Heterodimeric FC fusion protein

Abstract

The present invention provides Fc fusion protein constructs as monovalent dimers that have a longer serum half-life compared to native / natural molecules, thus advantageous for achieving higher protein titers during production, greater stability during storage, and improved efficacy when used as therapeutics. Also provided are Fc fusion protein constructs with effector function-reducing mutations in the Fc region that have increased tumor growth inhibitory activity and are therefore advantageous when used as cancer therapeutics.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 749,489, filed October 23, 2018, U.S. Provisional Patent Application No. 62 / 781,898, filed December 19, 2018, U.S. Provisional Patent Application No. 62 / 788,499, filed January 4, 2019, U.S. Provisional Patent Application No. 62 / 827,347, filed April 1, 2019, and U.S. Provisional Patent Application No. 62 / 895,889, filed September 4, 2019, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing which has been provided electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on October 22, 2019, is named DFY-063WO_SL.txt, and is 946,355 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates generally to heterodimeric Fc-fusion proteins and pharmaceutical compositions comprising such proteins and methods of use in the treatment of diseases or disorders in human patients. [Background technology]

[0004] background Most physiologically active proteins have the drawback of having a short in vivo half-life. Attempts to overcome this drawback have been made by conjugating them with PEG (polyethylene glycol) or fusing them with an antibody Fc (fragment crystallizable) region. A protein consisting of two or more different subunits, which form a protein complex to exert physiological activity, can be fused with a wild-type Fc domain to prepare an Fc fusion protein form in which the two or more different subunits form a homodimer due to the homodimerization of Fc. A protein consisting of two or more different subunits, which form a protein complex to exert physiological activity, can also be fused with a heterodimeric Fc region derived from antibodies of other isotypes, such as IgG1, IgG2, IgG3, and IgG4, to form a heterodimeric Fc fusion protein. Thus, one or more subunits of a protein that is composed of two or more different subunits and exerts physiological activity by forming a protein complex in which the two or more subunits form a heterodimeric Fc variant region may be fused to the terminus of the heterodimeric Fc variant region to form an improved Fc fusion protein form.

[0005] Fc heterodimerization is a technique in which mutations are introduced into the Fc of two different CH3 domains by genetic engineering so that two Fc fragments form heterodimers with minimal sequence variation while having a tertiary structure that is highly similar to that of naturally occurring antibodies (see, for example, U.S. Patent No. 7,695,936). Summary of the Invention [Problem to be solved by the invention]

[0006] The invention described herein provides designs for improving Fc fusion protein configurations in which two subunits of a heterodimeric protein are linked to two Fc domains with different heterodimerization domains by introducing linkers of various lengths or by mutations in the CH2 and CH3 domains of the Fc. [Means for solving the problem]

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

[0008] In one embodiment, 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 and second antibody Fc domain polypeptides each comprise a different mutation that promotes heterodimerization, wherein the first and / or second antibody Fc domain polypeptide comprises one or more mutations that reduce an Fc effector function, and wherein the first subunit and the second, different subunit of the multi-subunit protein are associated with each other.

[0009] In certain embodiments, the effector function includes the ability of the Fc domain polypeptide to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). In certain embodiments, the first and second antibody Fc domain polypeptides are human antibody Fc domain polypeptides. In certain embodiments, the first and second antibody Fc domain polypeptides 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 certain embodiments, the first and / or second antibody Fc domain polypeptides comprise one or more mutations at positions 233, 234, 235, 236, 237, 297, 318, 320, 322, 329, 330, and / or 331 (EU numbering). In certain embodiments, the heterodimeric Fc fusion protein is fucosylated.

[0010] In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 Fc domain polypeptides. In certain embodiments, the first and / or second antibody Fc domain polypeptides comprise one or more mutations at positions 234, 235, 237, 329, 330, and / or 331 (EU numbering). In certain embodiments, the first and / or second antibody Fc domain polypeptides comprise one or more mutations selected from L234A, L235A, L235E, G237A, P329A, A330S, and P331S. In certain embodiments, the first and second antibody Fc domain polypeptides each comprise the mutations L234A and L235A. In certain embodiments, the first and second antibody Fc domain polypeptides each comprise the mutations L234A, L235A, and P329A. In certain embodiments, the first and second antibody Fc domain polypeptides each comprise the mutations L234A, L235E, G237A, A330S, and P331S. In certain embodiments, the first and second antibody Fc domain polypeptides each comprise the mutation C220S.

[0011] In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG4 Fc domain polypeptides. In certain embodiments, the first and / or second antibody Fc domain polypeptides comprise one or more mutations at positions 235 and / or 329 (EU numbering). In certain embodiments, the first and / or second antibody Fc domain polypeptides comprise one or more mutations selected from L235E and P329A. In certain embodiments, the first and second antibody Fc domain polypeptides each comprise the mutation L235E. In certain embodiments, the first and second antibody Fc domain polypeptides each comprise the mutation L235E and P329A. In certain embodiments, the first and second antibody Fc domain polypeptides each comprise the mutation S228P.

[0012] In another embodiment, 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 embodiment, the present invention provides a polypeptide comprising a subunit of a multisubunit cytokine and an immunoglobulin Fc domain polypeptide, wherein the Fc domain polypeptide comprises a mutation that promotes heterodimerization with a different immunoglobulin Fc domain polypeptide and one or more mutations that reduce an Fc effector function. For example, the Fc domain polypeptide comprising the mutations can be a human IgG1 antibody Fc domain polypeptide.

[0014] In certain embodiments, the one or more mutations that reduce Fc effector function are selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, as numbered according to the EU numbering system. For example, in certain embodiments, the mutations that reduce Fc effector function are L234A, L235A, and P329A, as numbered according to the EU numbering system.

[0015] In some embodiments, the heterodimerization-promoting mutations are K360E and K409W, as numbered by the EU numbering system. In other embodiments, the heterodimerization-promoting mutations are Q347R, D399V, and F405T, as numbered by the EU numbering system.

[0016] In some embodiments, the Fc domain polypeptide further comprises a mutation that promotes disulfide bond formation with a different immunoglobulin Fc domain polypeptide. For example, when the heterodimerization mutations are K360E and K409W, in some embodiments, the mutation that promotes disulfide bond formation is Y349C according to the EU numbering system. When the heterodimerization mutations are Q347R, D399V, and F405T, in some embodiments, the mutation that promotes disulfide bond formation is S354C according to the EU numbering system.

[0017] For example, in certain 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 other embodiments, the 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 other embodiments, the 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 certain embodiments, the invention provides a cell comprising an expression vector comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 290 or SEQ ID NO: 291 or 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 and a second, distinct Fc domain polypeptide of an immunoglobulin Fc and a first subunit and a second, distinct subunit of a multi-subunit protein, wherein the first subunit and the second, distinct subunit are linked to each other and to one or more of the N-terminus or C-terminus of the first Fc domain polypeptide and / or the second, distinct Fc domain polypeptide; and wherein the first Fc domain polypeptide and the second Fc domain polypeptide are mutated to promote heterodimeric Fc formation and reduce Fc effector function. In one embodiment, the multisubunit protein is IL-12 (e.g., human IL-12), and the p35 and p40 subunits of IL-12 are linked to each other and to one or more of the N-terminus or C-terminus of a first Fc domain polypeptide and / or a second Fc domain polypeptide in a heterodimeric Fc fusion protein; wherein the first Fc domain polypeptide and the second Fc domain polypeptide are mutated to promote heterodimeric Fc formation and reduce Fc effector function.

[0020] In one embodiment, the invention provides 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 linked to the first antibody Fc domain polypeptide, wherein the first polypeptide further comprises a first subunit of a multi-subunit protein fused to the first antibody Fc domain polypeptide by a linker comprising the amino acid sequence PKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 237) or EPKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 6), where X1 is L or A, X2 is L, E, or A, and X3 is A or G; a second, different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide, and the subunits of the multi-subunit protein are linked to each other; and when X1 is L and / or X2 is L, at least one of the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide comprises a Q347R mutation that promotes heterodimerization.

[0021] In certain embodiments, the linker linking the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc-fusion protein comprises the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9). In certain embodiments, the linker linking the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc-fusion protein consists of the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9).

[0022] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 244). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 244).

[0023] In certain embodiments, the linker joining the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc fusion protein comprises the amino acid sequence PKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 238) or EPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 7). In certain embodiments, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc fusion protein consists of the amino acid sequence PKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 238) or EPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 7).

[0024] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 8) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 241). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 8) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 241).

[0025] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 15) or GGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 242). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 15) or GGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 242).

[0026] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 16) or GGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 243). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 16) or GGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 243).

[0027] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 65) or GGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 245). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 65) or GGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 245).

[0028] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 66) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 246). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 66) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 246).

[0029] In certain embodiments, the linker joining the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc fusion protein comprises the amino acid sequence EPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 11) or PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 240). In certain embodiments, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc fusion protein consists of the amino acid sequence EPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 11) or PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 240).

[0030] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 247). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 247).

[0031] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67) or GGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 248). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67) or GGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 248).

[0032] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 68) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 249). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 68) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 249).

[0033] In another aspect, the invention provides a heterodimeric Fc fusion protein comprising subunits of a multisubunit cytokine joined to an immunoglobulin Fc domain polypeptide by a linker comprising the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239); and the Fc domain polypeptide comprises mutations that promote heterodimerization with a different immunoglobulin Fc domain polypeptide and L234A, L235A, and P329A substitutions to reduce Fc effector function.

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

[0035] In one embodiment, the invention provides 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 a multi-subunit protein, wherein the protein sequence is fused to the first antibody Fc domain polypeptide by a linker comprising the amino acid sequence RVESKYGPPCPPCPAPEFXGG (SEQ ID NO: 1) (wherein X is L or E); a second, different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide, wherein the subunits of the multi-subunit protein are linked to each other, wherein the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise different mutations that promote heterodimerization, and wherein the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide are linked to each other.

[0036] In certain embodiments, the heterodimeric Fc-fusion proteins of the present invention comprise 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 can comprise G4S (SEQ ID NO: 110), (G4S)2 (SEQ ID NO: 109), or (G4S)3 (SEQ ID NO: 108).

[0037] In certain embodiments, within the heterodimeric Fc fusion protein, the linker joining a subunit of the multi-subunit protein to the first antibody Fc domain polypeptide comprises the amino acid sequence RVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 2). In certain 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).

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

[0039] In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13).

[0040] In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 14). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 14).

[0041] In certain embodiments, within the heterodimeric Fc fusion protein, the linker joining a subunit of the multi-subunit protein to the first antibody Fc domain polypeptide comprises the amino acid sequence RVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 4). In certain 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 certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5).

[0043] In certain embodiments, a second, different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63). In certain embodiments, a second, different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63).

[0044] In certain embodiments, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64). In certain embodiments, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64).

[0045] Some heterodimeric Fc-fusion proteins described herein comprise a first IgG4 antibody Fc domain polypeptide and a second, different IgG4 antibody Fc domain polypeptide, each mutated to promote heterodimerization with each other. In certain 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 certain 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 certain embodiments, the first IgG4 antibody Fc domain polypeptide comprises mutations K370E and R409W, and the second, different IgG4 antibody Fc domain polypeptide comprises mutations E357N, D399V, and F405T. In certain 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 certain 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 certain 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 certain 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 one embodiment, 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 heterodimeric Fc-fusion proteins disclosed herein comprise a first IgG1 antibody Fc domain polypeptide and a second, different IgG1 antibody Fc domain polypeptide, each mutated to promote heterodimerization with each other. In certain embodiments, the first IgG1 Fc domain polypeptide comprises one or more mutations selected from K360E and K409W, and the second, different IgG1 Fc domain polypeptide comprises one or more mutations selected from Q347R, D399V, and F405T. In certain embodiments, the first IgG1 Fc domain polypeptide comprises one or more mutations selected from Q347R, D399V, and F405T, and the second, different IgG1 Fc domain polypeptide comprises one or more mutations selected from K360E and K409W. In certain embodiments, the first IgG1 antibody Fc domain polypeptide comprises mutations K360E and K409W, and the second, different IgG1 antibody Fc domain polypeptide comprises mutations Q347R, D399V, and F405T. In one embodiment, the first IgG1 antibody Fc domain polypeptide comprises the mutations Q347R, D399V and F405T, and the second, different IgG1 antibody Fc domain polypeptide comprises the mutations K360E and K409W.

[0047] In certain embodiments, the heterodimeric Fc-fusion proteins described herein comprise an IgG4 or IgG1 Fc domain polypeptide that has been further mutated to reduce effector function. In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise the mutation P329G or P329A. In certain embodiments, the first IgG4 antibody Fc domain polypeptide and the second, different IgG4 antibody Fc domain polypeptide each comprise the mutation P329G or P329A. In certain embodiments, the first IgG1 antibody Fc domain polypeptide and the second, different IgG1 antibody Fc domain polypeptide each comprise the mutation P329G or P329A. In certain embodiments, the first IgG4 antibody Fc domain polypeptide and the second, different IgG4 antibody Fc domain polypeptide each comprise the mutation P329A. In certain embodiments, the first IgG1 antibody Fc domain polypeptide and the second, different IgG1 antibody Fc domain polypeptide each comprise the mutation P329A.

[0048] In certain embodiments, a heterodimeric Fc-based fusion described herein incorporates a first IgG1 antibody Fc domain polypeptide and a second, different IgG1 antibody Fc domain polypeptide, each comprising a mutation selected from A330S and P331S. In certain embodiments, a heterodimeric Fc-based fusion described herein incorporates a first IgG1 antibody Fc domain polypeptide and a second, different IgG1 antibody Fc domain polypeptide, each comprising a mutation selected from A330S and P331S.

[0049] In certain embodiments, the heterodimeric Fc-based proteins described herein incorporate IgG4 or IgG1 Fc domain polypeptides that have been further mutated to introduce an interchain disulfide bond. In certain embodiments, a first IgG4 or IgG1 Fc domain polypeptide comprises the mutation Y349C and a second, different IgG4 or IgG1 Fc domain polypeptide comprises the mutation S354C. In certain embodiments, a first IgG4 or IgG1 Fc domain polypeptide comprises the mutation S354C and a second, different IgG4 or IgG1 Fc domain polypeptide comprises the mutation Y349C.

[0050] Some heterodimeric Fc-fusion proteins of the invention contain a native disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein. For example, in an exemplary embodiment, a heterodimeric Fc-fusion protein of the invention contains the native heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. Such proteins contain the native disulfide bond between C74 of p35 and C177 of p40.

[0051] Some heterodimeric Fc-fusion proteins of the invention comprise an artificial or engineered heterodimeric disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein. For example, in an exemplary embodiment, a heterodimeric Fc-fusion protein of the invention comprises an artificial or engineered heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. Such proteins comprise an artificial or engineered disulfide bond between V185C of p35 and Y292C of p40.

[0052] Some heterodimeric Fc-fusion proteins of the invention contain a native disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein, and an artificial or engineered heterodimeric disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein. For example, in an exemplary embodiment, a native heterodimeric disulfide bond between the p35 and p40 subunits of IL-12 and an artificial or engineered heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. Such proteins contain a native 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.

[0053] A portion of the heterodimeric Fc-fusion proteins of the present invention are engineered to remove native 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 of the present invention comprises IL-12 p35 in which the native C74 has been mutated to serine and IL-12 p40 in which the native C177 has been mutated to serine, thereby eliminating the native disulfide bond between the p35 and p40 subunits of IL-12. Two novel mutations, V185C in P35 and Y292C in P40, are introduced into this mutant IL-12, thereby introducing non-natural, artificial, or engineered disulfide bonds.

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

[0055] In other embodiments, 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, at least one antibody heavy chain variable domain is linked 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, heterodimeric Fc fusion proteins comprising at least one antibody variable domain of the present invention have a first subunit of a multi-subunit protein and a second, different subunit of a multi-subunit protein attached to the C-terminus of a first antibody Fc domain polypeptide and a second antibody Fc domain polypeptide, respectively.

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

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

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

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

[0061] In some embodiments, the heterodimeric Fc-fusion protein of the present invention further comprises a collagen-binding domain. In some embodiments, the collagen-binding domain binds to one or more collagens specifically expressed in 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 present 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 present invention provides a method of treating cancer in a patient. The method comprises administering to the patient, e.g., a patient in need thereof, a formulation comprising an effective amount of a heterodimeric Fc-fusion protein described herein or an effective amount of a multispecific binding protein described herein. For example, in certain embodiments, the method of treating cancer comprises administering to the patient, e.g., a patient in need thereof, a formulation comprising an effective amount of a heterodimeric Fc-fusion protein described herein and a pharmaceutically acceptable carrier.

[0064] In some embodiments, the present invention provides a method for treating cancer, comprising administering to a patient only a single dose of a heterodimeric IL-12-Fc fusion protein. In some embodiments, the single dose is sufficient to induce a complete response against the cancer. In some embodiments, the single dose is sufficient to delay or prevent recurrence of the cancer.

[0065] In certain embodiments, the present invention provides a method of treating cancer, comprising administering to a patient only a single dose of a formulation comprising a heterodimeric IL-12-Fc fusion protein or a heterodimeric IL-12-Fc fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291, and a pharmaceutically acceptable carrier. In certain embodiments, the single dose of a formulation comprising a heterodimeric IL-12-Fc fusion protein or a heterodimeric IL-12-Fc fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291, and a pharmaceutically acceptable carrier, is sufficient to induce a complete response against cancer. In certain embodiments, the single dose is sufficient to delay or prevent recurrence of cancer.

[0066] Another aspect of the present invention provides a method of treating acute radiation syndrome, comprising administering to a patient a heterodimeric Fc-fusion protein or formulation disclosed herein. In certain embodiments, the acute radiation syndrome comprises one or more syndromes selected from hematopoietic radiation syndrome, gastrointestinal radiation syndrome, neurovascular radiation syndrome, and cutaneous radiation syndrome. In certain embodiments, the acute radiation syndrome comprises a syndrome 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 multisubunit proteins that may exhibit longer serum half-lives, improved yields during manufacturing, enhanced stability during storage, and / or improved efficacy when used as therapeutics compared to native / natural multisubunit proteins. [Brief explanation of the drawings]

[0068] [Figure 1A] 1A depicts an exemplary heterodimeric Fc-fusion protein comprising a first subunit of a multi-subunit protein linked by a linker to a first antibody Fc domain polypeptide and a second, different subunit of the multi-subunit protein linked by 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 disulfide bonds between the Fc domain polypeptides stabilize the heterodimer. The linker linking the additional subunit to the second antibody Fc domain polypeptide may contain a (G4S)3 sequence (SEQ ID NO: 108).

[0069] [Figure 1B] FIG. 1B depicts an exemplary heterodimeric Fc fusion protein similar to the protein depicted in FIG. 1A, but also containing mutations in the Fc domain polypeptide to reduce FcγR binding.

[0070] [Figure 1C] FIG. 1C depicts an exemplary protein in which a second, different subunit of a multi-subunit protein is attached to a first antibody Fc domain by a linker and another subunit of the multi-subunit protein is attached to a second antibody Fc domain by another linker.

[0071] [Figure 1D] FIG. 1D depicts an exemplary heterodimeric Fc fusion protein similar to the protein depicted in FIG. 1C, but also containing mutations in the Fc domain polypeptide to reduce FcγR binding.

[0072] [Figure 1E] FIG. 1E depicts an exemplary heterodimeric Fc fusion protein similar to the protein depicted in FIG. 1B, except that a second, different subunit of the multi-subunit protein is located C-terminal to a second antibody Fc domain polypeptide in the second polypeptide.

[0073] [Figure 1F] FIG. 1F depicts an exemplary heterodimeric Fc fusion protein similar to the protein depicted in FIG. 1E, except that a second, different subunit of the multi-subunit protein is located C-terminal to the first antibody Fc domain polypeptide in the first polypeptide.

[0074] [Figure 2A]2A depicts an exemplary heterodimeric Fc-fusion protein comprising a first subunit of a multi-subunit protein attached by a linker to a first antibody Fc domain polypeptide and a second, different subunit of the multi-subunit protein attached by another linker to a second antibody Fc domain polypeptide, where 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 disulfide bonds between the Fc domain polypeptides stabilize the heterodimer. The depicted protein also contains mutations in the Fc domain polypeptides to reduce FcγR binding.

[0075] [Figure 2B] Figure 2B depicts an exemplary heterodimeric Fc-fusion protein comprising a first subunit of a multi-subunit protein linked by a linker to a first antibody Fc domain polypeptide and a second, different subunit of the multi-subunit protein linked by another linker to a second antibody Fc domain polypeptide, where the subunits are linked by one non-native disulfide bond. In the exemplary protein shown, the native disulfide bond has been removed and replaced with an artificial disulfide bond. For example, an IL-12 construct may 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 also contains mutations in the Fc domain polypeptides to reduce FcγR binding.

[0076] [Figure 3A]3A depicts an exemplary heterodimeric Fc-fusion protein comprising a first subunit of a multi-subunit protein linked by a linker to a first antibody Fc domain polypeptide and a second, different subunit of the multi-subunit protein linked by another linker having the same amino acid sequence to a second antibody Fc domain polypeptide, where the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each contain different mutations that promote heterodimerization, and disulfide bonds between the Fc domain polypeptides stabilize the heterodimer.

[0077] [Figure 3B] Figure 3B depicts an exemplary heterodimeric Fc-fusion protein comprising a first subunit of a multi-subunit protein linked by a linker to a first antibody Fc domain polypeptide and a second, different subunit of the multi-subunit protein linked by 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 disulfide bonds between the Fc domain polypeptides stabilize the heterodimer. The linker linking the second, different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide can comprise a (G4S)2 (SEQ ID NO: 109) or G4S (SEQ ID NO: 110) sequence.

[0078] [Figure 4] Figures 4A-4L depict exemplary scFv-fused heterodimeric Fc fusion protein constructs (Figures 4A-4H) and mAb-fused heterodimeric Fc fusion protein constructs (Figures 4I-4L) in which a first subunit of the multi-subunit protein is attached to a first antibody Fc domain polypeptide by a linker and a second, different subunit of the multi-subunit protein is attached to a second antibody Fc domain polypeptide by another linker.

[0079] Figures 4A-4D depict exemplary Fc fusion proteins comprising 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 second scFv can be the same or different (e.g., bind to different antigens or different epitopes of a single antigen). The exemplary Fc fusion proteins depicted in Figures 4A and 4C comprise different pairs of heterodimerized Fc variants. The exemplary Fc fusion proteins depicted in Figures 4B and 4D comprise different pairs of heterodimerized Fc variants. For example, when the heterodimerization mutation comprises a "knobs-into-holes" mutation, Figures 4A and 4D depict exemplary Fc-fusion proteins in which a first polypeptide comprises a "hole" mutation and a second polypeptide comprises a "knobs" mutation; Figures 4B and 4C depict exemplary Fc-fusion proteins in which a first polypeptide comprises a "knobs" mutation and a second polypeptide comprises a "hole" mutation. Figures 4A and 4B depict exemplary Fc-fusion proteins in which the protein sequence of a first subunit of a multi-subunit protein is attached to the first polypeptide; Figures 4C and 4D depict exemplary Fc-fusion proteins in which a second, different subunit of a multi-subunit protein is attached to the first polypeptide. Heterodimeric Fc-fusion proteins with other types of heterodimerization mutations are also contemplated.

[0080] 4E and 4G depict exemplary Fc fusion proteins comprising an scFv linked to the C-terminus of a first antibody Fc domain polypeptide.

[0081] 4F and 4H depict exemplary Fc fusion proteins comprising an scFv linked to the C-terminus of a second antibody Fc domain polypeptide.

[0082] Figures 4I-4L depict exemplary Fc-fusion proteins comprising a first Fab attached to the N-terminus of a first antibody Fc domain polypeptide and a second Fab attached to the N-terminus of a second antibody Fc domain polypeptide. Figures 4I and 4K depict exemplary Fc-fusion proteins in which a first subunit of a multi-subunit protein is attached to the first polypeptide; Figures 4J and 4L depict exemplary Fc-fusion proteins in which a second, different subunit of the multi-subunit protein is attached to the first polypeptide. Figure 4K differs from Figure 4I in having a longer amino acid sequence (e.g., a spacer peptide disclosed herein) connecting the antibody Fc domain polypeptide and the first subunit of the multi-subunit protein; Figure 4J differs from Figure 4L in having a longer amino acid sequence (e.g., a spacer peptide disclosed herein) connecting the antibody Fc domain polypeptide and the first subunit of the multi-subunit protein.

[0083] [Figure 5] Figures 5A-5C are graphs showing tumor growth curves for 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] Figure 6 is a graph showing Kaplan-Meier survival curves for mice inoculated with CT26 tumor cells and treated weekly with rmIL-12, DF-mIL-12-Fc wt, DF-mIL-12-Fc si, or mIgG2a isotype control.

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

[0086] [Figure 8] Figure 8 is a graph showing Kaplan-Meier survival curves for mice inoculated with CT26 tumor cells and treated weekly with DF-mIL-12-Fc wt at the molar equivalent of 1 μg rmIL-12, DF-mIL-12-Fc si at the molar equivalent of 1 μg rmIL-12, DF-mIL-12-Fc wt at the molar equivalent of 0.1 μg rmIL-12, DF-mIL-12-Fc si at the molar equivalent of 0.1 μg rmIL-12, or mIgG2a isotype control.

[0087] [Figure 9] Figures 9A-9C are graphs showing tumor growth curves for individual mice inoculated with B16F10 melanoma cells and treated weekly with rmIL-12 (Figure 9A), DF-mIL-12-Fc wt (Figure 9B), DF-mIL-12-Fc si (Figure 9C), or mIgG2a isotype control.

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

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

[0090] [Figure 12] Figure 12 is a graph showing Kaplan-Meier survival curves for mice inoculated with B16F10 melanoma cells and treated weekly with DF-mIL-12-Fc wt at the molar equivalent of 0.5 μg rmIL-12, DF-mIL-12-Fc si at the molar equivalent of 0.5 μg rmIL-12, DF-mIL-12-Fc wt at the molar equivalent of 0.1 μg rmIL-12, DF-mIL-12-Fc si at the molar equivalent of 0.1 μg rmIL-12, or mIgG2a isotype control.

[0091] [Figure 13] Figure 13A is a graph showing the IL-12 response to treatment with DF-hIL-12-Fc si (DF IL-12-Fc) or recombinant human IL-12 (rhIL-12) using a HEK-Blue IL-12 reporter assay.

[0092] FIG. 13B is a graph showing IFNγ production by 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]Figure 14 is a graph showing the relative plasma concentrations of DF-hIL-12-Fc si, rhIL-12, and IFNγ in cynomolgus monkey K2 EDTA plasma after a single intravenous administration of equimolar amounts of DF-hIL-12-Fc si or wild-type rhIL-12 at 10 μg / kg.

[0094] [Figure 15] Figures 15A-15B are graphs showing the PK / PD profiles of rmIL-12 (Figure 15A) and DF-mIL-12-Fc si (Figure 15B) in naive Balb / c mice. 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. Serum IL-12 and IFNγ levels were analyzed by ELISA. Figure 15C is a graph showing the PK / PD profile of DF-mIL-12-Fc si administered intravenously to naive Balb / c mice. Figure 15D is a graph showing the PK / PD profile of DF-mIL-12-Fc si administered intraperitoneally to naive Balb / c mice. Figure 15E is a graph showing the PK / PD profile of DF-mIL-12-Fc si administered subcutaneously to naive Balb / c mice. Mean serum levels represent the mean ± SEM.

[0095] [Figure 16] Figures 16A-16C are graphs showing tumor growth curves of B16F10 tumor-bearing mice treated with DF-mIL-12-Fc si, anti-PD-1, or their combination. Mice were treated intraperitoneally with 0.5 μg isotype control or 0.5 μg 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). Animals were injected with DF-mIL-12-Fc si once a week and anti-PD-1 twice a week as described above. Tumor growth was assessed for 60 days. Graphs show tumor growth curves for individual mice.

[0096] [Figure 17] Figures 17A-17B are graphs showing the survival and body weight of B16F10 tumor-bearing mice 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 with 0.5 μg DF-mIL-12-Fc si once a week and 200 μg anti-PD-1 or isotype twice a week. Figure 17A shows the Kaplan-Meier survival curve. Figure 17B shows mouse body weight as the mean ± standard deviation.

[0097] [Figure 18] Figures 18A-18C are graphs showing tumor growth curves of B16F10 tumor-bearing mice treated with DF-mIL-12-Fc si, mcFAE-C26.99 TriNKET, or their combination. Mice were treated intraperitoneally with 150 μg isotype control or 0.5 μg DF-mIL-12-Fc si (Figure 18A), isotype control or 150 μg TriNKET (Figure 18B), and isotype control or DF-mIL-12-Fc si / TriNKET (Figure 18C). Animals were injected with DF-mIL-12-Fc si once a week and TriNKET three times a week as described above. Tumor growth was assessed for 72 days. Graphs show tumor growth curves for individual mice.

[0098] [Figure 19] Figures 19A-19B are graphs showing the survival and body weight of B16F10 tumor-bearing mice treated with DF-mIL-12-Fc si, mcFAE-C26.99 TriNKET, or their combination. 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 with 0.5 μg DF-mIL-12-Fc si once a week and 150 μg TriNKET or isotype three times a week. Figure 19A shows the Kaplan-Meier survival curve. Figure 19B shows mouse body weight as the mean ± standard deviation.

[0099] [Figure 20] Figure 20 is a graph showing tumor growth curves of complete responder (CR) mice from the B16F10 tumor model experiment in Figure 18 treated with DF-mIL-12-Fc si / TriNKET combination therapy (n=3) and rechallenged with 2x10 B16F10 melanoma cell implants.

[0100] [Figure 21A] FIG. 21A is a graph showing tumor growth curves for individual mice inoculated with CT26 tumor cells and given a single dose of DF-mIL-12-Fc si or mIgG2a isotype.

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

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

[0103] [Figure 22] Figures 22A-22B are graphs showing tumor growth curves for individual mice inoculated with CT26 tumor cells and administered weekly DF-mIL-12-Fc si or mIgG2a isotype intraperitoneally (IP) (Figure 22A) or subcutaneously (SC) (Figure 22B).

[0104] [Figure 23] FIG. 23 is a graph showing tumor growth curves for individual mice inoculated with B16F10 melanoma cells and given a single dose of DF-mIL-12-Fc si or mIgG2a isotype.

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

[0106] [Figure 25] Figures 25A-25B are graphs showing tumor growth curves for individual mice inoculated with CT26 tumor cells and administered DF-mIL-12-Fc si or mIgG2A isotype at the molar equivalent of 1 μg of rMIL-12 once (Figure 25A) or weekly.

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

[0108] [Figure 27] Figures 27A-27C are graphs showing IFNγ (Figure 27A), CXCL9 (Figure 27B), and CXCL10 (Figure 27C) levels in blood (left) and tumor (right) samples in B16F10 tumor-bearing C57BL / 6 mice 72 hours after a single dose of DF-mIL-12-Fc si.

[0109] [Figure 28]Figures 28A-28C are line graphs 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, and 2741 refer to individual cynomolgus monkey subjects.

[0110] [Figure 29] Figures 29A-29F are line graphs showing IFNγ and IP10 / CXCL10 concentrations in cynomolgus monkeys treated with a single subcutaneous dose of DF-hIL-12-Fc si. Figures 29A, 29C, and 29E show IFNγ concentrations / expression levels in cynomolgus monkeys treated with 1 μg / kg, 2 μg / kg, and 4 μg / kg of DF-hIL-12-Fc si, respectively. Figures 29B, 29D, and 29F show IP10 / CXCL10 concentrations / expression levels in cynomolgus monkeys treated with 1 μg / kg, 2 μg / kg, and 4 μg / kg of DF-hIL-12-Fc si, respectively. 3240, 3241, 3740, and 3741 refer to individual cynomolgus monkey subjects.

[0111] [Figure 30] Figure 30 is a graph showing tumor growth curves for individual mice inoculated with breast cancer cells and administered weekly monotherapy (isotype control, DF-mIL-12-Fc si, Doxil® (chemotherapy) or 10 Gy irradiation) or combination therapy (DF-mIL-12-Fc si in combination with Doxil® or irradiation).

[0112] [Figure 31]Figure 31A is a graph showing tumor growth curves for individual mice inoculated with CT26-Tyrp1 tumor cells and treated (twice weekly) with an isotype control or anti-PD-1 antibody. Figure 31B is a graph showing tumor growth curves for Balb / c mice inoculated with CT26-Tyrp1 tumor cells and treated (twice weekly) with an isotype control or anti-PD-1 antibody. Figure 31B also shows tumor growth curves for individual mice previously treated with anti-PD-1 antibody administered with weekly treatment of 1 μg of DF-mIL-12-Fc si.

[0113] [Figure 32] Figure 32A is a graph showing tumor growth curves for treated (Tr) tumors in individual mice inoculated with CT26-Tyrp1 tumor cells and treated intratumorally once (weekly) with isotype control or DF-mIL-12-Fc si. Figure 32B is a graph showing tumor growth curves for non-treated (NT) CT26-Tyrp1 tumors in individual mice described in Figure 32A.

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

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

[0116] Detailed Description The present invention provides improved heterodimeric Fc-fusion proteins, pharmaceutical compositions comprising such proteins, and therapeutic methods using such proteins and pharmaceutical compositions, including the treatment of cancer.

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

[0118] As used herein, the terms "a," "an," and "the" mean "one or more" and include plural, unless the context clearly indicates otherwise.

[0119] As used herein, the terms "subject" and "patient" refer to an organism treated with the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, etc.), and more preferably include, but are not limited to, 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 exert a beneficial or desired result (e.g., a desired prophylactic or therapeutic effect). An effective amount can be administered in one or more administrations, applications, or doses, and is not intended to be limited to a particular formulation or route of administration. As used herein, the term "treating" includes any effect, e.g., alleviation, reduction, modulation, amelioration, or elimination, that results in an improvement of a condition, disease, disorder, etc., or an improvement in the symptoms thereof.

[0121] As used herein, the term "pharmaceutical composition" refers to a combination of a drug substance with an active or inert carrier to make it particularly suitable for in vivo or ex vivo diagnosis or therapy.

[0122] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, 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 contain 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., with an 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 known to those skilled in the art, "salts" of the compounds of the present invention can be derived from inorganic or organic acids and 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, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Other acids, such as oxalic acid, while not themselves pharmaceutically acceptable, may be used in the preparation of salts useful as intermediates for obtaining the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

[0124] Exemplary bases include alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and bases of formula NW4 + (Wherein, W is C 1-4 This includes, but is not limited to, compounds of the formula (e.g., alkyl).

[0125] Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, phenylpropionate, picrate, pyruvate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. + , NH4 + and NW4 + (Wherein, W is C 1-4 and the anions of the compounds of the present invention associated with suitable cations such as alkyl groups.

[0126] For therapeutic use, the salts of the compounds of the invention are intended to be pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also be useful, for example, in the preparation or purification of a pharmaceutically acceptable compound.

[0127] Throughout the specification, when compositions are described as having, comprising or including particular components, or when processes and methods are described as having, comprising or including particular steps, it is further intended that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods of the invention that consist essentially of or consist of the recited processing steps.

[0128] As a general matter, compositions specified in percentages are by weight unless otherwise specified. Further, if no definition is accompanying a variable, the preceding definition of that variable will control.

[0129] I. Protein The present invention provides Fc-fusion protein constructs comprising the amino acid sequences of multi-subunit proteins. These fusion protein constructs may exhibit longer serum half-lives, improved yields during manufacturing, enhanced stability during storage, and / or improved efficacy when used as therapeutics compared to native / natural multi-subunit proteins.

[0130] IgG1 Fc fusion protein In one embodiment, the present invention provides a heterodimeric IgG1 Fc 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 linked to the first antibody Fc domain, wherein the first polypeptide further comprises a first subunit of a multi-subunit protein fused to the first antibody Fc domain polypeptide by a linker comprising the amino acid sequence PKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 237) or EPKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 6) (wherein X1 is L or A, X2 is L, E, or A, and X3 is A or G); a second, different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide, and the subunits of the multi-subunit protein are linked to each other; and when X1 is L and / or X2 is L, at least one of the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide comprises a Q347R mutation that promotes heterodimerization.

[0131] In one embodiment, within the heterodimeric Fc fusion protein, the linker joining 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), where X1 is L or A, X2 is L, E, or A, and X3 is 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, hi 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, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker comprising the sequence PKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 237) or EPKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 6) (wherein X1 is L or A, X2 is L, E or A, and X3 is A or G) and a spacer peptide. In certain embodiments, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence PKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 237) or EPKSSDKTHTCPPCPAPEX1X2GX3 (SEQ ID NO: 6) (wherein X1 is L or A, X2 is L, E or A, and X3 is A or G). In certain embodiments, the amino acid sequence of the linker connecting a second, different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide is identical to the amino acid sequence of the linker connecting a subunit of the multi-subunit protein to the first antibody Fc domain polypeptide.

[0134] Any of the spacer peptides listed under the heading "Spacer Peptides" can be used. For example, in certain embodiments, the spacer peptide comprises the amino acid sequence set forth in any of SEQ ID NOs: 107-120. In certain embodiments, the spacer peptide consists of the amino acid sequence set forth in any of SEQ ID NOs: 107-120. In certain embodiments, the linker linking a subunit of a multi-subunit protein to a 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 linking a second, different subunit of a multi-subunit protein to a 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 one or both of the linkers.

[0135] In certain embodiments, the linker linking the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc-fusion protein comprises the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9). In certain embodiments, the linker linking the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc-fusion protein consists of the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239) or EPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 9).

[0136] In certain embodiments, the linker linking the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc-fusion protein comprises the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239). In certain embodiments, the linker linking the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc-fusion protein consists of the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239).

[0137] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 244). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 244).

[0138] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10).

[0139] In certain embodiments, the linker joining a subunit of a multi-subunit protein to a first antibody Fc domain polypeptide in a heterodimeric Fc fusion protein comprises the amino acid sequence PKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 238) or EPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 7). In certain embodiments, the linker fusing a first subunit of a multi-subunit protein to a first antibody Fc domain polypeptide in a heterodimeric Fc fusion protein consists of the amino acid sequence PKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 238) or EPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 7).

[0140] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 8) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 241). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 8) or GGGGSGGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 241).

[0141] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 15) or GGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 242). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 15) or GGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 242).

[0142] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 16) or GGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 243). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 16) or GGGGSGGGGSPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 243).

[0143] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 65) or GGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 245). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 65) or GGGGGSPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 245).

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

[0145] In certain embodiments, the linker joining the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc fusion protein comprises the amino acid sequence EPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 11) or PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 240). In certain embodiments, the linker fusing the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric Fc fusion protein consists of the amino acid sequence EPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 11) or PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 240).

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

[0147] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67) or GGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 248). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67) or GGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 248).

[0148] In certain embodiments, within the heterodimeric Fc fusion protein, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 68) or GGGGSGGGGSPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 249). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting 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 from an IgG1 Fc. In certain 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 IgG1 Fc domain polypeptides that promote heterodimerization with each other. For example, if the Fc domain is derived from the Fc of human IgG1, the Fc domain may be at least 90% identical to amino acids 234 to 332 of a human IgG1 antibody and may contain a different amino acid sequence at one or more positions selected from the group consisting of 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 certain embodiments, the antibody constant domain is at least 90% identical to amino acids 234-332 of a human IgG1 antibody and is selected from the group consisting of Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, Q362E, S364K, S364E, S364H, S364D, T366V, 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 domain or hinge region disclosed herein are numbered according to EU numbering.

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

[0152] In certain embodiments, the heterodimeric Fc-fusion proteins of the present invention having an IgG1 Fc comprise one or more mutations in the first and / or second polypeptide that reduce binding to FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB) or a complement component (e.g., C1q). Such mutations are useful for reducing effector function. For example, proteins of the present disclosure comprise a LALA (L234A and L235A) mutation, a LALAPA (L234A, L235A, and P329A) mutation, a LALAPG (L234A, L235A, and P329G) mutation, or a LALEGAASPS (L234A, L235E, G237A, A330S, and P331S) mutation.

[0153] In certain embodiments, the heterodimeric Fc-fusion protein of 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 P329G or P329A. In a specific embodiment, the heterodimeric Fc-fusion protein of 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 certain embodiments, the first IgG1 antibody Fc domain polypeptide and the second, different IgG1 antibody Fc domain polypeptide each comprise a mutation selected from A330S and P331S, In certain embodiments, the first IgG1 antibody Fc domain polypeptide and the second, different IgG1 antibody Fc domain polypeptide each comprise the mutations A330S and P331S.

[0155] In certain embodiments, an additional disulfide bond between IgG1 Fc monomers is introduced, improving the stability of the heterodimer. In an exemplary embodiment, a first antibody Fc domain polypeptide fused to a first subunit of a multi-subunit protein contains a Y349C substitution in the CH3 domain, which forms a disulfide bond with a S354C substitution in a second antibody Fc domain polypeptide fused to a second, different subunit of the multi-subunit protein. Alternatively, a first antibody Fc domain polypeptide fused to a first subunit of a multi-subunit protein contains a S354C substitution in the CH3 domain, which forms a disulfide bond with a Y349C substitution in a second antibody Fc domain polypeptide fused to a second, different subunit of the multi-subunit protein.

[0156] Any of the IgG1 antibody Fc domain polypeptides provided in Table 2 below may be used in combination with any of the IgG1 hinge sequences provided in Table 1 below (which, in the present invention, are part or all of the linker connecting the protein sequence of a first subunit of a multi-subunit protein to a first IgG1 antibody Fc domain polypeptide or the linker connecting an additional subunit to a second, different IgG1 antibody Fc domain polypeptide). Exemplary IgG1 hinge-Fc domain polypeptides are provided in Table 3 below. In certain embodiments, the first and second polypeptides of the Fc fusion protein comprise the amino acid sequences of SEQ ID NOs: 212 and 212; 213 and 214; 215 and 216; 217 and 218; 214 and 213; 216 and 215; or 218 and 217, respectively. In certain embodiments, the first and second polypeptides of the Fc fusion protein comprise the amino acid sequences of SEQ ID NOs: 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.

[0157] IgG4 Fc fusion protein In certain embodiments, the present invention provides an improved multi-subunit protein. In certain embodiments, the present invention provides a heterodimeric IgG4 Fc 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 linked to the first antibody Fc domain polypeptide, wherein the first polypeptide further comprises a first subunit of the multi-subunit protein fused to the first antibody IgG4 Fc domain polypeptide via a linker comprising the amino acid sequence RVESKYGPPCPPCPAPEFXGG (SEQ ID NO: 1), where X is 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 are linked to each other; and the first antibody Fc domain polypeptide and the second antibody IgG4 Fc domain polypeptide each comprise a different mutation that promotes heterodimerization.

[0158] In one embodiment, the linker connecting the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide in the heterodimeric IgG4 Fc fusion protein consists of the amino acid sequence RVESKYGPPCPPCPAPEFXGG (where X is L or E) (SEQ ID NO: 1).

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

[0160] In certain embodiments, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker comprising the sequence RVESKYGPPCPPCPAPEFXGG (where X is L or E) (SEQ ID NO: 1) and a spacer peptide. In certain embodiments, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence RVESKYGPPCPPCPAPEFXGG (where X is L or E) (SEQ ID NO: 1). In certain embodiments, the amino acid sequence of the linker connecting the second different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide is identical to the amino acid sequence of the linker connecting the subunit of the multi-subunit protein to the first antibody Fc domain polypeptide.

[0161] Any spacer peptide described under the heading "Spacer Peptide" can be used. For example, in certain embodiments, the spacer peptide comprises the amino acid sequence set forth in any of SEQ ID NOs: 107-120. In certain embodiments, the spacer peptide consists of the amino acid sequence set forth in any of SEQ ID NOs: 107-120. In certain embodiments, the linker linking the subunits of the multi-subunit protein to the first antibody Fc domain polypeptide consists of or consists essentially of a spacer peptide disclosed herein and SEQ ID NO: 1. In certain embodiments, the linker consists of or consists essentially of a spacer peptide disclosed herein and SEQ ID NO: 1. In certain embodiments, the spacer peptide is N-terminal to the first linker and / or the second linker.

[0162] In certain embodiments, within the heterodimeric Fc fusion protein, the linker joining a subunit of the multi-subunit protein to the first antibody Fc domain polypeptide comprises the amino acid sequence RVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 2). In certain 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 certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 3). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 3).

[0164] In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13).

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

[0166] In certain embodiments, within the heterodimeric Fc fusion protein, the linker joining 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 certain 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 certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5). In certain embodiments, a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5).

[0168] In certain embodiments, a second, different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63). In certain embodiments, a second, different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63).

[0169] In certain embodiments, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64). In certain embodiments, a second different subunit of the multi-subunit protein is fused to the second antibody Fc domain polypeptide by a linker consisting of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64).

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

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

[0172] In certain 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 certain embodiments, the first antibody IgG4 Fc domain polypeptide comprises mutations K370E and R409W, and the second antibody IgG4 Fc domain polypeptide comprises mutations E357N, D399V, and F405T. In certain embodiments, the first antibody IgG4 Fc domain polypeptide comprises mutations E357N, D399V, and F405T, and the second antibody IgG4 Fc domain polypeptide comprises mutations K370E and R409W. In certain embodiments, the first antibody IgG4 Fc domain polypeptide comprises mutations K360E and R409W, and the second antibody IgG4 Fc domain polypeptide comprises mutations Q347R, D399V, and F405T. In one embodiment, 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 certain embodiments, the heterodimeric Fc-fusion proteins of the present invention having an IgG4 Fc comprise one or more mutations in the first and / or second polypeptide that reduce binding to FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB) or a complement component (e.g., C1q). Such mutations are useful for reducing effector function. For example, proteins of the present disclosure comprise the SPLE (S228P and L235E) mutation, the SPLEPA (S228P, L235E, and P329A) mutation, or the SPLEPG (S228P, L235E, and P329G) mutation.

[0174] Any of the IgG4 antibody Fc domain polypeptides provided in Table 2 may be used in combination with any of the IgG4 hinge sequences provided in Table 1 (which, in the present invention, are part or all of a linker connecting a first subunit of a multi-subunit protein to a first IgG4 antibody Fc domain polypeptide or a linker connecting a second, different subunit of a multi-subunit protein to a second, different IgG4 antibody Fc domain polypeptide). Exemplary IgG4 hinge-Fc domain polypeptides are provided in Table 3. In certain embodiments, the first and second polypeptides of the Fc fusion protein comprise the amino acid sequences of SEQ ID NOs: 205 and 205; 206 and 207; 208 and 209; 210 and 211; 207 and 206; 209 and 208; or 211 and 210, respectively. In certain embodiments, the first and second polypeptides of the Fc fusion protein comprise the amino acid sequences of SEQ ID NOs: 219 and 219; 220 and 221; 222 and 223; 224 and 225; 226 and 227; 221 and 220; 223 and 222; 225 and 224; or 227 and 226, respectively.

[0175] Disulfide bonds Some heterodimeric Fc-fusion proteins of the invention contain a native heterodimeric disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein. For example, in an exemplary embodiment, a heterodimeric Fc-fusion protein of the invention contains a native heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. Such proteins contain a native disulfide bond between C74 of p35 and C177 of p40.

[0176] Some heterodimeric Fc-fusion proteins of the invention comprise an artificial or engineered heterodimeric disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein. For example, in an exemplary embodiment, a heterodimeric Fc-fusion protein of the invention comprises an artificial or engineered heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. Such proteins comprise an artificial or engineered disulfide bond between V185C of p35 and Y292C of p40.

[0177] Some heterodimeric Fc-fusion proteins of the invention contain a native heterodimeric disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein, and an artificial or engineered heterodimeric disulfide bond between a first subunit of a multisubunit protein and a second, different subunit of the multisubunit protein. For example, in exemplary embodiments, a protein contains a native heterodimeric disulfide bond between the p35 and p40 subunits of IL-12, and an artificial or engineered heterodimeric disulfide bond between the p35 and p40 subunits of IL-12. Such proteins contain a native 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] A portion of the heterodimeric Fc-fusion proteins of the invention are engineered to remove native disulfide bonds and replace them with non-natural, artificial, or engineered disulfide bonds. For example, in an exemplary embodiment, a heterodimeric Fc-fusion protein of the invention comprises IL-12 p35 in which the native C74 has been mutated to serine and IL-12 p40 in which the native C177 has been mutated to serine, thereby eliminating the native disulfide bond between the p35 and p40 subunits of IL-12. Two novel mutations have been introduced into this mutant IL-12: V185C in P35 and Y292C in P40, thereby introducing non-natural, artificial, or engineered disulfide bonds.

[0179] Sequence of the components of the Fc fusion polypeptide Exemplary heterodimeric Fc-fusion proteins of the invention are constructed using any of the IgG1 or IgG4 Fc variant sequences and any of the corresponding linker sequences set forth below in Tables 1-2. The fusion protein constructs of the invention may provide longer serum half-life compared to native / natural multi-subunit proteins, improved protein yield during manufacturing, enhanced stability during storage, and / or improved efficacy when used as therapeutic agents.

[0180] Tables 4 and 5 list the amino acid sequences of exemplary protein constructs of the present 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 respective N-terminal amino acids of these subunits.

[0181] Any of the IgG4 antibody Fc variant domain polypeptides provided in Table 2 below may be used in combination with any of the IgG4 hinge sequences provided in Table 1 below. Similarly, any of the IgG1 antibody Fc variant domain polypeptides provided in Table 2 below may be used in combination with any of the IgG1 hinge sequences provided in Table 1 below. Exemplary IgG1 hinge-Fc domain polypeptides are provided in Table 3 below. [Table 1] [Table 2-1] [Table 2-2] [Table 2-3] [Table 3-1] [Table 3-2] Table 3-3 Table 3-4 Table 3-5 Table 4-1 Table 4-2 Table 4-3 Table 4-4 Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10 Table 4-11 Table 4-12 Table 4-13 Table 4-14 Table 4-15 Table 4-16 Table 4-17 Table 4-18 Table 4-19 Table 4-20 Table 4-21 Table 4-22 Table 4-23 Table 4-24 Table 4-25 Table 4-26 Table 4-27 Table 4-28 Table 4-29 Table 4-30 Table 4-31 Table 4-32 Table 4-33 Table 4-34 Table 4-35 Table 4-36 Table 4-37 Table 4-38 Table 4-39 Table 4-40 Table 4-41 Table 4-42 Table 4-43 Table 4-44 Table 4-45 Table 4-46 Table 4-47 Table 5-1 Table 5-2 Table 5-3 Table 5-4 Table 5-5 Table 5-6 Table 5-7 Table 5-8 Table 5-9 Table 5-10 Table 5-11 Table 5-12 Table 5-13 Table 5-14 Table 5-15 Table 5-16 Table 5-17 Table 5-18 Table 5-19 Table 5-20 Table 5-21 Table 5-22 Table 5-23 Table 5-24 Table 5-25 Table 5-26 Table 5-27 Table 5-28 Table 5-29 Table 5-30 Table 5-31 Table 5-32 Table 5-33 Table 5-34 Table 5-35 [Table 5-36] [Table 5-37] [Table 5-38] [Table 5-39] [Table 5-40] [Table 5-41]

[0182] IL-12 subunits IL-12 is a multisubunit protein comprising a p40 subunit and a p35 subunit. The amino acid sequence of mature wild-type IL-12 p40 is amino acids 23-328 of GenBank Accession No. NP_002178.2, SEQ ID NO: 127, shown below. The amino acid sequence of mature wild-type IL-12 p35 is amino acids 57-253 of GenBank Accession No. NP_000873.2, SEQ ID NO: 128, shown below. As used herein, the numbering of amino acid residues in p40 and p35 corresponds to the mature wild-type protein sequence. As used herein, an IL-12 p40 subunit comprises an amino acid sequence at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 127. As used herein, an IL-12 p35 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 NOs: 121 and 122; 127 and 128; 201 and 202; 203 and 204; 123 and 124; or 125 and 126, respectively. In certain embodiments, the first polypeptide comprises the amino acid sequence of the p40 subunit of IL-12, and the second polypeptide comprises the amino acid sequence of the p35 subunit of IL-12. In certain embodiments, the first polypeptide comprises the amino acid sequence of the p35 subunit of IL-12, and the second polypeptide comprises the amino acid sequence of the p40 subunit of IL-12.

[0184] In certain embodiments, the present invention provides 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 by a linker; and a second, distinct subunit of IL-12 fused to the second antibody Fc domain polypeptide, wherein the first and second distinct subunits of IL-12 are linked to each other, wherein the first and second antibody Fc domain polypeptides each comprise a different mutation that promotes heterodimerization, wherein the first and second antibody Fc domain polypeptides are linked to each other, and wherein the first subunit of IL-12 is a p40 subunit with a Y292C substitution and the second, distinct subunit of IL-12 is a p35 subunit with a V185C substitution. In certain embodiments, the first and second distinct subunits of IL-12 comprise the amino acid sequences of SEQ ID NOs: 125 and 126, respectively.

[0185] The first subunit and second distinct subunit of IL-12 may be fused to any of the antibody Fc domain polypeptides via any linker disclosed herein to form Fc fusion proteins having sequences including, but not limited to, constructs 120, 120-1, 120-2, 120-3, 120-4, 120-5, 120-6 and 120-7 set forth 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 set forth 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 substituted with S, and C74 in the p35 subunit of IL-12 is substituted with S. In certain embodiments, the p40 and p35 subunits of IL-12 comprise the amino acid sequences of SEQ ID NOs: 123 and 124, respectively.

[0187] The first subunit and second distinct subunit of IL-12 may be fused to any of the antibody Fc domain polypeptides via any linker disclosed herein to form Fc fusion proteins having sequences including, but not limited to, constructs 119, 119-1, 119-2, 119-3, 119-4, 119-5, 119-6, 119-7 and 119-8 set forth 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 set forth in Table 5. [Table 6-1] [Table 6-2] [Table 6-3]

[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 a 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 each Fc domain polypeptide sequence to promote heterodimerization between the Fc domains.

[0189] In certain embodiments, the first polypeptide sequence comprises a first antibody Fc domain polypeptide (human IgG1) sequence comprising K360E and K409W substitutions. In certain embodiments, the second polypeptide sequence comprises a second antibody Fc domain polypeptide (human IgG1) sequence comprising Q347R, D399V, and F405T substitutions. In certain embodiments, the first and second polypeptide amino acid sequences comprise one or more mutations to reduce effector function. In certain embodiments, the heterodimeric Fc fusion protein of the present invention comprises LALAPA (L234A, L235A, and P329A) mutations.

[0190] In one embodiment, in the first polypeptide of the heterodimeric Fc fusion protein of the invention (SEQ ID NO: 290), the p40 subunit of human IL-12 is fused to the first antibody Fc domain polypeptide by a first linker comprising a first amino acid sequence, and in the second polypeptide of the heterodimeric Fc fusion protein of the invention (SEQ ID NO: 291), the p35 subunit of human IL-12 is fused to the second antibody Fc domain polypeptide by 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. [ka]

[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. [ka]

[0193] The first and second polypeptides represented by the amino acid sequences of SEQ ID NO: 290 and SEQ ID NO: 291, respectively, form a disulfide bond via the Y349C mutation (bold and underlined) in the CH3 domain of the first antibody Fc domain polypeptide sequence (human IgG1) in SEQ ID NO: 290 and the S354C mutation (bold and underlined) in the CH3 domain of the second antibody Fc domain polypeptide sequence (human IgG1) in 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, a first antibody Fc domain polypeptide sequence of SEQ ID NO: 290 (human IgG1) comprises K360E and K409W substitutions in the CH3 domain, and a second, different Fc domain polypeptide sequence of SEQ ID NO: 291 (human IgG1) comprises Q347R, D399V and F405T substitutions in the CH3 domain (Fc numbering according to the EU system).

[0195] The first antibody Fc domain polypeptide sequence of SEQ ID NO: 290 and SEQ ID NO: 291 and a second, different Fc domain polypeptide sequence (human IgG1) comprise L234A, L235A and P329A (LALAPA) mutations to reduce 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 a first polypeptide of the invention, a first subunit of the multi-subunit protein is fused in an amino to carboxyl direction 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 disclosed in Table 2), and within a second polypeptide of the invention, a second, different subunit of the multi-subunit protein is fused in an amino to carboxyl direction 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 disclosed in Table 2).

[0198] In certain embodiments, a first subunit of a multi-subunit protein of the invention is fused to a first antibody Fc domain sequence via a linker, wherein the linker comprises or consists of a spacer peptide L1 and the amino acid sequence of SEQ ID NO: 1, 2, 4, 6, 7, 9, 11, 237, 238, 239 or 240. In certain embodiments, a second, different subunit of a multi-subunit protein is fused to a second antibody Fc domain polypeptide via a linker, wherein the linker comprises or consists of a spacer peptide L2 and the amino acid sequence of 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 to 50 amino acid residues. In certain embodiments, L1 consists of 4 to 50 amino acid residues. In certain embodiments, L1 consists of 4 to 20 amino acid residues. In certain embodiments, L2 consists of 4 to 50 amino acid residues. In certain embodiments, L2 consists of about 4 to 20 amino acid residues. In certain embodiments, L1 and L2 each independently consist of about 4 to 50 amino acid residues. In certain embodiments, L1 and L2 each independently consist of 4 to 20 amino acid residues.

[0200] In some embodiments, L1 and L2 have optimized length and / or amino acid composition. In some embodiments, L1 and L2 are the same length and have the same amino acid composition. In other embodiments, L1 and L2 are different.

[0201] In some embodiments, L1 and L2 have the same number of amino acids; in some embodiments, L1 is longer (ie, has more amino acids) than L2; in some embodiments, L1 is shorter (ie, has fewer amino acids) than L2.

[0202] In some embodiments, L1 and / or L2 are "short," e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid residues. Thus, in some cases, the spacer peptide consists of about 12 or fewer amino acid residues. In the case of 0 amino acid residues, the spacer peptide is a peptide bond. In some embodiments, L1 and / or L2 are "long," e.g., 15, 20, or 25 amino acid residues. In some embodiments, the spacer peptide consists of about 3 to about 15, e.g., 8, 9, or 10 consecutive amino acid residues. Regarding the amino acid composition of L1 and L2, the peptides are selected for their properties of imparting flexibility to the first and second polypeptides of the protein of the present invention, not interfering with the binding of the first and second subunits to each other, and resisting protease cleavage. For example, glycine and serine residues generally provide protease resistance. A spacer peptide suitable for linking a first subunit of a 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 a second, different subunit of a 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 be used as part of a linker, such as (GS) n , (GGS) n , (GGGS) n , (GGSG) n , (GGSGG) n and (GGGGS) nIn some embodiments, L1 and / or L2 independently comprise a (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 as part of the linker selected from 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 listed in Table 7. ... n=3 (SEQ ID NO: 108), (GGGGS) n=2 (SEQ ID NO: 109) or (GGGGS) n=1 (SEQ ID NO: 110). [Table 7]

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

[0204] In certain embodiments, only L2 comprises, as part of a linker, the sequence set forth 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, neither L1 nor L2 comprises, as part of a linker, the sequence set forth 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 include a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, where a linker comprising GGGGSGGGSGGGGGS (SEQ ID NO: 118; (G4S)3) joins the first subunit of the multi-subunit protein to the first antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide. Some heterodimeric Fc-fusion proteins of the invention include a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, where a linker comprising GGGGSGGGSGGGGGS (SEQ ID NO: 118; (G4S)3) joins the second, different subunit of the multi-subunit protein to the second antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide.

[0206] In one embodiment, a portion of a heterodimeric Fc-fusion protein of the invention includes a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, where a linker comprising GGGGSGGGSGGGGGS (SEQ ID NO: 118; (G4S)3) joins 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, where the additional subunit is joined to the second antibody Fc domain polypeptide by a linker that does not comprise GGGGSGGGSGGGGGS (SEQ ID NO: 118; (G4S)3).

[0207] In one embodiment, a portion of the heterodimeric Fc-fusion proteins of the invention includes 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 contain GGGGSGGGSGGGGGS (SEQ ID NO: 118; (G4S)3) joins the first subunit of the multi-subunit protein to the Fc domain polypeptide and a second, different subunit of the multi-subunit protein, and a second polypeptide comprising a second antibody Fc domain polypeptide, wherein the second, different subunit of the multi-subunit protein is joined to the second antibody Fc domain polypeptide by a linker that contains GGGGSGGGSGGGGGS (SEQ ID NO: 118; (G4S)3).

[0208] A portion of the heterodimeric Fc fusion proteins of the invention includes a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n=2A linker comprising (GGGGS) (SEQ ID NO: 109) connects the first subunit of the multi-subunit protein to a first antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide. Some heterodimeric Fc-fusion proteins of the invention include a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein (GGGGS) n=2 A linker comprising (SEQ ID NO: 109) connects a second, different subunit of the multi-subunit protein to a second antibody Fc domain polypeptide, for example, an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide.

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

[0210] In one embodiment, a portion of a heterodimeric Fc-fusion protein of the invention is a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n=2 (SEQ ID NO: 109) connects a first subunit of a multisubunit protein to an Fc domain polypeptide, and a second polypeptide comprising a second, different subunit of the multisubunit protein and a second antibody Fc domain polypeptide, wherein the additional subunit of the multisubunit protein is (GGGGS) n=2 (SEQ ID NO: 109) is linked to the second antibody Fc domain polypeptide by a linker that does not include the Fc domain polypeptide of the present invention.

[0211] In one embodiment, a portion of a heterodimeric Fc-fusion protein of the invention is a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n=2 a linker that does not include (SEQ ID NO: 109) connecting a first subunit of a multi-subunit protein to an Fc domain polypeptide and a second, different subunit of the multi-subunit protein, and a second polypeptide comprising a second antibody Fc domain polypeptide, wherein the second, different subunit of the multi-subunit protein is (GGGGS) n=2 (SEQ ID NO: 109) linked to the second antibody Fc domain polypeptide.

[0212] A portion of the heterodimeric Fc fusion proteins of the invention includes a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n=1 A linker comprising (GGGGS) (SEQ ID NO: 110) connects the first subunit of the multi-subunit protein to a first antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide. Some heterodimeric Fc fusion proteins of the invention include a second polypeptide comprising a second, different subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein (GGGGS) n=1 A linker comprising (SEQ ID NO: 110) connects a second, different subunit of the multi-subunit protein to a second antibody Fc domain polypeptide, for example, an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide.

[0213] Some of the heterodimeric Fc fusion proteins of the present disclosure are (GGGGS) n=1(SEQ ID NO: 110), which links a first subunit of the multi-subunit protein to a first antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide, and links a second, different subunit of the multi-subunit protein to a second antibody Fc domain polypeptide, e.g., an IgG4 antibody Fc domain polypeptide or an IgG1 antibody Fc domain polypeptide.

[0214] In one embodiment, a portion of a heterodimeric Fc-fusion protein of the invention is a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n=1 (SEQ ID NO: 110) connects a first subunit of a multisubunit protein to an Fc domain polypeptide, and a second polypeptide comprising a second, distinct subunit of the multisubunit protein and a second antibody Fc domain polypeptide, wherein the second, distinct subunit of the multisubunit protein is (GGGGS) n=1 (SEQ ID NO: 110) and is linked to the second antibody Fc domain polypeptide via a linker that does not contain the Fc domain of the second antibody.

[0215] In one embodiment, a portion of a heterodimeric Fc-fusion protein of the invention is a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n=1 a linker not comprising (SEQ ID NO: 110) connecting a first subunit of a multi-subunit protein to an Fc domain polypeptide and a second, distinct subunit of the multi-subunit protein, and a second polypeptide comprising a second antibody Fc domain polypeptide, wherein the second, distinct subunit of the multi-subunit protein is (GGGGS) n=1 (SEQ ID NO: 110) linked to the second antibody Fc domain polypeptide.

[0216] In one embodiment, a portion of a heterodimeric Fc-fusion protein of the invention is a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n=1 (SEQ ID NO: 110) linker connecting a first subunit of the multi-subunit protein to an Fc domain polypeptide, and a second polypeptide comprising a second, distinct subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second, distinct subunit of the multi-subunit protein is (GGGGS) n=2 (SEQ ID NO: 109) which is linked to the second antibody Fc domain polypeptide.

[0217] In one embodiment, a portion of a heterodimeric Fc-fusion protein of the invention is a first polypeptide comprising a first subunit of a multi-subunit protein and a first antibody Fc domain polypeptide, wherein (GGGGS) n=2 (SEQ ID NO: 109) links a first subunit of the multi-subunit protein to an Fc domain polypeptide, and a second polypeptide comprising a second, distinct subunit of the multi-subunit protein and a second antibody Fc domain polypeptide, wherein the second, distinct subunit of the multi-subunit protein is (GGGGS) n=1 (SEQ ID NO: 110) which links the second antibody Fc domain polypeptide.

[0218] Fc domains and substitutions to promote heterodimerization Assembly of the proteins of the invention can be achieved by expressing in the same cell a first polypeptide comprising a first subunit sequence of a multi-subunit protein fused to a first antibody Fc domain polypeptide (e.g., an IgG4 antibody Fc variant sequence or an IgG1 antibody Fc variant sequence disclosed in Table 2) and a second polypeptide comprising a second, different subunit sequence of a multi-subunit protein fused to a second antibody Fc domain polypeptide (e.g., an IgG4 antibody Fc variant sequence or an IgG1 antibody Fc variant sequence disclosed in Table 2), resulting in assembly of the heterodimeric Fc-fusion proteins of the invention. The assembled protein comprises a heterodimeric Fc domain polypeptide comprising the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide linked to each other. Promotion of preferential assembly of Fc heterodimers can be achieved by incorporating various mutations into the CH3 domain of each antibody heavy chain constant region, as described in US 13 / 494870, US 16 / 028850, US 11 / 533709, US 12 / 875015, US 13 / 289934, US 14 / 773418, US 12 / 811207, US 13 / 866756, US 14 / 647480, and US 14 / 830336. For example, mutations can be made in the CH3 domain based on human IgG1 by incorporating distinct pairs of amino acid substitutions into the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide, which allow these two chains to selectively heterodimerize with each other. The positions of the amino acid substitutions described below are all numbered according to the EU index as in Kabat.

[0219] In one scenario, the amino acid substitutions in the first antibody Fc domain polypeptide replace the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W), and the amino acid substitutions in at least one second antibody Fc domain polypeptide replace the original amino acid with a smaller amino acid selected from alanine (A), serine (S), threonine (T), or valine (V), such that the larger amino acid substitutions (bulges) fit into the surface of the smaller amino acid substitutions (cavities). For example, one antibody Fc domain polypeptide may incorporate a T366W substitution, while the other may incorporate three substitutions including T366S, L368A, and Y407V.

[0220] A first polypeptide comprising a first subunit sequence of a multi-subunit protein of the present invention or a second polypeptide comprising a second, different subunit sequence of a multi-subunit protein can optionally be linked 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 comprising a hinge, CH2, and CH3 domain, with or without a CH1 domain. In certain 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, IgG2, IgG3, or IgG4 constant region. In certain 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 that of an antibody constant region from another mammal, such as a rabbit, dog, cat, mouse, or horse. Compared to the human IgG1 constant region, one or more mutations can be incorporated into the 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, Includes 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 may be introduced into CH1 of the human IgG1 constant region may be at amino acids V125, F126, P127, T135, T139, A140, F170, P171, and / or V173. In certain embodiments, mutations that may be introduced into Cκ of the human IgG1 constant region may be at amino acids E123, F116, S176, V163, S174, and / or T164.

[0222] The amino acid substitutions are selected from the following set of substitutions shown in Table 8: [Table 8]

[0223] Alternatively, the amino acid substitutions are selected from the following set of substitutions shown in Table 9: [Table 9]

[0224] Alternatively, the amino acid substitutions are selected from the following set of substitutions shown in Table 10: [Table 10]

[0225] Alternatively, the polypeptide chains of each polypeptide chain are selected from Table 11. [Table 11]

[0226] Alternatively, at least one amino acid substitution can be selected from the following set of substitutions in Table 12, where the position shown in the first polypeptide column is substituted with any known negatively charged amino acid and the position shown in the second polypeptide column is substituted with any known positively charged amino acid. [Table 12]

[0227] Alternatively, at least one amino acid substitution can be selected from the following set of Table 13, where the position shown in the first polypeptide column is substituted with any known positively charged amino acid and the position shown in the second polypeptide column is substituted with any known negatively charged amino acid. [Table 13]

[0228] Alternatively, amino acid substitutions can be selected from the following set in Table 14: [Table 14]

[0229] Alternatively or additionally, the structural stability of the heterodimeric Fc fusion proteins of the present invention can be enhanced by introducing S354C into either the first or second polypeptide chain and Y349C into the opposite polypeptide chain, which form an artificial disulfide bond within the interface of the two polypeptides.

[0230] In one embodiment, 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 one embodiment, 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 one embodiment, 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 one embodiment, 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 one embodiment, 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 one embodiment, 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 certain 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 one embodiment, 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 one embodiment, 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 one embodiment, 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 one embodiment, 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 one embodiment, 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 certain 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 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 one embodiment, 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 a S354C substitution.

[0244] In one embodiment, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by 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 an IgG1 constant region by O347R, D399V, and F405T substitutions.

[0245] In one embodiment, 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 one embodiment, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by a T366W substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs from the amino acid sequence of an IgG1 constant region by T366S, T368A, and Y407V substitutions.

[0247] In one embodiment, 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 one embodiment, 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 one embodiment, 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 recognize that during protein production and / or storage, the N-terminal glutamic acid (E) or glutamine (Q) can cyclize to form a lactam (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Thus, in certain embodiments, when the N-terminal residue of the amino acid sequence of a polypeptide is E or Q, the amino acid sequence corresponding to E or Q substituted with pyroglutamic acid is also contemplated herein.

[0251] Those skilled in the art will recognize that during protein production and / or storage, the C-terminal lysine (K) of a protein may be removed (e.g., spontaneously or catalyzed by an enzyme present during production and / or storage). Such K removal is often observed in proteins that contain an Fc domain at the C-terminus. Thus, in some embodiments, when the C-terminal residue of the amino acid sequence of a polypeptide (e.g., an Fc domain sequence) is K, the amino acid sequence corresponding to the removed K is also contemplated herein.

[0252] Mutations to reduce effector function In one embodiment, 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 multisubunit protein; and (b) a second polypeptide comprising a second antibody Fc domain polypeptide and a second, different subunit of the multisubunit protein, wherein the first and second antibody Fc domain polypeptides each comprise a different mutation that promotes heterodimerization, wherein the first and / or second antibody Fc domain polypeptides comprise one or more mutations that reduce Fc effector function, and wherein the first subunit and the second, different subunit of the multisubunit protein bind to each other. In one embodiment, the heterodimeric Fc fusion protein disclosed herein comprising one or more mutations that reduce Fc effector function exhibits greater tumor growth inhibitory activity than its counterpart lacking the effector-reducing Fc mutation(s). Contemplated mutations 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 and / or second antibody Fc domain polypeptides contain one or more mutations that reduce the ability of the Fc domain polypeptides to induce antibody-dependent cellular cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP). ADCC and ADCP are generally mediated by Fc receptors. For example, in certain embodiments, the first and second antibody Fc domain polypeptides are human IgG (e.g., human IgG1, human IgG2, human IgG3, or human IgG4) antibody sequences. Fc receptors for human IgG, also known as Fc gamma receptors (FcγR), include, but are not limited to, activating Fc gamma receptors FcγRI (CD64), FcγRIIA (CD32A), FcγRIIIA (CD16 or CD16A), and FcγRIIIB (CD16B), and inhibitory Fc gamma receptor FcγRIIB (CD32B). Thus, in certain embodiments, the heterodimeric Fc-fusion proteins of the invention comprise one or more mutations in the first and / or second polypeptide that reduce binding to an activating FcγR (e.g., FcγRI, FcγRIIA, FcγRIIIA, or FcγRIIIB). In certain embodiments, the heterodimeric Fc-fusion proteins of the invention comprise one or more mutations in the first and / or second polypeptide that increase binding to an inhibitory FcγR (e.g., FcγRIIB).

[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 and Fc region, CD16 binding is mediated by the hinge region and CH2 domain. For example, in human IgG1, interaction with CD16 is primarily concentrated in the amino acid residues Asp265-Glu269, Asn297-Thr299, Ala327-Ile332, Leu234-Ser239 of the CH2 domain and the carbohydrate residue N-acetyl-D-glucosamine (see Sondermann et al., Nature, 406 (6793):267-273). Based on known domains, mutations can be selected to enhance or reduce binding affinity to CD16, such as by using a phage display library or a yeast surface-displayed cDNA library, or can be designed based on the known three-dimensional structure of the interaction.

[0255] As reviewed in Wang et al., Protein Cell (2018) 9(1):63-73, regions including amino acid positions 232-239, 265-270, 296-299, and 325-332 are involved in activating FcγR binding according to the crystal structure of human IgG1 Fc. Wang et al. also disclose that the L235E and F234A / L235A mutations in human IgG4, the L234A / L235A mutation in human IgG1, and the N297 mutation (e.g., N297A, N297Q, N297G, or N297D) in IgG antibodies reduce activating FcγR binding. As disclosed in U.S. Patent 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 and second antibody Fc domain polypeptides comprise a mutation (e.g., a substitution relative to wild-type human IgG1) at one or more positions selected from 233, 234, 235, 297, and 329. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising 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 and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutations L234A and L235A. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutations L234A, L235A, and P329A. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG4 antibody Fc domain polypeptides comprising the mutations L235E and P329A. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutations L235E and P329A.

[0257] In certain embodiments, the first and / or second antibody Fc domain polypeptide comprises one or more mutations that reduce the ability of the Fc domain polypeptide to induce complement-dependent cytotoxicity (CDC). CDC is generally mediated by complement components (e.g., C1q). Accordingly, in certain embodiments, the heterodimeric Fc-fusion proteins of the invention comprise one or more mutations in the first and / or second polypeptide that reduce binding to complement components (e.g., C1q).

[0258] Fc mutations that reduce C1q binding are known in the art. For example, as disclosed in U.S. Patents 5,648,260 and 5,624,821, the 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 Fc positions 270 (e.g., D270A), 322 (K322A), 329 (e.g., P329A), and 331 (e.g., P331A, P331S, or P331G) reduce C1q binding.

[0259] Thus, in certain embodiments, the first and second antibody Fc domain polypeptides 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 and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutations G237A, A330S, P331S, and / or P329A. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutations G237A, A330S, and P331S. In certain embodiments, the first and second antibody Fc domain polypeptides are human IgG1 antibody Fc domain polypeptides comprising the mutation P329A.

[0260] Mutations that reduce ADCC and / or ADCP and mutations that reduce CDC may be combined. In certain embodiments, the first and / or second antibody Fc domain polypeptides comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce ADCC and / or ADCP, and further comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce CDC. In certain embodiments, the first and second antibody Fc domain polypeptides each comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce ADCC and / or ADCP, and further comprise one or more mutations that reduce the ability of the Fc domain polypeptide to induce CDC.

[0261] In certain embodiments, the heterodimeric Fc-fusion proteins of the present invention having an IgG4 Fc comprise one or more mutations in the first and / or second polypeptide that reduce binding to FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB) or a complement component (e.g., C1q). Such mutations are useful for reducing effector function. For example, proteins of the present disclosure may comprise the SPLE (S228P and L235E) mutation, the SPLEPA (S228P, L235E, and P329A) mutation, or the SPLEPG (S228P, L235E, and P329G) mutation.

[0262] In certain embodiments, the heterodimeric Fc-fusion proteins of the present invention having an IgG1 Fc comprise one or more mutations in the first and / or second polypeptide that reduce binding to FcγR (e.g., FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, or FcγRIIIB) or a complement component (e.g., C1q). Such mutations are useful for reducing effector function. For example, proteins of the present disclosure may comprise a LALA (L234A and L235A) mutation, a LALAPA (L234A, L235A, and P329A) mutation, a LALAPG (L234A, L235A, and P329G) mutation, or a LALEGAASPS (L234A, L235E, G237A, A330S, and P331S) mutation.

[0263] In certain embodiments, the heterodimeric Fc-fusion protein of the invention comprises a first antibody IgG4 or IgG1 Fc domain polypeptide and a second antibody IgG4 or IgG1 Fc domain polypeptide comprising the mutation P329G or P329A, respectively.

[0264] In certain embodiments, the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide each comprise a mutation selected from A330S and P331S.

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

[0266] In certain embodiments, in the first polypeptide of the heterodimeric Fc fusion protein of the invention, a first subunit of the multi-subunit protein is fused to a first antibody Fc domain polypeptide by a first linker. In certain embodiments, in the second polypeptide of the heterodimeric Fc fusion protein of the invention, a second, different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker. The 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 and / or 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 and second linkers each comprise the amino acid sequence ESKYGPPCPPCPAPEFXGG (wherein X is L or E) (SEQ ID NO: 280) or SKYGPPCPPCPAPEFXGG (wherein X is L or E) (SEQ ID NO: 281). In some embodiments, the first and second linkers each comprise the amino acid sequence ESKYGPPCPPCPAPEFLGG (SEQ ID NO: 282) or SKYGPPCPPCPAPEFLGG (SEQ ID NO: 283). In some embodiments, the first and second linkers each comprise the amino acid sequence ESKYGPPCPPCPAPEFEGG (SEQ ID NO: 284) or SKYGPPCPPCPAPEFEGG (SEQ ID NO: 285).

[0267] Serum half-life The heterodimeric Fc-fusion proteins of the present invention have pharmacokinetic properties suitable for therapeutic use. For example, in some embodiments, the heterodimeric Fc-fusion proteins of the present invention have a serum half-life of at least about 50 hours. In some embodiments, the heterodimeric Fc-fusion proteins of the present invention have 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 a heterodimeric Fc-fusion protein of the invention is at least 10% of the serum concentration of the protein of the invention 1 hour after administration to said subject.

[0269] In certain embodiments, heterodimeric Fc-fusion proteins of the invention have a serum half-life that is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% longer than a multi-subunit protein that is not fused to an Fc domain polypeptide. In certain embodiments, heterodimeric Fc-fusion proteins comprising the protein sequence of a multi-subunit protein of the invention have a serum half-life that is at least 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, or 20-fold longer than a multi-subunit protein that is not fused to an Fc domain polypeptide.

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

[0271] In certain embodiments, the proteoglycan binding domain contained in the heterodimeric Fc-fusion protein binds to one or more proteoglycans specifically expressed in tumors (e.g., on the surface of tumor cells, in the pericellular matrix of tumors, or in the extracellular matrix of tumors). In certain embodiments, the collagen binding domain contained in the heterodimeric Fc-fusion protein binds to one or more collagens specifically expressed in tumors (e.g., on the surface of tumor cells, in the pericellular matrix of tumors, or in the extracellular matrix of tumors). Such heterodimeric Fc-fusion proteins may be concentrated in tumors after administration (e.g., intravenous, subcutaneous, or pulmonary administration), enhancing tumor retention and thereby allowing for lower doses and / or more frequent administration.

[0272] In some embodiments, the heterodimeric Fc-fusion proteins of the present invention further comprise a proteoglycan-binding domain that binds to one or more proteoglycans selected from syndecan, chondroitin sulfate proteoglycan 4 (CSPG4), betaglycan, phosphacan, glypican, perlecan, agrin, collagen (e.g., collagen IX, IXI, XV, or XVIII), hyalectan, aggrecan, versican, neurocan, brevican, and small leucine-rich proteoglycans (SLRPs). 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), perlecan, versican, brevican, and SLRPs (e.g., decorin, biglycan, asporin, fibromodulin, and lumican). Thus, in certain embodiments, the proteoglycan binding domain comprised 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), perlecan, versican, brevican, and SLPR. In certain embodiments, the proteoglycan binding domain comprised in the heterodimeric Fc-fusion protein binds to one or more SLPRs 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 its antigen-binding fragment), a peptide (e.g., a portion of a proteoglycan binding protein or a variant thereof), an aptamer, a small molecule, or a combination thereof. Proteoglycan binding domains are also known in the literature. For example, syndecan binding domains are disclosed in U.S. Patents 6,566,489, 8,647,828, and 10,124,038; U.S. Patent Application Publication No. 2009 / 0297479; and PCT Patent Application Publication No. WO2018199176A1. CSPG4 binding domains are disclosed in U.S. Patents 9,801,928 and 10,093,745; and U.S. Patent Application Publication Nos. 2016 / 0032007, 2017 / 0342151, and 2018 / 0072811. β-glycan binding domains are disclosed in U.S. Patent 7,455,839. Glypican binding domains are disclosed in U.S. Patents 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 / 012 3998, 2018 / 0244805, 2018 / 0230230, and 2018 / 0346592; European Patent 2270509; and PCT Patent Application Publications WO2017053619A1, WO2018026533A1, WO2018165344A1, and WO2018199318A1. Perlecan-binding domains are disclosed in U.S. Patent 10,166,304. Decorin-binding domains are disclosed in U.S. Patent 6,517,838 and PCT Patent Application Publications WO2000021989A1, WO2000077041A2, and WO2000078800A2.

[0274] In some embodiments, the heterodimeric Fc-fusion protein of the present invention further comprises a collagen-binding domain. Collagen is a group of proteins, with at least 28 different types identified in vertebrates. Each type of collagen has its own unique structural characteristics and distribution pattern, as described in Fang et al., Tumor Biol. (2014) 35:2871-82 and Xiong et al., J. Cancer Metasta. Treat. (2016) 2:357-64. Various types of collagen, including Col3A1, Col5A2, Col6, Col7A1, Col15A1, Col19A1, and Col22A1, are involved in cancer. The collagen-binding domain can be a protein (e.g., an antibody or its antigen-binding fragment), 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. Patents 5,788,966, 5,587,360, 5,851,794, 5,741,670, 5,849,701, 6,288,214, 6,387,663, 6,908,994, 7,169,902, 7,488,792, 7,820,401, 8,956,612, 8,642,728, and 8,906,649 and U.S. Patent Application Publication Nos. 2007 / 0161062, 2009 / 0142345, and 2012 / 0100106.

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

[0276] If present, the proteoglycan-binding domain, collagen-binding domain, and / or hyaluronic acid-binding domain may be located at any position in the heterodimeric Fc-fusion protein. For example, in certain embodiments, when the IL-12 subunit is located N-terminal to the antibody Fc domain polypeptide, the proteoglycan-binding domain, collagen-binding domain, and / or hyaluronic acid-binding domain disclosed herein may be fused to the C-terminus of the first antibody Fc domain polypeptide and / or the C-terminus of the second antibody Fc domain polypeptide. In certain embodiments, when the IL-12 subunit is located C-terminal to the antibody Fc domain polypeptide, the proteoglycan-binding domain, collagen-binding domain, and / or hyaluronic acid-binding domain disclosed herein may be fused to the N-terminus of the first antibody Fc domain polypeptide and / or the N-terminus of the second antibody Fc domain polypeptide.

[0277] If present, the proteoglycan-binding domain, collagen-binding domain, and / or hyaluronic acid-binding domain may be linked to the remainder of the heterodimeric Fc-fusion protein via a linker. In some embodiments, the proteoglycan-binding domain is fused to the remainder of the heterodimeric Fc-fusion protein via a peptide linker. In some embodiments, the peptide linker comprises a spacer peptide disclosed herein.

[0278] II. Manufacturing method The proteins of the present invention can be produced using recombinant DNA technology well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first polypeptide comprising a first subunit sequence of a multi-subunit protein fused to a first antibody Fc domain polypeptide can be cloned into a first expression vector; a second nucleic acid sequence encoding a second polypeptide comprising a second, different subunit of the multi-subunit fused to a second antibody Fc domain polypeptide can be cloned into a second expression vector; and the first and second expression vectors can then be stably transfected together into a host cell to produce the multimeric protein.

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

[0280] Clones can be cultured under conditions suitable for bioreactor scale-up and sustained expression of the proteins of the invention. 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.

[0281] III. Pharmaceutical Compositions The present invention also relates to pharmaceutical compositions comprising 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 additives or carriers can also be included in the composition for appropriate formulation. Suitable formulations for use in the present invention can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).

[0282] The intravenous drug delivery formulation of the present invention may be contained in a bag, pen, or syringe. In some embodiments, the bag may be connected to a fluid path including tubing and / or a needle. In some embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In some embodiments, the formulation may be freeze-dried (lyophilized) and contained in approximately 12 to 60 vials. In some embodiments, the formulation may be freeze-dried, and 45 mg of the freeze-dried formulation may be contained in one vial. In some embodiments, about 40 mg to about 100 mg of the freeze-dried formulation may be contained in one vial. In some embodiments, 12, 27, or 45 vials of the freeze-dried formulation are combined to obtain a therapeutic dose of protein for the intravenous drug formulation. In some embodiments, the formulation is a liquid formulation and may be stored at about 250 mg / vial to about 1000 mg / vial. In some embodiments, the formulation is a liquid formulation and may be stored at about 600 mg / vial. In some embodiments, the formulation is a liquid formulation and may be stored at about 250 mg / vial.

[0283] The heterodimeric Fc-fusion proteins of the present invention are contained in a liquid, aqueous pharmaceutical formulation comprising an effective amount of the protein in a buffered solution to form the formulation.

[0284] These compositions can be sterilized by conventional sterilization techniques or sterile filtered. The resulting aqueous solutions can be packaged as is or lyophilized, with the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation is generally 3 to 11, more preferably 5 to 9 or 6 to 8, and most preferably 7 to 8, e.g., 7 to 7.5. The resulting solid compositions can be packaged as multiple single-dose units, each containing a fixed amount of one or more of the above-mentioned agents. The solid compositions can also be packaged in containers that allow for flexible quantities.

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

[0286] In certain embodiments, aqueous formulations are prepared containing the heterodimeric Fc-fusion proteins of the present disclosure in a pH-buffered solution. The buffers of the present invention 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 may have a pH ranging from about 5.0 to about 5.2. pH values ​​intermediate to the above pH ranges are also intended to be part of the present invention. For example, ranges of values ​​using any combination of the values ​​listed above as upper and / or lower limits are intended to be encompassed. Examples of buffers that control the pH within this range include acetate (e.g., sodium acetate), succinate (e.g., sodium succinate), gluconate, histidine, citrate, and other organic acid buffers.

[0287] In some embodiments, the formulation includes a buffer system containing citric acid and phosphate to maintain a pH in the range of about 4 to about 8. In some embodiments, the pH range can be about 4.5 to about 6.0, about pH 4.8 to about 5.5, or about pH 5.0 to about 5.2. In some embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In some embodiments, the buffer system includes about 1.3 mg / mL citric acid (e.g., 1.305 mg / mL), about 0.3 mg / mL sodium citrate (e.g., 0.305 mg / mL), about 1.5 mg / mL disodium phosphate dihydrate (e.g., 1.53 mg / mL), about 0.9 mg / mL sodium dihydrogen phosphate dihydrate (e.g., 0.86 mg / mL), and about 6.2 mg / mL sodium chloride (e.g., 6.165 mg / mL). In some embodiments, the buffer system comprises 1-1.5 mg / mL citric acid, 0.25-0.5 mg / mL sodium citrate, 1.25-1.75 mg / mL disodium phosphate dihydrate, 0.7-1.1 mg / mL sodium dihydrogen phosphate dihydrate, and 6.0-6.4 mg / mL sodium chloride. In some embodiments, the pH of the formulation is adjusted with sodium hydroxide.

[0288] A polyol, which can act as an isotonicity agent and stabilize the antibody, can also be included in the formulation. 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, the amount of monosaccharides (e.g., mannitol) added can be smaller than the amount of disaccharides (e.g., trehalose). In certain embodiments, the polyol that can be used as an isotonicity 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 mannitol concentration can be about 7.5 to 15 mg / mL. In certain embodiments, the mannitol concentration can be about 10 to 14 mg / mL. In certain embodiments, the mannitol concentration can be about 12 mg / mL. In certain embodiments, the polyol sorbitol can be included in the formulation.

[0289] A surfactant or surface-active substance may also be added to the formulation. Exemplary surfactants include non-ionic surfactants such as polysorbates (e.g., polysorbate 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of surfactant added is such that it reduces aggregation of the formulated antibody and / or minimizes particle formation in the formulation and / or reduces adsorption. In certain embodiments, the formulation may contain a surfactant that is a polysorbate. In certain embodiments, the formulation may contain the surfactant polysorbate 80 or Tween 80. Tween 80 is the name used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). In certain embodiments, the formulation may contain about 0.1 mg / mL to about 10 mg / mL of polysorbate 80 or about 0.5 mg / mL to about 5 mg / mL. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

[0290] In some embodiments, the protein product of the present invention is formulated as a liquid formulation. The liquid formulation can be present at a concentration of 10 mg / mL in a USP or Ph Eur Type I 50R vial equipped with a rubber stopper and sealed with an aluminum crimp seal. The stopper is made of an elastomer that complies with USP and Ph Eur. In some embodiments, 61.2 mL of protein product solution is placed in the vial, allowing for an extractable volume of 60 mL. In some embodiments, the liquid formulation can be diluted with 0.9% saline solution.

[0291] In some embodiments, the liquid formulation of the present invention can be prepared as a 10mg / mL solution by combining with stabilizing sugar levels.In some embodiments, the liquid formulation can be prepared in an aqueous carrier.In some embodiments, the stabilizer can be added in an amount that does not exceed the amount that can cause undesirable or inappropriate viscosity for intravenous administration.In some embodiments, the sugar can be a disaccharide, such as sucrose.In some embodiments, the liquid formulation can also include one or more of buffering agent, surfactant and preservative.

[0292] In some embodiments, the pH of the liquid formulation can be adjusted by adding a pharmaceutically acceptable acid and / or base. In some embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In some embodiments, the base can be sodium hydroxide.

[0293] In addition to aggregation, deamidation is a common denaturation of peptides and proteins that can occur during fermentation, harvesting / cell clarification, purification, drug substance / drug product storage, and sample analysis. Deamidation is the loss of NH3 from proteins, forming a succinimide intermediate that undergoes hydrolysis. The succinimide intermediate results in a 17-dalton mass loss from the parent peptide. Subsequent hydrolysis results in an 18-dalton mass increase. Isolation of the succinimide intermediate is difficult due to its instability under aqueous conditions. Therefore, deamidation is generally detectable as a 1-dalton mass increase. Deamidation of asparagine results in asparagine or isoaspartic acid. Parameters that affect the rate of deamidation include pH, temperature, solvent dielectric constant, ionic strength, primary sequence, local polypeptide conformation, and three-dimensional structure. The amino acid residue adjacent to Asn in the peptide chain affects the deamidation rate. Gly and Ser following Asn in the protein sequence are highly susceptible to deamidation.

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

[0295] Aqueous carriers of interest herein are those that are pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

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

[0297] An intravenous (IV) formulation may be the preferred route of administration in certain cases, such as when a patient is hospitalized after transplant and receives all medication via the IV route. In some embodiments, the liquid formulation is diluted with 0.9% sodium chloride solution prior to administration. In some embodiments, the diluted drug product for injection is isotonic and suitable for administration via intravenous infusion.

[0298] In some embodiments, salts or buffer components may be added in amounts of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from a variety of known acids (inorganic and organic) and "base-forming" metals or amines. In some embodiments, the buffer may be a phosphate buffer. In some embodiments, the buffer may be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may serve as counterions.

[0299] Preservatives may optionally be added to the formulations herein to inhibit bacterial activity. The addition of a preservative may, for example, facilitate the production of a multi-use (multi-dose) formulation.

[0300] Aqueous carriers of interest herein are those that are pharmaceutically acceptable (safe and non-toxic for administration to humans) and useful for preparing liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

[0301] The protein of the present disclosure can be in a lyophilized formulation comprising the protein and a cryoprotectant. The cryoprotectant can be a sugar, for example, a disaccharide. In some embodiments, the cryoprotectant can be sucrose or maltose. The lyophilized formulation can also comprise one or more of a buffer, a surfactant, a bulking agent, and / or a preservative.

[0302] The amount of sucrose or maltose useful for stabilizing a lyophilized drug product can be at least a 1:2 protein:sucrose or maltose weight ratio. In some embodiments, the protein:sucrose or maltose weight ratio can be 1:2 to 1:5.

[0303] In some embodiments, the pH of the formulation can be adjusted by adding a pharmaceutically acceptable acid and / or base before lyophilization. In some embodiments, the pharmaceutically acceptable acid can be hydrochloric acid. In some embodiments, the pharmaceutically acceptable base can be sodium hydroxide.

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

[0305] In some embodiments, salts or buffer components may be added in amounts of 10 mM to 200 mM. The salts and / or buffers are pharmaceutically acceptable and are derived from a variety of known acids (inorganic and organic) and "base-forming" metals or amines. In some embodiments, the buffer may be a phosphate buffer. In some embodiments, the buffer may be a glycinate, carbonate, or citrate buffer, in which case sodium, potassium, or ammonium ions may serve as counterions.

[0306] In some embodiments, a "bulking agent" may be added. A "bulking agent" is a compound that increases the volume of the lyophilization mixture and contributes to maintaining the physical form of the lyophilized cake (e.g., facilitating the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. The lyophilized formulations of the present invention may contain such bulking agents.

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

[0308] In some embodiments, the lyophilized drug product may be comprised of an aqueous carrier, where the flavor-masking aqueous carrier is pharmaceutically acceptable (e.g., safe and non-toxic for human administration) and useful for preparing a liquid formulation after lyophilization. Exemplary diluents include SWFI, BWFI, pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solution, Ringer's solution, or dextrose solution.

[0309] In certain embodiments, the lyophilized drug product of the invention is reconstituted with USP SWFI, or USP 0.9% Sodium Chloride Injection. During reconstitution, the lyophilized powder dissolves in solution.

[0310] In one embodiment, the lyophilized protein product of the present invention is made up in about 4.5 mL water for injection and diluted with 0.9% saline solution (sodium chloride solution).

[0311] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without being toxic to the patient.

[0312] A specific dose may be a uniform dose for each patient, e.g., 50-5000 mg of protein. Alternatively, a patient's dose may be adapted to the patient's approximate body weight or surface area. Other factors determining the appropriate dosage may include the disease or condition being treated or prevented, disease severity, route of administration, and the patient's age, sex, and medical condition. Further refinement of the calculations necessary to determine the appropriate dosage for treatment can be readily accomplished by one of skill in the art, particularly in light of the dosage information and assays disclosed herein. Dosages may also be determined through the use of known assays for determining dosages used in conjunction with appropriate dose-response data. Dosages for individual patients may be adjusted by monitoring disease progression. Patient blood levels of the targetable construct or complex can be measured to determine whether dosage adjustments are necessary to reach or maintain effective concentrations. Pharmacogenetics can be used to determine which targetable constructs and / or complexes and their dosages are most likely to be effective in an individual (Schmitz et al., Clinica Chimica Acta 308: 43-53, 2001; Steimer et al., Clinica Chimica Acta 308: 33-41, 2001).

[0313] Generally, the dosage based on body weight is about 0.01 μg to about 100 mg / kg body weight, e.g., 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 g~about 10μg / kg body weight, about 0.1μg~about 1μg / kg body weight, about 1μg~about 100mg / kg body weight, about 1μg~about 50mg / kg body weight, about 1μg~about 10mg / kg body weight, about 1μg~about 1mg / kg body weight, about 1μg~about 10 0 μg / kg body weight, about 1 μg to about 50 μg / kg body weight, about 1 μg to about 10 μg / kg body weight, about 10 μg to about 100 mg / kg body weight, about 10 μg to about 50 mg / kg body weight, about 10 μg to about 10 mg / kg body weight, about 10 μg to about 1 mg / kg body weight, about 10 μg to about 100 μg / kg body weight, about 10 μg to about 50 μg / kg body weight, about 50 μg to about 100 mg / kg body weight, about 50 μg to about 50 mg / kg body weight, about 50 μg to about 10 mg / kg body weight, about 50 μg to about 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, and about 50 mg to about 100 mg / kg body weight.

[0314] Doses may be administered once or more daily, weekly, monthly, or yearly, or once every 2 to 20 years. Those skilled in the art can easily estimate repetition rates for administration based on the measured residence time and concentration of the targetable construct or complex in bodily fluids or tissues. Administration of the present invention may be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, or intracavity via perfusion through a catheter or direct intralesional injection. Administration may be once or more daily, once or more weekly, once or more monthly, or once or more yearly. In certain embodiments, the heterodimeric Fc fusion protein (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) is administered intratumorally. In certain embodiments, the heterodimeric Fc fusion protein is administered intratumorally at a dose or frequency less than systemically. In certain embodiments, the heterodimeric Fc fusion protein is administered intratumorally after administration of local anesthesia. In certain embodiments, the heterodimeric Fc fusion protein is administered intratumorally under direct palpation of the tumor mass.

[0315] IV. Therapeutic applications The present invention provides methods for treating cancer using the heterodimeric Fc fusion binding proteins described herein and / or the pharmaceutical compositions described herein. The methods can be used to treat a variety of cancers by administering to a patient in need thereof an effective amount of the heterodimeric Fc fusion proteins described herein. In certain embodiments, the cancer treated by the methods disclosed herein is a locally advanced malignant tumor. In certain embodiments, the locally advanced malignant tumor can be completely resected. In certain embodiments, the locally advanced malignant tumor is completely resected, and the treatment of the present invention is provided following the resection.

[0316] In certain embodiments, the heterodimeric Fc fusion proteins of the present invention (e.g., heterodimeric Fc fusion proteins comprising an IL-12 subunit) are used as monotherapy to treat advanced malignancies. In certain embodiments, the heterodimeric Fc fusion proteins of the present invention (e.g., heterodimeric Fc fusion proteins comprising an IL-12 subunit) are used as adjuvants for active immunotherapy of severe infectious diseases. In certain embodiments, the heterodimeric Fc fusion proteins of the present invention (e.g., heterodimeric Fc fusion proteins comprising an IL-12 subunit) are used as adjuvants for prophylactic vaccination. In certain embodiments, the heterodimeric Fc fusion proteins of the present invention are used to treat post-radiotherapy myelosuppression.

[0317] The present invention also provides a method for reducing hematopoietic toxicity. Hematopoietic toxicity can be due to genetic, infectious, or environmental causes, including, but not limited to, radiation and chemotherapy. For example, in certain embodiments, the present invention provides a method for treating a disease or disorder associated with irradiation (e.g., ionizing radiation, alpha radiation, beta radiation, gamma radiation, X-ray radiation, or neutron radiation). For example, in certain embodiments, the present invention provides a method for suppressing bone marrow suppression after radiation therapy, comprising administering to a patient in need thereof an effective amount of a heterodimeric Fc-fusion protein or formulation described herein. In certain embodiments, the radiation therapy dose is at least 1 Gy, 5 Gy, 10 Gy, 15 Gy, or 20 Gy. In certain embodiments, radiation therapy damages a system, organ, or tissue selected from the group consisting of bone marrow, lymphatic system, immune system, mucosal tissue, mucosal immune system, digestive system, cardiovascular system, nervous system, reproductive organs, prostate, ovaries, lungs, kidneys, skin, and brain. In certain embodiments, the method of the present invention reduces damage.

[0318] The methods provided herein are useful for treating myelosuppression caused by radiation. Accordingly, in certain embodiments, the present invention provides a method for treating myelosuppression that occurs in the setting of accidental exposure to radiation, comprising administering to a patient in need thereof a therapeutically effective amount of a heterodimeric Fc-fusion protein or formulation described herein.

[0319] In certain embodiments, the present invention provides a method for treating acute radiation syndrome (ARS), comprising administering to a patient in need thereof an effective amount of a 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 due, at least in part, to depletion of the hematopoietic stem cell pool and manifests as lymphopenia and granulocytopenia. Gastrointestinal syndrome is due, at least in part, to damage to crypt-located stem and progenitor cells and failure to replace cells at the villus surface and manifests as watery diarrhea, dehydration, electrolyte loss, gastrointestinal bleeding, and perforation. In certain embodiments, the treatment methods provided herein are performed during the prodromal phase of ARS. The prodromal phase is the initial phase of acute illness, generally within 1 to 3 days after radiation exposure, characterized by symptoms of nausea, vomiting, anorexia, fever, headache, and / or early skin erythema. In some embodiments, the treatment methods provided herein are administered during the latent phase of ARS, a phase in which symptoms improve but laboratory values ​​show lymphopenia and granulocytopenia, and which can last from several hours to several weeks depending on the radiation dose. Treatment during the prodromal or latent phase can alleviate the onset of symptoms in affected systems, organs, and / or tissues. In some embodiments, the treatment methods provided herein are administered during the manifest disease phase of ARS. Treatment during this phase can still promote recovery from ARS.

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

[0321] In certain embodiments, the heterodimeric Fc fusion proteins of the present 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 comprises 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 monotherapy 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 monotherapy to a subject diagnosed with melanoma. In certain embodiments, the cancer is breast cancer. In certain embodiments, the heterodimeric Fc-fusion protein is administered as a monotherapy to a subject diagnosed with breast cancer.

[0322] In some embodiments, the present invention provides a method for treating cancer, comprising administering to a patient only a single dose of a heterodimeric IL-12-Fc fusion protein. In some embodiments, the single dose is sufficient to induce a complete response against the cancer. In some embodiments, the single dose is sufficient to delay or prevent recurrence of the cancer.

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

[0324] In certain embodiments, a single dose of a heterodimeric IL-12-Fc fusion protein comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291 or a 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 cancer. In certain embodiments, the single dose is sufficient to delay or prevent recurrence of 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 present invention) is delayed or prevented for 6 to 72 months or longer (e.g., 6 months, 12 months, 24 months, 36 months, 48 ​​months, 60 months, 72 months, 84 months, or 96 months).

[0325] In certain embodiments, the cancer treated with one or more heterodimeric IL-12-Fc fusion proteins (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291) is a metastatic cancer. In certain embodiments, the metastatic cancer is a local, regional, or distant metastatic cancer. In certain embodiments, one or more heterodimeric IL-12-Fc fusion proteins (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291) treat a distant cancer that is not the organ or tissue of origin of the primary cancer and / or that is not the direct target of the treatment regimen via an abscopal effect. In certain embodiments, the abscopal effect of the heterodimeric IL-12-Fc fusion protein is enhanced during and / or after a treatment regimen including radiation and / or chemotherapy. In certain embodiments, one or more heterodimeric IL-12-Fc fusion proteins (e.g., comprising a first polypeptide comprising the amino acid sequence of SEQ ID NO: 290 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 291) treat cancer in a patient by inducing a systemic anti-tumor response, e.g., as determined by increased expression of IFNy, CXCL9, and / or CXCL10 in the patient's serum and / or tumor.

[0326] V. Combination Therapy 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 some embodiments, the heterodimeric Fc fusion protein of the present invention (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) is administered as a combination therapy to treat a subject diagnosed with cancer. In some 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 some embodiments, the cancer is colon cancer. In some embodiments, the heterodimeric Fc fusion protein is administered as a combination therapy to a subject diagnosed with colon cancer. In some embodiments, the cancer is melanoma. In some embodiments, the heterodimeric Fc fusion protein is administered as a combination therapy to a subject diagnosed with melanoma. In some 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 certain embodiments, the heterodimeric Fc fusion proteins of the present invention (e.g., heterodimeric Fc fusion proteins comprising an IL-12 subunit) are used in the treatment of advanced malignancies in combination with other therapeutic agents selected from: cytotoxic chemotherapy; radiation therapy; antibodies targeting molecules involved in anti-tumor immune responses, such as CTLA-4, PD-1, PD-L1, or TGF-β; antibodies that act by ADCC at tumor-associated antigens; multispecific antibodies that bind to NKG2D, CD16, and a tumor-associated antigen, 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.

[0329] In certain embodiments, the heterodimeric Fc fusion proteins of the present invention (e.g., heterodimeric Fc fusion proteins comprising an IL-12 subunit) are used in combination with other therapies, 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, radiation therapy, adoptive NK therapy, stem cell transplant (SCT) therapy, agonist antibodies, chimeric antigen receptor (CAR) T-cell therapy, T-cell receptor (TCR) engineered therapy, multispecific binding protein (TriNKET), agents that induce cellular senescence, and vaccines and / or oncolytic virus therapy, to treat malignancies (e.g., advanced malignancies). In certain embodiments, the heterodimeric Fc-fusion proteins of the present invention are used to treat a malignancy (e.g., an advanced malignancy) in combination with two or more additional therapies selected from 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, radiation therapy, adoptive NK therapy, stem cell transplant (SCT) therapy, agonist antibody, chimeric antigen receptor (CAR) T-cell therapy, T-cell receptor (TCR) engineered therapy, multispecific binding protein (TriNKET), an agent that induces cellular senescence, and a vaccine and / or oncolytic virus therapy.

[0330] In certain embodiments, the heterodimeric Fc fusion proteins of the present invention (e.g., heterodimeric Fc fusion proteins comprising IL-12) are used in combination with cancer vaccines or antibodies targeting PD-1 or PD-L1 to treat locally advanced malignancies that can be completely resected.

[0331] The proteins of the present invention may also be used as an adjunct to surgical removal of the primary lesion.

[0332] The amounts and relative timing of administration of the heterodimeric Fc-fusion protein of the present invention (e.g., a heterodimeric Fc-fusion protein comprising IL-12) and an additional therapeutic agent can be selected to achieve a desired combined therapeutic effect. For example, when administering a combination therapy to a patient in need of such administration, the therapeutic agents in the combination or one or more pharmaceutical compositions comprising the therapeutic agents can be administered in any order, e.g., sequentially, simultaneously, concomitantly, or together. Furthermore, for example, the heterodimeric Fc-fusion protein can be administered at the same time that the additional therapeutic agent is exerting a prophylactic or therapeutic effect, or vice versa.

[0333] As disclosed herein, the methods of the invention involve the co-administration of a combination of a heterodimeric Fc-fusion protein (e.g., a heterodimeric Fc-fusion protein comprising an IL-12 subunit) and an additional therapeutic agent.As disclosed herein, the methods of the invention involve the co-administration of a combination of a heterodimeric Fc-fusion protein comprising an IL-12 subunit and an additional therapeutic agent.

[0334] Co-administration encompasses methods in which the heterodimeric Fc fusion protein (e.g., a heterodimeric Fc fusion protein comprising an IL-12 subunit) and the additional therapeutic agent are administered simultaneously, sequentially, and intermittently or continuously, or any combination of simultaneous, sequential, intermittent, and / or continuous administration of either the heterodimeric Fc fusion protein or the additional therapeutic agent. Those skilled in the art will recognize that intermittent administration is not necessarily the same as continuous, as intermittent administration also includes the initial administration of one agent followed by subsequent administration of that very agent. Furthermore, those skilled in the art will understand that in certain embodiments, intermittent administration also includes sequential administration, as intermittent administration does not include interrupting the initial administration of one agent with the administration of another agent before the first agent is re-administered. Furthermore, those skilled in the art will also recognize that continuous administration can be achieved by numerous routes, including intravenous drip (IV drip) or feeding tube.

[0335] Additionally and more generally, the term "co-administration" encompasses any and all methods in which the individual administration of a heterodimeric Fc-fusion protein and the individual administration of an additional therapeutic agent to a subject overlap in any timeframe.

[0336] The frequency of administration of the heterodimeric Fc-fusion protein or additional therapeutic agent to a subject is known in the art as Qnd or qnd (where n is the frequency in days for consecutive administration of the agent). For example, Q3d is administration of the agent once every three days. In certain embodiments, the method comprises administering either or both of the heterodimeric Fc-fusion protein and / or additional therapeutic agent, or a combination thereof, to a subject at Qld, Q2d, Q3d, Q4d, Q5d, Q6d, Q7d, Q8d, Q9d, Q10d, Q14d, Q21d, Q28d, Q30d, Q90d, Q120d, Q240d, or Q365d.

[0337] In certain embodiments, one or both of the heterodimeric Fc-fusion protein and / or additional therapeutic agents are administered intermittently. In certain embodiments, the methods comprise administering one or both of the heterodimeric Fc-fusion protein or additional therapeutic agents to a subject with a delay of at least 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, or 4 weeks between administrations. In certain embodiments, the administration with a delay follows a pattern in which one or both of the heterodimeric Fc-fusion protein and / or additional therapeutic agents, or any combination thereof, are administered consecutively for a period of 10 minutes to about 365 days, followed by a period of no administration for about 10 minutes to about 30 days.

[0338] In certain embodiments, one or any combination of the heterodimeric Fc-fusion protein and / or additional therapeutic agent is administered intermittently, and the other is administered continuously. In certain embodiments, a first effective amount of a combination of heterodimeric Fc-fusion proteins is administered sequentially with a second effective amount of an additional therapeutic agent.

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

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

[0341] In certain embodiments, the 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, where a first effective amount of the heterodimeric Fc fusion protein is administered in a separate formulation from a second effective amount of the additional therapeutic agent. In certain embodiments, the combination is administered in two (2) compositions, where a first effective amount of the heterodimeric Fc fusion protein is administered in a separate formulation from a second effective amount of the additional therapeutic agent. In certain embodiments, the first effective amount of the heterodimeric Fc fusion protein is administered sequentially after the second effective amount of the additional therapeutic agent. In certain embodiments, the second effective amount of the additional therapeutic agent is administered sequentially after the first effective amount of the heterodimeric Fc fusion protein. In certain embodiments, the heterodimeric Fc fusion protein and the additional therapeutic agent are administered, and then both the heterodimeric Fc fusion protein and the additional therapeutic agent are administered intermittently for at least twenty-four (24) hours. In certain embodiments, the heterodimeric Fc-fusion protein and the additional therapeutic agent are administered on a non-overlapping every other day schedule.

[0342] In certain embodiments, the first effective amount of the heterodimeric Fc-fusion protein is administered no less than 4 hours after the second effective amount of the additional therapeutic agent. In certain embodiments, the first effective amount of the heterodimeric Fc-fusion protein is administered no less than 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 60 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, 14 days, 21 days, or 30 days after the administration of the second effective amount of the additional therapeutic agent. In certain embodiments, the second effective amount of an additional therapeutic agent is administered at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 60 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, at least 24 hours, at least 2 days, at least 4 days, at least 6 days, at least 8 days, at least 10 days, at least 12 days, at least 14 days, at least 21 days, or at least 30 days after administration of the first effective amount of the heterodimeric Fc-fusion protein.

[0343] In some embodiments, one or both of the heterodimeric Fc-fusion protein and / or additional therapeutic agent(s) are administered by a route selected from the group consisting of intravenous, subcutaneous, cutaneous, oral, intramuscular, and intraperitoneal. In some embodiments, one or both of the heterodimeric Fc-fusion protein and / or additional therapeutic agent(s) are administered intravenously. In some embodiments, one or both of the heterodimeric Fc-fusion protein and / or additional therapeutic agent(s), or any combination thereof, are administered orally.

[0344] Those skilled in the art will understand that the unit dosage forms of the present invention can be administered in the same or different physical forms, i.e., via capsules or tablets, orally and / or as a liquid via IV infusion. Furthermore, the unit dosage form for each administration may vary depending on the specific route of administration. Several different dosage forms may exist for one or both of the heterodimeric Fc-fusion protein and additional therapeutic agent combinations. Because different medical conditions may require different routes of administration, the same components of the combinations described herein may be identical in composition and physical form, but may still need to be administered in different ways, perhaps at different times, to alleviate the condition. For example, conditions such as persistent nausea, particularly with vomiting, may make the use of oral dosage forms difficult, and in such cases, they may need to be administered in other unit dosage forms, perhaps the same as other dosage forms used before or after, instead of or as well as by inhalation, buccal, sublingual, or suppository routes. A specific dosage form may be necessary for a particular combination of heterodimeric Fc-fusion protein and additional therapeutic agent because of potential issues with various factors, such as chemical stability or pharmacokinetics.

[0345] NK targeting therapy In some embodiments, heterodimeric Fc fusion protein therapy is combined with NK targeting therapy.For example, in some embodiments, heterodimeric Fc fusion protein is co-administered with a therapeutic agent that targets NKp46.In some embodiments, the therapeutic agent that targets NKp46 also binds to CD16, one or more tumor-associated antigens, or a combination thereof.The exemplary therapeutic agent that targets NKp46 is described in more detail in US Patent Application US20170198038A1, which is incorporated herein by reference for all purposes.

[0346] In one embodiment, heterodimeric Fc fusion protein therapy is combined with bispecific and trispecific killer engager (BiKE and TriKE) therapy, including BiKE and TriKE therapy targeting NK cells. BiKE and TriKE are constructed from a single heavy (VH) and light (VL) chain of the variable region of each antibody of interest. The VH and VL domains are connected by a short, flexible polypeptide linker to prevent dissociation. BiKE and TriKE are described in more detail in U.S. Patent Applications US20180282386A1 and US20180258396A1, which are incorporated herein by reference for all purposes. BiKE and TriKE may contain a binding domain specific for NK cells.

[0347] In some embodiments, BiKE and TriKE therapy is used in combination with 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 some embodiments, BiKE and TriKE therapy is administered as a single course of a 3-week treatment block. In some embodiments, the treatment block comprises four consecutive 24-hour continuous infusions (approximately 96 hours) followed by a 72-hour rest period. In some embodiments, BiKE and TriKE therapy is 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 some embodiments, BiKE and TriKE therapy is 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 some embodiments, BiKE and TriKE therapy is administered at a dose of at least 1 μg / kg / day. In some embodiments, BiKE and TriKE therapy is administered at a dose of at least 5 μg / kg / day. In some embodiments, BiKE and TriKE therapy is administered at a dose of at least 200 μg / kg / day. In some embodiments, BiKE and TriKE therapy is administered at a dose of at least 500 μg / kg / day. In some embodiments, BiKE and TriKE therapy is administered at a dose of at least 1000 μg / kg / day. In some embodiments, BiKE and TriKE therapy is administered at a dose of 200 μg / kg / day or less. In some embodiments, BiKE and TriKE therapy is administered at a dose of 500 μg / kg / day or less. In some embodiments, BiKE and TriKE therapy is administered at a dose of 1000 μg / kg / day or less. In some embodiments, BiKE and TriKE therapy is administered at a dose of 1-200 μg / kg / day. In some embodiments, BiKE and TriKE therapy is administered at a dose of 5-200 μg / kg / day.In some embodiments, BiKE and TriKE therapy is administered at a dose of 1-500 μg / kg / day. In some embodiments, BiKE and TriKE therapy is administered at a dose of 1-1000 μg / kg / day. In some embodiments, BiKE and TriKE therapy is administered at a dose of 5-500 μg / kg / day. In some embodiments, BiKE and TriKE therapy is administered at a dose of 5-1000 μg / kg / day. In some embodiments, BiKE and TriKE therapy is administered at the maximum tolerated dose. In some embodiments, BiKE and TriKE therapy is administered at less than the maximum tolerated dose.

[0348] Multispecific binding protein (“TriNKET”) therapy In certain embodiments, exemplary tumor-associated antigens of the heterodimeric Fc fusion protein 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 weight-based dose of a multispecific binding protein. For example, the weight-based dose of the multispecific binding protein may be from about 0.01 μg to about 100 mg / kg body weight, e.g., from about 0.01 μg to about 100 mg / kg body weight, from about 0.01 μg to about 50 mg / kg body weight, from about 0.01 μg to about 10 mg / kg body weight, from about 0.01 μg to about 1 mg / kg body weight, from about 0.01 μg to about 100 μg / kg body weight, from about 0.01 μg to about 50 μg / kg body weight, from about 0.01 μg to about 10 μg / kg body weight, from about 0.01 μg to about 1 μg / kg body weight, or from about 0.01 μg to about 1 μg / kg body weight. μg ~ approx. 0.1 μg / kg body weight, approx. 0.1 μg ~ approx. 100 mg / kg body weight, approx. 0.1 μg ~ approx. 50 mg / kg body weight, approx. 0.1 μg ~ approx. 10 mg / kg body weight, approx. 0.1 μg ~ approx. 1 mg / kg body weight, approx. 0.1 μg ~ approx. 100 μg / kg Body weight, about 0.1 μg to about 10 μg / kg body weight, about 0.1 μg to about 1 μg / kg body weight, about 1 μg to about 100 mg / kg body weight, about 1 μg to about 50 mg / kg body weight, about 1 μg to about 10 mg / kg body weight, about 1 μg to about 1 mg / kg body weight, about 1 μg~about 100μg / kg body weight, about 1μg~about 50μg / kg body weight, about 1μg~about 10μg / kg body weight, about 10μg~about 100mg / kg body weight, about 10μg~about 50mg / kg body weight, about 10μg~about 10mg / kg body weight, about 10μ g~about 1mg / kg body weight, about 10μg~about 100μg / kg body weight, about 10μg~about 50μg / kg body weight, about 50μg~about 100mg / kg body weight, about 50μg~about 50mg / kg body weight, about 50μg~about 10mg / kg body weight, about 50μ g to about 1 mg / kg body weight, about 50 μg to about 100 μg / kg body weight, about 100 μg to about 100 mg / kg body weight, about 100 μg to about 50 mg / kg body weight, about 100 μg to about 10 mg / kg body weight, about 100 μg to about 1 mg / kg body weight, about 1 mg to about 100 mg / kg body weight, about 1 mg to about 50 mg / kg body weight, about 1 mg to about 10 mg / kg body weight, about 10 mg to about 100 mg / kg body weight, about 10 mg to about 50 mg / kg body weight, and about 50 mg to about 100 mg / kg body weight.

[0350] In some embodiments, heterodimeric Fc-fusion protein therapy is combined with a therapy comprising a dose of a multispecific binding protein administered one or more times daily, weekly, monthly, or yearly, or once every 2 to 20 years. Those skilled in the art can readily estimate the repetition rate of administration based on measurements of residence time and concentration of the targetable construct or complex in bodily fluids or tissues. Administration of the multispecific binding protein can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, or intracavity, via catheter perfusion or direct intralesional injection. Administration can be once or more times daily, weekly, monthly, or yearly.

[0351] Chimeric antigen receptor (CAR) therapy In one embodiment, heterodimeric Fc-fusion protein therapy is combined with CAR therapy. The term "chimeric antigen receptor," or "CAR," refers to a recombinant polypeptide construct that includes at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain (also referred to herein as a "primary signaling domain") that includes a functional signaling domain derived from a stimulatory molecule.

[0352] Thus, in some embodiments, the CAR comprises an extracellular antigen-binding site that binds to a tumor-associated antigen, an intracellular signaling domain that comprises a transmembrane domain and a primary signaling domain. In some embodiments, the CAR further comprises a functional signaling domain (also referred to as a "costimulatory signaling domain") derived from at least one or more costimulatory molecules.

[0353] In some embodiments, the CAR comprises a chimeric fusion protein comprising a tumor-associated antigen binding domain (e.g., a tumor-associated antigen-binding scFv domain) comprising a heavy chain variable domain and a light chain variable domain as the extracellular antigen-binding domain, an intracellular signaling domain comprising a transmembrane domain and a primary signaling domain. In some embodiments, the CAR comprises a chimeric fusion protein comprising a tumor-associated antigen binding domain (e.g., a tumor-associated antigen-binding scFv domain) comprising a heavy chain variable domain and a light chain variable domain as the extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain comprising a costimulatory signaling domain and a primary signaling domain. In some embodiments, the CAR comprises a chimeric fusion protein comprising a tumor-associated antigen binding domain (e.g., a tumor-associated antigen-binding scFv domain) comprising a heavy chain variable domain and a light chain variable domain as the extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain comprising two costimulatory signaling domains and a primary signaling domain. In some embodiments, the CAR comprises a chimeric fusion protein comprising a tumor-associated antigen binding domain (e.g., a tumor-associated antigen-binding scFv domain) comprising a heavy chain variable domain and a light chain variable domain as the extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two costimulatory signaling domains and a primary signaling domain.

[0354] Regarding the transmembrane domain, in various embodiments, the CAR is designed to include a transmembrane domain fused to the extracellular domain of the CAR. In some embodiments, the transmembrane domain is naturally associated with one of the domains of the CAR. In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein to minimize interaction with other members of the receptor complex. In other embodiments, the transmembrane domain can homodimerize with other CARs on the surface of the CAR T cell. In other embodiments, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interaction with the binding domain of a natural binding partner present on the same CAR T cell.

[0355] The transmembrane domain can be derived from any naturally occurring membrane-bound or transmembrane protein.In some embodiments, the transmembrane region can transmit signals to the intracellular domain whenever CAR binds to the target.In some embodiments, the transmembrane domain comprises the transmembrane region of one or more proteins selected from the group consisting of TCR alpha chain, TCR beta chain, TCR zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154. In some embodiments, the transmembrane domain is selected from the group consisting of KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, IT The transmembrane domains include one or more proteins selected from the group consisting of GAX, 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 by a hinge region. A variety of hinges can be used, including, but not limited to, human Ig hinges (e.g., IgG4 hinges, IgD hinges), Gly-Ser linkers, (G4S)4 linkers, KIR2DS2 hinges, and CD8α hinges.

[0357] The intracellular signaling domain of a CAR is responsible for activating at least one specialized function of an immune cell in which the CAR is placed (e.g., cytolytic activity or helper activity, including cytokine secretion, of a T cell). Thus, as used herein, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as it transmits the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[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 costimulatory signaling domains (i.e., a functional signaling domain derived from at least one costimulatory 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 cytoplasmic signaling sequences that control immune cell activation in a stimulatory direction for at least some aspect of an immune cell signaling pathway. In certain embodiments, the signal is a primary signal that is initiated, e.g., by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, and leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc.

[0360] The primary signaling domain that acts in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of ITAM-containing cytoplasmic signaling sequences that are particularly useful in the present invention include CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In some embodiments, the primary signaling domain in any one or more CARs comprises a cytoplasmic signaling sequence derived from CD3-zeta.

[0361] In certain embodiments, the primary signaling domain is a functional signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, 4-1BB, and / or CD3-zeta. In certain embodiments, the intracellular signaling domain comprises a functional signaling domain of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (Fc epsilon R1b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and / or DAP12. In certain embodiments, the primary signaling domain is a functional signaling domain of the zeta chain associated with the T cell receptor complex.

[0362] As used herein, the term "costimulatory molecule" refers to a cognate binding partner of a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, including, 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 an efficient response of lymphocytes to antigens. 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 ligands that specifically bind to CD83. Further examples of such costimulatory molecules include CD5, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, 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, CD These include ligands that specifically bind to 18, 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 CD83.In one embodiment, the costimulatory signaling domain of the CAR is a functional signaling domain of a costimulatory molecule described herein, e.g., 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 a functional portion of a protein that affects signal transduction within a cell to control cellular activity via a defined signaling pathway, either by producing second messengers or by functioning as an effector in response to such messengers.

[0364] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other randomly or in a specific order. Optionally, a short oligo- or polypeptide linker, e.g., 2-10 amino acids in length, can form the linkage.

[0365] antibody treatment In certain embodiments, heterodimeric Fc-fusion protein therapy is combined with antibody therapy for the treatment of subjects known or suspected to have cancer.

[0366] In some embodiments, the heterodimeric Fc-fusion protein is combined with a treatment comprising an anti-HER2 binding domain, such as an anti-HER2 antibody or an anti-HER2 antibody platform (e.g., a bispecific or trispecific antibody 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 (Herceptin® - Roche / Genentech; Kanjinti - Amgen), pertuzumab (PERJETA® - Roche / Genentech), and MGAH22 (described in detail in U.S. Patent 8,802,093, which is incorporated herein by reference for all purposes). Anti-HER2 antibody platforms include, but are not limited to, elitumaxomab (REXOMUN® - Creative Biolabs) and trastuzumab emtansine (ado-trastuzumab emtansine / T-DM1; KADCYLA® - Roche / Genentech). In some embodiments, anti-HER2 binding domain therapy is used in combination with heterodimeric Fc-fusion protein therapy to treat subjects known or suspected to have cancer. In some embodiments, anti-HER2 binding domain therapy is administered by IV infusion. In some embodiments, 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, or 10 mg / kg / day. In some embodiments, 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, or at least 10 mg / kg / day. In certain embodiments, the anti-HER2 binding domain therapy is administered at a dose of less than 1 mg / kg / day.

[0367] In some embodiments, anti-HER2 binding domain therapy is used in combination with heterodimeric Fc-fusion protein therapy to treat subjects known or suspected to have breast cancer, for example, subjects diagnosed with metastatic HER2-overexpressing breast cancer. In some embodiments, anti-HER2 binding domain therapy is administered at 4 mg / kg / day. In some embodiments, anti-HER2 binding domain therapy is administered at 4 mg / kg / day via a 90-minute IV infusion. In some embodiments, anti-HER2 binding domain therapy is administered at 2 mg / kg / day. In some embodiments, anti-HER2 binding domain therapy is administered at 2 mg / kg / day via a 30-minute IV infusion. In some embodiments, anti-HER2 binding domain therapy is administered at an initial dose of 4 mg / kg / day, followed by weekly doses of 2 mg / kg / day. In some embodiments, anti-HER2 binding domain therapy is administered at an initial dose of 4 mg / kg / day, followed by weekly doses of 2 mg / kg / day for 52 weeks.

[0368] In some embodiments, anti-HER2 binding domain therapy is used in combination with heterodimeric Fc fusion protein therapy to treat subjects known or suspected to have gastric cancer, e.g., subjects diagnosed with metastatic HER2-overexpressing gastric cancer. In some embodiments, anti-HER2 binding domain therapy is administered at 8 mg / kg / day. In some embodiments, anti-HER2 binding domain therapy is administered at 8 mg / kg / day via a 90-minute IV infusion. In some embodiments, anti-HER2 binding domain therapy is administered at 6 mg / kg / day. In some embodiments, anti-HER2 binding domain therapy is administered at 6 mg / kg / day via a 30-90 minute IV infusion. In some embodiments, anti-HER2 binding domain therapy is administered at an initial dose of 8 mg / kg / day, followed by weekly dosing at 6 mg / kg / day. In some embodiments, anti-HER2 binding domain therapy is administered at an initial dose of 8 mg / kg / day, followed by weekly dosing at 6 mg / kg / day for 52 weeks.

[0369] In some embodiments, the heterodimeric Fc-fusion protein therapy is combined with a therapy comprising an anti-CD20 binding domain, such as an anti-CD20 antibody or 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 (Rituxan® - Roche / Genentech), ocrelizumab (OCREVUS® - Roche / Genentech), obinutuzumab (GAZYVA® - Roche / Genentech), ofatumumab (ARZERRA® - Novartis), and veltuzumab. In some embodiments, the anti-CD20 binding domain therapy is used in combination with the heterodimeric Fc-fusion protein therapy to treat subjects known or suspected to have cancer. In some embodiments, the anti-CD20 binding domain therapy is administered by IV infusion. In one embodiment, the anti-CD20 binding domain treatment is at 100 mg / m 2 , 200 mg / m 2 , 300 mg / m 2 , 400 mg / m 2 , 500 mg / m 2 , 600 mg / m 2 , 700 mg / m 2 , 800 mg / m 2 , 900 mg / m 2 or 1000 mg / m 2 In one embodiment, the anti-CD20 binding domain treatment is administered at a dose of 375 mg / m 2 In certain embodiments, the anti-CD20 binding domain treatment is administered at a dose of at least 100 mg / m 2 , at least 200 mg / m 2 , at least 300 mg / m 2 , at least 400 mg / m 2 , at least 500 mg / m 2 , at least 600 mg / m 2 , at least 700 mg / m 2 , at least 800 mg / m 2, at least 900 mg / m 2 or at least 1000 mg / m 2 In one embodiment, the anti-CD20 binding domain treatment is administered at a dose of 400 mg / m 2 In one embodiment, the anti-CD20 binding domain treatment is administered at a dose of less than 375 mg / m 2 It is administered in doses less than 100 mg / kg.

[0370] In one embodiment, an anti-CD20 binding domain therapy is used in combination with a heterodimeric Fc-fusion protein therapy for the treatment of a subject known or suspected to have non-Hodgkin's lymphoma (NHL). In one embodiment, the anti-CD20 binding domain therapy is administered at a dose of 375 mg / m IV infusion. 2 In one embodiment, the anti-CD20 binding domain treatment is administered at a dose of 375 mg / m 2 It is administered in doses less than 100 mg / kg.

[0371] In certain embodiments, anti-CD20 binding domain therapy is used in combination with heterodimeric Fc-fusion protein therapy for the treatment of subjects known or suspected to have chronic lymphocytic leukemia (CLL). In certain embodiments, anti-CD20 binding domain therapy is administered at a dose of 375 mg / m in the first cycle. 2 IV infusion at a dose of 500 mg / m per cycle for an additional 2 to 6 cycles 2 In one embodiment, the anti-CD20 binding domain treatment is administered at a dose of 375 mg / m IV infusion. 2 The combined anti-CD20 binding domain and heterodimeric Fc fusion protein therapy may be used in combination with fludarabine and cyclophosphamide (FC).

[0372] In some embodiments, anti-CD20 binding domain therapy is used in combination with heterodimeric Fc fusion protein therapy to treat subjects known or suspected to have rheumatoid arthritis (RA). In some embodiments, anti-CD20 binding domain therapy is administered by IV infusion at 1000 mg twice, 2 weeks apart. In some embodiments, anti-CD20 binding domain therapy is administered by IV infusion at 1000 mg twice, 2 weeks apart, for up to 24 weeks. In some embodiments, the combined anti-CD20 binding domain and heterodimeric Fc fusion protein therapy is co-administered with methotrexate.

[0373] In some embodiments, heterodimeric Fc fusion protein therapy is combined with therapy comprising an antibody therapy comprising an agonist antibody. In some 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 some embodiments, the agonist antibody is a bispecific antibody. In some embodiments, the agonist antibody is a multispecific antibody, for example, a bispecific antibody, comprising 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 example is a bispecific agonist antibody that targets 4-1BB and CD137, such as utomilumab (Pfizer).

[0374] Checkpoint inhibitor therapy In some embodiments, heterodimeric Fc fusion protein therapy can be combined with checkpoint inhibitor therapy.Exemplary immune checkpoint molecules that can be targeted for blocking or inhibition include but are not limited to CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules, and all NK, gamma delta and memory CD8+ (alpha beta) 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 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 nivolumab (anti-PD-1; Opdivo® - BMS), AMP224 (anti-PD-1; NCI), pembrolizumab (anti-PD-1; MK-3475 / Keytruda® - Merck), pidilizumab (anti-PD-1 antibody; CT-011 - Teva / CureTech), atezolizumab (anti-PD-L1; Tecentriq® - Roche / Genentech), durvalumab (anti-PD-L1; MEDI4736 / Imfinzi® - Medimmune / AstraZeneca), avelumab (anti-PD-L1; Bavencio® - Pfizer), BMS-936559 (anti-PD-L1 - BMS), ipilimumab (anti-CTLA-4; Yervoy® - BMS), tremelimumab (anti-CTLA-4; Mediimmune / AstraZeneca), lirilumab (anti-KIR; BMS), monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca), BY55 (anti-CD160), anti-OX40, anti-TIM3, and anti-LAG3.

[0375] In some embodiments, checkpoint inhibitor therapy is used in combination with heterodimeric Fc-fusion protein therapy to treat subjects known or suspected to have cancer. In some embodiments, checkpoint inhibitor therapy is administered by IV infusion. In some embodiments, checkpoint inhibitor therapy is administered by IV infusion over 30 minutes. In some embodiments, checkpoint inhibitor therapy is administered every 3 weeks. In some embodiments, 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 some embodiments, checkpoint inhibitor therapy is administered at a dose of 200 mg. In some embodiments, 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 some embodiments, checkpoint inhibitor therapy is administered at a dose of less than 200 mg. In some embodiments, checkpoint inhibitor therapy is used in combination with heterodimeric Fc-fusion protein therapy to treat a subject known or suspected to have melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite unstable high cancer, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), or renal cell carcinoma (RCC).

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

[0377] In certain embodiments, the cytokine treatment includes a cytokine that is an interleukin (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 certain embodiments, the cytokine treatment includes a cytokine that is a growth factor (e.g., tumor necrosis factor (TNF), LT, EMAP-II, GM-CSF, FGF, and PDGF). In certain embodiments, the cytokine treatment includes an anti-inflammatory cytokine (e.g., IL-4, IL-10, IL-11, IL-13, and TGF).

[0378] In some embodiments, the chemokine treatment comprises a pro-inflammatory chemokine (e.g., GRO-α, GRO-b, LIX, GCP-2, MIG, IP10, I-TAC and MCP-1, RANTES, eotaxin, SDF-1 and MIP3a). In some embodiments, the chemokine treatment comprises a chemokine receptor. In some embodiments, the chemokine treatment comprises a C-X-C chemokine receptor (e.g., CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and CXCR7), a C-C chemokine receptor (e.g., CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10 and CCR11), a C-X-C chemokine receptor (e.g., CX3C11) or an X-C chemokine receptor (e.g., XCR1). In certain embodiments, the chemokine therapy comprises a G protein-bridged transmembrane receptor.

[0379] In some embodiments, the cytokine treatment includes a cytokine treatment that synergizes with IL-12 signaling. In some embodiments, the cytokine treatment includes an IL-2 cytokine or a derivative thereof. In some embodiments, the IL-2 treatment is aldesleukin (Proleukin - Prometheus Therapeutics). In some embodiments, the IL-2 treatment and / or aldesleukin are administered intravenously. In some embodiments, the cytokine treatment includes an IL-15 cytokine or a derivative thereof. In some embodiments, the IL-15 treatment is ALT-803 (Altor Bioscience) or NKTR-255 (Nektar). In some embodiments, the IL-15 treatment, NKTR-255 and / or ALT-803 are administered subcutaneously. In some embodiments, the chemokine treatment includes a CXCL9 chemokine, a CXCL10 chemokine, or a derivative thereof.

[0380] In some embodiments, cytokine or chemokine therapy comprises administration of a cytokine or chemokine to a subject. In some embodiments, cytokine or chemokine therapy comprises administration of a recombinant cytokine or chemokine to a subject. In some embodiments, cytokine or chemokine therapy comprises engineering cells to produce a cytokine or chemokine. In some embodiments, cytokine or chemokine therapy comprises engineering cells ex vivo, in vitro, or in vivo to produce a cytokine or chemokine.

[0381] In certain embodiments, cytokine or chemokine therapy involves engineering cells to produce cytokines or chemokines using viral vector-based delivery platforms designed to target specific cell types or receptors, such as vaccinia, fowlpox, self-replicating alphaviruses, Maraba virus, adenovirus (e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), lentiviruses, including but not limited to second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation (e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1): 45-61, Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue of Splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873-9880) or U.S. Patent 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 adeno-associated virus ("AAV") as detailed in Samuiski et al, J. Virol. 63:03822-3828 (1989). In certain embodiments, cytokine or chemokine therapy involves engineering cells to produce cytokines or chemokines using LNPs, liposomes, or exosomes. In some embodiments, cytokine or chemokine therapy involves engineering cells to produce cytokines or chemokines using genome editing, such as using a nuclease-based genome editing system (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) family, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), and homing endonuclease (HE)-based genome editing systems or derivatives thereof). In some embodiments, cytokine or chemokine therapy involves engineering cells to produce cytokines or chemokines using electroporation.

[0382] Innate immune system agonist therapy In certain 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 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 lintatolimod (AMPLIGEN® - Hemispherx Biopharma) or poly IC-LC (e.g., Hirutonol®). 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 an ssRNA or derivative or an imidazoquinoline derivative, including, but not limited to, resiquimod (also known as R848), imiquimod (ZYCLARA®, Aldara-Medicis), and gardikimod. In some embodiments, the TLR7 agonist is also a TLR8 agonist, such as imiquimod or Medi-9197 (AstraZeneca / Medimmune). In some embodiments, the TLR9 agonist comprises a CpG-containing oligodeoxynucleotide (CpG-ODN) or SD-101 (Dynavax).

[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 diAMP, cyclic diGMP or cyclic-GMP-AMP (cGAMP). In some embodiments, the STING agonist comprises a nucleic acid (e.g., DNA or RNA) that stimulates cGAS. In some embodiments, the STING agonist is ADU-S100 (also known as MIW815 - Aduro / Novartis).

[0385] Chemotherapy In certain embodiments, the heterodimeric Fc-fusion protein therapy is combined with one or more chemotherapeutics. In certain embodiments, the heterodimeric Fc-fusion protein therapy is combined with one or more chemotherapeutics to treat a subject diagnosed with cancer. Examples of chemotherapeutic agents include aldesleukin, alvocidib, antineoplaston AS2-1, antineoplaston A10, antithymocyte globulin, amifostine trihydrate, aminocamptothecin, arsenic trioxide, beta-arretin, Bcl-2 family protein inhibitors ABT-263, ABT-199, BMS-345541, bortezomib (Velcade®), bryostatin 1, busulfan, carboplatin, Cambus-1H, CC-5103, carmustine, caspofungin acetate, clofarabine, cisplatin, cladribine (Leustatin®), chlorambucil (Leukeran®), curcumin, cyclosporine, cyclophosphamide (Cyloxan, Endoxana, Cyclostin), cytarabine, denileukin diftitox, dexamethasone, DT PACE, docetaxel, dolastatin 10, doxorubicin (Adriamycin®, ADRIBLASTINE®), doxorubicin hydrochloride, enzastaurin, epoetin alfa, etoposide, everolimus (RAD001), fenretinide, filgrastim, melphalan, mesna, flavopiridol, fludarabine (Fludara®), geldanamycin (17-AAG), ifosfamide, irinotecan hydrochloride, ixabepilone, lenalidomide (Revlimid®, CC-5013), lymphokine-activated killer cells, melphalan, methotrexate, mitoxantrone hydrochloride, motexanin gadolinium, mycophenolate mofetil, nelarabine, oblimersen (GENASENSE®)Obatoclax (GX15-070), oblimersen, octreotide acetate, omega-3 fatty acids, oxaliplatin, paclitaxel, PD0332991, pegylated liposomal doxorubicin hydrochloride, pegfilgrastim, pentostatin (NIPENT®), perifosine, prednisolone, prednisone, R-roscovitine (SELICILIB®, CYC202), recombinant interferon alpha, recombinant interleukin (IL-1), Interleukin-12, recombinant interleukin-11, recombinant flt3 ligand, recombinant human thrombopoietin, rituximab, sargramostim, ludenafil citrate, simvastatin, sirolimus, styryl sulfone, tacrolimus, tanespimycin, temsirolimus (CC1-779), thalidomide, therapeutic allogeneic lymphocytes, thiotepa, tipifarnib, Velcade® (bortezomib® or PS-341), vincristine ( Incovin®), vincristine sulfate, vinorelbine bitartrate, vorinostat (SAHA) and FR (fludarabine, rituximab), CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), CVP (cyclophosphamide, vincristine and prednisone), FCM (fludarabine, cyclophosphamide, mitoxantrone), FCR (fludarabine, cyclophosphamide, rituximab), hyperCVAD (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 some embodiments, heterodimeric Fc-fusion protein therapy is combined with one or more chemotherapies to treat a subject diagnosed with colon cancer, rectal cancer, or colorectal cancer (CRC). In some embodiments, the chemotherapies include FOLFOX (5-FU, leucovorin, and oxaliplatin / Eloxatin), FOLFIRI (leucovorin, 5-FU, and irinotecan / Camptosar), FOLFOXIRI (leucovorin, 5-FU, oxaliplatin, and irinotecan), CapeOx (capecitabine and oxaliplatin), 5-FU and leucovorin co-administration, capecitabine (Xeloda®) alone, or trifluridine and tipiracil (Lonsurf®). In some embodiments, the chemotherapy comprises a VEGF-targeted agent, such as bevacizumab (Avastin®), ziv-aflibercept (Zaltrap®), ramucirumab (CYRAMZA®), or regorafenib (Stivarga®), or an EGFR-targeted agent, such as cetuximab (Erbitux) or panitumumab (Vectibix®). In some embodiments, the chemotherapy is co-administered with chemotherapy selected from FOLFOX, FOLFIRI, FOLFOXIRI, CapeOx, 5-FU with leucovorin, capecitabine alone, and trifluridine / tipiracil together with a VEGF-targeted agent or an EGFR-targeted agent.

[0387] In some embodiments, heterodimeric Fc-fusion protein therapy is combined with one or more chemotherapeutics to treat a subject diagnosed with breast cancer. In some embodiments, the chemotherapeutics include doxorubicin (Adriamycin®), pegylated liposomal doxorubicin, epirubicin (ELLENCE®), paclitaxel (Taxol), docetaxel (Taxotere®), albumin-bound paclitaxel (Abraxane®), 5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin (Paraplatin®), cisplatin, vinorelbine (Navelbine®), capecitabine (Xeloda), gemcitabine (Gemzar®), ixabepilone (Ixempra®), or eribulin (Halaven). In some embodiments, the chemotherapy comprises a combination of two or more chemotherapies selected from doxorubicin (Adriamycin®), pegylated liposomal doxorubicin, epirubicin (ELLENCE®), paclitaxel (Taxol), docetaxel (Taxotere®), albumin-bound paclitaxel (Abraxane®), 5-fluorouracil (5-FU), cyclophosphamide (Cytoxan®), carboplatin (Paraplatin®), cisplatin, vinorelbine (Navelbine®), capecitabine (Xeloda®), gemcitabine (Gemzar®), ixabepilone (Ixempra®), and eribulin (Halaven®).

[0388] In some embodiments, the heterodimeric Fc-fusion protein therapy is combined with one or more chemotherapies to treat a subject diagnosed with melanoma / skin cancer. In some embodiments, the chemotherapies include dacarbazine (also known as DTIC), temozolomide, nab-paclitaxel, paclitaxel, cisplatin, carboplatin, or vinblastine.

[0389] Targeted drug therapy In some embodiments, heterodimeric Fc fusion protein therapy is combined with one or more targeting agents.Targeting agents generally act on specific molecular targets, such as targets associated with cancer.Targeting agents are distinct from standard chemotherapy in that standard chemotherapy acts on all rapidly dividing normal and cancerous cells.Targeting agents include, but are not limited to, hormone therapy, signal transduction inhibitors, gene expression modulators, apoptosis inducers, angiogenesis inhibitors, immunotherapy, toxin delivery molecules (e.g., antibody drug conjugates) and kinase inhibitors.In some embodiments, the targeting agent comprises a receptor agonist or ligand.

[0390] In some embodiments, the heterodimeric Fc-fusion protein therapy is combined with one or more targeting agents to treat subjects diagnosed with colon cancer, rectal cancer, or colorectal cancer (CRC). In some embodiments, the targeting agents include cetuximab (Erbitux®), panitumumab (Vectibix®), bevacizumab (Avastin®), ziv-aflibercept (Zaltrap®), regorafenib (Stivarga®), ramucirumab (CYRAMZA®), nivolumab (Opdivo®), or ipilimumab (Yervoy®).

[0391] In some embodiments, heterodimeric Fc-fusion protein therapy is combined with one or more targeting agents to treat a subject diagnosed with breast cancer. In some embodiments, the targeting agent is selected from the group consisting of everolimus (Afinitor®), tamoxifen (Nolvadex®), toremifene (Fareston®), trastuzumab (Herceptin®), fulvestrant (Faslodex®), anastrozole (Arimidex®), exemestane (Aromasin®), lapatinib (Tykerb®), letrozole (Femara®), pertuzumab (PERJETA®), ado-trastuzumab emtansine (Kadcyla®), palbociclib (Ibrance®), ribociclib (KISQALI®), neratinib maleate (NERLYNX®), and others. TM ), abemaciclib (Verzenio) TM ), olaparib (LYNPARZA TM ), atezolizumab (Tecentriq®), or alpelisib (PIQRAY®).

[0392] In some embodiments, the heterodimeric Fc-fusion protein therapy is combined with one or more targeting agents to treat a subject diagnosed with melanoma / skin cancer. In some embodiments, the targeting agent is selected from the group consisting of vismodegib (Elivege®), sonidegib (Odomzo®), ipilimumab (Yervoy®), vemurafenib (Zelboraf®), trametinib (MEKINIST®), dabrafenib (Tafinlar®), pembrolizumab (Keytruda®), nivolumab (Opdivo®), cobimetinib (COTELLIC®), and others. TM ), alitretinoin (PANRETIN®), avelumab (BAVENCIO®), encorafenib (Braftobi®), TM ), binimetinib (Mektovi®), or cemiplimab-rwlc (LIBTAYO®).

[0393] In some embodiments, the heterodimeric Fc fusion protein treatment is combined with a receptor agonist or ligand treatment. In some embodiments, the receptor agonist or ligand treatment comprises an agonist antibody. In some embodiments, the receptor agonist or ligand treatment comprises a receptor ligand, such as 4-1BBL or CD40L.

[0394] Radiation therapy In some embodiments, heterodimeric Fc-fusion protein therapy is combined with radiation therapy. In some embodiments, heterodimeric Fc-fusion protein therapy is combined with radioisotope particles, such as indium In-111, yttrium Y-90, or iodine I-131. Examples of combination therapy include, but are not limited to, iodine-131 tositumomab (Bexar®), yttrium-90 ibritumomab tiuxetan (Zevalin®), and Bexar® with CHOP. In some embodiments, radiation therapy includes external beam radiation therapy (EBRT), interstitial radiation therapy (brachytherapy), intracavitary radiation therapy, interstitial brachytherapy, radioembolization, hypofractionated radiation therapy, intraoperative radiation therapy (IORT), 3D-conformal radiation therapy, stereotactic radiosurgery (SRS), or stereotactic body radiation therapy (SBRT).

[0395] In some embodiments, heterodimeric Fc-fusion protein therapy is combined with one or more radiation therapies to treat subjects diagnosed with colon cancer, rectal cancer, or colorectal cancer (CRC). In some embodiments, radiation therapy includes external beam radiation therapy (EBRT), interstitial radiation therapy (brachytherapy), intracavitary radiation therapy, interstitial brachytherapy, or radioembolization therapy.

[0396] In some embodiments, the heterodimeric Fc-fusion protein therapy is combined with one or more radiation therapies to treat a subject diagnosed with breast cancer. In some embodiments, the radiation therapy comprises external beam radiation therapy, hypofractionated radiation therapy, intraoperative radiation therapy (IORT), or 3D-conformal radiation therapy.

[0397] In some embodiments, heterodimeric Fc fusion protein therapy is combined with one or more radiation therapies to treat subjects diagnosed with melanoma / skin cancer.In some embodiments, radiation therapy comprises stereotactic radiosurgery (SRS; for example, using gamma knife or linear accelerator) or stereotactic body radiation therapy (SBRT).

[0398] Vaccines and Oncolytic Virus Therapies In certain embodiments, the heterodimeric Fc-fusion protein therapy is combined with one or more immunogenic compositions, such as vaccine compositions or oncolytic viruses, that are capable of eliciting a specific immune response, such as a tumor-specific immune response.

[0399] In some embodiments, the heterodimeric Fc-fusion protein therapy is combined with a vaccine composition. The vaccine composition generally contains multiple antigens and / or neoantigens specific to the tumor to be targeted. The vaccine composition may also be referred to as a vaccine.

[0400] In some embodiments, the vaccine composition further comprises an adjuvant and / or a carrier. In some embodiments, the vaccine composition is accompanied by a carrier, such as a protein or an antigen-presenting cell, such as a dendritic cell (DC), that can present a peptide to T cells. In some embodiments, the carrier is a scaffold, such as a polypeptide or polysaccharide, to which an antigen or neoantigen can be bound.

[0401] Generally, an adjuvant is any substance that, when incorporated into a vaccine composition, enhances or otherwise modifies the immune response to an antigen or neoantigen. Adjuvants are covalently or non-covalently conjugated, as desired. The ability of an adjuvant to increase the immune response to an antigen is generally manifested by a significant or substantial increase in immune-mediated reactions or a reduction in disease symptoms. For example, an increase in humoral immunity is generally manifested by an increase in the titer of antibodies elicited against the antigen, and T cell activity is generally manifested by increased cell proliferation or cytotoxicity or cytokine secretion. Adjuvants can also alter the immune response, for example, by altering a primary humoral or Th response to a primary cellular or Th response. Suitable adjuvants include 1018 ISS, alum, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS patch, ISS, ISCO matrix, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF Adjuvants include, but are not limited to, Aquila's QS21 Stimulon (Aquila Biotech, Worcester, Mass., USA), which is derived from trap, R848, beta-glucan, Pam3Cys, saponins, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as incomplete Freund's or GM-CSF are also useful.Several immunological adjuvants specific for dendritic cells and their preparation (e.g., MF59) 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. Some cytokines are directly involved in the migration of dendritic cells to lymphoid tissues (e.g., TNF-alpha), 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 5,849,589, specifically incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI, et al., J Immunother Emphasis Tumor Immunol. 1996 (6):414-418). In certain embodiments, the adjuvant comprises a CpG immunostimulatory oligonucleotide. In certain 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., polyi:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies that can act as therapeutic agents and / or adjuvants, such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175. The amounts and concentrations of adjuvants and additives can be easily determined by those skilled 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 above, or a combination 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 additive) is present 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 extend serum half-life. In some embodiments, the carrier aids in presenting the peptide to T cells. In some embodiments, the carrier comprises any suitable carrier known to those skilled in the art, such as a protein or antigen-presenting cell. Examples of carrier proteins include, but are not limited to, keyhole limpet hemocyanin, transferrin, bovine serum albumin, human serum albumin, serum proteins such as thyroglobulin or ovalbumin, immunoglobulins, or hormones such as insulin or palmitic acid. For human immunization, the carrier is generally a physiologically acceptable carrier that is tolerated and safe by humans. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier is dextran, such as Sepharose.

[0405] In some embodiments, the vaccine is a viral vector-based vaccine platform designed to target a specific cell type or receptor, such as vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or a lentivirus, including, but not limited to, second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1): 45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human (See, for example, the 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.) Generally, upon introduction into a host, infected cells express the antigen or neoantigen, thereby eliciting a host immune (e.g., CTL) response against the peptide.

[0406] Due to the packaging capabilities of the viral vector-based vaccine platform, in some embodiments, the vaccine composition comprises one or more viral vectors. In some embodiments, the viral vector comprises a sequence flanked by non-mutated sequences separated by a linker or preceded by one or more sequences that target intracellular compartments (see, e.g., 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 protocols are described, for example, in U.S. Patent 4,722,848. Another vector is Bacillus Calmette-Guerin (BCG). BCG vectors are described by Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vaccine vectors useful for therapeutic administration or immunization of neoantigens, such as Salmonella typhi vectors, will be apparent to those skilled in the art from the disclosure herein.

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

[0408] In certain embodiments, heterodimeric Fc-fusion protein therapy is combined with oncolytic virus therapy to treat a subject diagnosed with melanoma / skin cancer. In certain embodiments, the oncolytic virus comprises talimogene laherparepvec (IMLYGIC®), also known as T-VEC. In certain embodiments, a heterodimeric Fc-fusion protein comprising a subunit of IL-12 is combined with an oncolytic virus (e.g., talimogene laherparepvec (IMLYGIC®)). (登録商標) ) or T-VEC) for the treatment of cancer (e.g., advanced malignancies).

[0409] The above description describes several aspects and embodiments of the present invention, and this patent application specifically contemplates all combinations and permutations of these aspects and embodiments. The present disclosure relates, for example, to the following: [Section 1] (a) a first polypeptide comprising a first antibody Fc domain polypeptide and a first subunit of a multisubunit protein; and (b) a second polypeptide comprising a second antibody Fc domain polypeptide and a second, different subunit of a multisubunit protein. A heterodimeric Fc fusion protein comprising: wherein the first and second antibody Fc domain polypeptides each contain a different mutation that promotes heterodimerization; wherein the first and / or second antibody Fc domain polypeptide comprises one or more mutations that reduce an Fc effector function, and A heterodimeric Fc fusion protein in which a first subunit and a second different subunit of a multisubunit protein are linked to each other. [Section 2] The heterodimeric Fc fusion protein of paragraph 1, wherein the effector function comprises the ability of the Fc to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). [Section 3] 3. The heterodimeric Fc fusion protein of paragraph 1 or 2, wherein the heterodimeric Fc fusion protein is fucosylated. [Section 4] Item 4. The heterodimeric Fc fusion protein according to any one of Items 1 to 3, wherein the first and second antibody Fc domain polypeptides are human antibody Fc domain polypeptides. [Section 5] Item 5. The heterodimeric Fc fusion protein according to any one of Items 1 to 4, wherein the first and second antibody Fc domain polypeptides are IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, or IgE Fc domain polypeptides. [Section 6] Item 6. The heterodimeric Fc fusion protein according to any one of Items 1 to 5, wherein the first and / or second antibody Fc domain polypeptide comprises one or more mutations at positions 233, 234, 235, 236, 237, 297, 318, 320, 322, 329, 330, and / or 331, as numbered according to the EU numbering system. [Section 7] Item 7. The heterodimeric Fc fusion protein according to any one of Items 1 to 6, wherein the first and second antibody Fc domain polypeptides are human IgG1 Fc domain polypeptides. [Section 8] 8. The heterodimeric Fc fusion protein of claim 7, wherein the first and / or second antibody Fc domain polypeptide comprises one or more mutations at positions 234, 235, 237, 329, 330, and / or 331 according to the EU numbering system. [Section 9] 9. The heterodimeric Fc fusion protein of paragraph 8, wherein the first and / or second antibody Fc domain polypeptide comprises one or more mutations selected from L234A, L235A, L235E, G237A, P329A, A330S, and P331S. [Section 10] 10. The heterodimeric Fc fusion protein of paragraph 9, wherein the first and second antibody Fc domain polypeptides each comprise the mutations L234A, L235A, and P329A. [Section 11] 10. The heterodimeric Fc fusion protein of paragraph 9, wherein the first and second antibody Fc domain polypeptides each comprise the mutations L234A and L235A. [Section 12] 10. The heterodimeric Fc fusion protein of paragraph 9, wherein the first and second antibody Fc domain polypeptides each comprise the mutations L234A, L235E, G237A, A330S, and P331S. [Section 13] 13. The heterodimeric Fc fusion protein according to any one of Items 7 to 12, wherein the first and second antibody Fc domain polypeptides each contain the C220S mutation. [Section 14] Item 7. The heterodimeric Fc fusion protein according to any one of Items 1 to 6, wherein the first and second antibody Fc domain polypeptides are human IgG4 Fc domain polypeptides. [Section 15] 15. The heterodimeric Fc fusion protein of claim 14, wherein the first and / or second antibody Fc domain polypeptide comprises one or more mutations at positions 235 and / or 329, as numbered according to the EU numbering system. [Section 16] 16. The heterodimeric Fc fusion protein of claim 15, wherein the first and / or second antibody Fc domain polypeptide comprises one or more mutations selected from L235E and P329A. [Section 17] 17. The heterodimeric Fc fusion protein of paragraph 16, wherein the first and second antibody Fc domain polypeptides each comprise the mutation L235E. [Section 18] 17. The heterodimeric Fc fusion protein of paragraph 16, wherein the first and second antibody Fc domain polypeptides each comprise the mutations L235E and P329A. [Section 19] 19. The heterodimeric Fc fusion protein according to any one of Items 14 to 18, wherein the first and second antibody Fc domain polypeptides each contain the mutation S228P. [Section 20] 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. [Section 21] A polypeptide comprising a subunit of a multisubunit cytokine and an immunoglobulin Fc domain polypeptide, wherein the Fc domain polypeptide comprises a mutation that promotes heterodimerization with a different immunoglobulin Fc domain polypeptide and one or more mutations that reduce an Fc effector function. [Section 22] 22. The polypeptide of claim 21, wherein the Fc domain is a human IgG1 antibody Fc domain. [Section 23] 23. The polypeptide of claim 22, wherein the one or more mutations that reduce the effector function of Fc are selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, as numbered according to the EU numbering system. [Section 24] 24. The polypeptide of claim 22 or 23, wherein the mutations that reduce the effector function of Fc are L234A, L235A, and P329A, as numbered according to the EU numbering system. [Section 25] 25. The polypeptide according to any one of Items 22 to 24, wherein the mutations that promote heterodimerization are K360E and K409W according to the EU numbering system. [Section 26] 25. The polypeptide according to any one of Items 22 to 24, wherein the mutations that promote heterodimerization are Q347R, D399V, and F405T according to the EU numbering system. [Section 27] 27. The polypeptide of paragraph 25 or 26, further comprising a mutation in the Fc domain that promotes disulfide bond formation with a different immunoglobulin Fc domain polypeptide. [Section 28] 28. The polypeptide of claim 27, wherein when the heterodimerization mutations are K360E and K409W, the mutation that promotes disulfide bond formation is Y349C, as numbered according to the EU numbering system. [Section 29] 28. The polypeptide of claim 27, wherein when the heterodimerization mutations are Q347R, D399V, and F405T, the mutation that promotes disulfide bond formation is S354C, as numbered according to the EU numbering system. [Section 30] 29. The polypeptide of claim 28, comprising the amino acid sequence of SEQ ID NO: 290. [Section 31] 30. The polypeptide of claim 29, comprising the amino acid sequence of SEQ ID NO: 291. [Section 32] A nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 290 or SEQ ID NO: 291. [Section 33] 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. [Section 34] A cell comprising the nucleic acid of paragraph 32 or the expression vector of paragraph 33. [Section 35] (a) a first polypeptide comprising a first antibody Fc domain polypeptide and a first subunit of a multisubunit protein; and (b) a second polypeptide comprising a second antibody Fc domain polypeptide and a second, different subunit of a multisubunit protein. A heterodimeric Fc fusion protein comprising: wherein the first and second antibody Fc domain polypeptides each contain a different mutation that promotes heterodimerization; wherein a first subunit of the multi-subunit protein is fused to a first antibody Fc domain polypeptide by a first linker, wherein the first linker comprises a first amino acid sequence, wherein the first amino acid sequence is the amino acid sequence PKSSDKTHTCPPCPAPEX 1 X 2 GX 3 (SEQ ID NO: 237) (wherein X 1 is L or A, and X 2 is L, E or A, and X 3 is A or G), where X 1 is L and / or X 2 is L, at least one of the first antibody Fc domain polypeptide and the second antibody Fc domain polypeptide contains a Q347R mutation that promotes heterodimerization. Here, a heterodimeric Fc fusion protein in which a first subunit and a second different subunit of a multisubunit protein are bound to each other. [Section 36] 36. The heterodimeric Fc fusion protein of paragraph 35, wherein the first amino acid sequence comprises the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239). [Section 37] 37. The heterodimeric Fc fusion protein of paragraph 36, wherein the first amino acid sequence consists of the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239). [Section 38] 38. The heterodimeric Fc fusion protein of paragraph 36 or 37, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10). [Section 39] 39. The heterodimeric Fc fusion protein of paragraph 38, wherein the second amino acid sequence consists of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10). [Section 40] 36. The heterodimeric Fc fusion protein of clause 35, wherein the first amino acid sequence comprises the amino acid sequence PKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 238). [Section 41] 41. The heterodimeric Fc fusion protein of paragraph 40, wherein the first amino acid sequence consists of the amino acid sequence PKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 238). [Section 42] 42. The heterodimeric Fc fusion protein of paragraph 40 or 41, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 8). [Section 43] 43. The heterodimeric Fc fusion protein of paragraph 42, wherein the second amino acid sequence consists of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 8). [Section 44] 42. The heterodimeric Fc fusion protein of paragraph 40 or 41, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 15). [Section 45] 45. The heterodimeric Fc fusion protein of paragraph 44, wherein the second amino acid sequence consists of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 15). [Section 46] 42. The heterodimeric Fc fusion protein of paragraph 40 or 41, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 16). [Section 47] 47. The heterodimeric Fc fusion protein of paragraph 46, wherein the second amino acid sequence consists of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPELLGG (SEQ ID NO: 16). [Section 48] 42. The heterodimeric Fc fusion protein of paragraph 40 or 41, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 65). [Section 49] 49. The heterodimeric Fc fusion protein of paragraph 48, wherein the second amino acid sequence consists of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 65). [Section 50] 42. The heterodimeric Fc fusion protein of paragraph 40 or 41, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 66). [Section 51] 51. The heterodimeric Fc fusion protein of paragraph 50, wherein the second amino acid sequence consists of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 66). [Section 52] 36. The heterodimeric Fc fusion protein of paragraph 35, wherein the first amino acid sequence comprises the amino acid sequence PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 240). [Section 53] 53. The heterodimeric Fc fusion protein of paragraph 52, wherein the first amino acid sequence consists of the amino acid sequence PKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 240). [Section 54] 54. The heterodimeric Fc fusion protein of paragraph 52 or 53, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12). [Section 55] 55. The heterodimeric Fc fusion protein of paragraph 54, wherein the second amino acid sequence consists of the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 12). [Section 56] 54. The heterodimeric Fc fusion protein of paragraph 52 or 53, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67). [Section 57] 57. The heterodimeric Fc fusion protein of paragraph 56, wherein the second amino acid sequence consists of the amino acid sequence GGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 67). [Section 58] 54. The heterodimeric Fc fusion protein of paragraph 52 or 53, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 68). [Section 59] 59. The heterodimeric Fc fusion protein of paragraph 58, wherein the second amino acid sequence consists of the amino acid sequence GGGGSGGGGSEPKSSDKTHTCPPCPAPEAEGA (SEQ ID NO: 68). [Section 60] (a) a first polypeptide comprising a first antibody Fc domain polypeptide and a first subunit of a multisubunit protein; and (b) a second polypeptide comprising a second antibody Fc domain polypeptide and a second, different subunit of a multisubunit protein. A heterodimeric Fc fusion protein comprising: wherein the first and second antibody Fc domain polypeptides each contain a different mutation that promotes heterodimerization; wherein a first subunit of the multi-subunit protein is fused to a first antibody Fc domain polypeptide by a first linker, wherein the first linker comprises a first amino acid sequence, wherein the first amino acid sequence comprises the amino acid sequence RVESKYGPPCPPCPAPEFXGG (SEQ ID NO: 1) (wherein X is L or E); and A heterodimeric Fc fusion protein in which a first subunit and a second different subunit of a multisubunit protein are linked to each other. [Section 61] 61. The heterodimeric Fc fusion protein of paragraph 60, wherein the first amino acid sequence comprises the amino acid sequence RVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 2). [Section 62] 62. The heterodimeric Fc fusion protein of paragraph 61, wherein the first amino acid sequence consists of the amino acid sequence RVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 2). [Section 63] 63. The heterodimeric Fc fusion protein of paragraph 61 or 62, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 3). [Section 64] 64. The heterodimeric Fc fusion protein of paragraph 63, wherein the second amino acid sequence consists of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 3). [Section 65] 63. The heterodimeric Fc fusion protein of paragraph 61 or 62, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13). [Section 66] 66. The heterodimeric Fc fusion protein of paragraph 65, wherein the second amino acid sequence consists of the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 13). [Section 67] 63. The heterodimeric Fc fusion protein of paragraph 61 or 62, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 14). [Section 68] 68. The heterodimeric Fc fusion protein of paragraph 67, wherein the second amino acid sequence consists of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFLGG (SEQ ID NO: 14). [Section 69] 61. The heterodimeric Fc fusion protein of paragraph 60, wherein the first amino acid sequence comprises the amino acid sequence RVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 4). [Section 70] 70. The heterodimeric Fc fusion protein of paragraph 69, wherein the first amino acid sequence consists of the amino acid sequence RVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 4). [Section 71] 71. The heterodimeric Fc fusion protein of paragraph 69 or 70, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5). [Section 72] 72. The heterodimeric Fc fusion protein of paragraph 71, wherein the second amino acid sequence consists of the amino acid sequence GGGGSGGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 5). [Section 73] 71. The heterodimeric Fc fusion protein of paragraph 69 or 70, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63). [Section 74] 74. The heterodimeric Fc fusion protein of paragraph 73, wherein the second amino acid sequence consists of the amino acid sequence GGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 63). [Section 75] 71. The heterodimeric Fc fusion protein of paragraph 69 or 70, wherein a second different subunit of the multi-subunit protein is fused to a second antibody Fc domain polypeptide by a second linker comprising a second amino acid sequence, and wherein the second amino acid sequence comprises the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64). [Section 76] 76. The heterodimeric Fc fusion protein of paragraph 75, wherein the second amino acid sequence consists of the amino acid sequence GGGGSGGGGSRVESKYGPPCPPCPAPEFEGG (SEQ ID NO: 64). [Section 77] 77. The heterodimeric Fc fusion protein of any of Items 35 to 76, wherein the first and / or second polypeptide comprises one or more mutations that reduce an effector function of the Fc. [Section 78] 78. The heterodimeric Fc fusion protein of paragraph 77, wherein the effector function comprises the ability of the Fc to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC). [Section 79] 79. The heterodimeric Fc fusion protein of paragraph 77 or 78, wherein the first and second antibody Fc domain polypeptides are human IgG1 Fc domain polypeptides. [Section 80] 80. The heterodimeric Fc fusion protein of paragraph 79, wherein the first and second antibody Fc domain polypeptides each comprise one or more mutations selected from L234A, L235A or L235E, G237A, P329A, A330S, and P331S, as numbered according to the EU numbering system. [Section 81] 81. The heterodimeric Fc fusion protein of paragraph 80, wherein the first and second antibody Fc domain polypeptides each comprise the mutations L234A, L235A and P329A. [Section 82] 81. The heterodimeric Fc fusion protein of paragraph 80, wherein the first and second antibody Fc domain polypeptides each comprise the mutations L234A and L235A. [Section 83] 81. The heterodimeric Fc fusion protein of paragraph 80, wherein the first and second antibody Fc domain polypeptides each comprise the mutations L234A, L235E, G237A, A330S and P331S. [Section 84] 84. The heterodimeric Fc fusion protein of any of Items 79 to 83, wherein the first and second antibody Fc domain polypeptides each contain the mutation C220S. [Section 85] 79. The heterodimeric Fc fusion protein of paragraph 77 or 78, wherein the first and second antibody Fc domain polypeptides are human IgG4 Fc domain polypeptides. [Section 86] 86. The heterodimeric Fc fusion protein of paragraph 85, wherein the first and second antibody Fc domain polypeptides comprise one or more mutations selected from L235E and P329A, as numbered according to the EU numbering system. [Section 87] 87. The heterodimeric Fc fusion protein of paragraph 86, wherein the first and second antibody Fc domain polypeptides each comprise the mutation L235E. [Section 88] 87. The heterodimeric Fc fusion protein of paragraph 86, wherein the first and second antibody Fc domain polypeptides each comprise the mutations L235E and P329A. [Section 89] 89. The heterodimeric Fc fusion protein of any of Items 85 to 88, wherein the first and second antibody Fc domain polypeptides each contain the mutation S228P. [Section 90] 85. The heterodimeric Fc fusion protein according to any one of Items 7 to 13, 35 to 59, and 79 to 84, wherein the first antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of K360E, K409W, and a combination thereof, and the second antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of Q347R, D399V, F405T, and any combination thereof. [Section 91] 91. The heterodimeric Fc fusion protein of paragraph 90, wherein the first antibody Fc domain polypeptide comprises the mutations K360E and K409W, and the second antibody Fc domain polypeptide comprises the mutations Q347R, D399V, and F405T. [Section 92] 85. The heterodimeric Fc fusion protein according to any one of Items 7 to 13, 35 to 59, and 79 to 84, wherein the first antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of Q347R, D399V, F405T, and any combination thereof, and the second antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of K360E, K409W, and any combination thereof. [Section 93] 93. The heterodimeric Fc fusion protein of paragraph 92, wherein the first antibody Fc domain polypeptide comprises the mutations Q347R, D399V and F405T, and the second antibody Fc domain polypeptide comprises the mutations K360E and K409W. [Section 94] 90. The heterodimeric Fc fusion protein according to any one of Items 14 to 19, 60 to 76, and 85 to 89, wherein the first antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of K370E, R409W, and a combination thereof, and the second antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of E357N, D399V, F405T, and any combination thereof. [Section 95] 95. The heterodimeric Fc fusion protein of paragraph 94, wherein the first antibody Fc domain polypeptide comprises the mutations K370E and R409W, and the second antibody Fc domain polypeptide comprises the mutations E357N, D399V, and F405T. [Section 96] 90. The heterodimeric Fc fusion protein according to any one of Items 14 to 19, 60 to 76, and 85 to 89, wherein the first antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of E357N, D399V, F405T, and any combination thereof, and the second antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of K370E, R409W, and any combination thereof. [Section 97] 97. The heterodimeric Fc fusion protein of paragraph 96, wherein the first antibody Fc domain polypeptide comprises the mutations E357N, D399V and F405T, and the second antibody Fc domain polypeptide comprises the mutations K370E and R409W. [Section 98] 90. The heterodimeric Fc fusion protein according to any one of Items 14 to 19, 60 to 76, and 85 to 89, wherein the first antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of K360E, R409W, and a combination thereof, and the second antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of Q347R, D399V, F405T, and any combination thereof. [Section 99] 99. The heterodimeric Fc fusion protein of paragraph 98, wherein the first antibody Fc domain polypeptide comprises the mutations K360E and R409W, and the second antibody Fc domain polypeptide comprises the mutations Q347R, D399V, and F405T. [Section 100] 90. The heterodimeric Fc fusion protein according to any one of Items 14 to 19, 60 to 76, and 85 to 89, wherein the first antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of Q347R, D399V, F405T, and any combination thereof, and the second antibody Fc domain polypeptide comprises one or more mutations selected from the group consisting of K360E, R409W, and any combination thereof. [Section 101] 101. The heterodimeric Fc fusion protein of paragraph 100, wherein the first antibody Fc domain polypeptide comprises the mutations Q347R, D399V and F405T, and the second antibody Fc domain polypeptide comprises the mutations K360E and R409W. [Section 102] 102. The heterodimeric Fc fusion protein of any of paragraphs 1 to 19 or 35 to 101, wherein the first and second antibody Fc domain polypeptides are further mutated to introduce an interchain disulfide bond. [Section 103] 103. The heterodimeric Fc fusion protein of paragraph 102, wherein the first antibody Fc domain polypeptide comprises the mutation Y349C and the second antibody Fc domain polypeptide comprises the mutation S354C. [Section 104] 103. The heterodimeric Fc fusion protein of paragraph 102, wherein the first antibody Fc domain polypeptide comprises the mutation S354C and the second antibody Fc domain polypeptide comprises the mutation Y349C. [Section 105] The heterodimeric Fc fusion protein according to any one of Items 1 to 19 or 35 to 104, wherein the multisubunit protein is a multisubunit cytokine. [Section 106] The heterodimeric Fc fusion protein of paragraph 105, wherein the multi-subunit cytokine is IL-12. [Section 107] 107. The heterodimeric Fc fusion protein of paragraph 106, wherein a first subunit of the multisubunit cytokine is the p40 subunit of human IL-12 and a second, different subunit of the multisubunit cytokine is the p35 subunit of human IL-12. [Section 108] 107. The heterodimeric Fc fusion protein of paragraph 106, wherein a first subunit of the multisubunit cytokine is the p35 subunit of human IL-12 and a second, different subunit of the multisubunit cytokine is the p40 subunit of human IL-12. [Section 109] 109. The heterodimeric Fc fusion protein of paragraph 107 or 108, wherein C74 corresponding to the wild-type p35 sequence is substituted with serine and C177 corresponding to wild-type p40 is substituted with serine, these substitutions eliminating the native disulfide bond formed between the wild-type p35 and wild-type p40 subunits of human IL-12. [Section 110] 110. The heterodimeric Fc fusion protein of paragraph 109, wherein the p35 subunit of human IL-12 comprises a V185C substitution and the p40 subunit of human IL-12 comprises a Y292C substitution, which forms a non-native disulfide bond between the p35 and p40 subunits of human IL-12. [Section 111] Item 111. The heterodimeric Fc fusion protein according to any one of Items 1 to 19 or 35 to 110, further comprising an antibody variable domain. [Section 112] The heterodimeric Fc fusion protein of paragraph 111, wherein the antibody variable domain comprises an antibody heavy chain variable domain. [Section 113] The heterodimeric Fc fusion protein of paragraph 112, wherein the antibody heavy chain variable domain is fused to the N-terminus of the first antibody Fc domain polypeptide or the second antibody Fc domain polypeptide. [Section 114] The heterodimeric Fc fusion protein of paragraph 113, wherein an antibody heavy chain variable domain is linked to an antibody light chain variable domain to form a Fab. [Section 115] 114. The heterodimeric Fc fusion protein of paragraph 113, wherein an antibody heavy chain variable domain is further fused to an antibody light chain variable domain to form an scFv. [Section 116] 116. The heterodimeric Fc fusion protein according to any one of Items 113 to 115, wherein a first subunit and a second different subunit of the multi-subunit protein are fused to the C-terminus of the first antibody Fc domain polypeptide and the C-terminus of the second antibody Fc domain polypeptide, respectively. [Section 117] The heterodimeric Fc fusion protein of paragraph 112, wherein the antibody heavy chain variable domain is fused to the C-terminus of the first antibody Fc domain polypeptide or the second antibody Fc domain polypeptide. [Section 118] 118. The heterodimeric Fc fusion protein of paragraph 117, wherein an antibody heavy chain variable domain is further fused to an antibody light chain variable domain to form an scFv. [Section 119] 119. The heterodimeric Fc fusion protein of paragraph 117 or 118, wherein a first subunit and a second different subunit of the multi-subunit protein are fused to the N-terminus of a first antibody Fc domain polypeptide and the N-terminus of a second antibody Fc domain polypeptide, respectively. [Section 120] 32. The polypeptide according to any one of Items 21 to 31, further comprising an antibody variable domain. [Section 121] The polypeptide of Paragraph 120, wherein the antibody variable domain comprises an antibody heavy chain variable domain. [Section 122] 122. The polypeptide of paragraph 121, wherein the antibody heavy chain variable domain is fused to the N-terminus of the polypeptide. [Section 123] 123. The polypeptide of paragraph 122, wherein an antibody heavy chain variable domain is linked to an antibody light chain variable domain to form a Fab. [Section 124] 123. The polypeptide of paragraph 122, wherein the antibody heavy chain variable domain is further fused to an antibody light chain variable domain to form an scFv. [Section 125] 125. The polypeptide of any of Items 122 to 124, wherein a subunit of a multisubunit cytokine is fused to the C-terminus of an immunoglobulin Fc domain. [Section 126] 122. The polypeptide of paragraph 121, wherein the antibody heavy chain variable domain is fused to the C-terminus of the polypeptide. [Section 127] 127. The polypeptide of paragraph 126, wherein the antibody heavy chain variable domain is further fused to an antibody light chain variable domain to form an scFv. [Section 128] 128. The polypeptide of paragraph 126 or 127, wherein a subunit of the multisubunit cytokine is fused to the N-terminus of an immunoglobulin Fc domain. [Section 129] Item 111. The heterodimeric Fc fusion protein according to any one of Items 1 to 19 or 35 to 110, which does not contain an antibody variable domain. [Section 130] Item 130. The heterodimeric Fc fusion protein of any one of items 1 to 19, 35 to 110, or 129, further comprising a proteoglycan binding domain. [Section 131] 131. The heterodimeric Fc fusion protein of paragraph 130, wherein the proteoglycan binding domain binds to one or more proteoglycans that are specifically expressed in tumors. [Section 132] The heterodimeric Fc fusion protein of paragraph 131, wherein the proteoglycan binding domain binds to one or more proteoglycan...

Claims

1. (a) a first polypeptide comprising a first human IgG1 Fc domain polypeptide and a p40 subunit of human IL-12; and (b) a second polypeptide comprising a second human IgG1 Fc domain polypeptide and a p35 subunit of human IL-12; A heterodimeric Fc fusion protein comprising: (i) the first human IgG1 Fc domain polypeptide comprises the mutations Y349C, K360E and K409W, numbered according to the EU numbering system; (ii) the second human IgG1 Fc domain polypeptide comprises the mutations Q347R, S354C, D399V and F405T, numbered according to the EU numbering system; (iii) each of the first and second human IgG1 Fc domain polypeptides comprises the mutations L234A, L235A and P329A, or L234A, L235A and P329G, as numbered according to the EU numbering system, which reduces an Fc effector function; (iv) each of the first and second human IgG1 Fc domain polypeptides has at least 90% identity to the amino acid sequence of SEQ ID NO: 212; and (v) The p40 and p35 subunits of human IL-12 are bound to each other; Heterodimeric Fc fusion proteins.

2. 2. The heterodimeric Fc-fusion protein of claim 1, wherein the p40 subunit of human IL-12 is fused to the first human IgG1 Fc domain polypeptide by a linker.

3. 3. The heterodimeric Fc-fusion protein of claim 2, wherein the linker comprises the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239).

4. 2. The heterodimeric Fc-fusion protein of claim 1, wherein the p35 subunit of human IL-12 is fused to a second human IgG1 Fc domain polypeptide by a linker.

5. 5. The heterodimeric Fc fusion protein of claim 4, wherein the linker comprises the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10).

6. (a) the p40 subunit of human IL-12 is fused to a first human IgG1 Fc domain polypeptide by a linker comprising the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO:239); and (b) the p35 subunit of human IL-12 is fused to a second human IgG1 Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10); The heterodimeric Fc fusion protein of claim 1.

7. The heterodimeric Fc-fusion protein according to any one of claims 1 to 6, wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:

290.

8. The heterodimeric Fc-fusion protein of any one of claims 1 to 7, wherein the second polypeptide comprises the amino acid sequence of SEQ ID NO:

291.

9. 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.

10. The heterodimeric Fc-fusion protein of any one of claims 1 to 8, wherein the effector function comprises the ability of the Fc to induce antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC).

11. The heterodimeric Fc-fusion protein according to any one of claims 1 to 10, wherein the heterodimeric Fc-fusion protein is fucosylated.

12. 1. A nucleic acid encoding a human IgG1 Fc domain polypeptide and a polypeptide comprising a p40 subunit of human IL-12, wherein the human IgG1 Fc domain polypeptide comprises: (a) Mutations Y349C, K360E and K409W, numbered according to the EU numbering system; (b) the mutations L234A, L235A and P329A, or L234A, L235A and P329G, numbered according to the EU numbering system, which reduce Fc effector function; and (c) at least 90% identical to the amino acid sequence of SEQ ID NO:

212. Including, nucleic acid.

13. 1. A nucleic acid encoding a polypeptide comprising a human IgG1 Fc domain polypeptide and a p35 subunit of human IL-12, wherein the human IgG1 Fc domain polypeptide comprises: (a) the mutations Q347R, S354C, D399V and F405T, numbered according to the EU numbering system; (b) the mutations L234A, L235A and P329A, or L234A, L235A and P329G, numbered according to the EU numbering system, which reduce Fc effector function; and (c) at least 90% identical to the amino acid sequence of SEQ ID NO:

212. Including, nucleic acid.

14. (a) the p40 subunit of human IL-12 is fused to a human IgG1 Fc domain polypeptide by a linker comprising the amino acid sequence PKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 239); and / or (b) the p35 subunit of human IL-12 is fused to a human IgG1 Fc domain polypeptide by a linker comprising the amino acid sequence GGGGSGGGGSGGGGSEPKSSDKTHTCPPCPAPEAAGG (SEQ ID NO: 10); A nucleic acid according to claim 12 or 13.

15. A nucleic acid according to any one of claims 12 to 14, wherein the nucleic acid encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:290 or SEQ ID NO:

291.

16. A nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:290 or SEQ ID NO:

291.

17. One or more expression vectors comprising the nucleic acid of any one of claims 12 to 16.

18. 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.

19. A formulation comprising the heterodimeric Fc-fusion protein of any one of claims 1 to 11 and a pharma- ceutically acceptable carrier.

20. 20. The formulation of claim 19, wherein the formulation is suitable for intravenous, subcutaneous, intraperitoneal, or intratumoral administration.

21. A cell comprising one or more nucleic acids encoding the heterodimeric Fc-fusion protein of any one of claims 1 to 11.

22. A cell comprising one or more nucleic acids according to any one of claims 12 to 16 or one or more expression vectors according to claim 17 or 18.

23. A pharmaceutical composition comprising the heterodimeric Fc-fusion protein of any one of claims 1 to 11, for use in treating cancer or acute radiation syndrome.

24. 24. The pharmaceutical composition of claim 23, wherein the acute radiation syndrome comprises one or more syndromes selected from hematopoietic radiation syndrome, gastrointestinal radiation syndrome, neurovascular radiation syndrome, cutaneous radiation syndrome, and any combination thereof.

25. 24. The pharmaceutical composition of claim 23, (a) the cancer is selected from the group consisting of T cell lymphoma, B cell lymphoma, diffuse large B cell lymphoma, follicular lymphoma, breast cancer, colorectal cancer, pancreatic cancer, acute myeloid leukemia, acute myelomonocytic leukemia, bladder cancer, esophageal cancer, Ewing's sarcoma, gastric cancer, gastrointestinal cancer, gastrointestinal stromal tumors, glioblastoma, head and neck cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), skin cancer, melanoma, mesothelioma, multiple myeloma, myelodysplastic syndrome, renal cell carcinoma, neuroblastoma, non-small cell lung cancer, neuroendocrine tumors, ovarian cancer, prostate cancer, sarcoma, small cell lung cancer, testicular cancer, thymic carcinoma, thyroid cancer, urothelial cancer, cancer infiltrated by myeloid derived suppressor cells, cancer with extracellular matrix deposition, cancer with high levels of reactive stroma, and cancer with neovascularization; and / or (b) the cancer is a metastatic cancer, wherein the metastatic cancer is a local, regional, or distant metastatic cancer; Pharmaceutical compositions.

26. (a) a checkpoint inhibitor, (b) cytokines, (c) a TLR agonist; (d) a STING agonist, (e) a chemotherapeutic agent selected from the group consisting of paclitaxel, nab-paclitaxel, doxorubicin, gemcitabine, and cyclophosphamide; (f) a cancer targeting agent; (g) oncolytic viruses, (h) vaccines; (i) irradiation, (j) Adoptive NK treatment, (k) Stem cell transplantation (SCT) treatment, (l) agonist antibodies, (m) chimeric antigen receptor (CAR) T cell therapy; (n) multispecific binding protein (TriNKET), and (o) an agent that induces cellular senescence; The pharmaceutical composition of any one of claims 23 to 25, further comprising a second therapeutic agent selected from:

27. (a) a checkpoint inhibitor: (i) anti-PD1 antibody, anti-PD-L1 antibody, anti-CTLA4 antibody, anti-KIR antibody, anti-NKG2A antibody, anti-LAG3 antibody and anti-TIM3 antibody; or (ii) nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, ipilimumab, tremelimumab, lirilumab and monalizumab, Selected from: (b) the cytokine is selected from IL-2, IL-15, CXCL9 and CXCL10; (c) a TLR agonist comprising: (i) a TLR7 agonist, a TLR8 agonist, a TLR7 / 8 agonist, a TLR9 agonist, a TLR4 agonist, and a TLR3 agonist; or (ii) R848 / resiquimod, VTX-2337, imiquimod and CpG oligodeoxynucleotides; Selected from: (d) the STING agonist is ADU-S100; and (e) the agonist antibody is selected from an anti-4-IBB / CD137 antibody, an anti-OX40 antibody, an anti-CD40 antibody, an anti-CD40L antibody, an anti-GITR antibody, and an anti-CD27 antibody; 27. The pharmaceutical composition of claim 26.

28. The pharmaceutical composition described in claim 26, wherein the checkpoint inhibitor is an anti-PD1 antibody.

29. TriNKET: (i) a first antigen-binding site that binds to NKG2D; (ii) a second antigen-binding site that binds to a tumor-associated antigen; and (iii) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen-binding site that binds CD16; 27. The pharmaceutical composition of claim 26, comprising:

30. 30. The pharmaceutical composition of claim 29, wherein the tumor associated antigen is selected from the group consisting of EpCAM, CD2, CD19, CD20, CD30, CD38, CD40, CD52, CD70, EGFR / ERBB1, IGF1R, HER3 / ERBB3, HER4 / ERBB4, MUC1, cMET, SLAMF7, PSCA, MICA, MICB, TRAILR1, TRAILR2, MAGE-A3, B7.1, B7.2, CTLA4 and PD-L1.

31. A pharmaceutical composition according to any one of claims 23 to 30, prepared for intravenous, subcutaneous, intraperitoneal or intratumoral administration to a patient.

32. Use of the heterodimeric Fc-fusion protein according to any one of claims 1 to 11 for the manufacture of a pharmaceutical composition used for treating patients with cancer or acute radiation syndrome.

33. (a) introducing into a cell one or more polynucleotides encoding the heterodimeric Fc-fusion protein of any one of claims 1 to 11; and (b) culturing the cells for a period of time sufficient to express the heterodimeric Fc-fusion protein; 21. A method for producing the heterodimeric Fc-fusion protein of any one of claims 1 to 11, or the formulation of claim 19 or 20, comprising:

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