Hinge-modified bispecific antibodies

Abstract

Provided herein are bispecific agonistic antibodies having modified hinge regions and methods of using the antibodies.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 458,045, filed April 7, 2023, and U.S. Provisional Patent Application Serial No. 63 / 596,905, filed November 7, 2023, the entire disclosure of which is incorporated herein by reference. Background Technology

[0003] The antibody consists of two Fab regions, which are linked to a crystallizable fragment (Fc) via a flexible hinge region. The hinge region is a flexible amino acid segment in the middle of the heavy chain of the IgG and IgA immunoglobulin classes, which connects the two heavy chains via disulfide bonds.

[0004] The antibody hinge can be divided into three regions: the upper hinge, the core hinge, and the lower hinge, each with a different function. On the N-terminal side, the upper hinge allows the movement and rotation of the antigen-binding fragment domain (Fab). The central core hinge contains a variable number (depending on the IgG subtype) of cysteine ​​residues, which form disulfide bonds, thereby stabilizing HC binding. On the C-terminal side is the lower hinge, which allows Fc movement relative to the Fab, and whose amino acid residues can participate in Fcγ receptor (FcγR) binding.

[0005] Human IgG isotypes exhibit significant differences in the hinge number of residues and the number of possible disulfide bridges between the two heavy chains. This contributes to the overall stability of the antibody. For example, among all IgGs, IgG4 is the only isotype that undergoes a natural Fab arm exchange to produce a bispecific antibody molecule. Furthermore, this variability (including differences in amino acid sequence) contributes in part to the strength of the interaction between IgG and FcγR. Summary of the Invention

[0006] This disclosure improves upon the prior art by providing heteropolymer antibodies with modified hinge regions, thereby enhancing the agonistic activity of the antibodies.

[0007] In one aspect, this document provides a bispecific antibody exhibiting agonistic activity, the bispecific antibody 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 of up to 7 amino acids in length or lacks an upper hinge region, and a middle hinge and a lower hinge region, wherein the lower hinge region is connected 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 receptor subunit and the second receptor subunit. In some embodiments, the first receptor subunit and the second receptor subunit are different subunits forming a heterodimer. In some embodiments, the first receptor subunit and the second receptor subunit are the same subunit forming a homodimer. In some implementations, the first and second receptor subunits are selected from: tumor necrosis factor superfamily (TNFSF) receptors, type I interleukin receptors, type II interleukin receptors, Ig superfamily (IGSF) receptors, receptor tyrosine kinases (RTKs), growth hormone receptors, transforming growth factor β (TGFβ) receptor superfamily, type C lectin-like receptors, interferon receptors, phosphatase receptors (i.e., receptor-type protein tyrosine phosphatases), and integrin receptors.

[0008] In some embodiments, the bispecific antibody further comprises a second modified hinge region attached to the N-terminus of the second heavy chain Fc domain. In some embodiments, the second modified hinge region comprises an upper hinge region of up to 7 amino acids in length, or no upper hinge region, and a middle hinge region and a lower hinge region, wherein the lower hinge region is attached to the N-terminus of the second heavy chain Fc domain. In some embodiments, the upper hinge region is attached to the N-terminus of the second heavy chain Fc domain.

[0009] In some embodiments, the first modified hinge region and the upper hinge region of the second modified hinge region are identical. In some embodiments, the first modified hinge region and the upper hinge region of the second modified hinge region are different. In some embodiments, the upper hinge region comprises an amino acid sequence derived from the upper hinge region of a human IgG antibody. In some embodiments, the IgG antibody is selected from IgG1, IgG2, IgG3, and IgG4. In some embodiments, the IgG antibody is IgG1. In some embodiments, the upper hinge region comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the upper hinge region comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the IgG antibody is IgG4. In some embodiments, the upper hinge region comprises the amino acid sequence of SEQ ID NO: 4. In some embodiments, there is no upper hinge. In some embodiments, the middle hinge region and the lower hinge region comprise the amino acid sequence of SEQ ID NO: 6.

[0010] In some embodiments, the first modified hinge region and / or the second modified hinge region comprises the amino acid sequence of SEQ ID NO: 7. In some embodiments, the first modified hinge region and / or the second modified hinge region comprises the amino acid sequence of SEQ ID NO: 8. In some embodiments, the first modified hinge region and / or the second modified hinge region comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the first modified hinge region and / or the second modified hinge region comprises the amino acid sequence of SEQ ID NO: 9.

[0011] In some embodiments, the first heavy chain Fc domain and / or the second heavy chain Fc domain comprises human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the first heavy chain Fc domain and / or the second heavy chain Fc domain comprises the amino acid sequence of SEQ ID NO: 10.

[0012] In some embodiments, the first heavy chain Fc domain and / or the second heavy chain Fc domain comprises one or more amino acid substitutions. In some embodiments, according to EU designations, at least one heavy chain Fc domain comprises a substitution at amino acid position 234. In some embodiments, the substitution at amino acid position 234 is alanine (A). In some embodiments, according to EU designations, at least one heavy chain Fc domain comprises a substitution at amino acid position 235. In some embodiments, the substitution at amino acid position 235 is alanine (A). In some embodiments, according to EU designations, at least one heavy chain Fc domain comprises a substitution at amino acid position 237. In some embodiments, the substitution at amino acid position 237 is alanine (A). In some embodiments, according to EU designations, at least one heavy chain Fc domain comprises one or more substitutions at amino acid positions 234, 235, or 237. 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).

[0013] In some embodiments, the heavy chain Fc domain includes a heterodimerization mutation to promote heterodimerization of the first binding moiety with the second binding moiety. In some embodiments, the heterodimerization mutation is a mortar-and-stick (KIH) mutation. In some embodiments, the first heavy chain Fc domain includes a mortar-forming amino acid substitution at position 366, 368, or 407, and the second heavy chain Fc domain includes a mortar-forming amino acid substitution 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.

[0014] In some embodiments, the heterodimerization 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.

[0015] In some embodiments, the heterodimerization mutation comprises an engineered disulfide bond. In some embodiments, the engineered disulfide bond is formed by a first heavy chain Fc domain comprising an amino acid substitution Y349C and a second heavy chain Fc domain comprising an amino acid substitution S354C. In some embodiments, the engineered disulfide bond is formed by a C-terminal extension peptide fused to the C-terminus of each of the first and second heavy chain Fc domains. In some embodiments, the C-terminal extension of the first heavy chain Fc domain comprises the amino acid sequence GEC, and the C-terminal extension of the second heavy chain Fc domain comprises the amino acid sequence SCDKT.

[0016] In some embodiments, at least one heavy chain Fc domain contains one or more mutations to promote half-life extension. In some embodiments, according to EU designations, at least one heavy chain Fc domain contains one or more substitutions at amino acid positions 252, 254, or 256. 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).

[0017] In some embodiments, the first modified hinge region and the first heavy chain Fc domain are as shown 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 as shown 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 as shown 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 as shown in the amino acid sequence of SEQ ID NO: 14.

[0018] In some embodiments, the first antigen-binding domain is selected from single-chain Fv (scFv), VHH, or Fab. In some embodiments, the second antigen-binding domain is selected from single-chain Fv (scFv), VHH, or Fab.

[0019] In some embodiments, the bispecific antibody comprises, from the N-terminus to the C-terminus: ai) a first polypeptide chain including a first antigen-binding domain, a first modified hinge region, and a first Fc domain; and bi) a second polypeptide chain including a second antigen-binding domain, a second modified hinge region, and a second Fc domain; aii) a first polypeptide chain including 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 including 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.

[0020] In some implementations, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, and the fourth antigen-binding domain contain scFv.

[0021] In some implementations, (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 contains the Fab domain, and the second antigen-binding domain contains the VHH domain. (c) The first antigen-binding domain contains a VHH domain and the antigen-binding domain contains a Fab domain. (d) The first antigen-binding domain contains a Fab domain, and the second antigen-binding domain contains a Fab domain; (e) The first antigen-binding domain contains a Fab domain, and the second antigen-binding domain contains scFv; (f) The first antigen-binding domain contains scFv, and the second antigen-binding domain contains a Fab domain; (g) The first antigen-binding domain contains scFv, and the second antigen-binding domain contains scFv; (h) The first antigen-binding domain contains scFv, and the second antigen-binding domain contains VHH; or (i) The first antigen-binding domain contains VHH, and the second antigen-binding domain contains scFv.

[0022] In some embodiments, the first antibody-binding domain and / or the second antibody-binding domain are truncated at the C-terminus immediately adjacent to the upper hinge domain. In some embodiments, the C-terminus immediately adjacent to the upper hinge domain is truncated by at least one residue. In some embodiments, the C-terminus immediately adjacent to the upper hinge domain is truncated by at least two residues.

[0023] In some implementations, the bispecific antibody comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain contains VH1-(HX1)n-VH2-C-(HX2)n, where: VH1 is the first heavy chain variable structural domain; VH2 is a second-chain variable structural domain; C is a heavy-chain constant structural domain; HX1 is a connector; HX2 is the Fc region; and n is independently 0 or 1; and The second polypeptide chain contains VL1-(LX1)n-VL2-C-(LX2)n, in: VL1 is the first light chain variable structural domain; VL2 is the second light chain variable structural domain; C is a constant structural domain of the light chain; LX1 is a connector; LX2 does not include the Fc region; and n is independently 0 or 1.

[0024] In some embodiments, linker HX1 contains the amino acid sequence PLAP or PAPNLLGGP. In some embodiments, linker LX1 contains the amino acid sequence PLAP or PAPNLLGGP. In some embodiments, linker HX1 contains the amino acid sequence PLAP, and linker LX1 contains the amino acid sequence PLAP or PAPNLLGGP.

[0025] In some embodiments, the first receptor subunit comprises the IL18Rα subunit, and the second receptor subunit is the IL18Rβ subunit. In some embodiments, the first receptor subunit comprises the ALK1 receptor subunit, and the second receptor subunit is selected from BMPRII, ActRIIA, and ActRIIB.

[0026] In another aspect, this article provides bispecific antibodies exhibiting agonistic activity, the bispecific antibodies comprising: a) a first antigen-binding domain, a first modified hinge region, and a first heavy chain Fc domain; and b) The second antigen-binding domain and the second heavy chain Fc domain, wherein: The first modified hinge area includes: i) An upper hinge region with a length of at most 7 amino acids, or no upper hinge region; and ii) Lower hinge region, wherein the lower hinge region is connected to the N end of the first heavy chain Fc structural 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 inducing proximity between the first and second receptor subunits.

[0027] In some implementations, (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 contains the Fab domain, and the second antigen-binding domain contains the VHH domain. (c) The first antigen-binding domain contains a VHH domain and the antigen-binding domain contains a Fab domain. (d) The first antigen-binding domain contains a Fab domain, and the second antigen-binding domain contains a Fab domain; (e) The first antigen-binding domain contains a Fab domain, and the second antigen-binding domain contains scFv; (f) The first antigen-binding domain contains scFv, and the second antigen-binding domain contains a Fab domain; (g) The first antigen-binding domain contains scFv, and the second antigen-binding domain contains scFv; (h) The first antigen-binding domain contains scFv, and the second antigen-binding domain contains VHH; or (i) The first antigen-binding domain contains VHH, and the second antigen-binding domain contains scFv.

[0028] In some embodiments, the first receptor subunit comprises the IL18Rα subunit, and the second receptor subunit is the IL18Rβ subunit. In some embodiments, the first receptor subunit comprises the ALK1 receptor subunit, and the second receptor subunit is selected from BMPRII, ActRIIA, and ActRIIB.

[0029] In another aspect, this article provides bispecific antibodies exhibiting agonistic activity, the bispecific antibodies comprising: a) a first antigen-binding domain, a first modified hinge region, and a first heavy chain Fc domain; and b) The second antigen-binding domain and the second heavy chain Fc domain, wherein: The first modified hinge area includes: i) An upper hinge region with a length of at most 7 amino acids, or no upper hinge region; and ii) Lower hinge region, wherein the lower hinge region is connected to the N end of the first heavy chain Fc structural 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 inducing proximity between the first and second receptor subunits.

[0030] In another aspect, this article provides a multispecific binding protein comprising at least a first polypeptide chain, wherein: The first polypeptide chain includes a first variable heavy chain domain (VH1) connected to a second variable heavy chain domain (VH2) via at least one modified hinge region.

[0031] In some implementations, one or both of VH1 and VH2 are VH domains or VHH domains.

[0032] In some embodiments, the multispecific binding protein further comprises a second polypeptide chain, wherein the second polypeptide chain comprises a first variable light chain domain (VL1) connected to a second variable light chain domain (VL2) via at least one modified hinge region.

[0033] In some implementations, one or both of VH1 and VH2 are truncated at the C end.

[0034] In some implementations, the C-terminus is truncated by at least one residue.

[0035] In some implementations, the C-terminus is truncated by at least two residues.

[0036] In some implementations, the C-terminal SS amino acid residue is deleted.

[0037] In some embodiments, the multispecific binding protein comprises a first polypeptide chain of VH1-HX1-VH2-C-Fc, wherein: VH1 is the first heavy chain variable structural domain; VH2 is a second-chain variable structural domain; C is a heavy-chain constant structural domain; HX1 is a modified hinge area joint; and Fc is the Fc region; and The second polypeptide chain of VL1-LX1-VL2-C, in: VL1 is the first light chain variable structural domain; VL2 is the second light chain variable structural domain; C is a constant structural domain of the light chain; and LX1 is a modified hinge area joint.

[0038] In some embodiments, the modified hinge region contains or is composed of the amino acid sequence PLAP or PAPNLLGGP.

[0039] In some implementations, VH1 binds to the first receptor subunit and VH2 binds to the second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits.

[0040] In some embodiments, a first antigen-binding domain formed by VH1 and VL1 binds to a first receptor subunit, and a second antigen-binding domain formed by VH2 and VL2 binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits.

[0041] In some implementations, the first receptor subunit and the second receptor subunit are different subunits that form a heterodimer.

[0042] In some implementations, the first receptor subunit and the second receptor subunit are the same subunit that form the homodimer.

[0043] In some implementations, the first and second receptor subunits are selected from: tumor necrosis factor superfamily (TNFSF) receptors, type I interleukin receptors, type II interleukin receptors, Ig superfamily (IGSF) receptors, receptor tyrosine kinases (RTKs), growth hormone receptors, transforming growth factor β (TGFβ) receptor superfamily, type C lectin-like receptors, interferon receptors, phosphatase receptors (i.e., receptor-type protein tyrosine phosphatases), and integrin receptors.

[0044] In some implementations, the antigen-binding domain is a VHH containing a P14A amino acid substitution, according to the Kabat number.

[0045] In one aspect, this disclosure provides a multispecific binding protein comprising at least a first binding domain and a second binding domain, wherein the first binding domain is connected to the second binding domain via at least one modified hinge region.

[0046] In some implementations, 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).

[0047] In some implementations, one or both of VH1 and VH2 are VH domains or VHH domains.

[0048] In some embodiments, the multispecific binding protein further includes a first variable light chain domain (VL1) connected to a second variable light chain domain (VL2) via at least one modified hinge region.

[0049] In some implementations, the first binding domain is a first scFv, and the second binding domain is a second scFv.

[0050] In one aspect, this disclosure provides a multispecific binding protein comprising at least a first polypeptide chain, wherein: the first polypeptide chain comprises a first variable heavy chain domain (VH1) connected to a second variable heavy chain domain (VH2) via at least one modified hinge region.

[0051] In some implementations, one or both of VH1 and VH2 are VH domains or VHH domains.

[0052] In some embodiments, the multispecific binding protein further comprises a second polypeptide chain, wherein the second polypeptide chain comprises a first variable light chain domain (VL1) connected to a second variable light chain domain (VL2) via at least one modified hinge region.

[0053] In some implementations, one or both of VH1 and VH2 are truncated at the C end.

[0054] In some implementations, the C-terminus is truncated by at least one residue.

[0055] In some implementations, the C-terminus is truncated by at least two residues.

[0056] In some implementations, the C-terminal SS amino acid residue is deleted.

[0057] In some embodiments, the multispecific binding protein comprises a first polypeptide chain of VH1-HX1-VH2-C-Fc, wherein: VH1 is the first heavy chain variable structural domain; VH2 is a second-chain variable structural domain; C is a heavy-chain constant structural domain; HX1 is a modified hinge area joint; and Fc is the Fc region; and The second polypeptide chain of VL1-LX1-VL2-C, in: VL1 is the first light chain variable structural domain; VL2 is the second light chain variable structural domain; C is a constant structural domain of the light chain; and LX1 is a modified hinge area joint.

[0058] In some embodiments, the modified hinge region includes: i) an upper hinge region with a length of up to 7 amino acids or no upper hinge region; and ii) a lower hinge region.

[0059] In some embodiments, the modified hinge region contains or is composed of the amino acid sequence PLAP or PAPNLLGGP.

[0060] In some implementations, 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.

[0061] In some implementations, VH1 binds to the first receptor subunit and VH2 binds to the second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits.

[0062] In some embodiments, a first antigen-binding domain formed by VH1 and VL1 binds to a first receptor subunit, and a second antigen-binding domain formed by VH2 and VL2 binds to a second receptor subunit, thereby inducing signal transduction by inducing proximity between the first and second receptor subunits.

[0063] In some implementations, 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.

[0064] In some implementations, the first receptor subunit and the second receptor subunit are different subunits that form a heterodimer.

[0065] In some implementations, the first receptor subunit and the second receptor subunit are the same subunit that form the homodimer.

[0066] In some implementations, the first and second receptor subunits are selected from: tumor necrosis factor superfamily (TNFSF) receptors, type I interleukin receptors, type II interleukin receptors, Ig superfamily (IGSF) receptors, receptor tyrosine kinases (RTKs), growth hormone receptors, transforming growth factor β (TGFβ) receptor superfamily, type C lectin-like receptors, interferon receptors, phosphatase receptors (i.e., receptor-type protein tyrosine phosphatases), and integrin receptors.

[0067] In some implementations, the antigen-binding domain is a VHH containing a P14A amino acid substitution, according to the Kabat number.

[0068] In some implementations, the P14A amino acid substitution further stabilizes the multispecific binding protein.

[0069] In some implementations, P14A amino acid substitution enhances the agonist properties of multispecific binding proteins.

[0070] In one aspect, the present disclosure provides a multispecific binding protein comprising a first polypeptide chain and a second polypeptide chain, wherein each of the first and second polypeptide chains comprises, from the N-terminus to the C-terminus, a first single-chain variable fragment (scFv) linked to the second scFv; wherein the first scFv is linked to the second scFv via at least one modified hinge region.

[0071] In some embodiments, the modified hinge region includes: i) an upper hinge region with a length of up to 7 amino acids or no upper hinge region; and ii) a lower hinge region.

[0072] In some embodiments, the modified hinge region contains or is composed of the amino acid sequence PLAP or PAPNLLGGP.

[0073] In some implementations, 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.

[0074] In some implementations, the first receptor subunit and the second receptor subunit are different subunits that form a heterodimer.

[0075] In some implementations, the first receptor subunit and the second receptor subunit are the same subunit that form the homodimer.

[0076] In some implementations, the first and second receptor subunits are selected from: tumor necrosis factor superfamily (TNFSF) receptors, type I interleukin receptors, type II interleukin receptors, Ig superfamily (IGSF) receptors, receptor tyrosine kinases (RTKs), growth hormone receptors, transforming growth factor β (TGFβ) receptor superfamily, type C lectin-like receptors, interferon receptors, phosphatase receptors (i.e., receptor-type protein tyrosine phosphatases), and integrin receptors.

[0077] In some implementations, the multispecific binding protein also includes a heavy chain constant region.

[0078] In some implementations, according to the EU designation, the heavy chain constant region includes a substitution at amino acid position 234.

[0079] In some implementations, the substitution at amino acid position 234 is alanine (A).

[0080] In some implementations, according to the EU designation, the heavy chain constant region includes a substitution at amino acid position 235.

[0081] In some implementations, the substitution at amino acid position 235 is alanine (A).

[0082] In some implementations, according to the EU designation, the heavy chain constant region includes a substitution at amino acid position 237.

[0083] In some implementations, the substitution at amino acid position 237 is alanine (A).

[0084] In some implementations, according to the EU designation, the heavy chain constant region includes one or more substitutions at amino acid positions 234, 235, or 237.

[0085] 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).

[0086] In some implementations, the heavy chain constant region contains heterodimerization mutations to promote heterodimerization of the first binding moiety with the second binding moiety.

[0087] In some implementations, the heterodimerization mutation is the kilomerization (KIH) mutation.

[0088] In some embodiments, the first heavy chain constant region includes an amino acid substitution for producing a mortar at position 366, 368, or 407, and the second heavy chain constant region includes an amino acid substitution for producing a pestle at position 366.

[0089] In some implementations, the first heavy chain constant region contains amino acid substitutions T366S, L368A, or Y407V, and the second heavy chain constant region contains amino acid substitution T366W.

[0090] In some implementations, the heterodimerization mutation is a charge stabilization mutation.

[0091] In some implementations, the first heavy chain constant region contains amino acid substitution N297K, and the second heavy chain constant region contains amino acid substitution N297D.

[0092] In some implementations, the first heavy chain constant region contains the amino acid substitution T299K, and the second heavy chain constant region contains the amino acid substitution T299D.

[0093] In some implementations, the heterodimerization mutation includes engineered disulfide bonds.

[0094] In some implementations, the engineered disulfide bond is formed by a first heavy chain constant region containing amino acid substitution Y349C and a second heavy chain constant region containing amino acid substitution S354C.

[0095] In some implementations, the engineered disulfide bond is formed by a C-terminal extension peptide fused to the C-terminus of each of the first heavy chain constant region and the second heavy chain constant region.

[0096] In some embodiments, the C-terminal extension of the first heavy chain constant region contains the amino acid sequence GEC, and the C-terminal extension of the second heavy chain constant region contains the amino acid sequence SCDKT.

[0097] In some implementations, at least one heavy chain constant region contains one or more mutations to promote half-life extension.

[0098] In some implementations, according to the EU designation, at least one heavy chain constant region comprises one or more substitutions at amino acid positions 252, 254, or 256.

[0099] 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).

[0100] In some implementations, according to EU designations, at least one heavy chain constant region comprises one or more substitutions at amino acid positions 428 or 434.

[0101] In some implementations, according to the EU designation, at least one heavy chain constant region contains M428L and N434S substitutions.

[0102] In another aspect, this article provides a pharmaceutical composition comprising a bispecific antibody as provided herein and a pharmaceutically acceptable carrier.

[0103] In one aspect, this document provides isolated nucleic acid molecules encoding bispecific antibodies as provided herein. In some embodiments, the expression vector comprises the nucleic acid molecules provided herein. In some embodiments, the host cell comprises the expression vector.

[0104] In another aspect, this article provides a method for treating a disease or condition in a subject, the method comprising administering a bispecific antibody, as provided herein, to the subject in need.

[0105] In another respect, bispecific antibodies, as described herein, are used as pharmaceuticals. In yet another respect, bispecific antibodies, as described herein, are used as diagnostic reagents.

[0106] In another aspect, this disclosure provides a method for inducing signal transduction between a first receptor subunit and a second receptor subunit in a subject, the method comprising administering to the subject a multispecific binding protein provided herein.

[0107] In some implementations, multispecific binding proteins can induce signal transduction by inducing proximity between the first receptor subunit and the second receptor subunit.

[0108] In some embodiments, this multispecific binding protein exhibits stronger agonist activity compared to multispecific binding proteins lacking at least one modified hinge region.

[0109] In some embodiments, the multispecific binding protein-induced agonist activity 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 natural ligand for the first and second receptor subunits. Attached Figure Description

[0110] Figure 1 This is a diagram illustrating certain exemplary embodiments of the bispecific antibody format described herein.

[0111] Figure 2 This is a schematic diagram depicting the workflow for characterizing the bispecific antibodies of this disclosure.

[0112] Figures 3A-3C This is a diagram depicting arteriovenous malformations (AVMs) in the retina of an HHT mouse model. Figure 3A Mice treated with DGL288 (15 mg / kg / day) were shown compared to control mice (without bispecific antibody). Mice treated with DGL288 did not develop detectable AVMs compared to the control mice. Figure 3B Mice treated with 1 mg / kg / day of DGL292 did not develop AVMs compared to mice treated with control mice. Figure 3C This indicates that DGL288 administered at a dose of 1 mg / kg / day did not form AVMs, compared to mice treated with the control group.

[0113] Figure 4 This is a diagram depicting arteriovenous malformations (AVMs) in the retina of a HHT mouse model. Mice were treated with a control (without bispecific antibodies) compared to DGL292, DGL945, and DGL947 (1 mg / kg / day). Mice treated with DGL292, DGL945, and DGL947 did not develop detectable AVMs compared to the control.

[0114] Figure 5 This is a graph illustrating the agonistic effects of DGL207, DGL333, and DGL620 in the HEK Blue assay. Detailed Implementation

[0115] Before describing this disclosure, it should be understood that this disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive, as the scope of this disclosure will be limited only by the appended claims.

[0116] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0117] Although any methods and materials similar to or equivalent to those described herein may be used in practice with this disclosure, exemplary methods and materials are described hereafter. All publications mentioned herein are incorporated herein by reference for their full description.

[0118] As used herein, the term “antibody / antibodies” includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules containing antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinant antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intracellular antibodies, heterologous conjugates, antibody-drug conjugates, single-domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camel-derived antibodies, affinity antibodies, common light chain antibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), bivariate domain (DVD) antibodies, and antigen-binding fragments of any of the above. In some embodiments, the antibodies described herein refer to a population of polyclonal antibodies. Antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecules. In some embodiments, the antibodies described herein are IgG antibodies, or their class (e.g., human IgG1 or IgG4) or subclass. As used herein, the terms “VH” and “VL” refer to the variable domain of the antibody heavy chain and the variable domain of the light chain, 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.

[0119] As used herein, the terms “antigen-binding moiety,” “binding domain,” or “binding specificity” refer to a molecule that specifically binds to an antigen, as understood by those skilled in the art. For example, an antigen-binding moiety that specifically binds to an antigen typically binds to other molecules with lower affinity, as determined by, for example, immunoassays, BIAcore®, KinExA 3000 instruments (Sapidyne Instruments, Boise, ID), or other assays known in the art. In some embodiments, the Ka of the antigen-binding moiety that specifically binds to the antigen is at least two logarithms (e.g., multiples of 10), 2.5 logarithms, three logarithms, four logarithms, or more of the Ka of the molecule when it nonspecifically binds to another antigen.

[0120] As used herein, the term “VHH” refers to the heavy chain variable domain of camel-only heavy chain antibodies (HCAbs) and its humanized variants, as described in Hamers-Casterman C. et al., Nature (1993) 363:446–8.10.1038 / 363446a0, which is incorporated herein by reference in its entirety.

[0121] As used in this article, the term "VH / VL pair" refers to the combination of VH and VL that together form an antigen-binding site.

[0122] As used herein, the term “heavy chain” when used to refer to antibodies can refer to any different type based on the amino acid sequence of the constant region, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (µ), which produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, respectively, where IgG includes subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4.

[0123] As used in this article, the term "full-length antibody heavy chain" refers to an antibody heavy chain that includes VH, CH1 region, hinge region, CH2 domain and CH3 domain from the N-terminus to the C-terminus.

[0124] As used herein, the term "light chain" when used to refer to an antibody can refer to any different type of amino acid sequence based on a constant structural domain, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In a particular embodiment, the light chain is a human light chain. As used herein, the term "complementarity-determining region" or "CDR" refers to the sequence of amino acids within the variable region of the antibody that confers antigen specificity and binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Frame region" or "FR," as known in the art, refers to the non-CDR portion of the variable regions of both the heavy and light chains. Generally, there are four FRs (FR-H1, FR-H2, FR-H3 and FR-H4) in each heavy chain variable region and four FRs (FR-L1, FR-L2, FR-L3 and FR-L4) in each light chain variable region.

[0125] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of recognized schemes, including those described below: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD. (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., January 2003;27(1):55-77 (“IMGT” numbering scheme); and Honegger A. and Pluckthun A., “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J. Mol. Biol., June 2001. 8; 309(3):657-70, (Aho numbering scheme).

[0126] The boundaries of a given CDR or FR can vary depending on the scheme used for identification. For example, the Kabat scheme is based on sequence alignment, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes number antibodies based on the length of the most common antibody region sequence, where insertions are handled by an insertion letter (e.g., "30a"), while deletions occur in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in several ways.

[0127] As used herein, the term "single-chain variable fragment" (scFv) refers to a fusion protein comprising at least one antibody fragment containing a light chain variable region and at least one antibody fragment containing a heavy chain variable region, wherein the light chain variable region and the heavy chain variable region are sequentially linked via a short, flexible polypeptide linker and are capable of being expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise stated, as used herein, the scFv may have VL and VH variable regions in either order; for example, with respect to the N-terminus and C-terminus of the polypeptide, the scFv may comprise a VL-linker-VH or may comprise a VH-linker-VL.

[0128] As used herein, the term "human antibody" is intended to include antibodies having variable domains and Fc domains derived from human germline immunoglobulin sequences. Human mAbs of this disclosure may, for example, include amino acid residues in the CDR (particularly in CDR3) that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term "human antibody" as used herein is not intended to include mAbs in which a CDR sequence derived from another mammalian species (e.g., mouse) has been grafted onto a human FR sequence. The term includes antibodies recombinantly produced in non-human mammals or in the cells of non-human mammals. The term is not intended to include antibodies isolated or generated from human subjects.

[0129] As used herein, the term "multispecific antigen-binding molecule" refers to a bispecific, trispecific, or multispecific antigen-binding molecule and its antigen-binding fragment. A multispecific antigen-binding molecule may be specific to different epitopes of a target polypeptide, or may contain antigen-binding domains specific to epitopes of more than one target polypeptide. In some embodiments, the multispecific antigen-binding molecule of this disclosure comprises at least a first binding specificity against a subunit of a receptor and at least a second binding specificity against the subunit. A multispecific antigen-binding molecule may be a single multifunctional polypeptide, or it may be a multimeric complex of two or more polypeptides bound together covalently or non-covalently. The term "multispecific antigen-binding molecule" includes antibodies of this disclosure that can be linked to or co-expressed with another functional molecule (e.g., another peptide or protein). For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent binding, or other means) to one or more other molecular entities (such as proteins or fragments thereof) to produce a bispecific or multispecific antigen-binding molecule having a second binding specificity. According to this disclosure, the term "multispecific antigen-binding molecule" also includes bispecific, trispecific, or multispecific antibodies or their antigen-binding fragments. In some exemplary embodiments, the antibody function of this disclosure is linked to another antibody or its antigen-binding fragment to produce a bispecific antibody with a second binding specificity.

[0130] In exemplary embodiments, the heteropolymer antibody of this disclosure is a bispecific antibody. A bispecific antibody may be a monoclonal antibody having binding specificity against at least two different antigens, such as a human antibody or a humanized antibody. In some embodiments, the bispecific antibody of this disclosure comprises at least a first binding domain against a receptor subunit and at least a second binding domain against another receptor subunit.

[0131] Methods for preparing bispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537 (1983)). Due to the random allocation of the immunoglobulin heavy and light chains, hybridomas (tetragenic hybridomas) can produce a potential mixture of ten different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is typically accomplished via an affinity chromatography step. More modern bispecific antibody generation techniques employ heterodimerization domains that facilitate the desired pairing of the heavy chain of the antibody with the first specificity to the heavy chain of the antibody with the second specificity.

[0132] Antibody variable domains with the desired binding specificity can be fused with immunoglobulin constant domain sequences. The fusion typically contains an immunoglobulin heavy chain constant domain, which includes at least a portion of the hinge region, the CH2 region, and the CH3 region. At least one of the fusions may have a first heavy chain Fc domain (CH1) containing the site required for light chain binding. DNA encoding the immunoglobulin heavy chain fusion and (if desired) the immunoglobulin light chain is inserted into separate expression vectors and co-transformed into a suitable host organism. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Meth. Enzymol. 121:210 (1986).

[0133] 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 linked to antibody light chains (e.g., forming haptens) via disulfide bonds.

[0134] In some embodiments, the Fc chain begins in the hinge region, immediately upstream of the papain cleavage site, and terminates at the C-terminus of the antibody. Therefore, a complete Fc chain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, the Fc chain comprises at least one of the following: a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or variants, portions, or fragments thereof. In some embodiments, the Fc domain comprises the complete Fc chain (i.e., the hinge domain, the CH2 domain, and the CH3 domain). In some embodiments, the Fc chain comprises a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In some embodiments, the Fc chain comprises a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In some embodiments, the Fc chain consists of a CH3 domain or a portion thereof. In some embodiments, the Fc chain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In some embodiments, the Fc chain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In some embodiments, the Fc chain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In some embodiments, the Fc chain lacks at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). As used herein, Fc chain generally refers to a polypeptide comprising all or part 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, and fragments of such peptides comprising only, for example, the hinge, CH2, and CH3 domains. Fc chains can be derived 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 both native Fc and Fc variant molecules. As with Fc variants and native Fc, the term Fc chain includes molecules in monomeric or multimeric form, whether derived from digestion of a whole antibody or otherwise produced. In some embodiments, the Fc chain comprises the carboxyl-terminal portions of two heavy chains held together by disulfide bonds. In some embodiments, the Fc chain consists of a CH2 domain and a CH3 domain.

[0135] In some embodiments, the Fc polypeptide contains part or all of the wild-type hinge sequence (typically located at its N-terminus). In some embodiments, the Fc polypeptide does not contain a functional or wild-type hinge sequence.

[0136] 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). The term includes both naturally occurring CH1 domains and engineered variants of naturally occurring CH1 domains (e.g., CH1 domains containing one or more amino acid insertions, deletions, substitutions, or modifications relative to naturally occurring CH1 domains).

[0137] 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 both naturally occurring CH2 domains and engineered variants of naturally occurring CH2 domains (e.g., CH2 domains containing one or more amino acid insertions, deletions, substitutions, or modifications relative to naturally occurring CH2 domains).

[0138] As used herein, the term "CH3 domain" refers to the third constant domain of an antibody heavy chain (e.g., amino acid positions 341-447 of human IgG1 according to the EU index). This term includes both naturally occurring CH3 domains and engineered variants of naturally occurring CH3 domains (e.g., CH3 domains containing one or more amino acid insertions, deletions, substitutions, or modifications relative to naturally occurring CH3 domains).

[0139] As used herein, the term "hinge region" refers to a portion of the antibody heavy chain containing cysteine ​​residues (e.g., cysteine ​​residues at amino acid positions 226 and 229 of human IgG1, according to the EU index) that mediate disulfide bonding between the two heavy chains in a complete antibody. The hinge region can be divided into three peptide regions: the upper hinge, the middle hinge, and the lower hinge. The term includes naturally occurring hinge regions and engineered variations of naturally occurring hinge regions (e.g., hinge regions containing one or more amino acid insertions, deletions, substitutions, or modifications relative to naturally occurring hinge regions). According to the EU index, an exemplary full-length IgG1 hinge region contains amino acid positions 216-230 of human IgG1. The hinge region may consist of at least two (e.g., five, ten, fifteen, twenty, forty, sixty, or more) amino acids, resulting in flexible or semi-flexible connections between adjacent variable regions and / or constant domains within a single polypeptide molecule. In some implementations, the immunoglobulin-like hinge region may be derived from or originate from any IgG1, IgG2, IgG3 or IgG4 subtype, or from IgA, IgE, IgD or IgM, including their chimeric forms.

[0140] In some embodiments, the hinge region may originate from a human IgG1 subtype extending from amino acid 216 to amino acid 230 according to the EU index numbering system, or from a human IgG1 subtype extending from amino acid 226 to amino acid 243 according to the Kabat numbering system. Those skilled in the art may have different understandings of the exact amino acids corresponding to the various domains of the IgG molecule. Therefore, the N-terminus or C-terminus of the domains outlined above may be extended or shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, or even 10 amino acids.

[0141] As used herein, the term "upper hinge" generally refers to the last residue of the CH1 domain up to, but not including, the first heavy chain metacysteine. The upper hinge can sometimes be defined as the N-terminal sequence from position 216 to position 225 in an IgG1 antibody, according to the Kabat EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition. Public Health Service, National Institute of Health, Bethesda, Md., 1991). The term "middle hinge" refers to the region extending from the first heavy chain metacysteine ​​to the proline residue adjacent to the carboxyl terminus of the last middle hinge cysteine. The middle hinge can be the N-terminal sequence from position 226 to position 230, according to the Kabat EU numbering system. The term "lower hinge" refers to a highly conserved 7-8 amino acids. The lower hinge can be defined as the sequence from position 231 to 238 in an IgG1 antibody, according to the Kabat EU numbering system. In some embodiments, the antibody according to the invention actually comprises an upper hinge, a middle hinge, and a lower hinge.

[0142] As used herein, the term "modified hinge region" refers to a hinge region in which one or more characteristics of the hinge are altered relative to the wild-type hinge, including but not limited to flexibility, length, conformation, charge, and hydrophobicity. The modified hinge regions disclosed herein can be generated by methods well known in the art, such as, for example, introducing modifications into a wild-type hinge. In some embodiments, the hinge region may be modified with one or more amino acids. Modifications that can be used to generate modified hinge regions include, but are not limited to, amino acid insertions, deletions, substitutions, and rearrangements. The disclosed hinges and the modifications to the modified hinge regions are collectively referred to herein as "hinge modifications of the present invention," "modified hinges of the present invention," or simply "hinge modifications" or "modified hinges." The modified hinge regions disclosed herein may be incorporated into selected molecules (including but not limited to antibodies and fragments thereof). In some embodiments, the hinge region may be truncated and contain only a portion of the complete hinge region. As demonstrated herein, molecules containing modified hinges can exhibit altered (e.g., enhanced) agonistic activity when compared to molecules having the same amino acid sequence except for the modified hinge (such as molecules having the same amino acid sequence except for containing a wild-type hinge). In some implementations The protocol includes antibodies. The modified hinge region, wherein the upper hinge region is at most 7 amino acids in length. In some embodiments, the upper hinge region is absent. In some embodiments, the modified hinge is a modified IgG1 linker. In some embodiments, the modified IgG1 hinge is derived from the sequence PLAPDKTHT (SEQ ID NO: 1). In some embodiments, the modified IgG1 hinge comprises the sequence PLAP (SEQ ID NO: 2). In some embodiments, the modified IgG1 hinge comprises 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 comprises the sequence EKSYGPP (SEQ ID NO: 4). In some embodiments, the modified hinge is a Gly / Ser hinge. In some embodiments, the Gly / Ser hinge comprises the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3). In some embodiments, the C-terminal residues of the variable domain adjacent to the upper hinge are truncated. In some embodiments, at least one residue of the variable domain adjacent to the upper hinge is truncated. In some embodiments, at least two residues of the variable domain adjacent to the upper hinge are truncated. In some embodiments, the C-terminal SS amino acid of the heavy chain variable domain is deleted.

[0143] The modified hinge region of this disclosure can be used as a connector to connect one or more antigen-binding domains of this disclosure. In some embodiments, a first variable heavy chain domain (VH1) is connected to a second variable heavy chain domain (VH2) via at least one modified hinge region. In some embodiments, a first variable light chain domain (VL1) is connected to a second variable light chain domain (VL2) via at least one modified hinge region. VH1 and VL1 bind to form a first antigen-binding domain, and VH2 and VL2 bind to form a second antigen-binding domain. In other embodiments, a first scFv is connected to a second scFv via at least one modified hinge region.

[0144] In some 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) exhibit stronger agonist activity compared to multispecific binding proteins lacking at least one modified hinge region. For example, but not limited to, multispecific binding proteins 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 stronger agonist activity for target receptor pairs (e.g., VH1 / VL1 binding to a first receptor subunit and VH2 / VL2 binding to a second receptor subunit) compared to the same multispecific binding protein lacking at least one modified hinge region.

[0145] As used herein, the term “EU index” refers to the EU numbering convention for the Fc domain of antibodies, as described in Edelman, GM. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety. All amino acid positions of Fc peptides or fragments thereof used herein are numbered according to the EU index.

[0146] In some implementations, the term "linker" refers to 1-100 consecutive amino acid residues. Typically, linkers provide flexibility and spatial separation between two amino acids or between two polypeptide domains. Linkers can be inserted between VH, VL, CH, and / or CL domains to provide sufficient flexibility and mobility to the light and heavy chain domains, depending on the molecular format. At the amino sequence level, linkers are typically inserted at transitions between variable domains, between a variable domain and a knockout domain, or between a variable domain and a constant domain. Because the approximate size of immunoglobulin domains is well understood, transition regions between domains can be identified. The precise location of domain transition regions can be determined by locating peptides that do not form secondary structural elements (such as β-sheets or α-helices), as can be demonstrated by experimental data or determined by modeling or secondary structure prediction techniques.

[0147] As used herein, the terms “specific binding,” “specifically binding,” “binding specificity,” or “specific recognition” refer to an antigen-binding protein or antigen-binding fragment thereof that exhibits considerable affinity for an antigen (e.g., IL-18R antigen) and does not exhibit significant cross-reactivity with 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 antigen-binding fragment thereof and the epitope to which it binds. In some exemplary embodiments, affinity is measured by surface plasmon resonance (SPR), for example in a Biacore instrument. As will be readily understood by those skilled in the art, antigen-binding protein affinity can be reported as a dissociation constant (KD) in molar concentration (M). The antigen-binding protein or antigen-binding fragment thereof of this disclosure has approximately 10 -5 M to approximately 10 -12 M (i.e., the range from micromoles to picomoles), approximately 10 -7 M to 10 -11 M, approximately 10 -8 M to approximately 10 -10 M, approximately 10 -9 KD values ​​within the M range. In some embodiments, the antigen-binding protein or its antigen-binding fragment has a KD value of approximately 10. -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 -12 The binding affinity of M. In some embodiments, the antigen-binding protein or its antigen-binding fragment has approximately 10. -7 M to approximately 10 -9 Binding affinity in the M (nanomolar range).

[0148] Specific binding can be determined using any means recognized in the art for determining such binding. In some embodiments, specific binding is determined by a competitive binding assay (e.g., ELISA) or a Biacore assay. In some embodiments, the assay is performed at about 20°C, 25°C, 30°C, or 37°C.

[0149] As used herein, “administer / administration” means the act of delivering a substance present outside the body (e.g., the isolated binding peptides provided herein) to a patient by injection or other physical means, such as through, but not limited to, the lungs (e.g., inhalation), mucous membranes (e.g., intranasal), intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When a disease or its symptoms are being managed or treated, the administration of the substance typically occurs after the onset of the disease or its symptoms. When a disease or its symptoms are being prevented, the substance is typically administered before the onset of the disease or its symptoms, and may be administered continuously over a long period to delay or reduce the onset or intensity of disease-related symptoms.

[0150] As used herein, the term "composition" is intended to cover products containing, optionally in specified amounts, specified components (e.g., isolated binding polypeptides provided herein), and any product produced directly or indirectly from a combination of specified components optionally in specified amounts.

[0151] "Effective amount" means the amount of an active pharmaceutical agent (e.g., the isolated binding polypeptide of this disclosure) sufficient to achieve the desired physiological outcome in an individual who requires the agent. Effective amounts can vary from individual to individual, depending on the health and physical condition of the individual being treated, the individual's taxonomy, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0152] As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, a subject may be a mammal, such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, mice, etc.) or a primate (e.g., monkeys and humans). In some embodiments, as used herein, the term “subject” refers to a vertebrate, such as a mammal. Mammals include, but are not limited to, humans, non-human primates, wild animals, rewilded animals, farm animals, sporting animals, and pets.

[0153] As used herein, the term "therapy" means any protocol, method, and / or agent that can be used to prevent, manage, treat, and / or improve a disease or related symptoms. In some embodiments, the term "therapy" means any protocol, method, and / or agent that can be used to modulate a subject's immune response to an infection or related symptoms. In some embodiments, the term "therapies" means biological therapies, supportive therapies, and / or other therapies known to those skilled in the art (such as medical personnel) that can be used to prevent, manage, treat, and / or improve a disease or related symptoms. In other embodiments, the term "therapies" means biological therapies, supportive therapies, and / or other therapies known to those skilled in the art (such as medical personnel) that can be used to modulate a subject's immune response to an infection or related symptoms.

[0154] As used herein, the term "treat / treatment / treating" refers to the reduction / shortening or improvement of the progression, severity, and / or duration of disease or related symptoms resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, such as the isolated binding peptides provided herein). The term "treatment" as used herein may also refer to altering the course of disease in the treated subject. The therapeutic effects of treatment include, but are not limited to: preventing the onset or recurrence of disease, alleviating symptoms, weakening the direct or indirect pathological consequences of disease, slowing the rate of disease progression, improving or alleviating disease status, and mitigating or improving prognosis.

[0155] The term “about” or “approximately” means about 20% of a given value or range, such as about 10%, about 5%, or about 1% or less.

[0156] Combined structural domain One component of the bispecific antibody disclosed herein is one or more antigen-binding domains or binding specificities that bind to a first cell surface target and a second cell surface target. In some embodiments, the first cell surface target is a first receptor subunit, and the second cell surface target is a second receptor subunit.

[0157] Any type of binding moiety that specifically binds to a particular receptor subunit may be used in the bispecific antibodies disclosed herein. In some embodiments, the binding moiety includes an antibody variable domain. Exemplary binding moieties including antibody variable domains include, but are not limited to, VH, VL, VHH, VH / VL pairs, scFv, biantibodies, or Fab. Other suitable binding modalities include, but are not limited to, lipid transport proteins (see, for example, Gebauer M. et al., 2012, Method Enzymol. 503:157–188, which is incorporated herein by reference in its entirety), adnectin (see, for example, Lipovsek D., 2011, Protein Eng. Des. Sel. 24:3–9, which is incorporated herein by reference in its entirety), avimer (see, for example, Silverman J et al., 2005, Nat. Biotechnol. 23:1556–1561, which is incorporated herein by reference in its entirety), fynomer (see, for example, Schlatter D et al., 2012, mAbs 4:497–508, which is incorporated herein by reference in its entirety), and kunitz domains (see, for example, Hosse RJ et al., 2006, Protein Sci.). 15:14–27, which is incorporated herein by reference in its entirety, knottin (see, for example, Kintzing JR et al., 2016, Curr. Opin. Chem. Biol. 34:143–150, which is incorporated herein by reference in its entirety), affinity peptides (see, for example, Feldwisch J et al., 2010 J. Mol. Biol. 398:232–247, which is incorporated herein by reference in its entirety), and DARPin (see, for example, Pluckthun A., 2015, Annu. Rev. Pharmacol. Toxicol. 55:489–511, which is incorporated herein by reference in its entirety).

[0158] In some embodiments, the binding domain comprises a heavy chain and / or light chain variable region of a conventional antibody or its antigen-binding fragment (e.g., Fab or scFv), wherein the term "conventional antibody" is used herein to describe a heterotetrameric antibody containing heavy and light chain immunoglobulins arranged according to a "Y" conformation. Such conventional antibodies may be derived from any suitable species, including but not limited to antibodies derived from llamas, alpacas, camels, mice, rats, rabbits, goats, hamsters, chickens, monkeys, or humans. In some exemplary embodiments, the conventional antibody comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH and / or VL domains, or one or more of their complementarity-determining regions (CDRs), are derived from the same antibody. In some embodiments, the antigen-binding region of the conventional antibody may be referred to as "Fab" (antigen-binding fragment). Fab comprises a constant domain and a variable domain from each of the heavy and light chains. The variable heavy and light chains contain CDRs responsible for antigen binding.

[0159] In other embodiments, the specific receptor subunit binding subunit contains at least the CDR or VHH domain of the VHH antibody or nanobody. VHH antibodies are heavy-chain antibodies derived from camels, consisting of two heavy chains and lacking a light chain (Hamers-Casterman et al., Nature. 1993; 363; 446-8). Each heavy chain of the VHH antibody has a variable domain at its N-terminus, and these variable domains are referred to in the art as “VHH” domains to distinguish them from the variable domains (i.e., VH domains) of the heavy chains of conventional antibodies. Similar to conventional antibodies, the VHH domain of this molecule contains HCDR1, HCDR2, and HCDR3 regions that confer antigen-binding specificity, and therefore VHH antibodies or fragments (such as isolated VHH domains) are suitable as components of the multispecific binding proteins of this disclosure.

[0160] Multispecific binding protein In some embodiments, the first and second binding domains disclosed herein may be paired or effectively linked to generate a multispecific binding protein capable of crosslinking a first and second subunit of a given receptor (e.g., human IL-18 receptor). In some embodiments, the first specific binding domain (e.g., VHH or scFv) is effectively linked (directly or indirectly) to the N-terminus and / or C-terminus of the first Fc domain or peptide, and the second specific binding domain is effectively linked to the N-terminus and / or C-terminus of the second Fc domain or peptide, such that the first and second Fc domains promote heterodimerization of the first and second specific binding domains.

[0161] As used herein, the term "dual variable domain" or "DVD" refers to a binding protein containing two or more antigen-binding sites and is tetravalent or multivalent. DVDs can be monospecific, meaning they bind to one antigen, or multispecific, meaning they bind to two or more antigens. DVD-binding proteins containing two heavy-chain DVD peptides and two light-chain DVD peptides are referred to as "DVD immunoglobulins" or "DVD-Ig." Each half of a DVD-Ig contains a heavy-chain DVD peptide and a light-chain DVD peptide, as well as two or more antigen-binding sites. Each binding site contains a heavy-chain variable domain and a light-chain variable domain, and each antigen-binding site has a total of six CDRs involved in antigen binding.

[0162] The design, expression, and characterization of the DVD-Ig molecule are described in PCT Publication No. WO 2007 / 024715, U.S. Patent No. 7,612,181, and Wu et al., Nature Biotechnol., 25: 1290-1297 (2007). Preferred examples of such DVD-Ig molecules include: a heavy chain comprising the structural formula VD1-(X1)n-VD2-C-(X2)n, wherein 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, wherein 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 a DVD-Ig may contain two such heavy chains and two such light chains, each chain containing tandemly linked variable domains and constant regions without spacers between the variable domains, wherein the heavy and light chains bind to form tandemly linked functional antigen-binding sites, and one pair of heavy and light chains may bind to another pair of heavy and light chains to form a tetrameric binding protein with four functional antigen-binding sites. In another example, a DVD-Ig molecule may contain heavy and light chains, each containing three tandemly linked variable domains (VD1, VD2, VD3) and constant regions without spacers between the variable domains, wherein one pair of heavy and light chains may bind to form three antigen-binding sites, and wherein one pair of heavy and light chains may bind to another pair of heavy and light chains to form a tetrameric binding protein with six antigen-binding sites.

[0163] In embodiments, this disclosure provides a binding protein comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein: 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 independently 0 or 1; and wherein the second polypeptide chain comprises a second VD1-(X1)n-VD2-C-(X2)n, wherein: 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 independently 0 or 1.

[0164] Regarding the construction of DVD-Ig or other binding protein molecules, the term "linker" is used to refer to a single amino acid or polypeptide ("linker polypeptide") containing two or more amino acid residues linked by peptide bonds and 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)). Flexible linkers may be used, which are typically composed of small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. Exemplary flexible joints include, but are not limited to, GGGGSG (SEQ ID NO: ##), GGSGG (SEQ ID NO: ##), GGGGSGGGGS (SEQ ID NO: ##), GGSGGGGSGS (SEQ ID NO: ##), GGSGGGGSGGGGS (SEQ ID NO: ##), GGGGSGGGGSGGG (SEQ ID NO: ##), GGGGSGGGGSGGGGS (SEQ ID NO: ##), and RADAAAAGGGGSGGGGGSGGGGSGGGGS (SEQ ID NO: ##).

[0165] Alternatively, rigid connectors can be used to attach one or more antigen-binding proteins. The rigid connector allows maintaining a fixed distance between the attached antigen-binding proteins, thereby promoting the activity of each individual protein. Rigid connectors can be given rigidity using one of a variety of proline amino acids. Exemplary flexible joints include, but are not limited to, 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: ##), RADAAAAGGPGS (SEQ ID NO: ##), SAKTTPKLEEGEFSEARV (SEQ ID NO: ##), ADAAP (SEQ ID NO: ##), ADAAPTVSIFPP (SEQ ID NO: ##), QPKAAP (SEQ ID NO: ##), and others. ##), QPKAAPSVTLFPP (SEQ ID NO: ##), AKTTPP (SEQ ID NO: ##), AKTTPPSVTPLAP (SEQ ID NO: ##), AKTTAP (SEQ ID NO: ##), AKTTAPSVYPLAP (SEQ ID NO: ##), GENKVEYAPALMALS (SEQ ID NO: ##), GPAKELTPLKEAKVS (SEQ ID NO: ##) and GHEAAAVMQVQYPAS (SEQ ID NO: ##).

[0166] In some implementations, the joint includes a modified hinge area as described herein.

[0167] In some implementations, the connector comprises PLAP, PAPNLLGGP, PLAPDKTHT, EKSYGPP, or DKTHT, or a combination thereof.

[0168] In some embodiments, the multispecific binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises VH1-(HX1)n-VH2-C-(HX2)n, wherein: VH1 is the first heavy chain variable structure domain; VH2 is the second heavy chain variable structure domain; C is the heavy chain constant structure domain; HX1 is the linker; HX2 is the Fc region; and n is independently 0 or 1; and The second polypeptide chain comprises VL1-(LX1)n-VL2-C-(LX2)n, wherein: VL1 is the first variable structural domain of the light chain; VL2 is the second variable structural domain of the light chain; C is the constant structural domain of the light chain; LX1 is the connector; LX2 does not contain the Fc region; and n is independently 0 or 1.

[0169] In some implementations, the linker HX1 contains an amino acid sequence of PLAP or PAPNLLGGP.

[0170] In some implementations, the linker LX1 contains the amino acid sequence PLAP or PAPNLLGGP.

[0171] In some embodiments, the linker HX1 contains the amino acid sequence of PLAP, and the linker LX1 contains the amino acid sequence of PLAP or PAPNLLGGP.

[0172] In some embodiments, the multispecific binding protein comprises two polypeptide chains, VH1-(HX1)n-VH2-C-(HX2)n and VL1-(LX1)n-VL2-C-(LX2)n.

[0173] In some implementations, n is 1 for (HX1)n and n is 1 for (HX2)n.

[0174] In some implementations, n is 1 for (LX1)n and n is 0 for (LX2)n.

[0175] In some embodiments, the multispecific binding protein comprises a first polypeptide chain and a second polypeptide chain, wherein: The first polypeptide chain comprises VH1-(HX1)n-VH2-C-Fc, wherein: VH1 is the first heavy chain variable structure domain; VH2 is the second heavy chain variable structure domain; C is the heavy chain constant structure domain; HX1 is the connector; Fc is the Fc region; and n is independently 0 or 1; and The second polypeptide chain comprises VL1-(LX1)n-VL2-C, wherein: VL1 is the first variable structural domain of the light chain; VL2 is the second variable structural domain of the light chain; C is the constant structural domain of the light chain; LX1 is the connector; and n is independently 0 or 1.

[0176] Non-DVD-Ig format In another aspect of this disclosure, 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 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 polypeptide chain, which includes a second linker (e.g., a modified hinge region) and a second constant region, or no linker is present; aiii) A first polypeptide chain comprising a first linker (e.g., a modified hinge region) and a first constant region, or without a linker; and 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 The second polypeptide chain (biv) includes 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.

[0177] In some implementations, the first antigen-binding domain comprises scFv, VHH, Fab, F(ab')2, or a single-domain antibody.

[0178] In some implementations, the second antigen-binding domain comprises scFv, VHH, Fab, F(ab')2, or a single-domain antibody.

[0179] In some implementations, the third antigen-binding domain comprises scFv, VHH, Fab, F(ab')2, or a single-domain antibody.

[0180] In some implementations, the fourth antigen-binding domain comprises scFv, VHH, Fab, F(ab')2, or a single-domain antibody.

[0181] In some implementations, 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 contain scFv, VHH, Fab, F(ab')2, or a single-domain antibody.

[0182] In some embodiments, the first antigen-binding domain, the second antigen-binding domain, the third antigen-binding domain, and the fourth antigen-binding domain each contain an scFv. In some exemplary embodiments, the multispecific binding proteins of this disclosure are agonistic to any given signaling pathway, i.e., they are not antagonistic to that pathway. In some embodiments, agonistic activity can be measured using a specific receptor potency assay (e.g., the HEK-Blue™ potency assay (InVivogen)). The potency assay (e.g., HEK-Blue) involves a cell line expressing a target receptor (e.g., HEK293). Binding of a bispecific antibody to the receptor triggers a signaling cascade, resulting 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.

[0183] As used herein, the term "inducing proximity between the first and second subunits of a given receptor" refers to facilitating the binding of the first and second subunits, thereby activating a subsequent signaling cascade. In some embodiments, the proximity induced by the multispecific binding protein of this disclosure is the same as or similar to the proximity induced when a natural ligand facilitates the binding of the first and second subunits of a target receptor.

[0184] The bispecific antibody disclosed herein may employ at least one modified hinge region. The modified hinge region serves as a linker connecting the different domains of the bispecific antibody. In some embodiments, the modified hinge region links a first variable heavy chain domain (VH1) to a second variable heavy chain domain (VH2), and / or links a 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 some embodiments, the modified hinge region comprises: i) an upper hinge region of at most 7 amino acids in length or the absence of an upper hinge region; and ii) a lower hinge region. In some embodiments, the modified hinge region comprises an amino acid sequence of PLAP or PAPNLLGGP or consists of the above.

[0185] Compared to bispecific antibodies lacking at least one modified hinge region, the bispecific antibodies of this disclosure (e.g., multispecific binding proteins) exhibit stronger agonist activity. Agonist activity can be measured using a specific receptor potency assay (e.g., the Pathhunter U2OS dimerization assay (DiscoverX)). The potency assay (e.g., Pathhunter) involves a cell line expressing the target receptor (e.g., U2OS). Binding of the bispecific antibody to the receptor triggers a signal transduction cascade, resulting in quantifiable reporter gene expression.

[0186] In some embodiments, the multispecific binding protein-induced agonist activity 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 natural ligand for the first and second receptor subunits.

[0187] The bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces agonist activity of at least about 35% of the activity of the natural ligand with respect to the first and second receptor subunits. In some embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 40% of the activity of the natural ligand with respect to the first and second receptor subunits. In some embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 40% of the activity of the natural ligand with respect to the first and second receptor subunits. In some embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 45% of the activity of the natural ligand with respect to the first and second receptor subunits. In some embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 50% of the activity of the natural ligand with respect to the first and second receptor subunits. In some embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 55% of the activity of BMP9. In some embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 60% activity of the natural ligand against the first and second receptor subunits. In some embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 65% activity of the natural ligand against the first and second receptor subunits. In some embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 70% activity of the natural ligand against the first and second receptor subunits. In some embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 75% activity of the natural ligand against the first and second receptor subunits. In some embodiments, the bispecific antibody (e.g., a multispecific binding protein) of this disclosure induces at least about 80% activity of the natural ligand against the first and second receptor subunits.

[0188] In some implementations, the activity of the natural ligand is determined by measuring the activation of the protein activated by the target receptor. This activation can be measured in cells incubated with multispecific binding proteins and / or in cells incubated with the natural ligand. Enzyme-linked immunosorbent assay (ELISA) can be used to detect the activation of the protein activated by the target receptor.

[0189] In some embodiments, the activity of the natural ligand is determined by measuring changes in gene expression of genes known to be expressed when the target receptor is activated. The detection of gene expression of genes known to be expressed when the target receptor is activated can be achieved using standard molecular biology techniques and PCR. Briefly, a first cell population is incubated with the bispecific antibody of this disclosure, and a second cell population is incubated with the natural ligand. After incubation for a period of time, mRNA is isolated from the cells, cDNA is generated, and PCR is performed to detect gene levels relative to a control gene (such as GAPDH). The gene levels in the first cell population are then compared with the gene levels in the second cell population.

[0190] Fc structural domain The heavy chain Fc domain used in the bispecific antibodies disclosed herein typically comprises a CH2 domain and a CH3 domain, wherein the C-terminus of the CH2 domain is (directly or indirectly) linked to the N-terminus of the CH3 domain. Any naturally occurring or modified CH2 and / or CH3 domains can be used. For example, in some embodiments, the CH2 and / or CH3 domains are naturally occurring CH2 or CH3 domains from IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chains (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chains). The CH2 and CH3 domains may originate from the same or different antibody heavy chains. In some embodiments, the Fc polypeptide comprises a portion from a single antibody heavy chain containing both the CH2 and CH3 domains. In some embodiments, the CH2 and / or CH3 domains are variants of the naturally occurring CH2 or CH3 domains, respectively. In some embodiments, the CH2 domain and / or CH3 domain are variants comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to the naturally occurring CH2 domain or CH3 domain, respectively. In some embodiments, the CH2 domain and / or CH3 domain are chimeras of one or more CH2 domains or CH3 domains, respectively. In some embodiments, according to the EU index, the CH2 domain comprises amino acid positions 231-340 of a naturally occurring hinge region (e.g., human IgG1). In some embodiments, according to the EU index, the CH3 domain comprises amino acid positions 341-447 of a naturally occurring hinge region (e.g., human IgG1).

[0191] In some embodiments, the Fc peptide further comprises a hinge region, wherein the C-terminus of the hinge region is (directly or indirectly) connected to the N-terminus of the CH2 domain. For example, in some embodiments, the hinge region is a naturally occurring hinge region derived from an antibody heavy chain of IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain). The hinge region may be derived from an antibody heavy chain that is the same as or different from the CH2 and / or CH3 domains. In some embodiments, the hinge region is a variant comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to a naturally occurring hinge region. In some embodiments, the hinge region is a chimera of one or more hinge regions. In some embodiments, according to the EU index, the hinge region comprises amino acid positions 226-229 of a naturally occurring hinge region (e.g., human IgG1). In some embodiments, according to the EU index, the hinge region comprises amino acid positions 216-230 of a naturally occurring hinge region (e.g., human IgG1). In some embodiments, according to the EU index, the hinge region comprises amino acid positions 216-230 of a naturally occurring hinge region (e.g., human IgG1). In some embodiments, according to the EU index, the hinge region is the IgG4 hinge region containing serine (S) at amino acid position 228.

[0192] In some embodiments, the Fc peptide further comprises a CH1 domain, wherein the C-terminus of the CH1 domain is (directly or indirectly) connected to the N-terminus of the hinge region. For example, in some embodiments, the CH1 domain is a naturally occurring CH1 domain from an antibody heavy chain of IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 (e.g., human IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 antibody heavy chain). The CH1 domain may be derived from the same or different antibody heavy chain as the hinge region, CH2 domain, and / or CH3 domain. In some embodiments, the CH1 domain is a variant comprising one or more amino acid insertions, deletions, substitutions, or modifications relative to the naturally occurring CH1 domain. In some embodiments, the CH1 domain is a chimera of one or more CH1 domains. In some embodiments, according to the EU index, the CH1 domain comprises amino acid positions 118-215 of the naturally occurring hinge region (e.g., human IgG1).

[0193] In some embodiments, the Fc peptide lacks the CH1 domain, or contains a mutation located in the CH1 domain or a variable domain of the heavy chain that prevents the heavy chain from binding to the antibody light chain. In some embodiments, the antibody heavy chain lacks a portion of its hinge region.

[0194] Heterodimerization motif In some exemplary embodiments, the first Fc domain and the second Fc domain are further engineered to enhance the heterodimerization of the first specific binding domain and the second specific binding domain, and to minimize the impact of mischain pairing (i.e., pairing of the IL-18Rα binding domain or the same IL-18Rβ domain).

[0195] Any method recognized in the art for addressing mispairing can be employed to improve the production of desired multispecific antibodies. For example, US2010 / 0254989 A1 describes the construction of a bispecific cMet-ErbB1 antibody in which the VH and VL of a single antibody are fused via a GlySer adapter gene. For bispecific antibodies including an Fc domain, mutations can be introduced into the Fc to promote partially correct heterodimerization of the Fc. Several such approaches are reviewed in Klein et al. (mAbs (2012) 4:6, 1-11), the contents of which are incorporated herein by reference in their entirety.

[0196] In some implementations, the first and second specific binding specificities of a multispecific antibody are heterodimerized via a club-and-mortar (KiH) pairing of the Fc domain. This dimerization technique utilizes "protrusions" or "clumps" and "recesses" or "mortars" engineered into the interface of the CH3 domain. When a club or mortar with suitable position and size is present at the interface of the first or second CH3 domain, the corresponding club or mortar only needs to be engineered at adjacent interfaces, thus promoting and enhancing Fc domain pairing at the CH3 / CH3 domain interface. The IgG Fc domain fused with VHH has a club, and the IgG Fc domain of a conventional antibody has a mortar designed to accommodate the club, and vice versa. A "clump" refers to at least one amino acid side chain, typically a larger side chain, protruding from the interface of the CH3 portion of the first Fc domain. The protrusion creates a "clump" that is complementary to and accommodated by a "mortar" in the CH3 portion of the second Fc domain. A “mortise” is at least one amino acid side chain, typically a smaller one, recessed from the interface of the CH3 portion of the second Fc domain. This technique is described, for example, in U.S. Patent Nos. 5,821,333, 5,731,168, and 8,216,805; Ridgway et al., Protein Engineering (1996) 9:617-621; and Carter PJ Immunol. Methods (2001) 248: 7-15, which are incorporated herein by reference.

[0197] Exemplary amino acid residues that can act as pestles include arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W). Existing amino acid residues in the CH3 domain may be replaced or substituted by pestle amino acid residues. Preferred substituted amino acids may include any amino acid with a small side chain, such as alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), or valine (V).

[0198] Exemplary amino acid residues that can serve as mordane include alanine (A), serine (S), threonine (T), or valine (V). Existing amino acid residues in the CH3 domain may be replaced or substituted with mordane amino acid residues. Preferred substituted amino acids may include any amino acid with a large side chain, such as arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W).

[0199] The CH3 domain is preferably derived from human IgG1 antibody. Exemplary amino acid substitutions for the CH3 domain include Y349C, S354C, T366S, T366Y, T366W, F405A, F405W, Y407T, Y407A, Y407V, T394S, or combinations thereof. Preferred exemplary combinations are S354C, T366Y, or T366W for a mortar mutation in the first CH3 domain and Y349C, T366S, L368A, Y407T, or Y407V for a mortar mutation in the second CH3 domain.

[0200] In some embodiments, the two Fc domains of the antigen-binding construct are heterodimerized via Fab arm exchange (FAE). Human IgG1 with a P228S hinge mutation may contain either an F405L or K409R CH3 domain mutation. These two antibodies are mixed with a reducing agent to generate the FAE. This technique is described in U.S. Patents 9,212,230 and Labrijn AF PNAS (2013) 110(13):5145-5150, which are incorporated herein by reference.

[0201] In other embodiments, the two Fc domains of the antigen-binding construct are heterodimerized via an electrostatic guiding effect. This dimerization technique utilizes electrostatic guidance to promote and enhance Fc domain pairing at the CH3 / CH3 domain interface. The charge complementarity between the two CH3 domains is altered to favor heterodimerization (opposite charge pairing) rather than homodimerization (same charge pairing). In this approach, electrostatic repulsion prevents homodimerization. Some exemplary amino acid residue substitutions that confer the electrostatic guiding effect include K409D, K392D, and / or K370D in the first CH3 domain and D399K, E356K, and / or E357K in the second CH3 domain. This technique is described in U.S. Patent Publication No. 2014 / 0154254 A1 and Gunasekaran K. JBC (2010) 285(25):19637-19646, which are incorporated herein by reference.

[0202] In other embodiments, charge complementarity is formed by a first Fc domain containing N297K and / or T299K mutations and a second Fc domain containing N297D and / or T299D mutations.

[0203] In aspects of this invention, the two Fc domains of the antigen-binding construct are heterodimerized through hydrophobic interactions. This dimerization technique utilizes hydrophobic interactions, rather than electrostatic interactions, to promote and enhance Fc domain pairing at the CH3 / CH3 domain interface. Exemplary amino acid residue substitutions may include K409W, K360E, Q347E, Y349S, and / or S354C located in the first CH3 domain and D399V, F405T, Q347R, E357W, and / or Y349C located in the second CH3 domain. Preferred amino acid residue substitution pairs between the first and second CH3 domains include K409W:D399V, K409W:F405T, K360E:Q347R, Y349S:E357W, and S354C:Y349C. This technique is described in U.S. Patent Publication No. 2015 / 0307628 A1.

[0204] In aspects of this invention, heterodimerization can be mediated by using leucine zipper fusion. Leucine zipper domains fused to the C-terminus of each CH3 domain of the antibody chain drive heterodimerization. This technique is described in Wranik B. JBC (2012) 287(52):43331-43339.

[0205] In aspects of this invention, heterodimerization can be mediated by using a chain-exchange engineered domain (SEED) body. CH3 domains derived from IgG and IgA formats drive heterodimerization. This technique is described in Muda M. PEDS (2011) 24(5): 447-454.

[0206] In other embodiments, the heterodimerization motif may comprise a non-natural disulfide bond formed from engineered cysteine ​​residues. In some embodiments, the first set of disulfide bonds may consist of a Y349C mutation in the first Fc domain and an S354C mutation in the second Fc domain. In other embodiments, engineered disulfide bonds may be introduced by fusing a C-terminal extension peptide having engineered cysteine ​​residues to the C-terminus of each of the two Fc domains. In some embodiments, the first Fc domain may comprise a substitution of "PGK" at the carboxyl terminus with "GEC", and the second Fc domain may comprise a substitution of "PGK" at the carboxyl terminus with "KSCDKT".

[0207] In another approach, multispecific antibodies can employ the CrossMab principle (as reviewed by Klein et al.), which involves domain exchange between the heavy and light chains to facilitate proper pairing. Yet another approach involves engineering the interface between the paired VH-VL domains or paired CH1-CL domains of the heavy and light chains to enhance the affinity between the heavy chain and its homologous light chain (Lewis et al., Nature Biotechnology (2014) 32: 191-198).

[0208] An alternative approach for producing multispecific antibody formulations with the correct antigen specificity is to develop methods for enriching antibodies with the correct heavy-light chain pairing. For example, Spiess et al. (Nature Biotechnology (2013) 31: 753-758) described a method for producing MET-EGFR bispecific antibodies from a bacterial co-culture expressing two different half-antibodies.

[0209] A method is also described in which the Fc domain of at least one of the heavy chains of a bispecific antibody is mutated to alter its binding affinity to an affinity agent (e.g., protein A). This allows for the separation of correctly paired heavy chain heterodimers based on purification techniques that utilize the differential binding of the two heavy chains to the affinity agent (see US2010 / 0331527, WO2013 / 136186).

[0210] International patent application number PCT / EP2012 / 071866 (WO2013 / 064701) addresses the problem of chain pairing errors using a multispecific antibody separation method based on the use of anti-idiotype binders (particularly anti-idiotype antibodies). The anti-idiotype binder is used in a two-step selection process, where a first agent captures antibodies with VH-VL domains specific to a first antigen, and a second agent subsequently captures antibodies with a second VH-VL domain specific to a second antigen.

[0211] In another embodiment, the multispecific antibody employs a first binding specificity having a conventional Fab binding region and a second binding specificity containing a single-domain antibody (VHH) binding region. The heterodimerization method used drives the heavy chain region of Fab to bind only to the complete heavy chain of the VHH. Since the VHH chain does not bind to the light chain, the light chain region of the Fab portion will bind only to its corresponding heavy chain.

[0212] In some other embodiments, the multispecific binding proteins described herein also comprise a common light chain. As used herein, the term "common light chain" refers to a light chain capable of pairing with a first heavy chain of an antibody bound to a first antigen to form a binding site specifically binding to the first antigen, and also capable of pairing with a second heavy chain of an antibody bound to a second antigen to form a binding site specifically binding to the second antigen. The common light chain is a polypeptide comprising a variable domain (VL) and a constant domain (CL) of an antibody light chain from the N-terminus to the C-terminus, and is also abbreviated herein as "VL-CL". Multispecific binding proteins having a common light chain require heterodimerization of the different heavy chains. In some embodiments, the heterodimerization methods listed above may be used in conjunction with the common light chain. In some exemplary embodiments, the heterodimerization motif may comprise a non-natural disulfide bond formed by engineered cysteine ​​residues. Adding a disulfide bond between the heavy and light chains of the antibody has been shown to improve stability. In addition, disulfide bonds have also been used as a solution to improve light chain pairing within bispecific antibodies (Geddie ML et al., mABs (2022) 14(1)).

[0213] Unless otherwise stated, all antibody Fc domain numbers used in this paper correspond to the EU numbering scheme as described in Edelman et al. (Proc. Natl. Acad. Sci. 63(1): 78-85. 1969).

[0214] Other methods for the heterodimerization of heavy and / or light chains, as well as the generation and purification of asymmetric antibodies, are known in the art. See, for example, Klein C. mAbs (2012) 4(6): 653-663 and U.S. Patent 9,499,634, each of which is incorporated herein by reference.

[0215] Effector functional mutation As discussed above, the multispecific binding proteins of this disclosure are available in various isotypes with different Fc domains. The multispecifically binding Fc region primarily determines its effector functions in Fc binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent phagocytosis (ADCP) activity. Unlike effector T cell functions, these "cellular effector functions" involve recruiting Fc receptor-carrying cells to the location of target cells, thereby leading to the killing of antibody-bound cells.

[0216] Antibodies according to the invention may be antibodies exhibiting reduced effector function. In some embodiments, one or more mutations reduce one or more of antibody-dependent cytotoxicity (ADCC), antibody-dependent cytophagy (ADCP), or complement-dependent cytotoxicity (CDC). In some embodiments, antibodies according to the invention may lack ADCC, ADCP, and / or CDC activity. In any case, antibodies according to the 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 and the presence or absence of FcR binding and cellular effector function allow antibodies to be tailored for specific therapeutic purposes, as discussed elsewhere herein.

[0217] In some embodiments, the first and second Fc domains contain one or more mutations that reduce Fc effector function. In some embodiments, the first and second Fc domains each contain L234A and L235A mutations. These IgG1 mutations are also known as “LALA” mutations and are described in more detail by Xu et al. (Cell Immunol. 2000; 200:16–26). In some embodiments, the first and second Fc domains each contain L234A, L235A, G237A, and / or P329G mutations. The amino acid positions of the Fc domains mentioned herein are based on EU antibody numbers. Alternatively, the antibody may have an effector-free Fc domain. The antibody may have a heavy chain Fc domain that does not bind to the Fcγ receptor; for example, the Fc domain may contain an L235E mutation. Another optional mutation in the heavy chain Fc domain is S228P, which enhances stability. The heavy chain Fc domain can be IgG4 containing both the L235E and S228P mutations. This “IgG4-PE” heavy chain Fc domain is non-functional. The inactivated IgG1 heavy chain Fc domain is also non-functional. The inactivated IgG1 heavy chain Fc domain may contain alanine residues at positions 234, 235, and / or 237 (EU index number), for example, it can be an IgG1 sequence containing the L234A, L235A, and / or G237A mutations (“LALAGA”).

[0218] Human IgG1 Fc domains containing specific mutations or altered glycosylations (e.g., N297Q, N297D, and N297K, EU index numbers) at residue Asn297 have been shown to reduce binding to the Fc receptor.

[0219] In other embodiments, it may be desirable to enhance this binding of the Fc region of a multispecific antibody to the human Fc γ receptor IIIA (FcγRIIIA) relative to the binding of the corresponding naturally occurring antibody's Fc region. In some embodiments, the Fc domain can be engineered to achieve enhanced ADCC and / or CDC and / or ADCP. The potency of Fc-mediated effects can be enhanced by engineering the Fc domain using various established techniques. Such approaches enhance affinity for certain Fc receptors, resulting in a potentially diverse spectrum of activation enhancements. This can be achieved by modifying one or more amino acid residues. Exemplary mutations are one or more residues selected from the human IgG1 Fc domain, 332, and 330 (or equivalent positions in other IgG isotypes). Thus, the antibody may comprise a human IgG1 Fc domain having one or more mutations independently selected from S239D, I332E, and A330L (EU index number).

[0220] Affinity to the Fc receptor can also be enhanced by altering the native glycosylation profile of the Fc domain (e.g., by generating variants that are under- or de-fucosylated). Non-fucosylated antibodies contain a trimannose core structure of an Fc complex N-glycan lacking fucoose residues. These glycoengineered antibodies, lacking the core fucoose residues from the Fc N-glycan, may exhibit stronger ADCC than fucosylated equivalents due to enhanced FcγRIIIA binding. For example, to increase ADCC, residues in the hinge region can be altered to enhance binding to FcγRIIIA. Therefore, antibodies may contain a human IgG heavy chain Fc domain as a variant of the wild-type human IgG heavy chain Fc domain. In some embodiments, the variant human IgG heavy chain Fc domain binds to the human Fcγ receptor selected from the group consisting of FcγRIIB and FcγRIIA with a higher affinity for human FcγRIIIA than the wild-type human IgG heavy chain Fc domain. The antibody may contain a human IgG heavy chain Fc domain, which is a variant of the wild-type human IgG heavy chain Fc domain, wherein the variant human IgG heavy chain Fc domain binds human FcγRIIB with a higher affinity than the wild-type human IgG heavy chain Fc domain. The variant human IgG heavy chain Fc domain may be a variant human IgG1, variant human IgG2, or variant human IgG4 heavy chain Fc domain. 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 following groups: 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).

[0221] Enhancement of CDC can be achieved through amino acid changes that increase affinity for C1q (the first component of the classical complement activation cascade). Another approach is to generate a chimeric Fc domain composed of fragments of human IgG1 and human IgG3 to utilize the higher affinity of IgG3 for C1q. The antibodies of the present invention may contain mutant amino acids located at residues 329, 331, and / or 322 to alter C1q binding and / or reduce or eliminate CDC activity. In another embodiment, the antibodies or antibody fragments disclosed herein may contain a modified Fc region at residues 231 and 239, whereby the amino acids are replaced to alter the antibody's ability to fix complement. In one embodiment, the antibody or fragment has an Fc domain containing one or more mutations selected from E345K, E430G, R344D, and D356R, particularly containing a double mutation of R344D and D356R (EU index numbering system).

[0222] The functional properties of multispecific binding proteins can be further modulated by combining amino acid substitutions that alter Fc binding affinity with those that affect binding to FcRn. Binding proteins with amino acid substitutions that affect binding to FcRn (also referred to herein as "FcRn variants") may, in some cases, prolong the in vivo serum half-life compared to unmodified binding proteins. Understandably, any combination of Fc and FcRn variants can be used to modulate the clearance of antigen-antibody complexes. Suitable FcRn variants that can be combined with any of the Fc variants described herein include, but are not limited to, N434A, N434S, M428L, V308F, V259I, M428L / N434S, V259I / V308F, Y436I / M428L, Y436I / N434S, Y436V / N434S, Y436V / M428L, M252Y, M252Y / S254T / T256E, and V259I / V308F / M428L.

[0223] Expression of antigen-binding proteins In one aspect, polynucleotides encoding binding proteins disclosed herein (e.g., antigen-binding proteins and their antigen-binding fragments) are provided. A method for preparing binding proteins, comprising expressing these polynucleotides, is also provided.

[0224] Polynucleotides encoding the binding proteins disclosed herein are typically inserted into expression vectors for introduction into host cells, which can then be used to produce the desired amount of the binding protein. Therefore, in some aspects, this disclosure provides expression vectors comprising the polynucleotides disclosed herein, and host cells comprising these vectors and the polynucleotides.

[0225] The terms "vector" or "expression vector" are used herein to refer to a vector used according to this disclosure as a delivery tool for introducing a desired gene into a cell and expressing that gene in the cell. As known to those skilled in the art, such vectors are readily selected from the group consisting of plasmids, bacteriophages, viruses, and retroviruses. Generally, vectors compatible with this disclosure include selection markers, appropriate restriction sites to facilitate the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0226] Numerous expression vector systems can be used for the purposes of this disclosure. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Other vectors involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells that have integrated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. These markers can provide protrophic phenotypes to auxotrophic hosts, provide resistance to biocides (e.g., antibiotics), or resistance to heavy metals (such as copper). Selective marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cell via co-transformation. Other elements may also be required to achieve optimal mRNA synthesis. These elements may include signal sequences, splicing signals, and transcription promoters, enhancers, and termination signals. In some embodiments, a cloned variable region gene is inserted into the expression vector along with heavy and light chain Fc domain genes (e.g., human Fc domain genes) synthesized as discussed above.

[0227] In other embodiments, polycistronic constructs can be used to express binding proteins. In such expression systems, a variety of target gene products, such as the heavy and light chains of antibodies, can be generated from a single polycistronic construct. These systems advantageously utilize internal ribosome entry sites (IRES) to provide relatively high levels of peptides in eukaryotic host cells. A compatible IRES sequence is disclosed in U.S. Patent No. 6,193,980, which is incorporated herein by reference in its entirety for all purposes. Those skilled in the art will understand that such expression systems can be used to efficiently generate the full range of peptides disclosed in this application.

[0228] In summary, once a vector or DNA sequence encoding a binding protein (e.g., an antibody or fragment thereof) has been prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Plasmids can be introduced into host cells using a variety of techniques well known to those skilled in the art. These techniques include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion using enveloped DNA, microinjection, and full viral infection. See Ridgway, AAG, “Mammalian Expression Vectors,” Chapter 24.2, pp. 470-472, Vectors, eds. Rodriguez and Denhardt (Butterworths, Boston, Mass. 1988). Plasmids can be introduced into the host via electroporation. Transformed cells are then grown under conditions suitable for the production of light and heavy chains, and the synthesis of heavy and / or light chain proteins in the cells is measured. Exemplary assays include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), immunohistochemistry, etc.

[0229] As used in this article, the term "transformation" should be used in a broad sense to refer to the introduction of DNA into recipient host cells, thereby altering the genotype.

[0230] Similarly, "host cell" refers to a cell transformed using a vector constructed using recombinant DNA technology that encodes at least one heterologous gene. Unless otherwise explicitly stated, the terms "cell" and "cell culture" are used interchangeably to indicate the source of the antibody when describing the process of isolating peptides from a recombinant host. In other words, recovering peptides from "cells" can mean recovering them from intact cells after centrifugation, from the supernatant of lysed cell cultures, or from cell cultures containing both culture medium and suspended cells.

[0231] 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 for expressing the desired gene product. Exemplary host cell lines include, but are not limited to, GS-CHO and CHO-K1 (Chinese hamster ovary cell lines), DG44 and DUXB11 (Chinese hamster ovary cell lines, DHFR-deficient), HELA (human cervical cancer cells), CV-1 (monkey kidney cell line), COS (CV-1-derived cell line with SV40 T antigen), R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HEK (human kidney cell line), SP2 / O (mouse myeloma cells), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney cell line). In one embodiment, the cell line alters the glycosylation of the antibody expressed therein, for example, by eliminating fucosylation (e.g., PER.C6® (Crucell) or FUT8 knockout CHO cell lines (POTELLIGENT® cells) (Biowa, Princeton, NJ)). In one embodiment, NSO cells may be used. CHO cells are particularly useful. Host cell lines are generally available from commercial service providers (e.g., the U.S. Center for Tissue Cultures) or from authors of published literature.

[0232] In vitro production allows for scale-up to obtain large quantities of the desired peptide. Techniques for culturing mammalian cells under tissue culture conditions are known in the art, including homogeneous suspension culture (e.g., in an airlift reactor or a continuous stirred reactor) or immobilized or embedded cell culture (e.g., in hollow fibers, microcapsules, agarose beads, or ceramic columns). If necessary and / or required, the peptide solution can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, DEAE-cellulose chromatography, and / or (immuno)affinity chromatography.

[0233] The genes encoding the binding proteins specifically described in this disclosure can also be expressed in non-mammalian cells, such as bacteria, yeast, or plant cells. In this respect, it should be understood that various single-celled non-mammalian microorganisms, such as bacteria, can also be transformed; that is, those microorganisms capable of growth in a culture or fermentation environment. Susceptible bacteria include members of the Enterobacteriaceae family, such as *Escherichia coli*. Escherichia coli ) or Salmonella ( Salmonella strains of Bacillus; Bacillus family ( Bacillaceae ), such as Bacillus subtilis ( Bacillus subtilis ); Pneumococcus ( Pneumococcus Streptococcus ( Streptococcus) and Haemophilus influenzae ( Haemophilus influenzae It should also be understood that, when expressed in bacteria, the binding protein may become part of an inclusion body. In some embodiments, the binding protein is subsequently isolated, purified, and assembled into a functional molecule. In some embodiments, the binding protein of this disclosure is expressed in bacterial host cells. In some embodiments, bacterial host cells are transformed with an expression vector containing a nucleic acid molecule encoding the binding protein of this disclosure.

[0234] Besides prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae (Saccharomyces cerevisiae) Saccharomyces cerevisiae Yeast (or common baker's yeast) is the most commonly used eukaryotic microorganism, but many other strains are also readily available. To [discuss] yeasts (…), Saccharomyces Expression is typically achieved using the plasmid Yrp7, for example (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)). This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow in tryptophan (e.g., ATCC number 44076 or PEP4-1) (Jones, Genetics, 85:12 (1977)). Therefore, the presence of trpl damage in the yeast host cell genome provides an efficient environment for detecting transformation by growing in the absence of tryptophan.

[0235] Formulation / Pharmaceutical Composition In some embodiments, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of the antigen-binding protein described herein. Some embodiments include a pharmaceutical composition comprising a therapeutically effective amount of any of the binding proteins or binding protein-drug conjugates described herein, and mixed with a pharmaceutically or physiologically acceptable formulation selected according to suitability for administration.

[0236] Acceptable formulations are generally non-toxic to recipients at the dosage and concentration used.

[0237] In some embodiments, the pharmaceutical composition may contain formulation materials for altering, maintaining, or preserving properties of the composition, such as pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability. Suitable formulation materials include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), swelling agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin), proteins (such as serum albumin, gelatin, or immunoglobulins), colorants, flavoring agents, and diluents, emulsifiers, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight peptides, salt-forming counterions (such as...). Preservatives (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronic; PEG; sorbitol esters; polysorbate esters, such as polysorbate 20 or polysorbate 80; triaton; tromethamine; lecithin; cholesterol, or teroxaprol), stability enhancers (such as sucrose or sorbitol), tension enhancers (such as alkali metal halides, such as sodium chloride or potassium chloride, or mannitol and sorbitol), delivery media, diluents, excipients, and / or pharmaceutical adjuvants (see, for example, REMINGTON'S PHARMACEUTICAL). SCIENCES (18th edition, edited by AR Gennaro, Mack Publishing Company, 1990) and subsequent editions thereof, which are incorporated herein by reference for any purpose.

[0238] In some embodiments, the optimal pharmaceutical composition will be determined by a technician based on, for example, the intended route of administration, delivery format, and required dosage. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[0239] In some embodiments, the primary medium or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, suitable mediums or carriers for injection may be water, physiological saline solutions, or artificial cerebrospinal fluid, possibly supplemented with other materials commonly found in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are other exemplary mediums. Other exemplary pharmaceutical compositions contain Tris buffer with a pH of about 7.0-8.5 or acetate buffer with a pH of about 4.0-5.5, and may also contain sorbitol or suitable alternatives. In one embodiment of this disclosure, a binding protein composition for storage can be prepared by mixing a selected composition having the desired purity with an optional formulation in the form of a lyophilized cake or aqueous solution. Furthermore, a suitable excipient (such as sucrose) can be used to formulate the binding protein as a lyophilized product.

[0240] In some embodiments, the pharmaceutical compositions disclosed herein may be selected for parenteral or subcutaneous delivery. Alternatively, the compositions may be selected for inhalation or delivery via the digestive tract, such as oral delivery. The preparation of such pharmaceutically acceptable compositions is within the scope of the art.

[0241] In some embodiments, the formulation components are present at a concentration acceptable at the application site. For example, a buffer solution is used to maintain the composition at a physiological pH or slightly lower, typically in the pH range of about 5 to about 8.

[0242] When considering parenteral administration, the therapeutic composition may be in the form of a pyrogen-free, parenterically acceptable aqueous solution containing the desired binding protein in a pharmaceutically acceptable medium. A particularly suitable medium for parenteral injection is sterile distilled water in which the binding protein is formulated as a properly preserved sterile isotonic solution. Another preparation may involve formulating the desired molecule with a medium providing controlled or sustained release of the product (such as injectable microspheres, biodegradable particles, polymeric compounds such as polylactic acid or polyglycolic acid, beads, or liposomes), followed by delivery via reservoir injection. Hyaluronic acid may also be used, and it may have the effect of promoting a prolonged duration of circulation. Other suitable means for introducing the desired molecule include implantable drug delivery devices.

[0243] In one embodiment, the pharmaceutical composition may be formulated for inhalation. For example, the binding protein may be formulated as a dry powder for inhalation. The binding protein inhalation solution may also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution may be nebulized.

[0244] It should also be understood that some formulations can be administered orally. In one embodiment of this disclosure, the multispecific binding protein administered in this manner may or may not be formulated using carriers conventionally used in compound solid dosage forms, such as tablets and capsules. For example, capsules may be designed to release the active portion of the formulation at a site in the gastrointestinal tract where bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to promote the absorption of the binding protein. Diluents, flavoring agents, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0245] Another pharmaceutical composition may involve an effective amount of a multispecific binding protein mixed with a nontoxic excipient suitable for manufacturing tablets. A solution in unit dose form can be prepared by dissolving the tablet in sterile water or another suitable medium. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate, or sodium bicarbonate, lactose, or calcium phosphate; or binders such as starch, gelatin, or gum arabic; or lubricants such as magnesium stearate, stearic acid, or talc.

[0246] Other pharmaceutical compositions disclosed herein will be apparent to those skilled in the art, including formulations of binding proteins to form sustained-release or controlled-release delivery agents. Techniques for formulating various other sustained-release or controlled-release delivery methods are also known to those skilled in the art, such as liposome carriers, biodegradable microparticles or porous beads, and reservoir injections. Other examples of sustained-release formulations include semi-permeable polymer matrices in the form of shaped articles, such as films or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactide, copolymers of L-glutamic acid and L-glutamic acid γ-ethyl ester, poly(2-hydroxyethyl methacrylate), ethylene-vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes, which may be prepared by any of several methods known in the art.

[0247] In some embodiments, pharmaceutical compositions intended for internal administration must generally be sterile. This can be achieved by filtration through a sterile filter membrane. In the case of lyophilization of the composition, sterilization using this method can be performed before or after lyophilization and reconstitution. Compositions intended for parenteral administration can be stored in lyophilized or solution form. Additionally, parenteral compositions are typically placed in containers with sterile access points, such as intravenous bags or vials with stoppers that can be punctured by a hypodermic needle.

[0248] Once the pharmaceutical composition is formulated, it can be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such formulations can be stored in ready-to-use form or in a form that requires reconstitution before administration (e.g., lyophilized form).

[0249] This disclosure also covers kits for producing single-dose administration units. Each kit may contain a first container with a dried multispecific binding protein and a second container with an aqueous formulation. Kits containing single-lumen and multi-lumen pre-filled syringes (e.g., liquid syringes and lyophilized powder syringes) are also included within the scope of this disclosure.

[0250] The effective amount of the binding protein drug composition to be used in treatment will depend, for example, on the treatment context and purpose. Those skilled in the art will understand that the appropriate dose level for treatment will therefore vary in part depending on the molecule delivered, the indication for use of the binding protein, the route of administration, and the patient's body size (weight, body surface area, or organ size) and condition (age and overall health). Therefore, clinicians may adjust the dose and modify the route of administration to achieve the best therapeutic effect.

[0251] The frequency of administration will depend on the pharmacokinetic parameters of the binding protein in the formulation. Typically, clinicians will administer the composition until a dose is reached to achieve the desired effect. Therefore, the composition can be administered as a single dose, as two or more doses over time (each dose may contain the same or different amounts of the desired molecule), or as a continuous infusion via an implanted device or catheter. Those skilled in the art will typically refine the appropriate dosage further, and this falls within the scope of their routine duties. The appropriate dosage can be determined by using appropriate dose-response data.

[0252] The pharmaceutical composition may be administered by known methods, such as oral administration, intravenous, intraperitoneal, intracerebral (parenchymal), intraventricular, intramuscular, intraocular, intraarterial, portal vein, or intralesional injection, via a sustained-release system, or via an implantable device. If necessary, the composition may be administered by bolus injection, continuous infusion, or implantation.

[0253] In some embodiments, the composition can also be applied topically via implantation of a membrane, sponge, or other suitable material that has absorbed or encapsulated the desired molecules. When an implantable device is used, it can be implanted into any suitable tissue or organ, and the delivery of the desired molecules can be achieved via diffusion, timed release bolus, or continuous administration.

[0254] The multispecific binding proteins disclosed herein can be formulated as aerosols for external application (such as by inhalation) (see, for example, U.S. Patent Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivering steroids that can be used to treat inflammatory diseases, particularly asthma, and are incorporated herein by reference in their entirety). These formulations for administration to the respiratory tract can be, alone or in combination with an inert carrier (such as lactose), in the form of an aerosol or solution for a nebulizer, or as a fine powder for inhalation. In this case, the particles of the formulation will have a diameter of less than 50 micrometers in one embodiment and less than 10 micrometers in another embodiment.

[0255] The multispecific binding proteins disclosed herein can be formulated for topical or external application, such as for application to the skin and mucous membranes in the form of gels, creams, and lotions, such as for application to the eye, and for application to the eye, or for application to the cisterns or spine. External application is considered for transdermal delivery, and also for application to the eye or mucous membranes, or for inhalation therapy. Nasal solutions of heterodimeric proteins, alone or in combination with other pharmaceutically acceptable excipients, can also be administered.

[0256] Transdermal patches (including iontophoresis and electrophoresis devices) are well known to those skilled in the art and can be used to administer heterodimeric proteins. Such patches are disclosed, for example, in U.S. Patent Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of which are incorporated herein by reference in their entirety.

[0257] In some embodiments, the pharmaceutical composition comprising the multispecific binding protein described herein is a lyophilized powder that can be reconstituted for application as a solution, emulsion, and other mixture. The lyophilized powder can also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the heterodimeric protein described herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In some embodiments, the lyophilized powder is sterile. The solvent may contain excipients that improve the stability of the powder or a reconstituted solution prepared from the powder, or other pharmacological components. Excipients that may be used include, but are not limited to, dextran, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also contain a buffer solution, such as citrate, sodium phosphate, or potassium phosphate, or other such buffer solutions known to those skilled in the art, in one embodiment being at approximately neutral pH. The solution is then sterilely filtered and subsequently lyophilized under standard conditions known to those skilled in the art to obtain the desired formulation. In one embodiment, the resulting solution is aliquoted into vials for lyophilization. Each vial will contain a single or multiple doses of the compound. The lyophilized powder can be stored under suitable conditions, such as from about 4°C to room temperature. The lyophilized powder is reconstituted with water for injection to obtain a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends on the selected compound. This amount can be determined empirically. The multispecific binding proteins provided herein can also be formulated to target specific tissues, receptors, or other areas of the body of the subject to be treated. Many such targeting methods are well known to those skilled in the art. This document contemplates the use of all such targeting methods in the compositions of this invention. For non-limiting examples of targeting methods, see, for example, U.S. Patent Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are incorporated herein by reference in their entirety. In a specific embodiment, the heterodimeric protein described herein is targeted to a tumor.

[0258] Treatment methods / uses Another aspect of this disclosure is bispecific antibodies and / or antigen-binding proteins used as medicines as described herein.

[0259] In a specific implementation, a method for treating a disease or condition by stimulating its activity is provided, the method comprising administering an effective amount of the antigen-binding protein described herein to a subject in need.

[0260] This binding protein can be used in any known assay method (such as competitive binding assay, direct and indirect sandwich assay, and immunoprecipitation assay) to detect and quantify one or more target antigens. The binding protein will bind to the one or more target antigens with an affinity suitable for the assay method employed.

[0261] For diagnostic applications, in some implementations, a detectable portion can be used to label the binding protein. This detectable portion can be any portion capable of directly or indirectly generating a detectable signal. For example, the detectable portion can be a radioactive isotope, such as... 3 H, 14 C 32 P, 35 S, 125 I, 99 Tc, 111 In or 67 Ga; fluorescent or chemiluminescent compounds, such as fluorescein isothiocyanate, rhodamine, or luciferin; or enzymes, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[0262] This binding protein can also be used for in vivo imaging. The binding protein, labeled with a detectable portion, can be administered to an animal, for example, into the bloodstream, and the presence and localization of the labeled antibody in the host can be determined. The binding protein can be labeled with any portion that is detectable in animals (whether by MRI, radiology, or other detection methods known in the art).

[0263] This disclosure also relates to a kit comprising a binding protein and other reagents for detecting levels of a target antigen in a biological sample. Such reagents may include detectable markers, blocked sera, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition containing any of the binding proteins, polynucleotides, carriers, carrier systems, and / or host cells described herein. In some embodiments, the kit includes a container and a label or instructions affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container may be formed from a variety of materials, such as glass or plastic. The container contains a composition that, on its own or in combination with another composition, is effective in treating, preventing, and / or diagnosing a condition, and may have a sterile access point (e.g., the container may be an intravenous infusion bag or vial with a stopper that can be punctured by a hypodermic needle). In some embodiments, the label or instructions indicate that the composition is used for the prevention, diagnosis, and / or treatment of the selected condition. Alternatively or additionally, the finished product or kit may also include a second (or third) container containing pharmaceutically acceptable buffers, such as water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may also include other materials required from a commercial and user perspective, including additional buffers, diluents, filters, needles, and syringes.

[0264] In some embodiments, this disclosure relates to a method for preventing and / or treating a disease or condition (e.g., cancer). In some embodiments, the method includes administering to a patient a therapeutically effective amount of at least one of the binding proteins or associated pharmaceutical compositions described herein. In some embodiments, the patient is a person.

[0265] The contents of any articles, patents and patent applications, and all other documents and electronic information mentioned or cited herein are hereby incorporated in their entirety by reference as if each separate publication were specifically and individually indicated to be incorporated by reference. The applicant reserves the right to incorporate any and all materials and information from any such articles, patents, patent applications or other physical and electronic documents into this application in their entirety.

[0266] While this disclosure has been described with reference to specific embodiments thereof, those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of this disclosure. It will be apparent to those skilled in the art that other suitable modifications and alterations can be made to the methods described herein using suitable equivalents without departing from the scope of the embodiments disclosed herein. Furthermore, various modifications can be made to adapt specific circumstances, materials, composition, processes, or one or more process steps to the purpose, spirit, and scope of this disclosure. All such modifications are intended to be within the scope of the appended claims. Certain embodiments have been described in detail and will be more clearly understood by referring to the following examples, which are included for illustrative purposes only and are not intended to be limiting.

[0267] Example The following embodiments are provided to provide a complete disclosure and description to those skilled in the art of how to manufacture and use the methods and compositions particularly described in this invention, and are not intended to limit the scope of what the inventors consider their invention. Efforts have been made to ensure the accuracy of the numerical values ​​used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should also be taken into account. Unless otherwise stated, parts are parts by weight, molecular weights are average molecular weights, temperatures are degrees Celsius, and pressures are atmospheric pressure or close to atmospheric pressure.

[0268] Example 1. Bispecific agonist antibody with a modified hinge IL-18 receptor Bispecific agonist antibodies targeting the IL18 receptor subunits IL18Rα and IL18Rβ are designed with modified hinge regions.

[0269] Table 1. Hinge region and Fc domain sequence

[0270] Table 2a. Bispecific antibodies with hinge variants

[0271] Table 2b. Exemplary IL-18R VHH sequences with modified hinge regions.

[0272]

[0273]

[0274] The agonist activity of the bispecific antibodies was screened. HEK-Blue™ IL-18 cells were purchased from Invivogen (hbk-hmil18). These cell lines overexpress IL-18Rα and IL-18Rβ, while blocking responses to TNFα and IL-1β. Reporter cells were resuscitated and cultured according to the supplier's recommendations. Cells were washed with PBS and added to 96-well plates at a density of approximately 50,000 cells / well. 20 μL of control or heteropolymer antibody was added to each well. The plates were incubated in a CO2 incubator at 37°C for 20–24 h. QUANTI-Blue™ (Invivogen) solution was prepared according to the manufacturer's instructions, and 180 μL was added to new wells. 20 μL of induced HEK-Blue IL-18 supernatant was added to each well, and the plates were incubated at 37°C for 3 h, after which readings were taken at 630 nm on a spectrophotometer (Clariostar). Bispecific antibodies with modified hinges are compared with antibodies without modified hinges (e.g., WT IgG1 hinges).

[0275] Table 3. HEK-Blue assay results, representing human IL18 activity.

[0276]

[0277] Bispecific constructs DGL207 and DGL209 contain modified hinges. DGL207 contains hinge 1 (without an upper hinge region), and DGL209 contains hinge 3 (PLAP; SEQ ID NO: 2). The bispecific construct with the modified hinge is superior to its counterpart (DGL093) which has a wild-type IgG1 hinge.

[0278] Table 4. HEK-Blue assay results, representing human IL18 activity.

[0279]

[0280] 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 (SEQ ID NO: 1). Hinge 3 contains the upper hinge sequence of PLA (SEQ ID NO: 2). Hinge 4 contains the upper hinge sequence of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 3). Hinge 5 contains the upper hinge sequence of EKSYGPP (SEQ ID NO: 4). Hinge 6 contains the upper hinge sequence of DKTHT (SEQ ID NO: 5). DGL212 with hinge 6 is similar to DGL093, except that it contains a fully human frame 4 (FW4).

[0281] Activity was also measured in IL-18R bispecific antibodies with an asymmetric hinge (a hinge variant on a polypeptide of a bispecific antibody).

[0282] Table 5. HEK-Blue assay results of IL18R bispecific antibodies with hinge variants on peptides targeting IL18Rβ.

[0283]

[0284] Table 6. HEK-Blue assay results of IL18R bispecific antibodies with hinge variants on peptides targeting IL18Rα.

[0285]

[0286] Example 2. Bispecific antibody against BMPR type I and II receptors with modified hinges A bispecific antibody targeting BMRP type I receptor ALK1 and BMRP type II receptor BMPRII was designed, and its sequence is as follows.

[0287] Table 7. Bispecific antibodies against ALK1 and BMPRII with modified hinges

[0288]

[0289]

[0290] The agonist activity of the bispecific antibodies was screened. PathHunter U2Os ALK-1 / BMPR-2 dimerization assays were obtained from DiscoverX Corporation (93-0962C3). Protein-protein interactions were assessed using enzyme fragment complementation (EFC) technology with β-galactosidase fragments. Reporter cells were resuscitated and cultured according to the supplier's recommendations. The bispecific antibodies were compared with the natural ligand BMP-9.

[0291] The same assay can be used to detect Alk-1 / ActRIIA agonist (93-1069C3) and Alk1 / ActRIIB agonist (93-0964C3).

[0292] Table 7b: Agonist activity of bispecific antibody constructs

[0293] Example 3. Engineering of scFv containing an ALK1 / BMPRII bispecific agonist antibody with an optimized hinge.

[0294] The agonist activity of heteropolymers with modified hinges, identified via the DIAGONAL platform, was also tested. A variant of DGL288, DGL809, was designed using hinge 1. DGL809 was designed, expressed, and purified according to the manufacturer's instructions using the Expi293 (Thermo) system. Cells were harvested six days post-transfection and batch purified using mabSelect resin. The purity of the final product was assessed using SDS-PAGE and analytical gel filtration. The heteropolymers were tested using a DiscoverX assay. DGL809 was superior to the parental DGL288, as shown in Table 9 (which displays the average of two different experiments), demonstrating its activity level relative to BMP9 at a 100 nM antibody concentration.

[0295] Table 8. Sequences of DGL288 and DGL809.

[0296]

[0297] Table 9. Agonist activity of DGL288 and DGL809 relative to BMP9.

[0298]

[0299] The agonist activity of additional bispecific antibodies was screened. PathHunter U2Os ALK-1 / BMPR-2 dimerization assays were obtained from DiscoverX Corporation (93-0962C3). Protein-protein interactions were assessed using enzyme fragment complementation (EFC) technology with β-galactosidase fragments. Reporter cells were resuscitated and cultured according to the supplier's recommendations. The bispecific antibodies were compared with natural ligands BMP9 and BMP10.

[0300] For the assay, cells were dissociated from the culture flask and removed using a cell dissociation reagent (DiscoverX, 92-0009). Cells were centrifuged at 300g for four minutes and resuspended at a density of 250 K / ml in assay medium (DiscoverX 93-0563R22A). 20 μL of the suspension was plated into each well of a 384-well plate and incubated at 37°C for 24 hours. The bispecific antibody was prepared to a final concentration of 5-fold. A 1:10 dilution was used for 12-point titration to generate a curve. 5 μL of the bispecific antibody was added to each well of the 384-well plate and incubated for three hours. 25 μL of flash assay reagent (DiscoverX, 93-0247) was added to each well, and the plate was read on a Verilux Skan after 60 minutes. Data were analyzed using PRISM.

[0301] Table 10: Agonist activity of bispecific antibody constructs

[0302] It was observed that tetravalent bispecific antibodies (i.e., both binding domains of the first receptor subunit (e.g., ALK1) and both binding domains of the second receptor subunit (e.g., BMPRII)) elicited stronger agonistic effects than bivalent bispecific antibodies (i.e., one binding domain of the first receptor subunit and one binding domain of the second receptor subunit). The bivalent bispecific antibodies were DGL266-DGL271, which had 0-46% BMP9 activity, while tetravalent bispecific antibodies such as DGL285-DGL292 consistently produced higher values.

[0303] The agonist activity of bispecific antibodies was determined by measuring the downstream effects of target receptor activation. In the context of ALK1 / BMPRII signaling, receptor activation leads to phosphorylation of SMAD1 (pSMAD1).

[0304] To measure pSMAD1 levels, HUVEC cells (CRL-1730) from ATCC were seeded at 15K cells per well in 96-well plates, and each well was in 100 μL of complete HUVEC medium (F12K (Corning, 10-025-CV), 10% FBS (Gibco, A31605-02), ECGS (30 μg / ml, Corning, 356006), 0.1 mg / ml heparin (Sigma, H3393), 1x Pen / Strep). Cells were incubated overnight in (Gibco, 15140-122). The following morning, cells were starved for 4 hours by replacing the medium with 50 μL of serum-free / ECGS-free F12K medium. Cells were then treated with 50 μL of serum-free / ECGS-free medium containing a dose curve of 2x concentration of bispecific antibody or BMP ligand. At different time points (5 min, 15 min, 30 min, 60 min), the medium was removed from the cells, and 50 μL of lysis buffer (Abcam ELISA kit, AB186037) was added to each well. After lysis, the buffer from every four wells was combined into a 200 μL lysis sample for each condition, which was frozen and subsequently detected on an ELISA measuring total SMAD1 (Abcam, AB186037) or pSMAD1 (Abcam, AB186036). As a negative control, an anti-HEL antibody with the LALA-PG mutation (BioXCell, CP149) was used.

[0305] Table 11. Phosphorylation of SMAD1 after treatment with bispecific antibodies.

[0306]

[0307] The agonist activity of the bispecific antibody in vivo was also determined. The agonist activity of the antibody was measured in an HHT mouse model, where circulating BMP9 / BMP10 was neutralized by anti-BMP9 / 10 antibody (Ruiz S et al., Scientific Reports, 2016 Nov 22: 5:37366). These mice exhibited vascular defects in the retina after birth. Three animals were administered DGL288 or negative control antibodies (anti-HEL, LALA-PG, BioXCell, CP149) at 15 mg / kg / day for two days, i.e., P3 and P4. BMP9 / 10 antibody was administered on the same day. Analysis was performed at P6. The retina was dissected and prepared into whole slides, which were then stained with both isolectin B4 and SMA to label the retinal vascular system and detect arteriovenous malformations (AVMs). Results were... Figure 3AIn mice treated with DGL288, no AVMs were observed, while the negative control group showed an average of 4.8 AVMs per retina.

[0308] For the second group of experiments, all animals were administered BMP9 / 10 antibody at points P3 and P4. DGL288, DGL292, or PBS controls were administered at 1 mg / kg / day at points P4 and P5. Analysis was performed on point P6 for DGL288 and littermate negative controls, and on point P7 for DGL292 and littermates administered with PBS controls. Retinas were dissected and prepared into whole slides, then stained with both isolectin B4 and SMA to detect AVMs. Mice administered DGL292 did not develop AVMs compared to the control group's average of 5.7 AVMs per retina. Figure 3B Mice treated with DGL288 did not develop AVMs, compared to an average of 4.5 AVMs per retina in the control group. Figure 3C No difference in body weight was observed, indicating that the agonist was well tolerated.

[0309] Example 4. Engineering bispecific agonist antibodies using optimized linkers in DVD-Ig format.

[0310] An alternative approach to rigidifying antibodies is to optimize the linker between IgG and the additional variable domain in the DVD-Ig format. To investigate whether the DVD-Ig format is a viable format for bispecific agonist antibodies, the activity of heteropolymers with modified VH-to-IgG hinges, identified via the DIAGONAL platform, was measured. Variants of DGL292 (DGL810, DGL811, and DGL812) were designed, expressed, and purified as described above. The heteropolymers were tested using DiscoverX assays, and all variants outperformed the parental DGL292, as shown in Table 14 (showing the average of two different experiments).

[0311] Table 12. Connector sequences used in DVD-Ig format.

[0312]

[0313] Table 13. Heavy and light chain sequences of DVD-Ig antibody.

[0314]

[0315]

[0316] Table 14. Agonist activity of the DGL292 variant compared to BMP9.

[0317]

[0318] Table 15. Sequences.

[0319]

[0320]

[0321]

[0322] The agonist activity of additional bispecific antibodies was screened. PathHunter U2Os ALK-1 / BMPR-2 dimerization assays were obtained from DiscoverX Corporation (93-0962C3). Protein-protein interactions were assessed using enzyme fragment complementation (EFC) technology with β-galactosidase fragments. Reporter cells were resuscitated and cultured according to the supplier's recommendations. The bispecific antibodies were compared with natural ligands BMP9 and BMP10.

[0323] For the assay, cells were dissociated from the culture flask and removed using a cell dissociation reagent (DiscoverX, 92-0009). Cells were centrifuged at 300g for four minutes and resuspended at a density of 250 K / ml in assay plating medium (DiscoverX 93-0563R22A). 20 µl of the suspension was plated into each well of a 384-well plate and incubated at 37°C for 24 hours. Bispecific antibodies were prepared 5 times the final concentration. A 1:10 dilution was used for 12-point titration to generate a curve. 5 µl of the bispecific antibody was added to each well of the 384-well plate and incubated for three hours. 25 µl of flash assay reagent (DiscoverX, 93-0247) was added to each well, and the plate was read on a Verilux Skan after 60 minutes. Data were analyzed using PRISM. The results are shown in Table 16 below. The data demonstrate that each of the tested bispecific antibodies possesses potent agonist activity.

[0324] Table 16. Agonist activity in DiscoverX assays

[0325] The agonistic activity of antibodies was measured in a HHT mouse model, in which circulating BMP9 / BMP10 was neutralized by anti-BMP9 / 10 antibodies (Ruiz S et al., Scientific Reports, 2016 Nov 22: 5:37366). These mice exhibited vascular defects in the retina after birth. Three animals were administered DGL292, DGL945, DGL947, or negative control antibodies (anti-HEL, LALA-PG, BioXCell, CP149) at 1 mg / kg / day for two days, i.e., P3 and P4. BMP9 / 10 antibodies were administered on the same day. Analysis was performed at P6. The retina was dissected and prepared into whole slides, which were then stained with both isolectin B4 and SMA to label the retinal vascular system and detect arteriovenous malformations (AVMs). Results were shown in Figure 4 In mice, administration of any ALK1-BMPRII agonist showed a significant reduction in AVM formation, while the negative control showed an average of 4.5 AVMs per retina.

[0326] Example 5. Humanization of IL-18R binder.

[0327] The conjugates previously identified with high activity levels using HEK Blue assays were humanized and optimized for therapeutic use. VHH conjugates targeting IL-18Rα and IL-18Rβ were computer-modeled with the antigens using the DIAGONAL platform. Residues not essential for epitope recognition were replaced with human sequences. Reverse mutations were added solely to maintain antigen binding and stability. Affinity of the constructs was measured using Carterra, and activity was measured using HEK Blue assays. Furthermore, in some cases, proline at position 14 of the VHH binding domain was replaced with alanine to enhance the stability and agonistic effect of the conjugates.

[0328] When DGL207 was humanized, decreased potency and affinity for IL-18Rβ (DGL333) were observed. Using the DIAGONAL platform, it was observed that the proline at position 14 (P14) may destabilize the molecule in the context of a rigid hinge (hinge 1). Reverting this mutation to alanine, present in llama lines, enhanced the affinity and activity of the bispecific antibody in the HEK Blue assay. Figure 5 Therefore, according to Kabat, the alanine substitution at position 14 of VHH enhances agonist activity.

[0329] In addition to humanizing VHH, Fc was engineered for optimal therapeutic use (e.g., LALAGA, mortis, and YTE mutations).

[0330] Table 17. Optimized IL-18R antibody.

[0331]

[0332] Table 18. Optimized sequences of agonist antibodies against IL-18R.

[0333]

Claims

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

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

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

5. The multispecific binding protein of 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, wherein: The first polypeptide chain includes a first variable heavy chain domain (VH1) connected to a second variable heavy chain domain (VH2) via at least one modified hinge region.

7. The multispecific binding protein of claim 66, wherein one or both of VH1 and VH2 are VH domains or VHH domains.

8. The multispecific binding protein of claim 6, further comprising a second polypeptide chain, wherein the second polypeptide chain comprises a first variable light chain domain (VL1) connected 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 6-8, wherein one or both of VH1 and VH2 are truncated at the C-terminus.

10. The multispecific binding protein of claim 9, wherein the C-terminus is truncated by at least one residue.

11. The multispecific binding protein of claim 9 or 10, wherein the C-terminus is truncated by at least two residues.

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

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

14. The multispecific binding protein of any one of claims 1-13, wherein the modified hinge region comprises: i) An upper hinge region with a length of at most 7 amino acids, or no upper hinge region; and ii) Lower hinge area.

15. The multispecific binding protein of any one of claims 1-14, wherein the modified hinge region comprises or is composed of the amino acid sequence PLAP or PAPNLLGGP.

16. The multispecific binding protein of any one of claims 1-15, 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 of any one of claims 6-16, wherein the VH1 binds to a first receptor subunit and the 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 of any one of claims 8-16, wherein a first antigen-binding domain formed by the VH1 and the VL1 binds to a first receptor subunit, and a second antigen-binding domain formed by the VH2 and the 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 multispecific binding protein of any one of claims 5-16, wherein the first scFv binds to the first receptor subunit and the second scFv binds to the second receptor subunit, thereby inducing signal transduction by inducing proximity between the first receptor subunit and the second receptor subunit.

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

21. The multispecific binding protein of any one of claims 16-19, wherein the first receptor subunit and the second receptor subunit are the same subunit that form a homodimer.

22. The multispecific binding protein of any one of claims 16-21, wherein the first receptor subunit and the second receptor subunit are selected from: tumor necrosis factor superfamily (TNFSF) receptor, type I interleukin receptor, type II interleukin receptor, Ig superfamily (IGSF) receptor, receptor tyrosine kinase (RTK), growth hormone receptor, transforming growth factor β (TGFβ) receptor superfamily, type C lectin-like receptor, interferon receptor, phosphatase receptor (i.e., receptor-type protein tyrosine phosphatase), and integrin receptor.

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

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

25. The multispecific binding protein of claim 24, wherein the modified hinge region comprises: i) An upper hinge region with a length of at most 7 amino acids, or no upper hinge region; and ii) Lower hinge area.

26. The multispecific binding protein of claim 24 or 25, wherein the modified hinge region comprises or is composed of the amino acid sequence of PLAP or PAPNLLGGP.

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

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

29. The multispecific binding protein of claim 27, wherein the first receptor subunit and the second receptor subunit are the same subunit that form a homodimer.

30. The multispecific binding protein of any one of claims 27-29, wherein the first receptor subunit and the second receptor subunit are selected from: tumor necrosis factor superfamily (TNFSF) receptor, type I interleukin receptor, type II interleukin receptor, Ig superfamily (IGSF) receptor, receptor tyrosine kinase (RTK), growth hormone receptor, transforming growth factor β (TGFβ) receptor superfamily, C-type lectin-like receptor, interferon receptor, phosphatase receptor (i.e., receptor-type protein tyrosine phosphatase), and integrin receptor.

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

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

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

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

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

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

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

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

39. The multispecific binding protein as described in claim 38, 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).

40. The multispecific binding protein of any one of claims 31-39, wherein the heavy chain constant region comprises a heterodimerization mutation to promote heterodimerization of the first binding moiety and the second binding moiety.

41. The multispecific binding protein of claim 40, wherein the heterodimerization mutation is a kilomerization (KIH) mutation.

42. The multispecific binding protein of claim 41, wherein the first heavy chain constant region comprises a mordant-generating amino acid substitution at position 366, 368 or 407, and the second heavy chain constant region comprises a mordant-generating amino acid substitution at position 366.

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

44. The multispecific binding protein of claim 40, wherein the heterodimerization mutation is a charge stabilization mutation.

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

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

47. The multispecific binding protein of claim 40, wherein the heterodimerization mutation comprises engineered disulfide bonds.

48. The multispecific binding protein of claim 47, wherein the engineered disulfide bond is formed by a first heavy chain constant region comprising an amino acid substitution Y349C and a second heavy chain constant region comprising an amino acid substitution S354C.

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

50. The multispecific binding protein of 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 of any one of claims 31-50, wherein at least one heavy chain constant region contains one or more mutations to promote a prolonged half-life.

52. The multispecific binding protein of claim 51, wherein, according to EU designation, at least one heavy chain constant region comprises one or more substitutions at amino acid positions 252, 254, or 256.

53. The multispecific binding protein as described in claim 52, 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).

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

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

56. A pharmaceutical composition comprising a multispecific binding protein as described in any one of the preceding claims and a pharmaceutically acceptable carrier.

57. An isolated nucleic acid molecule encoding a multispecific binding protein as described in any one of claims 1-55.

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

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

60. A method for treating a disease or condition in a subject, comprising administering to a subject in need the multispecific binding protein as described in any one of claims 1-55.

61. The multispecific binding protein as described in any one of claims 1-55, used as a drug.

62. The multispecific binding protein as described in any one of claims 1-55, which is used as a diagnostic reagent.

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 as described in any one of claims 1-55.

64. The method of claim 63, wherein the multispecific binding protein is capable of inducing signal transduction by inducing proximity between the first receptor subunit and the second receptor subunit.

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

66. The method of any one of claims 63-65, wherein the multispecific binding protein induces agonist activity, the agonist activity being at least about 35% of the activity of the natural ligand for the first receptor subunit and the second receptor subunit.

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