria, it will also be understood that the binding proteins may become part of an inclusion body. In some embodiments, the binding proteins are subsequently isolated, purified, and assembled into a functional molecule. In some embodiments, the binding proteins of this disclosure are expressed in bacterial host cells. In some embodiments, bacterial host cells are transformed with an expression vector containing a nucleic acid molecule encoding the binding proteins of this disclosure.

[0229] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, but several other strains are also commonly available. For expression in Saccharomyces, for example, 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 number 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). Therefore, the presence of trpl damage, a characteristic of the yeast host cell genome, provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0230] Pharmaceutical formulations / pharmaceutical compositions In certain embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an antigen-binding protein described herein. Some embodiments include a pharmaceutical composition comprising a therapeutically effective amount of any one of the binding proteins described herein, or a binding protein-drug conjugate, mixed with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.

[0231] Acceptable formulation materials are typically non-toxic to the recipient at the dosage and concentration used.

[0232] In some embodiments, the pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving the composition's pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution rate or release rate, adsorption, or osmosis. 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), fillers (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 immunoglobulin), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (e.g., polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (e.g., sodium), and preservatives (e.g., benzalcohol chloride). (e.g., sodium benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (e.g., glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (e.g., mannitol or sorbitol), suspending agents, surfactants or wetting agents (e.g., Pluronic acid; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancers (e.g., sucrose or sorbitol), tonicity enhancers (e.g., alkali metal halides such as sodium chloride or potassium chloride, or mannitol sorbitol), delivery media, diluents, excipients and / or pharmaceutically adjuvants (e.g., REMINGTON'S PHARMACEUTICAL SCIENCES (18) as incorporated herein by reference for any purpose). thThis includes, but is not limited to, the edition of the same book (Ed., Argennaro, ed., Mack Publishing Company 1990) and subsequent editions of the same book.

[0233] In some embodiments, the optimal pharmaceutical composition is determined by those skilled in the art, for example, depending on the intended route of administration, the mode of delivery, and the desired dose. Such a composition may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[0234] In some embodiments, the primary medium or carrier in the pharmaceutical composition may be either aqueous or non-aqueous. For example, suitable mediums or carriers for injection may be water, saline solution, or artificial cerebrospinal fluid, and other materials common in compositions for parenteral administration may be added. Neutral buffered saline, or saline mixed with serum albumin, are further exemplary mediums. Other exemplary pharmaceutical compositions include Tris buffer at pH about 7.0–8.5, or acetate buffer at pH 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 may be prepared for storage by mixing a selected composition having a desired purity with an optional formulation agent in the form of a lyophilized cake or aqueous solution. Furthermore, the binding protein may be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0235] In some embodiments, the pharmaceutical compositions of the present disclosure may be selected for parenteral or subcutaneous delivery. Alternatively, the compositions may be selected for inhalation or for delivery via the gastrointestinal tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the scope of the art of those skilled in the art.

[0236] In some embodiments, the components of the formulation are present at the administration site at an acceptable concentration. For example, a buffer is used to maintain the composition at a physiological pH or slightly lower, typically within a pH range of about 5 to about 8.

[0237] When parenteral administration is intended, the therapeutic composition for use may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the desired binding protein in a pharmaceutically acceptable medium. A particularly preferred medium for parenteral injection is sterile distilled water, which is properly stored and contains the binding protein as a sterile isotonic solution. Further preparations may involve formulating the desired molecule using agents such as injection microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, resulting in controlled or sustained release of the product, which can then be delivered by depot injection. Hyaluronic acid may also be used, which may have the effect of promoting the duration of action in the circulatory system. Other preferred means for introducing the desired molecule include implantable drug delivery devices.

[0238] In one embodiment, the pharmaceutical composition may be formulated for inhalation. For example, the binding protein may be formulated as a dry powder for inhalation. The inhalation solution of the binding protein may also be formulated using a propellant for aerosol delivery. In yet another embodiment, the solution may be sprayed.

[0239] Furthermore, it is intended that certain formulations may be administered orally. In one embodiment of this disclosure, the multispecific binding protein administered in this manner may be formulated with or without carriers conventionally used in the formulation of solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at a point when bioavailability is maximized in the gastrointestinal tract and pre-circulatory degradation is minimized. Additional agents may be included to facilitate the absorption of the binding protein. Diluents, flavorings, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0240] Another pharmaceutical composition may contain an effective amount of multispecific binding protein in a mixture with a non-toxic excipient suitable for the manufacture of tablets. The solution can be prepared in unit dose form by dissolving the tablets in sterile water or another suitable medium. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate; binders such as starch, gelatin, or acacia; or lubricants such as magnesium stearate, stearic acid, or talc.

[0241] Further pharmaceutical compositions of the present disclosure, including formulations containing binding proteins to sustained-release or controlled-release formulations, will be apparent to those skilled in the art. Techniques for formulating various other sustained-release or controlled-release means, such as liposome carriers, biodegradable 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 articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gammaethyl-L-glutamic acid, poly(2-hydroxyethyl-methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Liposomes may also be given as sustained-release compositions, which may be prepared by any of several methods known in the art.

[0242] 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 may be performed either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or as a solution. Furthermore, parenteral compositions are generally placed in containers with sterile access ports, such as intravenous solution bags or vials with stoppers that can be punctured by a subcutaneous needle.

[0243] Once formulated, pharmaceutical compositions may be stored in sterile vials as solutions, suspensions, gels, emulsions, solids, or dehydrated or lyophilized powders. Such formulations may be stored in a form ready for immediate use or in a form that requires reconstitution before administration (e.g., lyophilized).

[0244] This disclosure also encompasses kits for producing single-dose units. Each kit may include both a first container containing a dry multispecific binding protein and a second container containing an aqueous formulation. Kits including single-chamber and multi-chamber prefilled syringes (e.g., liquid syringes and lyosyringes) are also included within the scope of this disclosure.

[0245] The effective amount of a binding protein pharmaceutical composition used in treatment depends, for example, on the situation and purpose of the treatment. Therefore, it will be understood by those skilled in the art that the appropriate dosage level for a treatment depends to some extent on the molecules being delivered, the indications for which the binding protein is intended, the route of administration, and the patient's size (body weight, body surface or organ size) and condition (age and overall health). Accordingly, clinicians may titrate the dosage and modify the route of administration to achieve the optimal therapeutic effect.

[0246] The frequency of administration is determined by the pharmacokinetic parameters of the binding protein in the formulation used. Typically, the clinician will administer the composition until the desired effect is achieved. Therefore, the composition may be administered as a single dose, as two or more doses over time (with or without equal amounts of the desired molecule), or as a continuous infusion via an implantable device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and falls within the scope of routine tasks performed by those skilled in the art. The appropriate dosage can be confirmed by using appropriate dose-response data.

[0247] The administration route of the pharmaceutical composition follows known methods, such as by oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intrafocal injection, by continuous release system, or by implantable device. If desired, the composition may be administered by bolus injection, continuously by infusion, or by implantable device.

[0248] In some embodiments, the composition can also be administered topically via implantation of a membrane, sponge, or other suitable material that absorbs or encapsulates the desired molecule. When an implantable device is used, the device can be implanted in any suitable tissue or organ, and the desired molecule can be delivered by diffusion, sustained-release bolus, or continuous administration.

[0249] The multispecific binding proteins disclosed herein may be formulated as aerosols for topical application, such as by inhalation (see, for example, U.S. Patents 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 entirety). These formulations for administration to the airways may be in the form of aerosols or solutions for nebulizers, or ultrafine powders for blowing, either alone or in combination with an inert carrier such as lactose. In such cases, the particles of the formulations may have a diameter of less than 50 microns in one embodiment and less than 10 microns in another embodiment.

[0250] The multispecific binding proteins disclosed herein may be formulated in the form of gels, creams, and lotions for topical or local application, e.g., to the skin and mucous membranes, e.g., to the eyes, and for application to the eyes, or to the capsule or intrathecal cavity. Topical administration is intended for transdermal delivery, administration to the eyes or mucous membranes, or inhalation therapy. Heterodimeric proteins may also be administered as nasal drops, either alone or in combination with other pharmaceutically acceptable excipients.

[0251] Transdermal patches, including ionophoresis and electrophoresis 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. Patents 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference in their entirety.

[0252] In certain embodiments, the pharmaceutical composition comprising the multispecific binding protein described herein is a lyophilized powder and can be reconstituted for administration as a solution, emulsion, and other mixture. It can also be reconstituted and formulated as a solid or gel. 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 or other pharmacological components of the powder to improve stability, or a reconstituted solution prepared from the powder. Excipients that may 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, e.g., citrate buffer, sodium phosphate buffer, or potassium phosphate buffer, or other such buffers known to those skilled in the art; in one embodiment, a buffer with a pH of approximately neutral. The solution is then sterile filtered under standard conditions known to those skilled in the art, followed by lyophilization, to provide the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial contains a single-dose or multi-dose dose of the compound. The lyophilized powder can be stored under suitable conditions, such as room temperature, at approximately 4°C. Reconstitution of this lyophilized powder with water for injection provides a formulation for parenteral administration. When reconstituting, the lyophilized powder is added to sterile water or another suitable carrier. The exact amount depends on the selected compound. Such an 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 being 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 composition.For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,97,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874. All of these are hereby incorporated by reference in their entirety. In certain embodiments, the heterodimeric proteins described herein target tumors.

[0253] Methods of treatment / use Another aspect of the disclosure is the bispecific antibodies and / or antigen-binding proteins described herein for use as a medicament.

[0254] In certain embodiments, a method of treating a disease or disorder by agonist activity is provided, the method comprising administering to a subject that needs it an effective amount of the antigen-binding protein described herein.

[0255] The binding protein 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 protein binds to one or more target antigens with an affinity appropriate for the assay method used.

[0256] For diagnostic use, in some embodiments, the binding protein can be labeled with a detectable moiety. The detectable moiety can be anything that is 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 may be a radioactive isotope 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.

[0257] Binding proteins are also useful for in vivo imaging. Binding proteins labeled at a detectable region can be administered to animals (e.g., into the bloodstream) to assay the presence and location of the labeled antibody in the host. Binding proteins can be labeled at any region detectable in animals, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

[0258] This disclosure also relates to a kit comprising a binding protein and other reagents useful for detecting the level of a target antigen in a biological sample. Such reagents include detectable labels, 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 accompanying information attached to or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. Containers may be formed from a variety of materials such as glass or plastic. Containers may hold the composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing a disease, and may have a sterile access port (for example, the container may be an IV solution bag or vial with a stopper that can be punctured by a subcutaneous needle). In some embodiments, the label or accompanying information indicates that the composition is used for the prevention, diagnosis, and / or treatment of a selected disease. Alternatively, or furthermore, the product or kit may further comprise a second (or third) container containing pharmaceutically acceptable buffers such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further comprise other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0259] In some embodiments, this disclosure relates to methods for preventing and / or treating a disease or disorder (e.g., cancer). In some embodiments, the method involves 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 a human.

[0260] The content of any papers, patents, and patent applications, as well as all other documents and electronically available information, mentioned or cited herein are incorporated herein by reference to the same extent as each individual publication specifically and individually indicates that it is incorporated by reference. The applicant reserves the right to physically incorporate any material and information from any such papers, patents, patent applications, or other physical and electronic documents into this application. While this disclosure has been described in terms of its specific embodiments, those skilled in the art will understand that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of this disclosure. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt specific circumstances, materials, compositions, processes, or treatment steps to the purpose, spirit, and scope of this disclosure. All such modifications are intended to fall within the scope of the claims attached herein. While specific embodiments have been described in detail up to this point, embodiments will be more clearly understood by referring to the following examples. These examples are included for illustrative purposes only and are not intended to be limiting. [Examples]

[0261] The following examples are provided to those skilled in the art to provide a complete disclosure and explanation of the methods and compositions discussed herein, and are not intended to limit the scope of what the inventors consider to be their invention. While efforts have been made to ensure accuracy in the figures used (e.g., quantities, temperatures, etc.), some experimental error and deviation should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is Celsius, and pressure is atmospheric pressure or near atmospheric pressure.

[0262] Example 1. Bispecific agonist antibody against IL-18 receptor with modified hinge Bispecific agonist antibodies against IL18Rα and IL18Rβ, which are IL18 receptor subunits, were designed taking into account the modified hinge region. [Table 1-1] [Table 1-2] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0263] Bispecific antibodies were screened 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 the response to TNFα and IL-1β. Reporter cells were revived and cultured according to the supplier's recommendations. The cells were washed with PBS and added to a 96-well plate at a density of approximately 50,000 cells / well. 20 μl of either a control or a heteromeric antibody was added to the wells. The plate was incubated at 37 °C in a CO2 incubator for 20 - 24 hours. QUANTI-Blue™ (Invivogen) solution was prepared using the manufacturer's instructions, and 180 μl of the solution was added to the fresh plate. 20 μl of induced HEK-Blue IL-18 supernatant was added to each well, and the plate was incubated at 37 °C for 3 hours and read at 630 nm using a spectrophotometer (Clariostar). Bispecific antibodies with a modified hinge were compared to antibodies without a modified hinge (e.g., WT IgG1 hinge).

Table 4

[0264] Bispecific constructs DGL207 and DGL209 contain a modified hinge. DGL207 contains hinge 1 (without the upper hinge region), and DGL209 contains hinge 3 (PLAP, SEQ ID NO: 2). Bispecific constructs with a modified hinge performed better than their counterparts with a wild-type IgG1 hinge (DGL093).

Table 5

[0265] <The IL-18R bispecific DGL207 agonist with hinge variant 1 (hinge 1; no hinge) performed best among the hinge variants. Hinge 2 contains the upper hinge sequence of PLAPDKTHT (sequence ID 1). Hinge 3 contains the upper hinge sequence of PLAP (sequence ID 2). Hinge 4 contains the upper hinge sequence of GGGGSGGGGSGGGGSGGGGS (sequence ID 3). Hinge 5 contains the upper hinge sequence of EKSYGPP (sequence ID 4). Hinge 6 contains the upper hinge sequence of DKTHT (sequence ID 5). DGL212 with hinge 6 is similar to DGL093 except that it contains the full human framework 4 (FW4).

[0266] Activity was also measured with an IL-18R bispecific antibody having an asymmetric hinge (a hinge variant on one of the bispecific polypeptides). [Table 6] [Table 7]

[0267] Example 2. Bispecific antibodies against BMPRI and type II receptors with modified hinges. Bispecific antibodies targeting the BMPRI receptor ALK1 and the BMPRII receptor BMPRII were designed using the sequences provided below. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5]

[0268] Bispecific antibodies were screened for agonist activity using PathHunter U2O ALK-1 / BMPR-2 dimerization assay cells obtained from DiscoverX Corporation (93-0962C3). These cells were prepared using enzyme fragment complementarity (EFC) technology, employing β-galactosidase fragments to evaluate protein-protein interactions. Reporter cells were revived and cultured according to the supplier's recommendations. Bispecific antibodies were compared to their natural ligand, BMP-9.

[0269] The same assay can be used to detect Alk-1 / ActRIIA agonism (93-1069C3) and Alk1 / ActRIIB agonism (93-0964C3). [Table 9]

[0270] Example 3. Engineering of scFv containing an ALK1 / BMPRII bispecific agonist antibody with an optimized hinge. The agonist activity of heteromer antibodies with modified hinges identified by the DIAGONAL platform was also tested. Variants of DGL288 and DGL809 were designed with hinge 1. DGL809 was designed, expressed, and purified using the Expi293 (Thermo) system according to the manufacturer's instructions. Cells were harvested 6 days after transfection and collected using batch purification with mabSelect resin. The purity of the final product was assessed using SDS-PAGE and analytical gel filtration. The heteromer antibodies were tested using the DiscoverX assay. As shown in Table 9 (showing mean values ​​across two different experiments), DGL809 performed better than the parent DGL288, with activity levels compared to BMP9 at an antibody concentration of 100 nM. [Table 10] [Table 11]

[0271] Further bispecific antibodies were screened for agonist activity. The PathHunter U2O ALK-1 / BMPR-2 dimerization assay was obtained from DiscoverX Corporation (93-0962C3). These cells were prepared using enzyme fragment complementarity (EFC) technology, employing β-galactosidase fragments to evaluate protein-protein interactions. Reporter cells were revived and cultured according to the supplier's recommendations. Bispecific antibodies were compared to their natural ligands, BMP9 and BMP10.

[0272] To perform the assay, cells were isolated and removed from the flask using cell separation reagent (DiscoverX, 92-0009). The cells were centrifuged at 300 g for 4 minutes and resuspended in assay plating medium (DiscoverX 93-0563R22A) at a density of 250 K / ml. 20 μl of the suspension was plated per well of a 384-well plate and incubated at 37°C for 24 hours. A bispecificity was prepared at 5 times the final concentration. A 12-stop titration was performed using a 1:10 dilution to generate a curve. 5 μl of bispecificity was added to the 384-well plate and incubated for 3 hours. 25 μl of flash detection reagent (DiscoverX, 93-0247) was added per well, and the plate was read at 60 minutes using Verilux Skan. Data were analyzed using PRISM. [Table 12]

[0273] It was observed that tetravalent bispecific antibodies (i.e., two binding domains for the first receptor subunit (e.g., ALK1) and two binding domains for the second receptor subunit (e.g., BMPRII)) elicited stronger agonism than bivalent bispecific antibodies (i.e., one binding domain for the first receptor subunit and one binding domain for the second receptor subunit). The bivalent bispecific antibodies, DGL266-DGL271, had 0-46% of BMP9 activity, while tetravalent bispecific antibodies, such as DGL285-DGL292, consistently yielded higher values.

[0274] The agonist activity of bispecific antibodies was determined separately by measuring their downstream effects on target receptor activation. In the context of ALK1 / BMPRII signaling, receptor activation leads to SMAD1 (pSMAD1) phosphorylation.

[0275] To measure pSMAD1 levels, HUVEC cells derived from ATCC (CRL-1730) were plated overnight in 100 µl of complete HUVEC medium in a 96-well plate with 15K cells per well (F12K (Corning, 10-025-CV), 10% FBS (Gibco, A31605-02), ECGS (30 µg / ml, Corning, 356006), and 0.1 mg / ml heparin (Sigma, H3). 393), 1×Pen / Strep (Gibco, 15140-122). The following morning, the cells were starved for 4 hours by replacing the medium with 50 ul of serum-free / ECGS-free F12K medium. The cells were then treated with 50 ul of serum-free / ECGS-free medium containing a dose curve of bispecific or 2x concentration BMP ligand. The medium was removed from the cells at various time points (5 min, 15 min, 30 min, 60 min) and 50 ul of lysis buffer (Abcam). ELISA kit (AB186037) was added to each well. After lysis, buffer from the four wells was conditionally pooled for a single 200 μl lysed sample, frozen, and subsequently subjected to ELISA to measure either total SMAD1 (Abcam, AB186037) or pSMAD1 (Abcam, AB186036). An anti-HEL antibody with a LALA-PG mutation (BioXCell, CP149) was used as a negative control. [Table 13]

[0276] The agonist activity of the bispecific antibodies in vivo was also determined. The antibodies were measured for agonist activity in a mouse model of HHT in which circulating BMP9 / BMP10 was neutralized by an anti-BMP9 / 10 antibody (Ruiz S, et al, Scientific Reports, 2016 Nov 22:5:37366). These mice developed retinal vascular defects postnatally. Three animals were administered either DGL288 or a negative control antibody (anti-HEL, LALA-PG, BioXCell, CP149) at a dose of 15 mg / kg / day for two days in phases 3 and 4. The BMP9 / 10 antibody was administered on the same day. Analysis was completed in phase 6. The retinas were dissected, whole mounts were prepared, and then stained with both isolectin B4 and SMA to label the retinal vascular system and detect arteriovenous malformations (AVMs). The results are shown in Figure 3A. Mice treated with DGL288 did not show AVM formation, while negative controls showed an average AVM / retina ratio of 4.8.

[0277] In the second set of experiments, all animals were administered BMP9 / 10 antibodies at P3 and P4. DGL288, DGL292, or PBS control were administered at 1 mg / kg / day at P4 and P5. Analysis was completed at P6 for DGL288 and litter-negative control animals, or at P7 for littermates administered DGL292 and PBS control. The retinas were dissected, whole mounts were prepared, and then stained with both isolectin B4 and SMA to detect AVMs. Mice administered with DGL292 did not develop AVMs, compared to a mean of 5.7 / retina in the control group (Figure 3B). Mice administered with DGL288 did not develop AVMs, compared to a mean of 4.5 / retina in the control group (Figure 3C). The absence of observed weight differences suggests good tolerability of the agonists.

[0278] Example 4. Engineering of a bispecific agonist antibody with an optimized linker in DVD-Ig format. An alternative method for rigidifying antibodies is to optimize the linker between IgG and additional variable domains in the DVD-Ig format. To determine whether the DVD-Ig format is a viable format for bispecific agonist antibodies, the activity of heteromeric antibodies with modified VH vs. IgG hinge linkers identified by the DIAGONAL platform was measured. Variants of DGL292 (DGL810, DGL811, and DGL812) were designed, expressed, and purified as described above. When the heteromeric antibodies were tested using the Discovery X assay, all variants performed better than the parent DGL292, as seen in Table 14 (showing mean values ​​across two different experiments). [Table 14] [Table 15-1] [Table 15-2] [Table 16] [Table 17-1] [Table 17-2] [Table 17-3]

[0279] Further bispecific antibodies were screened for agonist activity. The PathHunter U2O ALK-1 / BMPR-2 dimerization assay was obtained from DiscoverX Corporation (93-0962C3). These cells were prepared using enzyme fragment complementarity (EFC) technology, employing β-galactosidase fragments to evaluate protein-protein interactions. Reporter cells were revived and cultured according to the supplier's recommendations. Bispecific antibodies were compared to their natural ligands, BMP9 and BMP10.

[0280] To perform the assay, cells were isolated and removed from the flask using cell separation reagent (DiscoverX, 92-0009). The cells were centrifuged at 300 g for 4 minutes and resuspended in assay plating medium (DiscoverX 93-0563R22A) at a density of 250 K / ml. 20 μl of the suspension was plated per well of a 384-well plate and incubated at 37°C for 24 hours. Bispecificity was prepared at 5-fold the final concentration. A 12-stop titration was performed using a 1:10 dilution to generate a curve. 5 μl of bispecificity was added to the 384-well plate and incubated for 3 hours. 25 μl of flash detection reagent (DiscoverX, 93-0247) was added per well, and the plate was read at 60 minutes using Verilux Skan. Data were analyzed using PRISM. The results are presented in Table 16 below. This data indicates that each of the bispecificity antibodies tested possessed potent agonist activity. [Table 18]

[0281] The antibody was measured for agonist activity in a mouse model of HHT in which circulating BMP9 / BMP10 was neutralized by an anti-BMP9 / 10 antibody (Ruiz S, et al, Scientific Reports, 2016 Nov 22:5:37366). These mice developed postnatal retinal vascular defects. Three animals were administered DGL292, DGL945, DGL947, or a negative control antibody (anti-HEL, LALA-PG, BioXCell, CP149) at a dose of 1 mg / kg / day for 2 days in phases 3 and 4. The BMP9 / 10 antibody was administered on the same day. Analysis was completed in phase 6. The retinas were dissected, whole mounts were prepared, and then stained with both isolectin B4 and SMA to label the retinal vascular system and detect arteriovenous malformations (AVMs). The results are shown in Figure 4. Mice treated with any ALK1-BMPRII agonist showed a significant reduction in AVM formation, while negative controls had a mean AVM / retina of 4.5.

[0282] Example 5. Humanization of IL-18R binder. Previously identified binders with high levels of activity were humanized using the HEK Blue assay and optimized for therapeutic applications. VHH binders against IL-18Rα and IL-18Rβ were computationally modeled using antigens with the DIAGONAL platform. Non-essential residues for epitope recognition were replaced with human sequences. Reverse mutations were added solely to maintain antigen binding and stability. Construct affinity was measured using Carterra, and activity was measured using the HEK Blue assay. In addition, in some cases, proline at position 14 of the VHH binding domain was replaced with alanine to improve binder stability and agonism.

[0283] Humanization of DGL207 resulted in reduced potency and affinity for IL-18Rβ (DGL333). Using the DIAGONAL platform, it was observed that the proline at position 14 (P14) could destabilize the molecule in association with a rigid hinge (hinge 1). Reverting this mutation to alanine found in the llama germline improved both the affinity and activity of the bispecific compound in the HEK Blue assay (Figure 5). Therefore, according to Kabat, alanine substitution at position 14 of VHH enhances agonist activity.

[0284] In addition to humanizing VHH, Fc was manipulated for optimal therapeutic applications (e.g., LALAGA, nob-in-hole, and YTE mutations). [Table 19] [Table 20-1] [Table 20-2]

Claims

1. A multispecific binding protein comprising at least a first binding domain and a second binding domain, wherein the first binding domain is linked to the second binding domain via at least one modified hinge region.

2. The multispecific binding protein according to claim 1, wherein the first binding domain is a first variable heavy chain domain (VH1), and the second binding domain is a second variable heavy chain domain (VH2).

3. The multispecific binding protein according to claim 2, wherein one or both of VH1 and VH2 are VH domains or VHH domains.

4. The multispecificity binding protein according to any one of claims 1 to 3, further comprising a first variable light chain domain (VL1) linked to a second variable light chain domain (VL2) via at least one modified hinge region.

5. The multispecific binding protein according to claim 1, wherein the first binding domain is a first scFv and the second binding domain is a second scFv.

6. A multispecific binding protein comprising at least a first polypeptide chain, The multispecificity binding protein wherein the first polypeptide chain comprises a first variable heavy chain domain (VH1) linked to a second variable heavy chain domain (VH2) via at least one modified hinge region.

7. The multispecific binding protein according to claim 6, wherein one or both of VH1 and VH2 are VH domains or VHH domains.

8. The multispecificity binding protein according to claim 6, further comprising a second polypeptide chain, the second polypeptide chain comprising a first variable light chain domain (VL1) linked to a second variable light chain domain (VL2) via at least one modified hinge region.

9. The multispecific binding protein according to any one of claims 6 to 8, wherein one or both of VH1 and VH2 are cleaved at the C-terminus.

10. The multispecificity binding protein according to claim 9, wherein the C-terminus is cleaved by at least one residue.

11. The multispecificity binding protein according to claim 9 or 10, wherein the C-terminus is cleaved by at least two residues.

12. The multispecific binding protein according to any one of claims 9 to 11, wherein the C-terminal SS amino acid residue is deleted.

13. The first polypeptide chain is VH1-HX1-VH2-C-Fc, where, VH1 is the first heavy chain variable domain, VH2 is the second heavy chain variable domain, C is the heavy chain constant domain, HX1 is a linker in the modified hinge region, Fc is the Fc region of the first polypeptide chain, and The second polypeptide chain of VL1-LX1-VL2-C, Here, VL1 is the first light chain variable domain, VL2 is the second light chain variable domain, C is a light chain constant domain, and The multispecific binding protein according to any one of claims 6 to 12, comprising the second polypeptide chain in which LX1 is a linker of a modified hinge region.

14. The modified hinge region is i) Upper hinge region that is or does not exist and is at most 7 amino acids long, ii) A multispecific binding protein according to any one of claims 1 to 13, comprising a lower hinge region.

15. The multispecific binding protein according to any one of claims 1 to 14, wherein the modified hinge region comprises or consists of the amino acid sequence of PLAP or PAPNLLLGGP.

16. The multispecific binding protein according to any one of claims 1 to 15, wherein the first binding domain binds to a first receptor subunit, and the second binding domain binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first receptor subunit and the second receptor subunit.

17. The multispecific binding protein according to any one of claims 6 to 16, wherein VH1 binds to a first receptor subunit, and VH2 binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first receptor subunit and the second receptor subunit.

18. The multispecific binding protein according to any one of claims 8 to 16, wherein a first antigen-binding domain formed from VH1 and VL1 binds to a first receptor subunit, and a second antigen-binding domain formed from VH2 and VL2 binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first receptor subunit and the second receptor subunit.

19. The multispecificity binding protein according to any one of claims 5 to 16, wherein the first scFv binds to a first receptor subunit, and the second scFv binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first receptor subunit and the second receptor subunit.

20. The multispecificity binding protein according to any one of claims 16 to 19, wherein the first receptor subunit and the second receptor subunit are different subunits that form a heterodimer.

21. The multispecificity binding protein according to any one of claims 16 to 19, wherein the first receptor subunit and the second receptor subunit are the same subunit that forms a heterodimer.

22. The multispecific binding protein according to any one of claims 16 to 21, wherein the first and second receptor subunits are selected from tumor necrosis factor superfamily (TNFSF) receptors, interleukin type I receptors, interleukin type II receptors, Ig superfamily (IGSF) receptors, receptor tyrosine kinases (RTKs), growth hormone receptors, transforming growth factor beta (TGFβ) receptor superfamily, type C lectin-like receptors, interferon receptors, phosphatase receptors (i.e., receptor protein tyrosine phosphatases), and integrin receptors.

23. The multispecific binding protein according to any one of claims 1 to 22, wherein the antigen-binding domain is a VHH containing a P14A amino acid substitution according to Kabat numbering.

24. A multispecific binding protein comprising a first polypeptide chain and a second polypeptide chain, wherein each of the first and second polypeptide chains comprises a first scFv linked from the N-terminus to the C-terminus of a second single-chain variable fragment (scFv), wherein the first scFv is linked to the second scFv via at least one modified hinge region.

25. The modified hinge region is i) Upper hinge region that is or does not exist and is at most 7 amino acids long, ii) The multispecific binding protein according to claim 24, comprising a lower hinge region.

26. The multispecific binding protein according to claim 24 or 25, wherein the modified hinge region comprises or consists of the amino acid sequence of PLAP or PAPNLLLGGP.

27. The multispecific binding protein according to any one of claims 24 to 26, wherein the first scFv binds to a first receptor subunit, and the second scFv binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first receptor subunit and the second receptor subunit.

28. The multispecificity binding protein according to claim 27, wherein the first receptor subunit and the second receptor subunit are different subunits that form a heterodimer.

29. The multispecificity binding protein according to claim 27, wherein the first receptor subunit and the second receptor subunit are the same subunit that forms a heterodimer.

30. The multispecific binding protein according to any one of claims 27 to 29, wherein the first and second receptor subunits are selected from tumor necrosis factor superfamily (TNFSF) receptors, interleukin type I receptors, interleukin type II receptors, Ig superfamily (IGSF) receptors, receptor tyrosine kinases (RTKs), growth hormone receptors, transforming growth factor beta (TGFβ) receptor superfamily, type C lectin-like receptors, interferon receptors, phosphatase receptors (i.e., receptor protein tyrosine phosphatases), and integrin receptors.

31. A multispecific binding protein according to any one of claims 1 to 30, further comprising a heavy chain constant region.

32. The multispecific binding protein according to claim 31, wherein the heavy chain constant region includes a substitution at the 234th amino acid position according to EU numbering.

33. The multispecific binding protein according to claim 32, wherein the substitution at the 234th amino acid position is alanine (A).

34. The multispecific binding protein according to claim 31, wherein the heavy chain constant region includes a substitution at the 235th amino acid position according to EU numbering.

35. The multispecific binding protein according to claim 34, wherein the substitution at the 235th amino acid position is alanine (A).

36. The multispecific binding protein according to claim 31, wherein the heavy chain constant region includes a substitution at the 237th amino acid position according to EU numbering.

37. The multispecific binding protein according to claim 36, wherein the substitution at the 237th amino acid position is alanine (A).

38. The multispecific binding protein according to claim 31, wherein the heavy chain constant region includes one or more substitutions at amino acid positions 234, 235, or 237, according to EU numbering.

39. A multispecific binding protein according to claim 38, The aforementioned substitution at the 234th position of the amino acid is alanine (A), The aforementioned substitution at the 235th position of the amino acid is alanine (A), and The multispecific binding protein wherein the substitution at the 237th amino acid position is alanine (A).

40. The multispecific binding protein according to any one of claims 31 to 39, wherein the heavy chain constant region includes a heterodimerization mutation that promotes heterodimerization between the first binding portion and the second binding portion.

41. The multispecific binding protein according to claim 40, wherein the heterodimerizing mutation is a knob-in-hole (KIH) mutation.

42. The multispecific binding protein according to claim 41, wherein the first heavy chain constant region includes an amino acid substitution that creates a hole at position 366, 368, or 407, and the second heavy chain constant region includes an amino acid substitution that creates a knob at position 366.

43. The multispecific binding protein according to claim 42, wherein the first heavy chain constant region comprises an amino acid substitution T366S, L368A, or Y407V, and the second heavy chain constant region comprises an amino acid substitution T366W.

44. The multispecific binding protein according to claim 40, wherein the heterodimerizing mutation is a charge-stabilizing mutation.

45. The multispecific binding protein according to claim 44, wherein the first heavy chain constant region comprises the amino acid substitution N297K, and the second heavy chain constant region comprises the amino acid substitution N297D.

46. The multispecific binding protein according to claim 44, wherein the first heavy chain constant region comprises the amino acid substitution T299K, and the second heavy chain constant region comprises the amino acid substitution T299D.

47. The multispecific binding protein according to claim 40, wherein the heterodimerizing mutation comprises an engineered disulfide bond.

48. The multispecific binding protein according to claim 47, wherein the manipulated disulfide bond is formed by a first heavy chain constant region containing the amino acid substitution Y349C and a second heavy chain constant region containing the amino acid substitution S354C.

49. The multispecific binding protein according to claim 47 or 48, wherein the manipulated disulfide bond is formed by a C-terminal extension peptide fused to the C-terminus of each of the first and second heavy chain constant regions.

50. The multispecificity binding protein according to claim 49, wherein the C-terminal extension of the first heavy chain constant region comprises the amino acid sequence GEC, and the C-terminal extension of the second heavy chain constant region comprises the amino acid sequence SCDKT.

51. The multispecific binding protein according to any one of claims 31 to 50, wherein at least one heavy chain constant region contains one or more mutations that promote an increase in half-life.

52. The multispecificity binding protein according to claim 51, wherein at least one heavy chain constant region comprises one or more substitutions at the 252nd, 254th, or 256th amino acid positions according to EU numbering.

53. A multispecific binding protein according to claim 52, The aforementioned substitution at the 252nd position of the amino acid is tyrosine (Y), The aforementioned substitution at the 254th position of the amino acid is threonine (T), and The multispecific binding protein wherein the substitution at the 256th amino acid position is glutamic acid (E).

54. The multispecificity binding protein according to claim 51, wherein at least one heavy chain constant region comprises one or more substitutions at the 428th or 434th amino acid position according to EU numbering.

55. The multispecific binding protein according to claim 54, wherein at least one heavy chain constant region comprises substitutions of M428L and N434S according to EU numbering.

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

57. An isolated nucleic acid molecule encoding a multispecific binding protein according to any one of claims 1 to 55.

58. An expression vector comprising the nucleic acid molecule described in claim 57.

59. A host cell comprising the expression vector described in claim 58.

60. A method for treating a disease or disorder in a subject, comprising administering a multispecific binding protein according to any one of claims 1 to 55 to a subject in need thereof.

61. A multispecific binding protein according to any one of claims 1 to 55, for use as a pharmaceutical product.

62. A multispecific binding protein according to any one of claims 1 to 55, for use as a diagnostic tool.

63. A method for inducing signal transduction between a first receptor subunit and a second receptor subunit in a subject, comprising administering to the subject a multispecific binding protein according to any one of claims 1 to 55.

64. The method according to claim 63, wherein the multispecific binding protein can induce signal transduction by inducing proximity between the first receptor subunit and the second receptor subunit.

65. The method according to claim 63 or 64, wherein the multispecific binding protein has higher agonist activity compared to a multispecific binding protein lacking at least one modified hinge region.

66. The method according to any one of claims 63 to 65, wherein the multispecific binding protein induces agonist activity that is at least about 35% of the activity of the innate ligand to the first receptor subunit and the second receptor subunit.

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