Heterodimeric Fc fusion protein

Abstract

The present invention provides Fc fusion protein constructs that have a higher serum half-life as a monovalent dimer compared to native / natural molecules, thereby facilitating higher protein titers during production, higher stability during storage, and improved efficacy when used as therapeutic agents. Also provided are Fc fusion protein constructs with mutations in the Fc region that reduce effector function, which have increased activity in inhibiting tumor growth, and are therefore advantageous when used as cancer therapy.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 749,489 filed on October 23, 2018, U.S. Provisional Patent Application No. 62 / 781,898 filed on December 19, 2018, U.S. Provisional Patent Application No. 62 / 788,499 filed on January 4, 2019, U.S. Provisional Patent Application No. 62 / 827,347 filed on April 1, 2019, and U.S. Provisional Patent Application No. 62 / 895,889 filed on September 4, 2019, the disclosures of which are hereby incorporated by reference in their entireties for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on October 22, 2019 is named DFY-063WO_SL.txt and is 946,355 bytes in size. Technical Field

[0005] The present invention generally relates to heterodimeric Fc fusion proteins and pharmaceutical compositions comprising such proteins, and methods of use in treating diseases or disorders in human patients. Background Art

[0006] Physiologically active proteins mostly have the disadvantage of short half-life in vivo. In order to solve this shortcoming, attempts have been made to conjugate them with PEG (polyethylene glycol) etc., or to fuse them with antibody Fc (crystallizable fragment) region. The protein (wherein the two or more different subunits form a protein complex to show physiological activity) composed of two or more different subunits can be fused with wild-type Fc domains to prepare Fc fusion protein form, due to the homodimer nature of Fc, thereby forming a homodimer. The protein (wherein the two or more different subunits form a protein complex to show physiological activity) composed of two or more different subunits can also be fused with the heterodimer Fc region not only derived from IgG1 but also derived from other isotype antibodies (such as IgG2, IgG3 and IgG4), to form heterodimer Fc fusion protein. Therefore, one or more subunits of the protein composed of two or more different subunits and wherein two or more subunits show physiological activity by forming a protein complex can be fused with the end of the heterodimer Fc variant region, to form an improved Fc fusion protein form.

[0007] Fc heterodimerization is a technology that induces mutations in two different CH3 domains of Fc by genetic engineering so that the two Fc fragments form a heterodimer with minimal sequence variation while having a tertiary structure very similar to that of a naturally occurring antibody (see, e.g., U.S. Pat. No. 7,695,936).

[0008] The invention described in this disclosure provides a design for improving the Fc fusion protein format, in which the two subunits of the heterodimeric protein are connected to two Fc domains with different heterodimerization domains by introducing different length linkers or mutations in the CH2 and CH3 domains of Fc. Summary of the invention

[0009] The present invention generally relates to heterodimeric Fc fusion proteins and pharmaceutical compositions comprising such proteins.

[0010] In one aspect, the present invention provides a heterodimeric Fc fusion protein, the fusion protein comprising a first polypeptide comprising a first antibody Fc domain polypeptide and a first subunit of a multi-subunit protein; and a second polypeptide comprising a second antibody Fc domain polypeptide and a second different subunit of the multi-subunit protein, wherein the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise a different mutation that promotes heterodimerization, wherein the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide comprise one or more mutations that reduce the effector function of Fc, and wherein the first subunit and the second different subunit of the multi-subunit protein are bound to each other.

[0011] In some embodiments, the effector function includes the ability of Fc domain polypeptides to induce antibody-dependent cellular toxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human antibody Fc domain polypeptides. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD or IgE Fc domain polypeptides (e.g., human IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD or IgE Fc domain polypeptides). In some embodiments, the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide are included in one or more positions 233, 234, 235, 236, 237, 297, 318, 320, 322, 329, 330 and / or 331 according to EU numbering. One or more mutations at. In some embodiments, the heterodimeric Fc fusion protein is fucosylated.

[0012] In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG1 Fc domain polypeptides. In some embodiments, the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide are included in one or more mutations at positions 234, 235, 237, 329, 330 and / or 331 according to EU numbering. In some embodiments, the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide include one or more mutations selected from 234A, L235A, L235E, G237A, P329A, A330S and P331S. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each include mutations L234A and L235A. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each include mutations L234A, L235A and P329A. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise mutations L234A, L235E, G237A, A330S and P331 S. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise mutation C220S.

[0013] In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG4 Fc domain polypeptides. In some embodiments, the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide are included in one or more mutations at one or more positions 235 and / or 329 according to EU numbering. In some embodiments, the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide include one or more mutations selected from L235E and P329A. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each include mutation L235E. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each include mutation L235E and P329A. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each include mutation S228P.

[0014] In another aspect, the present invention provides a heterodimeric Fc fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291.

[0015] In yet another aspect, the present invention provides a polypeptide comprising a subunit of a multi-subunit cytokine and an immunoglobulin Fc domain polypeptide; the Fc domain polypeptide comprises a mutation for promoting heterodimerization with a different immunoglobulin Fc domain polypeptide and one or more mutations that reduce the effector function of Fc. For example, the Fc domain polypeptide comprising the mutation can be a human IgG1 antibody Fc domain polypeptide.

[0016] In some embodiments, the one or more mutations that reduce the effector function of Fc are selected from L234A, L235A or L235E, G237A, P329A, A330S and P331S, numbered according to the EU numbering system. For example, in some embodiments, the mutations that reduce the effector function of Fc are L234A, L235A and P329A, numbered according to the EU numbering system.

[0017] In some embodiments, the mutations for promoting heterodimerization are K360E and K409W, numbered according to the EU numbering system. In some other embodiments, the mutations for promoting heterodimerization are Q347R, D399V and F405T, numbered according to the EU numbering system.

[0018] In some embodiments, the Fc domain polypeptide further comprises a mutation for promoting disulfide bond formation with a different immunoglobulin Fc domain polypeptide. For example, when the heterodimerization mutations are K360E and K409W, in some embodiments, the mutation for promoting disulfide bond formation is Y349C, numbered according to the EU numbering system. When the heterodimerization mutations are Q347R, D399V and F405T, in some embodiments, the mutation for promoting disulfide bond formation is S354C, numbered according to the EU numbering system.

[0019] For example, in some embodiments, the polypeptide comprising a subunit of a multi-subunit cytokine and an immunoglobulin Fc domain polypeptide comprises the amino acid sequence of SEQ ID NO:290 or the amino acid sequence of SEQ ID NO:291.

[0020] In another aspect, the present invention provides a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 290 or SEQ ID NO: 291. In yet another aspect, the present invention provides an expression vector comprising a nucleic acid comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 290 or SEQ ID NO: 291. In one aspect, the present invention provides a cell comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 290 or SEQ ID NO: 291; or an expression vector comprising a nucleic acid comprising a sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 290 or SEQ ID NO: 291.

[0021] In another aspect, the present invention provides a heterodimeric Fc fusion protein comprising a first Fc domain polypeptide and a second different Fc domain polypeptide of an immunoglobulin Fc, and a first subunit and a second different subunit of a multi-subunit protein, wherein the first subunit and the second different subunit are bound to each other and connected to one or more ends of the N-terminus or C-terminus of the first Fc domain polypeptide and / or the second different Fc domain polypeptide; wherein the first Fc domain polypeptide and the second Fc domain polypeptide are mutated to promote heterodimeric Fc formation and reduce the effector function of Fc. In some embodiments, the multi-subunit protein is IL-12 (e.g., human IL-12), so that in the heterodimeric Fc fusion protein, the p35 and p40 subunits of IL-12 are bound to each other and connected to one or more ends of the N-terminus or C-terminus of the first Fc domain polypeptide and / or the second Fc domain polypeptide; wherein the first Fc domain polypeptide and the second Fc domain polypeptide are mutated to promote heterodimeric Fc formation and reduce the effector function of Fc.

[0022] In one aspect, the present invention provides a heterodimeric Fc fusion protein, the fusion protein comprising: a first polypeptide comprising a first antibody Fc domain polypeptide; and a second polypeptide comprising a second antibody Fc domain polypeptide bound to the first antibody Fc domain polypeptide, wherein the first polypeptide further comprises a first subunit of a multi-subunit protein, the first subunit being fused to the first antibody Fc domain polypeptide via a linker comprising the amino acid sequence PKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 237) or EPKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 6), wherein X1 represents L or A, X2 represents L, E or A, and X3 represents A or G; a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide, and the subunits of the multi-subunit protein are bound to each other; when X1 represents L and / or X2 represents L, at least one of the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide comprises a Q347R mutation for promoting heterodimerization.

[0023] In some embodiments, in the heterodimeric Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide comprises the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9). In some embodiments, in the heterodimeric Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide consists of the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9).

[0024] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 244). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 244).

[0025] In some embodiments, within the heterodimeric Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide comprises the amino acid sequence PKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 238) or EPKSSDKTHTCPPCPAPELL GG (SEQ ID NO: 7). In some embodiments, within the heterodimeric Fc fusion protein, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of the amino acid sequence PKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 238) or EPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 7).

[0026] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 8) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 241). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 8) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 241).

[0027] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 15) or GGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 242). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 15) or GGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 242).

[0028] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 16) or GGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 243). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 16) or GGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 243).

[0029] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 65) or GGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 245). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 65) or GGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 245).

[0030] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 66) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 246). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 66) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 246).

[0031] In some embodiments, within the heterodimeric Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide comprises the amino acid sequence EPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 11) or PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 240). In some embodiments, within the heterodimeric Fc fusion protein, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of the amino acid sequence EPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 11) or PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 240).

[0032] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 247). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 247).

[0033] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67) or GGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 248). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67) or GGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 248).

[0034] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 68) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 249). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 68) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 249).

[0035] In another aspect, the present invention provides a heterodimeric Fc fusion protein, which comprises subunits of a multi-subunit cytokine, wherein the subunits are connected to an immunoglobulin Fc domain polypeptide via a linker, wherein the linker comprises the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239); the Fc domain polypeptide comprises mutations for promoting heterodimerization with different immunoglobulin Fc domain polypeptides, and L234A, L235A and P329A substitutions for reducing the effector function of Fc.

[0036] In some embodiments, the present invention provides a heterodimeric Fc fusion protein, comprising a p40 subunit of human IL-12, which is connected to a first human IgG1 (hIgG1) Fc domain polypeptide via a linker, wherein the linker comprises or consists of the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239); and a p35 subunit of human IL-12, which is connected to a second-hIgG1 Fc domain polypeptide via a linker, wherein the linker comprises or consists of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10); the first Fc domain polypeptide and the second Fc domain polypeptide comprise mutations that promote heterodimerization, and L234A, L235A and P329A substitutions for reducing the effector function of hIgG1 Fc.

[0037] In one aspect, the present invention provides a heterodimeric Fc fusion protein, the fusion protein comprising: a first polypeptide comprising a first antibody Fc domain polypeptide; and a second polypeptide comprising a second antibody Fc domain polypeptide, wherein the first polypeptide also comprises a first subunit of a multi-subunit protein, wherein the protein sequence is fused to the first antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence RVESKYGPPCPPCPAPEFXGG (SEQ ID NO: 1), wherein X represents L or E; a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide, and the subunits of the multi-subunit protein are bound to each other, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain different mutations that promote heterodimerization, and the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are bound to each other.

[0038] In some embodiments, the heterodimeric Fc fusion protein of the present invention comprises a first polypeptide comprising a first antibody Fc domain polypeptide; and a second polypeptide comprising a second antibody Fc domain polypeptide, wherein the first polypeptide further comprises a first subunit of a multi-subunit protein, wherein the protein sequence is fused to the first antibody Fc domain polypeptide via a linker; and the second polypeptide further comprises a second different subunit of the multi-subunit protein, wherein the protein sequence is fused to the second antibody Fc domain polypeptide via a linker. The linker connecting the protein sequence of the second different subunit of the multi-subunit protein with the second antibody Fc domain polypeptide may include G4S (SEQ ID NO: 110), (G4S)2 (SEQ ID NO: 109), or (G4S)3 (SEQ ID NO: 108).

[0039] In some embodiments, within the heterodimeric Fc fusion protein, the linker connecting the subunits of the multi-subunit protein to the first antibody Fc domain polypeptide comprises the amino acid sequence RVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 2). In some embodiments, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of the amino acid sequence RVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 2).

[0040] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 3). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 3).

[0041] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13).

[0042] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 14). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 14).

[0043] In some embodiments, within the heterodimeric Fc fusion protein, the linker connecting the subunits of the multi-subunit protein to the first antibody Fc domain polypeptide comprises the amino acid sequence RVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 4). In some embodiments, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of the amino acid sequence RVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 4).

[0044] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5).

[0045] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63).

[0046] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64).

[0047] Some heterodimeric Fc fusion proteins described herein include a first IgG4 antibody Fc domain polypeptide and a second different IgG4 antibody Fc domain polypeptide, each of which is mutated to promote heterodimerization with each other. In some embodiments, the first IgG4 antibody Fc domain polypeptide includes one or more mutations selected from K370E and R409W, and the second different IgG4 antibody Fc domain polypeptide includes one or more mutations selected from E357N, D399V and F405T. In some embodiments, the first IgG4 antibody Fc domain polypeptide includes one or more mutations selected from E357N, D399V and F405T, and the second different IgG4 antibody Fc domain polypeptide includes one or more mutations selected from K370E and R409W. In some embodiments, the first IgG4 antibody Fc domain polypeptide includes mutations K370E and R409W, and the second different IgG4 antibody Fc domain polypeptide includes mutations E357N, D399V and F405T. In some embodiments, the first IgG4 antibody Fc domain polypeptide comprises mutations E357N, D399V, and F405T, and the second different IgG4 antibody Fc domain polypeptide comprises mutations K370E and R409W. In some embodiments, the first IgG4 Fc domain polypeptide comprises one or more mutations selected from K360E and R409W, and the second different IgG4 Fc domain polypeptide comprises one or more mutations selected from Q347R, D399V, and F405T. In some embodiments, the first IgG4 Fc domain polypeptide comprises one or more mutations selected from Q347R, D399V, and F405T, and the second different IgG4 Fc domain polypeptide comprises one or more mutations selected from K360E and R409W. In some embodiments, the first IgG4 Fc domain polypeptide comprises mutations K360E and R409W, and the second different IgG4 Fc domain polypeptide comprises mutations Q347R, D399V, and F405T. In some embodiments, the first IgG4 Fc domain polypeptide comprises mutations Q347R, D399V, and F405T, and the second, different IgG4 Fc domain polypeptide comprises mutations K360E and R409W.

[0048] Some heterodimeric Fc fusion proteins disclosed herein include a first IgG1 antibody Fc domain polypeptide and a second different IgG1 antibody Fc domain polypeptide, each of which is mutated to promote heterodimerization with each other. In some embodiments, the first IgG1 Fc domain polypeptide includes one or more mutations selected from K360E and K409W, and the second different IgG1 Fc domain polypeptide includes one or more mutations selected from Q347R, D399V and F405T. In some embodiments, the first IgG1 Fc domain polypeptide includes one or more mutations selected from Q347R, D399V and F405T, and the second different IgG1 Fc domain polypeptide includes one or more mutations selected from K360E and K409W. In some embodiments, the first IgG1 antibody Fc domain polypeptide includes mutations K360E and K409W, and the second different IgG1 antibody Fc domain polypeptide includes mutations Q347R, D399V and F405T. In some embodiments, the first IgG1 antibody Fc domain polypeptide comprises mutations Q347R, D399V, and F405T, and the second, different IgG1 antibody Fc domain polypeptide comprises mutations K360E and K409W.

[0049] In some embodiments, the heterodimeric Fc fusion proteins described herein comprise an IgG4 or IgG1 Fc domain polypeptide that is further mutated to reduce effector function. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain a mutation P329G or P329A. In some embodiments, the first IgG4 antibody Fc domain polypeptide and the second different IgG4 antibody Fc domain polypeptide each contain a mutation P329G or P329A. In some embodiments, the first IgG1 antibody Fc domain polypeptide and the second different IgG1 antibody Fc domain polypeptide each contain a mutation P329G or P329A. In some embodiments, the first IgG4 antibody Fc domain polypeptide and the second different IgG4 antibody Fc domain polypeptide each contain a mutation P329A. In some embodiments, the first IgG1 antibody Fc domain polypeptide and the second different IgG1 antibody Fc domain polypeptide each contain a mutation P329A.

[0050] In some embodiments, the Fc-based heterodimeric fusions described herein incorporate a first IgG1 antibody Fc domain polypeptide and a second different IgG1 antibody Fc domain polypeptide, each of which contains a mutation selected from A330S and P331S. In some embodiments, the Fc-based heterodimeric fusions described herein incorporate a first IgG1 antibody Fc domain polypeptide and a second different IgG1 antibody Fc domain polypeptide, each of which contains mutations A330S and P331S.

[0051] In some embodiments, the Fc-based heterodimeric proteins described herein incorporate an IgG4 or IgG1 Fc domain polypeptide that is further mutated to introduce an interchain disulfide bond. In some embodiments, the first IgG4 or IgG1 Fc domain polypeptide comprises the mutation Y349C, and the second different IgG4 or IgG1 Fc domain polypeptide comprises the mutation S354C. In some embodiments, the first IgG4 or IgG1 Fc domain polypeptide comprises the mutation S354C, and the second different IgG4 or IgG1 Fc domain polypeptide comprises the mutation Y349C.

[0052] Some heterodimeric Fc fusion proteins of the present invention include a native disulfide bond between a first subunit of a multi-subunit protein and a second, different subunit of the multi-subunit protein. For example, in an exemplary embodiment, a heterodimeric Fc fusion protein according to the present invention includes a native heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. This protein includes a native disulfide bond between C74 of p35 and C177 of p40.

[0053] Some heterodimeric Fc fusion proteins of the present invention include an artificial or engineered heterodimeric disulfide bond between a first subunit of a multi-subunit protein and a second, different subunit of the multi-subunit protein. For example, in an exemplary embodiment, a heterodimeric Fc fusion protein according to the present invention includes an artificial or engineered heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. This protein includes an artificial or engineered disulfide bond between V185C of p35 and Y292C of p40.

[0054] Some heterodimeric Fc fusion proteins of the present invention include a natural disulfide bond between a first subunit of a multi-subunit protein and a second different subunit of the multi-subunit protein, and an artificial or engineered heterodimeric disulfide bond between a first subunit of a multi-subunit protein and a second different subunit of the multi-subunit protein. For example, in an exemplary embodiment, a natural heterodimeric disulfide bond between p35 and p40 subunits of IL-12, and an artificial or engineered heterodimeric disulfide bond between p35 and p40 subunits of IL-12. This protein includes a natural disulfide bond between C74 of p35 and C177 of p40, and an artificial or engineered disulfide bond between V185C of p35 and Y292C of p40.

[0055] Some heterodimeric Fc fusion proteins of the present invention are engineered to remove natural disulfide bonds, which are then replaced by non-natural artificial or engineered disulfide bonds. For example, in an exemplary embodiment, a heterodimeric Fc fusion protein according to the present invention includes p35 of IL-12 in which the native C74 is mutated to serine, and p40 of IL-12 in which the native C177 is mutated to serine, thereby removing the natural disulfide bond between the p35 and p40 subunits of IL-12. Two new mutations, V185C on p35 and Y292C on p40, are introduced into the mutant IL-12, thereby introducing non-natural artificial or engineered disulfide bonds.

[0056] In the heterodimeric Fc fusion protein of the present invention, the first polypeptide and the second different polypeptide respectively comprise the first subunit and the second different subunit of the multi-subunit cytokine. In the heterodimeric Fc fusion protein of the present invention, the first polypeptide and the second different polypeptide respectively comprise the second different subunit and the first subunit of the multi-subunit cytokine. In an exemplary embodiment, the cytokine is IL-12 (e.g., human IL-12). Also provided are preparations containing any one of the heterodimeric Fc fusion proteins described herein, cells containing one or more nucleic acids expressing the heterodimeric Fc fusion proteins or one or more vectors expressing the heterodimeric Fc fusion proteins, and methods for enhancing tumor cell death using the heterodimeric Fc fusion proteins. In some embodiments, the present invention provides a preparation comprising the heterodimeric Fc fusion protein described herein and a pharmaceutically acceptable carrier.

[0057] In another aspect, the heterodimer Fc fusion protein of the present invention further comprises at least one antibody variable domain (e.g., antibody heavy chain variable domain). In certain embodiments, the at least one antibody heavy chain variable domain is combined with an antibody light chain variable region to form a Fab, and the heavy chain variable domain or light chain variable domain of the Fab is fused at the N-terminus of the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide.

[0058] In certain embodiments, the heterodimeric Fc fusion protein of the present invention comprising at least one antibody variable domain has a first subunit of a multimeric protein and a second different subunit of the multimeric protein connected to the C-termini of the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide, respectively.

[0059] In certain embodiments, the heterodimeric Fc fusion protein of the present invention comprising at least one antibody variable domain has a first subunit of a multimeric protein and a second different subunit of the multimeric protein connected to the C-termini of the second antibody Fc domain polypeptide and the first antibody Fc domain polypeptide, respectively.

[0060] In certain embodiments, the heterodimeric Fc fusion protein of the present invention comprises an antibody heavy chain variable domain located at the C-terminus of the first antibody Fc domain polypeptide relative to the first polypeptide. In certain embodiments, the heterodimeric Fc fusion protein of the present invention comprises an antibody heavy chain variable domain located at the C-terminus of the second antibody Fc domain polypeptide relative to the second polypeptide. In certain embodiments, the antibody heavy chain variable region is combined with the antibody light chain variable region to form an scFv. In certain embodiments, the first subunit of the multi-subunit protein and the second different subunit of the multi-subunit protein are respectively connected to the N-terminus of the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide. In certain embodiments, the first subunit of the multi-subunit protein and the second different subunit of the multi-subunit protein are respectively connected to the N-terminus of the second antibody Fc domain polypeptide and the first antibody Fc domain polypeptide.

[0061] In some embodiments, the heterodimeric Fc fusion protein of the present invention does not include an antibody variable domain. For example, in some embodiments, the heterodimeric Fc fusion protein consists of or essentially consists of: a first subunit of a multi-subunit protein (e.g., an IL-12 protein), a linker optionally comprising a spacer peptide, and a first antibody Fc domain polypeptide; and a second different subunit of the multi-subunit protein (e.g., an IL-12 protein), a linker optionally comprising a spacer peptide, and a second antibody Fc domain polypeptide.

[0062] In some embodiments, the heterodimeric Fc fusion protein of the present invention further comprises a proteoglycan binding domain. In some embodiments, the proteoglycan binding domain binds to one or more proteoglycans specifically expressed in tumors. In some embodiments, the proteoglycan binding domain binds to one or more proteoglycans selected from the following: cohesin, serglycan, CSPG4, beta-proteoglycan, phosphatidylinositol proteoglycan, leucine, versican, short proteoglycan and small leucine-rich proteoglycan (SLRP). In some embodiments, the SLRP is selected from decorin, biglycan, asporidin, fibromodulin and aphrodisiac. In some embodiments, the proteoglycan binding domain is connected to the C-terminus of the first antibody Fc domain polypeptide. In some embodiments, the proteoglycan binding domain is connected to the C-terminus of the second antibody Fc domain polypeptide.

[0063] In some embodiments, the heterodimeric Fc fusion protein of the present invention further comprises a collagen binding domain. In some embodiments, the collagen binding domain binds to one or more collagens specifically expressed in a tumor. In some embodiments, the collagen binding domain is connected to the C-terminus of the first antibody Fc domain polypeptide. In some embodiments, the collagen binding domain is connected to the C-terminus of the second antibody Fc domain polypeptide.

[0064] In some embodiments, the heterodimer Fc fusion protein of the present invention further comprises a hyaluronic acid binding domain. In some embodiments, the hyaluronic acid binding domain is connected to the C-terminal end of the first antibody Fc domain polypeptide. In some embodiments, the hyaluronic acid binding domain is connected to the C-terminal end of the second antibody Fc domain polypeptide.

[0065] Another aspect of the present invention provides a method for treating cancer in a patient. The method comprises administering to a patient (e.g., a patient in need thereof) an effective amount of a heterodimeric Fc fusion protein as described herein or a formulation comprising an effective amount of a multispecific binding protein as described herein. For example, in some embodiments, the method for treating cancer comprises administering to a patient (e.g., a patient in need thereof) a formulation comprising an effective amount of a heterodimeric Fc fusion protein as described herein and a pharmaceutically acceptable carrier.

[0066] In certain embodiments, the disclosure provides a method of treating cancer, comprising administering to a patient only a single dose of a heterodimeric IL-12-Fc fusion protein. In certain embodiments, the amount of the single dose is sufficient to induce a complete response to the cancer. In certain embodiments, the amount of the single dose is sufficient to delay or prevent the recurrence of the cancer.

[0067] In certain embodiments, the present disclosure provides a method of treating cancer, the method comprising administering to a patient only a single dose of a heterodimeric IL-12-Fc fusion protein, the fusion protein comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 291; or a formulation comprising the heterodimeric IL-12-Fc fusion protein and a pharmaceutically acceptable carrier. In certain embodiments, the amount of a single dose of a heterodimeric IL-12-Fc fusion protein comprising a first polypeptide comprising an amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising an amino acid sequence of SEQ ID NO: 291 or a formulation comprising the heterodimeric IL-12-Fc fusion protein and a pharmaceutically acceptable carrier is sufficient to induce a complete response to the cancer. In certain embodiments, the amount of the single dose is sufficient to delay or prevent the recurrence of the cancer.

[0068] Another aspect of the present invention provides a method for treating acute radiation syndrome, wherein the method comprises administering to a patient a heterodimeric Fc fusion protein or formulation disclosed herein. In some embodiments, the acute radiation syndrome comprises one or more syndromes selected from the following: hematopoietic radiation syndrome, gastrointestinal radiation syndrome, neurovascular radiation syndrome, and cutaneous radiation syndrome. In some embodiments, the acute radiation syndrome comprises a syndrome selected from the following: hematopoietic radiation syndrome, gastrointestinal radiation syndrome, neurovascular radiation syndrome, and cutaneous radiation syndrome.

[0069] In summary, the present invention provides heterodimeric Fc fusion protein constructs of multi-subunit proteins. These fusion protein constructs can show higher serum half-life compared to native / natural multi-subunit proteins, improved yield during production, enhanced stability during storage and / or improved efficacy when used as a therapeutic agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1AAn exemplary heterodimeric Fc fusion protein is shown, which comprises a first subunit of a multi-subunit protein, the subunit being connected to a first antibody Fc domain polypeptide by a joint; and a second different subunit of the multi-subunit protein, the subunit being connected to a second antibody Fc domain polypeptide by a joint. The first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain different mutations that promote heterodimerization, and the disulfide bonds between the Fc domain polypeptides stabilize the heterodimer. The joint connecting the other subunit and the second antibody Fc domain polypeptide may include a (G4S)3 sequence (SEQ ID NO: 108).

[0071] Figure 1B Exemplary heterodimeric Fc fusion proteins are shown, which are similar to Figure 1A The proteins displayed, but also included mutations in the Fc domain polypeptide to reduce FcγR binding.

[0072] Figure 1C Exemplary proteins are shown in which a second, different subunit of the multimeric protein is linked to a first antibody Fc domain polypeptide via a linker, and another subunit of the multimeric protein is linked to a second antibody Fc domain polypeptide via another linker.

[0073] Figure 1D Exemplary heterodimeric Fc fusion proteins are shown, which are similar to Figure 1C The proteins displayed, but also included mutations in the Fc domain polypeptide to reduce FcγR binding.

[0074] Figure 1E Shown with Figure 1B The displayed proteins are similar to exemplary heterodimeric Fc fusion proteins, except that the second, distinct subunit of the multi-subunit protein is located C-terminally to the second antibody Fc domain polypeptide in the second polypeptide.

[0075] Figure 1F Shown with Figure 1E The displayed proteins are similar to exemplary heterodimeric Fc fusion proteins, except that the second, distinct subunit of the multi-subunit protein is located C-terminally to the first antibody Fc domain polypeptide in the first polypeptide.

[0076] Figure 2AAn exemplary heterodimeric Fc fusion protein is shown, comprising a first subunit of a multi-subunit protein, the subunit being connected to a first antibody Fc domain polypeptide via a linker; and a second different subunit of the multi-subunit protein, the subunit being connected to a second antibody Fc domain polypeptide via another linker, wherein the subunits are connected via two disulfide bonds. The first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain different mutations that promote heterodimerization, and the disulfide bonds between the Fc domain polypeptides stabilize the heterodimers. The displayed protein also includes mutations in the Fc domain polypeptide to reduce FcγR binding.

[0077] Figure 2B An exemplary heterodimeric Fc fusion protein is shown, which comprises a first subunit of a multi-subunit protein, which is connected to a first antibody Fc domain polypeptide by a linker; and a second different subunit of the multi-subunit protein, which is connected to a second antibody Fc domain polypeptide by another linker, wherein the subunits are connected by a non-natural disulfide bond. In the exemplary protein shown, the natural disulfide bonds have been removed and replaced with artificial disulfide bonds. For example, the IL-12 construct can incorporate mutations p35-V185C / C74S and p40-Y292C / C177S. The first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain different mutations that promote heterodimerization, and the disulfide bonds between the Fc domain polypeptides stabilize the heterodimer. The protein also includes mutations in the Fc domain polypeptide to reduce FcγR binding.

[0078] Figure 3A An exemplary heterodimeric Fc fusion protein is shown, which comprises a first subunit of a multi-subunit protein, the subunit being connected to a first antibody Fc domain polypeptide via a linker; and a second different subunit of the multi-subunit protein, the subunit being connected to a second antibody Fc domain polypeptide via another linker having the same amino acid sequence. The first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain different mutations that promote heterodimerization, and the disulfide bonds between the Fc domain polypeptides stabilize the heterodimer.

[0079] Figure 3B An exemplary heterodimeric Fc fusion protein is shown, which comprises a first subunit of a multi-subunit protein, the subunit being connected to a first antibody Fc domain polypeptide by a joint; and a second different subunit of the multi-subunit protein, the subunit being connected to a second antibody Fc domain polypeptide by another joint. The first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain different mutations that promote heterodimerization, and the disulfide bonds between the Fc domain polypeptides stabilize the heterodimer. The joint connecting the second different subunit of the multi-subunit protein and the second antibody Fc domain polypeptide can include (G4S)2 (SEQ ID NO: 109) or G4S (SEQ ID NO: 110).

[0080] Figures 4A-4L Exemplary scFv fusion heterodimer Fc fusion protein constructs are shown ( Figures 4A-4H ) and mAb fusion heterodimer Fc fusion protein constructs ( Figure 4I-4L ), wherein a first subunit of the multimeric protein is connected to a first antibody Fc domain polypeptide via a linker, and a second different subunit of the multimeric protein is connected to a second antibody Fc domain polypeptide via another linker.

[0081] Figures 4A-4D An exemplary Fc fusion protein is shown, which includes a first scFv connected to the C-terminus of a first antibody Fc domain polypeptide and a second scFv connected to the C-terminus of a second antibody Fc domain polypeptide. The first scFv and the second scFv can be the same or different (e.g., bind to different antigens or bind to different epitopes on a single antigen). Figure 4A and Figure 4C The exemplary Fc fusion proteins shown comprise different pairs of heterodimeric Fc variants. Figure 4B and Figure 4D The exemplary Fc fusion proteins shown include different pairs of heterodimerization Fc variants. For example, in the case where the heterodimerization mutations include "knobs-into-holes" mutations, Figure 4A and Figure 4D Exemplary Fc fusion proteins are shown, wherein the first polypeptide comprises one or more "hole" mutations and the second polypeptide comprises one or more "knob" mutations; Figure 4B and Figure 4C Exemplary Fc fusion proteins are shown in which a first polypeptide comprises one or more "knob" mutations and a second polypeptide comprises one or more "hole" mutations. Figure 4A and Figure 4B Exemplary Fc fusion proteins are shown in which the protein sequence of a first subunit of a multi-subunit protein is linked to a first polypeptide; Figure 4C and Figure 4D Exemplary Fc fusion proteins are shown in which a second, different subunit of the multi-subunit protein is linked to a first polypeptide. Heterodimeric Fc fusion proteins with other types of heterodimerization mutations are similarly contemplated.

[0082] Figure 4E and 4G Exemplary Fc fusion proteins are shown that include a scFv linked to the C-terminus of a first antibody Fc domain polypeptide.

[0083] Figure 4F and 4H Exemplary Fc fusion proteins are shown that include a scFv linked to the C-terminus of a second antibody Fc domain polypeptide.

[0084] Figure 4I-4L Exemplary Fc fusion proteins are shown that include a first Fab linked to the N-terminus of a first antibody Fc domain polypeptide and a second Fab linked to the N-terminus of a second antibody Fc domain polypeptide. Fig. 4I and Figure 4K Exemplary Fc fusion proteins are shown in which a first subunit of a multi-subunit protein is linked to a first polypeptide; Figure 4J and Figure 4L Exemplary Fc fusion proteins are shown in which a second, distinct subunit of the multi-subunit protein is linked to a first polypeptide. Figure 4K and Fig. 4I The difference is that it has a longer amino acid sequence (e.g., a spacer peptide disclosed herein) that connects the antibody Fc domain polypeptide to the first subunit of the multi-subunit protein; Figure 4J and Figure 4L The difference is that there is a longer amino acid sequence (eg, a spacer peptide disclosed herein) that connects the antibody Fc domain polypeptide to the first subunit of the multi-subunit protein.

[0085] Figures 5A-5C Figure 2 shows the inoculation of CT26 tumor cells and the treatment with recombinant mouse IL-12 (rmIL-12) ( Figure 5A )、DF-mIL-12-Fc wt( Figure 5B )、DF-mIL-12-Fc si( Figure 5C ) or mIgG2a isotype control once weekly.

[0086] Figure 6 is a graph showing Kaplan-Meier survival curves for mice inoculated with CT26 tumor cells and treated weekly with rmIL-12, DF-mIL-12-Fc wt, DF-mIL-12-Fc si, or mIgG2a isotype control.

[0087] Figures 7A-7D Figure 2 shows the inoculation of CT26 tumor cells and the inoculation of DF-mIL-12-Fc wt with a molar equivalent of 1 μg rmIL-12 ( Fig. 7A ), DF-mIL-12-Fc si was used as the molar equivalent of 1 μg rmIL-12 ( Figure 7B ), DF-mIL-12-Fc wt was used with a molar equivalent of 0.1 μg of mIL-12 ( Figure 7C ), DF-mIL-12-Fc si was used as the molar equivalent of 0.1 μg rmIL-12 ( Fig.7D ) or mIgG2a isotype control once weekly.

[0088] Figure 8 Graph showing Kaplan-Meier survival curves for mice inoculated with CT26 tumor cells and treated weekly with DF-mIL-12-Fc wt at a molar equivalent of 1 μg rmIL-12, DF-mIL-12-Fc si at a molar equivalent of 1 μg rmIL-12, DF-mIL-12-Fc wt at a molar equivalent of 0.1 μg rmIL-12, DF-mIL-12-Fc si at a molar equivalent of 0.1 μg rmIL-12, or mIgG2a isotype control.

[0089] Figures 9A-9C Figure 2 shows the inoculation of B16F10 melanoma cells and the treatment with rmIL-12 ( Fig.9A )、DF-mIL-12-Fc wt( Fig. 9B )、DF-mIL-12-Fc si( Fig. 9C ) or mIgG2a isotype control once weekly.

[0090] Fig.10 Graph showing Kaplan-Meier survival curves for mice inoculated with B16F10 melanoma cells and treated weekly with rmIL-12, DF-mIL-12-Fc wt, DF-mIL-12-Fc si, or mIgG2a isotype control.

[0091] Figures 11A-11D Figure 2 shows the inoculation of B16F10 melanoma cells and the inoculation of DF-mIL-12-Fc wt with a molar equivalent of 0.5 μg rmIL-12 ( Fig.11A ), DF-mIL-12-Fc si was used as the molar equivalent of 0.5 μg rmIL-12 ( Fig. 11B ), DF-mIL-12-Fc wt was used as the molar equivalent of 0.1 μg rmIL-12 ( Fig. 11C ), DF-mIL-12-Fc si was used as the molar equivalent of 0.1 μg rmIL-12 ( Fig.11D ) or mIgG2a isotype control once weekly.

[0092] Fig.12Graph showing Kaplan-Meier survival curves of mice inoculated with B16F10 melanoma cells and treated weekly with DF-mIL-12-Fc wt at a molar equivalent of 0.5 μg rmIL-12, DF-mIL-12-Fc si at a molar equivalent of 0.5 μg rmIL-12, DF-mIL-12-Fc wt at a molar equivalent of 0.1 μg rmIL-12, DF-mIL-12-Fc si at a molar equivalent of 0.1 μg rmIL-12, or mIgG2a isotype control.

[0093] Fig.13A Graph showing IL-12 response to treatment with DF-hIL-12-Fc si (DFIL-12-Fc) or recombinant human IL-12 (rhIL-12) obtained using a HEK-Blue IL-12 reporter assay.

[0094] Fig. 13B is a graph showing IFNγ production in response to peripheral blood mononuclear cells (PBMCs) treated with DF-hIL-12-Fc si (DF IL-12-Fc) and rhIL-12.

[0095] Fig.14 is a graph showing the relative plasma concentrations of DF-hIL-12-Fc si, rhIL-12, and IFNγ in cynomolgus monkey K2 EDTA plasma following a single intravenous dose of equimolar amounts of DF-hIL-12-Fc si or wild-type rhIL-12 at 10 μg / kg.

[0096] Figures 15A-15B Is showing childishness In Balb / c mice, rmIL-12 ( Fig.15A ) and DF-mIL-12-Fc si( Fig. 15B )'s PK / PD profile. Fig.15A The PK / PD profile of rmIL-12 in naive Balb / c mice is shown, and Fig. 15B Shown is the PK / PD profile of DF-mIL-12-Fc si in naive Balb / c mice IL-12. IL-12 and IFNγ levels in serum were analyzed by ELISA. Fig. 15C is a graph showing the PK / PD profile of DF-mIL-12-Fc si administered intravenously in naive Balb / c mice. Fig.15D is a graph showing the PK / PD profile of DF-mIL-12-Fc si administered intraperitoneally in naive Balb / c mice. Fig.15Eis a graph showing the PK / PD profile of DF-mIL-12-Fc si administered subcutaneously in naive Balb / c mice. Mean serum levels represent mean ± SEM.

[0097] Figures 16A-16C Graph showing tumor growth curves of B16F10 tumor-bearing mice treated with DF-mIL-12-Fc si, anti-PD-1, or a combination thereof. Fig.16A ), isotype control or anti-PD-1 ( Fig. 16B ) and isotype control or DF-mIL-12-Fc si / anti-PD-1 ( Fig. 16C ) Mice were treated intraperitoneally. Animals were injected with DF-mIL-12-Fc si once a week as indicated above, and anti-PD-1 was injected twice a week. Tumor growth was assessed for 60 days. The figure shows the tumor growth curve of individual mice.

[0098] Figures 17A-17B Is a graph showing the survival and body weight of mice bearing B16F10 tumors treated with DF-mIL-12-Fc si, anti-PD-1, or a combination thereof. Mice were treated with isotype, DF-mIL-12-Fc si, anti-PD-1, or a combination of DF-mIL-12-Fc si and anti-PD-1. Animals were injected with 0.5 μg DF-mIL-12-Fc si once a week, and animals were injected with 200 μg anti-PD-1 or isotype twice a week. Fig.17A Kaplan-Meier survival curves are shown. Fig. 17B Body weights of mice are shown as mean ± standard deviation.

[0099] Figures 18A-18C Figure 2 is a graph showing tumor growth curves of B16F10 tumor-bearing mice treated with DF-mIL-12-Fc si, mcFAE-C26.99 TriNKET, or a combination thereof. Fig.18A ), isotype control or 150 μg TriNKET ( Fig.18B ) and isotype control or DF-mIL-12-Fc si / TriNKET ( Fig.18C ) Mice were treated intraperitoneally. Animals were injected with DF-mIL-12-Fc si once a week and TriNKET three times a week as indicated above. Tumor growth was assessed for 72 days. The figure shows the tumor growth curves of individual mice.

[0100] Figures 19A-19BIs a graph showing the survival and body weight of mice bearing B16F10 tumors treated with DF-mIL-12-Fc si, mcFAE-C26.99 TriNKET or a combination thereof. Mice were treated with isotypes, DF-mIL-12-Fc si, TriNKET or a combination of DF-mIL-12-Fc si and TriNKET. Animals were injected with 0.5 μg DF-mIL-12-Fc si once a week and 150 μg TriNKET or isotypes three times a week. Fig.19A Kaplan-Meier survival curves are shown. Fig.19B Body weights of mice are shown as mean + standard deviation.

[0101] Fig. 20 is a graph showing the tumor growth curve of complete responder (CR) mice from the B16F10 tumor model experiment in FIG. 18 , wherein the mice were treated with DF-mIL-12-Fc si / TriNKET combination therapy (n=3) by implanting 2×10 5 B16F10 melanoma cells were re-stimulated.

[0102] Fig.21A is a graph showing tumor growth curves of individual mice inoculated with CT26 tumor cells and administered a single dose of DF-mIL-12-Fc si or mIgG2a isotype.

[0103] Fig. 21B is a graph showing body weight ± standard deviation of mice inoculated with CT26 tumor cells and administered weekly doses of DF-mIL-12-Fc si, mIgG2a isotype, or rmIL-12.

[0104] Fig. 21C is a graph showing tumor growth curves of individual mice re-challenged as naive or complete responders (CR) when previously administered a single dose of DF-mIL-12-Fc si in the CT26 tumor model.

[0105] Figures 22A-22B Figure 2 shows the inoculation of CT26 tumor cells and intraperitoneal (IP) ( Fig.22A ) or subcutaneous (SC) ( Fig. 22B ) Graph of tumor growth curves of individual mice administered weekly doses of DF-mIL-12-Fc si or mIgG2a isotype.

[0106] Fig.23 is a graph showing tumor growth curves of individual mice inoculated with B16F10 melanoma cells and administered a single dose of DF-mIL-12-Fc si or mIgG2a isotype.

[0107] Figures 24A-24B Figure 2 shows B16F10 melanoma cells inoculated intraperitoneally (IP) ( Fig.24A ) or subcutaneous (SC) ( Fig. 24B ) Graph of tumor growth curves of individual mice administered weekly doses of DF-mIL-12-Fc si or mIgG2a isotype.

[0108] Figures 25A-25B Figure 2 shows the inoculation of CT26 tumor cells and the intraperitoneal administration of a single dose of 1 μg rmIL-12 ( Fig.25A ) or once-weekly dose ( Fig.25B ) of the tumor growth curves of mice harboring either the DF-mIL-12-Fc si or the mIgG2A isotype alone.

[0109] Figures 26A-26B is a graph showing tumor growth curves of individual mice inoculated with CT26 tumor cells and administered subcutaneously once weekly doses of DF-mIL-12-Fcsi. Fig.26A is a graph showing tumor growth curves of individual mice inoculated with CT26-Tyrpl tumor cells and treated once (weekly) with 2 μg of mIgG2a isotype control or 1 μg of DF-mIL-12-Fc si. Fig.26B is a graph showing tumor growth curves of individual mice inoculated with CT26-Tyrpl tumor cells and treated once (weekly) with 2 μg of mIgG2a isotype control or 2 μg of DF-mIL-12-Fc si.

[0110] Figures 27A-27C Figure 2 shows the expression of IFNγ in blood (left) and tumor (right) samples 72 hours after a single dose of DF-mIL-12-Fc si in C57BL / 6 mice bearing B16F10 tumors. Fig.27A )、CXCL9( Fig.27B ) and CXCL10( Fig.27C )Horizontal graph.

[0111] Figures 28A-28C A single subcutaneous dose of 1 μg / kg ( Fig.28A )、2μg / kg( Fig.28B ) or 4 μg / kg ( Fig.28C ) in DF-hIL-12-Fc si-treated cynomolgus monkeys. 2240, 2241, 2740, 2741 represent individual cynomolgus monkey subjects.

[0112] Figures 29A-29Fis a line graph showing the concentrations of IFNγ and IP10 / CXCL10 in cynomolgus monkeys treated with a single subcutaneous dose of DF-hIL-12-Fc si. Fig.29A , 29C and 29E show the IFNγ concentration / expression level in cynomolgus monkeys treated with 1 μg / kg, 2 μg / kg and 4 μg / kg of DF-hIL-12-Fc si, respectively. Fig.29B , 29D 29F and 29F show IP10 / CXCL10 concentration / expression levels in cynomolgus monkeys treated with 1 μg / kg, 2 μg / kg and 4 μg / kg of DF-hIL-12-Fc si, respectively. 3240, 3241, 3740, 3741 represent individual cynomolgus monkey subjects.

[0113] Fig.30 Figure 2 shows the inoculation of breast cancer cells and administration of weekly doses of monotherapy (isotype control, DF-mIL-12-Fc si, (chemotherapy) or irradiation with 10 Gy) or combination therapy (DF-mIL-12-Fc si and Figure 3 is a graph of tumor growth curves for individual mice treated with either or radiation combination.

[0114] Fig.31A is a graph showing tumor growth curves of individual mice inoculated with CT26-Tyrpl tumor cells and treated (every two weeks) with isotype control or anti-PD-1 antibody. Fig.31B is a graph showing tumor growth curves of Balb / c mice inoculated with CT26-Tyrpl tumor cells and treated (every two weeks) with isotype control or anti-PD-1 antibody. Fig.31B Also shown are tumor growth curves of separate mice previously treated with anti-PD-1 antibody to which the anti-PD-1 antibody was administered (every two weeks) along with weekly treatments of 1 μg DF-mIL-12-Fc si.

[0115] Fig.32A Graph showing tumor growth curves of treated (Tr) tumors of individual mice inoculated with CT26-Tyrpl tumor cells and treated intratumorally once (weekly) with isotype control or DF-mIL-12-Fc si. Fig.32B It is shown Fig.32A Graph of tumor growth curves of untreated (NT) CT26-Tyrp1 tumors in separate mice as described in .

[0116] Fig.33A is a graph showing tumor growth curves of individual mice inoculated with CT26-Tyrpl tumor cells and treated once with 2 μg of mIgG2a isotype control or 2 μg of DF-mIL-12-Fc si. Fig.33B is a graph showing mean tumor growth curves of individual mice inoculated with CT26-Tyrp1 tumor cells and treated with 2 μg mIgG2a isotype control, 1 μg DF-mIL-12-Fc si (administered weekly), 2 μg DF-mIL-12-Fc si (administered weekly), or 2 μg DF-mIL-12-Fc si (once).

[0117] Fig.34A is a graph showing IFNγ production by PHA-stimulated PBMCs treated with DF hIL-12-Fc-si having L234A, L235A, and P329A mutations (LALAPA) or L234A, L235A, P329G mutations (LALAPG). Fig.34B Shown are flow cytometry histograms of fluorophore-conjugated hIgG1 binding to THP-1 cells in the presence or absence of DF hIL-12-Fc-si with LALAPA mutation or LALAPG mutation. DETAILED DESCRIPTION

[0118] The present invention provides improvements in heterodimeric Fc fusion proteins, pharmaceutical compositions comprising such proteins, and therapeutic methods using such proteins and pharmaceutical compositions, including for the treatment of cancer.

[0119] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0120] As used herein, the terms "a" and "an" mean "one or more" and include the plural unless the context is inappropriate.

[0121] As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, apes, horses, cattle, pigs, dogs, cats, etc.), and more preferably include humans.

[0122] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a compound of the present invention) sufficient to produce a beneficial or desired result (e.g., a desired preventive or therapeutic effect). An effective amount can be administered in one or more administrations, one or more applications, or one or more doses, and is not intended to be limited to a specific formulation or route of administration. As used herein, the term "treatment" includes any effect that leads to improvement of a condition, disease, disorder, etc. or alleviates its symptoms, such as reduction, lowering, regulating, alleviating, or eliminating.

[0123] As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use, either in vivo or ex vivo.

[0124] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions (such as, for example, oil / water or water / oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Edition, Mack Publ. Co., Easton, PA

[1975] .

[0125] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention, which can provide a compound of the present invention or its active metabolite or residue when administered to a subject. As known to those skilled in the art, the "salt" of a compound of the present invention can be derived from an inorganic acid or an organic acid and an inorganic base or an organic base. Exemplary acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Although other acids (such as oxalic acid) are not pharmaceutically acceptable per se, they can be used to prepare salts, which can be used as intermediates when obtaining a compound of the present invention and a pharmaceutically acceptable acid addition salt thereof.

[0126] Exemplary bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NW4 + Compounds (wherein W is C 1-4 Alkyl) etc.

[0127] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmitate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and the like. Other examples of salts include pyrophosphate, ... +NH4 + and NW4 + (W is C 1-4 The anion of the compound of the present invention is complexed with an alkyl group).

[0128] For therapeutic use, salts of the compounds of the invention are considered pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also be useful, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0129] Throughout this specification, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that there are additional compositions of the invention consisting essentially of, or consisting of, the recited components, as well as processes and methods according to the invention consisting essentially of, or consisting of, the recited processing steps.

[0130] In general, compositions specifying percentages are by weight unless otherwise specified. In addition, if a variable is not accompanied by a definition, the preceding definition for the variable controls.

[0131] I. Protein

[0132] The present invention provides Fc fusion protein constructs, which include the amino acid sequence of a multi-subunit protein. These fusion protein constructs can show a higher serum half-life compared to native / natural multi-subunit proteins, improved yields during production, enhanced stability during storage and / or improved efficacy when used as a therapeutic agent.

[0133] IgG1 Fc fusion protein

[0134] In one aspect, the present invention provides a heterodimeric IgG1 Fc fusion protein, the fusion protein comprising: a first polypeptide comprising a first antibody IgG1 Fc domain polypeptide; and a second polypeptide comprising a second antibody IgG1 Fc domain polypeptide bound to the first antibody Fc domain, wherein the first polypeptide further comprises a first subunit of a multi-subunit protein, the first subunit being fused to the first antibody Fc domain polypeptide via a linker comprising the amino acid sequence PKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 237) or EPKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 6), wherein X1 represents L or A, X2 represents L, E or A, and X3 represents A or G; a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide, and the subunits of the multi-subunit protein are bound to each other; when X1 represents L and / or X2 represents L, at least one of the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide comprises a Q347R mutation for promoting heterodimerization.

[0135] In some embodiments, within the heterodimeric Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide consists of the amino acid sequence PKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 237) or EPKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 6), wherein X1 represents L or A, X2 represents L, E or A, and X3 represents A or G.

[0136] In certain embodiments, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide further comprises a spacer peptide. In certain embodiments, the linker comprises a sequence of SEQ ID NO: 237 or SEQ ID NO: 6 and a spacer peptide.

[0137] In certain embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the sequence PKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 237) or EPKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 6) (wherein X1 represents L or A, X2 represents L, E or A, and X3 represents A or G) and a spacer peptide. In certain embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence PKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 237) or EPKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 6) (wherein X1 represents L or A, X2 represents L, E or A, and X3 represents A or G). In certain embodiments, the amino acid sequence of the linker connecting the second different subunit of the multimeric protein to the second antibody Fc domain polypeptide is the same as the amino acid sequence of the linker connecting the subunit of the multimeric protein to the first antibody Fc domain polypeptide.

[0138] Any of the spacer peptides described under the heading "Spacer Peptide" may be employed. For example, in certain embodiments, the spacer peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 107-120. In certain embodiments, the spacer peptide consists of the amino acid sequence set forth in any one of SEQ ID NOs: 107-120. In certain embodiments, the linker connecting the subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of, or essentially consists of, a spacer peptide disclosed herein and a peptide having a sequence of SEQ ID NO: 237 or SEQ ID NO: 6. In certain embodiments, the linker connecting the second different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide consists of, or essentially consists of, a spacer peptide disclosed herein and a peptide having a sequence of SEQ ID NO: 237 or SEQ ID NO: 6. In certain embodiments, the spacer peptide is at the N-terminus of either or both of the linkers.

[0139] In some embodiments, in the heterodimeric Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide comprises the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9). In some embodiments, in the heterodimeric Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide consists of the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9).

[0140] In some embodiments, within the heterodimeric Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide comprises the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQID NO: 239). In some embodiments, within the heterodimeric Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide consists of the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQID NO: 239).

[0141] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 244). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 244).

[0142] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10).

[0143] In some embodiments, within the heterodimeric Fc fusion protein, the linker connecting the subunit of the multi-subunit protein to the first antibody Fc domain polypeptide comprises the amino acid sequence PKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 238) or EPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 7). In some embodiments, within the heterodimeric Fc fusion protein, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of the amino acid sequence PKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 238) or EPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 7).

[0144] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multiunit protein is fused to the second antibody Fc domain polypeptide via a linker comprising the amino acid sequence GGGGSGGGGSGGGGSPPKSSDKTHTCPPCPAPELLGG ((SEQ ID NO: 8) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 241). In some embodiments, the second different subunit of the multiunit protein is fused to the second antibody Fc domain polypeptide via a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 8) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 241).

[0145] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 15) or GGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 242). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 15) or GGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 242).

[0146] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 16) or GGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 243). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 16) or GGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 243).

[0147] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 65) or GGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 245). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 65) or GGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 245).

[0148] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 66) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 246). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 66) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 246).

[0149] In some embodiments, within the heterodimeric Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide comprises the amino acid sequence EPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 11) or PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 240). In some embodiments, within the heterodimeric Fc fusion protein, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of the amino acid sequence EPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 11) or PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 240).

[0150] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 247). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 217).

[0151] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67) or GGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 248). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67) or GGGGSPKSSDKTHTCPPCPAPFAEGA (SEQ ID NO: 248).

[0152] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 68) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 249). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 68) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 249).

[0153] In certain embodiments, the Fc domain polypeptide is an Fc domain polypeptide of IgG1 Fc. In some embodiments, the protein of the present invention includes a first antibody Fc domain polypeptide and a second antibody Fc domain polypeptide, both of which are mutated IgG1 Fc domain polypeptides that promote heterodimerization with each other. For example, if the Fc domain is derived from the Fc of human IgG1, the Fc domain may comprise an amino acid sequence that is at least 90% identical to amino acids 234-332 of a human IgG1 antibody and is different at one or more positions selected from the following: Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439.

[0154] In some embodiments, the antibody constant domain may comprise an amino acid sequence that is at least 90% identical to amino acids 234-332 of a human IgG1 antibody and differs by one or more substitutions selected from the group consisting of: Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, 96A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D and K439E. All amino acid positions in the Fc domain or hinge region disclosed herein are numbered according to EU numbering.

[0155] In some embodiments, the first antibody IgG1 Fc domain polypeptide comprises one or more mutations selected from K360E and K409W, and the second antibody IgG1 Fc domain polypeptide comprises one or more mutations selected from Q347R, D399V and F405T. In some embodiments, the first antibody IgG1 Fc domain polypeptide comprises one or more mutations selected from Q347R, D399V and F405T, and the second antibody IgG1 Fc domain polypeptide comprises one or more mutations selected from K360E and K409W. In some embodiments, the first antibody IgG1 Fc domain polypeptide comprises mutations K360E and K409W, and the second antibody IgG1 Fc domain polypeptide comprises mutations Q347R, D399V and F405T. In some embodiments, the first antibody IgG1 Fc domain polypeptide comprises mutations Q347R, D399V and F405T, and the second antibody IgG1 Fc domain polypeptide comprises mutations K360E and K409W.

[0156] In some embodiments, the heterodimeric Fc fusion protein with IgG1 Fc of the present invention includes one or more mutations in the first polypeptide and / or the second polypeptide to reduce binding to FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB) or complement components (e.g., C1q). Such mutations can be used to reduce effector function. For example, the protein of the present disclosure includes LALA (L234A and L235A) mutations, LALAPA (L234A, L235A, and P329A) mutations, LALAPG (L234A, L235A, and P329G) mutations, or LALEGAASPS (L234A, L235E, G237A, A330S, and P331S) mutations.

[0157] In some embodiments, the heterodimeric Fc fusion protein according to the present invention comprises a first antibody IgG4 or IgG1 Fc domain polypeptide and a second antibody IgG4 or IgG1 Fc domain polypeptide, each of which contains a mutation P329G or P329A. In a specific embodiment, the heterodimeric Fc fusion protein according to the present invention comprises a first antibody IgG4 or IgG1 Fc domain polypeptide and a second antibody IgG4 or IgG1 Fc domain polypeptide, each of which contains a mutation P329A.

[0158] In some embodiments, the first IgG1 antibody Fc domain polypeptide and the second different IgG1 antibody Fc domain polypeptide each contain a mutation selected from A330S and P331 S. In some embodiments, the first IgG1 antibody Fc domain polypeptide and the second different IgG1 antibody Fc domain polypeptide each contain mutations A330S and P331 S.

[0159] In certain embodiments, additional disulfide bonds are introduced between IgG1 Fc monomers, which improves the stability of the heterodimer. In an exemplary embodiment, the first antibody Fc domain polypeptide fused to the first subunit of the multi-subunit protein includes a Y349C substitution in the CH3 domain, which replaces the S354C on the second antibody Fc domain polypeptide fused to the second different subunit of the multi-subunit protein to form a disulfide bond. Alternatively, the first antibody Fc domain polypeptide fused to the first subunit of the multi-subunit protein includes a S354C substitution in the CH3 domain, which replaces the Y349C on the second antibody Fc domain polypeptide fused to the second different subunit of the multi-subunit protein to form a disulfide bond.

[0160] Any IgG1 antibody Fc domain polypeptide provided in Table 2 below can be used in combination with any IgG1 hinge sequence provided in Table 1 below (which in the present invention is a linker connecting the protein sequence of the first subunit of the multi-subunit protein with the first IgG1 antibody Fc domain polypeptide or a part or all of a linker connecting the additional subunit with the second different IgG1 antibody Fc domain polypeptide). Exemplary IgG1 hinge-Fc domain polypeptides are provided in Table 3 below. In certain embodiments, the first polypeptide and the second polypeptide of the Fc fusion protein comprise the amino acid sequences of SEQ ID NOs: 212 and 212, 213 and 214, 215 and 216, 217 and 218, 214 and 213, 216 and 215, or 218 and 217, respectively. In certain embodiments, the first polypeptide and the second polypeptide of the Fc fusion protein comprise the amino acid sequences of SEQ ID NOs: 212 and 212, 213 and 214, 215 and 216, 217 and 218, 214 and 213, 216 and 215, or 218 and 217, respectively. NO:228 and 228; 229 and 230; 231 and 232; 233 and 234; 235 and 236; 230 and 229; 232 and 231; 234 and 233; 236 and 235; 228 and 250; 250 and 228; 250 and 250; 229 and 252; 252 and 229; 251 and 230; 230 and 251; 253 and 232; 232 and 253; 231 and 254; 254 and 231; 255 and 234; 234 and 255; 233 and 256; 256 and 233; 257 and 236; 236 and 257; 258 and 235; or the amino acid sequence of 235 and 258.

[0161] IgG4 Fc fusion protein

[0162] In one aspect, the present invention provides improvements to multi-subunit proteins. In one aspect, the present invention provides a heterodimeric IgG4 Fc fusion protein, the fusion protein comprising: a first polypeptide comprising a first antibody IgG4 Fc domain polypeptide; and a second polypeptide comprising a second different antibody IgG4 Fc domain polypeptide bound to the first antibody Fc domain polypeptide, wherein the first polypeptide also comprises a first subunit of the multi-subunit protein, the first subunit is fused to the first antibody IgG4 Fc domain polypeptide via a linker comprising the amino acid sequence RVESKYGPPCPPCPAPEFXGG (SEQ ID NO: 1), wherein X represents L or E; the second different subunit of the multi-subunit protein is fused to the second antibody IgG4 Fc domain polypeptide, and the subunits of the multi-subunit protein are bound to each other; the first antibody Fc domain polypeptide and the second antibody IgG4 Fc domain polypeptide each contain different mutations that promote heterodimerization.

[0163] In some embodiments, within the heterodimeric IgG4 Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide consists of the amino acid sequence RVESKYGPPCPPCPAPEFXGG (wherein X represents L or E) (SEQ ID NO: 1).

[0164] In certain embodiments, the linker connecting the protein sequence of the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide further comprises a spacer peptide. In certain embodiments, the linker comprises a sequence of SEQ ID NO: 1 and a spacer peptide.

[0165] In certain embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker comprising the sequence RVESKYGPPCPPCPAPEFXGG (wherein X represents L or E) (SEQ ID NO: 1) and a spacer peptide. In certain embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, the linker consisting of the amino acid sequence RVESKYGPPCPPCPAPEFXGG (wherein X represents L or E) (SEQ ID NO: 1). In certain embodiments, the amino acid sequence of the linker connecting the second different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide is the same as the amino acid sequence of the linker connecting the subunit of the multi-subunit protein to the first antibody Fc domain polypeptide.

[0166] Any spacer peptide described under the heading "Spacer Peptide" can be used. For example, in certain embodiments, the spacer peptide comprises the amino acid sequence shown in any one of SEQ ID NOs: 107-120. In certain embodiments, the spacer peptide consists of the amino acid sequence shown in any one of SEQ ID NOs: 107-120. In certain embodiments, the linker connecting the subunits of the multi-subunit protein to the first antibody Fc domain polypeptide consists of, or essentially consists of, a spacer peptide disclosed herein and SEQ ID NO: 1. In certain embodiments, the linker consists of, or essentially consists of, a spacer peptide disclosed herein and SEQ ID NO: 1. In certain embodiments, the spacer peptide is at the N-terminus of the first linker and / or the second linker.

[0167] In some embodiments, within the heterodimeric Fc fusion protein, the linker connecting the subunits of the multi-subunit protein to the first antibody Fc domain polypeptide comprises the amino acid sequence RVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 2). In some embodiments, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of the amino acid sequence RVESKYGPPCPPCPAPEELGG (SEQ ID NO: 2).

[0168] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 3). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 3).

[0169] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13).

[0170] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 14). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 14).

[0171] In some embodiments, within the heterodimeric Fc fusion protein, the linker connecting the first subunit of the multi-subunit protein and the first antibody Fc domain polypeptide comprises the amino acid sequence RVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 4). In some embodiments, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of the amino acid sequence RVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 4).

[0172] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5).

[0173] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSRVHSKYGPPCPPCPAPEFEGG (SEQ ID NO: 63). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63).

[0174] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker comprises the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64). In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, and the linker consists of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64).

[0175] In certain embodiments, the Fc domain polypeptide is the Fc domain polypeptide of IgG4 Fc.IgG4 is an unstable dimer, which can exchange and pair Fab arms with other IgG4 antibodies in vivo.In certain embodiments, S228P mutation is introduced into hinge (it is the joint of the first subunit of the multi-subunit protein and the first IgG4 antibody Fc domain polypeptide or the joint of the other subunit and the second different IgG4 antibody Fc domain polypeptide in the present invention), which increases the stability of hinge region and reduces the chance of Fab arm exchange.In certain embodiments, other disulfide bonds are introduced between Fc domain polypeptide monomers, which improves the stability of the heterodimer.In an exemplary embodiment, the first antibody Fc domain polypeptide connected to the first subunit of the multi-subunit protein includes a Y349C substitution in the CH3 domain, which replaces with the S354C on the second antibody Fc domain polypeptide of the second different subunit connected to the multi-subunit protein second antibody Fc domain polypeptide to form a disulfide bond. Alternatively, a first antibody Fc domain polypeptide linked to a first subunit of said multimeric protein comprises a S354C substitution in the CH3 domain which forms a disulfide bond with a Y349C substitution on a second antibody Fc domain polypeptide linked to a second, different subunit of said multimeric protein.

[0176] In some embodiments, proteins of the invention include a first antibody Fc domain polypeptide and a second antibody Fc domain polypeptide, both of which are mutated IgG4 Fc domain polypeptides that promote heterodimerization with each other.

[0177] In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises one or more mutations selected from K360E, K370E and R409W, and the second antibody IgG4 Fc domain polypeptide comprises one or more mutations selected from E357N, Q347R, D399V and F405T. In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises mutations K370E and R409W, and the second antibody IgG4 Fc domain polypeptide comprises mutations E357N, D399V and F405T. In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises mutations E357N, D399V and F405T, and the second antibody IgG4 Fc domain polypeptide comprises mutations K370E and R409W. In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises mutations K360E and R409W, and the second antibody IgG4 Fc domain polypeptide comprises mutations Q347R, D399V and F405T. In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises mutations Q347R, D399V, and F405T, and the second antibody IgG4 Fc domain polypeptide comprises mutations K360E and R409W.

[0178] In some embodiments, the heterodimeric Fc fusion protein with IgG4 Fc of the present invention includes one or more mutations in the first polypeptide and / or the second polypeptide to reduce binding to FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA or FcγRIIIB) or complement components (e.g., C1q). Such mutations can be used to reduce effector function. For example, the protein of the present disclosure includes SPLE (S228P and L235E) mutations, SPLEPA (S228P, L235E and P329A) mutations, or SPLEPG (S228P, L235E and P329G) mutations.

[0179] Any IgG4 antibody Fc domain polypeptide provided in Table 2 can be used in combination with any IgG4 hinge sequence provided in Table 1 (which in the present invention is part or all of a linker connecting the first subunit of the multi-subunit protein with the first IgG4 antibody Fc domain polypeptide or a linker connecting the second different subunit of the multi-subunit protein with the second different IgG4 antibody Fc domain polypeptide). Exemplary IgG4 hinge-Fc domain polypeptides are provided in Table 3. In certain embodiments, the first polypeptide and the second polypeptide of the Fc fusion protein comprise the amino acid sequences of SEQ ID NOs: 205 and 205; 206 and 207; 208 and 209; 210 and 211; 207 and 206; 209 and 208; or 211 and 210, respectively. In certain embodiments, the first polypeptide and the second polypeptide of the Fc fusion protein comprise the amino acid sequences of SEQ ID NOs: 219 and 219; 220 and 221; 222 and 223; 224 and 225; 226 and 227; 221 and 220; 223 and 222; 225 and 224; or 227 and 226, respectively.

[0180] Disulfide bonds

[0181] Some heterodimeric Fc fusion proteins of the present invention include a native heterodimeric disulfide bond between a first subunit of a multi-subunit protein and a second, different subunit of the multi-subunit protein. For example, in an exemplary embodiment, a heterodimeric Fc fusion protein according to the present invention includes a native heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. This protein includes a native disulfide bond between C74 of p35 and C177 of p40.

[0182] Some heterodimeric Fc fusion proteins of the present invention include an artificial or engineered heterodimeric disulfide bond between a first subunit of a multi-subunit protein and a second, different subunit of the multi-subunit protein. For example, in an exemplary embodiment, a heterodimeric Fc fusion protein according to the present invention includes an artificial or engineered heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. This protein includes an artificial or engineered disulfide bond between V185C of p35 and Y292C of p40.

[0183] Some heterodimeric Fc fusion proteins of the present invention include a natural heterodimeric disulfide bond between a first subunit of a multi-subunit protein and a second different subunit of the multi-subunit protein, and an artificial or engineered heterodimeric disulfide bond between a first subunit of a multi-subunit protein and a second different subunit of the multi-subunit protein. For example, in an exemplary embodiment, a natural heterodimeric disulfide bond between p35 and p40 subunits of IL-12, and an artificial or engineered heterodimeric disulfide bond between p35 and p40 subunits of IL-12. This protein includes a natural disulfide bond between C74 of p35 and C177 of p40, and an artificial or engineered disulfide bond between V185C of p35 and Y292C of p40.

[0184] Some heterodimeric Fc fusion proteins of the present invention are engineered to remove the native disulfide bond, which is then replaced with a non-natural artificial or engineered disulfide bond. For example, in an exemplary embodiment, a heterodimeric Fc fusion protein according to the present invention includes p35 of IL-12 in which the native C74 is mutated to serine, and p40 of IL-12 in which the native C177 is mutated to serine, thereby removing the native disulfide bond between the p35 and p40 subunits of IL-12. Two new mutations, V185C on p35 and Y292C on p40, are introduced into the mutant IL-12, thereby introducing a non-natural artificial or engineered disulfide bond.

[0185] Sequences of components of Fc fusion polypeptides

[0186] Exemplary heterodimeric Fc fusion proteins of the present invention are constructed with any of the IgG1 or IgG4 Fc variant sequences and any of the corresponding linker sequences described in Tables 1-2 below. The fusion protein constructs of the present invention can confer a higher serum half-life compared to native / natural multi-subunit proteins, improve protein yield during production, enhance stability during storage, and / or improve efficacy when used as a therapeutic agent.

[0187] Tables 4 and 5 list the amino acid sequences of exemplary protein constructs of the invention. All mutations / substitutions in the Fc domain polypeptides are numbered according to the EU numbering system. Mutations in the p35 and p40 subunits are numbered starting from the corresponding N-terminal amino acids of these subunits.

[0188] Any IgG4 antibody Fc variant domain polypeptide provided in Table 2 below can be used in combination with any IgG4 hinge sequence provided in Table 1 below. Similarly, any IgG1 antibody Fc variant domain polypeptide provided in Table 2 below can be used in combination with any IgG1 hinge sequence provided in Table 1 below. Exemplary IgG1 hinge-Fc domain polypeptides are provided in Table 3 below.

[0189] Table 1: Linker variants

[0190]

[0191] Table 2: IgG4 Fc and IgG1 Fc wild-type sequences; and exemplary IgG4 antibody Fc variant and IgG1 antibody Fc variant sequences (amino acid substitutions are indicated in bold)

[0192]

[0193]

[0194] *The amino acid sequence may also contain lysine (K) at the C-terminus.

[0195] Table 3: S228P mutated IgG4 hinge-Fc (wild type); exemplary S228P mutated IgG4 hinge-Fc variants or hinge portion-Fc variants; C220S mutated IgG1 hinge-Fc (wild type); exemplary C220S mutated IgG1 hinge-Fc variants or hinge portion-Fc variants

[0196]

[0197]

[0198]

[0199]

[0200]

[0201] *The amino acid sequence may also contain lysine (K) at the C-terminus.

[0202] Table 4: Exemplary heterodimeric Fc fusion polypeptide constructs

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240] *The amino acid sequence may also contain lysine (K) at the C-terminus.

[0241] Table 5: Exemplary dimeric Fc fusion proteins

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274] *The amino acid sequence may also contain lysine (K) at the C-terminus.

[0275] IL-12 subunits

[0276] IL-12 is a multi-subunit protein including a p40 subunit and a p35 subunit. The amino acid sequence of mature wild-type IL-12p40 is amino acids 23-328 of GenBank Accession No. NP_002178.2, shown in SEQ ID NO: 127 below. The amino acid sequence of mature wild-type IL-12p35 is amino acids 57-253 of GenBank Accession No. NP_000873.2, shown in SEQ ID NO: 128 below. The numbering of the amino acid residues of p40 and p35 used herein corresponds to the mature wild-type protein sequence. As used herein, the IL-12p40 subunit comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 127. As used herein, an IL-12p35 subunit comprises an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO:128.

[0277] In certain embodiments of any of the foregoing aspects, the p40 and p35 subunits of IL-12 comprise the amino acid sequences of SEQ ID NOs: 121 and 122; 127 and 128; 201 and 202; 203 and 204; 123 and 124; or 125 and 126, respectively. In certain embodiments, the first polypeptide comprises the amino acid sequence of the p40 subunit of IL-12, and the second polypeptide comprises the amino acid sequence of the p35 subunit of IL-12. In certain embodiments, the first polypeptide comprises the amino acid sequence of the p35 subunit of IL-12, and the second polypeptide comprises the amino acid sequence of the p40 subunit of IL-12.

[0278] In certain embodiments, the present disclosure includes a heterodimeric Fc fusion protein comprising: a first polypeptide comprising a first antibody Fc domain polypeptide; and a second polypeptide comprising a second antibody Fc domain polypeptide, wherein the first polypeptide further comprises a first subunit of IL-12 fused to the first antibody Fc domain polypeptide via a linker; and a second different subunit of IL-12 is fused to the second antibody Fc domain polypeptide, wherein the first subunit and the second different subunit of IL-12 are bound to each other, wherein the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain a different mutation that promotes heterodimerization, wherein the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are bound to each other, and wherein the first subunit of IL-12 is a p40 subunit having a Y292C substitution, and the second different subunit of IL-12 is a p35 subunit having a V185C substitution. In certain embodiments, the first subunit and the second different subunit of IL-12 comprise the amino acid sequences of SEQ ID NOs: 125 and 126, respectively.

[0279] The first subunit and the second different subunit of IL-12 can be fused to any antibody Fc domain polypeptide via any linker disclosed herein to form an Fc fusion protein, whose sequences include but are not limited to constructs 120, 120-1, 120-2, 120-3, 120-4, 120-5, 120-6 and 120-7 as described in Table 4 and constructs 20, 20-1, 20-2, 20-3, 20-4, 20-5, 20-6, 20-7, 20-8 and 20-9 as described in Table 5.

[0280] In certain embodiments, the p40 subunit of IL-12 further comprises a replacement of C177, and the p35 subunit of IL-12 further comprises a replacement of C74. In certain embodiments, C177 in the p40 subunit of IL-12 is replaced by S, and C74 in the p35 subunit of IL-12 is replaced by S. In certain embodiments, the p40 and p35 subunits of IL-12 comprise the amino acid sequences of SEQ ID NOs: 123 and 124, respectively.

[0281] The first subunit and the second different subunit of IL-12 can be fused to any antibody Fc domain polypeptide via any linker disclosed herein to form an Fc fusion protein, whose sequences include but are not limited to constructs 119, 119-1, 119-2, 119-3, 119-4, 119-5, 119-6, 119-7 and 119-8 as described in Table 4 and constructs 19, 19-1, 19-2, 19-3, 19-4, 19-5, 19-6, 19-7, 19-8, 19-9 and 19-10 as described in Table 5.

[0282] Table 6: Human IL-12p40 and p35 amino acid sequences

[0283]

[0284]

[0285]

[0286] In certain embodiments, the heterodimeric Fc fusion protein of the present invention comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290; and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291. In certain embodiments, the heterodimeric Fc fusion protein of the present invention comprising SEQ ID NO: 290 and SEQ ID NO: 291 comprises a Y349C mutation in the CH3 domain of the first antibody Fc domain polypeptide and an S354C mutation in the CH3 domain of the second antibody Fc domain polypeptide. In certain embodiments, the heterodimeric Fc fusion protein of the present invention comprising SEQ ID NO: 290 and SEQ ID NO: 291 comprises different mutations in the corresponding Fc domain polypeptide sequences for promoting heterodimerization between the Fc domains.

[0287] In certain embodiments, the first polypeptide sequence comprises a first antibody Fc domain polypeptide (human IgG1) sequence comprising K360E and K409W substitutions. In certain embodiments, the second polypeptide sequence comprises a second antibody Fc domain polypeptide (human IgG1) sequence comprising Q347R, D399V and F405T substitutions. In certain embodiments, the first polypeptide amino acid sequence and the second polypeptide amino acid sequence comprise one or more mutations for reducing effector function. In certain embodiments, the heterodimer Fc fusion protein of the present invention comprises LALAPA (L234A, L235A and P329A) mutations.

[0288] In certain embodiments, in the first polypeptide of the heterodimeric Fc fusion protein of the present invention (SEQ ID NO: 290), the p40 subunit of human IL-12 is fused to the first antibody Fc domain polypeptide via a first linker comprising a first amino acid sequence, and in the second polypeptide of the heterodimeric Fc fusion protein of the present invention (SEQ ID NO: 291), the p35 subunit of human IL-12 is fused to the second antibody Fc domain polypeptide via a second linker comprising a second amino acid sequence.

[0289] SEQ ID NO: 290 is the sequence of the p40 subunit of human IL-12 (underlined amino acids) fused to a human IgG1 Fc domain polypeptide. Mutations are shown in bold.

[0290]

[0291] SEQ ID NO: 291 is the sequence of the p35 subunit of human IL-12 (underlined amino acids) fused to a human IgG1 Fc domain polypeptide. Mutations are shown in bold.

[0292]

[0293] The first polypeptide and the second polypeptide represented by the amino acid sequences of SEQ ID NO: 290 and SEQ ID NO: 291, respectively, form a disulfide bond due to the Y349C mutation (bold and underlined) in the CH3 domain of the first antibody Fc domain polypeptide sequence (human IgG1) shown in SEQ ID NO: 290 and the S354C mutation (bold and underlined) in the CH3 domain of the second antibody Fc domain polypeptide sequence (human IgG1) shown in SEQ ID NO: 291, which confers stability to the heterodimeric Fc fusion protein (Fc numbering according to the EU system).

[0294] In order to promote heterodimerization between the two Fc domain polypeptides of the heterodimeric Fc fusion protein, the first antibody Fc domain polypeptide sequence shown in SEQ ID NO: 290 (human IgG1) includes K360E and K409W substitutions in the CH3 domain, and the second different Fc domain polypeptide sequence shown in SEQ ID NO: 291 (human IgG1) includes Q347R, D399V and F405T substitutions in the CH3 domain (Fc numbering according to the EU system).

[0295] The first antibody Fc domain polypeptide sequence and the second different Fc domain polypeptide sequence (human IgG1) shown in SEQ ID NO: 290 and SEQ ID NO: 291 further include L234A, L235A and P329A (LALAPA) mutations for reducing effector function.

[0296] Spacer peptide

[0297] Exemplary spacer peptide sequences are provided in Table 7, and exemplary full-length linker sequences are provided in Tables 4 and 5.

[0298] In the first polypeptide of the present invention, the first subunit of the multi-subunit protein is fused to the first antibody Fc domain polypeptide (e.g., an IgG4 antibody Fc variant sequence or an IgG1 antibody Fc variant sequence as disclosed in Table 2) via a linker in the amino to carboxyl direction. And in the second polypeptide of the present invention, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide (e.g., an IgG4 antibody Fc variant sequence or an IgG1 antibody Fc variant sequence as disclosed in Table 2) via a linker in the amino to carboxyl direction.

[0299] In some embodiments, the first subunit of the multi-subunit protein of the present invention is fused to the first antibody Fc domain sequence via a linker, wherein the linker comprises a spacer peptide L1 and an amino acid sequence of SEQ ID NO: 1, 2, 4, 6, 7, 9, 11, 237, 238, 239 or 240, or consists thereof. In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker, wherein the linker comprises a spacer peptide L2 and an amino acid sequence of SEQ ID NO: 1, 2, 4, 6, 7, 9, 11, 237, 238, 239 or 240, or consists thereof.

[0300] In certain embodiments, L1 and L2 are peptide linkers, for example, L1 and / or L2 include 4-50 amino acid residues. In certain embodiments, L1 consists of 4-50 amino acid residues. In certain embodiments, L1 consists of 4-20 amino acid residues. In certain embodiments, L2 consists of 4-50 amino acid residues. In certain embodiments, L2 consists of about 4-20 amino acid residues. In certain embodiments, L1 and L2 each independently consist of about 4-50 amino acid residues. In certain embodiments, L1 and L2 each independently consist of 4-20 amino acid residues.

[0301] In some embodiments, L1 and L2 have optimized length and / or amino acid composition. In some embodiments, L1 and L2 have the same length and have the same amino acid composition. In other embodiments, L1 and L2 are different.

[0302] In certain embodiments, L1 has the same number of amino acids as L2; ​​in certain embodiments, L1 is longer (ie, has a greater number of amino acids) than L2; in certain embodiments, L1 is shorter (ie, has a fewer number of amino acids) than L2.

[0303] In certain embodiments, L1 and / or L2 are "short", for example, consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 amino acid residues. Therefore, in some cases, the spacer peptide consists of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the spacer peptide is a peptide bond. In certain embodiments, L1 and / or L2 are "long", for example, consisting of 15, 20 or 25 amino acid residues. In some embodiments, the spacer peptide consists of about 3 to about 15 (e.g., 8, 9 or 10) consecutive amino acid residues. Regarding the amino acid composition of L1 and L2, peptides with the characteristics of conferring flexibility to the first polypeptide and the second polypeptide of the protein of the present invention, not interfering with the binding of the first subunit and the second different subunit to each other, and resisting the cleavage of proteases are selected. For example, glycine and serine residues generally provide protease resistance. As part of the linker, a spacer peptide suitable for connecting the first subunit of the multi-subunit protein to the amino acid sequence of SEQ ID NO: 1, 2, 4, 6, 7, 9, 11, 237, 238, 239 or 240 and / or suitable for connecting the second different subunit of the multi-subunit protein to the amino acid sequence of SEQ ID NO: 1, 2, 4, 6, 7, 9, 11, 237, 238, 239 or 240 may include (GS) n 、(GGS) n 、(GGGS) n 、(GGSG) n 、(GGSGG)n and (GGGGS) n Sequence, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In some embodiments, L1 and / or L2 independently include (GGGGS)4 (SEQ ID NO: 107) or (GGGGS)3 (SEQ ID NO: 108) sequence as part of the linker. In other embodiments, L1 and / or L2 independently include a peptide sequence as shown in the sequence selected from the group consisting of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120 as listed in Table 7. In some embodiments, L1 and / or L2 independently include (GGGGS)4 (SEQ ID NO: 107) or (GGGGS)3 (SEQ ID NO: 108) sequence as part of the linker. In other embodiments, L1 and / or L2 independently include a peptide sequence as shown in the sequence selected from the group consisting of SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 and SEQ ID NO: 120 as listed in Table 7. n＝3 (SEQ ID NO: 108), (GGGGS) n＝2 (SEQ ID NO: 109) or (GGGGS) n＝1 (SEQ ID NO:110).

[0304] Table 7

[0305]

[0306] In certain embodiments, L1 comprises the sequence (GGGGS) n＝3 (SEQ ID NO: 108) sequence as part of the linker, and L2 includes (GGGGS) n =2 (SEQ ID NO: 109) or (GGGGS) n＝1 (SEQ ID NO: 110) sequence as part of the linker. In certain embodiments, L2 comprises the sequence (GGGGS) n＝3 (SEQ ID NO: 108) sequence as part of the linker, and L1 includes (GGGGS) n＝2 (SEQ ID NO: 109) or (GGGGS) n＝1(SEQ ID NO: 110) sequence as part of the linker. In certain embodiments, as part of the linker, L1 does not include the sequence shown in SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 or SEQ ID NO: 120.

[0307] In certain embodiments, as part of the linker, only L2 includes a sequence as shown in SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 120. In certain embodiments, as part of the linker sequence, neither L1 nor L2 includes a sequence as shown in SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 120.

[0308] Some heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide, which comprises a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker comprising GGGGSGGGGSGGGGS (SEQ ID NO: 118; (G4S) 3) connects the first subunit of the multi-subunit protein with the first antibody Fc domain polypeptide, such as an Fc domain polypeptide of an IgG4 antibody or an Fc domain polypeptide of an IgG1 antibody. Some heterodimeric Fc fusion proteins of the present invention comprise a second polypeptide, which comprises a second different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein a linker comprising GGGGSGGGGSGGGGS (SEQ ID NO: 118; (G4S) 3) connects the second different subunit of the multi-subunit protein with the second antibody Fc domain polypeptide, such as an Fc domain polypeptide of an IgG4 antibody or an Fc domain polypeptide of an IgG1 antibody.

[0309] In certain embodiments, some heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of a multisubunit protein and a first antibody Fc domain polypeptide, wherein a linker comprising GGGGSGGGGSGGGGS (SEQ ID NO: 118; (G4S) 3) connects the first subunit of the multisubunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second different subunit of the multisubunit protein and a second antibody Fc domain polypeptide, wherein the additional subunit is connected to the second antibody Fc domain polypeptide using a linker that does not comprise GGGGSGGGGSGGGGS (SEQ ID NO: 118; (G4S) 3).

[0310] In certain embodiments, some heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of a multisubunit protein and a first antibody Fc domain polypeptide, wherein a linker that does not comprise GGGGSGGGGSGGGGS (SEQ ID NO: 118; (G4S) 3) connects the first subunit of the multisubunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second different subunit of the multisubunit protein and a second antibody Fc domain polypeptide, wherein the second different subunit of the multisubunit protein is connected to the second antibody Fc domain polypeptide using a linker comprising GGGGSGGGGSGGGGS (SEQ ID NO: 118; (G4S) 3).

[0311] Some heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n＝2(SEQ ID NO: 109) connects the first subunit of the multi-subunit protein with the first antibody Fc domain polypeptide, such as the Fc domain polypeptide of an IgG4 antibody or the Fc domain polypeptide of an IgG1 antibody. Some heterodimeric Fc fusion proteins of the present invention comprise a second polypeptide comprising a second different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein (GGGGS) n＝2 The linker (SEQ ID NO: 109) connects the second different subunit of the multi-subunit protein with the second antibody Fc domain polypeptide, such as the Fc domain polypeptide of an IgG4 antibody or the Fc domain polypeptide of an IgG1 antibody.

[0312] Some heterodimeric Fc fusion proteins of the present disclosure include a linker comprising (GGGGS)n=2 (SEQ ID NO: 109), which connects a first subunit of the multi-subunit protein with a first antibody Fc domain polypeptide, such as an Fc domain polypeptide of an IgG4 antibody or an Fc domain polypeptide of an IgG1 antibody; and connects a second different subunit of the multi-subunit protein with a second antibody Fc domain polypeptide, such as an Fc domain polypeptide of an IgG4 antibody or an Fc domain polypeptide of an IgG1 antibody.

[0313] In certain embodiments, some heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n＝2 (SEQ ID NO: 109) connecting the first subunit of the multi-subunit protein with the Fc domain polypeptide; and a second polypeptide comprising a second different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the additional subunit of the multi-subunit protein is not comprised of (GGGGS) n＝2 The linker of (SEQ ID NO: 109) is connected to the second antibody Fc domain polypeptide.

[0314] In certain embodiments, some heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, which does not comprise (GGGGS) n＝2 (SEQ ID NO: 109) connecting the first subunit of the multi-subunit protein with the Fc domain polypeptide; and a second polypeptide comprising a second different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second different subunit of the multi-subunit protein is connected with a linker comprising (GGGGS) n＝2 The linker of (SEQ ID NO: 109) is connected to the second antibody Fc domain polypeptide.

[0315] Some heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n＝1 (SEQ ID NO: 110) is used to connect the first subunit of the multi-subunit protein with the first antibody Fc domain polypeptide, such as an Fc domain polypeptide of an IgG4 antibody or an Fc domain polypeptide of an IgG1 antibody. Some heterodimeric Fc fusion proteins of the present invention comprise a second polypeptide comprising a second different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein (GGGGS) n＝1 The linker (SEQ ID NO: 110) connects the second different subunit of the multi-subunit protein with the second antibody Fc domain polypeptide, such as the Fc domain polypeptide of an IgG4 antibody or the Fc domain polypeptide of an IgG1 antibody.

[0316] Some heterodimeric Fc fusion proteins of the present disclosure include (GGGGS) n＝1 (SEQ ID NO: 110), which connects a first subunit of the multi-subunit protein with a first antibody Fc domain polypeptide, such as an Fc domain polypeptide of an IgG4 antibody or an Fc domain polypeptide of an IgG1 antibody; and connects a second different subunit of the multi-subunit protein with a second antibody Fc domain polypeptide, such as an Fc domain polypeptide of an IgG4 antibody or an Fc domain polypeptide of an IgG1 antibody.

[0317] In certain embodiments, some heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n＝1 (SEQ ID NO: 110) connecting the first subunit of the multi-subunit protein with the Fc domain polypeptide; and a second polypeptide comprising a second different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second different subunit of the multi-subunit protein is connected with a linker not comprising (GGGGS) n＝1 The linker of (SEQ ID NO: 110) is connected to the second antibody Fc domain polypeptide.

[0318] In certain embodiments, some heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, which does not comprise (GGGGS) n＝1 (SEQ ID NO: 110) connecting the first subunit of the multi-subunit protein with the Fc domain polypeptide; and a second polypeptide comprising a second different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second different subunit of the multi-subunit protein is connected with a linker comprising (GGGGS) n＝1The linker of (SEQ ID NO: 110) is connected to the second antibody Fc domain polypeptide.

[0319] In certain embodiments, some heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n＝1 (SEQ ID NO: 110) sequence linker connecting the first subunit of the multi-subunit protein and the Fc domain polypeptide; and a second polypeptide, which comprises a second different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second different subunit of the multi-subunit protein is connected with a linker comprising (GGGGS) n＝2 The linker having the sequence of (SEQ ID NO: 109) is connected to the second antibody Fc domain polypeptide.

[0320] In certain embodiments, some heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n＝2 (SEQ ID NO: 109) sequence linker connecting the first subunit of the multi-subunit protein and the Fc domain polypeptide; and a second polypeptide, which comprises a second different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second different subunit of the multi-subunit protein is connected with a linker comprising (GGGGS) n＝1 The linker having the sequence of (SEQ ID NO: 110) is connected to the second antibody Fc domain polypeptide.

[0321] Fc domains and substitutions to promote heterodimerization

[0322] The assembly of the protein of the present invention can be accomplished by expressing in the same cell a first polypeptide of a first subunit comprising a multi-subunit protein sequence fused to a first antibody Fc domain polypeptide (e.g., an IgG4 antibody Fc variant sequence or an IgG1 antibody Fc variant sequence as disclosed in Table 2) and a second polypeptide of a second different subunit comprising a multi-subunit protein sequence fused to a second antibody Fc domain polypeptide (e.g., an IgG4 antibody Fc variant sequence or an IgG1 antibody Fc variant sequence as disclosed in Table 2), which results in the assembly of a heterodimeric Fc fusion protein according to the present invention. The assembled protein has a heterodimeric Fc domain polypeptide, wherein the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are bound to each other. The preferential assembly of the heterodimer promoting Fc can be completed by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region, as shown in US 13 / 494870, US 16 / 028850, US 11 / 533709, US 12 / 875015, US 13 / 289934, US 14 / 773418, US 12 / 811207, US 13 / 866756, US 14 / 647480 and US 14 / 830336. For example, mutations can be made in the CH3 domain based on human IgG1, and different amino acid substitutions are incorporated into the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide, which allow the two chains to selectively heterodimerize with each other. The positions of the amino acid substitutions shown below are all numbered according to the EU index (as in Kabat).

[0323] In one context, the amino acid substitution in the first antibody Fc domain polypeptide replaces the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y) or tryptophan (W), and at least one amino acid substitution in the second antibody Fc domain polypeptide replaces one or more original amino acids with one or more smaller amino acids selected from alanine (A), serine (S), threonine (T) or valine (V), so that the larger amino acid substitution (protrusion) fits the surface of the smaller amino acid substitution (cavity). For example, one antibody Fc domain polypeptide can incorporate a T366W substitution, and another can incorporate three substitutions, including T366S, L368A and Y407V.

[0324] The first polypeptide of the first subunit comprising a multi-subunit protein sequence of the present invention or the second polypeptide comprising a second different subunit of the multi-subunit protein sequence can be optionally coupled to an amino acid sequence that is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to an antibody constant region (e.g., an IgG constant region comprising a hinge, CH2, and CH3 domains with or without a CH1 domain). In some embodiments, the amino acid sequence of the constant region is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to a human antibody constant region (e.g., a human IgG1 constant region, an IgG2 constant region, an IgG3 constant region, or an IgG4 constant region). In some other embodiments, the amino acid sequence of the constant region is at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to an antibody constant region from another mammal, such as a rabbit, dog, cat, mouse, or horse. One or more mutations can be incorporated into the constant region compared to a human IgG1 constant region, for example, at Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and / or K439. Exemplary substitutions include, e.g., Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366 S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, T394W, D399R, D399K , D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D and K439E.

[0325] In certain embodiments, the mutations that may be incorporated into the CH1 of the human IgG1 constant region may be at amino acids V125, F126, P127, T135, T139, A140, F170, P171 and / or V173. In certain embodiments, the mutations that may be incorporated into the CK of the human IgG1 constant region may be at amino acids E123, F116, S176, V163, S174 and / or T164.

[0326] Amino acid substitutions can be selected from the following substitution groups shown in Table 8.

[0327]

[0328] Alternatively, the amino acid substitutions may be selected from the following substitution groups shown in Table 9.

[0329]

[0330] Alternatively, the amino acid substitutions may be selected from the following substitution groups shown in Table 10.

[0331]

[0332]

[0333] Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 11.

[0334]

[0335] Alternatively, at least one amino acid substitution can be selected from the following substitution group in Table 12, wherein one or more positions indicated in the first polypeptide column are replaced by any known negatively charged amino acid, and one or more positions indicated in the second polypeptide column are replaced by any known positively charged amino acid.

[0336]

[0337] Alternatively, at least one amino acid substitution can be selected from the following group in Table 13, wherein one or more positions indicated in the first polypeptide column are replaced by any known positively charged amino acid, and one or more positions indicated in the second polypeptide column are replaced by any known negatively charged amino acid.

[0338]

[0339]

[0340] Alternatively, the amino acid substitutions may be selected from the following groups shown in Table 14.

[0341]

[0342] Alternatively, or in addition, the structural stability of the heterodimeric Fc fusion protein according to the present invention can be increased by introducing S354C on either the first or second polypeptide chain and Y349C on the opposite polypeptide chain, which forms an artificial disulfide bond within the interface of the two polypeptides.

[0343] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, L368 and Y407.

[0344] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, L368 and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366.

[0345] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from E357, K360, Q362, S364, L368, K370, T394, D401, F405 and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, E357, S364, L368, K370, T394, D401, F405 and T411.

[0346] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, E357, S364, L368, K370, T394, D401, F405 and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from E357, K360, Q362, S364, L368, K370, T394, D401, F405 and T411.

[0347] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, D399, S400 and Y407, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, N390, K392, K409 and T411.

[0348] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from T366, N390, K392, K409 and T411, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, D399, S400 and Y407.

[0349] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Q347, Y349, K360 and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Q347, E357, D399 and F405.

[0350] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Q347, E357, D399 and F405, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, K360, Q347 and K409.

[0351] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from K370, K392, K409 and K439, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from D356, E357 and D399.

[0352] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from D356, E357 and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from K370, K392, K409 and K439.

[0353] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, E356, T366 and D399, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, L351, L368, K392 and K409.

[0354] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from Y349, L351, L368, K392 and K409, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from L351, E356, T366 and D399.

[0355] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a S354C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a Y349C substitution.

[0356] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a Y349C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a S354C substitution.

[0357] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by K360E and K409W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by O347R, D399V and F405T substitutions.

[0358] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by O347R, D399V and F405T substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by K360E and K409W substitutions.

[0359] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by a T366W substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T366S, T368A, and Y407V substitutions.

[0360] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T366S, T368A, and Y407V substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T366W substitution.

[0361] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, L351Y, F405A and Y407V substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, T366L, K392L and T394W substitutions.

[0362] In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, T366L, K392L and T394W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T350V, L351Y, F405A and Y407V substitutions.

[0363] Those skilled in the art will appreciate that during the production and / or storage of the protein, the N-terminal glutamic acid (E) or glutamine (Q) may be cyclized to form a lactam (e.g., spontaneously or by catalysis of an enzyme present during production and / or storage). Therefore, in some embodiments, where the N-terminal residue of the amino acid sequence of the polypeptide is E or Q, the corresponding amino acid sequence in which E or Q is replaced with pyroglutamic acid is also contemplated herein.

[0364] Those skilled in the art will also appreciate that during protein production and / or storage, the C-terminal lysine (K) of a protein may be removed (e.g., spontaneously or by catalysis by enzymes present during production and / or storage). This removal of K is often observed in the case of proteins comprising an Fc domain at their C-terminus. Therefore, in some embodiments, where the C-terminal residue of the amino acid sequence of a polypeptide (e.g., an Fc domain sequence) is K, the corresponding amino acid sequence with K removed is also contemplated herein.

[0365] Mutations to reduce effector function

[0366] In one aspect, the present invention provides a heterodimeric Fc fusion protein, the fusion protein comprising (a) a first polypeptide, which comprises a first antibody Fc domain polypeptide and a first subunit of a multi-subunit protein; and (b) a second polypeptide, which comprises a second antibody Fc domain polypeptide and a second different subunit of the multi-subunit protein, wherein the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise different mutations that promote heterodimerization, wherein the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide comprise one or more mutations that reduce the effector function of Fc, and wherein the first subunit and the second different subunit of the multi-subunit protein are bound to each other. In certain embodiments, the heterodimeric Fc fusion protein disclosed herein comprising one or more mutations that reduce the effector function of Fc has an increased activity of inhibiting tumor growth than its counterpart without the one or more Fc mutations that reduce the effector function. Mutations considered herein include substitutions, insertions and deletions of amino acid residues. All amino acid positions in the Fc domain or hinge region disclosed herein are numbered according to EU numbering.

[0367] In certain embodiments, the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce antibody-dependent cellular toxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). ADCC and ADCP are generally mediated by Fc receptors. For example, in certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG (e.g., human IgG1, human IgG2, human IgG3 or human IgG4) antibody sequences. The Fc receptors of human IgG (also referred to as Fcγ receptors (FcγR)) include, but are not limited to, activating Fcγ receptors FcγRI (CD64), FcγRIIA (CD32A), FcγRIIIA (CD16 or CD16A) and FcγRIIIB (CD16B), and inhibitor Fcγ receptors FcγRIIB (CD32B). Therefore, in some embodiments, the heterodimeric Fc fusion protein of the present invention includes one or more mutations to reduce binding to the activating FcγR (e.g., FcγRI, FcγRIIA, FcγRIIIA or FcγRIIIB) in the first polypeptide and / or the second polypeptide. In some embodiments, the heterodimeric Fc fusion protein of the present invention includes one or more mutations to increase binding to the inhibitory FcγR (e.g., FcγRIIB) in the first polypeptide and / or the second polypeptide.

[0368] Fc mutations that reduce binding to activating FcγRs and / or increase binding to inhibitory FcγRs are known in the art. For example, in the hinge region and Fc region, CD16 binding is mediated by the hinge region and the CH2 domain. For example, in human IgG1, the interaction with CD16 is mainly concentrated on the amino acid residues Asp 265-Glu 269, Asn 297-Thr299, Ala 327-Ile 332, Leu 234-Ser 239 and the carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see, Sondermann et al., Nature, 406 (6793): 267-273). Based on the known domains, mutations can be selected to enhance or reduce binding affinity to CD16, such as by using a phage display library or a yeast surface display cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0369] As reviewed in Want et al., Protein Cell (2018) 9(1): 63-73, according to the crystal structure of human IgG1 Fc, the region including amino acid positions 232-239, 265-270, 296-299 and 325-332 is involved in activating FcγR binding. Wang et al. also disclosed that L235E and F234A / L235A mutations of human IgG4, L234A / L235A mutations of human IgG1 and N297 mutations of IgG antibodies (e.g., N297A, N297Q, N297G or N297D) reduce activating FcγR binding. As disclosed in U.S. Pat. No. 8,969,526, mutations at position 329 (e.g., P329A, P329G or P329R) also reduce activating FcγR binding. Additional amino acid positions and mutations involved in activating FcγR binding (eg, the E233P mutation) are disclosed in US Pat. No. 7,943,743 and Isaacs et al., J. Immunol. (1998) 161:3862-69.

[0370] Thus, in certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide comprise a mutation at one or more positions selected from 233, 234, 235, 297, and 329 (e.g., a substitution relative to wild-type human IgG1). In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG1 antibody Fc domain polypeptides comprising one or more mutations E233P; L234A (human IgG1) or F234A (human IgG4); L235A or L235E; N297A, N297Q, N297G, or N297D; and / or P329A, P329G, or P329R. In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG1 antibody Fc domain polypeptides comprising mutations L234A and L235A. In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG1 antibody Fc domain polypeptides comprising mutations L234A, L235A and P329A. In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG4 antibody Fc domain polypeptides comprising mutations L235E. In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG1 antibody Fc domain polypeptides comprising mutations L235E and P329A.

[0371] In certain embodiments, the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce complement dependent cytotoxicity (CDC). CDC is typically mediated by complement components (e.g., C1q). Therefore, in certain embodiments, the heterodimeric Fc fusion protein of the present invention comprises one or more mutations to reduce binding to complement components (e.g., C1q) in the first polypeptide and / or the second polypeptide.

[0372] Fc mutations that reduce binding to C1q are known in the art. For example, as disclosed in U.S. Pat. Nos. 5,648,260 and 5,624,821, amino acid residues at positions 234, 235, 236, 237, 297, 318, 320, and 322 of Fc are involved in C1q binding. As disclosed in Tao et al., J. Exp. Med. (1993) 178: 661-667 and Brekke et al., Eur. J. Immunol. (1994) 24: 2542-47, residue Pro at position 331 is involved in C1q binding. As disclosed in Idusogie et al., J. Immunol. (2000) 164:4178-84, mutations at positions 270 (eg, D270A), 322 (K322A), 329 (eg, P329A), and 331 (eg, P331A, P331S, or P331G) of Fc reduce CIq binding.

[0373] Therefore, in certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are included in a mutation (e.g., a substitution relative to wild-type human IgG1) at one or more positions selected from 234, 235, 236, 237, 270, 297, 318, 320, 322, 329 and 331. In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG1 antibody Fc domain polypeptides, which contain one or more mutations G237A, A330S, P331S and / or P329A. In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG1 antibody Fc domain polypeptides, which contain mutations G237A, A330S and P331S. In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG1 antibody Fc domain polypeptides, which contain mutations P329A.

[0374] Mutations that reduce ADCC and / or ADCP and mutations that reduce CDC can be combined. In certain embodiments, the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce ADCC and / or ADCP, and also comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce CDC. In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce ADCC and / or ADCP, and also comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce CDC.

[0375] In some embodiments, the heterodimeric Fc fusion protein of the present invention with IgG4 Fc includes one or more mutations in the first polypeptide and / or the second polypeptide to reduce binding to FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA or FcγRIIIB) or complement components (e.g., C1q). Such mutations can be used to reduce effector function. For example, a protein of the present disclosure can include a SPLE (S228P and L235E) mutation, a SPLEPA (S228P, L235E and P329A) mutation, or a SPLEPG (S228P, L235E and P329G) mutation.

[0376] In some embodiments, the heterodimeric Fc fusion protein with IgG1 Fc of the present invention includes one or more mutations in the first polypeptide and / or the second polypeptide to reduce binding to FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA or FcγRIIIB) or complement components (e.g., C1q). Such mutations can be used to reduce effector function. For example, the protein of the present disclosure can include LALA (L234A and L235A) mutations, LALAPA (L234A, L235A and P329A) mutations, LALAPG (L234A, L235A and P329G) mutations, or LALEGAASPS (L234A, L235E, G237A, A330S and P331S) mutations.

[0377] In some embodiments, the heterodimeric Fc fusion protein according to the present invention comprises a first antibody IgG4 or IgG1 Fc domain polypeptide and a second antibody IgG4 or IgG1 Fc domain polypeptide, each of which contains the mutation P329G or P329A.

[0378] In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain a mutation selected from A330S and P331S.

[0379] In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain the mutations A330S and P331S.

[0380] In certain embodiments, in the first polypeptide of the heterodimeric Fc fusion protein of the present invention, the first subunit of the multi-subunit protein is fused to the first antibody Fc domain polypeptide through a first linker. In certain embodiments, in the second polypeptide of the heterodimeric Fc fusion protein of the present invention, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide through a second linker. The amino acid sequences of the linkers suitable for this use are described under the headings "IgG4 constructs" and "IgG1 constructs". Other linker sequences suitable for use in the first polypeptide and / or the second polypeptide include, but are not limited to, wild-type IgG (e.g., human IgG1, human IgG2, human IgG3 or human IgG4) hinge sequences and mutant forms thereof. For example, in certain embodiments, the first linker and the second linker each comprise the amino acid sequence ESKYGPPCPPCPAPEFXGG (wherein X is L or E) (SEQ ID NO: 280) or SKYGPPCPPCPAPEFXGG (wherein X is L or E) (SEQ ID NO: 281). In certain embodiments, the first linker and the second linker each comprise the amino acid sequence ESKYGPPCPPCPAPEFLGG (SEQ ID NO: 282) or sKYGPPCPPCPAPEFLGG (SEQ ID NO: 283). In certain embodiments, the first linker and the second linker each comprise the amino acid sequence ESKYGPPCPPCPAPEFEGG (SEQ ID NO: 284) or SKYGPPCPPCPAPEFEGG (SEQ ID NO: 285).

[0381] Serum half-life

[0382] The heterodimeric Fc fusion proteins according to the present invention have pharmacokinetic properties suitable for therapeutic use. For example, in certain embodiments, the heterodimeric Fc fusion proteins according to the present invention have a serum half-life of at least about 50 hours. In certain embodiments, the heterodimeric Fc fusion proteins according to the present invention have a serum half-life of at least about 100 hours.

[0383] In certain embodiments, 50 hours after intravenous administration to a subject, the serum concentration of a heterodimeric Fc fusion protein according to the invention is at least 10% of the serum concentration of a protein of the invention in said subject 1 hour after administration.

[0384] In certain embodiments, the heterodimeric Fc fusion protein according to the present invention has a serum half-life that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% longer than the multi-subunit protein not fused with the Fc domain polypeptide. In certain embodiments, the heterodimeric Fc fusion protein of the protein sequence comprising the multi-subunit protein according to the present invention has a serum half-life that is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times or 20 times longer than the multi-subunit protein not fused with the Fc domain polypeptide.

[0385] Tumor preservation

[0386] The heterodimer Fc fusion protein of the present invention can optionally incorporate additional features to enhance the retention of the protein at the tumor site. For example, in certain embodiments of the present invention, the heterodimer Fc fusion protein also includes a proteoglycan binding domain, a collagen binding domain and / or a hyaluronic acid binding domain. In certain embodiments, the heterodimer Fc fusion protein also includes a proteoglycan binding domain that binds one or more proteoglycans (e.g., proteoglycans known in the art, such as Lozzo et al., Matrix Bio (2015) 42: 11-55; and Nikitovic et al., Frontiers in Endocrinology (2018) 9: 69 disclosed in) a proteoglycan binding domain, the proteoglycan is present in a tumor (e.g., on the surface of a tumor cell, in the pericellular matrix of a tumor, or in the extracellular matrix of a tumor). In certain embodiments, the collagen binding domain binds one or more collagens, and the collagen is present in a tumor (e.g., on the surface of a tumor cell, in the pericellular matrix of a tumor, or in the extracellular matrix of a tumor). In certain embodiments, the heterodimeric Fc fusion protein further comprises an hyaluronic acid binding domain that binds to one or more hyaluronic acids present in the tumor. Such heterodimeric Fc fusion proteins have enhanced retention in tumors and can be administered intratumorally to subjects at lower dosages and / or frequencies.

[0387] In certain embodiments, the proteoglycan binding domain contained in the heterodimer Fc fusion protein binds one or more proteoglycans that are specifically expressed in a tumor (e.g., on the surface of a tumor cell, in the pericellular matrix of a tumor, or in the extracellular matrix of a tumor). In certain embodiments, the collagen binding domain contained in the heterodimer Fc fusion protein binds one or more collagens that are specifically expressed in a tumor (e.g., on the surface of a tumor cell, in the pericellular matrix of a tumor, or in the extracellular matrix of a tumor). Such heterodimer Fc fusion proteins can be enriched in a tumor after administration (e.g., intravenous, subcutaneous, or pulmonary administration) and have enhanced tumor retention, thereby allowing administration at lower doses and / or frequencies.

[0388] In certain embodiments, the heterodimeric Fc fusion protein of the present invention further comprises a proteoglycan binding domain that binds one or more proteoglycans selected from the group consisting of syndecan, chondroitin sulfate proteoglycan 4 (CSPG4), beta-proteoglycan, phosphacan, glypican, synaptobrevin, collagen (e.g., collagen IX, XII, XV, or XVIII), pergelcan, aggrecan, versican, neurocan, brevican, and small leucine-rich proteoglycan (SLRP). Proteoglycans implicated in cancer include, but are not limited to, collagen, syndecan (e.g., syndecan-1 or syndecan-2), serglycin, CSPG4, betaglycan, glypican (e.g., glypican-1 or glypican-3), leucine, versican, brevican, and SLPR (e.g., decorin, biglycan, asporidin, fibromodulin, and lumican). Thus, in certain embodiments, the proteoglycan binding domain contained in the heterodimeric Fc fusion protein binds one or more proteoglycans selected from the group consisting of syndecan (e.g., syndecan-1 or syndecan-2), serglycin, CSPG4, betacan, glypican (e.g., glypican-1 or glypican-3), leucine, versican, brevican, and SLPR. In certain embodiments, the proteoglycan binding domain contained in the heterodimeric Fc fusion protein binds one or more SLPRs selected from the group consisting of decorin, biglycan, asporin, fibromodulin, and lumican.

[0389] The proteoglycan binding domain contained in the heterodimeric Fc fusion protein can be a protein (e.g., an antibody or an antigen-binding fragment thereof), a peptide (e.g., a portion of a proteoglycan binding protein or a variant thereof), an aptamer, a small molecule, or a combination thereof. Proteoglycan binding domains are also known in the art. For example, the cohesin binding domain is disclosed in U.S. Patent Nos. 6,566,489, 8,647,828, and 10,124,038; U.S. Patent Application Publication No. 2009 / 0297479; and PCT Patent Application Publication No. WO 2018199176 A1. The CSPG4 binding domain is disclosed in U.S. Patent Nos. 9,801,928 and 10,093,745; and U.S. Patent Application Publication Nos. 2016 / 0032007, 2017 / 0342151, and 2018 / 0072811. The β-glycan binding domain is disclosed in U.S. Patent No. 7,455,839. Glypican binding domains are disclosed in U.S. Patent Nos. 7,919,086, 7,776,329, 8,680,247, 8,388,937, 9,260,492, 9,394,364, 9,790,267, 9,522,940, and 9,409,994; U.S. Patent Application Publication Nos. 2004 / 0236080, 2011 / 0123998, 2018 / 0244805, 2018 / 0230230, and 2018 / 0346592; European Patent No. 2270509; and PCT Patent Application Publication Nos. WO 2017053619 A1, WO 2018026533 A1, WO 2018165344 A1, and WO 2018199318 A1. Beaded protein glycan binding domains are disclosed in U.S. Patent No. 10,166,304. Decorin protein glycan binding domains are disclosed in U.S. Patent No. 6,517,838 and PCT Patent Application Publication Nos. WO 2000021989 A1, WO 2000077041 A2 and WO2000078800A2.

[0390] In certain embodiments, the heterodimer Fc fusion protein of the present invention further comprises a collagen binding domain. Collagen is a class of proteins with at least 28 different types identified in vertebrates. Each type of collagen has its unique structural characteristics and distribution pattern, as disclosed in Fang et al., Tumor Biol. (2014) 35: 2871-82 and Xiong et al., J. Cancer Metasta. Treat. (2016) 2: 357-64. Various types of collagen are involved in cancer, including but not limited to Col3A1, Col5A2, Col6, Col7A1, Col15A1, Col19A1 and Col22A1. The collagen binding domain can be a protein (e.g., an antibody or its antigen binding fragment), a peptide (e.g., a part of a collagen binding protein or a variant thereof), an aptamer, a small molecule or a combination thereof. Collagen binding domains are known in the art and are disclosed, for example, in U.S. Pat. Nos. 5,788,966, 5,587,360, 5,851,794, 5,741,670, 5,849,701, 6,288,214, 6,387,663, 6,908,994, 7,169,902, 7,488,792, 7,820,401, 8,956,612, 8,642,728, and 8,906,649, and U.S. Patent Application Publication Nos. 2007 / 0161062, 2009 / 0142345, and 2012 / 0100106.

[0391] In certain embodiments, heterodimer Fc fusion protein of the present invention also comprises hyaluronic acid binding domain.The hyaluronic acid binding domain can be protein (for example, antibody or its Fab), peptide (for example, a part of hyaluronic acid binding protein or its variant), aptamer, small molecule or its combination.Hyaluronic acid binding domain is known in the art, and is disclosed in for example U.S. Patent number 6,864,235,8,192,744,8,044,022,8,163,498,8,034,630,9,217,016,9,795,686 and 9,751,919, and in U.S. Patent Application Publication No. 2002 / 0055488 and 2007 / 0259380.

[0392] Proteoglycan binding domain, collagen binding domain and / or hyaluronic acid binding domain (if present) can be at any position of the heterodimer Fc fusion protein. For example, in certain embodiments, when the IL-12 subunit is located at the N-terminus of the antibody Fc domain polypeptide, the proteoglycan binding domain, collagen binding domain and / or hyaluronic acid binding domain as disclosed herein can be fused with the C-terminus of the first antibody Fc domain polypeptide and / or with the C-terminus of the second antibody Fc domain polypeptide. In certain embodiments, when the IL-12 subunit is located at the C-terminus of the antibody Fc domain polypeptide, the proteoglycan binding domain, collagen binding domain and / or hyaluronic acid binding domain as disclosed herein can be fused with the N-terminus of the first antibody Fc domain polypeptide and / or with the N-terminus of the second antibody Fc domain polypeptide.

[0393] Proteoglycan binding domain, collagen binding domain and / or hyaluronic acid binding domain (if present) can be fused with the rest of the heterodimer Fc fusion protein through a joint. In certain embodiments, the proteoglycan binding domain is fused with the rest of the heterodimer Fc fusion protein through a peptide joint. In certain embodiments, the peptide joint includes a spacer peptide disclosed herein.

[0394] II. Preparation Method

[0395] The protein of the present invention can be prepared using recombinant DNA techniques well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first polypeptide can be cloned into a first expression vector, wherein the first polypeptide comprises a first subunit of a multi-subunit protein sequence fused to a first antibody Fc domain polypeptide; a second nucleic acid sequence encoding a second polypeptide can be cloned into a second expression vector, wherein the second polypeptide comprises a second different subunit of a multi-subunit fused to a second antibody Fc domain polypeptide; and the first expression vector and the second expression vector can be stably transfected into a host cell together to produce the multimeric protein.

[0396] In order to achieve the highest yield of protein, different ratios of the first expression vector and the second expression vector can be explored to determine the optimal ratio for transfection into host cells. After transfection, individual clones can be isolated using methods known in the art (such as limiting dilution, ELISA, FACS, microscopy or Clonepix) to generate cell banks.

[0397] The clones can be cultured under conditions suitable for bioreactor scale-up and maintenance expression of the protein of the invention. The protein can be isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography.

[0398] III. Pharmaceutical Compositions

[0399] The disclosure also features pharmaceutical compositions containing an effective amount of the proteins described herein. The compositions can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the compositions for appropriate formulation. Suitable formulations for use in the disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th edition, 1985. For a brief review of drug delivery methods, see, for example, Langer (Science 249: 1527-1533, 1990).

[0400] The intravenous drug delivery preparation of the present disclosure can be contained in a bag, a pen or a syringe. In certain embodiments, the bag can be connected to a channel including a tube and / or a needle. In certain embodiments, the preparation can be a lyophilized preparation or a liquid preparation. In certain embodiments, the preparation can be freeze-dried (lyophilized) and contained in about 12-60 vials. In certain embodiments, the preparation can be freeze-dried, and the freeze-dried preparation of 45mg can be contained in a vial. In certain embodiments, the freeze-dried preparation of about 40mg-about 100mg can be contained in a vial. In certain embodiments, the freeze-dried preparation from 12, 27 or 45 vials is combined to obtain the therapeutic dose of protein in the intravenous drug preparation. In certain embodiments, the preparation can be a liquid preparation, and is stored as about 250mg / vial to about 1000mg / vial. In certain embodiments, the preparation can be a liquid preparation, and is stored as about 600mg / vial. In certain embodiments, the formulation may be a liquid formulation and stored at about 250 mg per vial.

[0401] The heterodimeric Fc fusion protein of the present invention may be present in a liquid aqueous pharmaceutical formulation comprising an effective amount of the protein in a buffer solution to form a formulation.

[0402] These compositions can be sterilized by conventional sterilization techniques, or can be aseptically filtered. The resulting aqueous solution can be packaged for use as is, or lyophilized, and the lyophilized preparation is combined with a sterile aqueous carrier before administration. The pH of the preparation will generally be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, most preferably between 7 and 8, such as 7 to 7.5. The resulting solid form composition can be packaged in multiple single dose units, each single dose unit containing a fixed amount of one or more of the above-mentioned medicaments. The solid form composition can also be packaged in a container for a flexible amount.

[0403] In certain embodiments, the present disclosure provides a formulation with an extended shelf life comprising a heterodimeric Fc fusion protein of the present disclosure in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0404] In certain embodiments, an aqueous formulation is prepared, which includes the heterodimeric Fc fusion protein of the present disclosure in a pH buffered solution. The buffer of the present invention can have a pH ranging from about 4 to about 8 (e.g., from about 4.5 to about 6.0 or from about 4.8 to about 5.5), or can have a pH of about 5.0 to about 5.2. Ranges in the middle of the above-mentioned pH are also intended to be part of the present disclosure. For example, it is intended to include a range of values ​​using a combination of any of the above-mentioned values ​​as an upper and / or lower limit. Examples of buffers that control pH within this range include acetates (e.g., sodium acetate), succinates (such as sodium succinate), gluconates, histidine, citrates, and other organic acid buffers.

[0405] In certain embodiments, the formulation includes a buffer system containing citrate and phosphate to maintain pH in the range of about 4 to about 8. In certain embodiments, the pH range can be from about 4.5 to about 6.0 or from about pH4.8 to about 5.5, or in the pH range of about 5.0 to about 5.2. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system includes about 1.3mg / mL of citric acid (e.g., 1.305mg / mL), about 0.3mg / mL of sodium citrate (e.g., 0.305mg / mL), about 1.5mg / mL of disodium phosphate dihydrate (e.g., 1.53mg / mL), about 0.9mg / mL of sodium dihydrogen phosphate dihydrate (e.g., 0.86) and about 6.2mg / mL of sodium chloride (e.g., 6.165mg / mL). In certain embodiments, the buffer system comprises 1-1.5 mg / mL of citric acid, 0.25 to 0.5 mg / mL of sodium citrate, 1.25 to 1.75 mg / mL of disodium phosphate dihydrate, 0.7 to 1.1 mg / mL of sodium dihydrogen phosphate dihydrate, and 6.0 to 6.4 mg / mL of sodium chloride. In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.

[0406] Polyols can also be included in the formulation, and the polyols serve as tonicity agents and can stabilize antibodies. The polyols are added to the formulation in an amount that is variable relative to the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation can be isotonic. The addition amount of polyols can also be changed relative to the molecular weight of the polyols. For example, compared with disaccharides (such as trehalose), a lower amount of monosaccharides (e.g., mannitol) can be added. In certain embodiments, the polyol that can be used as a tonicity agent in the formulation is mannitol. In certain embodiments, the mannitol concentration can be about 5 to about 20 mg / mL. In certain embodiments, the concentration of mannitol can be about 7.5 to 15 mg / mL. In certain embodiments, the concentration of mannitol can be about 10-14 mg / mL. In certain embodiments, the concentration of mannitol can be about 12 mg / mL. In certain embodiments, the polyol sorbitol can be included in the formulation.

[0407] Detergents or surfactants can also be added to the formulation. Exemplary detergents include nonionic detergents, such as polysorbates (e.g., polysorbates 20, 80, etc.) or poloxamer (e.g., poloxamer 188). The amount of detergent added is such that it reduces the aggregation of the formulated antibody and / or minimizes the formation of particles in the formulation and / or reduces adsorption. In certain embodiments, the formulation can include a surfactant, which is a polysorbate. In certain embodiments, the formulation can contain the detergent polysorbate 80 or Tween 80. Tween 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edition, 1996). In certain embodiments, the formulation may contain between about 0.1 mg / mL and about 10 mg / mL or between about 0.5 mg / mL and about 5 mg / mL polysorbate 80. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

[0408] In certain embodiments, the protein product of the present disclosure is formulated as a liquid formulation. The liquid formulation can be present in a USP / Ph Eur I type 50R vial at a concentration of 10 mg / mL, the vial being closed with a rubber stopper and sealed with an aluminum crimped cap. The stopper can be made of an elastomer that complies with USP and Ph Eur. In certain embodiments, the vial can be filled with 61.2 mL of protein product solution to allow for 60 mL of extractable volume. In certain embodiments, the liquid formulation can be diluted with a 0.9% saline solution.

[0409] In certain embodiments, the combination of the liquid formulation of the present disclosure and the sugar of stable level can be prepared as a solution of 10mg / mL concentration. In certain embodiments, the liquid formulation can be prepared in an aqueous carrier. In certain embodiments, the addition of stabilizer may be no greater than the amount that may cause viscosity to be undesirable or unsuitable for intravenous administration. In certain embodiments, the sugar may be a disaccharide, such as sucrose. In certain embodiments, the liquid formulation may also include one or more of a buffer, a surfactant, and a preservative.

[0410] In certain embodiments, the pH of the liquid formulation can be set by adding a pharmaceutically acceptable acid and / or base. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the base can be sodium hydroxide.

[0411] In addition to aggregation, deamidation is a common product variation of peptides and proteins that may occur during fermentation, harvest / cell clarification, purification, storage of drug substances / drug products, and during sample analysis. Deamidation is the loss of U from a protein, forming a succinimide intermediate that can undergo hydrolysis. The succinimide intermediate results in a mass decrease of 17 Daltons in the parent peptide. Subsequent hydrolysis results in a mass increase of 18 Daltons. Due to its instability under aqueous conditions, it is difficult to separate the succinimide intermediate. Therefore, deamidation can usually be detected as a mass increase of 1 Dalton. Deamidation of asparagine produces aspartic acid or isoaspartic acid. Parameters that affect the deamidation rate include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and tertiary structure. Amino acid residues adjacent to Asn in the peptide chain affect the deamidation rate. Gly and Ser after Asn in the protein sequence lead to higher deamidation sensitivity.

[0412] In certain embodiments, liquid formulations of the present disclosure may be stored under pH and humidity conditions that prevent deamidation of the protein product.

[0413] Aqueous carriers of interest herein are carriers that are pharmaceutically acceptable (safe and non-toxic for administration to humans) and can be used to prepare liquid formulations. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextrose solution.

[0414] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of preservatives may, for example, facilitate the production of multi-use (multi-dose) formulations.

[0415] In certain circumstances, such as when the patient receives all medications via the intravenous (IV) route during hospitalization following transplantation, an IV formulation may be the preferred route of administration. In certain embodiments, the liquid formulation is diluted with 0.9% sodium chloride solution prior to administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

[0416] In certain embodiments, salt or buffer components can be added in an amount of 10mM-200mM. The salt and / or buffer are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) and "alkali-forming" metals or amines. In certain embodiments, the buffer can be a phosphate buffer. In certain embodiments, the buffer can be a glycine salt, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions can be used as counterions.

[0417] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of preservatives may, for example, facilitate the production of multi-use (multi-dose) formulations.

[0418] Aqueous carriers of interest herein are carriers that are pharmaceutically acceptable (safe and non-toxic for administration to humans) and can be used to prepare liquid formulations. Illustrative carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextrose solution.

[0419] The protein of the present disclosure may be present in a lyophilized formulation comprising the protein and a lyoprotectant. The lyoprotectant may be a sugar, such as a disaccharide. In certain embodiments, the lyoprotectant may be sucrose or maltose. The lyoprotectant may also include one or more of a buffer, a surfactant, a filler, and / or a preservative.

[0420] The amount of sucrose or maltose that can be used to stabilize the lyophilized drug product can be at least a 1:2 weight ratio of protein to sucrose or maltose. In certain embodiments, the weight ratio of protein to sucrose or maltose can be from 1:2 to 1:5.

[0421] In certain embodiments, the pH of the formulation may be set by adding a pharmaceutically acceptable acid and / or base prior to lyophilization. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base may be sodium hydroxide.

[0422] Prior to lyophilization, the pH of the solution containing the protein of the present disclosure may be adjusted between 6 and 8. In certain embodiments, the pH of the lyophilized drug product may range from 7 to 8.

[0423] In certain embodiments, salt or buffer components can be added in an amount of 10mM-200mM. The salt and / or buffer are pharmaceutically acceptable and are derived from various known acids (inorganic and organic) and "alkali-forming" metals or amines. In certain embodiments, the buffer can be a phosphate buffer. In certain embodiments, the buffer can be a glycine salt, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions can be used as counterions.

[0424] In certain embodiments, a "filler" may be added. A "filler" is a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., promotes the production of a substantially uniform lyophilized cake that maintains an open pore structure). Illustrative fillers include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain such fillers.

[0425] Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of preservatives may, for example, facilitate the production of multi-use (multi-dose) formulations.

[0426] In certain embodiments, the lyophilized drug product can be made with an aqueous carrier. The aqueous carrier of interest herein is a carrier that is pharmaceutically acceptable (e.g., safe and nontoxic for administration to a human) and can be used to prepare a liquid formulation after lyophilization. Illustrative diluents include SWFI, BWFI, pH buffered solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solution, or dextrose solution.

[0427] In certain embodiments, the lyophilized drug product of the present disclosure is reconstituted with SWFI (USP) or 0.9% Sodium Chloride Injection (USP).During reconstitution, the lyophilized powder dissolves into solution.

[0428] In certain embodiments, the lyophilized protein product of the present disclosure is constituted with about 4.5 mL of water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0429] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0430] The specific dosage can be a uniform dosage per patient, such as 50-5000 mg protein. Alternatively, the dosage of the patient can be adjusted according to the patient's approximate body weight or surface area. Other factors determining the appropriate dosage may include the disease or condition to be treated or prevented, the severity of the disease, the route of administration, and the age, sex, and physical condition of the patient. Those skilled in the art routinely further refine the calculations necessary to determine the appropriate dosage for treatment, especially according to the dosage information and determination disclosed herein. The dosage can also be determined by using a known determination for determining the dosage used in combination with appropriate dose-response data. The dosage of individual patients can be adjusted when monitoring disease progression. The blood level of targetable constructs or complexes in patients can be measured to see if the dosage needs to be adjusted to reach or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or complexes and their dosages are most likely to be effective for a given individual (Schmitz et al., Clinica Chimica Acta 308:43-53, 2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).

[0431] Typically, a dosage based on body weight is from about 0.01 μg to about 100 mg / kg body weight, such as about 0.01 μg to about 100 mg / kg body weight, about 0.01 μg to about 50 mg / kg body weight, about 0.01 μg to about 10 mg / kg body weight, about 0.01 μg to about 1 mg / kg body weight, about 0.01 μg to about 100 μg / kg body weight, about 0.01 μg to about 50 μg / kg body weight, about 0.01 μg to about 10 μg / kg body weight, about 0.01 μg to about 1 μg / kg body weight. g body weight, about 0.1 μg to about 100 mg / kg body weight, about 0.1 μg to about 50 mg / kg body weight, about 0.1 μg to about 10 mg / kg body weight, about 0.1 μg to about 1 mg / kg body weight, about 0.1 μg to about 100 μg / kg body weight, about 0.1 μg to about 10 μg / kg body weight, about 0.1 μg to about 1 μg / kg body weight, about 1 μg to about 100 mg / kg body weight, about 1 μg to about 50 mg / kg body weight, about 1 μg to about 10 mg / kg body weight, about 1 μg to about 10 0 μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg to about 10 About 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, about 50 mg to about 100 mg / kg body weight.

[0432] The dosage can be given once or more every day, every week, every month or every year, or even once every 2 to 20 years. One of ordinary skill in the art can easily estimate the repetition rate for administration based on the measured residence time and the concentration of the targetable construct or complex in the body fluid or tissue. The administration of the present invention can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion via a catheter or by direct intralesional injection. This can be applied once or more every day, once or more every week, once or more every month, and once or more every year. In some embodiments, the heterodimer Fc fusion protein (e.g., a heterodimer Fc fusion protein comprising IL-12 subunits) is administered intratumorally. In some embodiments, the heterodimer Fc fusion protein is administered intratumorally at a dosage or frequency lower than that during systemic administration. In some embodiments, after applying a local anesthetic, the heterodimer Fc fusion protein is administered intratumorally. In some embodiments, the heterodimer Fc fusion protein is administered intratumorally according to direct palpation of a tumor mass.

[0433] IV. Therapeutic Applications

[0434] The present invention provides methods for treating cancer using heterodimeric Fc fusion binding proteins described herein and / or pharmaceutical compositions described herein. The method can be used to treat a variety of cancers by administering an effective amount of heterodimeric Fc fusion proteins described herein to patients in need. In some embodiments, the cancer treated by the methods disclosed herein is a locally advanced malignant tumor. In some embodiments, the locally advanced malignant tumor can be completely resected. In some embodiments, the locally advanced malignant tumor has been completely resected, and the treatment is provided after resection.

[0435] In some embodiments, the heterodimeric Fc fusion protein of the present invention (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) is used as a monotherapy for the treatment of advanced malignancies. In some embodiments, the heterodimeric Fc fusion protein of the present invention (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) is used as an adjuvant for active immunotherapy of severe infectious diseases. In some embodiments, the heterodimeric Fc fusion protein of the present invention (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) is used as an adjuvant for preventive vaccination. In some embodiments, the heterodimeric Fc fusion protein of the present invention is used to treat bone marrow suppression after radiotherapy.

[0436] The present invention also provides a method for reducing hematopoietic toxicity. Hematopoietic toxicity can be caused by genetic, infectious or environmental reasons (including but not limited to irradiation and chemotherapy). For example, in certain embodiments, the present invention provides a method for treating a disease or disorder associated with radiation (e.g., ionizing radiation, alpha radiation, beta radiation, gamma radiation, X-radiation or neutron radiation). For example, in certain embodiments, the present invention provides a method for treating bone marrow suppression after radiotherapy, the method comprising administering an effective amount of a heterodimer Fc fusion protein or formulation described herein to a patient in need. In certain embodiments, the dose of the radiotherapy is at least 1, 5, 10, 15 or 20 Gy. In certain embodiments, the radiotherapy causes damage to a system, organ or tissue selected from the following: bone marrow, lymphatic system, immune system, mucosal tissue, mucosal immune system, gastrointestinal system, cardiovascular system, nervous system, reproductive organs, prostate, ovary, lung, kidney, skin and brain. In certain embodiments, the method of the present invention reduces the damage.

[0437] The methods provided herein can be used to treat myelosuppression caused by radiation. Therefore, in certain embodiments, the present invention provides a method for treating myelosuppression occurring in the case of accidental exposure to radiation, the method comprising administering to a patient in need thereof a therapeutically effective amount of a heterodimeric Fc fusion protein or formulation described herein.

[0438] In certain embodiments, the present invention provides a method for treating acute radiation syndrome (ARS), comprising administering an effective amount of a heterodimeric Fc fusion protein or formulation described herein to a patient in need thereof. ARS includes, but is not limited to, hematopoietic radiation syndrome, gastrointestinal radiation syndrome, neurovascular radiation syndrome, and skin radiation syndrome. For example, hematopoietic radiation syndrome is caused at least in part by the depletion of the hematopoietic stem cell pool, and exhibits symptoms of lymphopenia and granulocytopenia. Gastrointestinal syndrome is caused at least in part by damage to stem cells and progenitor cells located in the crypts and the inability to replace cells in the villus surface, and exhibits symptoms of watery diarrhea, dehydration, electrolyte loss, gastrointestinal bleeding, and perforation. In certain embodiments, the treatment methods provided herein are performed in the prodromal phase of ARS. The prodromal phase is the initial stage of an acute illness, characterized by symptoms of nausea, vomiting, anorexia, fever, headache, and / or early skin erythema, typically within 1-3 days after exposure to radiation. In certain embodiments, the treatment methods provided herein are performed in the latent phase of ARS. The latent period is characterized by improved symptoms but shows lymphopenia and granulocytopenia in laboratory tests, and may last for hours to weeks depending on the exposure dose. Treatment in the prodromal or latent period may alleviate the development of syndrome in the affected system, organ and / or tissue. In certain embodiments, the therapeutic method provided herein is carried out in the obvious illness stage of ARS. Treatment in this stage may still promote recovery from ARS.

[0439] The present invention also provides a method for increasing the survival, proliferation, differentiation and / or activity of an immune cell, the method comprising contacting the immune cell with a heterodimeric Fc fusion protein or formulation disclosed herein. In certain embodiments, the immune cell is a T cell (e.g., CD4 + In certain embodiments, the immune cell is a NK cell.

[0440] In certain embodiments, the heterodimer Fc fusion protein of the present invention (e.g., a heterodimer Fc fusion protein comprising an IL-12 subunit) is used to treat a subject diagnosed with cancer. In certain embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, leukemia, lung cancer, lymphoma, mesothelioma, melanoma, myeloma, ovarian cancer, endometrial cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, non-small cell lung cancer, small cell lung cancer, brain cancer, sarcoma, neuroblastoma or squamous cell carcinoma of head and neck cancer cells. In certain embodiments, the cancer is colon cancer. In certain embodiments, the heterodimer Fc fusion protein is administered as a monotherapy to a subject diagnosed with colon cancer. In certain embodiments, the cancer is melanoma. In certain embodiments, the heterodimer Fc fusion protein is administered as a monotherapy to a subject diagnosed with melanoma. In certain embodiments, the cancer is breast cancer. In certain embodiments, the heterodimeric Fc fusion protein is administered as a monotherapy to a subject diagnosed with breast cancer.

[0441] In certain embodiments, the disclosure provides a method of treating cancer, comprising administering to a patient only a single dose of a heterodimeric IL-12-Fc fusion protein. In certain embodiments, the amount of the single dose is sufficient to induce a complete response to the cancer. In certain embodiments, the amount of the single dose is sufficient to delay or prevent the recurrence of the cancer.

[0442] In certain embodiments, the present disclosure provides a method for treating cancer, comprising administering to a patient only a single dose of a heterodimeric IL-12-Fc fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291; or a formulation comprising the heterodimeric IL-12-Fc fusion protein and a pharmaceutically acceptable carrier.

[0443] In certain embodiments, a single dose of a heterodimeric IL-12-Fc fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291 or a formulation comprising the heterodimeric IL-12-Fc fusion protein (e.g., comprising SEQ ID NO: 290 and SEQ ID NO: 291) and a pharmaceutically acceptable carrier is sufficient to induce a complete response to the cancer. In certain embodiments, the single dose is sufficient to delay or prevent the recurrence of the cancer. In certain embodiments, the recurrence of a completely treated cancer (e.g., a complete response after treatment with an IL-12-Fc fusion protein of the invention) is delayed or prevented for 6 to 72 months or longer (e.g., 6 months, 12 months, 24 months, 36 months, 48 ​​months, 60 months, 72 months, 84 months, or 96 months).

[0444] In certain embodiments, the cancer treated with a single dose or more of a heterodimeric IL-12-Fc fusion protein (e.g., comprising a first polypeptide containing an amino acid sequence of SEQ ID NO: 290 and a second polypeptide containing an amino acid sequence of SEQ ID NO: 291) is a metastatic cancer. In certain embodiments, the metastatic cancer is a local, regional or distal metastatic cancer. In certain embodiments, a single dose or multiple doses of a heterodimeric IL-12-Fc fusion protein (e.g., comprising a first polypeptide containing an amino acid sequence of SEQ ID NO: 290 and a second polypeptide containing an amino acid sequence of SEQ ID NO: 291) treats a distant cancer through a distant effect, which is not a direct target of the primary cancer of the source organ or tissue and / or the treatment regimen. In certain embodiments, the a distant effect of the heterodimeric IL-12-Fc fusion protein is enhanced during and / or after a treatment plan including radiation and / or chemotherapy. In certain embodiments, a single or multiple doses of a heterodimeric IL-12-Fc fusion protein (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291) treats a patient's cancer by inducing a systemic anti-tumor response, as determined, for example, by increased expression of IFNy, CXCL9, and / or CXCL10 in the patient's serum and / or tumor.

[0445] V. Combination Therapy

[0446] Another aspect of the invention provides combination therapies.The multispecific binding proteins described herein can be used in combination with additional therapeutic agents to treat cancer.

[0447] In certain embodiments, the heterodimer Fc fusion protein of the present invention (e.g., a heterodimer Fc fusion protein comprising an IL-12 subunit) is administered as a combination therapy to treat a subject diagnosed with cancer. In certain embodiments, the cancer is bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, hepatocellular carcinoma, leukemia, lung cancer, lymphoma, mesothelioma, melanoma, myeloma, ovarian cancer, endometrial cancer, prostate cancer, pancreatic cancer, renal cell carcinoma, non-small cell lung cancer, small cell lung cancer, brain cancer, sarcoma, neuroblastoma or squamous cell carcinoma of head and neck cancer cells. In certain embodiments, the cancer is colon cancer. In certain embodiments, the heterodimer Fc fusion protein is administered as a combination therapy to a subject diagnosed with colon cancer. In certain embodiments, the cancer is melanoma. In certain embodiments, the heterodimer Fc fusion protein is administered as a combination therapy to a subject diagnosed with melanoma. In certain embodiments, the cancer is breast cancer. In certain embodiments, the heterodimeric Fc fusion protein is administered as a combination therapy to a subject diagnosed with breast cancer.

[0448] In some embodiments, the heterodimeric Fc fusion proteins of the present invention (e.g., heterodimeric Fc fusion proteins comprising IL-12 subunits) are used in combination with another therapeutic agent selected from the following for the treatment of advanced malignancies: cytotoxic chemotherapy; radiotherapy; antibodies targeting molecules involved in anti-tumor immune responses (such as CTLA-4, PD-1, PD-L1 or TGF-β); antibodies that act on tumor-associated antigens through ADCC; multispecific antibodies that bind NKG2D, CD16 and tumor-associated antigens, optionally administered in combination with antibodies targeting PD-1 or PD-L1; personalized cancer vaccines; oncolytic cancer vaccines; and personalized vaccines administered in combination with antibodies targeting PD-1 or PD-L1.

[0449] In some embodiments, the heterodimeric Fc fusion protein of the present invention (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) is used in combination with another therapy to treat a malignancy (e.g., an advanced malignancy), wherein the other therapy includes but is not limited to NK targeted therapy (e.g., CAR-NK therapy), antibody therapy, checkpoint inhibitor therapy, additional cytokine therapy, innate immune system agonist therapy, chemotherapy, targeted agent therapy, radiotherapy, adoptive NK therapy, stem cell transplantation (SCT) therapy, agonistic antibodies, chimeric antigen receptor (CAR) T cell therapy, T cell receptor (TCR) engineering therapy, multispecific binding proteins (TriNKET), agents that induce cellular senescence, and vaccines and / or oncolytic virus therapy. In some embodiments, the heterodimeric Fc fusion proteins of the present invention are used to treat malignancies (e.g., advanced malignancies) in combination with two or more additional therapies selected from the following: NK targeted therapy (e.g., CAR-NK therapy), antibody therapy, checkpoint inhibitor therapy, additional cytokine therapy, innate immune system agonist therapy, chemotherapy, targeted agent therapy, radiotherapy, adoptive NK therapy, stem cell transplantation (SCT) therapy, agonistic antibodies, chimeric antigen receptor (CAR) T cell therapy, T cell receptor (TCR) engineering therapy, multispecific binding proteins (TriNKET), agents that induce cellular senescence, and vaccines and / or oncolytic virus therapy.

[0450] In some embodiments, the heterodimeric Fc fusion proteins of the present invention (e.g., heterodimeric Fc fusion proteins comprising IL-12) are used in combination with cancer vaccines or antibodies targeting PD-1 or PD-L1 to treat locally advanced malignancies that can be completely resected.

[0451] The proteins of the invention may also be used as a supplement to surgical resection of the primary lesion.

[0452] The amounts and relative timing of administration of the heterodimeric Fc fusion proteins of the present invention (e.g., heterodimeric Fc fusion proteins comprising IL-12) and additional therapeutic agents can be selected so as to achieve the desired combined therapeutic effect. For example, when administering a combination therapy to a patient in need of such administration, the therapeutic agents in the combination or one or more pharmaceutical compositions comprising the therapeutic agents can be administered in any order, such as, for example, sequentially, concurrently, together, simultaneously, etc. In addition, for example, the heterodimeric Fc fusion protein can be administered during the time when the additional one or more therapeutic agents exert their preventive or therapeutic effects, or vice versa.

[0453] As disclosed herein, the methods of the present invention include co-administering a combination of a heterodimeric Fc fusion protein (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) and an additional therapeutic agent. As disclosed herein, the methods of the present invention include co-administering a combination of a heterodimeric Fc fusion protein comprising an IL-12 subunit and an additional therapeutic agent.

[0454] Co-administration includes methods in which a heterodimeric Fc fusion protein (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) and an additional therapeutic agent are administered simultaneously, in which the heterodimeric Fc fusion protein and the additional therapeutic agent are administered sequentially, and in which any one or both of the heterodimeric Fc fusion protein and the additional therapeutic agent are administered intermittently or continuously or in any combination of the following: simultaneously, sequentially, intermittently and / or continuously. The skilled artisan will recognize that intermittent administration is not necessarily the same as sequential administration, because intermittent also includes the first administration of an agent, followed by another administration after the time of the same agent. In addition, the skilled artisan understands that, in some embodiments, intermittent administration also includes sequential administration, because intermittent administration does include interrupting the first administration of an agent and the administration of a different agent before the first agent is administered again. In addition, the skilled artisan will also know that continuous administration can be achieved by a variety of routes, including intravenous drip (IV infusion) or feeding tube, etc.

[0455] Furthermore, and in a more general manner, the term "co-administration" includes any and all methods in which separate administration of the heterodimeric Fc fusion protein and separate administration of the additional therapeutic agent to a subject overlap within any time frame.

[0456] The frequency of administering a heterodimeric Fc fusion protein or an additional therapeutic agent to a subject is known in the art as Qnd or qnd, where n is the frequency (in days) of continuous administration of the agent. For example, Q3d would be an administration of an agent every three (3) days. In certain embodiments, the method comprises administering any one or two or any combination of heterodimeric Fc fusion proteins and / or additional therapeutic agents to a subject at Q1d, Q2d, Q3d, Q4d, Q5d, Q6d, Q7d, Q8d, Q9d, Q10d, Q14d, Q21d, Q28d, Q30d, Q90d, Q120d, Q240d, or Q365d.

[0457] In certain embodiments, any one or both of the heterodimeric Fc fusion protein and / or the additional therapeutic agent is administered intermittently. In certain embodiments, the method comprises administering the heterodimeric Fc fusion protein and / or the additional therapeutic agent intermittently with at least ten (10) minutes, fifteen (15) minutes, twenty (20) minutes, thirty (30) minutes, forty (40) minutes, sixty (60) minutes, two (2) hours, three (3) hours, four (4) hours, six (6) hours, eight (8) hours, ten (10) hours, twelve (12) hours, fourteen (14) hours, eighteen (18) hours, twenty-four (24) hours, or more. In some embodiments, the administration with delay follows a pattern in which one or both of the heterodimeric Fc fusion protein and / or additional therapeutic agent or any combination thereof is administered continuously for a given period of time from about ten (10) minutes to about three hundred sixty-five (365) days, followed by no administration for a given period of time from about ten (10) minutes to about thirty (30) days.

[0458] In certain embodiments, any one or two or a combination of heterodimeric Fc fusion proteins and / or additional therapeutic agents are administered intermittently, while the other is administered continuously. In certain embodiments, a combination of a first effective amount of heterodimeric Fc fusion proteins and a second effective amount of additional therapeutic agents is administered sequentially.

[0459] In certain embodiments, the heterodimeric Fc fusion protein and the additional therapeutic agent are administered simultaneously. In certain embodiments, the combination of the first effective amount of the heterodimeric Fc fusion protein and the second effective amount of the additional therapeutic agent is administered sequentially. In such embodiments, the combination is also referred to as "co-administration" because the term includes any and all methods in which the subject is exposed to the two components in the combination. However, such embodiments are not limited to combinations given only in one formulation or composition. It is possible that certain concentrations of the heterodimeric Fc fusion protein and the additional therapeutic agent are delivered at certain intervals, and therefore, the first effective amount and the second effective amount can be changed according to the formulation administered.

[0460] In certain embodiments, the heterodimer Fc fusion protein and the additional therapeutic agent are administered simultaneously or sequentially. In certain embodiments, the first effective amount of heterodimer Fc fusion protein is administered sequentially after the second effective amount of additional therapeutic agent. In certain embodiments, the second effective amount of additional therapeutic agent is administered sequentially after the first effective amount of heterodimer Fc fusion protein.

[0461] In certain embodiments, a combination of a heterodimeric Fc fusion protein (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) and an additional therapeutic agent is administered in one formulation. In certain embodiments, the combination is administered in two (2) compositions, wherein a first effective amount of a heterodimeric Fc fusion protein is administered in a formulation separate from a second effective amount of an additional therapeutic agent. In certain embodiments, the combination is administered in two (2) compositions, wherein a first effective amount of a heterodimeric Fc fusion protein is administered in a formulation separate from a second effective amount of an additional therapeutic agent. In certain embodiments, a first effective amount of a heterodimeric Fc fusion protein is administered sequentially after a second effective amount of an additional therapeutic agent. In certain embodiments, a second effective amount of an additional therapeutic agent is administered sequentially after a first effective amount of a heterodimeric Fc fusion protein. In certain embodiments, a heterodimeric Fc fusion protein and the additional therapeutic agent are administered; subsequently, both the heterodimeric Fc fusion protein and the additional therapeutic agent are administered intermittently for at least twenty-four (24) hours. In certain embodiments, the heterodimeric Fc fusion protein and the additional therapeutic agent are administered on a non-overlapping every other day schedule.

[0462] In certain embodiments, the first effective amount of the heterodimeric Fc fusion protein is administered no less than four (4) hours after the second effective amount of the additional therapeutic agent. In certain embodiments, the first effective amount of the heterodimeric Fc fusion protein is administered no less than ten (10) minutes, no less than fifteen (15) minutes, no less than twenty (20) minutes, no less than thirty (30) minutes, no less than forty (40) minutes, no less than sixty (60) minutes, no less than one (1) hour, no less than two (2) hours, no less than four (4) hours, no less than six (6) hours, no less than eight (8) hours, no less than ten (10) hours, no less than twelve (12) hours, no less than twenty-four (24) hours, no less than two (2) days, no less than four (4) days, no less than six (6) days, no less than eight (8) days, no less than ten (10) days, no less than twelve (12) days, no less than fourteen (14) days, no less than twenty-one (21) days, or no less than thirty (30) days after the second effective amount of the additional therapeutic agent. In certain embodiments, the second effective amount of an additional therapeutic agent is administered no less than ten (10) minutes, no less than fifteen (15) minutes, no less than twenty (20) minutes, no less than thirty (30) minutes, no less than forty (40) minutes, no less than sixty (60) minutes, no less than one (1) hour, no less than two (2) hours, no less than four (4) hours, no less than six (6) hours, no less than eight (8) hours, no less than ten (10) hours, no less than twelve (12) hours, no less than twenty-four (24) hours, no less than two (2) days, no less than four (4) days, no less than six (6) days, no less than eight (8) days, no less than ten (10) days, no less than twelve (12) days, no less than fourteen (14) days, no less than twenty-one (21) days, or no less than thirty (30) days after the first effective amount of the heterodimeric Fc fusion protein.

[0463] In certain embodiments, any one or both of the heterodimeric Fc fusion protein and / or the additional therapeutic agent is administered by a route selected from the following: intravenous, subcutaneous, cutaneous, oral, intramuscular, and intraperitoneal. In certain embodiments, any one or both of the heterodimeric Fc fusion protein and / or the additional therapeutic agent is administered intravenously. In certain embodiments, any one or both of the heterodimeric Fc fusion protein and / or the additional therapeutic agent, orally, or any combination thereof.

[0464] It will be appreciated by the skilled artisan that the unit dosage forms of the present disclosure may be administered in the same or different physical forms, i.e., orally via capsules or tablets and / or via IV infusion via liquids, etc. In addition, the unit dosage form of each administration may vary depending on the specific route of administration. For any one or both of the combinations of heterodimeric Fc fusion proteins and additional therapeutic agents, there may be several different dosage forms. Because different medical conditions may warrant different routes of administration, the same components of the combinations described herein may be identical in composition and physical form, and may still need to be administered in different ways and at different times to alleviate the condition. For example, conditions such as persistent nausea (especially with vomiting) may make it difficult to use an oral dosage form, and in such cases, another unit dosage form may need to be administered alternatively or similarly, possibly even the same dosage form as other dosage forms used before or after, via inhalation, buccal, sublingual or suppository routes. A specific dosage form may be a requirement for certain combinations of heterodimeric Fc fusion proteins and additional therapeutic agents, because there may be problems associated with various factors such as chemical stability or pharmacokinetics.

[0465] NK-targeted therapy

[0466] In certain embodiments, the heterodimer Fc fusion protein therapy is combined with NK targeted therapy. For example, in one embodiment, the heterodimer Fc fusion protein is co-administered with a therapeutic agent targeting NKp46. In certain embodiments, the therapeutic agent targeting NKp46 also binds CD16, one or more tumor-associated antigens, or a combination thereof. Exemplary therapeutic agents targeting NKp46 are described in more detail in U.S. Application No. US 20170198038 A1 (incorporated herein by reference for all purposes).

[0467] In certain embodiments, the heterodimer Fc fusion protein therapy is combined with bispecific and trispecific killer conjugate (BiKE and TriKE) therapy (including BiKE and TriKE therapy targeting NK cells). BiKE and TriKE are composed of a single heavy chain (VH) and a light chain (VL) of the variable region of each antibody of interest. The VH and VL domains are connected by a short flexible polypeptide linker to prevent dissociation. BiKE and TriKE are described in more detail in U.S. Application Nos. US 20180282386 A1 and US20180258396A1 (incorporated herein by reference for all purposes). BiKE and TriKE may contain a binding domain specific for NK cells.

[0468] In certain embodiments, BiKE and TriKE therapy are used in combination with the heterodimer Fc fusion protein therapy to treat subjects known or suspected of having high-risk myelodysplastic syndrome, acute myeloid leukemia, systemic mastocytosis or mast cell leukemia. In certain embodiments, BiKE and TriKE therapy are administered as a single course of 3 weekly treatment blocks. In certain embodiments, the treatment block includes 4 consecutive 24-hour continuous infusions (approximately 96 hours), followed by 72 hours of rest. In certain embodiments, BiKE and TriKE therapy are administered at a dose of 5 μg / kg / day, 10 μg / kg / day, 25 μg / kg / day, 50 μg / kg / day, 100 μg / kg / day or 200 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of at least 5 μg / kg / day, at least 10 μg / kg / day, at least 25 μg / kg / day, at least 50 μg / kg / day, at least 100 μg / kg / day, or at least 200 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of at least 1 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of at least 5 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of at least 200 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of at least 500 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of at least 1000 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of 200 μg / kg / day or less. In certain embodiments, BiKE and TriKE therapies are administered at a dose of 500 μg / kg / day or less. In certain embodiments, BiKE and TriKE therapies are administered at a dose of 1000 μg / kg / day or less. In certain embodiments, BiKE and TriKE therapies are administered at a dose of 1-200 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of 5-200 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of 1-500 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of 1-1000 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of 5-500 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at a dose of 5-1000 μg / kg / day. In certain embodiments, BiKE and TriKE therapies are administered at the maximum tolerated dose. In certain embodiments, BiKE and TriKE therapies are administered at less than the maximum tolerated dose.

[0469] Multispecific Binding Protein (“TriNKET”) Therapeutics

[0470] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with a therapy comprising a multispecific binding protein comprising: (a) a first antigen binding site that binds NKG2D; (b) a second antigen binding site that binds a tumor-associated antigen; and (c) an antibody Fc domain or portion thereof sufficient to bind to CD16, or a third antigen binding site that binds CD16 ("TriNKET") (e.g., the multispecific binding proteins comprising various NKG2D binders and tumor-associated antigen binding sites described in International Publication No. WO 2019 / 157332, which is incorporated herein by reference for its contents relating to the multispecific binding proteins described therein) to treat a subject known or suspected of having cancer. Exemplary tumor-associated antigens include, but are not limited to, HER2, CD20, CD33, B cell maturation antigen (BCMA), EpCAM, CD2, CD19, CD25, CD30, CD38, CD40, CD52, CD70, CLL1 / CLEC12A, FLT3, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4, HLA-E, and PD-L1.

[0471] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with a therapy comprising a certain dose of a multispecific binding protein based on body weight. For example, a dose of a multispecific binding protein based on body weight is from about 0.01 μg to about 100 mg / kg body weight, such as about 0.01 μg to about 100 mg / kg body weight, about 0.01 μg to about 50 mg / kg body weight, about 0.01 μg to about 10 mg / kg body weight, about 0.01 μg to about 1 mg / kg body weight, about 0.01 μg to about 100 μg / kg body weight, about 0.01 μg to about 50 μg / kg body weight, about 0.01 μg to about 10 μg / kg body weight, about 0.01 μg to about 1 μg / kg body weight, about 0.01 μg to about 0. .1 μg / kg body weight, about 0.1 μg to about 100 mg / kg body weight, about 0.1 μg to about 50 mg / kg body weight, about 0.1 μg to about 10 mg / kg body weight, about 0.1 μg to about 1 mg / kg body weight, about 0.1 μg to about 100 μg / kg body weight, about 0.1 μg to about 10 μg / kg body weight, about 0.1 μg to about 1 μg / kg body weight, about 1 μg to about 100 mg / kg body weight, about 1 μg to about 50 mg / kg body weight, about 1 μg to about 10 mg / kg body weight, about 1 μg to about 1 mg / kg body weight, about 1 μg about 100 μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight g to about 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, about 50 mg to about 100 mg / kg body weight.

[0472] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with a therapy comprising a dose of a multispecific binding protein administered once or more daily, weekly, monthly or annually, or even once every 2 to 20 years. One of ordinary skill in the art can easily estimate the repetition rate for administration based on the measured residence time and concentration of the targetable construct or complex in the body fluid or tissue. Administration of the multispecific binding protein can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion via a catheter or by direct intralesional injection. This can be administered once or more daily, once or more weekly, once or more monthly, and once or more annually.

[0473] Chimeric Antigen Receptor (CAR) Therapy

[0474] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with CAR therapy. The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain containing a functional signaling domain derived from a stimulatory molecule (also referred to herein as a "primary signaling domain").

[0475] Therefore, in certain embodiments, the CAR comprises an extracellular antigen binding site that binds to a tumor-associated antigen, a transmembrane domain, and an intracellular signaling domain comprising a primary signaling domain. In certain embodiments, the CAR also comprises one or more functional signaling domains (also referred to as "co-stimulatory signaling domains") derived from at least one costimulatory molecule.

[0476] In one embodiment, the CAR comprises a chimeric fusion protein, which comprises a tumor-associated antigen binding domain (e.g., a tumor-associated antigen binding scFv domain) containing a heavy chain variable domain and a light chain variable domain as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain containing a primary signaling domain. In one embodiment, the CAR comprises a chimeric fusion protein, which comprises a tumor-associated antigen binding domain (e.g., a tumor-associated antigen binding scFv domain) containing a heavy chain variable domain and a light chain variable domain as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain containing a costimulatory signaling domain and a primary signaling domain. In certain embodiments, the CAR comprises a chimeric fusion protein, which comprises a tumor-associated antigen binding domain (e.g., a tumor-associated antigen binding scFv domain) containing a heavy chain variable domain and a light chain variable domain as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain containing two costimulatory signaling domains and a primary signaling domain. In one embodiment, the CAR comprises a chimeric fusion protein comprising a tumor associated antigen binding domain comprising a heavy chain variable domain and a light chain variable domain as an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two co-stimulatory signaling domains and a primary signaling domain.

[0477] Regarding the transmembrane domain, in various embodiments, the CAR is designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain is a domain naturally associated with a domain in the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins to minimize the interaction with other members of the receptor complex. In another embodiment, the transmembrane domain can be homodimerized with another CAR on the surface of the CAR T cell. In another embodiment, the amino acid sequence of the transmembrane domain can be modified or replaced to minimize the interaction with the binding domain of the natural binding partner present in the same CAR T cell.

[0478] The transmembrane domain can be derived from any naturally occurring membrane-bound or transmembrane protein. In one embodiment, whenever the CAR has been bound to a target, the transmembrane region can signal to the one or more intracellular domains. In some embodiments, the transmembrane domain includes one or more transmembrane regions of one or more proteins selected from the following: TCR α chain, TCR β chain, TCR ζ chain, CD28, CD3 ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In some embodiments, the transmembrane domain comprises one or more transmembrane regions of one or more proteins selected from the group consisting of KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL , CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​PAG / Cbp, NKG2D and NKG2C.

[0479] The extracellular tumor-associated antigen binding domain (e.g., a tumor-associated antigen binding scFv domain) can be connected to the transmembrane domain via a hinge region. A variety of hinges can be used, including but not limited to human Ig hinges (e.g., IgG4 hinges, IgD hinges), Gly-Ser joints, (G4S)4 joints, KIR2DS2 hinges, and CD8α hinges.

[0480] The intracellular signaling domain of the CAR is responsible for activating at least one specialized function of the immune cell in which the CAR has been placed (e.g., cytolytic activity or auxiliary activity of T cells, including secretion of cytokines). Therefore, as used herein, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and instructs cells to perform specialized functions. Although a complete intracellular signaling domain can generally be used, the entire chain does not need to be used in many cases. In terms of using a truncated portion of the intracellular signaling domain, such a truncated portion can be used to replace the complete chain as long as it transduces the effector function signal. Therefore, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0481] The intracellular signaling domain of the CAR comprises a primary signaling domain (i.e., a functional signaling domain derived from a stimulatory molecule) and one or more co-stimulatory signaling domains (i.e., a functional signaling domain derived from at least one co-stimulatory molecule).

[0482] As used herein, the term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., a T cell, a NK cell, or a b cell) that provides one or more cytoplasmic signaling sequences that regulate the activation of immune cells in a stimulatory manner for at least some aspect of the immune cell signaling pathway. In one embodiment, the signal is a primary signal that is initiated by, for example, the TCR / CD3 complex binding to an MHC molecule loaded with a peptide, and that results in the mediation of a T cell response (including but not limited to proliferation, activation, differentiation, etc.).

[0483] The primary signaling domain that acts in a stimulatory manner may contain a signaling motif (which is referred to as an immunoreceptor tyrosine-based activation motif or ITAM). Examples of cytoplasmic signaling sequences containing ITAMs that are particularly useful in the present disclosure include those derived from CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In one embodiment, the primary signaling domain in any one or more CARs comprises a cytoplasmic signaling sequence derived from CD3-ζ.

[0484] In some embodiments, the primary signaling domain is a functional signaling domain of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, 4-1BB and / or CD3-ζ. In one embodiment, the intracellular signaling domain comprises a functional signaling domain of CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10 and / or DAP12. In a specific embodiment, the primary signaling domain is a functional signaling domain of the ζ chain associated with the T cell receptor complex.

[0485] As used herein, the term "costimulatory molecule" refers to a cognate binding partner on a T cell, which specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of a T cell, such as but not limited to proliferation. Costimulatory molecules are cell surface molecules required for lymphocytes to effectively respond to antigens other than antigen receptors or their ligands. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD2, CD7, CD258 (LIGHT), NKG2C, B7-H3, and a ligand specifically bound to CD83, etc. Other examples of such co-stimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, L FA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp and ligands that specifically bind to CD83. In some embodiments, the costimulatory signaling domain of the CAR is a functional signaling domain of a costimulatory molecule described herein, such as OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD11a / CD18), ICOS, and 4-1BB (CD137), or any combination thereof.

[0486] As used herein, the term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within the cell to regulate cellular activity via a defined signaling pathway, either by generating second messengers or by acting as an effector in response to such messengers.

[0487] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be connected to each other in a random or specified order. Optionally, a short oligopeptide or polypeptide linker (e.g., a length between 2 and 10 amino acids) can form the connection.

[0488] Antibody therapy

[0489] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with antibody therapy to treat a subject known or suspected of having cancer.

[0490] In certain embodiments, the heterodimeric Fc fusion protein is combined with a therapy comprising an anti-HER2 binding domain, such as an anti-HER2 antibody or an anti-HER2 antibody platform (e.g., a bispecific or trispecific antibody, an anti-HER2 antibody-drug conjugate, or an anti-HER2 CAR comprising an anti-HER2 binding domain). Anti-HER2 antibodies include, but are not limited to, trastuzumab ( -Roche / Genentech; Kanjinti-Amgen), Pertuzumab ( -Roche / Genentech) and MGAH22 (described in detail in U.S. Pat. No. 8,802,093, incorporated herein by reference for all purposes). Anti-HER2 antibody platforms include, but are not limited to, ertumaxomab ( -Creative Biolabs) and trastuzumab emtansine (ado-trastuzumab emtansine / T-DM1; -Roche / Genentech). In certain embodiments, the anti-HER2 binding domain therapy is used in combination with the heterodimer Fc fusion protein therapy to treat subjects known or suspected of having cancer. In certain embodiments, the anti-HER2 binding domain therapy is administered by IV infusion. In certain embodiments, the anti-HER2 binding domain therapy is administered at a dose of 1 mg / kg / day, 2 mg / kg / day, 3 mg / kg / day, 4 mg / kg / day, 5 mg / kg / day, 6 mg / kg / day, 7 mg / kg / day, 8 mg / kg / day, 9 mg / kg / day, 10 mg / kg / day. In certain embodiments, the anti-HER2 binding domain therapy is administered at a dose of at least 1 mg / kg / day, at least 2 mg / kg / day, at least 3 mg / kg / day, at least 4 mg / kg / day, at least 5 mg / kg / day, at least 6 mg / kg / day, at least 7 mg / kg / day, at least 8 mg / kg / day, at least 9 mg / kg / day, at least 10 mg / kg / day. In certain embodiments, the anti-HER2 binding domain therapy is administered at a dose of less than 1 mg / kg / day.

[0491] In certain embodiments, the anti-HER2 binding domain therapy is used in combination with the heterodimer Fc fusion protein therapy to treat subjects known or suspected of having breast cancer, such as subjects diagnosed with metastatic breast cancer that overexpresses HER2. In certain embodiments, the anti-HER2 binding domain therapy is administered at 4 mg / kg / day. In certain embodiments, the anti-HER2 binding domain therapy is administered at 4 mg / kg / day by IV infusion over 90 minutes. In certain embodiments, the anti-HER2 binding domain therapy is administered at 2 mg / kg / day. In certain embodiments, the anti-HER2 binding domain therapy is administered at 2 mg / kg / day by IV infusion over 30 minutes. In certain embodiments, the anti-HER2 binding domain therapy is administered at an initial dose of 4 mg / kg / day, followed by weekly administration at 2 mg / kg / day. In certain embodiments, the anti-HER2 binding domain therapy is administered at an initial dose of 4 mg / kg / day, followed by weekly administration at 2 mg / kg / day for 52 weeks.

[0492] In certain embodiments, the anti-HER2 binding domain therapy is used in combination with the heterodimer Fc fusion protein therapy to treat subjects known or suspected of having gastric cancer, such as subjects diagnosed with metastatic gastric cancer that overexpresses HER2. In certain embodiments, the anti-HER2 binding domain therapy is administered at 8 mg / kg / day. In certain embodiments, the anti-HER2 binding domain therapy is administered at 8 mg / kg / day by IV infusion within 90 minutes. In certain embodiments, the anti-HER2 binding domain therapy is administered at 6 mg / kg / day. In certain embodiments, the anti-HER2 binding domain therapy is administered at 6 mg / kg / day by IV infusion within 30-90 minutes. In certain embodiments, the anti-HER2 binding domain therapy is administered at an initial dose of 8 mg / kg / day, followed by weekly administration at 6 mg / kg / day. In certain embodiments, the anti-HER2 binding domain therapy is administered at an initial dose of 8 mg / kg / day, followed by weekly administration at 6 mg / kg / day for 52 weeks.

[0493] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with a therapy comprising an anti-CD20 binding domain, such as an anti-CD20 antibody or an anti-CD20 antibody platform (e.g., a bispecific or trispecific antibody, an anti-CD20 antibody-drug conjugate, or an anti-CD20 CAR comprising an anti-CD20 binding domain). Anti-CD20 antibodies include but are not limited to rituximab ( -Roche / Genentech), ocrelizumab ( -Roche / Genentech), obinutuzumab ( -Roche / Genentech), ofatumumab ( -Novartis) and veltuzumab. In certain embodiments, the anti-CD20 binding domain therapy is used in combination with the heterodimeric Fc fusion protein therapy to treat a subject known or suspected of having cancer. In certain embodiments, the anti-CD20 binding domain therapy is administered by IV infusion. In certain embodiments, the anti-CD20 binding domain therapy is administered at 100 mg / m 2 , 200mg / m 2 , 300mg / m 2 , 400mg / m 2 , 500mg / m 2 , 600mg / m 2 , 700mg / m 2 , 800mg / m 2, 900mg / m 2 or 1000 mg / m 2 In certain embodiments, the anti-CD20 binding domain therapy is administered at a dose of 375 mg / m 2 In certain embodiments, the anti-CD20 binding domain therapy is administered at a dose of at least 100 mg / m 2 , at least 200 mg / m 2 , at least 300 mg / m 2 , at least 400 mg / m 2 , at least 500 mg / m 2 , at least 600 mg / m 2 , at least 700 mg / m 2 , at least 800 mg / m 2 , at least 900 mg / m 2 or at least 1000 mg / m 2 In certain embodiments, the anti-CD20 binding domain therapy is administered at a dose of less than 400 mg / m 2 In certain embodiments, the anti-CD20 binding domain therapy is administered at a dose of less than 375 mg / m 2 The anti-CD20 binding domain therapy is administered at a dose of

[0494] In certain embodiments, the anti-CD20 binding domain therapy is used in combination with the heterodimeric Fc fusion protein therapy to treat a subject known or suspected of having non-Hodgkin lymphoma (NHL). In certain embodiments, the anti-CD20 binding domain therapy is used in combination with the heterodimeric Fc fusion protein therapy to treat a subject known or suspected of having non-Hodgkin lymphoma (NHL). 2 In certain embodiments, the anti-CD20 binding domain therapy is administered by IV infusion at a dose of less than 375 mg / m 2 The anti-CD20 binding domain therapy is administered at a dose of

[0495] In certain embodiments, the anti-CD20 binding domain therapy is used in combination with the heterodimeric Fc fusion protein therapy to treat a subject known or suspected of having chronic lymphocytic leukemia (CLL). In certain embodiments, the anti-CD20 binding domain therapy is administered by IV infusion at 375 mg / m in the first cycle. 2 500 mg / m by IV infusion per cycle for an additional 2-6 cycles. 2 In certain embodiments, the drug is administered by IV infusion at a dose of less than 375 mg / m 2The anti-CD20 binding domain therapy is administered at a dose of . The combined anti-CD20 binding domain and heterodimeric Fc fusion protein therapy can be used in combination with fludarabine and cyclophosphamide (FC).

[0496] In certain embodiments, the anti-CD20 binding domain therapy is used in combination with the heterodimer Fc fusion protein therapy to treat subjects known or suspected of having rheumatoid arthritis (RA). In certain embodiments, the anti-CD20 binding domain therapy is administered by IV infusion as two doses of 1000 mg (dose separated by 2 weeks). In certain embodiments, the anti-CD20 binding domain therapy is administered by IV infusion as two doses of 1000 mg (dose separated by 2 weeks) for up to 24 weeks. In certain embodiments, the combined anti-CD20 binding domain and heterodimer Fc fusion protein therapy are co-administered with methotrexate.

[0497] In certain embodiments, the heterodimer Fc fusion protein therapy is combined with the therapy comprising an antibody therapy containing an agonist antibody. In certain embodiments, the agonist antibody is anti-4-1BB antibody, anti-CD137 antibody, anti-FAP antibody, anti-OX40 antibody, anti-CD40 antibody, anti-GITR antibody or anti-CD27 antibody. In certain embodiments, the agonist antibody is a bispecific antibody. In certain embodiments, the agonist antibody is a multispecific antibody (e.g., bispecific antibody), which includes two or more antigen-binding domains selected from anti-4-1BB antibody, anti-CD137 antibody, anti-FAP antibody, anti-OX40 antibody, anti-CD40 antibody, anti-GITR antibody or anti-CD27 antibody. An illustrative example is a bispecific agonist antibody targeting 4-1BB and CD137, such as utomilumab (Pfizer).

[0498] Checkpoint inhibitor therapy

[0499] In certain embodiments, the heterodimer Fc fusion protein therapy can be combined with checkpoint inhibitor therapy. Illustrative immune checkpoint molecules that can be targeted to block or inhibit include but are not limited to CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (belonging to CD2 family molecules and expressed on all NK, γδ and memory CD8+ (αβ) T cells), CD160 (also referred to as BY55) and CGEN-15049. Immune checkpoint inhibitors include antibodies or antigen-binding fragments thereof or other binding proteins that bind to one or more of the following and block or inhibit the activity of one or more of the following: CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4, CD160, and CGEN-15049. Illustrative immune checkpoint inhibitors include Illustrative immune checkpoint inhibitors include nivolumab (anti-PD-1; -BMS), AMP224 (anti-PD-1; NCI), pembrolizumab (anti-PD-1; MK-3475 / -Merck), pidilizumab (anti-PD-1 antibody; CT-011 - Teva / CureTech), atezolizumab (anti-PD-L1; -Roche / Genentech), durvalumab (anti-PD-L1; MEDI4736 / -Medimmune / AstraZeneca), avelumab (anti-PD-L1; -Pfizer), BMS-936559 (anti-PD-L1-BMS), Ipilimumab (anti-CTLA-4; -BMS), tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), lirilumab (anti-KIR; BMS), monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca), BY55 (anti-CD160), anti-OX40, anti-TIM3, and anti-LAG3.

[0500] In certain embodiments, the check point inhibitor therapy is used in combination with the heterodimer Fc fusion protein therapy to treat subjects known or suspected of having cancer. In certain embodiments, the check point inhibitor therapy is administered by IV infusion. In certain embodiments, the check point inhibitor therapy is administered by IV infusion within 30 minutes. In certain embodiments, the check point inhibitor therapy is administered every 3 weeks. In certain embodiments, the check point inhibitor therapy is administered at a dose of 100mg, 200mg, 300mg, 400mg, 500mg, 600mg, 700mg, 800mg, 900mg or 1000mg. In certain embodiments, the check point inhibitor therapy is administered at a dose of 200mg. In certain embodiments, the check point inhibitor therapy is administered at a dose of at least 100mg, at least 200mg, at least 300mg, at least 400mg, at least 500mg, at least 600mg, at least 700mg, at least 800mg, at least 900mg or at least 1000mg. In certain embodiments, the checkpoint inhibitor therapy is administered at a dose of less than 200 mg. In certain embodiments, the checkpoint inhibitor therapy is used in combination with the heterodimer Fc fusion protein therapy to treat subjects known or suspected of having melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, high microsatellite instability cancer, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC).

[0501] Additional cytokine therapy

[0502] In some embodiments, the heterodimer Fc fusion protein therapy is combined with one or more additional cytokine therapies, one or more chemokine therapies, or a combination thereof. In some embodiments, the heterodimer Fc fusion protein therapy is combined with one or more additional cytokine therapies. In some embodiments, the heterodimer Fc fusion protein therapy is combined with one or more chemokine therapies. In some embodiments, the cytokine therapy comprises proinflammatory cytokines, Th1 cytokines, or Th2 cytokines. In some embodiments, the cytokine therapy comprises recombinant human cytokines or chemokines.

[0503] In some embodiments, the cytokine therapy includes cytokines as interleukins (e.g., IL-1, IL-2, IL-6, IL-7, IL-8, IL-12, IL-13, IL-15, IL-16, IL-18, IL-21, and IL-22). In some embodiments, the cytokine therapy includes cytokines as growth factors (e.g., tumor necrosis factor (TNF), LT, EMAP-II, GM-CSF, FGF, and PDGF). In some embodiments, the cytokine therapy comprises anti-inflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13, and TGF).

[0504] In some embodiments, the chemokine therapy includes proinflammatory chemokines (e.g., GRO-α, GRO-b, LIX, GCP-2, MIG, IP10, I-TAC, and MCP-1, RANTES, eotaxin, SDF-1, and MIP3a). In some embodiments, the chemokine therapy includes chemokine receptors. In some embodiments, the chemokine therapy includes CXC chemokine receptors (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and CXCR7), CC chemokine receptors (e.g., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11), CX3C chemokine receptors (e.g., CX3C11) or XC chemokine receptors (e.g., XCR1). In some embodiments, the chemokine therapy includes transmembrane receptors connected to G proteins.

[0505] In some embodiments, the cytokine therapy comprises a cytokine therapy coordinated with IL-12 signaling. In some embodiments, the cytokine therapy comprises an IL-2 cytokine or a derivative thereof. In some embodiments, the IL-2 therapy is aldesleukin (Proleukin-Prometheus Therapeutics). In some embodiments, the IL-2 therapy and / or aldesleukin are administered intravenously. In some embodiments, the cytokine therapy comprises an IL-15 cytokine or a derivative thereof. In some embodiments, the IL-15 therapy is ALT-803 (AltorBioscience) or NKTR-255 (Nektar). In some embodiments, the IL-15 therapy, NKTR-255 and / or ALT-803 are administered subcutaneously. In some embodiments, the chemokine therapy comprises CXCL9 chemokines, CXCL10 chemokines or derivatives thereof.

[0506] In some embodiments, the cytokine or chemokine therapy comprises administering a cytokine or chemokine to a subject. In some embodiments, the cytokine or chemokine therapy comprises administering a recombinant cytokine or chemokine to a subject. In some embodiments, the cytokine or chemokine therapy comprises engineering cells to produce the cytokine or chemokine. In some embodiments, the cytokine or chemokine therapy comprises engineering cells ex vivo, in vitro, or in vivo to produce the cytokine or chemokine.

[0507] In some embodiments, the cytokine or chemokine therapy comprises engineering cells to produce the cytokine or chemokine using a viral vector-based delivery platform, such as vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629); lentiviruses, including but not limited to second, third or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intronloss maximizes expression in lentiviral vectors containing the humanubiquitin C promoter, Nucl. Acids Res. (2015) 43(1): 682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72(12): 9873-9880); or adeno-associated virus ("AAV") vectors, such as U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5: 3251-3260 (1985); Tratschin et al., Mol. Cell, Biol. 4: 2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:64666470 (1984); and Samuiski et al., J. Virol. 63:03822-3828 (1989) described in more detail. In some embodiments, the cytokine or chemokine therapy comprises engineering cells to produce the cytokine or chemokine using LNPs, liposomes, or exosomes.In some embodiments, the cytokine or chemokine therapy includes the use of genome editing, such as using a nuclease-based genome editing system (e.g., a genome editing system based on clustered regularly interspaced short palindromic repeats (CRISPR) family, transcription activator-like effector nuclease (TALEN), zinc finger nuclease (ZFN) and homing endonuclease (HE) or a derivative thereof) to engineer cells to produce the cytokine or chemokine. In some embodiments, the cytokine or chemokine therapy includes the use of electroporation to engineer cells to produce the cytokine or chemokine.

[0508] Innate immune system agonist therapy

[0509] In some embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more innate immune system agonists.

[0510] In some embodiments, the innate immune system agonist comprises a toll-like receptor (TLR) agonist. In some embodiments, the TLR agonist comprises a TLR1 / 2, TLR2 / 6, TLR3, TLR4, TLR7, TLR8, TLR7 / 8, or TLR9 agonist. In some embodiments, the TLR2 / 6 agonist comprises a lipoprotein, such as a bacterial lipoprotein or derivative, such as Pam2CSK4. In some embodiments, the TLR1 / 2 agonist comprises a lipoprotein. In some embodiments, the TLR3 agonist comprises a dsRNA analog, such as rintatolimod ( -Hemispherx Biopharma) or polyIC-LC (e.g. In some embodiments, the TLR4 agonist comprises lipopolysaccharide (LPS, also known as endotoxin) or a derivative, such as lipid A. In some embodiments, the TLR7 agonist comprises ssRNA or a derivative, or an imidazoquinoline derivative, including but not limited to resiquimod (also known as R848), imiquimod ( In some embodiments, the TLR7 agonist is also a TLR8 agonist, such as imiquimod or Medi-9197 (AstraZeneca / MedImmune). In some embodiments, the TLR9 agonist comprises a CpG-containing oligodeoxynucleotide (CpG-ODN) or SD-101 (Dynavax).

[0511] In some embodiments, the innate immune system agonist comprises a stimulator of interferon genes (STING) agonist. In some embodiments, the STING agonist comprises a cyclic dinucleotide (CDN). In some embodiments, the CDN comprises cyclic diAMP, cyclic diGMP, or cyclic GMP-AMP (cGAMP). In some embodiments, the STING agonist comprises a nucleic acid (e.g., DNA or RNA) that stimulates cGAS. In some embodiments, the STING agonist is ADU-S100 (also known as MIW815-Aduro / Novartis).

[0512] Chemotherapy

[0513] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more chemotherapy. In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more chemotherapy to treat subjects diagnosed with cancer. Examples of chemotherapeutic agents include aldesleukin, alvocidib, antineoplastic AS2-1, antineoplastic A10, antithymocyte globulin, amifostine trihydrate, aminocamptothecin, arsenic trioxide, betaalethine, Bcl-2 family protein inhibitor ABT-263, ABT-199, BMS-345541, bortezomib, Bryostatin 1, busulfan, carboplatin, campath-1H, CC-5103, carmustine, caspofungin acetate, clofarabine, cisplatin, cladribine Chlorambucil Curcumin, cyclosporine, cyclophosphamide (Cyloxan, Endoxan, Endoxana, Cyclostin), cytarabine, denileukin, dexamethasone, DT PACE, docetaxel, Aplysia 10, doxorubicin Doxorubicin hydrochloride, enzastaurin, epoetin alfa, etoposide, everolimus (RAD001), retinamide, filgrastim, melphalan, mesna, flavopiridol, fludarabine Geldanamycin (17-AAG), ifosfamide, irinotecan hydrochloride, ixabepilone, lenalidomide ( CC-5013), lymphokine-activated killer cells, melphalan, methotrexate, mitoxantrone hydrochloride, motexafin gadolinium, mycophenolate mofetil, nelarabine, oblimersen Obatoclax (GX15-070), obimersen, octreotide acetate, omega-3 fatty acids, oxaliplatin, paclitaxel, PD0332991, pegylated liposomal doxorubicin hydrochloride, pegfilgrastim, pentostatin Perifosine, prednisolone, prednisone, R-roscovitine ( CYC202), recombinant interferon α, recombinant interleukin-12, recombinant interleukin-11, recombinant flt3 ligand, recombinant human thrombopoietin, rituximab, sargramostim, sildenafil citrate, simvastatin, sirolimus, styryl sulfone, tacrolimus, tanespimycin, temsirolimus (CC1-779), thalidomide, therapeutic allogeneic lymphocytes, thiotepa, tipifamib, ( or PS-341), vincristine Vincristine sulfate, vinorelbine ditartrate, vorinostat (SAHA), and FR (fludarabine, rituximab), CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), CVP (cyclophosphamide, vincristine and prednisone), FCM (fludarabine, cyclophosphamide, mitoxantrone), FCR (fludarabine, cyclophosphamide, rituximab), hyper-CVAD (hyperfractionated cyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate, cytarabine), ICE (ifosfamide, carboplatin and etoposide), MCP (mitoxantrone, chlorambucil and prednisolone), R-CHOP (rituximab + CHOP), R-CVP (rituximab + CVP), R-FCM (rituximab + FCM), R-ICE (rituximab-ICE) and R-MCP (R-MCP).

[0514] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more chemotherapy to treat subjects diagnosed with colon cancer, rectal cancer or colorectal cancer (CRC). In certain embodiments, the chemotherapy comprises FOLFOX (5-FU, folinic acid and oxaliplatin / lorxadin), FOLFIRI (folinic acid, 5-FU and irinotecan / Camptosar), FOLFOXIRI (folinic acid, 5-FU, oxaliplatin and irinotecan), CapeOx (capecitabine and oxaliplatin), 5-FU co-administered with folinic acid, capecitabine alone or trifluridine and tipiracil In certain embodiments, the chemotherapy comprises a VEGF targeting agent, such as bevacizumab Aflibercept Ramucirumab Regorafenib or EGFR-targeted agents, such as cetuximab (Erbitux) or panitumumab In certain embodiments, the chemotherapy is co-administered with a VEGF targeting agent or an EGFR targeting agent with a chemotherapy selected from the group consisting of FOLFOX, FOLFIRI, FOLFOXIRI, CapeOx, 5-FU co-administered with folinic acid, capecitabine alone, and trifluridine / dipifrine.

[0515] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more chemotherapies to treat a subject diagnosed with breast cancer. In certain embodiments, the chemotherapies comprise doxorubicin. PEGylated liposomal doxorubicin, epirubicin Paclitaxel (Taxol), docetaxel Albumin-bound paclitaxel 5-fluorouracil (5-FU), cyclophosphamide Carboplatin Cisplatin, vinorelbine Capecitabine (Xeloda), gemcitabine Ixapilone or eribulin (HALAVEN). In certain embodiments, the chemotherapy comprises a combination of two or more chemotherapies selected from: doxorubicin PEGylated liposomal doxorubicin, epirubicin Paclitaxel (Taxol), docetaxel Albumin-bound paclitaxel 5-fluorouracil (5-FU), cyclophosphamide Carboplatin Cisplatin, vinorelbine Capecitabine Gemcitabine Ixapilone Eribulin

[0516] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more chemotherapy to treat subjects diagnosed with melanoma / skin cancer. In certain embodiments, the chemotherapy comprises dacarbazine (also known as DTIC), temozolomide, albumin-bound paclitaxel (nab-paclitaxel), paclitaxel, cisplatin, carboplatin, or vinblastine.

[0517] Targeted agent therapy

[0518] In certain embodiments, the heterodimer Fc fusion protein therapy is combined with one or more targeting agents. Typically, targeting agents act on specific molecular targets, such as targets associated with cancer. The difference between targeting agents and standard chemotherapy is that standard chemotherapy acts on all rapidly dividing normal cells and cancer cells. Targeting agents include but are not limited to hormone therapy, signal transduction inhibitors, gene expression regulators, apoptosis inducing agents, angiogenesis inhibitors, immunotherapy, toxin delivery molecules (e.g., antibody-drug conjugates) and kinase inhibitors. In certain embodiments, the targeting agent comprises a receptor agonist or a ligand.

[0519] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more targeting agents to treat subjects diagnosed with colon cancer, rectal cancer, or colorectal cancer (CRC). In certain embodiments, the targeting agent comprises cetuximab Panitumumab Bevacizumab Aflibercept Regorafenib Ramucirumab Nivolumab Ipilimumab

[0520] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more targeting agents to treat a subject diagnosed with breast cancer. In certain embodiments, the targeting agent comprises everolimus. Tamoxifen Toremifene Trastuzumab Fulvestrant Anastrozole Exemestane Lapatinib Letrozole Pertuzumab Enmetazocortuzumab conjugate Pabocini Riboxini Neratinib maleate (NERLYNXTM), abemaciclib (VERZENIO TM ), Olaparib (LYNPARZATM), Atezolizumab or alpelisib

[0521] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more targeting agents to treat subjects diagnosed with melanoma / skin cancer. In certain embodiments, the targeting agent comprises Vismodegib sonidegib Ipilimumab Vemurafenib trametinib Dabrafenib Pembrolizumab Nivolumab Cobimetinib (COTELLICTM), alitretinoin Avelumab encorafenib (BRAFTOVITM), binimetinib or cemiplimab (cemiplimab-rwlc)

[0522] In certain embodiments, the heterodimer Fc fusion protein therapy is combined with a receptor agonist or ligand therapy. In certain embodiments, the receptor agonist or ligand therapy comprises an agonist antibody. In certain embodiments, the receptor agonist or ligand therapy comprises a receptor ligand, such as 4-1BBL or CD40L.

[0523] Radiation therapy

[0524] In some embodiments, the heterodimer Fc fusion protein therapy is combined with radiotherapy. In certain embodiments, the heterodimer Fc fusion protein therapy is combined with radioisotope particles (such as indium In-111, yttrium Y-90 or iodine I-131). Examples of combination therapy include but are not limited to iodine-131 tositumomab Yttrium-90 Ibritumomab Tiuxetan and In certain embodiments, the radiation therapy comprises external beam radiation therapy (EBRT), internal radiation therapy (brachytherapy), intracavitary radiation therapy, interstitial brachytherapy, radioembolization, hypofractionated radiation therapy, intraoperative radiation therapy (IORT), three-dimensional conformal radiation therapy, stereotactic radiosurgery (SRS), or stereotactic body radiation therapy (SBRT).

[0525] In certain embodiments, the heterodimer Fc fusion protein therapy is combined with one or more radiotherapy to treat subjects diagnosed with colon cancer, rectal cancer or colorectal cancer (CRC). In certain embodiments, the radiotherapy comprises external beam radiation therapy (EBRT), internal radiation therapy (brachytherapy), intracavitary radiation therapy, interstitial brachytherapy or radioembolization.

[0526] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more radiotherapy to treat subjects diagnosed with breast cancer. In certain embodiments, the radiotherapy comprises external beam radiotherapy, hypofractionated radiotherapy, intraoperative radiotherapy (IORT), or three-dimensional conformal radiotherapy.

[0527] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more radiotherapy to treat subjects diagnosed with melanoma / skin cancer. In certain embodiments, the radiotherapy comprises stereotactic radiosurgery (SRS; e.g., using a gamma knife or linear accelerator) or stereotactic body radiation therapy (SBRT).

[0528] Vaccines and oncolytic virus therapy

[0529] In some embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more immunogenic compositions (e.g., vaccine compositions) or oncolytic viruses capable of generating a specific immune response (e.g., a tumor-specific immune response).

[0530] In some embodiments, the heterodimeric Fc fusion protein therapy is combined with a vaccine composition. Vaccine compositions generally contain multiple antigens and or neoantigens that are specific to the tumor to be targeted. Vaccine compositions may also be referred to as vaccines.

[0531] In some embodiments, the vaccine composition further comprises an adjuvant and / or a carrier. In some embodiments, the vaccine composition is associated with a carrier (such as a protein) or an antigen presenting cell (such as a dendritic cell (DC)) capable of presenting the peptide to T cells. In some embodiments, the carrier is a scaffold structure capable of associating with an antigen or neoantigen, such as a polypeptide or polysaccharide.

[0532] Generally, an adjuvant is any substance that is mixed into a vaccine composition to increase or otherwise alter the immune response to an antigen or neoantigen. Optionally, the adjuvant is covalently or non-covalently conjugated. The ability of an adjuvant to increase the immune response to an antigen is generally manifested as a significant or substantial increase in an immune-mediated response, or a reduction in disease symptoms. For example, an increase in humoral immunity is generally manifested as a significant increase in the titer of antibodies produced against an antigen, and an increase in T cell activity is generally manifested as an increase in cell proliferation or cytotoxicity or cytokine secretion. An adjuvant can also alter the immune response, for example by changing a primarily humoral or Th response to a primarily cellular or Th response. Suitable adjuvants include, but are not limited to, 1018ISS, alum, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech, Worcester, MA, USA) (which is derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics) and other proprietary adjuvants such as Ribi's Detox, Quil or Superfos. Adjuvants such as incomplete Freund's adjuvant or GM-CSF are useful. Several immunoadjuvants specific for dendritic cells (eg, MF59) and their preparation have been described previously (Dupuis M et al., Cell Immunol. 1998; 186(1): 18-27; Allison AC; Dev Biol Stand. 1998; 92: 3-11). Cytokines may also be used.Several cytokines have been directly linked to influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into potent antigen presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, expressly incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI et al., J Immunother Emphasis Tumor Immunol. 1996 (6): 414-418). In some embodiments, the adjuvant comprises a CpG immunostimulatory oligonucleotide. In some embodiments, the adjuvant comprises a TLR agonist.

[0533] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera); poly (I: C) (e.g., poly i: CI2U); non-CpG bacterial DNA or RNA; and immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tesimumab, and SC58175; which may act therapeutically and / or as an adjuvant. The amount and concentration of adjuvants and additives can be readily determined by a skilled artisan without undue experimentation. Additional adjuvants include colony stimulating factors such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0534] In some embodiments, the vaccine composition comprises more than one different adjuvant. In some embodiments, the vaccine composition comprises any adjuvant substance, including any of the above substances or combinations thereof. It is also contemplated that the vaccine and adjuvant may be administered together or separately in any appropriate order.

[0535] In some embodiments, the carrier (or excipient) exists independently of the adjuvant. In some embodiments, the function of the carrier is to increase molecular weight, increase activity or immunogenicity, confer stability, increase biological activity or increase serum half-life. In some embodiments, the carrier helps present peptides to T cells. In some embodiments, the carrier comprises any suitable carrier known to those skilled in the art, such as protein or antigen presenting cells. Examples of carrier proteins include, but are not limited to, keyhole limpet hemocyanin, serum proteins (such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin), immunoglobulins or hormones (such as insulin) or palmitic acid. For immune humans, the carrier is generally a physiologically acceptable carrier that is acceptable to humans and safe. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier can be a dextran, such as agarose.

[0536] In some embodiments, the vaccine comprises a viral vector-based vaccine platform, such as vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629); or a lentivirus, including but not limited to second, third or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873-9880). Typically, when introduced into a host, the infected cells express the antigen or neoantigen, thereby inducing a host immune (e.g., CTL) response against the one or more peptides.

[0537] Depending on the packaging capacity of the vaccine platform based on the viral vector mentioned above, in some embodiments, the vaccine composition comprises one or more viral vectors. In some embodiments, the viral vector comprises a sequence flanked by a non-mutated sequence, separated by a joint or preceded by one or more sequences targeting a subcellular compartment (see, for example, Gros et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22 (4): 433-8; Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science. (2016) 352 (6291): 1337-41; Lu et al., Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions, Clin Cancer Res. (2014) 20 (13): 3401-10). Vaccinia vectors and methods that can be used for immunization schemes are described in, for example, U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351: 456-460 (1991)). Based on the description herein, a wide variety of other vaccine vectors (e.g., Salmonella typhi vectors, etc.) that can be used for therapeutic administration or immunization of new antigens will be clear to those skilled in the art.

[0538] In some embodiments, the heterodimer Fc fusion protein therapy is combined with oncolytic virus therapy. Typically, oncolytic viruses are viruses that are engineered to primarily infect and kill cancer cells. In some embodiments, in addition to the oncolytic virus that kills cancer cells, the oncolytic virus also induces an immune response to the cancer cells.

[0539] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with oncolytic virus therapy to treat subjects diagnosed with melanoma / skin cancer. In certain embodiments, the oncolytic virus comprises talimogenelaherparepvec Also known as T-VEC. In some embodiments, a heterodimeric Fc fusion protein comprising a subunit of IL-12 is co-administered with an oncolytic virus (e.g., Talimogene Laherparepvec or T-VEC) in combination for treating cancer (e.g., advanced malignancies).

[0540] The above description describes various aspects and embodiments of the present invention. This patent application expressly contemplates all combinations and permutations of the aspects and embodiments.

[0541] Example

[0542] Having now generally described the invention, the invention will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain aspects and embodiments of the invention and are not intended to limit the invention.

[0543] Example 1 - Preparation method

[0544] The proteins of the present invention are generally prepared using recombinant DNA technology. In an exemplary embodiment, a first nucleic acid sequence encoding a first polypeptide is cloned into a first expression vector (pET-pSURE-Puro), wherein the first polypeptide comprises a first subunit of a multi-subunit protein fused to a first antibody Fc domain polypeptide (the p40 subunit of human IL-12); a second nucleic acid sequence encoding a second polypeptide is cloned into a second expression vector (pET-pSURE-Puro), wherein the second polypeptide comprises a second different subunit of a multi-subunit protein fused to a second antibody Fc domain polypeptide (the p35 subunit of human IL-12); and the first and second expression vectors are stably transfected together into a host cell to produce a heterodimeric Fc fusion protein.

[0545] An exemplary amino acid sequence encoded by the first expression vector is shown in SEQ ID NO: 292. The first expression vector encodes a first polypeptide comprising a p40 subunit of human IL-12 fused to a human IgG1 Fc sequence containing a Y349C mutation. The first polypeptide also includes K360E and K409W mutations that promote heterodimerization, and LALAPA (L234A, L235A, and P329A) mutations that reduce effector function. In SEQ ID NO: 292, the leader sequence is shown in italics, the p40 subunit sequence of human IL-12 is underlined, and the mutations are shown in bold.

[0546]

[0547] An exemplary amino acid sequence encoded by the second expression vector is shown in SEQ ID NO: 293. The second expression vector encodes a second polypeptide comprising a p35 subunit of human IL-12 fused to a human IgG1 Fc sequence containing an S354C mutation. The second polypeptide also includes Q347R, D399V, and F405T mutations that promote heterodimerization, and LALAPA (L234A, L235A, and P329A) mutations that reduce effector function. In SEQ ID NO: 293, the leader sequence is shown in italics, the p35 subunit sequence of human IL-12 is underlined, and the mutations are shown in bold.

[0548]

[0549] In order to achieve the highest yield of protein, different ratios of the first and second expression vectors are explored to determine the optimal ratio for transfection into host cells. After transfection, individual clones are isolated using methods known in the art (such as limiting dilution, ELISA, FACS, microscopy or Clonepix) to generate cell banks.

[0550] The clones are cultured under conditions suitable for bioreactor scale-up and sustained expression of the protein. The protein is isolated and purified using methods known in the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography.

[0551] Example 2 - Tumor inhibition by IL-12 fused to a silent Fc domain polypeptide in the CT26 tumor model

[0552] This example describes the relative ability of two IL-12-Fc fusion constructs of recombinant mouse IL-12 (rmIL-12) to control tumor progression in a mouse colon cancer model. The two IL-12-Fc fusion variants used in this example are mIL-12-Fc wild type (DF-mIL-12-Fc wt), which includes wild type mouse IL-12p40 and p35 subunits fused to the N-terminus of wild type mouse IgG2a Fc domain polypeptides; and mIL-12-Fc silence (DF-mIL-12-Fc si), which includes wild type mouse IL-12p40 and p35 subunits fused to the N-terminus of mouse IgG2a Fc domain polypeptides with mutations L234A, L235A and P329G. The amino acid sequence of the protein is shown below:

[0553] mIL-12-p40-mIgG2A-EW (DF-mIL-12-Fc wt first chain)

[0554] MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTEKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSWLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG(SEQ ID NO: 286)

[0555] mIL-12-p35-mIgG2A-RVT (the second chain of DF-mIL-12-Fc wt)

[0556] RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINRVMGYLSSAGGGGSGGGGSGGGGSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPRVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLVSDGSYTMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG(SEQ ID NO: 287)

[0557] mIL-12-p40-mIgG2A-EW-LALAPG (the first strand of DF-mIL-12-Fc si)

[0558] MWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRSPRGPTIKPCPPCKCPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIOHQDWMSGKEFKCKVNNKDLGAPIERTISKPKGSVRAPQVYVLPPPEEEMTEKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSWLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG (SEQ ID NO: 288)

[0559] mIL-12-p35-mIgG2A-RVT-LALAPG (the second strand of DF-mIL-12-Fc si)

[0560] RVIPVSGPARCLSQSRNLLKTTDDMVKTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRETSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQT EFQAINAALQNHNHQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLCILLHAFSTRVVTINNRVMGYLSSAGGGGSGGGGSGGGGSPRGPTIKPCPPCK CPAPNAAGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLGAPIERTISKP KGSVRAPRVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLVSDGSYTMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG(SEQ IDNO:289)

[0561] In short, 10 6 CT26-Tyrp1 colon cancer cells were subcutaneously injected into the flank of Balb / c mice. On day 14 after tumor inoculation, when the tumor volume reached 270 mm 3 At 1 hr, mice were randomized into different treatment groups (n=10 per group) and treated intraperitoneally once a week with 1 μg of rmIL-12, DF-mIL-12-Fc wt at a molar dose equal to 1 μg rmIL-12, DF-mIL-12-Fc si at a molar dose equal to 1 μg rmIL-12, or 1 μg of mIgG2a isotype control. Tumor growth was evaluated for 60 days.

[0562] like Figures 5A-5C As shown, although IL-12 ( Figure 5A ) and DF-mIL-12-Fc wt( Figure 5B ) effectively controlled tumor progression in some mice, but only DF-mIL-12-Fc si induced robust tumor regression and produced 100% complete tumor regression ( Figure 5C In addition, treatment with DF-mIL-12-Fc si therapy significantly prolonged overall survival - 100% of treated mice were still alive at day 60, while the median survival times of mice treated with isotype control, DF-mIL-12-Fc wt, and IL-12 were 27 days, 33 days, and 46 days, respectively ( Figure 6).

[0563] Next, different doses of DF-mIL-12-Fc wt and DF-mIL-12-Fc si were compared in terms of controlling tumor progression. 6 CT26-Tyrp1 colon cancer cells were subcutaneously injected into the flank of Balb / c mice. On day 14 after tumor inoculation, when the tumor volume reached 300 mm 3 At 4 hr, mice were randomized into different treatment groups (n=10 per group) and treated intraperitoneally once a week with DF-mIL-12-Fc wt at a molar dose equivalent to 1 μg or 0.1 μg rmIL-12 or DF-mIL-12-Fc si at a molar dose equivalent to 1 μg or 0.1 μg IL-12. Tumor growth was evaluated for 55 days.

[0564] like Figures 7A-7D As shown, at a 1 μg rmIL-12 molar equivalent dose, treatment with DF-mIL-12-Fc wt resulted in reduced tumor progression in some mice and complete regression in two mice ( Fig. 7A ), but no tumor inhibition was observed at a 0.1 μg IL-12 molar equivalent dose ( Figure 7C In contrast, DF-mIL-12-Fc si treatment at a 1 μg IL-12 molar equivalent dose resulted in 100% complete tumor regression ( Figure 7B ), and induced robust tumor growth delay at a lower dose of 0.1 μg IL-12 molar equivalent ( Fig.7D The median survival of mice treated with 1 μg IL-12 molar equivalent of DF-mIL-12-Fc wt was 32 days, similar to the median survival of 34 days of mice treated with 0.1 μg IL-12 molar equivalent of DF-mIL-12-Fc si, indicating that DF-mIL-12-Fc si is 10 times more potent than its wild-type variant ( Figure 8 DF-mIL-12-Fc wt was ineffective at a dose of 0.1 μg IL-12 molar equivalent and showed the same median survival of 24 days as the isotype-treated group.

[0565] Next, the in vivo efficacy of different routes of administration of DF-mIL-12-Fc si was compared. 6 CT26-Tyrp1 colon cancer cells were subcutaneously injected into the flank of Balb / c mice. On day 14 after tumor inoculation, when the tumor volume reached 270 mm 3At 4 hr, mice were randomized into different treatment groups (n=10 per group) and treated once a week with DF-mIL-12-Fc si at a molar dose equivalent to 1 μg IL-12 or a molar equivalent of mIgG2a isotype control intraperitoneally or subcutaneously. Tumor growth was evaluated for more than 60 days.

[0566] like Figures 22A-22B As shown, intraperitoneal ( Fig.22A ) and subcutaneous ( Fig. 22B ) administration of DF-mIL-12-Fc si induced robust tumor regression and resulted in 100% complete tumor regression. Thus, DF-mIL-12-Fc si treatment demonstrated the efficacy of using different administration routes.

[0567] Example 3 - Tumor inhibition by IL-12 fused to a silent Fc domain polypeptide in the B16F10 tumor model

[0568] This example describes the relative ability of DF-mIL-12-Fc wt and DF-mTT-12-Fc si to control tumor progression in a mouse melanoma model. 6 B16F10 melanoma cells were subcutaneously injected into C57BL / 6 mice. On day 8 after tumor inoculation, when the tumor volume reached 250 mm 3 At 14: 20, mice were randomized into different treatment groups (n=10) and treated once a week with 0.5 μg of IL-12, DF-mIL-12-Fc wt at a molar dose equal to 0.5 μg IL-12, DF-mIL-12-Fc si at a molar dose equal to 0.5 μg IL-12, or 0.5 μg of mIgG2a isotype control. Tumor growth was evaluated for 32 days.

[0569] like Figures 9A-9C As shown, although each IL-12-Fc construct tested delayed tumor progression, DF-mIL-12-Fc si was the most effective in controlling tumor growth. The median survival time of mice treated with DF-mIL-12-Fc si was 29 days, which was longer than the median survival time of mice treated with isotype control, DF-mIL-12-Fc wt, and IL-12, which were 16 days, 26 days, and 22 days, respectively ( Fig.10 ).

[0570] Next, different doses of DF-mIL-12-Fc wt and DF-mIL-12-Fc si were compared in terms of controlling tumor progression. 6B16F10 melanoma cells were subcutaneously injected into the flank of C57BL / 6 mice. On the 8th day after tumor inoculation, mice were randomly divided into different treatment groups (n=10 in each group) and treated intraperitoneally once a week with 0.5 μg or 0.1 μg IL-12 molar equivalent of DF-mIL-12-Fc wt or 0.5 μg or 0.1 μg IL-12 molar equivalent of DF-mIL-12-Fc si. Tumor growth was assessed for 30 days.

[0571] like Figures 11A-11D As shown, at both doses, DF-mIL-12-Fc si was superior to DF-mIL-12-Fc wt in inhibiting tumor growth. In addition, at each dose, the median survival of mice treated with DF-mIL-12-Fc wt was 20 days. In contrast, the median survival of mice treated with 0.1 μg IL-12 molar equivalent of DF-mIL-12-Fc si was 21 days, and the median survival of mice treated with 0.5 μg IL-12 molar equivalent of DF-mIL-12-Fc si was 28 days ( Fig.12 ). These results demonstrate that a high dose (0.5 μg IL-12 molar equivalent) of DF-mIL-12-Fc si significantly increased mouse survival compared to its wild-type counterpart or isotype control.

[0572] Next, single-dose administration of DF-mIL-12-Fc si treatment was compared with weekly treatment as described previously. 6 B16F10 melanoma cells were subcutaneously injected into C57BL / 6 mice. On day 8 after tumor inoculation, when the tumor volume reached 200 mm 3 At 4 d, mice were randomized into different treatment groups (n=10) and treated once a week with a molar dose of DF-mIL-12-Fc si equal to 0.5 μg IL-12 or a molar equivalent of mIgG2a isotype control. Tumor growth was evaluated for 39 days.

[0573] like Fig.23 As shown, a single administration of DF-mIL-12-Fc si resulted in reduced tumor outgrowth in 100% of the mice, although tumor outgrowth occurred more rapidly compared to weekly administration ( Fig. 9C In addition, mice showed temporary weight loss, but only after the first dose (data not shown). Thus, a single administration of DF-mIL-12-Fc si showed initial efficacy in a difficult-to-treat tumor model, although subsequent weekly administrations better delayed tumor outgrowth in this model.

[0574] Next, the in vivo efficacy of different routes of administration of DF-mIL-12-Fc si was compared. 6 B16F10 melanoma cells were injected subcutaneously into C57BL / 6 mice. On day 7 after tumor inoculation, when the tumor volume reached 260 mm 3 At 4 d, mice were randomized into different treatment groups (n=10) and treated once a week with DF-mIL-12-Fc si at a molar dose equivalent to 1 μg IL-12 or a molar equivalent of mIgG2a isotype control intraperitoneally or subcutaneously. Tumor growth was assessed for 40 days.

[0575] like Figures 24A-24B As shown, intraperitoneal ( Fig.24A ) and subcutaneous ( Fig. 24B ) Administration of DF-mIL-12-Fc si induced tumor regression in 100% of mice. Thus, DF-mIL-12-Fc si treatment demonstrated efficacy using different administration routes.

[0576] Example 4 - In vitro efficacy of DF-hIL-12-Fc wt and rhIL-12

[0577] The potency of DF-hIL-12-Fc si compared to rhIL-12 was evaluated using an in vitro bioassay.

[0578] IL-12 potency was assessed using the HEK-Blue IL-12 reporter assay. IL-12R+HEK-Blue reporter cells (InvivoGen) were harvested from culture and adjusted to 1 x 10 6 Cells / mL.DF-hIL-12-Fc si (DF IL-12-Fc) and recombinant human IL-12 (rhIL-12; PeproTech) were diluted in culture medium. 100 μL of PBMC suspension was mixed with 100 μL of diluted test article and incubated for 48 hours. The supernatant was harvested, and the engagement of the signal transduction components stably expressed by IL-12 receptor and reporter cells was detected by measuring the embryonic alkaline phosphatase secreted by the cells according to the manufacturer's instructions. In short, 25 μL of sample supernatant was mixed with 200 μL of QUANTI-Blue reagent, and incubated at room temperature in the dark for 10 minutes. The plate was then read at 620 nM with a SpectraMax i3x plate reader, and the optical density was reported to represent relative IL-12 activity.

[0579] like Fig.13AAs shown, SEAP production by IL-12R+HEK reporter cells increased with increasing concentrations of DF-hIL-12-Fc si or rhIL-12. At the concentrations examined, the IL-12 response measured in the HEK-Blue reporter assay was comparable between DF-hIL-12-Fc si and rhIL-12.

[0580] Next, IL-12 potency was assessed by quantifying IFNγ production by human PBMCs. PBMCs were isolated from human peripheral blood buffy coats using density gradient centrifugation and adjusted to 1 x 10 6 The IFNγ content in the sample wells was estimated by interpolation of the sample readings from the assay standard curve.

[0581] like Fig. 13B As shown, when human PBMCs were cultured with DF-hIL-12-Fc si or rhIL-12, IFNγ production was increased, while treatment with 5 μg / ml PHA amplified the magnitude of the IFNγ response. At the concentrations examined, IFN-γ production following IL-12 stimulation was comparable between DF-hIL-12-Fc si and rhIL-12.

[0582] Thus, despite the EC50 values ​​for the two cell types and stimulation conditions differing by more than an order of magnitude, comparable activity of DF-hIL-12-Fc si and rhIL-12 was demonstrated in both assays, indicating that the potency of the DF-hIL-12-Fc si construct exhibits potency similar to that of native recombinant human IL-12.

[0583] Example 5 - IL-12, DF-hIL-12-Fcsi and IFNγ concentrations in monkey plasma after IV infusion of DF-hIL-12-Fcsi or rhIL-12

[0584] Pharmacodynamics (PD) and pharmacokinetics (PK) were evaluated in cynomolgus monkeys following IV infusion of DF-hIL-12-Fc si or rhIL-12.

[0585] DF-hIL-12-Fc si and recombinant human IL-12 were administered to cynomolgus monkeys at 10 μg / kg by IV infusion.

[0586] Immunoassay was used to detect DF-hIL-12-Fc si and human IL-12 based on the Quantikine ELISA Human IL-12p70 Immunoassay Kit: This assay uses a quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for human IL-12p70 was used as solid phase capture, and detection was accomplished using an antibody HRP-labeled reporter. Standards and QCs spiked with rhIL-12 or DF-hIL-12-Fc si reference standards and test samples were pipetted into the wells of a microtiter plate, and any IL-12p70 present in the sample was bound by the immobilized antibody on the solid phase. Unbound material was washed away, and an enzyme-linked polyclonal antibody specific for human IL-12p70 was added to the wells. Unbound antibody-enzyme reagent was washed away, and TMB substrate was added to each well. The resulting enzyme reaction produced a blue product that turned yellow when an acid stop solution was added. The color intensity measured in each hole is directly proportional to the amount of rhIL-12 or DF-hIL-12-Fc si combined in the initial step. Plates are read at 450nm on the SpectraMax microplate reader with data collection software SoftMax Pro Enterprise Edition 4.6, using 540nm as a reference. Data are converted to text files and imported / processed in Watson LIMS v.7.2.0.02. Logistic (automatic estimation) curve fitting with a weighting factor of 1 is used for regression.

[0587] DF-hIL-12-Fc si was also detected using an immunoassay (meso scale discovery (MSD) - ELISA-like immunoassay) that involves coating untreated MSD microtiter plates with monkey adsorbed goat anti-human IgG and incubating at room temperature. The plates were washed, blocked, washed, and incubated with a standard curve and quality control samples spiked with DF-hIL-12-Fc si reference standards and test samples. After this incubation, the plates were washed and biotin anti-human IL-12 / IL-23p40 was added to the plates as the first detection antibody. After another washing step, a streptavidin-conjugated sulfo-tag (Sulfo-Tag) was added as the second detection antibody. The plates were washed for the last time, MSD read buffer T was added to the plates, and the plates were read using an MSD Sector imager S600. The raw MSD data was exported as a text file and then converted to a Watson LIMS compatible file using a custom-designed programming Excel spreadsheet from Envigo. Data were imported and regressed in Watson LIMS software v.7.2.0.02.

[0588] A mesoscale discovery method was performed for relative quantitative measurement of NHP pro-inflammatory biomarkers in cynomolgus monkey plasma. The method uses a sandwich immunoassay procedure to measure relative quantitatively the pro-inflammatory group 1 biomarkers in cynomolgus monkey K2 EDTA plasma (referred to as monkey plasma): IFNγ, IL-1β, IL-2, IL-6IL-8 and IL-10. The method is based on the MSD non-human primate (NHP) kit for V-PLEX and V-PLEX Plus, catalog numbers: K15056D-1, K15056D-2, K15056D-4, K15056D-6, K15056G-1, K15056G-2, K15056G-4, K15056G-6. The method uses human capture and detection antibodies that react with cynomolgus monkeys. The kit provides a plate pre-coated with capture antibodies on independent well-defined spots in each well of a 96-well multi-spot plate. The plate was incubated with monkey plasma samples, washed, and then incubated with detection antibodies (specific for each analyte) conjugated with electrochemiluminescent (ECL) labels (MSD sulfo tags). The analytes in the sample bind to the capture antibodies immobilized on the surface of the working electrode; the bound analytes recruit the detection antibodies to complete the sandwich. The plate was washed, and MSD read buffer was added to create the appropriate chemical environment for electrochemiluminescence (ECL). The plate was loaded into the MSD Sector Imager 600 (SI600) instrument, where a voltage was applied to the plate electrodes, causing the captured labels to emit light. The instrument measures the intensity of the emitted light expressed in relative light units (RLU) to provide a relative quantitative measurement of the analytes in the sample. The raw RLU data was exported as a text file and then converted to a Watson LIMS compatible file using a custom-designed, programmed Excel spreadsheet from Envigo. The data was then imported and regressed in Watson LIMS software v.7.2.0.02.

[0589] Fig.14 The relative plasma concentrations of DF-hIL-12-Fc si and recombinant human IL-12 over time after IV administration are shown. The data show that, as expected, the concentrations of DF-hIL-12-Fc si and rhIL-12 decrease over time. However, over time, DF-hIL-12-Fc si exhibits an extended half-life and greater exposure overall compared to rhIL-12.

[0590] Fig.14Also shown is the relative concentration (PD) of IFNγ in monkey plasma after IV administration. The data show that the pharmacodynamics of DF-hIL-12-Fc si and rhIL-12, as assessed by IFNγ production, exhibited activity after IV administration. However, compared with rhIL-12, DF-hIL-12-Fc si exhibited higher peak activity and longer duration.

[0591] Example 6 - Pharmacological Characterization of Mouse Surrogate DF-mIL-12-Fc si

[0592] The serum half-life and in vivo pharmacodynamics of a murine IL-12 variant with extended half-life, termed DF-mIL-12-Fc si, were examined.

[0593] DF-mIL-12-Fc si corresponding to the equivalent molar amount of 1 μg IL-12 was injected intravenously in non-tumor-bearing Balb / c mice, and the PK / PD characteristics were compared with IL-12. 1 μg DF-mIL-12-Fc si and IL-12 (equivalent molar is 1 μg IL-12) were injected intravenously to naive Balb / c (n=6). Blood was sampled at 0.017, 0.5, 3, 6, 24, 48, 72, 96, 144 and 219 hours after injection. As previously described, IL-12 and IFNγ levels in serum were analyzed by ELISA.

[0594] like Fig.15A and 15B As shown and quantified in Table 15, DF-mIL-12-Fc si exhibited a long-lasting serum half-life of approximately 30 hours ( Fig. 15B , DF-mIL-12-Fc si T 1 / 2 = 29.85 hours), which is the serum half-life of IL-12 ( Fig.15A IL-12T 1 / 2 In addition to the prolonged half-life, DF-mIL-12-Fc si-mediated IFNγ production (AUC = 916654) was also prolonged compared to IL-12 (AUC = 20304).

[0595] Next, the PK / PD characteristics of different routes of administration of DF-mIL-12-Fc si were compared. DF-mIL-12-Fc si was injected as a single dose in non-tumor-bearing Balb / c mice by intravenous, intraperitoneal or subcutaneous administration, and the PK / PD characteristics were evaluated as described.

[0596] like Figures 15C-15E As shown and quantified in Table 16, intravenous ( Fig. 15C ), intraperitoneal ( Fig.15D ) or subcutaneous ( Fig.15E ) administration all resulted in DF-mIL-12-Fc si-mediated IFNγ production comparable between different administration routes. It is noteworthy that subcutaneous administration resulted in a lower IL-12Cmax. Therefore, the pharmacokinetic properties (e.g., IL-12 concentration) of DF-mIL-12-Fc si administration vary depending on the administration route, while the pharmacodynamic properties (IFNγ production) remain persistent and relatively comparable between different routes.

[0597] Table 15 - Pharmacological characteristics of DF-mIL-12-Fc si and rmIL-12

[0598]

[0599] Table 16 - Pharmacological characteristics of DF-mIL-12-Fc si via IV, IP and SC

[0600]

[0601] Example 7 - Combination of DF-mIL-12-Fc si and PD-1 blockade in the B16F10 mouse model

[0602] Combination therapy of DF-mIL-12-Fc si and PD-1 blockade was performed to analyze whether the antitumor immune response could be amplified in established B16F10 tumors.

[0603] C57BL / 6 mice were injected subcutaneously for 10 6 B16F10 melanoma cells were inoculated into the flanks of mice. On day 8 after tumor inoculation, mice were randomly divided into groups (n=10 per group). When the average tumor volume reached approximately 245 mm 3 Mice were treated intraperitoneally with 0.5 μg isotype control, 0.5 μg DF-mIL-12-Fc si, 200 μg anti-PD-1 clone RMP1-14 or combined DF-mIL-12-Fc si / anti-PD-1 at 1:10 pm. Animals were injected with DF-mIL-12-Fc si once a week and anti-PD-1 twice a week. Tumor growth was assessed for 60 days, and survival and body weight were monitored.

[0604] like Figures 16A-16C As shown, although DF-mIL-12-Fc si alone delayed tumor regression ( Fig.16A ) and administration of PD-1 alone had minimal effect on tumor growth ( Fig. 16B ), but the combination of DF-mIL-12-Fc si and PD-1 blockade further delayed tumor growth ( Fig. 16C ), indicating that anti-PD-1 treatment further amplified the antitumor response to DF-mIL-12-Fc si treatment.

[0605] like Fig.17A and 17B As shown, overall survival was prolonged in the setting of DF-mIL-12-Fc si therapy and in combination with PD-1 blockade, showing a median survival of 29 days (DF-mIL-12-Fc si monotherapy) and 36 days (combination), compared to 15 days for isotype-treated mice and 17.5 days for 200 μg anti-PD-1-treated mice ( Fig.17A ). Notably, despite the high response rate, the DF-mIL-12-Fc si and combination therapy regimens appeared to be well tolerated by B16F10 tumor-bearing mice ( Fig. 17B ).

[0606] Thus, the combination therapy of DF-mIL-12-Fc si and PD-1 blockade demonstrated improved efficacy compared to either treatment alone.

[0607] Example 8 - Combination of DF-mIL-12-Fc si and mcFAE-C26.99 TriNKET in the B16F10 mouse model

[0608] Combination therapy of DF-mIL-12-Fc si and mcFAE-C26.99 TriNKET was performed to analyze whether the anti-tumor immune response could be amplified in established B16F10 tumors.

[0609] C57BL / 6 mice were injected subcutaneously for 10 6 B16F10 melanoma cells were inoculated into the flanks of mice. On day 7 after tumor inocu...

Claims

1. A heterodimeric Fc fusion protein, comprising a first polypeptide consisting of the amino acid sequence of SEQ ID NO: 290; and a second polypeptide consisting of the amino acid sequence of SEQ ID NO:

291.

2. One or more nucleic acids encoding the first and second polypeptides of the heterodimeric Fc fusion protein of claim 1.

3. One or more expression vectors comprising: a nucleic acid comprising a sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 290 and a nucleic acid comprising a sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO:

291. 4 . A formulation comprising the heterodimeric Fc fusion protein according to claim 1 and a pharmaceutically acceptable carrier.

5. The formulation of claim 4, wherein the formulation is suitable for intravenous, subcutaneous, intraperitoneal or intratumoral administration.

6. A cell comprising one or more nucleic acids encoding the heterodimeric Fc fusion protein according to claim 1.

7. A cell comprising one or more nucleic acids according to claim 2.

8. A cell comprising one or more expression vectors according to claim 3.

9. Use of the heterodimeric Fc fusion protein according to claim 1 or the formulation according to claim 4 or 5 in the preparation of a medicament for treating colon cancer or melanoma.

10. The use according to claim 9, wherein the medicament further comprises a second therapeutic agent selected from the group consisting of: (a) Checkpoint blockers, (b) cytokines, (c) TLR agonists, (d) STING agonists, (e) a chemotherapeutic agent selected from the group consisting of paclitaxel, nab-paclitaxel, doxorubicin, gemcitabine and cyclophosphamide, (f) cancer targeting agents, (g) oncolytic viruses, (h) vaccines, (i) radiation, (j) Adoptive NK therapy, (k) Stem cell transplantation (SCT) therapy, (l) agonistic antibodies, (m) Chimeric antigen receptor (CAR) T cell therapy, (n) a multispecific binding protein (TriNKET), and (o) Agents that induce cellular senescence.

11. The use according to claim 10, wherein the checkpoint blocker is selected from: Anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, anti-KIR antibody, anti-NKG2A antibody, anti-LAG3 antibody and anti-TIM3 antibody.

12. The use according to claim 10, wherein the checkpoint blocker is selected from nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, ipilimumab, tesimumab, lirilumab and monazumab.

13. The use according to claim 10, wherein the cytokine is selected from IL-2, IL-15, CXCL9 and CXCL10.

14. The use according to claim 10, wherein the TLR agonist is selected from: TLR7 agonist, TLR8 agonist, TLR7 / 8 agonist, TLR9 agonist, TLR4 agonist and TLR3 agonist.

15. The use according to claim 10, wherein the TLR agonist is selected from R848 / Resiquimod, VTX-2337, Imiquimod and CpG oligodeoxynucleotides.

16. The use according to claim 10, wherein the STING agonist is ADU-S100.

17. The use according to claim 10, wherein the agonistic antibody is selected from anti-4-1BB / CD137 antibody, anti-OX40 antibody, anti-CD40 antibody, anti-CD40L antibody, anti-GITR antibody and anti-CD27 antibody.

18. The use according to claim 10, wherein the TriNKET comprises a binding site for a tumor-associated antigen selected from the group consisting of EpCAM, CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4 and PD-L1.

19. The use according to claim 9, wherein the medicament is formulated for intravenous, subcutaneous, intraperitoneal or intratumoral administration to a patient.

20. A method for producing the heterodimeric Fc fusion protein of claim 1 or the formulation of claim 4, the method comprising: (a) introducing one or more polynucleotides encoding the heterodimeric Fc fusion protein of claim 1 into a cell; as well as (b) culturing the cells for a sufficient period of time to express the heterodimeric Fc fusion protein.

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