Heterodimeric Fc fusion proteins

Abstract

The present invention provides an Fc fusion protein construct which has a higher serum half-life compared to native / native molecules as a monovalent dimer, thus facilitating obtaining a higher protein titer during production, obtaining higher stability during storage, and obtaining improved efficacy when used as a therapeutic agent. Also provided are Fc fusion protein constructs having mutations that reduce effector function in the Fc region, which have increased activity to inhibit tumor growth, and thus are advantageous when used as cancer therapy.

Description

This application is a divisional application of the invention application with an application date of October 23, 2019, a Chinese application number of 201980083496.4, and an invention name of "Heterodimeric Fc Fusion Protein". Cross-reference to related applications

[0001] This application claims the benefit and priority of 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 entirety for all purposes. Sequence Listing

[0002] This application contains a sequence listing that has been electronically submitted 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

[0003] The present invention generally relates to heterodimeric Fc fusion proteins and pharmaceutical compositions containing such proteins, as well as methods of use in treating diseases or disorders in human patients. Background Art

[0004] Most physiologically active proteins have the disadvantage of a short in vivo half-life. To address this disadvantage, attempts have been made to conjugate them with PEG (polyethylene glycol) or to fuse them with the antibody Fc (fragment crystallizable) region. A protein composed of two or more different subunits (wherein the two or more different subunits form a protein complex to exhibit physiological activity) can be fused with a wild-type Fc domain to prepare an Fc fusion protein form, resulting in a homodimer due to the homodimeric nature of Fc. A protein composed of two or more different subunits (wherein the two or more different subunits form a protein complex to exhibit physiological activity) can also be fused with a heterodimeric Fc region derived not only from IgG1 but also from other isotype antibodies such as IgG2, IgG3, and IgG4 to form a heterodimeric Fc fusion protein. Thus, one or more subunits of a protein composed of two or more different subunits and wherein two or more subunits exhibit physiological activity by forming a protein complex can be fused to the end of a heterodimeric Fc variant region to form an improved Fc fusion protein form.

[0005] Fc heterodimerization is a technique that induces mutations in two different CH3 domains of Fc through genetic engineering, such 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, for example, U.S. Patent No. 7,695,936).

[0006] The invention described in the present disclosure provides for an improved design of the Fc fusion protein form, wherein by introducing linkers or mutations of different lengths in the CH2 and CH3 domains of Fc, the two subunits of the heterodimeric protein are linked to two Fc domains having different heterodimerization domains. Summary of the Invention

[0007] The present invention generally relates to heterodimeric Fc fusion proteins and pharmaceutical compositions comprising such proteins.

[0008] In one aspect, the present invention provides a heterodimeric Fc 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 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 bind to each other.

[0009] In some embodiments, the effector function includes the ability of the Fc domain polypeptide to induce antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell 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 comprise one or more mutations at one or more positions 233, 234, 235, 236, 237, 297, 318, 320, 322, 329, 330, and / or 331 according to EU numbering. In some embodiments, the heterodimeric Fc fusion protein is fucosylated.

[0010] 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 comprise one or more mutations at one or more 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 comprise 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 comprise the mutations L234A and L235A. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise the mutations L234A, L235A, and P329A. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise the mutations L234A, L235E, G237A, A330S, and P331S. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise the mutation C220S.

[0011] 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 comprise 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 comprise 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 comprise the mutation L235E. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise the mutations L235E and P329A. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise the mutation S228P.

[0012] 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.

[0013] 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 for reducing the effector function of Fc. For example, the Fc domain polypeptide comprising the mutation can be a human IgG1 antibody Fc domain polypeptide.

[0014] In some embodiments, the one or more mutations for reducing 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 for reducing the effector function of Fc are L234A, L235A, and P329A, numbered according to the EU numbering system.

[0015] 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.

[0016] In some embodiments, the Fc domain polypeptide further comprises a mutation for promoting the formation of a disulfide bond 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.

[0017] 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.

[0018] 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.

[0019] In another aspect, the present invention provides a heterodimeric Fc fusion protein comprising a first Fc domain polypeptide of an immunoglobulin Fc and a second different Fc domain polypeptide, and a first subunit and a second different subunit of a multi-subunit protein, wherein the first subunit and the second different subunit bind to each other and are linked to one or more termini of the N-terminus and / or the 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 the Fc. In some embodiments, the multi-subunit protein is IL-12 (e.g., human IL-12), such that in the heterodimeric Fc fusion protein, the p35 and p40 subunits of IL-12 bind to each other and are linked to one or more termini of the N-terminus and / or the 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 the Fc.

[0020] 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 that binds 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), where 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 bind 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.

[0021] 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 PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9). 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 consists of the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9).

[0022] 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).

[0023] 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 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 PKSSDKTHTCPPCPAPEKKGG (SEQ ID NO: 238) or EPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 7).

[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 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, the linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 8) or GGGGSGGGGSGGGGSPKSSKDTHTCPPCPAPELLGG (SEQ ID NO: 241).

[0025] 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, and the linker comprises the amino acid sequence GGGGGSEPKSSKDTHTCPPCPAPELLGG (SEQ ID NO: 15) or GGGGGSPKSSDKTHTCPPCPAPELLGG (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, and the linker consists of the amino acid sequence GGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 15) or GGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 242).

[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, and the linker comprises 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, and the linker consists of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 16) or GGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 243).

[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, and the linker comprises the amino acid sequence GGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 64) or GGGGGSPKSSDKTHTCPPCPAPEAAGG (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, and the linker consists of the amino acid sequence GGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 65) or GGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 245).

[0028] In some embodiments, within the heterodimeric Fc fusion protein, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 66) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 246). In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that consists of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 66) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 246).

[0029] In some embodiments, within the heterodimeric Fc fusion protein, the linker that connects the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide comprises the amino acid sequence EPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 246) or PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 240). In some embodiments, within the heterodimeric Fc fusion protein, the linker that fuses 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).

[0030] In some embodiments, within the heterodimeric Fc fusion protein, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 247). In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that consists of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 247).

[0031] In some embodiments, within the heterodimeric Fc fusion protein, the second distinct 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: 67) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 248). In some embodiments, the second distinct 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: 67) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 248).

[0032] In some embodiments, within the heterodimeric Fc fusion protein, the second distinct 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 distinct 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).

[0033] In another aspect, the present invention provides a heterodimeric Fc fusion protein, the fusion protein comprising a subunit of a multi-subunit cytokine, the subunit being linked to an immunoglobulin Fc domain polypeptide via a linker, the linker comprising the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239); the Fc domain polypeptide comprises mutations for promoting heterodimerization with a different immunoglobulin Fc domain polypeptide, and L234A, L235A, and P329A substitutions for reducing the effector function of Fc.

[0034] In some embodiments, the present invention provides a heterodimeric Fc fusion protein, the fusion protein comprising the p40 subunit of human IL-12, the subunit being linked to a first human IgG1 (hIgG1) Fc domain polypeptide via a linker, the linker comprising the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or consisting thereof; and the p35 subunit of human IL-12, the subunit being linked to a second hIgG1 Fc domain polypeptide via a linker, the linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10) or consisting thereof; the first Fc domain polypeptide and the second Fc domain polypeptide comprise mutations that promote heterodimerization, and the L234A, L235A, and P329A substitutions for reducing the effector function of hIgG1 Fc.

[0035] 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 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, the 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 Fc domain polypeptide, and the subunits of the multi-subunit protein bind 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 bind to each other.

[0036] 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 to 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).

[0037] In some embodiments, within the heterodimeric Fc fusion protein, the linker that connects the subunits of the multi-subunit protein to the first antibody Fc domain polypeptide comprises the amino acid sequence RVESKYGPPCPPCPAPEFKGG (SEQ ID NO: 2). In some embodiments, the linker that fuses 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).

[0038] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that 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 that consists of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 3).

[0039] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGS RVESKYGPPCPPCPAPEFLGG (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 that consists of the amino acid sequence GGGGS RVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13).

[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 that 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 that consists of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 14).

[0041] 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).

[0042] In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5). In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that consists of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5).

[0043] In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGS RVESKYGPPCPPCAPEFEGG (SEQ ID NO: 63). In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that consists of the amino acid sequence GGGGS RVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63).

[0044] In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64). In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that consists of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64).

[0045] Some of the 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 facilitate heterodimerization with each other. In some embodiments, the first IgG4 antibody Fc domain polypeptide comprises one or more mutations selected from K370E and R409W, and the second different IgG4 antibody Fc domain polypeptide comprises one or more mutations selected from E357N, D399V, and F405T. In some embodiments, the first IgG4 antibody Fc domain polypeptide comprises one or more mutations selected from E357N, D399V, and F405T, and the second different IgG4 antibody Fc domain polypeptide comprises one or more mutations selected from K370E and R409W. In some embodiments, the first IgG4 antibody Fc domain polypeptide comprises the mutations K370E and R409W, and the second different IgG4 antibody Fc domain polypeptide comprises the mutations E357N, D399V, and F405T. In some embodiments, the first IgG4 antibody Fc domain polypeptide comprises the mutations E357N, D399V, and F405T, and the second different IgG4 antibody Fc domain polypeptide comprises the 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 the mutations K360E and R409W, and the second different IgG4 Fc domain polypeptide comprises the mutations Q347R, D399V, and F405T. In some embodiments, the first IgG4 Fc domain polypeptide comprises the mutations Q347R, D399V, and F405T, and the second different IgG4 Fc domain polypeptide comprises the mutations K360E and R409W.

[0046] Some of the 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 the mutations K360E and K409W, and the second different IgG1 antibody Fc domain polypeptide includes the mutations Q347R, D399V, and F405T. In some embodiments, the first IgG1 antibody Fc domain polypeptide includes the mutations Q347R, D399V, and F405T, and the second different IgG1 antibody Fc domain polypeptide includes the mutations K360E and K409W.

[0047] In some embodiments, the heterodimeric Fc fusion proteins described herein comprise an IgG4 or IgG1 Fc domain polypeptide further mutated to reduce effector function. In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain the 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 the 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 the 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 the mutation P329A. In some embodiments, the first IgG1 antibody Fc domain polypeptide and the second different IgG1 antibody Fc domain polypeptide each contain the mutation P329A.

[0048] 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 containing 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 containing the mutations A330S and P331S.

[0049] In some embodiments, the Fc-based heterodimeric proteins described herein incorporate an IgG4 or IgG1 Fc domain polypeptide that has been further mutated to introduce interchain disulfide bonds. In some embodiments, the first IgG4 or IgG1 Fc domain polypeptide includes the mutation Y349C and the second, different IgG4 or IgG1 Fc domain polypeptide includes the mutation S354C. In some embodiments, the first IgG4 or IgG1 Fc domain polypeptide includes the mutation S354C and the second, different IgG4 or IgG1 Fc domain polypeptide includes the mutation Y349C.

[0050] Some heterodimeric Fc fusion proteins of the invention include native disulfide bonds between a first subunit of a multi-subunit protein and a second, different subunit of the multi-subunit protein. For example, in one exemplary embodiment, a heterodimeric Fc fusion protein according to the invention includes native heterodimeric disulfide bonds between the p35 and p40 subunits of IL-12. Such a protein includes native disulfide bonds between C74 of p35 and C177 of p40.

[0051] Some heterodimeric Fc fusion proteins of the invention include artificial or engineered heterodimeric disulfide bonds between a first subunit of a multi-subunit protein and a second, different subunit of the multi-subunit protein. For example, in one exemplary embodiment, a heterodimeric Fc fusion protein according to the invention includes artificial or engineered heterodimeric disulfide bonds between the p35 and p40 subunits of IL-12. Such a protein includes artificial or engineered disulfide bonds between V185C of p35 and Y292C of p40.

[0052] Some heterodimeric Fc fusion proteins of the invention include natural disulfide bonds between a first subunit of a multi-subunit protein and a second different subunit of the multi-subunit protein, as well as artificial or engineered heterodimeric disulfide bonds between a first subunit of the 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 the p35 and p40 subunits of IL-12 is included, and artificial or engineered heterodimeric disulfide bonds between the p35 and p40 subunits of IL-12 are included. Such a protein includes the natural disulfide bond between C74 of p35 and C177 of p40, and artificial or engineered disulfide bonds between V185C of p35 and Y292C of p40.

[0053] Some heterodimeric Fc fusion proteins of the invention are engineered to remove natural disulfide bonds and then replaced with non-natural artificial or engineered disulfide bonds. For example, in an exemplary embodiment, a heterodimeric Fc fusion protein according to the invention includes p35 of IL-12 in which the natural C74 has been mutated to serine, and p40 of Il-12 in which the natural C177 has been mutated to serine, thereby removing the natural disulfide bond between the p35 and p40 subunits of IL-12. Two new mutations are introduced into this mutated IL-12, namely V185C on p35 and Y292C on p40, thereby introducing non-natural artificial or engineered disulfide bonds.

[0054] Within the heterodimeric Fc fusion proteins of the invention, a first polypeptide and a second different polypeptide respectively comprise a first subunit and a second different subunit of a multi-subunit cytokine. Within the heterodimeric Fc fusion proteins of the invention, a first polypeptide and a second different polypeptide respectively comprise a second different subunit and a first subunit of a multi-subunit cytokine. In an exemplary embodiment, the cytokine is IL-12 (e.g., human IL-12). Also provided are formulations containing any one of the heterodimeric Fc fusion proteins described herein, cells containing one or more nucleic acids expressing the heterodimeric Fc fusion protein or one or more vectors expressing the heterodimeric Fc fusion protein, and methods of using the heterodimeric Fc fusion protein to enhance tumor cell death. In some embodiments, the invention provides a formulation comprising a heterodimeric Fc fusion protein described herein and a pharmaceutically acceptable carrier.

[0055] In another aspect, the heterodimeric Fc fusion proteins of the invention further comprise at least one antibody variable domain (e.g., an antibody heavy chain variable domain). In certain embodiments, the at least one antibody heavy chain variable domain binds to an antibody light chain variable region to form a Fab, and the heavy chain variable domain or the light chain variable domain of the Fab is fused to the N-terminus of the first antibody Fc domain polypeptide and / or the second antibody Fc domain polypeptide.

[0056] In certain embodiments, the heterodimeric Fc fusion protein of the invention comprising at least one antibody variable domain has a first subunit of a multi-subunit protein and a second different subunit of the multi-subunit protein that are respectively linked to the C-terminus of the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide.

[0057] In certain embodiments, the heterodimeric Fc fusion protein of the invention comprising at least one antibody variable domain has a first subunit of a multi-subunit protein and a second different subunit of the multi-subunit protein that are respectively linked to the C-terminus of the second antibody Fc domain polypeptide and the first antibody Fc domain polypeptide.

[0058] In certain embodiments, the heterodimeric Fc fusion protein of the invention comprises an antibody heavy chain variable domain located C-terminal to the first antibody Fc domain polypeptide with respect to the first polypeptide. In certain embodiments, the heterodimeric Fc fusion protein of the invention comprises an antibody heavy chain variable domain located C-terminal to the second antibody Fc domain polypeptide with respect to the second polypeptide. In certain embodiments, the antibody heavy chain variable region binds to an antibody light chain variable region to form a scFv. In certain embodiments, the first subunit of the multi-subunit protein and the second different subunit of the multi-subunit protein are respectively linked 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 linked to the N-terminus of the second antibody Fc domain polypeptide and the first antibody Fc domain polypeptide.

[0059] In some embodiments, the heterodimeric Fc fusion protein of the invention does not comprise an antibody variable domain. For example, in some embodiments, the heterodimeric Fc fusion protein consists of or consists essentially of: a first subunit of a multi-subunit protein (e.g., an IL-12 protein), an optional linker 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), an optional linker comprising a spacer peptide, and a second antibody Fc domain polypeptide.

[0060] In some embodiments, the heterodimeric Fc fusion protein of the invention further comprises a proteoglycan-binding domain. In some embodiments, the proteoglycan-binding domain binds to one or more proteoglycans that are specifically expressed in tumors. In some embodiments, the proteoglycan-binding domain binds to one or more proteoglycans selected from the group consisting of syndecan, serglycin, CSPG4, betaglycan, glypican, versican, perlecan, brevican, and small leucine-rich proteoglycan (SLRP). In some embodiments, the SLRP is selected from the group consisting of decorin, biglycan, asporin, fibrodulin, and lumican. In some embodiments, the proteoglycan-binding domain is linked to the C-terminus of the first antibody Fc domain polypeptide. In some embodiments, the proteoglycan-binding domain is linked to the C-terminus of the second antibody Fc domain polypeptide.

[0061] In some embodiments, the heterodimeric Fc fusion protein of the invention further comprises a collagen-binding domain. In some embodiments, the collagen-binding domain binds to one or more collagens that are specifically expressed in tumors. In some embodiments, the collagen-binding domain is linked to the C-terminus of the first antibody Fc domain polypeptide. In some embodiments, the collagen-binding domain is linked to the C-terminus of the second antibody Fc domain polypeptide.

[0062] In some embodiments, the heterodimeric Fc fusion protein of the invention further comprises a hyaluronic acid-binding domain. In some embodiments, the hyaluronic acid-binding domain is linked to the C-terminus of the first antibody Fc domain polypeptide. In some embodiments, the hyaluronic acid-binding domain is linked to the C-terminus of the second antibody Fc domain polypeptide.

[0063] Another aspect of the invention provides a method of treating cancer in a patient. The method comprises administering to the patient (e.g., a patient in need thereof) an effective amount of the heterodimeric Fc fusion protein described herein or a formulation comprising an effective amount of the multispecific binding protein described herein. For example, in some embodiments, the method of treating cancer comprises administering to the patient (e.g., a patient in need of treatment) a formulation comprising an effective amount of the heterodimeric Fc fusion protein described herein and a pharmaceutically acceptable carrier.

[0064] In certain embodiments, the present disclosure provides a method of treating cancer, the method comprising administering to a patient a single dose of an 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 recurrence of the cancer.

[0065] In certain embodiments, the present disclosure provides a method of treating cancer, the method comprising administering to a patient a single dose of an heterodimeric IL-12-Fc fusion protein, the fusion protein comprising a first polypeptide having the amino acid sequence set forth in SEQ ID NO:290 and a second polypeptide having the amino acid sequence set forth in 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 the heterodimeric IL-12-Fc fusion protein comprising a first polypeptide having the amino acid sequence set forth in SEQ ID NO:290 and a second polypeptide having the amino acid sequence set forth in 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 recurrence of the cancer.

[0066] Another aspect of the invention provides a method of treating acute radiation syndrome, wherein the method comprises administering to a patient the heterodimeric Fc fusion protein or formulation disclosed herein. In some embodiments, the acute radiation syndrome comprises one or more syndromes selected from the group consisting of: hematopoietic radiation syndrome, gastrointestinal radiation syndrome, neurovascular radiation syndrome, and cutaneous radiation syndrome. In some embodiments, the acute radiation syndrome comprises syndromes selected from the group consisting of: hematopoietic radiation syndrome, gastrointestinal radiation syndrome, neurovascular radiation syndrome, and cutaneous radiation syndrome.

[0067] In summary, the present invention provides heterodimeric Fc fusion protein constructs of multi-subunit proteins. These fusion protein constructs may exhibit a higher serum half-life, improved yield during production, enhanced stability during storage, and / or improved efficacy when used as a therapeutic agent, compared to the native / natural multi-subunit protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1ADisclosed is an exemplary heterodimeric Fc fusion protein that includes a first subunit of a multi-subunit protein, said subunit being linked via a linker to a first antibody Fc domain polypeptide; and a second, different subunit of the multi-subunit protein, said subunit being linked via a linker to a second antibody Fc domain polypeptide. The first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain different mutations that promote heterodimerization, and a disulfide bond between the Fc domain polypeptides stabilizes the heterodimer. The linker that links the additional subunit to the second antibody Fc domain polypeptide can include the (G4S)3 sequence (SEQ ID NO:108).

[0069] Figure 1B Disclosed is an exemplary heterodimeric Fc fusion protein that is similar to Figure 1A the protein shown, but also includes mutations in the Fc domain polypeptide to reduce FcγR binding.

[0070] Figure 1C Disclosed is an exemplary protein in which a second, different subunit of the multi-subunit protein is linked via a linker to a first antibody Fc domain polypeptide, and another subunit of the multi-subunit protein is linked via another linker to a second antibody Fc domain polypeptide.

[0071] Figure 1D Disclosed is an exemplary heterodimeric Fc fusion protein that is similar to Figure 1C the protein shown, but also includes mutations in the Fc domain polypeptide to reduce FcγR binding.

[0072] Figure 1E Disclosed is an exemplary heterodimeric Fc fusion protein similar to Figure 1B the protein shown, except that the second, different subunit of the multi-subunit protein is located at the C-terminus of the second antibody Fc domain polypeptide in the second polypeptide.

[0073] Figure 1F Disclosed is an exemplary heterodimeric Fc fusion protein similar to Figure 1E the protein shown, except that the second, different subunit of the multi-subunit protein is located at the C-terminus of the first antibody Fc domain polypeptide in the first polypeptide.

[0074] Figure 2ADisclosed is an exemplary heterodimeric Fc fusion protein that comprises a first subunit of a multi-subunit protein, which subunit is linked via a linker to a first antibody Fc domain polypeptide; and a second, different subunit of the multi-subunit protein, which subunit is linked via another linker to a second antibody Fc domain polypeptide, wherein the subunits are linked by 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 bond between the Fc domain polypeptides stabilizes the heterodimer. The disclosed protein further includes mutations in the Fc domain polypeptides to reduce FcγR binding.

[0075] Figure 2B Disclosed is an exemplary heterodimeric Fc fusion protein that comprises a first subunit of a multi-subunit protein, which subunit is linked via a linker to a first antibody Fc domain polypeptide; and a second, different subunit of the multi-subunit protein, which subunit is linked via another linker to a second antibody Fc domain polypeptide, wherein the subunits are linked by a non-native disulfide bond. In the exemplary protein shown, native disulfide bonds have been removed and replaced with artificial disulfide bonds. For example, an IL-12 construct can incorporate the 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 bond between the Fc domain polypeptides stabilizes the heterodimer. The protein further includes mutations in the Fc domain polypeptides to reduce FcγR binding.

[0076] Figure 3A Disclosed is an exemplary heterodimeric Fc fusion protein that comprises a first subunit of a multi-subunit protein, which subunit is linked via a linker to a first antibody Fc domain polypeptide; and a second, different subunit of the multi-subunit protein, which subunit is linked via another linker having the same amino acid sequence to a second antibody Fc domain polypeptide. The first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain different mutations that promote heterodimerization, and the disulfide bond between the Fc domain polypeptides stabilizes the heterodimer.

[0077] Figure 3B Disclosed is an exemplary heterodimeric Fc fusion protein that comprises a first subunit of a multi-subunit protein, which subunit is linked via a linker to a first antibody Fc domain polypeptide; and a second, different subunit of the multi-subunit protein, which subunit is linked via another linker to a second antibody Fc domain polypeptide. The first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain different mutations that promote heterodimerization, and the disulfide bond between the Fc domain polypeptides stabilizes the heterodimer. The linker that links the second, different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide can include (G4S)2 (SEQ ID NO:109) or G4S (SEQ ID NO:110).

[0078] Figures 4A - 4L show exemplary scFv fusion heterodimeric Fc fusion protein constructs ( Figures 4A - 4H ) and mAb fusion heterodimeric Fc fusion protein constructs ( Figures 4I - 4L ), wherein a first subunit of the multimeric protein is linked via a linker to a first antibody Fc domain polypeptide, and a second different subunit of the multimeric protein is linked via another linker to a second antibody Fc domain polypeptide.

[0079] Figures 4A - 4D show exemplary Fc fusion proteins that include a first scFv linked to the C-terminus of a first antibody Fc domain polypeptide and a second scFv linked 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 to different epitopes on a single antigen). Figure 4A and Figure 4C the exemplary Fc fusion proteins shown contain different pairs of heterodimerizing Fc variants. Figure 4B and Figure 4D the exemplary Fc fusion proteins shown contain different pairs of heterodimerizing Fc variants. For example, in cases where the heterodimerizing mutations include "knobs-into-holes" mutations, Figure 4A and Figure 4D show exemplary Fc fusion proteins in which a first polypeptide contains one or more "hole" mutations and a second polypeptide contains one or more "knob" mutations; Figure 4B and Figure 4C show exemplary Fc fusion proteins in which a first polypeptide contains one or more "knob" mutations and a second polypeptide contains one or more "hole" mutations. Figure 4A and Figure 4B show exemplary Fc fusion proteins in which the protein sequence of a first subunit of the multimeric protein is linked to a first polypeptide; Figure 4C and Figure 4D show exemplary Fc fusion proteins in which a second different subunit of the multimeric protein is linked to a first polypeptide. Similarly, heterodimeric Fc fusion proteins having other types of heterodimerizing mutations are contemplated.

[0080] Figure 4E and Figure 4G show exemplary Fc fusion proteins that include a scFv linked to the C-terminus of a first antibody Fc domain polypeptide.

[0081] Figure 4F and Figure 4H show exemplary Fc fusion proteins that include a scFv linked to the C-terminus of a second antibody Fc domain polypeptide.

[0082] Figures 4I - 4L Shows an exemplary Fc fusion protein comprising 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. Figure 4I and Figure 4K Shows an exemplary Fc fusion protein in which a first subunit of a multi-subunit protein is linked to a first polypeptide; Figure 4J and Figure 4L Shows an exemplary Fc fusion protein in which a second different subunit of a multi-subunit protein is linked to a first polypeptide. Figure 4K Differing from Figure 4I by having a longer amino acid sequence (e.g., a spacer peptide disclosed herein) that links the antibody Fc domain polypeptide to the first subunit of the multi-subunit protein; Figure 4J Differing from Figure 4L by having a longer amino acid sequence (e.g., a spacer peptide disclosed herein) that links the antibody Fc domain polypeptide to the first subunit of the multi-subunit protein.

[0083] Figures 5A - 5C Is a graph showing the tumor growth curves of individual mice inoculated with CT26 tumor cells and treated weekly with recombinant murine IL-12 (rmIL-12) ( Figure 5A ), DF-mIL-12-Fc wt ( Figure 5B ), DF-mIL-12-Fc si ( Figure 5C ) or mIgG2a isotype control.

[0084] Figure 6 Is a graph showing the Kaplan-Meier survival curves of 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.

[0085] Figures 7A - 7D Is a graph showing the tumor growth curves of individual mice inoculated with CT26 tumor cells and treated weekly with DF-mIL-12-Fc wt at a molar equivalent of 1 μg rmIL-12 ( Figure 7A ), DF-mIL-12-Fc si at a molar equivalent of 1 μg rmIL-12 ( Figure 7B ), DF-mIL-12-Fc wt at a molar equivalent of 0.1 μg rmIL-12 ( Figure 7C ), DF-mIL-12-Fc si at a molar equivalent of 0.1 μg rmIL-12 ( Figure 7D ) or mIgG2a isotype control.

[0086] Figure 8 It is a graph showing the Kaplan-Meier survival curves of mice inoculated with CT26 tumor cells and treated once a week 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.

[0087] Figures 9A - 9C It is a graph showing mice inoculated with B16F10 melanoma cells and treated with rmIL-12 ( Figure 9A ), DF-mIL-12-Fcwt ( Figure 9B ), DF-mIL-12-Fc si ( Figure 9C ), or mIgG2a isotype control once a week, showing the tumor growth curves of individual mice.

[0088] Figure 10 It is a graph showing the Kaplan-Meier survival curves of mice inoculated with B16F10 melanoma cells and treated once a week with rmIL-12, DF-mIL-12-Fc wt, DF-mIL-12-Fc si, or mIgG2a isotype control.

[0089] Figures 11A - 11D It is a graph showing mice inoculated with B16F10 melanoma cells and treated once a week with DF-mIL-12-Fc wt at a molar equivalent of 0.5 μg rmIL-12 ( Figure 11A ), DF-mIL-12-Fc si at a molar equivalent of 0.5 μg rmIL-12 ( Figure 11B ), DF-mIL-12-Fc wt at a molar equivalent of 0.1 μg rmIL-12 ( Figure 11C ), DF-mIL-12-Fc si at a molar equivalent of 0.1 μg rmIL-12 ( Figure 11D ), or mIgG2a isotype control, showing the tumor growth curves of individual mice.

[0090] Figure 12Graph showing the Kaplan-Meier survival curves of mice inoculated with B16F10 melanoma cells and treated once a week 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.

[0091] Figure 13A Graph showing the IL-12 responses measured using the HEK-Blue IL-12 reporter assay in response to treatment with DF-hIL-12-Fc si (DFIL-12-Fc) or recombinant human IL-12 (rhIL-12).

[0092] Figure 13B Graph showing IFNγ production in peripheral blood mononuclear cells (PBMCs) in response to treatment with DF-hIL-12-Fc si (DF IL-12-Fc) and rhIL-12.

[0093] Figure 14 Graph showing the relative plasma concentrations of DF-hIL-12-Fc si, rhIL-12, and IFNγ in cynomolgus monkey K2 EDTA plasma after a single intravenous dose of 10 μg / kg equimolar amounts of DF-hIL-12-Fc si or wild-type rhIL-12.

[0094] Figures 15A - 15B Graph showing naive Balb / c mice for the PK / PD profiles of rmIL-12 ( Figure 15A ) and DF-mIL-12-Fcsi ( Figure 15B ). Figure 15A Shows the PK / PD profile of rmIL-12 in naive Balb / c mice, and Figure 15B shows the PK / PD profile of DF-mIL-12-Fc si in naive Balb / c mice for IL-12. Serum IL-12 and IFNγ levels were analyzed by ELISA. Figure 15C Graph showing the PK / PD profile of intravenously administered DF-mIL-12-Fc si in naive Balb / c mice. Figure 15D Graph showing the PK / PD profile of intraperitoneally administered DF-mIL-12-Fc si in naive Balb / c mice. Figure 15EFigure showing the PK / PD profile of subcutaneously administered DF-mIL-12-Fc si in naive Balb / c mice. Mean serum levels represent mean ± SEM.

[0095] Figures 16A - 16C Figure showing the tumor growth curves of mice bearing B16F10 tumors treated with DF-mIL-12-Fc si, anti-PD-1, or their combination. Mice were treated intraperitoneally with 0.5 μg of isotype control or 0.5 μg of DF-mIL-12-Fc si( Figure 16A ), isotype control or anti-PD-1( Figure 16B ), and isotype control or DF-mIL-12-Fc si / anti-PD-1( Figure 16C ). As indicated above, animals were injected once a week with DF-mIL-12-Fc si and twice a week with anti-PD-1. Tumor growth was evaluated for 60 days. The figure shows the tumor growth curves of individual mice.

[0096] Figures 17A - 17B Figure showing the survival and body weights of mice bearing B16F10 tumors treated with DF-mIL-12-Fc si, anti-PD-1, or their combination. 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 once a week with 0.5 μg of DF-mIL-12-Fc si and twice a week with 200 μg of anti-PD-1 or isotype. Figure 17A Shows the Kaplan-Meier survival curve. Figure 17B Shows the body weight of the mice as mean ± standard deviation.

[0097] Figures 18A - 18C Figure showing the tumor growth curves of mice bearing B16F10 tumors treated with DF-mIL-12-Fc si, mcFAE-C26.99TriNKET, or their combination. Mice were treated intraperitoneally with 150 μg of isotype control or 0.5 μg of DF-mIL-12-Fc si( Figure 18A ), isotype control or 150 μg of TriNKET( Figure 18B ), and isotype control or DF-mIL-12-Fc si / TriNKET( Figure 18C ). As indicated above, animals were injected once a week with DF-mIL-12-Fc si and three times a week with TriNKET. Tumor growth was evaluated for 72 days. The figure shows the tumor growth curves of individual mice.

[0098] Figures 19A - 19BFigure showing the survival and body weight of mice bearing B16F10 tumors treated with DF-mIL-12-Fc si, mcFAE-C26.99TriNKET, or a combination thereof. Mice were treated with isotype, DF-mIL-12-Fc si, TriNKET, or a combination of DF-mIL-12-Fc si and TriNKET. Animals were injected once a week with 0.5 μg of DF-mIL-12-Fc si and three times a week with 150 μg of TriNKET or isotype. Figure 19A Shows the Kaplan-Meier survival curve. Figure 19B Shows the body weight of the mice as mean + standard deviation.

[0099] Figure 20 Figure showing the tumor growth curve of complete responder (CR) mice from the B16F10 tumor model experiment of Figure 18, the mice being treated with DF-mIL-12-Fc si / TriNKET combination therapy (n = 3) and re-challenged by implanting 2 x 10 5 B16F10 melanoma cells.

[0100] Figure 21A Figure showing the tumor growth curve of individual mice inoculated with CT26 tumor cells and administered a single dose of DF-mIL-12-Fc si or mIgG2a isotype.

[0101] Figure 21B Figure showing the body weight ± standard deviation of mice inoculated with CT26 tumor cells and administered a weekly dose of DF-mIL-12-Fc si, mIgG2a isotype, or rmIL-12.

[0102] Figure 21C Figure showing the tumor growth curve of individual re-challenged mice that were naive or complete responders (CR) when a single dose of DF-mIL-12-Fc si was previously administered in the CT26 tumor model.

[0103] Figures 22A - 22B Figure showing the tumor growth curve of individual mice inoculated with CT26 tumor cells and administered a weekly dose of DF-mIL-12-Fc si intraperitoneally (IP) ( Figure 22A ) or subcutaneously (SC) ( Figure 22B ).

[0104] Figure 23 Figure showing the tumor growth curve of individual mice inoculated with B16F10 melanoma cells and administered a single dose of DF-mIL-12-Fc si or mIgG2a isotype.

[0105] Figures 24A - 24B is a graph showing tumor growth curves of individual mice inoculated with B16F10 melanoma cells and administered a weekly dose of DF-mIL-12-Fc si or the mIgG2a isotype intraperitoneally (IP)( Figure 24A ) or subcutaneously (SC)( Figure 24B ).

[0106] Figures 25A - 25B is a graph showing tumor growth curves of individual mice inoculated with CT26 tumor cells and administered a single dose ( Figure 25A ) or a weekly dose ( Figure 25B ) of DF-mIL-12-Fc si or the mIgG2A isotype intraperitoneally at the molar equivalent of 1 μg rmIL-12(

[0107] Figures 26A - 26B is a graph showing tumor growth curves of individual mice inoculated with CT26 tumor cells and administered a weekly dose of DF-mIL-12-Fc si subcutaneously. Figure 26A is a graph showing tumor growth curves of individual mice inoculated with CT26-Tyrp1 tumor cells and treated once (weekly) with 2 μg of the mIgG2a isotype control or 1 μg of DF-mIL-12-Fc si. Figure 26B is a graph showing tumor growth curves of individual mice inoculated with CT26-Tyrp1 tumor cells and treated once (weekly) with 2 μg of the mIgG2a isotype control or 2 μg of DF-mIL-12-Fc si.

[0108] Figures 27A - 27C is a graph showing IFNγ( Figure 27A ), CXCL9( Figure 27B ), and CXCL10( Figure 27C ) levels 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.

[0109] Figures 28A - 28C is a linear graph showing the pharmacokinetics of DF-hIL-12-Fc si in cynomolgus monkeys treated with a single subcutaneous dose of 1 μg / kg( Figure 28A ), 2 μg / kg( Figure 28B ), or 4 μg / kg( Figure 28C ) of DF-hIL-12-Fc si. 2240, 2241, 2740, 2741 represent individual cynomolgus monkey subjects.

[0110] 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. Figure 29A 、 29C And 29E respectively show the IFNγ concentration / expression levels in cynomolgus monkeys treated with 1 μg / kg, 2 μg / kg, and 4 μg / kg of DF-hIL-12-Fc si. Figure 29B 、 Figure 29D And Figure 29F Respectively show the 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. 3240, 3241, 3740, 3741 represent individual cynomolgus monkey subjects.

[0111] Figure 30 Is a graph showing the tumor growth curves of individual mice inoculated with breast cancer cells and administered a weekly dose of single therapy (isotype control, DF-mIL-12-Fc si, (chemotherapy) or irradiated with 10 Gy) or combination therapy (DF-mIL-12-Fc si combined with or radiation).

[0112] Figure 31A Is a graph showing the tumor growth curves of individual mice inoculated with CT26-Tyrp1 tumor cells and treated (every two weeks) with an isotype control or an anti-PD-1 antibody. Figure 31B Is a graph showing the tumor growth curves of Balb / c mice inoculated with CT26-Tyrp1 tumor cells and treated (every two weeks) with an isotype control or an anti-PD-1 antibody. Figure 31B Also shows the tumor growth curves of individual mice previously treated with an anti-PD-1 antibody, to which the anti-PD-1 antibody (every two weeks) and a weekly treatment of 1 μg of DF-mIL-12-Fc si were administered.

[0113] Figure 32A Is a graph showing the tumor growth curves of the treated (Tr) tumors of individual mice inoculated with CT26-Tyrp1 tumor cells and treated (once a week) intratumorally with an isotype control or DF-mIL-12-Fc si. Figure 32B Is a graph showing Figure 32A The tumor growth curves of the untreated (NT) CT26-Tyrp1 tumors in the individual mice described in

[0114] Figure 33AIt is a graph showing the tumor growth curves of individual mice inoculated with CT26-Tyrp1 tumor cells and treated once with 2 μg of mIgG2a isotype control or 2 μg of DF-mIL-12-Fc si. Figure 33B It is a graph showing the average tumor growth curves of individual mice inoculated with CT26-Tyrp1 tumor cells and treated with 2 μg of mIgG2a isotype control, 1 μg of DF-mIL-12-Fc si (administered weekly), 2 μg of DF-mIL-12-Fc si (administered weekly), or 2 μg of DF-mIL-12-Fc si (once).

[0115] Figure 34A It is a graph showing the IFNγ production of PHA-stimulated PBMCs treated with DF hIL-12-Fc-si having L234A, L235A, and P329A mutations (LALAPA) or L234A, L235A, P329G mutations (LALAPG). Figure 34B It shows flow cytometry histograms of the binding of fluorophore-conjugated hIgG1 to THP-1 cells in the presence or absence of DF hIL-12-Fc-si having LALAPA mutations or LALAPG mutations. Detailed Description

[0116] The present invention provides improvements to heterodimeric Fc fusion proteins, pharmaceutical compositions comprising such proteins, and methods of treatment using such proteins and pharmaceutical compositions, including for the treatment of cancer.

[0117] To facilitate understanding of the present invention, a number of terms and phrases are defined below.

[0118] Unless the context is inappropriate, the terms "a" and "an" as used herein mean "one or more" and include the plural.

[0119] 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., mouse, ape, horse, cow, pig, dog, cat, etc.), and more preferably include humans.

[0120] 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 prophylactic or therapeutic effect). The 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 particular formulation or route of administration. As used herein, the term "treatment" includes any action that results in the improvement of a disorder, disease, condition, etc. or the alleviation of its symptoms, e.g., reducing, lowering, modulating, alleviating, or eliminating.

[0121] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier such that the composition is particularly suitable for diagnostic or therapeutic use in vivo or in vitro.

[0122] As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier such as phosphate buffered saline solution, water, emulsions (e.g., 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, e.g., Martin, Remington's Pharmaceutical Sciences, 15th ed., Mack Publ. Co., Easton, PA

[1975] .

[0123] 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 that, upon administration to a subject, is capable of providing the compound of the present invention or its active metabolite or residue. As is known to those skilled in the art, the "salts" of the compounds of the present invention can be derived from inorganic acids or organic acids and inorganic bases or organic bases. 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. Other acids (such as oxalic acid), although not pharmaceutically acceptable per se, can be used to prepare salts that can be used as intermediates in obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

[0124] 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 the formula NW4 + wherein W is C 1-4 alkyl, etc.

[0125] Exemplary salts include, but are not limited to: acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, bisulfates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, esulfonates, fumarates, flucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxyesulfonates, lactates, maleates, mesylates, 2-naphthalenesulfonates, nicotinates, oxalates, palmitates, pectates, persulfates, phenylpropionates, picrates, pivalates, propionates, succinates, tartrates, thiocyanates, toluenesulfonates, undecanoates, etc. Other examples of salts include anions of the compounds of the present invention complexed with suitable cations such as Na + , NH4 + and NW4 + (wherein W is C 1-4 alkyl), etc.

[0126] For therapeutic use, salts of the compounds of the present invention are considered pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases can also be used, for example, in the preparation or purification of pharmaceutically acceptable compounds.

[0127] Throughout the specification, where a composition is described as having, including, or containing a particular component, or where a process and method are described as having, including, or containing a particular step, compositions of the present invention consisting essentially of or consisting of the recited components are contemplated, as well as processes and methods according to the present invention consisting essentially of or consisting of the recited processing steps.

[0128] Generally, unless otherwise specified, percentages of compositions are by weight. Additionally, if a variable is not accompanied by a definition, the previous definition of the variable shall apply. I. Protein

[0129] The present invention provides Fc fusion protein constructs that contain the amino acid sequences of multi-subunit proteins. These fusion protein constructs can exhibit a higher serum half-life, improved yield during production, enhanced stability during storage, and / or improved efficacy when used as therapeutic agents, compared to the native / natural multi-subunit proteins. IgG1 Fc fusion protein

[0130] 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 that binds 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 bind 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.

[0131] 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 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.

[0132] In certain embodiments, the linker connecting the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide further comprises a spacer peptide. In certain embodiments, the linker comprises the sequence of SEQ ID NO: 237 or SEQ ID NO: 6 and a spacer peptide.

[0133] 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 that links 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 that links the subunit of the multi-subunit protein to the first antibody Fc domain polypeptide.

[0134] Any spacer peptide described under the heading "Spacer Peptide" can be employed. 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 that links the subunit of the multi-subunit protein to the first antibody Fc domain polypeptide consists of, or consists essentially of, a spacer peptide disclosed herein and a peptide having the sequence of SEQ ID NO: 237 or SEQ ID NO: 6. In certain embodiments, the linker that links the second different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide consists of, or consists essentially of, a spacer peptide disclosed herein and a peptide having the sequence of SEQ ID NO: 237 or SEQ ID NO: 6. In certain embodiments, the spacer peptide is at the N-terminus of any one or both of the linkers.

[0135] 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 PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9). 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 consists of the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9).

[0136] 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 PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239). 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 consists of the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239).

[0137] In some embodiments, within the heterodimeric Fc fusion protein, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 244). In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that consists of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 244).

[0138] 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 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 consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10).

[0139] 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).

[0140] 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).

[0141] In some embodiments, within the heterodimeric Fc fusion protein, the second distinct 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 GGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 242). In some embodiments, the second distinct 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).

[0142] In some embodiments, within the heterodimeric Fc fusion protein, the second distinct 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 distinct 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).

[0143] In some embodiments, within the heterodimeric Fc fusion protein, the second distinct 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 distinct 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).

[0144] In some embodiments, within the heterodimeric Fc fusion protein, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 66) or GGGGSGGGGSPLSSDLTJTCPPCPAPEAAGG (SEQ ID NO: 246). In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker consisting of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPCPAPEAAGG (SEQ ID NO: 66) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 246).

[0145] 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 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).

[0146] In some embodiments, within the heterodimeric Fc fusion protein, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12) or GGGGSGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 247). In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 247).

[0147] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multimeric protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67) or GGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 248). In some embodiments, the second different subunit of the multimeric protein is fused to the second antibody Fc domain polypeptide via a linker that consists of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67) or GGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 248).

[0148] In some embodiments, within the heterodimeric Fc fusion protein, the second different subunit of the multimeric protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 68) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 249). In some embodiments, the second different subunit of the multimeric protein is fused to the second antibody Fc domain polypeptide via a linker that consists of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 68) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 249).

[0149] In certain embodiments, the Fc domain polypeptide is the Fc domain polypeptide of IgG1 Fc. In some embodiments, the protein of the invention comprises a first antibody Fc domain polypeptide and a second antibody Fc domain polypeptide, both of which are mutant 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 can 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: Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411, and K439.

[0150] In some embodiments, the antibody constant domain can 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: Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, T366I, T366L, T366M, T366K, T366W, T366S, L368E, L368A, L368D, K370S, N390D, N390E, K392L, K392M, K392V, K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K439D, and K439E. All amino acid positions in the Fc domains or hinge regions disclosed herein are numbered according to EU numbering.

[0151] 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 the mutations K360E and K409W, and the second antibody IgG1 Fc domain polypeptide comprises the mutations Q347R, D399V, and F405T. In some embodiments, the first antibody IgG1 Fc domain polypeptide comprises the mutations Q347R, D399V, and F405T, and the second antibody IgG1 Fc domain polypeptide comprises the mutations K360E and K409W.

[0152] In some embodiments, the heterodimeric Fc fusion protein of the invention having an IgG1 Fc comprises 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 proteins of the present disclosure 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.

[0153] In some embodiments, the heterodimeric Fc fusion protein according to the invention comprises a first antibody IgG4 or IgG1 Fc domain polypeptide and a second antibody IgG4 or IgG1 Fc domain polypeptide, each containing the mutation P329G or P329A. In a specific embodiment, the heterodimeric Fc fusion protein according to the invention comprises a first antibody IgG4 or IgG1 Fc domain polypeptide and a second antibody IgG4 or IgG1 Fc domain polypeptide, each comprising the mutation P329A.

[0154] 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 P331S. In some embodiments, the first IgG1 antibody Fc domain polypeptide and the second different IgG1 antibody Fc domain polypeptide each contain the mutations A330S and P331S.

[0155] 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 comprises a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the second antibody Fc domain polypeptide fused to the second different subunit of the multi-subunit protein. Alternatively, the first antibody Fc domain polypeptide fused to the first subunit of the multi-subunit protein comprises an S354C substitution in the CH3 domain, which forms a disulfide bond with a Y349C substitution on the second antibody Fc domain polypeptide fused to the second different subunit of the multi-subunit protein.

[0156] 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 the portion or all of the linker that connects the protein sequence of the first subunit of the multi-subunit protein to the first IgG1 antibody Fc domain polypeptide or the linker that connects another subunit to 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 NO: 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 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 235 and 258, respectively. IgG4 Fc fusion protein

[0157] In one aspect, the present invention provides an improvement to a multi-subunit protein. 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 that binds to the first antibody Fc domain polypeptide, wherein the first polypeptide further comprises a first subunit of the multi-subunit protein, the first subunit being 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; a 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 bind to each other; and the first antibody Fc domain polypeptide and the second antibody IgG4 Fc domain polypeptide each contain different mutations that promote heterodimerization.

[0158] In some embodiments, within the heterodimeric IgG4 Fc fusion protein, the linker that connects the first subunit of the multi-subunit protein to the Fc domain polypeptide of the first antibody consists of the amino acid sequence RVESKYGPPCPPCPAPEFXGG, where X represents L or E (SEQ ID NO:1).

[0159] In certain embodiments, the linker that connects the protein sequence of the first subunit of the multi-subunit protein to the Fc domain polypeptide of the first antibody further comprises a spacer peptide. In certain embodiments, the linker comprises the sequence of SEQ ID NO:1 and a spacer peptide.

[0160] In certain embodiments, the second distinct subunit of the multi-subunit protein is fused to the Fc domain polypeptide of the second antibody via a linker that comprises the sequence RVESKYGPPCPPCPAPEFXGG, where X represents L or E (SEQ ID NO:1) and a spacer peptide. In certain embodiments, the second distinct subunit of the multi-subunit protein is fused to the Fc domain polypeptide of the second antibody via a linker that consists of the amino acid sequence RVESKYGPPCPPCPAPEFXGG, where X represents L or E (SEQ ID NO:1). In certain embodiments, the amino acid sequence of the linker that connects the second distinct subunit of the multi-subunit protein to the Fc domain polypeptide of the second antibody is the same as the amino acid sequence of the linker that connects the subunit of the multi-subunit protein to the Fc domain polypeptide of the first antibody.

[0161] Any spacer peptide described under the heading "Spacer Peptide" can be employed. 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 that connects the subunit of the multi-subunit protein to the Fc domain polypeptide of the first antibody consists of, or consists essentially of, the spacer peptide disclosed herein and SEQ ID NO:1. In certain embodiments, the linker consists of, or consists essentially of, the 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.

[0162] 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).

[0163] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPAPEFLGG (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 that consists of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 3).

[0164] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGS RVESKYGPPCPPCPAPEFLGG (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 that consists of the amino acid sequence GGGGS RVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13).

[0165] In some embodiments, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that 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 that consists of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 14).

[0166] 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 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).

[0167] In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5). In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that consists of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5).

[0168] In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGS RVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63). In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that consists of the amino acid sequence GGGGS RVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63).

[0169] In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that comprises the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64). In some embodiments, the second distinct subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a linker that consists of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64).

[0170] In certain embodiments, the Fc domain polypeptide is the Fc domain polypeptide of IgG4 Fc. IgG4 is an unstable dimer that can undergo Fab-arm exchange and pairing with other IgG4 antibodies in vivo. In certain embodiments, the S228P mutation is introduced into the hinge (which in the present invention is the portion or all of the linker connecting the first subunit of the multi-subunit protein to the Fc domain polypeptide of the first IgG4 antibody or the linker connecting the additional subunit to the Fc domain polypeptide of the second different IgG4 antibody), which increases the stability of the hinge region and reduces the chance of Fab-arm exchange. In certain embodiments, additional disulfide bonds are introduced between the Fc domain polypeptide monomers, which improves the stability of the heterodimer. In one exemplary embodiment, the first antibody Fc domain polypeptide linked to the first subunit of the multi-subunit protein includes a Y349C substitution in the CH3 domain, which forms a disulfide bond with an S354C substitution on the second antibody Fc domain polypeptide of the second different subunit linked to the multi-subunit protein. Alternatively, the first antibody Fc domain polypeptide linked to the first subunit of the multi-subunit protein includes an S354C substitution in the CH3 domain, which forms a disulfide bond with a Y349C substitution on the second antibody Fc domain polypeptide of the second different subunit linked to the multi-subunit protein.

[0171] In some embodiments, the protein of the present invention comprises a first antibody Fc domain polypeptide and a second antibody Fc domain polypeptide, both of which are mutant IgG4 Fc domain polypeptides that promote heterodimerization with each other.

[0172] 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 the mutations K370E and R409W, and the second antibody IgG4 Fc domain polypeptide comprises the mutations E357N, D399V, and F405T. In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises the mutations E357N, D399V, and F405T, and the second antibody IgG4 Fc domain polypeptide comprises the mutations K370E and R409W. In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises the mutations K360E and R409W, and the second antibody IgG4 Fc domain polypeptide comprises the mutations Q347R, D399V, and F405T. In some embodiments, the first antibody IgG4 Fc domain polypeptide comprises the mutations Q347R, D399V, and F405T, and the second antibody IgG4 Fc domain polypeptide comprises the mutations K360E and R409W.

[0173] In some embodiments, the heterodimeric Fc fusion protein of the invention having an IgG4 Fc comprises 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 proteins of the present disclosure include the SPLE (S228P and L235E) mutation, the SPLEPA (S228P, L235E, and P329A) mutation, or the SPLEPG (S228P, L235E, and P329G) mutation.

[0174] 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 is part or all of the linker that connects the first subunit of the multi-subunit protein to the first IgG4 antibody Fc domain polypeptide or connects the second different subunit of the multi-subunit protein to the second different IgG4 antibody Fc domain polypeptide in the present invention. 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 NO: 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 NO: 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. Disulfide bond

[0175] Some heterodimeric Fc fusion proteins of the present invention include native heterodimeric disulfide bonds between the first subunit of a multi-subunit protein and the second different subunit of the multi-subunit protein. For example, in one exemplary embodiment, the heterodimeric Fc fusion protein according to the present invention includes the native heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. Such a protein includes the native disulfide bond between C74 of p35 and C177 of p40.

[0176] Some heterodimeric Fc fusion proteins of the present invention include artificial or engineered heterodimeric disulfide bonds between the first subunit of a multi-subunit protein and the second different subunit of the multi-subunit protein. For example, in one exemplary embodiment, the heterodimeric Fc fusion protein according to the present invention includes the artificial or engineered heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. Such a protein includes the artificial or engineered disulfide bond between V185C of p35 and Y292C of p40.

[0177] Some heterodimeric Fc fusion proteins of the invention include natural heterodimeric disulfide bonds between a first subunit of a multi-subunit protein and a second different subunit of the multi-subunit protein, as well as artificial or engineered heterodimeric disulfide bonds between a first subunit of the multi-subunit protein and a second different subunit of the multi-subunit protein. For example, in an exemplary embodiment, natural heterodimeric disulfide bonds between the p35 and p40 subunits of IL-12 are included, and artificial or engineered heterodimeric disulfide bonds between the p35 and p40 subunits of IL-12 are included. Such a 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.

[0178] Some heterodimeric Fc fusion proteins of the invention are engineered to remove natural disulfide bonds and then replace them with non-natural artificial or engineered disulfide bonds. For example, in an exemplary embodiment, a heterodimeric Fc fusion protein according to the invention includes a p35 of IL-12 in which native C74 has been mutated to serine, and a p40 of IL-12 in which native C177 has been mutated to serine, thereby removing the natural disulfide bond between the p35 and p40 subunits of IL-12. Two new mutations, namely V185C on p35 and Y292C on p40, are introduced into this mutated IL-12, thereby introducing non-natural artificial or engineered disulfide bonds. Sequences of components of the Fc fusion polypeptide

[0179] Exemplary heterodimeric Fc fusion proteins of the invention are constructed using any one of the IgG1 or IgG4 Fc variant sequences described in Table 1-2 below and any one of the corresponding linker sequences. The fusion protein constructs of the invention can confer a higher serum half-life compared to the native / natural multi-subunit protein, improve protein yield during production, enhance stability during storage, and / or improve efficacy when used as a therapeutic agent.

[0180] Tables 4 and 5 list the amino acid sequences of exemplary protein constructs of the invention. All mutations / substitutions in the Fc domain polypeptide 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.

[0181] 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. Table 1: Linker variants Table 2: IgG4 Fc and IgG1 Fc wild-type sequences; and exemplary IgG4 antibody Fc variants and IgG1 antibody Fc variant sequences (amino acid substitutions are indicated in bold) * The amino acid sequence may also contain lysine (K) at the C-terminus. Table 3: IgG4 hinge-Fc with S228P mutation (wild-type); exemplary IgG4 hinge-Fc variants or hinge region-Fc variants with S228P mutation; IgG1 hinge-Fc with C220S mutation (wild-type); exemplary IgG1 hinge-Fc variants or hinge region-Fc variants with C220S mutation * The amino acid sequence may also contain lysine (K) at the C-terminus. Table 4: Exemplary heterodimeric Fc fusion polypeptide constructs * The amino acid sequence may also include lysine (K) at the C-terminus. Table 5: Exemplary dimeric Fc fusion proteins * The amino acid sequence may also include lysine (K) at the C-terminus. IL-12 subunit

[0182] 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 number 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 number NP_000873.2, shown in SEQ ID NO:128 below. The numbering of 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, the 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.

[0183] 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 NO: 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.

[0184] 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 bind to each other, wherein the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain different mutations that promote heterodimerization, wherein the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide bind 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 NO: 125 and 126, respectively.

[0185] 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, the sequences of which 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.

[0186] In certain embodiments, the p40 subunit of IL-12 further comprises a substitution at C177, and the p35 subunit of IL-12 further comprises a substitution at C74. In certain embodiments, C177 in the p40 subunit of IL-12 is replaced with S, and C74 in the p35 subunit of IL-12 is replaced with S. In certain embodiments, the p40 and p35 subunits of IL-12 comprise the amino acid sequences of SEQ ID NO:123 and 124, respectively.

[0187] The first and second different subunits of IL-12 can be fused to any antibody Fc domain polypeptide via any linker disclosed herein to form an Fc fusion protein, the sequences of which 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. Table 6: Amino acid sequences of human IL-12p40 and p35

[0188] 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.

[0189] In certain embodiments, the first polypeptide sequence comprises a first antibody Fc domain polypeptide (human IgG1) sequence that comprises K360E and K409W substitutions. In certain embodiments, the second polypeptide sequence comprises a second antibody Fc domain polypeptide (human IgG1) sequence that comprises 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 heterodimeric Fc fusion protein of the present invention comprises the LALAPA (L234A, L235A, and P329A) mutation.

[0190] In certain embodiments, in the first polypeptide (SEQ ID NO:290) of the heterodimeric Fc fusion protein of the present invention, 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 (SEQ ID NO:291) of the heterodimeric Fc fusion protein of the present invention, 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.

[0191] 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.

[0192] 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.

[0193] The first polypeptide and the second polypeptide represented by the amino acid sequences 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 as 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 as SEQ ID NO:291, which confers stability to the heterodimeric Fc fusion protein (Fc numbering according to the EU system).

[0194] To promote heterodimerization between the two Fc domain polypeptides of the heterodimeric Fc fusion protein, the first antibody Fc domain polypeptide sequence (human IgG1) shown as SEQ ID NO: 290 includes K360E and K409W substitutions in the CH3 domain, and the second different Fc domain polypeptide sequence (human IgG1) shown as SEQ ID NO: 291 includes Q347R, D399V, and F405T substitutions in the CH3 domain (Fc numbering according to the EU system).

[0195] The first antibody Fc domain polypeptide sequence and the second different Fc domain polypeptide sequence (human IgG1) shown as SEQ ID NO: 290 and SEQ ID NO: 291 also include L234A, L235A, and P329A (LALAPA) mutations for reducing effector function. Spacer peptide

[0196] Exemplary spacer peptide sequences are provided in Table 7, and exemplary full-length linker sequences are provided in Tables 4 and 5.

[0197] Within the first polypeptide of the present invention, in the amino to carboxyl direction, the first subunit of the multi-subunit protein is fused via a linker 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 within the second polypeptide of the present invention, in the amino to carboxyl direction, the second different subunit of the multi-subunit protein is fused via a linker 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).

[0198] In some embodiments, the first subunit of the multi-subunit protein of the present invention is fused via a linker to a first antibody Fc domain sequence, wherein the linker comprises or consists of the amino acid sequence of spacer peptide L1 and SEQ ID NO: 1, 2, 4, 6, 7, 9, 11, 237, 238, 239, or 240. In some embodiments, the second different subunit of the multi-subunit protein is fused via a linker to a second antibody Fc domain polypeptide, wherein the linker comprises or consists of the amino acid sequence of spacer peptide L2 and SEQ ID NO: 1, 2, 4, 6, 7, 9, 11, 237, 238, 239, or 240.

[0199] In certain embodiments, L1 and L2 are peptide linkers. For example, L1 and / or L2 comprise 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.

[0200] In some embodiments, L1 and L2 have an 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.

[0201] In certain embodiments, L1 has the same number of amino acids as L2; in certain embodiments, L1 is longer than L2 (i.e., has a greater number of amino acids); in certain embodiments, L1 is shorter than L2 (i.e., has a smaller number of amino acids).

[0202] In certain embodiments, L1 and / or L2 are "short", e.g., consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in certain 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", e.g., 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) contiguous amino acid residues. With respect to the amino acid composition of L1 and L2, peptides are selected that impart flexibility to the first and second polypeptides of the protein of the invention, do not interfere with the binding of the first subunit and the second different subunit to each other, and resist protease cleavage. For example, glycine and serine residues generally confer protease resistance. As part of the linker, a spacer peptide suitable for linking 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 linking 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, where 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 comprise the (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 comprise a peptide sequence shown as selected from the following sequences as part of the linker: 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 is (GGGGS) n＝3 (SEQ ID NO:108), (GGGGS) n＝2 (SEQ ID NO:109) or (GGGGS) n＝1 (SEQ ID NO:110). Table 7

[0203] In certain embodiments, L1 comprises the sequence (GGGGS) n＝3 (SEQ ID NO:108) sequence as part of the linker, and L2 comprises (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 comprises (GGGGS) n＝2 (SEQ ID NO:109) or (GGGGS) n＝1(SEQ ID NO:110) The sequence is part of the linker. In certain embodiments, as part of the linker, L1 does not include 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.

[0204] 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.

[0205] Some heterodimeric Fc fusion proteins of the invention comprise a first polypeptide comprising 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) links the first subunit of the multi-subunit protein to 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 invention comprise a second polypeptide comprising a second different subunit of a multi-subunit protein and a second antibody Fc domain polypeptide, wherein a linker comprising GGGGSGGGGSGGGGS (SEQ ID NO:118; (G4S)3) links the second different subunit of the multi-subunit protein to 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.

[0206] In certain embodiments, some heterodimeric Fc fusion proteins of the invention comprise a first polypeptide comprising 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) links the first subunit of the multi-subunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second different subunit of a multi-subunit protein and a second antibody Fc domain polypeptide, wherein the additional subunit is linked to the second antibody Fc domain polypeptide by a linker that does not comprise GGGGSGGGGSGGGGS (SEQ ID NO:118; (G4S)3).

[0207] In certain embodiments, some heterodimeric Fc fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker that does not comprise GGGGSGGGGSGGGGS (SEQ ID NO:118; (G4S)3) links the first subunit of the multi-subunit protein to the Fc domain polypeptide; and a second polypeptide comprising a second different subunit of a multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second different subunit of the multi-subunit protein is linked to the second antibody Fc domain polypeptide by a linker comprising GGGGSGGGGSGGGGS (SEQ ID NO:118; (G4S)3).

[0208] Some heterodimeric Fc fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) is included n＝2The linker of (SEQ ID NO:109) links the first subunit of the multi-subunit protein to the Fc domain polypeptide of the first antibody, such as the Fc domain polypeptide of an IgG4 antibody or the Fc domain polypeptide of an IgG1 antibody. Some of the 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) is included. n＝2 The linker of (SEQ ID NO:109) links the second different subunit of the multi-subunit protein to the Fc domain polypeptide of the second antibody, such as the Fc domain polypeptide of an IgG4 antibody or the Fc domain polypeptide of an IgG1 antibody.

[0209] Some of the heterodimeric Fc fusion proteins of the present disclosure comprise a linker of (GGGGS) n＝2 (SEQ ID NO:109) that links the first subunit of the multi-subunit protein to the Fc domain polypeptide of the first antibody, such as the Fc domain polypeptide of an IgG4 antibody or the Fc domain polypeptide of an IgG1 antibody; and links the second different subunit of the multi-subunit protein to the Fc domain polypeptide of the second antibody, such as the Fc domain polypeptide of an IgG4 antibody or the Fc domain polypeptide of an IgG1 antibody.

[0210] In certain embodiments, some of the heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of the multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker of (GGGGS) n＝2 (SEQ ID NO:109) links the first subunit of the multi-subunit protein to 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 additional subunits of the multi-subunit protein are linked to the second antibody Fc domain polypeptide with a linker that does not include (GGGGS) n＝2 (SEQ ID NO:109).

[0211] In certain embodiments, some of the heterodimeric Fc fusion proteins of the present invention comprise a first polypeptide comprising a first subunit of the multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker that does not include (GGGGS) n＝2 (SEQ ID NO:109) links the first subunit of the multi-subunit protein to 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 linked to the second antibody Fc domain polypeptide with a linker that includes (GGGGS) n＝2 (SEQ ID NO:109).

[0212] 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 a linker comprising (GGGGS) n＝1 (SEQ ID NO:110) links the first subunit of the multi-subunit protein to 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 a linker comprising (GGGGS) n＝1 (SEQ ID NO:110) links the second different subunit of the multi-subunit protein to 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.

[0213] Some heterodimeric Fc fusion proteins of the present disclosure comprise a linker comprising (GGGGS) n＝1 (SEQ ID NO:110) that links a first subunit of a multi-subunit protein to a first antibody Fc domain polypeptide, such as the Fc domain polypeptide of an IgG4 antibody or the Fc domain polypeptide of an IgG1 antibody; and links a second different subunit of the multi-subunit protein to a second antibody Fc domain polypeptide, such as the Fc domain polypeptide of an IgG4 antibody or the Fc domain polypeptide of an IgG1 antibody.

[0214] 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 a linker comprising (GGGGS) n＝1 (SEQ ID NO:110) links the first subunit of the multi-subunit protein to 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 linked to the second antibody Fc domain polypeptide by a linker that does not comprise (GGGGS) n＝1 (SEQ ID NO:110).

[0215] 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 a linker that does not comprise (GGGGS) n＝1 (SEQ ID NO:110) links the first subunit of the multi-subunit protein to 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 linked to the second antibody Fc domain polypeptide by a linker that comprises (GGGGS) n＝1The linker of (SEQ ID NO:110) is linked to the Fc domain polypeptide of the second antibody.

[0216] In certain embodiments, some of the heterodimeric Fc fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker comprising (GGGGS) n＝1 (SEQ ID NO:110) links the first subunit of the multi-subunit protein to 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 linked to the Fc domain polypeptide of the second antibody by a linker comprising (GGGGS) n＝2 (SEQ ID NO:109).

[0217] In certain embodiments, some of the heterodimeric Fc fusion proteins of the invention comprise a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein a linker comprising (GGGGS) n＝2 (SEQ ID NO:109) links the first subunit of the multi-subunit protein to 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 linked to the Fc domain polypeptide of the second antibody by a linker comprising (GGGGS) n＝1 (SEQ ID NO:110). Fc domains and substitutions for promoting heterodimerization

[0218] The assembly of the proteins 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 bind to each other. Facilitating the preferential assembly of the Fc heterodimer can be accomplished by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region, as shown in US13 / 494870, US16 / 028850, US11 / 533709, US12 / 875015, US13 / 289934, US14 / 773418, US12 / 811207, US13 / 866756, US14 / 647480, and US14 / 830336. For example, mutations can be made in the CH3 domain based on human IgG1, and different pairs of amino acid substitutions can be incorporated within 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 numbered according to the EU index (as in Kabat).

[0219] In one scenario, 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), such 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 the other can incorporate three substitutions, including T366S, L368A, and Y407V.

[0220] The first polypeptide comprising the first subunit of a multi-subunit protein sequence or the second polypeptide comprising a second, different subunit of a multi-subunit protein sequence may optionally be conjugated 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 (such as an IgG constant region including 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 (such as a human IgG1 constant region, IgG2 constant region, IgG3 constant region, or 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 may be incorporated into the constant region compared to the 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, for example, 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, T366S, 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.

[0221] In certain embodiments, mutations that can be incorporated into CH1 of the human IgG1 constant region can be at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that can be incorporated into CK of the human IgG1 constant region can be at amino acids E123, F116, S176, V163, S174, and / or T164.

[0222] The amino acid substitutions can be selected from the following groups of substitutions shown in Table 8.

[0223] Alternatively, the amino acid substitutions can be selected from the following groups of substitutions shown in Table 9.

[0224] Alternatively, the amino acid substitutions can be selected from the following groups of substitutions shown in Table 10.

[0225] Alternatively, at least one amino acid substitution in each polypeptide chain can be selected from Table 11.

[0226] Alternatively, at least one amino acid substitution can be selected from the following groups in Table 12, wherein one or more positions indicated in the first polypeptide column are replaced with any known negatively charged amino acid, and one or more positions indicated in the second polypeptide column are replaced with any known positively charged amino acid.

[0227] Alternatively, at least one amino acid substitution can be selected from the following groups in Table 13, wherein one or more positions indicated in the first polypeptide column are replaced with any known positively charged amino acid, and one or more positions indicated in the second polypeptide column are replaced with any known negatively charged amino acid.

[0228] Alternatively, the amino acid substitutions can be selected from the following groups shown in Table 14.

[0229] 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 the second polypeptide chain and Y349C on the opposite polypeptide chain, which forms an artificial disulfide bond within the interface of the two polypeptides.

[0230] 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.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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 an S354C 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 a Y349C substitution.

[0243] 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 Y349C 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 an S354C substitution.

[0244] 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.

[0245] 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.

[0246] 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.

[0247] 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 a T366W substitution.

[0248] 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.

[0249] 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.

[0250] Those skilled in the art will understand that during the production and / or storage of a protein, the N-terminal glutamate (E) or glutamine (Q) can be cyclized to form a lactam (e.g., spontaneously or through enzymatic catalysis by enzymes present during production and / or storage). Thus, in some embodiments, where the N-terminal residue of the amino acid sequence of a polypeptide is E or Q, the corresponding amino acid sequences with pyroglutamic acid replacing E or Q are also contemplated herein.

[0251] Those skilled in the art will also understand that during protein production and / or storage, the C-terminal lysine (K) of a protein can be removed (e.g., spontaneously or through enzymatic catalysis by enzymes present during production and / or storage). This removal of K is typically observed in the case of proteins that contain an Fc domain at their C-terminus. Thus, in some embodiments, where the C-terminal residue of the amino acid sequence of a polypeptide (e.g., Fc domain sequence) is K, the corresponding amino acid sequences with K removed are also contemplated herein. Mutations for reducing effector function

[0252] In one aspect, the present invention provides a heterodimeric Fc fusion protein comprising (a) a first polypeptide comprising a first antibody Fc domain polypeptide and a first subunit of a multi-subunit protein; and (b) 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 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 the Fc, and wherein the first subunit and the second different subunit of the multi-subunit protein bind to each other. In certain embodiments, the heterodimeric Fc fusion protein comprising one or more mutations that reduce the effector function of the Fc disclosed herein has increased activity in inhibiting tumor growth compared to its counterpart without the one or more Fc mutations that reduce effector function. Mutations contemplated 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.

[0253] 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 cell cytotoxicity (ADCC) and / or antibody-dependent cell phagocytosis (ADCP). ADCC and ADCP are typically 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. Fc receptors for human IgG (also referred to as Fcγ receptors (FcγR)) include, but are not limited to, the activating Fcγ receptors FcγRI (CD64), FcγRIIA (CD32A), FcγRIIIA (CD16 or CD16A), and FcγRIIIB (CD16B), and the inhibitory Fcγ receptor FcγRIIB (CD32B). Thus, in some embodiments, the heterodimeric Fc fusion protein of the present invention comprises one or more mutations to reduce binding to activating FcγRs (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 comprises one or more mutations to increase binding to inhibitory FcγRs (e.g., FcγRIIB) in the first polypeptide and / or the second polypeptide.

[0254] Fc mutations that reduce binding to activating FcγRs and / or increase binding to inhibitory FcγRs are known in the art. For example, within the hinge region and the Fc region, CD16 binding is mediated by the hinge region and the CH2 domain. For example, within 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 in the CH2 domain and the carbohydrate residue N - acetyl - D - glucosamine (see, Sondermann et al., Nature, 406(6793):267 - 273). Based on the known domains, mutations can be selected to enhance or reduce the binding affinity to CD16, such as by using phage display libraries or yeast surface display cDNA libraries, or can be designed based on the known three - dimensional structure of the interaction.

[0255] As reviewed by Want et al., Protein Cell (2018) 9(1):63 - 73, regions including amino acid positions 232 - 239, 265 - 270, 296 - 299, and 325 - 332 of the human IgG1 Fc are involved in activating FcγR binding. Wang et al. also disclosed that the L235E and F234A / L235A mutations of human IgG4, the L234A / L235A mutation of human IgG1, and the N297 mutation of IgG antibodies (e.g., N297A, N297Q, N297G, or N297D) reduce activating FcγR binding. As disclosed in U.S. Patent 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 (e.g., the E233P mutation) are disclosed in U.S. Patent No. 7,943,743 and Isaacs et al., J. Immunol. (1998) 161:3862 - 69.

[0256] Thus, in certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide comprise mutations (e.g., substitutions relative to wild-type human IgG1) at one or more positions selected from 233, 234, 235, 297, and 329. In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG1 antibody Fc domain polypeptides that comprise one or more of the 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 that comprise the 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 that comprise the 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 that comprise the mutation L235E. In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are human IgG1 antibody Fc domain polypeptides that comprise the mutations L235E and P329A.

[0257] 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). Thus, in certain embodiments, the heterodimeric Fc fusion protein of the invention comprises one or more mutations to reduce binding to complement components (e.g., C1q) in the first polypeptide and / or the second polypeptide.

[0258] Fc mutations that reduce binding to C1q are known in the art. For example, as disclosed in U.S. Patent 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, the 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 (e.g., D270A), 322 (K322A), 329 (e.g., P329A), and 331 (e.g., P331A, P331S, or P331G) of Fc reduce C1q binding.

[0259] Accordingly, in certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide comprise 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 that comprise one or more of the 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 that comprise the 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 that comprise the mutation P329A.

[0260] 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.

[0261] In some embodiments, the heterodimeric Fc fusion protein of the invention having an IgG4 Fc comprises 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 proteins of the present disclosure can comprise an SPLE (S228P and L235E) mutation, an SPLEPA (S228P, L235E, and P329A) mutation, or an SPLEPG (S228P, L235E, and P329G) mutation.

[0262] In some embodiments, the heterodimeric Fc fusion protein of the invention having an IgG1 Fc comprises 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 proteins of the present disclosure can comprise an LALA (L234A and L235A) mutation, an LALAPA (L234A, L235A, and P329A) mutation, an LALAPG (L234A, L235A, and P329G) mutation, or an LALEGAASPS (L234A, L235E, G237A, A330S, and P331S) mutation.

[0263] In some embodiments, the heterodimeric Fc fusion protein according to the invention comprises a first antibody IgG4 or IgG1 Fc domain polypeptide and a second antibody IgG4 or IgG1 Fc domain polypeptide, each containing the mutation P329G or P329A.

[0264] 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.

[0265] In some embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain the mutations A330S and P331S.

[0266] In certain embodiments, in the first polypeptide of the heterodimeric Fc fusion protein of the invention, the first subunit of the multi-subunit protein is fused to the first antibody Fc domain polypeptide via a first linker. In certain embodiments, in the second polypeptide of the heterodimeric Fc fusion protein of the invention, the second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide via a second linker. Amino acid sequences of linkers suitable for such use are described under the headings "IgG4 constructs" and "IgG1 constructs". Additional 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 (where X is L or E) (SEQ ID NO: 280) or SKYGPPCPPCPAPEFXGG (where 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). Serum half-life

[0267] The heterodimeric Fc fusion protein according to the invention has pharmacokinetic properties suitable for therapeutic use. For example, in certain embodiments, the heterodimeric Fc fusion protein according to the invention has a serum half-life of at least about 50 hours. In certain embodiments, the heterodimeric Fc fusion protein according to the invention has a serum half-life of at least about 100 hours.

[0268] In certain embodiments, 50 hours after intravenous administration to a subject, the serum concentration of the heterodimeric Fc fusion protein according to the invention is at least 10% of the serum concentration of the protein of the invention in the subject 1 hour after administration.

[0269] In certain embodiments, the heterodimeric Fc fusion protein according to the 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 to the Fc domain polypeptide. In certain embodiments, the heterodimeric Fc fusion protein comprising the protein sequence of the multi-subunit protein according to the invention has a serum half-life that is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold or 20-fold longer than the multi-subunit protein not fused to the Fc domain polypeptide. Tumor retention

[0270] The heterodimeric Fc fusion protein of the invention can optionally incorporate additional features to enhance the retention of the protein at the tumor site. For example, in certain embodiments of the invention, the heterodimeric Fc fusion protein further comprises a proteoglycan binding domain, a collagen binding domain, and / or a hyaluronic acid binding domain. In certain embodiments, the heterodimeric Fc fusion protein further comprises a proteoglycan binding domain that binds one or more proteoglycans (e.g., proteoglycans known in the art, such as those disclosed in Lozzo et al., Matrix Bio (2015) 42:11-55; and Nikitovic et al., Frontiers in Endocrinology (2018) 9:69), which are present in tumors (e.g., on the surface of tumor cells, in the pericellular matrix of the tumor, or in the extracellular matrix of the tumor). In certain embodiments, the collagen binding domain binds one or more collagens that are present in tumors (e.g., on the surface of tumor cells, in the pericellular matrix of the tumor, or in the extracellular matrix of the tumor). In certain embodiments, the heterodimeric Fc fusion protein further comprises an h acid binding domain that binds to one or more hyaluronic acids present in tumors. Such heterodimeric Fc fusion proteins have enhanced retention in tumors and can be administered intratumorally to a subject at a lower dose and / or frequency.

[0271] In certain embodiments, the proteoglycan-binding domain contained in the heterodimeric Fc fusion protein binds to one or more proteoglycans that are specifically expressed in a tumor (e.g., on the surface of tumor cells, in the pericellular matrix of the tumor, or in the extracellular matrix of the tumor). In certain embodiments, the collagen-binding domain contained in the heterodimeric Fc fusion protein binds to one or more collagens that are specifically expressed in a tumor (e.g., on the surface of tumor cells, in the pericellular matrix of the tumor, or in the extracellular matrix of the tumor). Such heterodimeric Fc fusion proteins can be enriched in tumors following administration (e.g., intravenous, subcutaneous, or pulmonary administration) and have enhanced tumor retention, thereby allowing administration at lower doses and / or frequencies.

[0272] In certain embodiments, the heterodimeric Fc fusion protein of the invention further comprises a proteoglycan-binding domain that binds to one or more proteoglycans selected from: syndecan, chondroitin sulfate proteoglycan 4 (CSPG4), betaglycan, phosphacan, glypican, versican, brevican, neurocan, perlecan, aggrecan, biglycan, decorin, fibromodulin, lumican, and small leucine-rich proteoglycan (SLRP). Proteoglycans involved 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), versican, biglycan, brevican, and SLPR (e.g., decorin, biglycan, asporin, fibromodulin, and lumican). Thus, in certain embodiments, the proteoglycan-binding domain contained in the heterodimeric Fc fusion protein binds to one or more proteoglycans selected from: syndecan (e.g., syndecan-1 or syndecan-2), serglycin, CSPG4, betaglycan, glypican (e.g., glypican-1 or glypican-3), versican, biglycan, brevican, and SLPR. In certain embodiments, the proteoglycan-binding domain contained in the heterodimeric Fc fusion protein binds to one or more SLPR selected from: decorin, biglycan, asporin, fibromodulin, and lumican.

[0273] 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 part 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 syndecan-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 betaglycan-binding domain is disclosed in U.S. Patent No. 7,455,839. The glypican-binding domain is 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 2018165344A1, and WO 2018199318 A1. The versican-binding domain is disclosed in U.S. Patent No. 10,166,304. The decorin-binding domain is disclosed in U.S. Patent No. 6,517,838 and PCT Patent Application Publication Nos. WO 2000021989 A1, WO 2000077041 A2, and WO2000078800A2.

[0274] In certain embodiments, the heterodimeric Fc fusion protein of the invention further comprises a collagen-binding domain. Collagen is a class of proteins identified in vertebrates that includes at least 28 different types. Each type of collagen has its unique structural features and distribution patterns, as disclosed in Fang et al., Tumor Biol. (2014) 35:2871-82 and Xiong et al., J. Cancer Metasta. Treat. (2016) 2:357-64. Multiple 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 an antigen-binding fragment thereof), a peptide (e.g., a portion 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. Patent 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, as well as U.S. Patent Application Publication Nos. 2007 / 0161062, 2009 / 0142345, and 2012 / 0100106.

[0275] In certain embodiments, the heterodimeric Fc fusion protein of the invention further comprises a hyaluronic acid-binding domain. The hyaluronic acid-binding domain can be a protein (e.g., an antibody or an antigen-binding fragment thereof), a peptide (e.g., a portion of a hyaluronic acid-binding protein or a variant thereof), an aptamer, a small molecule, or a combination thereof. Hyaluronic acid-binding domains are known in the art and are disclosed, for example, in U.S. Patent Nos. 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, as well as U.S. Patent Application Publication Nos. 2002 / 0055488 and 2007 / 0259380.

[0276] The proteoglycan-binding domain, collagen-binding domain, and / or hyaluronic acid-binding domain (if present) can be at any position of the heterodimeric Fc fusion protein. For example, in certain embodiments, where the IL-12 subunit is 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 to the C-terminus of the first antibody Fc domain polypeptide and / or to the C-terminus of the second antibody Fc domain polypeptide. In certain embodiments, where the IL-12 subunit is 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 to the N-terminus of the first antibody Fc domain polypeptide and / or to the N-terminus of the second antibody Fc domain polypeptide.

[0277] The proteoglycan-binding domain, collagen-binding domain, and / or hyaluronic acid-binding domain (if present) can be fused to the remainder of the heterodimeric Fc fusion protein via a linker. In certain embodiments, the proteoglycan-binding domain is fused to the remainder of the heterodimeric Fc fusion protein via a peptide linker. In certain embodiments, the peptide linker comprises a spacer peptide as disclosed herein. II. Preparation Method

[0278] The proteins 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, the first polypeptide comprising 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, the second polypeptide comprising a second different subunit of the multi-subunit fused to a second antibody Fc domain polypeptide; and the first expression vector and the second expression vector can be stably co-transfected into a host cell to produce the multimeric protein.

[0279] To achieve the highest yield of the protein, different ratios of the first expression vector and the second expression vector can be explored to determine the optimal ratio for transfection into the host cell. After transfection, single clones can be isolated using methods known in the art (such as limiting dilution, ELISA, FACS, microscopy, or Clonepix) to generate a cell bank.

[0280] Clones can be cultured under conditions suitable for bioreactor scale-up and maintenance expression of the proteins of the invention. The proteins 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. III. Pharmaceutical Compositions

[0281] The present 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 proper formulation. Suitable formulations for use in the present disclosure can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of drug delivery methods, see, e.g., Langer (Science 249:1527-1533, 1990).

[0282] The intravenous drug delivery formulations of the present disclosure can be contained in bags, pens, or syringes. In certain embodiments, the bags can be connected to a passageway including a tube and / or a needle. In certain embodiments, the formulations can be lyophilized formulations or liquid formulations. In certain embodiments, the formulations can be lyophilized (freeze-dried) and contained in about 12-60 vials. In certain embodiments, the formulations can be lyophilized, and 45 mg of the lyophilized formulation can be contained in one vial. In certain embodiments, about 40 mg - about 100 mg of the lyophilized formulation can be contained in one vial. In certain embodiments, the lyophilized formulations from 12, 27, or 45 vials are combined to obtain a therapeutic dose of the protein in the intravenous drug formulation. In certain embodiments, the formulations can be liquid formulations and stored at about 250 mg / vial to about 1000 mg / vial. In certain embodiments, the formulations can be liquid formulations and stored at about 600 mg / vial. In certain embodiments, the formulations can be liquid formulations and stored at about 250 mg / vial.

[0283] The heterodimeric Fc fusion proteins of the invention can be present in a liquid aqueous drug formulation comprising an effective amount of the protein in a buffer solution, thereby forming a formulation.

[0284] These compositions can be sterilized by conventional sterilization techniques or can be sterile filtered. The resulting aqueous solution can be packaged and used as is, or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to 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 of the composition can be packaged in multiple single-dose units, each single-dose unit containing a fixed amount of one or more of the above agents. The solid form of the composition can also be packaged in a container for flexible dosing.

[0285] In certain embodiments, the present disclosure provides a formulation having an extended shelf life, comprising a combination of the heterodimeric Fc fusion protein of the present disclosure with mannitol, citric acid monohydrate, sodium citrate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide.

[0286] In certain embodiments, an aqueous formulation is prepared that comprises the heterodimeric Fc fusion protein of the present disclosure in a pH buffer 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 intermediate to the above pH values are also intended to be part of the present disclosure. For example, ranges of values using combinations of any of the above values as upper and / or lower limits are intended to be included. Examples of buffers that control the pH within this range include acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffers.

[0287] In certain embodiments, the formulation comprises a buffer system containing citrate and phosphate to maintain the pH within a 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 pH 4.8 to about 5.5, or within a pH range of about 5.0 to about 5.2. In certain embodiments, the buffer system comprises citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3 mg / mL of citric acid (e.g., 1.305 mg / mL), about 0.3 mg / mL of sodium citrate (e.g., 0.305 mg / mL), about 1.5 mg / mL of disodium phosphate dihydrate (e.g., 1.53 mg / mL), about 0.9 mg / mL of sodium dihydrogen phosphate dihydrate (e.g., 0.86), and about 6.2 mg / mL of sodium chloride (e.g., 6.165 mg / 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.

[0288] A polyol can also be included in the formulation, and the polyol acts as a tonicity agent and can stabilize the antibody. The polyol is added to the formulation in an amount that can vary depending on the desired isotonicity of the formulation. In certain embodiments, the aqueous formulation can be isotonic. The amount of polyol added can also vary depending on the molecular weight of the polyol. For example, a lower amount of a monosaccharide (e.g., mannitol) can be added compared to a disaccharide (such as trehalose). 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.

[0289] A detergent or surfactant can also be added to the formulation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The detergent is added in an amount such that it reduces aggregation of the formulated antibody and / or minimizes 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 can contain polysorbate 80 in an amount between about 0.1 mg / mL and about 10 mg / mL or between about 0.5 mg / mL and about 5 mg / mL. In certain embodiments, about 0.1% polysorbate 80 can be added to the formulation.

[0290] 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 Type I 50R vial at a concentration of 10 mg / mL, the vial being closed with a rubber stopper and sealed with an aluminum crimp seal. The stopper can be made of an elastomer compliant with USP and Ph Eur. In certain embodiments, the vial can be filled with 61.2 mL of the protein product solution to allow for an extractable volume of 60 mL. In certain embodiments, the liquid formulation can be diluted with a 0.9% saline solution.

[0291] In certain embodiments, a combination of the liquid formulation of the present disclosure and a stabilizing level of sugar can be prepared as a solution at a concentration of 10 mg / mL. In certain embodiments, the liquid formulation can be prepared in an aqueous carrier. In certain embodiments, the stabilizer is added in an amount not greater than that which may result in an undesirably high viscosity or an unsuitability for intravenous administration. In certain embodiments, the sugar can be a disaccharide, such as sucrose. In certain embodiments, the liquid formulation can further include one or more of a buffer, a surfactant, and a preservative.

[0292] 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.

[0293] In addition to aggregation, deamidation is a common product variant of peptides and proteins, which can occur during fermentation, harvest / cell clarification, purification, drug substance / drug product storage, and during sample analysis. Deamidation is the loss of U from a protein, resulting in the formation of a succinimide intermediate that is subject to hydrolysis. The succinimide intermediate causes a 17 Dalton decrease in the mass of the parent peptide. Subsequent hydrolysis results in a 18 Dalton increase in mass. Due to its instability under aqueous conditions, it is difficult to isolate the succinimide intermediate. Therefore, deamidation is typically detectable as a 1 Dalton increase in mass. Deamidation of asparagine yields aspartic acid or isoaspartic acid. Parameters that affect the rate of deamidation 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 rate of deamidation. Gly and Ser following Asn in the protein sequence result in higher deamidation sensitivity.

[0294] In certain embodiments, the liquid formulations of the present disclosure can be stored under pH and humidity conditions that prevent deamidation of the protein product.

[0295] The 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 buffer solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solutions, or dextrose solutions.

[0296] A preservative can optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative can, for example, facilitate the production of multi-use (multi-dose) formulations.

[0297] In certain situations, such as when a patient is hospitalized after transplantation and receives all medications via the intravenous (IV) route, an IV formulation can 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.

[0298] In certain embodiments, a salt or buffer component can be added in an amount of 10 mM - 200 mM. The salt and / or buffer is pharmaceutically acceptable and is derived from various known acids (inorganic and organic) and "base-forming" metals or amines. In certain embodiments, the buffer can be a phosphate buffer. In certain embodiments, the buffer can be a glycinate, carbonate, citrate buffer, in which case sodium, potassium, or ammonium ions can be used as counterions.

[0299] A preservative can optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative can, for example, facilitate the production of multi-use (multi-dose) formulations.

[0300] 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 buffer solutions (e.g., phosphate buffered saline), sterile saline solutions, Ringer's solution, or dextrose solutions.

[0301] The proteins of the present disclosure can be present in a lyophilized formulation comprising the protein and a lyoprotectant. The lyoprotectant can be a sugar, such as a disaccharide. In certain embodiments, the lyoprotectant can be sucrose or maltose. The lyophilized formulation can also include one or more of a buffer, a surfactant, a filler, and / or a preservative.

[0302] 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.

[0303] In certain embodiments, the pH of the formulation can be set by adding a pharmaceutically acceptable acid and / or base prior to lyophilization. In certain embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base can be sodium hydroxide.

[0304] Prior to lyophilization, the pH of a solution containing the protein of the present disclosure can be adjusted between 6 and 8. In certain embodiments, the pH range of the lyophilized drug product can be from 7 to 8.

[0305] In certain embodiments, a salt or buffer component can be added in an amount of 10 mM - 200 mM. The salt and / or buffer is pharmaceutically acceptable and is derived from various known acids (inorganic and organic) and "base-forming" metals or amines. In certain embodiments, the buffer can be a phosphate buffer. In certain embodiments, the buffer can be a glycinate, carbonate, citrate buffer, in which case sodium, potassium, or ammonium ions can be used as counterions.

[0306] In certain embodiments, a "filler" can be added. A "filler" is a compound that adds mass to the lyophilization mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of a substantially uniform lyophilized cake that retains an open pore structure). Illustrative fillers include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention can contain such fillers.

[0307] A preservative can optionally be added to the formulations herein to reduce bacterial action. The addition of a preservative can, for example, facilitate the production of multi-use (multi-dose) formulations.

[0308] In certain embodiments, the lyophilized drug product can be constituted with an aqueous carrier. Aqueous carriers of interest herein are carriers that are pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) 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 solutions.

[0309] 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 a solution.

[0310] In certain embodiments, the lyophilized protein product of the present disclosure is constituted with approximately 4.5 mL of water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0311] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration and that is non-toxic to the patient.

[0312] The specific dose can be a uniform dose per patient, such as 50 - 5000 mg of protein. Alternatively, the dose for a patient can be adjusted according to the patient's approximate body weight or surface area. Other factors in determining an appropriate dose can include the disease or disorder to be treated or prevented, the severity of the disease, the route of administration, and the patient's age, sex, and physical condition. Those skilled in the art routinely further refine the calculations necessary to determine an appropriate dose for treatment, particularly in accordance with the dose information and assays disclosed herein. The dose can also be determined by using known assays for determining the dose to be used in conjunction with appropriate dose - response data. The dose of an individual patient can be adjusted while monitoring the progression of the disease. The blood level of the targetable construct or complex in the patient can be measured to see if the dose needs to be adjusted to achieve or maintain an effective concentration. Pharmacogenomics can be used to determine which targetable constructs and / or complexes and their doses 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).

[0313] Typically, the weight-based dose 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 to 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 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.

[0314] The dosage can be administered once or multiple times per day, per week, per month, or per year, or even once every 2 to 20 years. A person 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 body fluids or tissues. 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 administered once or multiple times per day, once or multiple times per week, once or multiple times per month, and once or multiple times per year. In some embodiments, the heterodimeric Fc fusion protein (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) is administered intratumorally. In some embodiments, the heterodimeric Fc fusion protein is administered intratumorally at a lower dose or frequency than when administered systemically. In some embodiments, the heterodimeric Fc fusion protein is administered intratumorally after administration of a local anesthetic. In some embodiments, the heterodimeric Fc fusion protein is administered intratumorally based on direct palpation of the tumor mass. IV. Therapeutic Applications

[0315] The present invention provides methods for treating cancer using the heterodimeric Fc fusion binding proteins described herein and / or the pharmaceutical compositions described herein. By administering an effective amount of the heterodimeric Fc fusion protein described herein to a patient in need, the methods can be used to treat a variety of cancers. In some embodiments, the cancer treated by the methods disclosed herein is locally advanced malignancy. In some embodiments, the locally advanced malignancy can be completely resected. In some embodiments, the locally advanced malignancy has been completely resected, and the treatment is provided after resection.

[0316] 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 single therapy for treating advanced malignancy. 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 prophylactic vaccination. In some embodiments, the heterodimeric Fc fusion protein of the present invention is used to treat myelosuppression after radiotherapy.

[0317] The present invention also provides methods for reducing hematotoxicity. Hematotoxicity can be caused by genetic, infectious, or environmental causes (including but not limited to irradiation and chemotherapy). For example, in certain embodiments, the present invention provides methods for treating diseases or disorders 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 methods for treating myelosuppression after radiotherapy, the methods comprising administering to a patient in need thereof an effective amount of the heterodimeric Fc fusion protein or formulation described herein. In certain embodiments, the dose of 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 methods of the present invention reduce such damage.

[0318] The methods provided herein can be used to treat myelosuppression caused by radiation. Thus, in certain embodiments, the present invention provides methods for treating myelosuppression that occurs in the case of accidental exposure to radiation, the methods comprising administering to a patient in need thereof a therapeutically effective amount of the heterodimeric Fc fusion protein or formulation described herein.

[0319] In certain embodiments, the present invention provides methods for treating acute radiation syndrome (ARS), the methods comprising administering to a patient in need thereof an effective amount of the heterodimeric Fc fusion protein or formulation described herein. ARS includes, but is not limited to, hematopoietic radiation syndrome, gastrointestinal radiation syndrome, neurovascular radiation syndrome, and cutaneous radiation syndrome. For example, hematopoietic radiation syndrome is caused at least in part by depletion of the hematopoietic stem cell pool and exhibits signs 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 signs of watery diarrhea, dehydration, electrolyte loss, gastrointestinal bleeding, and perforation. In certain embodiments, the treatment methods provided herein are carried out during 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 cutaneous erythema, typically within 1 - 3 days after exposure to radiation. In certain embodiments, the treatment methods provided herein are carried out during the latent phase of ARS. The latent phase is a stage characterized by improvement of symptoms but exhibiting lymphopenia and granulocytopenia in laboratory tests and may last from hours to weeks depending on the exposure dose. Treatment during the prodromal or latent phase may mitigate the development of the syndrome in the affected systems, organs, and / or tissues. In certain embodiments, the treatment methods provided herein are carried out during the manifest illness phase of ARS. Treatment during this phase may still facilitate recovery from ARS.

[0320] The present invention also provides methods for increasing the survival, proliferation, differentiation, and / or activity of immune cells, the methods comprising contacting the immune cells with the heterodimeric Fc fusion protein or formulation disclosed herein. In certain embodiments, the immune cells are T cells (e.g., CD4 + T cells). In certain embodiments, the immune cells are NK cells.

[0321] In certain embodiments, the heterodimeric Fc fusion proteins of the invention (e.g., heterodimeric Fc fusion proteins comprising an IL-12 subunit) are 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 heterodimeric Fc fusion protein is administered as a single therapy to a subject diagnosed with colon cancer. In certain embodiments, the cancer is melanoma. In certain embodiments, the heterodimeric Fc fusion protein is administered as a single 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 single therapy to a subject diagnosed with breast cancer.

[0322] In certain embodiments, the present disclosure provides a method of treating cancer, the method comprising administering to a patient a single dose of a heterodimeric IL-12-Fc fusion protein only. 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 recurrence of the cancer.

[0323] In certain embodiments, the present disclosure provides a method of treating cancer, the method comprising administering to a patient a single dose of a heterodimeric IL-12-Fc fusion protein, the fusion protein comprising a first polypeptide having the amino acid sequence set forth in SEQ ID NO:290 and a second polypeptide having the amino acid sequence set forth in SEQ ID NO:291; or a formulation comprising the heterodimeric IL-12-Fc fusion protein and a pharmaceutically acceptable carrier.

[0324] In certain embodiments, a single dose amount of a heterodimeric IL-12-Fc fusion protein comprising a first polypeptide having the amino acid sequence of SEQ ID NO:290 and a second polypeptide having 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 amount is sufficient to delay or prevent recurrence of the cancer. In certain embodiments, 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 months 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).

[0325] In certain embodiments, the cancer treated with a single dose or more of the heterodimeric IL-12-Fc fusion protein (e.g., comprising a first polypeptide having the amino acid sequence of SEQ ID NO:290 and a second polypeptide having the amino acid sequence of SEQ ID NO:291) is metastatic cancer. In certain embodiments, the metastatic cancer is local, regional, or distant metastatic cancer. In certain embodiments, a single dose or multiple doses of the heterodimeric IL-12-Fc fusion protein (e.g., comprising a first polypeptide having the amino acid sequence of SEQ ID NO:290 and a second polypeptide having the amino acid sequence of SEQ ID NO:291) treat distant cancers by a bystander effect, which are not the primary cancer of the source organ or tissue and / or the direct target of the treatment regimen. In certain embodiments, the bystander effect of the heterodimeric IL-12-Fc fusion protein is enhanced during and / or after a treatment regimen comprising radiation and / or chemotherapy. In certain embodiments, a single dose or multiple doses of the heterodimeric IL-12-Fc fusion protein (e.g., comprising a first polypeptide having the amino acid sequence of SEQ ID NO:290 and a second polypeptide having the amino acid sequence of SEQ ID NO:291) treat a patient's cancer by inducing a systemic anti-tumor response, such as determined by increased expression of IFNγ, CXCL9, and / or CXCL10 in the patient's serum and / or tumor. V. Combination Therapy

[0326] Another aspect of the invention provides combination therapy. The multispecific binding proteins described herein can be used in combination with additional therapeutic agents to treat cancer.

[0327] In certain embodiments, the heterodimeric Fc fusion proteins of the invention (e.g., heterodimeric Fc fusion proteins comprising an IL-12 subunit) are 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 heterodimeric 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 heterodimeric 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.

[0328] In some embodiments, the heterodimeric Fc fusion proteins of the invention (e.g., heterodimeric Fc fusion proteins comprising an IL-12 subunit) are combined with another therapeutic agent selected from the group consisting of: cytotoxic chemotherapy; radiotherapy; antibodies targeting molecules involved in the anti-tumor immune response (such as CTLA-4, PD-1, PD-L1, or TGF-β); antibodies that act on tumor-associated antigens via ADCC; multispecific antibodies that bind NKG2D, CD16, and tumor-associated antigens, optionally administered in combination with an antibody targeting PD-1 or PD-L1; personalized cancer vaccines; oncolytic cancer vaccines; and personalized vaccines administered in combination with an antibody targeting PD-1 or PD-L1 for the treatment of advanced malignancies.

[0329] In some embodiments, the heterodimeric Fc fusion proteins of the invention (e.g., heterodimeric Fc fusion proteins comprising an IL-12 subunit) are combined with another therapy for treating a malignancy (e.g., advanced malignancy), said another therapy including but 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 antibody, chimeric antigen receptor (CAR) T cell therapy, T cell receptor (TCR) engineered therapy, multispecific binding protein (TriNKET), agent for inducing cellular senescence, and vaccine and / or oncolytic virus therapy. In some embodiments, the heterodimeric Fc fusion proteins of the invention are combined with two or more additional therapies selected from the following for treating a malignancy (e.g., advanced malignancy): 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 antibody, chimeric antigen receptor (CAR) T cell therapy, T cell receptor (TCR) engineered therapy, multispecific binding protein (TriNKET), agent for inducing cellular senescence, and vaccine and / or oncolytic virus therapy.

[0330] In some embodiments, the heterodimeric Fc fusion proteins of the invention (e.g., heterodimeric Fc fusion proteins comprising IL-12) are combined with a cancer vaccine or an antibody targeting PD-1 or PD-L1 for treating locally advanced malignancies that can be completely resected.

[0331] The proteins of the invention can also be used as a supplement to surgical resection of the primary lesion.

[0332] The amounts and relative timing of administration of the heterodimeric Fc fusion proteins of the invention (e.g., heterodimeric Fc fusion proteins comprising IL-12) and additional therapeutic agents can be selected to achieve a desired combined therapeutic effect. For example, when a combination therapy is administered to a patient in need thereof, the therapeutic agents in the combination or one or more pharmaceutical compositions comprising the therapeutic agents can be administered in any order, such as sequentially, concurrently, together, simultaneously, etc. Further, for example, the heterodimeric Fc fusion protein can be administered during the time period in which the one or more additional therapeutic agents exert their prophylactic or therapeutic effect, or vice versa.

[0333] As disclosed herein, the methods of the invention include the combination of co-administering 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 invention include the combination of co-administering a heterodimeric Fc fusion protein comprising an IL-12 subunit and an additional therapeutic agent.

[0334] Co-administration includes methods where the heterodimeric Fc fusion protein (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) and the additional therapeutic agent are given simultaneously, where the heterodimeric Fc fusion protein and the additional therapeutic agent are given sequentially, and where either or both of the heterodimeric Fc fusion protein and the additional therapeutic agent are given intermittently or continuously or any combination of the following: simultaneously, sequentially, intermittently, and / or continuously. One of ordinary skill in the art will recognize that intermittent administration is not necessarily the same as sequential administration, as intermittent also includes a first administration of the agent and then another administration after a time for that same agent. Additionally, one of ordinary skill in the art understands that in some embodiments, intermittent administration also includes sequential administration, as intermittent administration does include interrupting the first administration of an agent with the administration of a different agent before re-administering the first agent. Additionally, one of ordinary skill in the art will also know that continuous administration can be achieved by a variety of routes (including intravenous drip (IV infusion) or feeding tube, etc.).

[0335] Furthermore, and in a more general manner, the term "co-administration" includes any and all methods where the separate administration of the heterodimeric Fc fusion protein to a subject and the separate administration of the additional therapeutic agent overlap within any time frame.

[0336] The frequency of administration of the heterodimeric Fc fusion protein or the additional therapeutic agent to a subject is known in the art as Qnd or qnd, where n is the frequency of consecutive administration of the agent (in days). For example, Q3d would be administration of the agent once every three (3) days. In certain embodiments, the methods include administering the heterodimeric Fc fusion protein and / or the additional therapeutic agent to the subject at Q1d, Q2d, Q3d, Q4d, Q5d, Q6d, Q7d, Q8d, Q9d, Q10d, Q14d, Q21d, Q28d, Q30d, Q90d, Q120d, Q240d, or Q365d or any combination of any one or two of them.

[0337] In certain embodiments, either or both of the heterodimeric Fc fusion protein and / or additional therapeutic agent are administered intermittently. In certain embodiments, the method comprises administering either or both of the heterodimeric Fc fusion protein or additional therapeutic agent to a subject with a delay of 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, thirty-six (36) hours, forty-eight (48) hours, three (3) days, four (4) days, five (5) days, six (6) days, seven (7) days, eight (8) days, nine (9) days, ten (10) days, eleven (11) days, twelve (12) days, thirteen (13) days, fourteen (14) days, three (3) weeks or four (4) weeks between administrations. In certain embodiments, the delayed administration follows a pattern wherein the consecutive administration of either or both of the heterodimeric Fc fusion protein and / or additional therapeutic agent or any combination thereof continues 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.

[0338] In certain embodiments, either or both of the heterodimeric Fc fusion protein and / or additional therapeutic agent or a combination thereof are administered intermittently while the other is administered continuously. In certain embodiments, a combination of a first effective amount of the heterodimeric Fc fusion protein and a second effective amount of the additional therapeutic agent are administered sequentially.

[0339] In certain embodiments, the heterodimeric Fc fusion protein and the additional therapeutic agent are administered simultaneously. In certain embodiments, a combination of a first effective amount of the heterodimeric Fc fusion protein and a second effective amount of the additional therapeutic agent are administered sequentially. In such embodiments, the combination is also referred to as "co-administered" as the term includes any and all methods wherein the subject is exposed to both components of the combination. However, such embodiments are not limited to combinations given only in one formulation or composition. It may be more advantageous for certain concentrations of the heterodimeric Fc fusion protein and the additional therapeutic agent to be delivered at intervals, and thus, the first and second effective amounts may vary depending on the formulation administered.

[0340] In certain embodiments, the heterodimeric Fc fusion protein and the additional therapeutic agent are administered simultaneously or sequentially. In certain embodiments, a first effective amount of the heterodimeric Fc fusion protein is administered sequentially after a second effective amount of the additional therapeutic agent. In certain embodiments, a second effective amount of the additional therapeutic agent is administered sequentially after a first effective amount of the heterodimeric Fc fusion protein.

[0341] In certain embodiments, a combination of the 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 the heterodimeric Fc fusion protein is administered in a formulation separate from the formulation of the second effective amount of the additional therapeutic agent. In certain embodiments, the combination is administered in two (2) compositions, wherein a first effective amount of the heterodimeric Fc fusion protein is administered in a formulation separate from the formulation of the second effective amount of the additional therapeutic agent. In certain embodiments, a first effective amount of the heterodimeric Fc fusion protein is administered sequentially after a second effective amount of the additional therapeutic agent. In certain embodiments, a second effective amount of the additional therapeutic agent is administered sequentially after a first effective amount of the heterodimeric Fc fusion protein. In certain embodiments, the 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 an every-other-day schedule with no overlap.

[0342] In certain embodiments, the first effective amount of the heterodimeric Fc fusion protein is administered not less than four (4) hours after a second effective amount of an additional therapeutic agent. In certain embodiments, the first effective amount of the heterodimeric Fc fusion protein is administered not less than ten (10) minutes, not less than fifteen (15) minutes, not less than twenty (20) minutes, not less than thirty (30) minutes, not less than forty (40) minutes, not less than sixty (60) minutes, not less than one (1) hour, not less than two (2) hours, not less than four (4) hours, not less than six (6) hours, not less than eight (8) hours, not less than ten (10) hours, not less than twelve (12) hours, not less than twenty-four (24) hours, not less than two (2) days, not less than four (4) days, not less than six (6) days, not less than eight (8) days, not less than ten (10) days, not less than twelve (12) days, not less than fourteen (14) days, not less than twenty-one (21) days or not less than thirty (30) days after a second effective amount of an additional therapeutic agent. In certain embodiments, the second effective amount of the additional therapeutic agent is administered not less than ten (10) minutes, not less than fifteen (15) minutes, not less than twenty (20) minutes, not less than thirty (30) minutes, not less than forty (40) minutes, not less than sixty (60) minutes, not less than one (1) hour, not less than two (2) hours, not less than four (4) hours, not less than six (6) hours, not less than eight (8) hours, not less than ten (10) hours, not less than twelve (12) hours, not less than twenty-four (24) hours, not less than two (2) days, not less than four (4) days, not less than six (6) days, not less than eight (8) days, not less than ten (10) days, not less than twelve (12) days, not less than fourteen (14) days, not less than twenty-one (21) days or not less than thirty (30) days after the first effective amount of the heterodimeric Fc fusion protein.

[0343] In certain embodiments, either or both of the heterodimeric Fc fusion protein and / or the additional therapeutic agent are administered by a route selected from: intravenous, subcutaneous, dermal, oral, intramuscular, and intraperitoneal. In certain embodiments, either or both of the heterodimeric Fc fusion protein and / or the additional therapeutic agent are administered intravenously. In certain embodiments, either or both of the heterodimeric Fc fusion protein and / or the additional therapeutic agent are administered orally or in any combination thereof.

[0344] One of ordinary skill in the art will appreciate that the unit dosage forms of the present disclosure may be administered in the same or different physical forms, i.e., orally administered via capsules or tablets and / or administered via IV infusion with a liquid, etc. Additionally, the unit dosage form for each administration may vary depending on the particular route of administration. For either or both of the heterodimeric Fc fusion protein and the additional therapeutic agent in the combination, there may be several different dosage forms. Since 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 yet may still need to be administered in different ways and possibly 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 instead or also via the inhalation, buccal, sublingual, or suppository route, even a dosage form that is the same as other dosage forms used previously or subsequently. A particular dosage form may be required for certain combinations of the heterodimeric Fc fusion protein and the additional therapeutic agent because there may be issues related to various factors such as chemical stability or pharmacokinetics. NK targeting therapy

[0345] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with an NK targeting therapy. For example, in one embodiment, the heterodimeric Fc fusion protein is co-administered with a therapeutic agent that targets NKp46. In certain embodiments, the therapeutic agent that targets NKp46 also binds to CD16, one or more tumor-associated antigens, or a combination thereof. Exemplary therapeutic agents that target NKp46 are described in more detail in U.S. Application No. US20170198038 A1 (incorporated herein by reference for all purposes).

[0346] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with bispecific and trispecific killer engagers (BiKEs and TriKEs) therapies, including BiKEs and TriKEs that target NK cells. BiKEs and TriKEs 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 linked by a short flexible polypeptide linker to prevent dissociation. BiKEs and TriKEs are described in more detail in U.S. Application Nos. US20180282386 A1 and US20180258396 A1 (incorporated herein by reference for all purposes). BiKEs and TriKEs may contain binding domains specific for NK cells.

[0347] In certain embodiments, the BiKE and TriKE therapies are used in combination with the heterodimeric Fc fusion protein therapy to treat subjects known or suspected to have high-risk myelodysplastic syndrome, acute myeloid leukemia, systemic mastocytosis, or mast cell leukemia. In certain embodiments, the BiKE and TriKE therapies are administered as a single course of 3 weekly treatment blocks. In certain embodiments, a treatment block comprises 4 consecutive 24-hour continuous infusions (about 96 hours), followed by a 72-hour rest. In certain embodiments, the BiKE and TriKE therapies 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, the 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, the BiKE and TriKE therapies are administered at a dose of at least 1 μg / kg / day. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of at least 5 μg / kg / day. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of at least 200 μg / kg / day. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of at least 500 μg / kg / day. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of at least 1000 μg / kg / day. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of 200 μg / kg / day or less. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of 500 μg / kg / day or less. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of 1000 μg / kg / day or less. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of 1-200 μg / kg / day. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of 5-200 μg / kg / day. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of 1-500 μg / kg / day. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of 1-1000 μg / kg / day. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of 5-500 μg / kg / day. In certain embodiments, the BiKE and TriKE therapies are administered at a dose of 5-1000 μg / kg / day. In certain embodiments, the BiKE and TriKE therapies are administered at the maximum tolerated dose. In certain embodiments, the BiKE and TriKE therapies are administered at less than the maximum tolerated dose. Multi-specific binding protein (“TriNKET”) therapy

[0348] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with a therapy comprising a multi-specific 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 that is sufficient to bind CD16, or a third antigen-binding site that binds CD16 (“TriNKET”) (e.g., a multi-specific binding protein comprising various NKG2D binders and tumor-associated antigen-binding sites described in International Publication No. WO 2019 / 157332, the content related to the multi-specific binding protein therein is incorporated herein by reference), 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.

[0349] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with a therapy comprising a body weight-based dose of a multispecific binding protein. For example, the body weight-based dose of the multispecific binding protein 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 to 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 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.

[0350] 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 than once daily, weekly, monthly, or annually, or even once every 2 to 20 years. One of ordinary skill in the art can readily estimate the rate of repetition for administration based on the measured residence time and concentration of the targetable construct or complex in body fluids or tissues. 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 than once daily, once or more than once weekly, once or more than once monthly, and once or more than once annually. Chimeric antigen receptor (CAR) therapy

[0351] 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 the "primary signaling domain").

[0352] Thus, 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 further comprises one or more functional signaling domains (also referred to as "costimulatory signaling domains") derived from at least one costimulatory molecule.

[0353] In one embodiment, the CAR comprises a chimeric fusion protein comprising 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 comprising 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 co-stimulatory signaling domain and a primary signaling domain. In certain embodiments, the CAR comprises a chimeric fusion protein comprising 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 co-stimulatory signaling domains and a primary signaling domain. In one embodiment, the CAR comprises a chimeric fusion protein comprising a tumor-associated antigen-binding 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 at least two co-stimulatory signaling domains and a primary signaling domain.

[0354] Regarding the transmembrane domain, in various embodiments, the CAR is designed to comprise a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain is a domain that is naturally associated with one of the domains in the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, to minimize interaction with other members of the receptor complex. In another embodiment, the transmembrane domain is capable of homodimerizing 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 substituted so as to minimize interaction with the binding domain of a natural binding partner present in the same CAR T cell.

[0355] The transmembrane domain can be derived from any naturally occurring membrane-binding or transmembrane protein. In one embodiment, whenever the CAR has bound to the target, the transmembrane region is capable of signaling to the one or more intracellular domains. In some embodiments, the transmembrane domain comprises one or more transmembrane regions of one or more proteins selected from: TCRα chain, TCRβ chain, TCRζ chain, CD28, CD3ε, 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: KIRDS2, OX40, CD2, CD27, LFA-l (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.

[0356] The extracellular tumor-associated antigen-binding domain (e.g., tumor-associated antigen-binding scFv domain) can be linked to the transmembrane domain via a hinge region. A variety of hinges can be employed, including but not limited to human Ig hinges (e.g., IgG4 hinge, IgD hinge), Gly-Ser linkers, (G4S)4 linkers, KIR2DS2 hinges, and CD8α hinges.

[0357] 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., the lytic activity or helper activity of a T cell, including the secretion of cytokines). Thus, as used herein, the term "intracellular signaling domain" refers to the part of a protein that transduces effector function signals and directs the cell to perform a specialized function. While the entire intracellular signaling domain can generally be employed, in many cases it is not necessary to use the entire chain. In terms of using truncated portions of the intracellular signaling domain, such truncated portions can be used in place of the full chain as long as they transduce effector function signals. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transduce effector function signals.

[0358] 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., functional signaling domains derived from at least one co-stimulatory molecule).

[0359] As used herein, the term "stimulatory molecule" refers to a molecule expressed by an immune cell (e.g., a T cell, NK cell, or B cell) that provides one or more cytoplasmic signaling sequences that modulate the activation of the immune cell 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, for example, by the binding of the TCR / CD3 complex to a peptide-loaded MHC molecule and that mediates T cell responses (including but not limited to proliferation, activation, differentiation, etc.).

[0360] The primary signaling domain that acts in a stimulatory manner can contain signaling motifs (which are referred to as immunoreceptor tyrosine-based activation motifs or ITAMs). 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.

[0361] In some embodiments, the primary signaling domain is the 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 the 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 the functional signaling domain of the ζ chain associated with the T cell receptor complex.

[0362] As used herein, the term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response in the T cell, such as, but not limited to, proliferation. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for a lymphocyte to respond effectively to an antigen. 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 that specifically binds to CD83, among others. Other examples of such costimulatory 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, LFA-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 a ligand that specifically binds to CD83. In some embodiments, the costimulatory signaling domain of the CAR is the functional signaling domain of a costimulatory molecule described herein, the costimulatory molecule being, for example, 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.

[0363] As used herein, the term "signaling domain" refers to the functional portion of a protein that functions by transmitting information within a cell to regulate cellular activity via a defined signaling pathway by generating a second messenger or by acting as an effector in response to such a messenger.

[0364] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specified order. Optionally, short oligopeptide or polypeptide linkers (e.g., between 2 and 10 amino acids in length) can form the linkage. Antibody therapy

[0365] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with antibody therapy to treat a subject known or suspected of having cancer.

[0366] In certain embodiments, the heterodimeric Fc fusion protein is combined with a therapy comprising an anti-HER2 binding domain, which is an anti-HER2 antibody or anti-HER2 antibody platform (e.g., a bispecific or trispecific antibody comprising an anti-HER2 binding domain, an anti-HER2 antibody-drug conjugate, or an anti-HER2 CAR). 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. Patent 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 heterodimeric Fc fusion protein therapy to treat a subject 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 less than 1 mg / kg / day.

[0367] In certain embodiments, the anti-HER2 binding domain therapy is used in combination with the heterodimeric Fc fusion protein therapy to treat a subject known or suspected of having breast cancer, such as a subject diagnosed with metastatic breast cancer overexpressing 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, and then subsequently at 2 mg / kg / day weekly. In certain embodiments, the anti-HER2 binding domain therapy is administered at an initial dose of 4 mg / kg / day, and then subsequently at 2 mg / kg / day weekly for 52 weeks.

[0368] In certain embodiments, the anti-HER2 binding domain therapy is used in combination with the heterodimeric Fc fusion protein therapy to treat a subject known or suspected of having gastric cancer, such as a subject diagnosed with metastatic gastric cancer overexpressing 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 over 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 over 30 - 90 minutes. In certain embodiments, the anti-HER2 binding domain therapy is administered at an initial dose of 8 mg / kg / day, and then subsequently administered at 6 mg / kg / week. In certain embodiments, the anti-HER2 binding domain therapy is administered at an initial dose of 8 mg / kg / day, and then subsequently administered at 6 mg / kg / week for 52 weeks.

[0369] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with a therapy comprising an anti-CD20 binding domain, which is an anti-CD20 antibody or anti-CD20 antibody platform (e.g., a bispecific or trispecific antibody comprising an anti-CD20 binding domain, an anti-CD20 antibody-drug conjugate, or an anti-CD20 CAR). 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, it is administered at 100 mg / m 2 ², 200 mg / m 2 ², 300 mg / m 2 ², 400 mg / m 2 ², 500 mg / m 2 ², 600 mg / m 2 ², 700 mg / m 2 ², 800 mg / m 2, 900 mg / m 2 or 1000 mg / m 2 The anti-CD20 binding domain therapy is administered at a dose of. In certain embodiments, the anti-CD20 binding domain therapy is administered at a dose of 375 mg / m 2 The anti-CD20 binding domain therapy is administered at a dose of. 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 The anti-CD20 binding domain therapy is administered at a dose of. In certain embodiments, the anti-CD20 binding domain therapy is administered at a dose of less than 400 mg / m 2 The anti-CD20 binding domain therapy is administered at a dose of. 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.

[0370] 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 administered by IV infusion at a dose of 375 mg / m 2 The anti-CD20 binding domain therapy is administered at a dose of. 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.

[0371] 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 a dose of 375 mg / m 2 in the first cycle and at a dose of 500 mg / m 2 in each of the additional 2 - 6 cycles by IV infusion. In certain embodiments, the anti-CD20 binding domain therapy is administered by IV infusion at a dose of less than 375 mg / m 2Administer the anti-CD20 binding domain therapy at a dose. The combined anti-CD20 binding domain and heterodimeric Fc fusion protein therapy can be used in combination with fludarabine and cyclophosphamide (FC).

[0372] 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 to have rheumatoid arthritis (RA). In certain embodiments, the anti-CD20 binding domain therapy is administered by IV infusion as two doses of 1000 mg (doses separated by 2 weeks). In certain embodiments, the anti-CD20 binding domain therapy is administered by IV infusion as two doses of 1000 mg (doses separated by 2 weeks) for up to 24 weeks. In certain embodiments, the combined anti-CD20 binding domain and heterodimeric Fc fusion protein therapy is co-administered with methotrexate.

[0373] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with a therapy comprising an antibody therapy that includes an agonist antibody. In certain embodiments, the agonist antibody is an anti-4-1BB antibody, an anti-CD137 antibody, an anti-FAP antibody, an anti-OX40 antibody, an anti-CD40 antibody, an anti-GITR antibody, or an 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., a bispecific antibody) that includes two or more antigen-binding domains selected from an anti-4-1BB antibody, an anti-CD137 antibody, an anti-FAP antibody, an anti-OX40 antibody, an anti-CD40 antibody, an anti-GITR antibody, or an anti-CD27 antibody. An illustrative example is the bispecific agonist antibody that targets 4-1BB and CD137, such as utomilumab (Pfizer). Checkpoint inhibitor therapy

[0374] In certain embodiments, the heterodimeric Fc fusion protein therapy can be combined with checkpoint inhibitor therapy. Exemplary 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 (which belongs to the CD2 family of molecules and is expressed on all NK, γδ, and memory CD8+(αβ) T cells), CD160 (also known as BY55), and CGEN-15049. Immune checkpoint inhibitors include antibodies or antigen-binding fragments thereof or other binding proteins that bind to 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. Exemplary immune checkpoint inhibitors include nivolumamb (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.

[0375] In certain embodiments, the checkpoint inhibitor 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 checkpoint inhibitor therapy is administered by IV infusion. In certain embodiments, the checkpoint inhibitor therapy is administered by IV infusion within 30 minutes. In certain embodiments, the checkpoint inhibitor therapy is administered every 3 weeks. In certain embodiments, the checkpoint inhibitor therapy is administered at a dose of 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg. In certain embodiments, the checkpoint inhibitor therapy is administered at a dose of 200 mg. In certain embodiments, the checkpoint inhibitor therapy is administered at a dose of at least 100 mg, at least 200 mg, at least 300 mg, at least 400 mg, at least 500 mg, at least 600 mg, at least 700 mg, at least 800 mg, at least 900 mg, or at least 1000 mg. In certain embodiments, the checkpoint inhibitor therapy is administered at a dose less than 200 mg. In certain embodiments, the checkpoint inhibitor therapy is used in combination with the heterodimeric Fc fusion protein therapy to treat a subject 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). Additional cytokine therapy

[0376] In some embodiments, the heterodimeric 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 heterodimeric Fc fusion protein therapy is combined with one or more additional cytokine therapies. In some embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more chemokine therapies. In some embodiments, the cytokine therapy comprises a pro-inflammatory cytokine, a Th1 cytokine, or a Th2 cytokine. In some embodiments, the cytokine therapy comprises a recombinant human cytokine or chemokine.

[0377] In some embodiments, the cytokine therapy includes cytokines that are 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 that are 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).

[0378] In some embodiments, the chemokine therapy includes pro-inflammatory chemokines (e.g., GRO-α, GRO-b, LIX, GCP-2, MIG, IP10, I-TAC, and MCP-1, RANTES, eosinophil chemotactic factor, 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 comprises G protein-coupled transmembrane receptors.

[0379] In some embodiments, the cytokine therapy comprises a cytokine therapy that synergizes with IL-12 signaling. In some embodiments, the cytokine therapy includes the 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 is administered intravenously. In some embodiments, the cytokine therapy includes the IL-15 cytokine or a derivative thereof. In some embodiments, the IL-15 therapy is ALT-803 (Altor Bioscience) or NKTR-255 (Nektar). In some embodiments, the IL-15 therapy, NKTR-255, and / or ALT-803 is administered subcutaneously. In some embodiments, the chemokine therapy includes the CXCL9 chemokine, the CXCL10 chemokine, or a derivative thereof.

[0380] 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.

[0381] In some embodiments, the cytokine or chemokine therapy includes engineering cells to produce the cytokine or chemokine using: virus vector-based delivery platforms such as vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629); lentivirus, including but not limited to second, third or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation that are 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-l8; 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); or adeno-associated virus (“AAV”) vectors, as described in more detail in U.S. Patent 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). In some embodiments, the cytokine or chemokine therapy includes engineering cells to produce the cytokine or chemokine using LNP, liposome or exosome.In some embodiments, the cytokine or chemokine therapy includes using genome editing, such as engineering cells using nuclease-based genome editing systems (e.g., genome editing systems based on the clustered regularly interspaced short palindromic repeats (CRISPR) family, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and homing endonucleases (HEs) or derivatives thereof) to produce the cytokine or chemokine. In some embodiments, the cytokine or chemokine therapy includes engineering cells using electroporation to produce the cytokine or chemokine. Innate immune system agonist therapy

[0382] In some embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more innate immune system agonists.

[0383] 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 a 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 poly IC-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 ( Aldara-Medicis), and gardiquimod. 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 an oligodeoxynucleotide containing CpG (CpG-ODN) or SD-101 (Dynavax).

[0384] 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 di-AMP, cyclic di-GMP, 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). Chemotherapy

[0385] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more chemotherapies. In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more chemotherapies to treat a subject diagnosed with cancer. Examples of chemotherapeutic agents include Aldesleukin, Alvocidib, Antineoplaston AS2 - 1, Antineoplaston A10, Anti-thymocyte globulin, Amifostine trihydrate, Amonafide, Arsenic trioxide, Betaalethine, Bcl-2 family protein inhibitors 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, DTPACE, Docetaxel, Dolastatin 10, Doxorubicin ( ), Doxorubicin hydrochloride, Enzastaurin, Epoetin alfa, Etoposide, Everolimus (RAD001), Fenretinide, Filgrastim, Melphalan, Mesna, Flavopiridol, Fludarabine ( ), Geldanamycin (17-AAG), Ifosfamide, Irinotecan hydrochloride, Ixabepilone, Lenalidomide ( CC-5013), Lymphokine-activated killer cells, Melphalan, Methotrexate, Mitoxantrone hydrochloride, Gadolinium motexafin, 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).

[0386] 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 ( ) or regorafenib ( );or an EGFR targeting agent, such as cetuximab (Erbitux) or panitumumab ( ). In certain embodiments, the chemotherapy is co-administered with a VEGF targeting agent or an EGFR targeting agent and is selected from the group consisting of: FOLFOX, FOLFIRI, FOLFOXIRI, CapeOx, 5-FU co-administered with leucovorin, capecitabine alone, and trifluridine / tipiracil.

[0387] 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 chemotherapy comprises doxorubicin ( ), pegylated liposomal doxorubicin, epirubicin ( ), paclitaxel (Taxol), docetaxel ( ), albumin-bound paclitaxel ( ), 5-fluorouracil (5-FU), cyclophosphamide ( ), carboplatin ( ), cisplatin, vinorelbine ( ), capecitabine (Xeloda), gemcitabine ( ), ixabepilone ( ), or eribulin (Halaven). In certain embodiments, the chemotherapy comprises a combination of two or more chemotherapies selected from the group consisting of doxorubicin ( ), pegylated liposomal doxorubicin, epirubicin ( ), paclitaxel (Taxol), docetaxel ( ), albumin-bound paclitaxel ( ), 5-fluorouracil (5-FU), cyclophosphamide ( ), carboplatin ( ), cisplatin, vinorelbine ( ), capecitabine ( ), gemcitabine ( ), ixabepilone ( ), and eribulin ( ).

[0388] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more chemotherapies to treat a subject diagnosed with melanoma / carcinoma of the skin. In certain embodiments, the chemotherapy comprises dacarbazine (also known as DTIC), temozolomide, albumin-bound paclitaxel (nab-paclitaxel), paclitaxel, cisplatin, carboplatin, or vinblastine. Targeted agent therapy

[0389] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more targeted agents. Generally, a targeted agent acts on a specific molecular target, such as a target associated with cancer. The difference between a targeted agent and standard chemotherapy is that standard chemotherapy acts on all rapidly dividing normal and cancer cells. Targeted agents include, but are not limited to, hormone therapy, signal transduction inhibitors, gene expression regulators, apoptosis inducers, angiogenesis inhibitors, immunotherapy, toxin delivery molecules (e.g., antibody-drug conjugates), and kinase inhibitors. In certain embodiments, the targeted agent comprises a receptor agonist or ligand.

[0390] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more targeted agents to treat a subject diagnosed with colon cancer, rectal cancer, or colorectal cancer (CRC). In certain embodiments, the targeted agent comprises cetuximab ( ), panitumumab ( ), bevacizumab ( ), aflibercept ( ), regorafenib ( ), ramucirumab ( ), nivolumab ( ), or ipilimumab ( ).

[0391] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more targeted agents to treat a subject diagnosed with breast cancer. In certain embodiments, the targeted agent comprises everolimus ( ), tamoxifen ( ), toremifene ( ), trastuzumab ( ), fulvestrant ( ), anastrozole ( ), exemestane ( ), lapatinib ( ), letrozole ( ), pertuzumab ( ), ado-trastuzumab emtansine conjugate ( ), palbociclib ( ), ribociclib ( ) Neratinib maleate (NERLYNX TM ) Abemaciclib (VERZENIO TM ) Olaparib (LYNPARZA TM ) Atezolizumab ) or Alpelisib ( ).

[0392] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more targeting agents to treat a subject diagnosed with melanoma / carcinoma of the skin. In certain embodiments, the targeting agent comprises Vismodegib ( ), Sonidegib ( ), Ipilimumab ), Vemurafenib ( ), Trametinib ( ), Dabrafenib ( ), Pembrolizumab ( ), Nivolumab ( ), Cobimetinib (COTELLIC TM ), Tretinoin ( ), Avelumab ( ), Encorafenib (BRAFTOVI TM ), Binimetinib ( ), or Cemiplimab-rwlc ( ).

[0393] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with 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. Radiation therapy

[0394] In some embodiments, the heterodimeric Fc fusion protein therapy is combined with radiation therapy. In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with radioactive isotope particles (such as indium In-111, yttrium Y-90, or iodine I-131). Examples of combination therapies include, but are not limited to, iodine-131 tositumomab ), yttrium-90 ibritumomab tiuxetan ), and and CHOP. In certain embodiments, the radiotherapy comprises external beam radiation therapy (EBRT), brachytherapy (interstitial brachytherapy), intracavitary radiotherapy, interstitial brachytherapy, radioembolization, hypofractionated radiotherapy, intraoperative radiotherapy (IORT), three-dimensional conformal radiotherapy, stereotactic radiosurgery (SRS), or stereotactic body radiotherapy (SBRT).

[0395] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more radiotherapy regimens to treat a subject diagnosed with colon cancer, rectal cancer, or colorectal cancer (CRC). In certain embodiments, the radiotherapy comprises external beam radiation therapy (EBRT), brachytherapy (interstitial brachytherapy), intracavitary radiotherapy, interstitial brachytherapy, or radioembolization.

[0396] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more radiotherapy regimens to treat a subject diagnosed with breast cancer. In certain embodiments, the radiotherapy comprises external beam radiation therapy, hypofractionated radiotherapy, intraoperative radiotherapy (IORT), or three-dimensional conformal radiotherapy.

[0397] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with one or more radiotherapy regimens to treat a subject diagnosed with melanoma / cutaneous cancer. In certain embodiments, the radiotherapy comprises stereotactic radiosurgery (SRS; e.g., using a gamma knife or linear accelerator) or stereotactic body radiotherapy (SBRT). Vaccines and oncolytic virus therapies

[0398] 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).

[0399] In some embodiments, the heterodimeric Fc fusion protein therapy is combined with a vaccine composition. Vaccine compositions typically comprise multiple antigens and / or neoantigens specific to the tumor to be targeted. Vaccine compositions may also be referred to as vaccines.

[0400] 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 a polysaccharide.

[0401] Generally, an adjuvant is any substance that, when incorporated into a vaccine composition, increases or otherwise modifies the immune response to an antigen or neoantigen. Optionally, the adjuvant is conjugated covalently or non-covalently. 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 antibody titer produced against the 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 modify the immune response, for example, by changing a predominant humoral response or Th response into a predominant cellular response 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 IMS1312, 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 stimulator (Aquila Biotech, Worcester, Massachusetts, USA) (which is derived from saponin, mycobacterial extract, 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 (e.g., MF59) and their preparation have been previously described (Dupuis M et al., Cell Immunol. 1998; 186(1):18-27; Allison AC; Dev Biol Stand. 1998; 92:3-11). Cytokines can 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 efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Patent No. 5,849,589, which is hereby incorporated by reference in its entirety), and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich D I 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.

[0402] Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g., CpR, Idera); poly(I:C) (e.g., poly i:CI2U); non-CpG bacterial DNA or RNA; and immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, celecoxib, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, ticilimumab, and SC58175; which can have a therapeutic effect and / or act as an adjuvant. The amounts and concentrations of the adjuvants and additives can be readily determined by one of ordinary skill in the art without undue experimentation. Additional adjuvants include colony stimulating factors such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0403] 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 foregoing substances or combinations thereof. It is also contemplated that the vaccine and adjuvant can be administered together or separately in any suitable order.

[0404] 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 aids in the presentation of peptides to T cells. In some embodiments, the carrier comprises any suitable carrier known to those of skill in the art, such as a protein or an antigen presenting cell. 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 immunizing humans, the carrier is generally a physiologically acceptable carrier that is human acceptable and safe. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier can be dextran, such as agarose.

[0405] 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 lentivirus, including but not limited to second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation that are 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-l8; 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, upon introduction into the host, the infected cells express the antigen or neoantigen, thereby eliciting a host immune (e.g., CTL) response against the one or more peptides.

[0406] Depending on the packaging capacity of the virus vector-based vaccine platform mentioned above, in some embodiments, the vaccine composition comprises one or more virus vectors. In some embodiments, the virus vector comprises a sequence flanked by non-mutated sequences, sequences separated by a linker, or 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 useful in immunization regimens are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). The BCG vector is described in Stover et al. (Nature 351:456-460 (1991)). A variety of other vaccine vectors (e.g., Salmonella typhi vectors, etc.) useful for therapeutic administration or immunization against neoantigens will be apparent to those skilled in the art based on the description herein.

[0407] In some embodiments, the heterodimeric Fc fusion protein therapy is combined with an oncolytic virus therapy. Generally, an oncolytic virus is a virus that has been engineered to primarily infect and kill cancer cells. In some embodiments, in addition to killing cancer cells, the oncolytic virus also induces an immune response against the cancer cells.

[0408] In certain embodiments, the heterodimeric Fc fusion protein therapy is combined with an oncolytic virus therapy to treat a subject diagnosed with melanoma / carcinoma of the skin. In certain embodiments, the oncolytic virus comprises talimogenelaherparepvec( ), also known as T-VEC. In some embodiments, a heterodimeric Fc fusion protein comprising subunits of IL-12 is combined with an oncolytic virus (e.g., Talimogene Laherparepvec( ) or T-VEC) for the treatment of cancer (e.g., advanced malignancies).

[0409] The foregoing description has described various aspects and embodiments of the present invention. This patent application expressly contemplates all combinations and permutations of the described aspects and embodiments. Examples

[0410] The present invention is now generally described, and it will be more readily understood by reference to the following examples, which are included for the purpose of illustrating certain aspects and embodiments of the present invention only and are not intended to limit the present invention. Example 1 - Preparation Method

[0411] The proteins of the present invention are generally prepared using recombinant DNA techniques. In one exemplary embodiment, a first nucleic acid sequence encoding a first polypeptide is cloned into a first expression vector (pET-pSURE-Puro), the first polypeptide comprising a first subunit of a multi-subunit protein (the p40 subunit of human IL-12) fused to a first antibody Fc domain polypeptide; a second nucleic acid sequence encoding a second polypeptide is cloned into a second expression vector (pET-pSURE-Puro), the second polypeptide comprising a second different subunit of a multi-subunit protein (the p35 subunit of human IL-12) fused to a second antibody Fc domain polypeptide; and the first and second expression vectors are co-stably transfected into a host cell to produce a heterodimeric Fc fusion protein.

[0412] 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 that comprises the 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 a LALAPA (L234A, L235A, and P329A) mutation that reduces 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.

[0413] Exemplary amino acid sequences encoded by the second expression vector are shown in SEQ ID NO:293. The second expression vector encodes a second polypeptide that comprises the p35 subunit of human IL-12 fused to a human IgG1 Fc sequence containing the S354C mutation. The second polypeptide further includes the Q347R, D399V, and F405T mutations that promote heterodimerization, and the LALAPA (L234A, L235A, and P329A) mutations that reduce effector function. In SEQ ID NO:293, the leader sequence is shown in italics, the sequence of the p35 subunit of human IL-12 is underlined, and the mutations are shown in bold.

[0414] To achieve the highest protein yield, different ratios of the first and second expression vectors were explored to determine the optimal ratio to transfect into host cells. After transfection, single clones were isolated using methods known in the art, such as limiting dilution, ELISA, FACS, microscopy, or Clonepix, to generate a cell bank.

[0415] The clones were cultured under conditions suitable for bioreactor scale-up and maintenance expression of the protein. The protein was 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. Example 2 - Tumor Inhibition by IL-12 Fused to a Silenced Fc Domain Polypeptide in the CT26 Tumor Model

[0416] This example describes the relative ability of two IL-12-Fc fusion constructs of recombinant murine IL-12 (rmIL-12) to control tumor progression in a murine 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 comprises wild-type murine IL-12 p40 and p35 subunits fused to the N-terminus of a wild-type murine IgG2a Fc domain polypeptide; and mIL-12-Fc silenced (DF-mIL-12-Fc si), which comprises wild-type murine IL-12 p40 and p35 subunits fused to the N-terminus of a murine IgG2a Fc domain polypeptide having the L234A, L235A, and P329G mutations. The amino acid sequences of the proteins are shown below: mIL-12-p40-mIgG2A-EW (first chain of DF-mIL-12-Fc wt) mIL-12-p35-mIgG2A-RVT (second chain of DF-mIL-12-Fc wt) mIL-12-p40-mIgG2A-EW-LALAPG (the first strand of DF-mIL-12-Fc si) mIL-12-p35-mIgG2A-RVT-LALAPG (the second strand of DF-mIL-12-Fc si)

[0417] In short, 10 6 CT26-Tyrp1 colon cancer cells were subcutaneously injected into the flanks of Balb / c mice. On day 14 after tumor inoculation, when the tumor volume reached 270 mm 3 , the mice were randomly divided 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 with a molar dose equal to 1 μg rmIL-12, DF-mIL-12-Fc si with a molar dose equal to 1 μg rmIL-12, or 1 μg of mIgG2a isotype control. Tumor growth was evaluated for 60 days.

[0418] As Figures 5A - 5C shown, although IL-12 ( Figure 5A ) and DF-mIL-12-Fc wt ( Figure 5B ) effectively controlled tumor progression in some mice, 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 the overall survival - 100% of the treated mice were still alive on 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 ).

[0419] Next, different doses of DF-mIL-12-Fc wt and DF-mIL-12-Fc si were compared in terms of controlling tumor progression. In short, 10 6 CT26-Tyrp1 colon cancer cells were subcutaneously injected into the flanks of Balb / c mice. On day 14 after tumor inoculation, when the tumor volume reached 300 mm 3At that time, mice were randomly divided into different treatment groups (n = 10 per group), and were intraperitoneally treated once a week with DF-mIL-12-Fc wt at a molar dose equal to 1 μg or 0.1 μg rmIL-12, or DF-mIL-12-Fc si at a molar dose equal to 1 μg or 0.1 μg IL-12. Tumor growth was evaluated for 55 days.

[0420] As Figures 7A - 7D shown, treatment with DF-mIL-12-Fc wt at a molar dose of 1 μg rmIL-12 equivalent resulted in reduced tumor progression in some mice and complete regression in two mice ( Figure 7A ), but no tumor suppression was observed at a molar dose of 0.1 μg IL-12 equivalent ( Figure 7C ). In contrast, treatment with DF-mIL-12-Fc si at a molar dose of 1 μg IL-12 equivalent resulted in 100% complete tumor regression ( Figure 7B ), and induced a robust tumor growth delay at the lower dose of 0.1 μg IL-12 equivalent ( Figure 7D ). The median survival of mice treated with DF-mIL-12-Fc wt at 1 μg IL-12 equivalent was 32 days, similar to the 34-day median survival of mice treated with DF-mIL-12-Fc si at 0.1 μg IL-12 equivalent, indicating that the potency of DF-mIL-12-Fc si is 10 times that of its wild-type variant ( Figure 8 ). DF-mIL-12-Fc wt was ineffective at a dose of 0.1 μg IL-12 equivalent and showed a median survival of 24 days, the same as that of the isotype treatment group.

[0421] Next, the in vivo efficacy of different routes of administration of DF-mIL-12-Fc si was compared. Briefly, 10 6 CT26-Tyrp1 colon cancer cells were subcutaneously injected into the flanks of Balb / c mice. On day 14 after tumor inoculation, when the tumor volume reached 270 mm 3 , the mice were randomly divided into different treatment groups (n = 10 per group), and were intraperitoneally or subcutaneously treated once a week with DF-mIL-12-Fc si at a molar dose equal to 1 μg IL-12 or the molar equivalent mIgG2a isotype control. Tumor growth was evaluated for more than 60 days.

[0422] As Figures 22A - 22B shown, intraperitoneally ( Figure 22A ) and subcutaneously ( Figure 22B) Administration of DF-mIL-12-Fc si induced robust tumor regression and produced 100% complete tumor regression. Thus, DF-mIL-12-Fc si treatment demonstrated efficacy using different administration routes. Example 3 - Inhibition of Tumors by IL-12 Fused with a Silent Fc Domain Polypeptide in the B16F10 Tumor Model

[0423] This example describes the relative ability of DF-mIL-12-Fc wt and DF-mTT-12-Fc si to control tumor progression in a murine melanoma model. Briefly, 10 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 , the mice were randomly divided 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 of IL-12, DF-mIL-12-Fc si at a molar dose equal to 0.5 μg of IL-12, or 0.5 μg of mIgG2a isotype control. Tumor growth was evaluated for 32 days.

[0424] As Figures 9A - 9C 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 times of mice treated with isotype control, DF-mIL-12-Fc wt, and IL-12, which were 16 days, 26 days, and 22 days, respectively ( Figure 10 ).

[0425] Next, different doses of DF-mIL-12-Fc wt and DF-mIL-12-Fc si were compared in terms of controlling tumor progression. Briefly, 10 6 B16F10 melanoma cells were subcutaneously injected into the flanks of C57BL / 6 mice. On day 8 after tumor inoculation, the mice were randomly divided into different treatment groups (n = 10 per group) and treated intraperitoneally once a week with 0.5 μg or 0.1 μg of IL-12 molar equivalent of DF-mIL-12-Fc wt or 0.5 μg or 0.1 μg of IL-12 molar equivalent of DF-mIL-12-Fc si. Tumor growth was evaluated for 30 days.

[0426] As Figures 11A - 11DAs shown, at both doses, DF-mIL-12-Fc si was superior to DF-mIL-12-Fc wt in inhibiting tumor growth. Additionally, 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 DF-mIL-12-Fc si at 0.1 μg IL-12 molar equivalent was 21 days, and the median survival of mice treated with DF-mIL-12-Fc si at 0.5 μg IL-12 molar equivalent was 28 days( Figure 12 ). These results demonstrate that, compared to its wild-type counterpart or isotype control, high-dose (0.5 μg IL-12 molar equivalent) DF-mIL-12-Fc si significantly increased the survival of mice.

[0427] Next, single-dose administration of DF-mIL-12-Fc si was compared to the previously described weekly treatment. Briefly, 10 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 , the mice were randomly divided into different treatment groups (n = 10), and were treated weekly with DF-mIL-12-Fc si at a molar dose equal to 0.5 μg IL-12 or an equivalent molar amount of mIgG2a isotype control. Tumor growth was evaluated for 39 days.

[0428] As Figure 23 shown, single administration of DF-mIL-12-Fc si resulted in reduced outgrowth of tumors in 100% of the mice, although outgrowth of tumors occurred more rapidly compared to weekly administration( Figure 9C ). Additionally, the mice exhibited a temporary weight loss, but only after the first dose (data not shown). Thus, single administration of DF-mIL-12-Fc si demonstrated initial efficacy in a difficult-to-treat tumor model, although subsequent weekly administration better delayed outgrowth of tumors in this model.

[0429] Next, the in vivo efficacy of different routes of administration of DF-mIL-12-Fc si was compared. Briefly, 10 6 B16F10 melanoma cells were subcutaneously injected into C57BL / 6 mice. On day 7 after tumor inoculation, when the tumor volume reached 260 mm 3 , the mice were randomly divided into different treatment groups (n = 10), and were treated weekly intraperitoneally or subcutaneously with DF-mIL-12-Fc si at a molar dose equal to 1 μg IL-12 or an equivalent molar amount of mIgG2a isotype control. Tumor growth was evaluated for 40 days.

[0430] As Figures 24A - 24B shown, intraperitoneal( Figure 24A ) and subcutaneous( Figure 24B ) administration of DF-mIL-12-Fc si induced tumor regression in 100% of the mice. Thus, DF-mIL-12-Fc si treatment demonstrated efficacy using different administration routes. Example 4 - In vitro potency of DF-hIL-12-Fc wt and rhIL-12

[0431] The potency of DF-hIL-12-Fc si was evaluated compared to rhIL-12 using an in vitro bioassay.

[0432] IL-12 potency was evaluated 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 in medium. DF-hIL-12-Fc si (DF IL-12-Fc) and recombinant human IL-12 (rhIL-12; PeproTech) were diluted in medium. 100 μL of the PBMC suspension was mixed with 100 μL of the diluted test article and incubated for 48 hours. The supernatant was harvested and the engagement of the IL-12 receptor and the signaling components stably expressed by the reporter cells was detected by measuring the embryonic alkaline phosphatase secreted by the cells according to the manufacturer's instructions. Briefly, 25 μL of the sample supernatant was mixed with 200 μL of the QUANTI-Blue reagent and incubated for 10 minutes at room temperature in the dark. The plate was then read at 620 nM using a SpectraMax i3x plate reader and the optical density was reported to represent relative IL-12 activity.

[0433] As Figure 13A 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 responses measured in the HEK-Blue reporter assay were comparable between DF-hIL-12-Fc si and rhIL-12.

[0434] Next, IL-12 potency was evaluated by quantifying IFNγ production in human PBMC. PBMC were isolated from human peripheral blood buffy coat by density gradient centrifugation and adjusted to 1 x 10 6cells / mL. Dilute DF-hIL-12-Fc si and recombinant human IL-12 (rhIL-12) in the medium. Mix 100 μL of the PBMC suspension with 100 μL of the diluted test article and incubate for 48 hours. Harvest the supernatant and quantify IFNγ using the Human IFN-γ ELISA MAX kit (BioLegend). After the IFNγ ELISA plate develops color, read them using a SpectraMax i3x instrument at 450 nm and perform background subtraction at 540 nm. Approximate the IFNγ content in the sample wells by interpolating the sample readings from the assay standard curve.

[0435] As Figure 13B shown, when culturing human PBMCs with DF-hIL-12-Fc si or rhIL-12, the production of IFNγ increases, and treatment with 5 μg / ml of PHA simultaneously amplifies the magnitude of the IFNγ response. At the concentrations examined, IFN-γ production after IL-12 stimulation is comparable between DF-hIL-12-Fc si and rhIL-12.

[0436] Thus, although the EC50 values for the two cell types and stimulation conditions differ by more than an order of magnitude, comparable activities of DF-hIL-12-Fc si and rhIL-12 are exhibited in both assays, indicating that the potency of the DF-hIL-12-Fc si construct shows a potency similar to that of native recombinant human IL-12. Example 5 - Concentrations of IL-12, DF-hIL-12-Fc si, and IFNγ in Monkey Plasma after IV Infusion of DF-hIL-12-Fc si or rhIL-12

[0437] After IV infusion of DF-hIL-12-Fc si or rhIL-12, pharmacodynamics (PD) and pharmacokinetics (PK) were evaluated in cynomolgus monkeys.

[0438] Administer 10 μg / kg of DF-hIL-12-Fc si and recombinant human IL-12 to cynomolgus monkeys by IV infusion.

[0439] Use immunoassays 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. Monoclonal antibodies specific for human IL-12p70 are used as solid-phase capture, and detection is completed using an antibody HRP-labeled reporter. Standards and QCs spiked with rhIL-12 or DF-hIL-12-Fc si reference standards and test samples are transferred to the wells of a microtiter plate, and any IL-12p70 present in the samples is bound by the immobilized antibodies on the solid phase. Unbound materials are washed away, and an enzyme-conjugated polyclonal antibody specific for human IL-12p70 is added to the wells. Unbound antibody-enzyme reagent is washed away, and TMB substrate is added to each well. The resulting enzyme reaction produces a blue product that turns yellow when an acid stop solution is added. The color intensity measured in each well is proportional to the amount of rhIL-12 or DF-hIL-12-Fc si bound in the initial step. The plate is read at 450 nm on a SpectraMax microplate reader with data collection software SoftMax Pro Enterprise Edition 4.6, using 540 nm as a reference. The data is converted to a text file and imported / processed in Watson LIMS v.7.2.0.02. Regression is performed using a logistic (auto-estimate) curve fit with a weighting factor of 1.

[0440] An immunoassay (Meso Scale Discovery (MSD)-ELISA-like immunoassay) is also used to detect DF-hIL-12-Fc si, which involves coating an untreated MSD microtiter plate with monkey-adsorbed goat anti-human IgG and incubating at room temperature. The plate is 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 plate is washed, and biotinylated anti-human IL-12 / IL-23p40 is added to the plate as the first detection antibody. After another wash step, streptavidin-conjugated sulfo-tag (Sulfo-Tag) is added as the second detection antibody. The plate is washed one last time, MSD read buffer T is added to the plate, and the plate is read using an MSD Sector Imager S600. The raw MSD data is exported as a text file and then converted to a Watson LIMS-compatible file using an Envigo custom-designed programming Excel spreadsheet. The data is imported and regressed in Watson LIMS software v.7.2.0.02.

[0441] A mesoscale discovery method for the relative quantitative measurement of NHP pro-inflammatory biomarkers in cynomolgus monkey plasma. The method uses a sandwich immunoassay procedure for the relative quantitative measurement of pro-inflammatory panel 1 biomarkers in cynomolgus monkey K2 EDTA plasma (referred to as monkey plasma): IFNγ, IL-1β, IL-2, IL-6, IL-8, and IL-10. The method is based on the MSD non-human primate (NHP) kits 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 employs human capture and detection antibodies that react with cynomolgus monkeys. The kits provide plates pre-coated with capture antibodies on each well of a 96-well multi-spot plate at independent, well-defined spots. The plate is incubated with the monkey plasma sample, washed, and then incubated with detection antibodies (specific for each analyte) conjugated to an electrochemiluminescence (ECL) label (MSD sulfo-tag). The analyte in the sample binds to the capture antibody immobilized on the surface of the working electrode; the bound analyte recruits the detection antibody to complete the sandwich. The plate is washed, and MSD read buffer is added to create an appropriate chemical environment for electrochemiluminescence (ECL). The plate is loaded into an MSD Sector Imager 600 (SI600) instrument, where a voltage is applied to the plate electrodes, causing the captured label to emit light. The instrument measures the emitted light intensity, expressed in relative light units (RLU), to provide a relative quantitative measurement of the analyte in the sample. The raw RLU data is exported as a text file and then converted to a Watson LIMS-compatible file using an Envigo custom-designed programming Excel spreadsheet. Subsequently, the data is imported and regressed in Watson LIMS software v.7.2.0.02.

[0442] Figure 14 Shows the relative plasma concentrations of DF-hIL-12-Fc si and recombinant human IL-12 over time after IV administration. The data indicate 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 generally greater exposure compared to rhIL-12.

[0443] Figure 14Also shown is the relative concentration of IFNγ in monkey plasma after IV administration (PD). The data indicate that the pharmacodynamics of DF-hIL-12-Fc si and rhIL-12 both exhibited activity after IV administration, as evaluated by IFNγ production. However, compared to rhIL-12, DF-hIL-12-Fc si exhibited higher peak activity and a longer duration. Example 6 - Pharmacological Characterization of the Murine Substitute DF-mIL-12-Fc si

[0444] The serum half-life and in vivo pharmacodynamics of a murine IL-12 variant with an extended half-life (designated DF-mIL-12-Fc si) were examined.

[0445] DF-mIL-12-Fc si corresponding to an equimolar amount of 1 μg IL-12 was intravenously injected into non-tumor-bearing Balb / c mice, and the PK / PD profiles were compared with those of IL-12. Naïve Balb / c (n = 6) were intravenously injected with 1 μg DF-mIL-12-Fc si and IL-12 (equimolar to 1 μg IL-12). Blood samples were taken at 0.017, 0.5, 3, 6, 24, 48, 72, 96, 144, and 219 hours after injection. Serum levels of IL-12 and IFNγ were analyzed by ELISA as previously described.

[0446] As Figure 15A and Figure 15B shown and quantified in Table 15, DF-mIL-12-Fc si showed a persistent serum half-life of approximately 30 hours ( Figure 15B , DF-mIL-12-Fc si T 1 / 2 = 29.85 hours), which is 5-fold that of the serum half-life of IL-12 ( Figure 15A ; IL-12T 1 / 2 = 6.05 hours). In addition to the extended half-life, IFNγ production mediated by DF-mIL-12-Fc si (AUC = 916654) was also prolonged compared to that of IL-12 (AUC = 20304).

[0447] Next, the PK / PD profiles of different routes of administration of DF-mIL-12-Fc si were compared. DF-mIL-12-Fc si corresponding to an equimolar amount of 1 μg IL-12 was injected as a single dose into non-tumor-bearing Balb / c mice by intravenous, intraperitoneal, or subcutaneous administration, and the PK / PD profiles were evaluated as described.

[0448] As Figures 15C - 15E shown and quantified in Table 16, intravenous ( Figure 15C ), intraperitoneal (Figure 15D ) or subcutaneous Figure 15E ) administration both result in comparable DF-mIL-12-Fc si-mediated IFNγ production between different administration routes. Notably, subcutaneous administration results in a lower IL-12 Cmax. Thus, the pharmacokinetic properties (e.g., IL-12 concentration) of DF-mIL-12-Fc si administration vary according to the administration route, while the pharmacodynamic properties (IFNγ production) remain persistent and relatively comparable between different routes. Table 15 - Pharmacological characteristics of DF-mIL-12-Fc si and rmIL-12 Table 16 - Pharmacological characteristics of DF-mIL-12-Fc si via IV, IP, and SC Example 7 - Combination of DF-mIL-12-Fc si and PD-1 blockade in a B16F10 mouse model

[0449] A combination therapy of DF-mIL-12-Fc si and PD-1 blockade was conducted to analyze whether the anti-tumor immune response could be amplified in established B16F10 tumors.

[0450] 10 6 B16F10 melanoma cells were subcutaneously injected into the flanks of C57BL / 6 mice. On day 8 after tumor inoculation, the mice were randomly grouped (n = 10 per group). When the average tumor volume reached approximately 245 mm 3 , the mice were treated intraperitoneally with 0.5 μg of isotype control, 0.5 μg of DF-mIL-12-Fc si, 200 μg of anti-PD-1 clone RMP1-14, or the combination of DF-mIL-12-Fc si / anti-PD-1. DF-mIL-12-Fc si was injected into the animals once a week, and anti-PD-1 was injected into the animals twice a week. Tumor growth was evaluated for 60 days, and survival and body weight were monitored.

[0451] As Figures 16A - 16C shown, although the administration of DF-mIL-12-Fc si alone delayed tumor regression ( Figure 16A ) and the administration of PD-1 alone had a minimal effect on tumor growth ( Figure 16B ), the combination of DF-mIL-12-Fc si and PD-1 blockade further delayed tumor growth ( Figure 16C ), indicating that anti-PD-1 treatment further amplified the anti-tumor response to DF-mIL-12-Fc si treatment.

[0452] AsFigure 17A and Figure 17B As shown, in the case of the combination of DF-mIL-12-Fc si therapy and PD-1 blockade, the overall survival was prolonged, with 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 mice treated with 200 μg anti-PD-1 ( Figure 17A ). Notably, despite the high response rate, the regimens of DF-mIL-12-Fc si and combination therapy appear to be well tolerated in mice bearing B16F10 tumors ( Figure 17B ).

[0453] Thus, the combination therapy of DF-mIL-12-Fc si and PD-1 blockade demonstrated improved efficacy compared to either treatment alone. Example 8 - Combination of DF-mIL-12-Fc si with mcFAE-C26.99TriNKET in a B16F10 mouse model

[0454] 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.

[0455] 10 6 B16F10 melanoma cells were subcutaneously injected into the flanks of C57BL / 6 mice. On day 7 after tumor inoculation, the mice were randomly grouped (n = 10 per group). When the tumor mean reached 200 mm 3 , the mice were treated intraperitoneally with 150 μg isotype control or 0.5 μg DF-mIL-12-Fc si, 150 μg TriNKET, or the combination DF-mIL-12-Fc si / TriNKET. Tumor growth was evaluated for 60 days, and survival and body weight were monitored.

[0456] As Figure 18A shown, monotherapy with DF-mIL-12-Fc si led to a reduction in tumor growth. Treatment with mcFAE-C26.99 TriNKET as a single agent at an initial tumor volume of 200 mm 3 did not result in a delay in tumor progression ( Figure 18B ). In contrast, the combination of DF-mIL-12-Fc si and mcFAE-C26.99 further enhanced the anti-tumor response compared to DF-mIL-12-Fc si alone ( Figure 18C), and produced 30% complete responders (CR) (n = 3), indicating that TriNKET treatment further amplified the anti-tumor response to DF-mIL-12-Fc si treatment.

[0457] As Figure 19A shown, the overall survival was prolonged in the case of DF-mIL-12-Fc si therapy and in combination with mcFAE-C26.99 TriNKET, showing a median survival of 29 days (DF-mIL-12-Fc si monotherapy) and 60 days (TriNKET combination), compared to 16 days for isotype-treated mice and 17 days for TriNKET-treated mice. Notably, despite the high response rate, the regimens of DF-mIL-12-Fc si and combination therapy seemed to be well t...

Claims

1. An heterodimeric Fc fusion protein, said fusion protein comprising: (a) a first polypeptide comprising a first antibody Fc domain polypeptide and a first subunit of a multi-subunit protein; and (b) a second polypeptide comprising a second antibody Fc domain polypeptide and a second different subunit of said multi-subunit protein, wherein said first antibody Fc domain polypeptide and said second antibody Fc domain polypeptide each comprise different mutations that promote heterodimerization, wherein said first antibody Fc domain polypeptide and / or said 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 said multi-subunit protein bind to each other.

2. The heterodimeric Fc fusion protein according to claim 1, wherein said effector function comprises the ability of Fc to induce antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

3. The heterodimeric Fc fusion protein according to claim 1 or 2, wherein said heterodimeric Fc fusion protein is fucosylated.

4. The heterodimeric Fc fusion protein according to any one of claims 1-3, wherein said first antibody Fc domain polypeptide and said second antibody Fc domain polypeptide are human antibody Fc domain polypeptides.

5. The heterodimeric Fc fusion protein according to any one of claims 1-4, wherein said first antibody Fc domain polypeptide and said second antibody Fc domain polypeptide are IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD or IgE Fc domain polypeptides.

6. The heterodimeric Fc fusion protein according to any one of claims 1-5, wherein said first antibody Fc domain polypeptide and / or said second antibody Fc domain polypeptide comprise one or more mutations at one or more positions 233, 234, 235, 236, 237, 297, 318, 320, 322, 329, 330 and / or 331, numbered according to the EU numbering system.

7. The heterodimeric Fc fusion protein according to any one of claims 1-6, wherein said first antibody Fc domain polypeptide and said second antibody Fc domain polypeptide are human IgG1 Fc domain polypeptides.

8. The heterodimeric Fc fusion protein according to claim 7, wherein said first antibody Fc domain polypeptide and / or said second antibody Fc domain polypeptide comprise one or more mutations at one or more positions 234, 235, 237, 329, 330 and / or 331, according to the EU numbering system.

9. The heterodimeric Fc fusion protein according to claim 8, wherein said first antibody Fc domain polypeptide and / or said second antibody Fc domain polypeptide comprise one or more mutations selected from L234A, L235A, L235E, G237A, P329A, A330S and P331S.

10. The heterodimeric Fc fusion protein according to claim 9, wherein the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise the mutations L234A, L235A and P329A.

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